Casarett and Doull's Toxicology. The Basic Science of Poisons [7 ed.] 0071593519, 0071470514

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CASARETT AND DOULL’S

TOXICOLOGY T B S P

HE ASIC CIENCE OF OISONS

What is there that is not poison? All things are poison and nothing (is) without poison. Solely the dose determines that a thing is not a poison. Paracelsus (1493–1541)

NOTICE Medicine is an ever-changing science. As new research and clinical experience broaden our knowledge, changes in treatment and drug therapy are required. The authors and the publisher of this work have checked with sources believed to be reliable in their efforts to provide information that is complete and generally in accord with the standards accepted at the time of publication. However, in view of the possibility of human error or changes in medical sciences, neither the authors nor the publisher nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete, and they disclaim all responsibility for any errors or omissions or for the results obtained from use of the information contained in this work. Readers are encouraged to confirm the information contained herein with other sources. For example and in particular, readers are advised to check the product information sheet included in the package of each drug they plan to administer to be certain that the information contained in this work is accurate and that changes have not been made in the recommended dose or in the contraindications for administration. This recommendation is of particular importance in connection with new or infrequently used drugs.

C ASARETT AND DOULL’S

TOXICOLOGY T B S P HE ASIC CIENCE OF OISONS

Seventh Edition EDITOR

Curtis D. Klaassen, Ph.D. University Distinguished Professor and Chair Department of Pharmacology, Toxicology, and Therapeutics University of Kansas Medical Center Kansas City, Kansas

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Copyright © 2008 by The McGraw-Hill Companies, Inc. All rights reserved. Manufactured in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher. 0-07-159351-9 The material in this eBook also appears in the print version of this title: 0-07-147051-4. All trademarks are trademarks of their respective owners. Rather than put a trademark symbol after every occurrence of a trademarked name, we use names in an editorial fashion only, and to the benefit of the trademark owner, with no intention of infringement of the trademark. Where such designations appear in this book, they have been printed with initial caps. McGraw-Hill eBooks are available at special quantity discounts to use as premiums and sales promotions, or for use in corporate training programs. For more information, please contact George Hoare, Special Sales, at [email protected] or (212) 904-4069. TERMS OF USE This is a copyrighted work and The McGraw-Hill Companies, Inc. (“McGraw-Hill”) and its licensors reserve all rights in and to the work. Use of this work is subject to these terms. Except as permitted under the Copyright Act of 1976 and the right to store and retrieve one copy of the work, you may not decompile, disassemble, reverse engineer, reproduce, modify, create derivative works based upon, transmit, distribute, disseminate, sell, publish or sublicense the work or any part of it without McGraw-Hill’s prior consent. You may use the work for your own noncommercial and personal use; any other use of the work is strictly prohibited. Your right to use the work may be terminated if you fail to comply with these terms. THE WORK IS PROVIDED “AS IS.” McGRAW-HILL AND ITS LICENSORS MAKE NO GUARANTEES OR WARRANTIES AS TO THE ACCURACY, ADEQUACY OR COMPLETENESS OF OR RESULTS TO BE OBTAINED FROM USING THE WORK, INCLUDING ANY INFORMATION THAT CAN BE ACCESSED THROUGH THE WORK VIA HYPERLINK OR OTHERWISE, AND EXPRESSLY DISCLAIM ANY WARRANTY, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. McGraw-Hill and its licensors do not warrant or guarantee that the functions contained in the work will meet your requirements or that its operation will be uninterrupted or error free. Neither McGraw-Hill nor its licensors shall be liable to you or anyone else for any inaccuracy, error or omission, regardless of cause, in the work or for any damages resulting therefrom. McGraw-Hill has no responsibility for the content of any information accessed through the work. Under no circumstances shall McGraw-Hill and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise. DOI: 10.1036/0071470514

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CONTENTS See color insert between pages 760 and 761

Contributors

ix

Preface

xiii

Preface to the First Edition

U N I T

xv

1

GENERAL PRINCIPLES OF TOXICOLOGY

1

1

History and Scope of Toxicology Michael A. Gallo

3

2

Principles of Toxicology David L. Eaton and Steven G. Gilbert

11

3

Mechanisms of Toxicity Zolt´an Gregus

45

4

Risk Assessment Elaine M. Faustman and Gilbert S. Omenn

U N I T

107

2

DISPOSITION OF TOXICANTS

129

5

Absorption, Distribution, and Excretion of Toxicants Lois D. Lehman-McKeeman

131

6

Biotransformation of Xenobiotics Andrew Parkinson and Brian W. Ogilvie

161

7

Toxicokinetics Danny D. Shen

305

U N I T

3

NON-ORGAN-DIRECTED TOXICITY 8

327

Chemical Carcinogens James E. Klaunig and Lisa M. Kamendulis

329 v

vi

9

CONTENTS

Genetic Toxicology R. Julian Preston and George R. Hoffmann

10 Developmental Toxicology John M. Rogers and Robert J. Kavlock

U N I T

381 415

4

TARGET ORGAN TOXICITY

453

11 Toxic Responses of the Blood John C. Bloom and John T. Brandt

455

12 Toxic Responses of the Immune System Norbert E. Kaminski, Barbara L. Faubert Kaplan, and Michael P. Holsapple

485

13 Toxic Responses of the Liver Hartmut Jaeschke

557

14 Toxic Responses of the Kidney Rick G. Schnellmann

583

15 Toxic Responses of the Respiratory System Hanspeter R. Witschi, Kent E. Pinkerton, Laura S. Van Winkle, and Jerold A. Last

609

16 Toxic Responses of the Nervous System Virginia C. Moser, Michael Aschner, Rudy J. Richardson, and Martin A. Philbert

631

17 Toxic Responses of the Ocular and Visual System Donald A. Fox and William K. Boyes

665

18 Toxic Responses of the Heart and Vascular System Y. James Kang

699

19 Toxic Responses of the Skin Robert H. Rice and Theodora M. Mauro

741

20 Toxic Responses of the Reproductive System Paul M.D. Foster and L. Earl Gray Jr.

761

21 Toxic Responses of the Endocrine System Charles C. Capen

807

U N I T

5

TOXIC AGENTS

881

22 Toxic Effects of Pesticides Lucio G. Costa

883

23 Toxic Effects of Metals Jie Liu, Robert A. Goyer, and Michael P. Waalkes

931

CONTENTS

vii

24 Toxic Effects of Solvents and Vapors James V. Bruckner, S. Satheesh Anand, and D. Alan Warren

981

25 Health Effects of Radiation and Radioactive Materials Naomi H. Harley

1053

26 Properties and Toxicities of Animal Venoms John B. Watkins, III

1083

27 Toxic Effects of Plants Stata Norton

1103

U N I T

6

ENVIRONMENTAL TOXICOLOGY

1117

28 Air Pollution Daniel L. Costa

1119

29 Ecotoxicology Richard T. Di Giulio and Michael C. Newman

1157

U N I T

7

APPLICATIONS OF TOXICOLOGY

1189

30 Food Toxicology Frank N. Kotsonis and George A. Burdock

1191

31 Analytic/Forensic Toxicology Alphonse Poklis

1237

32 Clinical Toxicology Louis R. Cantilena Jr.

1257

33 Occupational Toxicology Peter S. Thorne

1273

Index

1293

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CONTRIBUTORS

Michael Aschner, Ph.D.

Louis R. Cantilena Jr.

Gray E.B. Stahlman Chair in Neuroscience Professor of Pediatrics and Pharmacology and Senior Investigator of the Kennedy Center Vanderbilt University Nashville, Tennessee Chapter 16

Professor of Medicine and Pharmacology Director, Division of Clinical Pharmacology and Medical Toxicology Uniformed Services University Bethesda, Maryland Chapter 32

S. Satheesh Anand, Ph.D.

Distinguished University Professor The Ohio State University Department of Veterinary Biosciences Columbus, Ohio Chapter 21

Charles C. Capen, D.V.M., M.Sc., Ph.D.

Research Toxicologist DuPont Haskell Laboratory for Health and Environmental Sciences Newark, Delaware Chapter 24

Luis G. Costa, Ph.D. John C. Bloom, V.M.D., Ph.D.

Professor, Department of Environmental and Occupational Health Sciences University of Washington Seattle, Washington Chapter 22

Executive Director Distinguished Medical Fellow, Diagnostic and Experimental Medicine Eli Lilly and Company Indianapolis, Indiana Chapter 11

Daniel L. Costa, Sc.D. National Program Director for Air Research Office of Research and Development Environmental Protection Agency Research Triangle Park, North Carolina Chapter 28

William K. Boyes, Ph.D. Neurotoxicology Division National Health and Environmential Effects Research Laboratory Office of Research and Development U.S. Environmental Protection Agency Durham, North Carolina Chapter 17

Robert T. Di Giulio, Ph.D. Professor, Nicholas School of the Environment and Earth Sciences Duke University Durham, North Carolina Chapter 29

John T. Brandt, M.D. Medical Fellow II Diagnostic and Experimental Medicine Eli Lilly and Company Indianapolis, Indiana Chapter 11

David L. Eaton, Ph.D. Professor of Environmental and Occupational Health Sciences and Public Health Genetics School of Public Health and Community Medicine and Associate Vice Provost for Research University of Washington Seattle, Washington Chapter 2

James V. Bruckner, Ph.D. Department of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens, Georgia Chapter 24

Elaine M. Faustman Professor and Director Institute for Risk Analysis and Risk Communication Department of Environmental and Occupational Health Sciences University of Washington Seattle, Washington Chapter 4

George A. Burdock, Ph.D., D.A.B.T., F.A.C.N. President, Burdock Group Vero Beach, Florida Chapter 30

ix Copyright © 2008 by The McGraw-Hill Companies, Inc. Click here for terms of use.

x

CONTRIBUTORS

Paul M. D. Foster, Ph.D.

Hartmut Jaeschke, Ph.D.

National Toxicology Program National Institute of Environmental Health Sciences Research Triangle Park, North Carolina Chapter 20

Professor, Department of Pharmacology, Toxicology and Therapeutics University of Kansas Medical Center Kansas City, Kansas Chapter 13

Donald A. Fox, Ph.D.

Lisa M. Kamendulis, Ph.D.

Professor of Vision Sciences, Biology and Biochemistry, and Pharmacology University of Houston Houston, Texas Chapter 17

Assistant Professor Department of Pharmacology and Toxicology Indiana University School of Medicine Indianapolis, Indiana Chapter 8

Michael A. Gallo, Ph.D.

Norbert E. Kaminski, Ph.D.

UMDNJ-Robert Wood Johnson Medical School Piscataway, New Jersey Chapter 1

Steven G. Gilbert, Ph.D., D.A.B.T. Director, Institute of Neurotoxicology and Neurological Discoders (INND) Affilate Associate Professor Department of Environmental and Occupational Health Sciences University of Washington Seattle, Washington Chapter 2

Robert A. Goyer, M.D. Professor Emeritus Department of Pathology University of Western Ontario London, Ontario, Canada Chapter 23

Professor, Pharmacology & Toxicology Director, Center for Integrative Toxicology Michigan State University East Lansing, Michigan Chapter 12

Y. James Kang, D.V.M., Ph.D., F.A.T.S. Professor, Departments of Medicine, and Pharmacology and Toxicolgy University of Louisville School of Medicine Louisville, Kentucky Chapter 18

Barbara L. Faubert Kaplan, Ph.D. Assistant Professor Center for Integrative Toxicology Michigan State University East Lansing, Michigan Chapter 12

Robert J. Kavlock, Ph.D. L. Earl Gray, Jr. Ph.D. NHEERL Reprotoxicology Division Endocrinology Branch U.S. Environmental Protection Agency Research Triangle Park, North Carolina Chapter 20

Zolt´an Gregus, M.D., Ph.D., D.Sc., D.A.B.T. Department of Pharmacology and Therapeutics Toxicology Section University of P´ecs, Medical School P´ecs, Hungary Chapter 3

Naomi H. Harley, Ph.D. New York University School of Medicine Department of Environmental Medicine New York, New York Chapter 25

George R. Hoffman, Ph.D. Professor, Department of Biology College of Holy Cross Worcester, Massachusetts Chapter 9

Michael P. Holsapple, Ph.D., F.A.T.S. Executive Director ILSI Health and Environmental Sciences Institute (HESI) Washington DC Chapter 12

National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park, North Carolina Chapter 10

James E. Klaunig, Ph.D. Robert B. Forney Professor of Toxicology; Director, Center for Environmental Health; Associate Director, IU Cancer Center, School of Medicine, Indiana University Indianapolis, Indiana Chapter 8

Frank N. Kostonis, Ph.D. Department of Food Microbiology and Toxicology Food Research Institute, University of Wisconsin Madison, Wisconsin Chapter 30

Jerold A. Last, Ph.D. Professor, Department of Pulmonary and Critical Care Medicine University of California Davis, California Chapter 15

Lois D. Lehman-McKeeman, Ph.D. Distinguished Research Fellow Discovery Toxicology Bristol-Myers Squibb Company Princeton, New Jersey Chapter 5

CONTRIBUTORS

Jie Liu, Ph.D.

Alphonse Poklis, Ph.D.

Staff Scientist, Inorganic Carcinogenesis Laboratory of Comparative Carcinogenesis National Cancer Institute at NIEHS Research Triangle Park, North Carolina Chapter 23

Professor of Pathology Director of Toxicology Department of Pathology Virginia Commonwealth University Chapter 31

Theodora M. Mauro, M.D.

R. Julian Preston, Ph.D.

Associate Professor in Residence and Vice Chairman Department of Dermatology University of California, San Francisco; and Service Chief, Department of Dermatology VA Medical Center San Francisco San Francisco, California Chapter 19

Associate Director for Health, National Health and Environmental Effects Laboratory U.S. Environmental Protection Agency Research Triangle Park North Carolina Chapter 9

Virginia C. Moser, Ph.D., D.A.B.T. Toxicologist, Neurotoxicology Division National Health and Environmental Effects Research Laboratory US Enviromental Protection Agency Research Triangle Park, North Carolina Chapter 16

Professor of Toxicology Department of Environmental Health Sciences School of Public Health University of Michigan Ann Arbor, Michigan Chapter 16

Michael D. Newman, Ph.D.

Robert H. Rice, Ph.D.

Professor of Marine Science School of Marine Science College of William and Mary Gloucester Point, Virginia Chapter 29

Professor, Department of Environmental Toxicology University of California, Davis Davis, California Chapter 19

Stata Norton, Ph.D.

Chief Developmental Biology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, North Carolina Chapter 10

Emeritus Professor Department of Pharmacology, Toxicology and Therapeutics University of Kansas Medical Center Kansas City, Kansas Chapter 27

Brian W. Ogilvie, B.A. Director of Drug Interactions XenoTech, LLC Lenexa, Kansas Chapter 6

Gilbert S. Omenn, M.D., Ph.D. Professor of Internal Medicine Human Genetics, Public Health and Computational Biology University of Michigan Ann Arbor, Michigan Chapter 4

Andrew Parkinson, Ph.D. Chief Executive Officer XenoTech, LLC Lenexa, Kansas Chapter 6

Martin A. Philbert, Ph.D. Professor and Senior Associate Dean for Research University of Michigan School of Public Health Ann Arbor, Michigan Chapter 16

Kent E. Pinkerton, Ph.D. Professor, Center for Health and the Environment University of California Davis, California Chapter 15

xi

Rudy J. Richardson, S.D., D.A.B.T.

John M. Rogers, Ph.D.

Rick G. Schnellmann, Ph.D. Professor and Chair Department of Pharmaceutical Sciences Medical University of South Carolina Charleston, South Carolina Chapter 14

Danny D. Shen, Ph.D. Professor, Department of Pharmacy and Pharmaceutics School of Pharmacy University of Washington Seattle, Washington Chapter 7

Peter S. Thorne, Ph.D. Professor and Director Environmental Health Sciences Research Center The University of Iowa Iowa City, Iowa Chapter 33

Laura S. Van Winkle, Ph.D. Associate Adjunct Professor Department of Anatomy Physiology and Cell Biology, School of Veteranary Medicine; and Center for Health and the Environment University of California at Davis Davis, California Chapter 15

xii

CONTRIBUTORS

Michael P. Waalkes, Ph.D.

John B. Watkins III, Ph.D., D.A.B.T.

Chief Inorganic Carcinogenesis Section Laboratory of Comparative Carcinogenesis National Cancer Institute at the National Institue of Environmental Health Sciences Research Triangle Park, North Carolina Chapter 23

Assistant Dean and Director Professor of Pharmacology and Toxicology Medical Sciences Program Indiana University School of Medicine Bloomington, Indiana Chapter 26

Hanspeter R. Witschi, M.D., D.A.B.T., F.A.T.S. D. Alan Warren, M.P.H., Ph.D. Program Director Environmental Health Science University of South Carolina Beaufort Beaufort, South Carolina Chapter 24

Professor of Toxicology Institute of Toxicology and Environmental Health and Department of Molecular Biosciences School of Veterinary Medicine University of California Davis, California Chapter 15

PREFACE

This edition reflects the marked progress made in toxicology during the last few years. For example, the importance of apoptosis, cytokines, growth factors, oncogenes, cell cycling, receptors, gene regulation, transcription factors, signaling pathways, transgenic animals, “knock-out” animals, polymorphisms, microarray technology, genomics, proteonomics, etc., in understanding the mechanisms of toxicity are included in this edition. More information on environmental hormones is also included. References in this edition include not only traditional journal and review articles, but, internet sites also. (Readers who would like a PowerPoint version of the figures and tables can obtain the same from the publisher.) The editor is grateful to his colleagues in academia, industry, and government who have made useful suggestions for improving this edition, both as a book and as a reference source. The editor is especially thankful to all the contributors, whose combined expertise has made possible a volume of this breadth. I especially recognize John Doull, the original editor of this book, for his continued support.

The seventh edition of Casarett and Doull’s Toxicology: The Basic Science of Poisons, as the previous six, is meant to serve primarily as a text for, or an adjunct to, graduate courses in toxicology. Because the six previous editions have been widely used in courses in environmental health and related areas, an attempt has been made to maintain those characteristics that make it useful to scientists from other disciplines. This edition will again provide information on the many facets of toxicology, especially the principles, concepts, and modes of thoughts that are the foundation of the discipline. Mechanisms of toxicity are emphasized. Research toxicologists will find this book an excellent reference source to find updated material in areas of their special or peripheral interests. The overall framework of the seventh edition is similar to the sixth edition. The seven units are “General Principles of Toxicology” (Unit 1), “Disposition of Toxicants” (Unit 2), “Non-Organ-Directed Toxicity” (carcinogenicity, mutagenicity, and teratogenicity) (Unit 3), “Target Organ Toxicity” (Unit 4), “Toxic Agents” (Unit 5), “Environmental Toxicology” (Unit 6), and “Applications of Toxicology” (Unit 7).

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PREFACE TO THE FIRST EDITION

by chemical or use characteristics. In the final section (Unit IV) an attempt has been made to illustrate the ramifications of toxicology into all areas of the health sciences and even beyond. This unit is intended to provide perspective for the nontoxicologist in the application of the results of toxicologic studies and a better understanding of the activities of those engaged in the various aspects of the discipline of toxicology. It will be obvious to the reader that the contents of this book represent a compromise between the basic, fundamental, mechanistic approach to toxicology and the desire to give a view of the broad horizons presented by the subject. While it is certain that the editors’ selectivity might have been more severe, it is equally certain that it could have been less so, and we hope that the balance struck will prove to be appropriate for both toxicologic training and the scientific interest of our colleague. L.J.C. J.D. Although the philosophy and design of this book evolved over a long period of friendship and mutual respect between the editors, the effort needed to convert ideas into reality was undertaken primarily by Louis J. Casarett. Thus, his death at a time when completion of the manuscript was in sight was particularly tragic. With the help and encouragement of his wife, Margaret G. Casarett, and the other contributors, we have finished Lou’s task. This volume is a fitting embodiment of Louis J. Casarett’s dedication to toxicology and to toxicologic education. J.D.

This volume has been designed primarily as a textbook for, or adjunct to, courses in toxicology. However, it should also be of interest to those not directly involved in toxicologic education. For example, the research scientist in toxicology will find sections containing current reports on the status of circumscribed areas of special interest. Those concerned with community health, agriculture, food technology, pharmacy, veterinary medicine, and related disciplines will discover the contents to be most useful as a source of concepts and modes of thought that are applicable to other types of investigative and applied sciences. For those further removed from the field of toxicology or for those who have not entered a specific field of endeavor, this book attempts to present a selectively representative view of the many facets of the subject. Toxicology: The Basic Science of Poisons has been organized to facilitate its use by these different types of users. The first section (Unit I) describes the elements of method and approach that identify toxicology. It includes those principles most frequently invoked in a full understanding of toxicologic events, such as dose-response, and is primarily mechanistically oriented. Mechanisms arc also stressed in the subsequent sections of the book, particularly when these are well identified and extend across classic forms of chemicals and systems. However, the major focus in the second section (Unit II) is on the systemic site of action of toxins. The intent therein is to provide answers to two questions: What kinds of injury are produced in specific organs or systems by toxic agents? What are the agents that produce these effects? A more conventional approach to toxicology has been utilized in the third section (Unit III), in which the toxic agents are grouped

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CASARETT AND DOULL’S

TOXICOLOGY T B S P

HE ASIC CIENCE OF OISONS

What is there that is not poison? All things are poison and nothing (is) without poison. Solely the dose determines that a thing is not a poison. Paracelsus (1493–1541)

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UNIT 1

GENERAL PRINCIPLES OF TOXICOLOGY

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CHAPTER 1

HISTORY AND SCOPE OF TOXICOLOGY Michael A. Gallo HISTORY OF TOXICOLOGY

MODERN TOXICOLOGY

Antiquity Middle Ages Age of Enlightenment

AFTER WORLD WAR II

Toxicology, like medicine, is both a science and an art. The science of toxicology is defined as the observational and data-gathering phase, whereas the art of toxicology consists of utilization of data to predict outcomes of exposure in human and animal populations. In most cases, these phases are linked because the facts generated by the science of toxicology are used to develop extrapolations and hypotheses to explain the adverse effects of chemical agents in situations where there is little or no information. For example, the observation that the administration of TCDD (2,3,7,8-tetrachlorodibenzop-dioxin) to female Sprague Dawley rats induces hepatocellular carcinoma is a fact. However, the conclusion that it will also have a similar affect in humans is unclear whether it is a prediction or hypothesis. Therefore, it is important to distinguish facts from predictions. When we fail to distinguish the science from the art, we confuse facts with predictions and argue that they have equal validity, which they clearly do not suggest. In toxicology, as in all sciences, theories have a higher level of certainty than do hypotheses, which in turn are more certain than speculations, opinions, conjectures, and guesses. An insight into modern toxicology and the roles, points of view, and activities of toxicologists can be obtained by examining the evolution of this discipline.

Toxicology has been defined as the study of the adverse effects of xenobiotics and thus is a borrowing science that has evolved from ancient poisoners. Modern toxicology goes beyond the study of the adverse effects of exogenous agents to the study of molecular biology, using toxicants as tools. Currently, many toxicologists are studying the mechanisms of endogenous compounds such as oxygen radicals and other reactive intermediates generated from xenobiotics and endobiotics. Historically, toxicology formed the basis of therapeutics and experimental medicine. Toxicology in this and last century (1900 to the present) continues to develop and expand by assimilating knowledge and techniques from most branches of biology, chemistry, mathematics, and physics. A recent addition to the field of toxicology (1975 to the present) is the application of this discipline to safety evaluation and risk assessment. The contributions and activities of toxicologists are diverse and widespread. In this biomedical area, toxicologists are concerned with mechanisms of action and exposure to chemicals as a cause of acute and chronic illness. Toxicologists contribute to physiology and pharmacology by using toxic chemicals to understand physiological phenomena. They are involved in the recognition, identification, and quantification of hazards resulting from occupational exposure to chemicals and the public health aspects of chemicals in air, water, other parts of the environment, food, and drugs. Traditionally, toxicologists have been intimately involved in the discovery and development of new drugs, food additives, and pesticides. Toxicologists also participate in the development of standards and regulations designed to protect human health and the environment from the adverse effects of chemicals. Environmental toxicologists (a relatively new subset of the discipline) have expanded toxicology to study the effects of chemicals on flora and fauna. Molecular toxicologists are studying the mechanisms by which toxicants modulate cell growth and differentiation and how cells respond to toxicants at the level of the gene. In all branches of toxicology, scientists explore the mechanisms and modes of action by which chemicals produce adverse effects in biological systems. Clinical toxicologists develop antidotes and treatment regimens to ameliorate poisonings from xenobiotic injury. Toxicologists carry out some or all of these activities as members of academic, industrial, and governmental organizations. In fact, these activities help them to share methodologies for obtaining data for toxicity of materials and to make reasonable predictions regarding the hazards of the material to people and the environment using this data. Although different, these complementary activities characterize the discipline of toxicology.

HISTORY OF TOXICOLOGY Antiquity Toxicology dates back to the earliest humans, who used animal venom and plant extracts for hunting, warfare, and assassination. The knowledge of these poisons must have predated recorded history. It is safe to assume that prehistoric humans categorized some plants as harmful and others as safe. The same is probably true for the classification of snakes and other animals. The Ebers papyrus (circa 1500 BC) contains information pertaining to many recognized poisons, including hemlock (the state poison of the Greeks), aconite (a Chinese arrow poison), opium (used as both a poison and an antidote), and metals such as lead, copper, and antimony. There is also an indication that plants containing substances similar to digitalis and belladonna alkaloids were known. Hippocrates (circa 400 BC) added a number of poisons and clinical toxicology principles pertaining to bioavailability in therapy and overdosage, while the Book of Job (circa 400 BC) speaks of poison arrows (Job 6:4). In the literature of ancient Greece, there are several references to poisons and their use. Some interpretations of Homer have Odysseus obtaining poisons for his arrows (Homer, circa 600 BC). Theophrastus (370–286 BC), a student of Aristotle, included numerous references to poisonous 3

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4

UNIT 1

GENERAL PRINCIPLES OF TOXICOLOGY

plants in De Historia Plantarum. Dioscorides, a Greek physician in the court of the Roman emperor Nero, made the first attempt to classify poisons, which was accompanied by descriptions and drawings. His classification into plant, animal, and mineral poisons not only remained a standard for 16 centuries but is still a convenient classification (Gunther, 1934). Dioscorides also dabbled in therapy, recognizing the use of emetics in poisoning and the use of caustic agents and cupping glasses in snakebite. Poisoning with plant and animal toxins was quite common. Perhaps the best-known recipient of poison used as a state method of execution was Socrates (470– 399 BC), whose cup of hemlock extract was apparently estimated to be the proper dose. Expeditious suicide on a voluntary basis also made use of toxicologic knowledge. Demosthenes (385–322 BC), who took poison hidden in his pen, was one of many examples. The mode of suicide calling for one to fall on his sword, although manly and noble, carried little appeal and less significance for the women of the day. Cleopatra’s (69–30 BC) knowledge of natural primitive toxicology permitted her to use the more genteel method of falling on her asp. The Romans too made considerable use of poisons in politics. One legend tells of King Mithridates VI of Pontus, whose numerous acute toxicity experiments on unfortunate criminals led to his eventual claim that he had discovered an antidote for every venomous reptile and poisonous substance (Guthrie, 1946). Mithridates was so fearful of poisons that he regularly ingested a mixture of 36 ingredients (Galen reports 54) as protection against assassination. On the occasion of his imminent capture by enemies, his attempts to kill himself with poison failed because of his successful antidote concoction, and he was forced to use a sword held by a servant. From this tale comes the term “mithridatic,” referring to an antidotal or protective mixture. The term “theriac” has also become synonymous with “antidote,” although the word comes from the poetic treatise Theriaca by Nicander of Colophon (204–135 BC), which dealt with poisonous animals; his poem “Alexipharmaca” was about antidotes. Poisonings in Rome reached epidemic proportions during the fourth century BC (Livy). It was during this period that a conspiracy of women to remove men from whose death they might profit was uncovered. Similar large-scale poisoning continued until Sulla issued the Lex Cornelia (circa 82 BC). This appears to be the first law against poisoning, and it later became a regulatory statute directed at careless dispensers of drugs. Nero (AD 37–68) used poisons to do away with his stepbrother Brittanicus and employed his slaves as food tasters to differentiate edible mushrooms from their more poisonous kin.

Middle Ages Come bitter pilot, now at once run on The dashing rocks thy seasick weary bark! Here’s to my love! O true apothecary! Thy drugs are quick. Thus with a kiss I die. (Romeo and Juliet, act 5, scene 3)

Before the Renaissance, the writings of Maimonides (Moses ben Maimon, AD 1135–1204) included a treatise on the treatment of poisonings from insects, snakes, and mad dogs (Poisons and Their Antidotes, 1198). Maimonides, like Hippocrates before him, wrote on the subject of bioavailability, noting that milk, butter, and cream could delay intestinal absorption. Malmonides also refuted many of the popular remedies of the day and stated his doubts about others. It is rumored that alchemists of this period (circa AD 1200), in search

of the universal antidote, learned to distill fermented products and made a 60% ethanol beverage that had many interesting powers. In the early Renaissance, the Italians, with characteristic pragmatism, brought the art of poisoning to its zenith. The poisoner became an integral part of the political scene. The records of the city councils of Florence, particularly those of the infamous Council of Ten of Venice, contain ample testimony about the political use of poisons. Victims were named, prices set, and contracts recorded; when the deed was accomplished, payment was made. An infamous figure of the time was a lady named Toffana who peddled specially prepared arsenic-containing cosmetics (Agua Toffana). Accompanying the product were appropriate instructions for its use. Toffana was succeeded by an imitator with organizational genius, Hieronyma Spara, who provided a new fillip by directing her activities toward specific marital and monetary objectives. A local club was formed of young, wealthy, married women, which soon became a club of eligible young wealthy widows, reminiscent of the matronly conspiracy of Rome centuries earlier. Incidentally, arseniccontaining cosmetics were reported to be responsible for deaths well into the twentieth century (Kallett and Schlink, 1933). Among the prominent families engaged in poisoning, the Borgias were the most notorious. However, many deaths that were attributed to poisoning are now recognized as having resulted from infectious diseases such as malaria. It appears true, however, that Alexander VI, his son Cesare, and Lucrezia Borgia were quite active. The deft application of poisons to men of stature in the Catholic Church swelled the holdings of the papacy, which was their prime heir. In this period Catherine de Medici exported her skills from Italy to France, where the prime targets of women were their husbands. However, unlike poisoners of an earlier period, the circle represented by Catherine and epitomized by the notorious Marchioness de Brinvillers depended on developing direct evidence to arrive at the most effective compounds for their purposes. Under the guise of delivering provender to the sick and the poor, Catherine tested toxic concoctions, carefully noting the rapidity of the toxic response (onset of action), the effectiveness of the compound (potency), the degree of response of the parts of the body (specificity, site of action), and the complaints of the victim (clinical signs and symptoms). The culmination of the practice in France is represented by the commercialization of the service by Catherine Deshayes, who earned the title “La Voisine.” Her business was dissolved by her execution. Her trial was one of the most famous of those held by the Chambre Ardente, a special judicial commission established by Louis XIV to try such cases without regard to age, sex, or national origin. La Voisine was convicted of many poisonings, with over 2000 infants among her victims.

Age of Enlightenment All substances are poisons; there is none which is not a poison. The right dose differentiates poison from a remedy. Paracelsus A significant figure in the history of science and medicine in the late Middle Ages was the renaissance man Philippus Aureolus Theophrastus Bombastus von Hohenheim-Paracelsus (1493–1541). Between the time of Aristotle and the age of Paracelsus, there was little substantial change in the biomedical sciences. In the sixteenth century, the revolt against the authority of the Catholic Church was accompanied by a parallel attack on the godlike authority exercised

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by the followers of Hippocrates and Galen. Paracelsus personally and professionally embodied the qualities that forced numerous changes in this period. He and his age were pivotal, standing between the philosophy and magic of classical antiquity and the philosophy and science willed to us by figures of the seventeenth and eighteenth centuries. Clearly, one can identify in Paracelsus’s approach, point of view, and breadth of interest numerous similarities to the discipline that is now called toxicology. Paracelsus, a physician-alchemist and the son of a physician, formulated many revolutionary views that remain an integral part of the structure of toxicology, pharmacology, and therapeutics today (Pagel, 1958). He promoted a focus on the “toxicon,” the primary toxic agent, as a chemical entity, as opposed to the Grecian concept of the mixture or blend. A view initiated by Paracelsus that became a lasting contribution held as corollaries that (1) experimentation is essential in the examination of responses to chemicals, (2) one should make a distinction between the therapeutic and toxic properties of chemicals, (3) these properties are sometimes but not always indistinguishable except by dose, and (4) one can ascertain a degree of specificity of chemicals and their therapeutic or toxic effects. These principles led Paracelsus to introduce mercury as the drug of choice for the treatment of syphilis, a practice that survived 300 years but led to his famous trial. This viewpoint presaged the “magic bullet” (arsphenamine) of Paul Ehrlich and the introduction of the therapeutic index. Further, in a very real sense, this was the first sound articulation of the dose–response relation, a bulwark of toxicology (Pachter, 1961). The tradition of the poisoners spread throughout Europe, and their deeds played a major role in the distribution of political power throughout the Middle Ages. Pharmacology as it is known today had its beginnings during the Middle Ages and early Renaissance. Concurrently, the study of the toxicity and the dose–response relationship of therapeutic agents was commencing. The occupational hazards associated with metalworking were recognized during the fifteenth century. Early publications by Ellenbog (circa 1480) warned of the toxicity of the mercury and lead exposures involved in goldsmithing. Agricola published a short treatise on mining diseases in 1556. However, the major work on the subject, On the Miners’ Sickness and Other Diseases of Miners (1567), was published by Paracelsus. This treatise addressed the etiology of miners’ disease, along with treatment and prevention strategies. Occupational toxicology was further advanced by the work of Bernardino Ramazzini. His classic, published in 1700 and entitled Discourse on the Diseases of Workers, set the standard for occupational medicine well into the nineteenth century. Ramazzini’s work broadened the field by discussing occupations ranging from miners to midwives and including printers, weavers, and potters. The developments of the Industrial Revolution stimulated a rise in many occupational diseases. Percival Pott’s (1775) recognition of the role of soot in scrotal cancer among chimney sweepers was the first reported example of polyaromatic hydrocarbon carcinogenicity, a problem that still plagues toxicologists today. These findings led to improved medical practices, particularly in prevention. It should be noted that Paracelsus and Ramazzini also pointed out the toxicity of smoke and soot. The nineteenth century dawned in a climate of industrial and political revolution. Organic chemistry was in its infancy in 1800, but by 1825 phosgene (COCl2 ) and mustard gas (bis[Bchloroethyl]sulfide) had been synthesized. These two chemicals were used in World War I as war gases, and as late as the Iraq– Iran War in the late twentieth century. By 1880 over 10,000 organic

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compounds had been synthesized including chloroform, carbon tetrachloride, diethyl ether, and carbonic acid, and petroleum and coal gasification by-products were used in trade (Zapp, 1982). The toxicity of benzene was established at the turn of the twentieth century. Determination of the toxicologic potential of these newly created chemicals became the underpinning of the science of toxicology as it is practiced today. However, there was little interest during the mid-nineteenth century in hampering industrial development. Hence, the impact of industrial toxicology discoveries was not felt until the passage of worker’s insurance laws, first in Germany (1883), then in England (1897), and later in the United States (1910). Experimental toxicology accompanied the growth of organic chemistry and developed rapidly during the nineteenth century. Magendie (1783–1885), Orfila (1787–1853), and Bernard (1813– 1878) carried out truly seminal research in experimental toxicology and laid the groundwork for pharmacology and experimental therapeutics as well as occupational toxicology. Orfila, a Spanish physician in the French court, was the first toxicologist to use autopsy material and chemical analysis systematically as legal proof of poisoning. His introduction of this detailed type of analysis survives as the underpinning of forensic toxicology (Orfila, 1818). Orfila published the first major work devoted expressly to the toxicity of natural agents (1815). Magendie, a physician and experimental physiologist, studied the mechanisms of action of emetine, strychnine, and “arrow poisons” (Olmsted, 1944). His research into the absorption and distribution of these compounds in the body remains a classic in toxicology and pharmacology. One of Magendie’s more famous students, Claude Bernard, continued the study of arrow poisons (Bernard, 1850) but also added works on the mechanism of action of carbon monoxide. Bernard’s treatise, An Introduction to the Study of Experimental Medicine (translated by Greene in 1949), is a classic in the development of toxicology. Many German scientists contributed greatly to the growth of toxicology in the late nineteenth and early twentieth centuries. Among the giants of the field are Oswald Schmiedeberg (1838– 1921) and Louis Lewin (1850–1929). Schmiedeberg made many contributions to the science of toxicology, not the least of which was the training of approximately 120 students who later populated the most important laboratories of pharmacology and toxicology throughout the world. Many of today’s toxicologists and pharmacologists can trace their scientific heritage back to Schmiedeberg. His research focused on the synthesis of hippuric acid in the liver and the detoxification mechanisms of the liver in several animal species (Schmiedeberg and Koppe, 1869). Lewin, who was educated originally in medicine and the natural sciences, trained in toxicology under Liebreich at the Pharmacological Institute of Berlin (1881). His contributions on the chronic toxicity of narcotics and other alkaloids remain a classic. Lewin also published much of the early work on the toxicity of methanol, glycerol, acrolein, and chloroform (Lewin, 1920, 1929).

MODERN TOXICOLOGY Toxicology has evolved rapidly during the 1900s. The exponential growth of the discipline can be traced to the World War II era with its marked increase in the production of drugs, pesticides, munitions, synthetic fibers, and industrial chemicals. The history of many sciences represents an orderly transition based on theory, hypothesis testing, and synthesis of new ideas. Toxicology, as a gathering and an applied science, has, by contrast, developed in fits and starts. Toxicology calls on almost all the basic sciences to test

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its hypotheses. This fact, coupled with the health and occupational regulations that have driven toxicology research since 1900, has made this discipline exceptional in the history of science. The differentiation of toxicology as an art and a science, though arbitrary, permits the presentation of historical highlights along two major lines. Modern toxicology can be viewed as a continuation of the development of the biological and physical sciences in the late nineteenth and twentieth centuries (Table 1-1). During the second half of the nineteenth century, the world witnessed an explosion in science that produced the beginning of the modern era of genetics, medicine, synthetic chemistry, physics, and biology. Toxicology has drawn its strength and diversity from its proclivity to borrowing. With the advent of anesthetics and disinfectants and the advancement of experimental pharmacology in the late 1850s, toxicology as it is currently understood got its start. The introduction of ether, chloroform, and carbonic acid led to several iatrogenic deaths. These unfortunate outcomes spurred research into the causes of the deaths and early experiments on the physiological mechanisms by which these compounds caused both beneficial and adverse effects. By the late nineteenth century the use of organic chemicals was becoming more

widespread, and benzene, toluene, and the xylenes went into largerscale commercial production. Interestingly, benzene was used as a drug to treat leukemia in the early 1900s. During this period, the use of “patent” medicines was prevalent, and there were several incidents of poisonings from these medicaments. The adverse reactions to patent medicines, coupled with the response to Upton Sinclair’s expos´e of the meat-packing industry in The Jungle, culminated in the passage of the Wiley Bill (1906), the first of many U.S. pure food and drug laws (see Hutt and Hutt, 1984, for regulatory history). A working hypothesis about the development of toxicology is that the discipline expands in response to legislation, which itself is a response to a real or perceived tragedy. The Wiley bill was the first such reaction in the area of food and drugs, and the worker’s compensation laws cited above were a response to occupational toxicities. In addition, the National Safety Council was established in 1911, and the Division of Industrial Hygiene was established by the U.S. Public Health Service in 1914. A corollary to this hypothesis might be that the founding of scientific journals and/or societies is sparked by the development of a new field. The Journal of Industrial Hygiene began in 1918. The major chemical manufacturers in

Table 1.1 Selection of Developments in Toxicology Development of early advances in analytic methods Marsh, 1836: development of method for arsenic analysis Reinsh, 1841: combined method for separation and analysis of As and Hg Fresenius, 1845, and von Babo, 1847: development of screening method for general poisons Stas-Otto, 1851: detection and identification of phosphorus Early mechanistic studies F. Magendie, 1809: study of “arrow poisons,” mechanism of action of emetine and strychnine C. Bernard, 1850: carbon monoxide combination with hemoglobin, study of mechanism of action of strychnine, site of action of curare R. Bohm, ca. 1890: active anthelmintics from fern, action of croton oil catharsis, poisonous mushrooms Introduction of new toxicants and antidotes R. A. Peters, L. A. Stocken, and R. H. S. Thompson, 1945: development of British Anti Lewisite BAL) as a relatively specific antidote for arsenic, toxicity of monofluorocarbon compounds K. K. Chen, 1934: introduction of modern antidotes (nitrite and thiosulfate) for cyanide toxicity C. Voegtlin, 1923: mechanism of action of As and other metals on the SH groups P. M¨uller, 1944–1946: introduction and study of DDT (dichlorodiphenyltrichloroethane) and related insecticide compounds G. Schrader, 1952: introduction and study of organophosphorus compounds R. N. Chopra, 1933: indigenous drugs of India Miscellaneous toxicologic studies R. T. Williams: study of detoxication mechanisms and species variation A. Rothstein: effects of uranium ion on cell membrane transport R. A. Kehoe: investigation of acute and chronic effects of lead A. Vorwald: studies of chronic respiratory disease (beryllium) H. Hardy: community and industrial poisoning (beryllium) A. Hamilton: introduction of modern industrial toxicology H. C. Hodge: toxicology of uranium, fluorides; standards of toxicity A. Hoffman: introduction of lysergic acid and derivatives; pscyhotomimetics R. A. Peters: biochemical lesions, lethal synthesis A. E. Garrod: inborn errors of metabolism T. T. Litchfield and F. Wilcoxon: simplified dose-response evaluation C. J. Bliss: method of probits, calculation of dosage-mortality curves

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the United States (Dow, Union Carbide, and DuPont) established internal toxicology research laboratories to help guide decisions on worker health and product safety. During the 1890s and early 1900s, the French scientists Becquerel and the Curies reported the discovery of “radioactivity.” This opened up for exploration a very large area in physics, biology, and medicine, but it would not affect the science of toxicology for another 40 years. However, another discovery, that of vitamins, or “vital amines,” was to lead to the use of the first large-scale bioassays (multiple animal studies) to determine whether these “new” chemicals were beneficial or harmful to laboratory animals. The initial work in this area took place at around the time of World War I in several laboratories, including the laboratory of Philip B. Hawk in Philadelphia. Hawk and a young associate, Bernard L. Oser, were responsible for the development and verification of many early toxicologic assays that are still used in a slightly amended form. Oser’s contributions to food and regulatory toxicology were extraordinary. These early bioassays were made possible by two major advances in toxicology: the availability of developed and refined strains of inbred laboratory rodents (Donaldson, 1912), and the analytical chemical capability to assay urine and blood for residues. The 1920s saw many events that began to mold the fledgling field of toxicology. The use of arsenicals for the treatment of diseases such as syphilis (arsenicals had been used in agriculture since the mid-nineteenth century) resulted in acute and chronic toxicity. Prohibition of alcoholic beverages in the United States opened the door for early studies of neurotoxicology, with the discovery that triorthocresyl phosphate (TOCP), methanol, and lead (all products of “bootleg” liquor) are neurotoxicants. TOCP, which until recently was a gasoline additive, caused a syndrome that became known as “ginger-jake” walk, a spastic gait resulting from drinking adulterated ginger beer. Mueller’s discovery of DDT (dichlorodiphenyl-trichloroethane) and several other organohalides, such as hexachlorobenzene and hexachlorocyclohexane, during the late 1920s resulted in wider use of insecticidal agents. Other scientists were hard at work attempting to elucidate the structures and activity of the estrogens and androgens. Work on the steroid hormones led to the use of several assays for the determination of the biological activity of organ extracts and synthetic compounds. Efforts to synthesize steroid-like chemicals were spearheaded by E. C. Dodds and his co-workers, one of whom was Leon Golberg, a young organic chemist. Dodds’s work on the bioactivity of the estrogenic compounds resulted in the synthesis of diethylstilbestrol (DES), hexestrol, and other stilbenes and the discovery of the strong estrogenic activity of substituted stilbenes; eventually leading to the Nobel Prize in Medicine. Golberg’s intimate involvement in this work stimulated his interest in biology, leading to degrees in biochemistry and medicine and a career in toxicology in which he oversaw the creation of the laboratories of the British Industrial Biological Research Association (BIBRA) and the Chemical Industry Institute of Toxicology (CIIT). Interestingly, the initial observations that led to the discovery of DES were the findings of feminization of animals treated with the experimental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The 1930s saw the world preparing for World War II and a major effort by the pharmaceutical and chemical industries in Germany and the United States to manufacture the first mass-produced antibiotics, and warfare agents. One of the first journals expressly dedicated to experimental toxicology, Archiv f¨ur Toxikologie, began publication in Europe in 1930, the same year that Herbert Hoover

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signed the act that established the National Institutes of Health (NIH) in the United States. The discovery of sulfanilamide was heralded as a major event in combating bacterial diseases. However, for a drug to be effective, there must be a reasonable delivery system, and sulfanilamide is highly insoluble in an aqueous medium. Therefore, it was originally prepared in ethanol (elixir). However, it was soon discovered that the drug was more soluble in diethylene glycol, which is a dihydroxy rather than a monohydroxy ethane. The drug was sold in the diethylene glycol solution but was labeled as an elixir, and several patients died of acute renal failure resulting from the metabolism of the glycol to oxalic acid and glycolic acid, with the acids, along with the active drug, crystallizing in the kidney tubules. This tragic event led to the passage of the Copeland bill in 1938, the second major bill involving the formation of the U.S. Food and Drug Administration (FDA). The sulfanilamide disaster played a critical role in the further development of toxicology, resulting in work by Eugene Maximillian Geiling (a direct scientific offspring of John Jacob Abel and Schmiedeberg) in the Pharmacology Department of the University of Chicago that elucidated the mechanism of toxicity of both sulfanilamide and ethylene glycol. Studies of the glycols were simultaneously carried out at the U.S. FDA by a group led by Arnold Lehman. The scientists associated with Lehman and Geiling were to become the leaders of toxicology over the next 40 years. With few exceptions, toxicology in the United States owes its heritage to Geiling’s innovativeness and ability to stimulate and direct young scientists and Lehman’s vision of the use of experimental toxicology in public health decision making. Because of Geiling’s reputation, the U.S. government turned to this group for help in the war effort. There were three main areas in which the Chicago group took part during World War II: the toxicology and pharmacology of organophosphate chemicals, antimalarial drugs, and radionuclides. Each of these areas produced teams of toxicologists who became academic, governmental, and industrial leaders in the field. It was also during this time that DDT and the phenoxy herbicides were developed for increased food production and, in the case of DDT, control of insect-borne diseases. These efforts between 1940 and 1946 led to an explosion in toxicology. Thus, in line with the hypothesis advanced above, the crisis of World War II caused the next major leap in the development of toxicology. If one traces the history of the toxicology of metals over the past 45 years, the role of the Chicago group, and Rochester, is quite apparent. This story commences with the use of uranium for the “bomb” and continues today with research on the role of metals in their interactions with DNA, RNA, and growth factors. Indeed, the Manhattan Project created a fertile environment that resulted in the initiation of quantitative biology, drug metabolism and structure activity relationships (with antimalarials), radiotracer technology, and inhalation toxicology. These innovations have revolutionized modern biology, chemistry, therapeutics, and toxicology. Inhalation toxicology began at the University of Rochester under the direction of Stafford Warren, who headed the Department of Radiology. He developed a program with colleagues such as Harold Hodge (pharmacologist), Herb Stokinger (chemist), Sid Laskin (inhalation toxicologist), and Lou and George Casarett (toxicologists). These young scientists were to go on to become giants in the field. The other sites for the study of radionuclides were Chicago for the “internal” effects of radioactivity and Oak Ridge, Tennessee, for the effects of “external” radiation. The work of the scientists on these teams gave the scientific community data that contributed to the early understanding of macromolecular binding of xenobiotics,

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cellular mutational events, methods for inhalation toxicology and therapy, and toxicological properties of trace metals, along with a better appreciation of the complexities of the dose–response curve. Another seminal event in toxicology that occurred during the World War II era was the discovery of organophosphate cholinesterase inhibitors. This class of chemicals, which was discovered by Willy Lange and Gerhard Schrader, was destined to become a driving force in the study of neurophysiology and toxicology for several decades. Again, the scientists in Chicago played major roles in elucidating the mechanisms of action of this new class of compounds. Geiling’s group, Kenneth Dubois in particular, were leaders in this area of toxicology and pharmacology. Dubois’s students, particularly Sheldon Murphy (and his students), continued to be in the forefront of this special area. The importance of the early research on the organophosphates has taken on special meaning in the years since 1960, when these non-bioaccumulating insecticides were destined to replace DDT and other organochlorine insectides. Early in the twentieth century, it was demonstrated experimentally that quinine has a marked effect on the malaria parasite [it had been known for centuries that chincona bark extract is efficacious for “Jesuit fever” (malaria)]. This discovery led to the development of quinine derivatives for the treatment of the disease and the formulation of the early principles of chemotherapy. The pharmacology department at Chicago was charged with the development of antimalarials for the war effort. The original protocols called for testing of efficacy and toxicity in rodents and perhaps dogs and then the testing of efficacy in human volunteers. One of the investigators charged with generating the data needed to move a candidate drug from animals to humans was Fredrick Coulston. This young parasitologist and his colleagues, working under Geiling, were to evaluate potential drugs in animal models and then establish human clinical trials. It was during these experiments that the use of nonhuman primates came into vogue for toxicology testing. It had been noted by Russian scientists that some antimalarial compounds caused retinopathies in humans but did not apparently have the same adverse effect in rodents and dogs. This finding led the Chicago team to add one more step in the development process: toxicity testing in rhesus monkeys just before efficacy studies in people. This resulted in the prevention of blindness in untold numbers of volunteers and perhaps some of the troops in the field. It also led to the school of thought that nonhuman primates may be one of the better models for humans and the establishment of primate colonies for the study of toxicity. Coulston pioneered this area of toxicology and remained committed to it until his death in 2003. Another area not traditionally thought of as toxicology but one that evolved during the 1940s as an exciting and innovative field is experimental pathology. This branch of experimental biology developed from bioassays of estrogens and early experiments in chemicaland radiation-induced carcinogenesis. It is from these early studies that hypotheses on tumor promotion and cancer progression have evolved. Toxicologists today owe a great deal to the researchers of chemical carcinogenesis of the 1940s. Much of today’s work can be traced to Elizabeth and James Miller at Wisconsin. This husband and wife team started under the mentorship of Professor Rusch, the director of the newly formed McArdle Laboratory for Cancer Research, and Professor Baumann. The seminal research of the Millers, and a young Allen Conney, led to the discovery of the role of reactive intermediates in carcinogenicity and that of mixed-function oxidases in the endoplasmic reticulum. Conney’s discovery of benzo(a)pyrene hydroxylase induction in the 1950s opened the field of chemical

metabolism that has resulted in the elucidation of the arylhydrocarbon receptor in the 1970s and 1980s.These findings, which initiated the great works on the cytochrome-P450 family of proteins, were aided by two other major discoveries for which toxicologists (and all other biological scientists) are deeply indebted: paper chromatography in 1944 and the use of radiolabeled dibenzanthracene in 1948. Other major events of note in drug metabolism included the work of Bernard Brodie on the metabolism of methyl orange in 1947. This piece of seminal research led to the examination of blood and urine for chemical and drug metabolites. It became the tool with which one could study the relationship between blood levels and biological action. The classic treatise of R. T. Williams, Detoxication Mechanisms, was published in 1947. This text described the many pathways and possible mechanisms of detoxication and opened the field to several new areas of study. The decade after World War II was not as boisterous as the period from 1935 to 1945. The first major U.S. Pesticide Act was signed into law in 1947. The significance of the initial Federal Insecticide, Fungicide, and Rodenticide Act was that for the first time in U.S. history a substance that was neither a drug nor a food had to be shown to be safe and efficacious. This decade, which coincided with the Eisenhower years, saw the dispersion of the groups from Chicago, Rochester, and Oak Ridge and the establishment of new centers of research. Adrian Albert’s classic Selective Toxicity was published in 1951. This treatise, which has appeared in several editions, presented a concise documentation of the principles of the site-specific action of chemicals.

AFTER WORLD WAR II You too can be a toxicologist in two easy lessons, each of ten years. Arnold Lehman (circa 1955) The mid-1950s witnessed the strengthening of the U.S. Food and Drug Administration’s commitment to toxicology under the guidance of Arnold Lehman. Lehman’s tutelage and influence are still felt today. The adage “You too can be a toxicologist...” is as important a summation of toxicology as the often-quoted statement of Paracelsus: “The dose makes the poison.” The period from 1955 to 1958 produced two major events that would have a long-lasting impact on toxicology as a science and a professional discipline. Lehman, Fitzhugh, and their co-workers formalized the experimental program for the appraisal of food, drug, and cosmetic safety in 1955, updated by the U.S. FDA in 1982, and the Gordon Research Conferences established a conference on toxicology and safety evaluation, with Bernard L. Oser as its initial chairman. These two events led to close relationships among toxicologists from several groups and brought toxicology into a new phase. At about the same time, the U.S. Congress passed and the President of the United States signed the additives amendments to the Food, Drug, and Cosmetic Act. The Delaney clause (1958) of these amendments stated broadly that any chemical found to be carcinogenic in laboratory animals or humans could not be added to the U.S. food supply. The impact of this food additive legislation cannot be overstated. Delaney became a battle cry for many groups and resulted in the inclusion at a new level of biostatisticians and mathematical modelers in the field of toxicology. It fostered the expansion of quantitative methods in toxicology and led to innumerable arguments about the “one-hit” theory of carcinogenesis. Regardless of one’s view of Delaney, it has served as an excellent starting point for understanding the complexity of the biological phenomenon of carcinogenicity and the development

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of risk assessment models. One must remember that at the time of Delaney, the analytic detection level for most chemicals was 20 to 100 ppm (today, parts per quadrillion). Interestingly, the Delaney clause has been invoked only on a few occasions, and it has been stated that Congress added little to the food and drug law with this clause (Hutt and Hutt, 1984). Shortly after the Delaney Amendment and after three successful Gordon Conferences, the first American journal Toxicology and Applied Pharmacology dedicated to toxicology was launched by Coulston, Lehman, and Hayes. Since 1960, over 50 journals and innumerable societies have been launched to disseminate toxicological information. The founding of the Society of Toxicology followed shortly afterward, and became its official publication. The Society’s founding members were Fredrick Coulston, William Deichmann, Kenneth DuBois, Victor Drill, Harry Hayes, Harold Hodge, Paul Larson, Arnold Lehman, and C. Boyd Shaffer. These researchers deserve a great deal of credit for the growth of toxicology. DuBois and Geiling published their Textbook of Toxicology in 1959. The text you are reading is a continuum of the DuBois and Geiling classic. The 1960s were a tumultuous time for society, and toxicology was swept up in the tide. Starting with the tragic thalidomide incident, in which several thousand children were born with serious birth defects, and the publication of Rachel Carson’s Silent Spring (1962), the field of toxicology developed at a feverish pitch. Attempts to understand the effects of chemicals on the embryo and fetus and on the environment as a whole gained momentum. New legislation was passed, and new journals were founded. The education of toxicologists spread from the deep traditions at Chicago and Rochester to Harvard, Miami, Albany, Iowa, Jefferson, and beyond. Geiling’s fledglings spread as Schmiedeberg’s had a half century before. Many new fields were influencing and being assimilated into the broad scope of toxicology, including environmental sciences, aquatic and avian biology, biostatistics, risk modeling, cell biology, analytic chemistry, and molecular genetics. During the 1960s, particularly the latter half of the decade, the analytic tools used in toxicology were developed to a level of sophistication that allowed the detection of chemicals in tissues and other substrates at part per billion concentrations (today, parts per quadrillion may be detected). Pioneering work in the development of point mutation assays that were replicable, quick, and inexpensive led to a better understanding of the genetic mechanisms of carcinogenicity (Ames, 1983). The combined work of Ames, the Millers (Elizabeth C. and James A.) at McArdle Laboratory, Cooney, and others allowed the toxicology community to make major contributions to the understanding of the carcinogenic process. The low levels of detection of chemicals and the ability to detect point mutations rapidly created several problems and opportunities for toxicologists and risk assessors that stemmed from interpretation of the Delaney amendment. Cellular and molecular toxicology developed as a subdiscipline, and risk assessment became a major product of toxicological investigations. The establishment of the National Center for Toxicologic Research (NCTR), the expansion of the role of the U.S. FDA, and the establishment of the U.S. Environmental Protection Agency (EPA) and the National Institute of Environmental Health Sciences (NIEHS) were considered clear messages that the government had taken a strong interest in toxicology. Several new journals appeared during the 1960s, and new legislation was written quickly after Silent Spring and the thalidomide disaster. The end of the 1960s witnessed the “discovery” of TCDD as a contaminant in the herbicide Agent Orange (the original discovery

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of TCDD toxicity, as the “Chick Edema Factor,” was reported in 1957). The research on the toxicity of this compound has produced seminal findings regarding signal transduction, and some very poor research in the field of toxicology. The discovery of a high-affinity cellular binding protein designated the “Ah” receptor (see Poland and Knutsen, 1982, for a review) at the McArdle Laboratory and work on the genetics of the receptor at NIH (Nebert and Gonzalez, 1987; Thomas et al., 1972) have revolutionized the field of toxicology. The importance of TCDD to toxicology lies in the fact that it forced researchers, regulators, and the legal community to look at the role of mechanisms of toxic action in a different fashion. The compound is a potent carcinogen in several species but is not a mutagen. At least two other events precipitated a great deal of legislation during the 1970s: Love Canal and Kepone in the James River. The “discovery” of Love Canal led to major concerns regarding hazardous wastes, chemical dump sites, and disclosure of information about those sites. Soon after Love Canal, the EPA listed several equally contaminated sites in the United States. The agency was given the responsibility to develop risk assessment methodology to determine health risks from exposure to effluents and to attempt to remediate these sites. These combined efforts led to broad-based support for research into the mechanisms of action of individual chemicals and complex mixtures. Love Canal and similar issues created the legislative environment that led to the Toxic Substances Control Act and eventually to the Superfund bill. These omnibus bills were created to cover the toxicology of chemicals from initial synthesis to disposal (cradle to grave). The expansion of legislation, journals, and new societies involved with toxicology was exponential during the 1970s and 1980s and shows no signs of slowing down. Currently, in the United States there are dozens of professional, governmental, and other scientific organizations with thousands of members and over 120 journals dedicated to toxicology and related disciplines. In addition, toxicology continues to expand in stature and in the number of programs worldwide. The International Congress of Toxicology is made up of toxicology societies from Europe, South America, Asia, Africa, and Australia and brings together the broadest representation of toxicologists. The original Gordon Conference series has changed to Mechanisms of Toxicity, and several other conferences related to special areas of toxicology are now in existence. The Society of Toxicology in the United States has formed specialty sections and regional chapters to accommodate the over 5000 scientists involved in toxicology today. The American College of Toxicology has developed into an excellent venue for regulatory and industrial toxicology, and two boards have been established to accredit and certify toxicologists (The Academy of Toxicological Sciences and the American Board of Toxicology). Texts and reference books for toxicology students and scientists abound. Toxicology has evolved from a borrowing science to a seminal discipline seeding the growth and development of several related fields of science and science policy. The history of toxicology has been interesting and varied but never dull. Perhaps as a science that has grown and prospered by borrowing from many disciplines, it has suffered from the absence of a single goal, but its diversification has allowed for the interspersion of ideas and concepts from higher education, industry, and government. As an example of this diversification, one now finds toxicology graduate programs in medical schools, schools of public health, and schools of pharmacy as well as programs in environmental science and engineering, as well as undergraduate programs in toxicology at several institutions. Surprisingly, courses in toxicology

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are now being offered in several liberal arts undergraduate schools as part of their biology and chemistry curricula. This has resulted in an exciting, innovative, and diversified field that is serving science and the community at large.

Few disciplines can point to both basic sciences and direct applications at the same time. Toxicology—the study of the adverse effects of xenobiotics (and more recently endobiotics)—may be unique in this regard.

REFERENCES Albert A: Selective Toxicity. London: Methuen, 1951. Ames BN: Dietary carcinogens and anticarcinogens. Science 221:1249– 1264, 1983. Bernard C: Action du curare et de la nicotine sur le systeme nerveux et sur le systme musculaire. CR Soc Biol 2:195, 1850. Bernard C: Introduction to the Study of Experimental Medicine, trans., in Greene HC, Schuman H (eds.) New York: Dover, 1949. Carson R: Silent Spring. Boston: Houghton Mifflin, 1962. Christison R: A Treatise on Poisons, 4th ed. Philadelphia: Barrington & Howell, 1845. Doll R, Peto R: The Causes of Cancer. New York: Oxford University Press, 1981. Donaldson HH: The history and zoological position of the albino rat. Natl Acad Sci 15:365–369, 1912. DuBois K, Geiling EMK: Textbook of Toxicology. New York: Oxford University Press, 1959. Gunther RT: The Greek Herbal of Dioscorides. New York: Oxford University Press, 1934. Guthrie DA: A History of Medicine. Philadelphia: Lippincott, 1946. Handler P: Some comments on risk assessment, in The National Research Council in 1979: Current Issues and Studies. Washington, DC: NAS, 1979. Hutt PB, Hutt PB II: A history of government regulation of adulteration and misbranding of food. Food Drug Cosmet J 39:2–73, 1984. Kallet A, Schlink FJ: 100,000,000 Guinea Pigs: Dangers in Everyday Foods, Drugs and Cosmetics. New York: Vanguard, 1933. Levey M: Medieval arabic toxicology: The book on poisons of Ibn Wahshiya and its relation to early Indian and Greek texts. Trans Am Philos Soc 56(7): 1966. Lewin L: Die Gifte in der Weltgeschichte: Toxikologische, allgemeinverstandliche Untersuchungen der historischen Quellen. Berlin: Springer, 1920. Lewin L: Gifte und Vergiftungen. Berlin: Stilke, 1929. Loomis TA: Essentials of Toxicology, 3rd ed. Philadelphia: Lea & Febiger, 1978. Macht DJ: Louis Lewin: Pharmacologist, toxicologist, medical historian. Ann Med Hist 3:179–194, 1931. Meek WJ: The Gentle Art of Poisoning. Medico-Historical Papers. Madision: University of Wisconsin, 1954; reprinted from Phi Beta Pi Quarterly, May 1928. Muller P: Uber zusammenhange zwischen Konstitution und insektizider Wirkung I. Helv Chim Acta 29:1560–1580, 1946.

Munter S (ed.): Treatise on Poisons and Their Antidotes. Vol. II of the Medical Writings of Moses Maimonides. Philadelphia: Lippincott, 1966. Nebert D, Gonzalez FJ: P450 genes: Structure, evolution and regulation. Annu Rev Biochem 56:945–993, 1987. Olmsted JMD: Franc¸ois Magendie: Pioneer in Experimental Physiology and Scientific Medicine in XIX Century France. New York: Schuman, 1944. Orfila MJB: Secours a Donner aux Personnes Empoisonees et Asphyxiees. Paris: Feugeroy, 1818. Orfila MJB: Traite des Poisons Tires des Regnes Mineral, Vegetal et Animal, ou, Toxicologie Generale Consideree sous les Rapports de la Physiologie, de la Pathologie et de la Medecine Legale. Paris: Crochard, 1814–1815. Pachter HM: Paracelsus: Magic into Science. New York: Collier, 1961. Pagel W: Paracelsus: An Introduction to Philosophical Medicine in the Era of the Renaissance. New York: Karger, 1958. Paracelsus (Theophrastus ex Hohenheim Eremita): Von der Besucht. Dillingen, 1567. Poland A, Knutson JC: 2,3,7,8-Tetrachlorodibenzo- p-dioxin and related halogenated aromatic hydrocarbons, examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22:517–554, 1982. Ramazzini B: De Morbis Artificum Diatriba. Modena: Typis Antonii Capponi, 1700. Robert R: Lehrbuch der Intoxikationen. Stuttgart: Enke, 1893. Schmiedeberg O, Koppe R: Das Muscarin das giftige Alkaloid des Fliegenpilzes. Leipzig: Vogel, 1869. Thomas PE, Kouri RE, Hutton JJ.The genetics of AHH induction in mice: a single gene difference between C57/6J and DBA/2J. Biochem Genet. 6:157–168,1972. Thompson CJS: Poisons and Poisoners: With Historical Accounts of Some Famous Mysteries in Ancient and Modern Times. London: Shaylor, 1931. U.S. FDA: Toxicologic Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. Washington, DC: U.S. Food and Drug Administration, Bureau of Foods, 1982. Voegtlin C, Dyer HA, Leonard CS: On the mechanism of the action of arsenic upon protoplasm. Public Health Rep 38:1882–1912, 1923. Williams RT: Detoxication Mechanisms, 2nd ed. New York: Wiley, 1959. Zapp JA Jr, Doull J: Industrial toxicology: Retrospect and prospect, in Clayton GD, Clayton FE (eds.): Patty’s Industrial Hygiene and Toxicology, 4th ed. New York: Wiley Interscience, 1993, pp. 1–23.

SUPPLEMENTAL READING Adams F (trans.): The Genuine Works of Hippocrates. Baltimore: Williams & Wilkins, 1939. Beeson BB: Orfila—pioneer toxicologist. Ann Med Hist 2:68–70, 1930. Bernard C: Analyse physiologique des proprietes des systemes musculaire et nerveux au moyen du curare. CR Acad Sci (Paris) 43:325–329, 1856. Bryan CP: The Papyrus Ebers. London: Geoffrey Bales, 1930. Clendening L: Source Book of Medical History. New York: Dover, 1942. Gaddum JH: Pharmacology, 5th ed. New York: Oxford University Press, 1959.

Garrison FH: An Introduction to the History of Medicine, 4th ed. Philadelphia: Saunders, 1929. Hamilton A: Exploring the Dangerous Trades. Boston: Little, Brown, 1943. (Reprinted by Northeastern University Press, Boston, 1985.) Hays HW: Society of Toxicology History, 1961–1986. Washington, DC: Society of Toxicology, 1986. Holmstedt B, Liljestrand G: Readings in Pharmacology. New York: Raven Press, 1981.

CHAPTER 2

PRINCIPLES OF TOXICOLOGY David L. Eaton and Steven G. Gilbert Assumptions in Deriving the Dose–Response Relationship Evaluating the Dose–Response Relationship Comparison of Dose Responses Therapeutic Index Margins of Safety and Exposure Potency versus Efficacy

INTRODUCTION TO TOXICOLOGY Different Areas of Toxicology Toxicology and Society General Characteristics of the Toxic Response CLASSIFICATION OF TOXIC AGENTS SPECTRUM OF UNDESIRED EFFECTS

VARIATION IN TOXIC RESPONSES

Allergic Reactions Idiosyncratic Reactions Immediate versus Delayed Toxicity Reversible versus Irreversible Toxic Effects Local versus Systemic Toxicity Interaction of Chemicals Tolerance

Selective Toxicity Species Differences Individual Differences in Response DESCRIPTIVE ANIMAL TOXICITY TESTS Acute Toxicity Testing Skin and Eye Irritations Sensitization Subacute (Repeated-Dose Study) Subchronic Chronic Developmental and Reproductive Toxicity Mutagenicity Oncogenicity Bioassays Neurotoxicity Assessment Immunotoxicity Assessment Other Descriptive Toxicity Tests

CHARACTERISTICS OF EXPOSURE Route and Site of Exposure Duration and Frequency of Exposure DOSE–RESPONSE RELATIONSHIP Individual, or Graded, Dose–Response Relationships Quantal Dose–Response Relationships Shape of the Dose–Response Curve Essential Nutrients Hormesis Threshold

TOXICOGENOMICS

INTRODUCTION TO TOXICOLOGY

Different Areas of Toxicology

Toxicology is the study of the adverse effects of chemical or physical agents on living organisms. A toxicologist is trained to examine and communicate the nature of those effects on human, animal, and environmental health. Toxicological research examines the cellular, biochemical, and molecular mechanisms of action as well as functional effects such as neurobehavioral and immunological, and assesses the probability of their occurrence. Fundamental to this process is characterizing the relation of exposure (or dose) to the response. Risk assessment is the quantitative estimate of the potential effects on human health and environmental significance of various types of chemical exposures (e.g., pesticide residues on food, contaminants in drinking water). The variety of potential adverse effects and the diversity of chemicals in the environment make toxicology a broad science, which often demands specialization in one area of toxicology. Our society’s dependence on chemicals and the need to assess potential hazards have made toxicologists an increasingly important part of the decision-making processes.

The professional activities of toxicologists fall into three main categories: descriptive, mechanistic, and regulatory (Fig. 2-1). Although each has distinctive characteristics, each contributes to the other, and all are vitally important to chemical risk assessment (see Chap. 4). A mechanistic toxicologist is concerned with identifying and understanding the cellular, biochemical, and molecular mechanisms by which chemicals exert toxic effects on living organisms (see Chap. 3 for a detailed discussion of mechanisms of toxicity). The results of mechanistic studies are very important in many areas of applied toxicology. In risk assessment, mechanistic data may be very useful in demonstrating that an adverse outcome (e.g., cancer, birth defects) observed in laboratory animals is directly relevant to humans. For example, the relative toxic potential of organophosphate insecticides in humans, rodents, and insects can be accurately predicted on the basis of an understanding of common mechanisms (inhibition of acetylcholinesterase) and differences in biotransformation for these insecticides among the different species. Similarly, mechanistic data may be very useful in identifying adverse

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Risk Assessment

Figure 2-1. Graphical representation of the interconnections between different areas of toxicology.

responses in experimental animals that may not be relevant to humans. For example, the propensity of the widely used artificial sweetener saccharin to cause bladder cancer in rats may not be relevant to humans at normal dietary intake rates. This is because mechanistic studies have demonstrated that bladder cancer is induced only under conditions where saccharin is at such a high concentration in the urine that it forms a crystalline precipitate (Cohen, 1998). Dose–response studies suggest that such high concentrations would not be achieved in the human bladder even after extensive dietary consumption. Mechanistic data are also useful in the design and production of safer alternative chemicals and in rational therapy for chemical poisoning and treatment of disease. For example, the drug thalidomide was originally marketed in Europe and Australia as a sedative agent for pregnant women. However, it was banned for clinical use in 1962 because of devastating birth defects that occurred if the drug was ingested during a critical period in pregnancy. But mechanistic studies over the past several decades have demonstrated that this drug may have a unique molecular mechanism of action that interferes with the expression of certain genes responsible for blood vessel formation (angiogenesis). With an understanding of this mechanism, thalidomide has been “rediscovered” as a valuable therapeutic agent that may be highly effective in the treatment of certain infectious diseases (e.g., leprosy and AIDS), a variety of inflammatory diseases, and some types of cancer. This provides an interesting example of how a highly toxic drug with selectivity toward a specific population (pregnant women) can be used safely with proper precautions. Following its approval for therapeutic use in 1998, a program was established that required all clinicians, pharmacists, and patients that receive thalidomide to enroll in a specific program (System for Thalidomide Education and Prescribing Safety, STEPS). The population at risk for the potential teratogenic effects of thalidomide (all women of childbearing age) were required to use two forms of birth control, and also have a negative pregnancy test within 24 hours of beginning therapy, and periodically the patients registered with the STEPS program, 6000 were females of childbearing age. Remarkably, after 6 years of use, only one patient actually received thalidomide during her pregnancy. She initially tested negative at the beginning of therapy; on a subsequent test she was identified

as positive, and the drug was stopped. The pregnancy ended up as a miscarriage (Uhl et al., 2006). Thus, a clear understanding of mechanism of action led to the development of strict prescribing guidelines and patient monitoring, thereby allowing a potentially dangerous drug to be used safely and effectively to treat disease in tens of thousands of patients who would otherwise not have benefited from the therapeutic actions of the drug (Lary et al., 1999). In addition to aiding directly in the identification, treatment, and prevention of chemical toxicity, an understanding of the mechanisms of toxic action contributes to the knowledge of basic physiology, pharmacology, cell biology, and biochemistry. The advent of new technologies in molecular biology and genomics now provide mechanistic toxicologists with the tools to explore exactly how humans may differ from laboratory animals in their response to toxic substances. These same tools are also being utilized to identify individuals who are genetically susceptible to factors in the environment or respond differently to a chemical exposure. For example, it is now recognized that a small percentage of the population genetically lacks the ability to detoxify the chemotherapeutic drug, 6-mercaptopurine, used in the treatment of some forms of leukemia. Young children with leukemia who are homozygous for this genetic trait (about one in 300) may experience serious toxic effects from a standard therapeutic dose of this drug. Numerous genetic tests for polymorphisms in drug metabolizing enzymes and transporters are now available that can identify genetically susceptible individuals in advance of pharmacological treatment (Eichelbaum et al., 2006). These new areas of “pharmacogenomics” and “toxicogenomics” provides an exciting opportunity in the future for mechanistic toxicologists to identify and protect genetically susceptible individuals from harmful environmental exposures, and to customize drug therapies that enhance efficacy and minimize toxicity, based on an individual’s genetic makeup. A descriptive toxicologist is concerned directly with toxicity testing, which provides information for safety evaluation and regulatory requirements. The appropriate toxicity tests (as described later in this chapter and other chapters) in cell culture systems or experimental animals are designed to yield information to evaluate risks posed to humans and the environment from exposure to specific chemicals. The concern may be limited to effects on humans, as in the case of drugs and food additives. Toxicologists in the chemical industry, however, must be concerned not only with the risk posed by a company’s chemicals (insecticides, herbicides, solvents, etc.) to humans but also with potential effects on fish, birds, and plants, as well as other factors that might disturb the balance of the ecosystem. Descriptive toxicology is of course not divorced from mechanistic studies, as such studies provide important clues to a chemical’s mechanism of action, and thus contribute to the development of mechanistic toxicology through hypothesis generation. Such studies are also a key component of risk assessments that are used by regulatory toxicologists. The recent advent of so-called “omics” technologies (genomics, transcriptomics, proteomics, metabonomics, etc.) form the basis of the emerging subdiscipline of toxicogenomics. The application of these new technologies to toxicity testing is in many ways “descriptive” in nature, yet affords great mechanistic insights into how chemicals produce their toxic effects. This exciting new area of toxicology is discussed in more detail later in the chapter. A regulatory toxicologist has the responsibility for deciding, on the basis of data provided by descriptive and mechanistic toxicologists, whether a drug or other chemical poses a sufficiently low risk to be marketed for a stated purpose or subsequent human or

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environmental exposure resulting from its use. The Food and Drug Administration (FDA) is responsible for allowing drugs, cosmetics, and food additives to be sold in the market according to the Federal Food, Drug and Cosmetic Act (FFDCA). The U.S. Environmental Protection Agency (EPA) is responsible for regulating most other chemicals according to the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the Toxic Substances Control Act (TSCA), the Resource Conservation and Recovery Act (RCRA), the Safe Drinking Water Act, and the Clean Air Act. In 1996, the U.S. Congress passed the Food Quality Protection Act (FQPA) which fundamentally changed the pesticide and food safety laws under FIFRA and FFDCA requiring stricter safety standards particularly for infants and children, who were recognized as more susceptible to health effects of pesticides. The EPA is also responsible for enforcing the Comprehensive Environmental Response, Compensation and Liability Act [CERCLA, later revised as the Superfund Amendments Reauthorization Act (SARA)], more commonly called the Superfund Act. This regulation provides direction and financial support for the cleanup of waste sites that contain toxic chemicals that may present a risk to human health or the environment. The Occupational Safety and Health Administration (OSHA) of the Department of Labor was established to ensure that safe and healthful conditions exist in the workplace. The National Institute for Occupational Safety and Health (NIOSH) as part of the Centers for Disease Control and Prevention (CDC) in the Department of Health and Human Services is responsible for conducting research and making recommendations for the prevention of work-related injury and illness. The Consumer Product Safety Commission is responsible for protecting consumers from hazardous household substances, whereas the Department of Transportation (DOT) ensures that materials shipped in interstate commerce are labeled and packaged in a manner consistent with the degree of hazard they present. Regulatory toxicologists are also involved in the establishment of standards for the amount of chemicals permitted in ambient air, industrial atmospheres, and drinking water, often integrating scientific information from basic descriptive and mechanistic toxicology studies with the principles and approaches used for risk assessment (see Chap. 4). In addition to the above categories, there are other specialized areas of toxicology such as forensic, clinical, and environmental toxicology. Forensic toxicology is a hybrid of analytic chemistry and fundamental toxicological principles. It is concerned primarily with the medicolegal aspects of the harmful effects of chemicals on humans and animals. The expertise of forensic toxicologists is invoked primarily to aid in establishing the cause of death and determining its circumstances in a postmortem investigation (see Chap. 31). Clinical toxicology designates an area of professional emphasis in the realm of medical science that is concerned with disease caused by or uniquely associated with toxic substances (see Chap. 32). Generally, clinical toxicologists are physicians who receive specialized training in emergency medicine and poison management. Efforts are directed at treating patients poisoned with drugs or other chemicals and at the development of new techniques to treat those intoxications. Public contact about treatment and prevention is often through the national network of poison control centers. Environmental toxicology focuses on the impacts of chemical pollutants in the environment on biological organisms. Although toxicologists concerned with the effects of environmental pollutants on human health fit into this definition, it is most commonly associated with studies on the impacts of chemicals on nonhuman organisms such as fish, birds, terrestrial animals, and plants. Ecotoxicology is a specialized area within environmental toxicology that focuses more specifically

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on the impacts of toxic substances on population dynamics in an ecosystem. The transport, fate, and interactions of chemicals in the environment constitute a critical component of both environmental toxicology and ecotoxicology.

Toxicology and Society Information from the toxicological sciences, gained by experience or research, has a growing influence on our personal lives as well as for human and environmental health across the globe. Knowledge about the toxicological effects of a compound affects consumer products, drugs, manufacturing processes, waste clean up, regulatory action, civil disputes, and broad policy decisions. The expanding influence of toxicology on societal issues is accompanied by the responsibility to be increasingly sensitive to the ethical, legal, and social implications of toxicological research and testing. The convergence of multiple elements has highlighted the evolving ethical dynamics of toxicology. First, experience and new discoveries in the biological sciences have emphasized our interconnectedness with nature and the need for well-articulated visions of human, animal, and environmental health. One vision is that we have “condition(s) that ensure that all living things have the best opportunity to reach and maintain their full genetic potential” (Gilbert, 2005a). Second, we have experience with the health consequences of exposure to such things as lead, asbestos, and tobacco, along with the detailed mechanistic research to understand the long-term risks to individuals and society. This has precipitated many regulatory and legal actions and public policy decisions, not to mention costly and time-consuming lawsuits. Third, we have an increasingly well-defined framework for discussing our social and ethical responsibilities. There is growing recognition that ethics play a crucial role in public health decision-making that involve conflicts between individual, corporate, and social justice goals (Callahan and Jennings, 2002; Kass, 2001; Lee, 2002). Fourth, is the appreciation that all research involving humans or animals must be conducted in a responsible and ethical manner. Fifth, is managing both the uncertainty and biological variability inherent in the biological sciences. Decision-making often includes making judgments with limited or uncertain information, which often includes an overlay of individual values and ethics. Finally, individuals involved in toxicological research must be aware of and accountable to their own individual biases and possible conflicts of interest and adhere to the highest ethical standards of the profession (Maurissen et al., 2005). Ethical reasoning and philosophy has a long and deep history, but more pragmatic bioethical reasoning can be traced to Aldo Leopold, who is arguably, America’s first bioethicist: “A thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. It is wrong when it tends otherwise.” (Leopold, 1949). The essence of toxicology is to understand the effects of chemicals on the biotic community. This broader definition of an ethic became more focused with examples such as the mercury poisoning in Minamata Bay, Japan, thalidomide, and the effects of pesticides as brought to public awareness by Rachel Carson’s “Silent Spring” (Carson, 1962). In the United States, these events supported the public and political will to establish the EPA, strengthening of the FDA and other regulations designed to protect human and environmental health. The appreciation that some segments of our society were deferentially at risk from chemical exposures evolved into an appreciation of environmental justice (Coburn, 2002; EPA, 2005; Lee, 2002; Morello-Frosch et al., 2002). The EPA defines

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environmental justice as “the fair treatment and meaningful involvement of all people regardless of race, color, national origin, or income with respect to the development, implementation, and enforcement of environmental laws, regulations, and policies. . . ” (EPA, 2005). Environmental justice is now an important component of numerous community-based programs of interest, and is relevant to the field of toxicology. There is growing recognition of the direct financial and indirect costs to individuals and society from environmental exposures that are not equally distributed across society (Landrigan et al., 2002). On a parallel track, biomedical ethics developed out of the lessons of World War II and related abuses of human subjects. The four principle of biomedical ethics—respect for autonomy, beneficence (do good), non-maleficence (do no harm), and justice (be fair)—became well established as a basis for decision-making in health care settings (Beauchamp and Childress, 1994). These principles formed the basis of rules and regulations regarding the conduct of human research. The demands of ethics and science made it clear that the highest standards of care produced the best results in both human and animal research. Rules and regulations regarding the housing and conduct of animal studies evolved similarly. Professional toxicology societies now require their members to adhere to the highest ethical standards when conducting research with humans or animals. A further refinement and expansion of biomedical ethical principles is the development of community-based participatory research that takes into consideration community needs to ensure the best results and benefit to the community (Arcury et al., 2001; Gilbert, 2006; O’Fallon and Dearry, 2002). A glance at the daily newspaper confirms the number of current, sometimes controversial issues that are relevant to the field of toxicology. Decisions and action are often demanded or required even when there is a certain level of uncertainty in the toxicological data. The classic example of this challenge is establishing causation of the health effects of tobacco products. In part to address issues related to the health effects of tobacco products, Bradford Hill defined criteria for determining causation (Hill, 1965). These criteria are briefly summarized below. 1. Strength of association (relationship between independent and dependent variables) 2. Consistency of findings (replication of results by different studies) 3. Biological gradient (strength of the dose-response relationship) 4. Temporal sequence (“cause” before effect) 5. Biologic or theoretical plausibility (mechanism of action) 6. Coherence with established knowledge (no competing hypotheses) 7. Specificity of association (cause is tightly linked to an outcome) Quantitative risk assessment was developed in part to address issues of uncertainty related to potential harm. The risk assessment process summarized data for risk managers and other decision makers, who must take into consideration to some degree the qualitative elements of ethical, social and political issues. Whereas risk management clearly has an ethical and values based aspect, risk assessment is not immune from the influence of one’s values, bias or perspective. Ultimately action is required and as Bradford Hill (1965) noted: ”All scientific work is incomplete—whether it be observational or experimental. All scientific work is liable to be upset or modified by advancing knowledge. That does not confer upon us a freedom to ignore the knowledge we already have or postpone the

action that it appears to demand at a given time.” These so-called “Bradford Hill criteria” were developed largely as a “weight of evidence” approach for interpreting a body of epidemiology data, yet are relevant as well to toxicology. Guzelian et al. (2005) provide a more detailed, evidence-based approach for determining causation in toxicology, primarily for application in the legal arena. Although the scientific data may be the same, there are substantial differences in how toxicological data are used in a regulatory framework to protect public health versus establishing individual causation in the courtroom (Eaton, 2003). The approach to regulatory decision-making is in part directed by policy. For example, the experience with thalidomide and other drugs motivated the U.S. Congress to give the FDA broad power to ensure the efficacy and safety of new medicines or medical procedures. In this situation the pharmaceutical company or proponents of an activity must invest in the appropriate animal and human studies to demonstrate safety of the product. We have instituted a very precautionary approach with regard to drugs and medical devices. The approach to industrial chemicals is defined by the Toxic Substance Control Act and does not stipulate such a rigorous approach when introducing a new chemical. Building on the work of Hill and others particularly from Europe, the Precautionary Principle was defined at the Wingspread Conference, in 1998: “When an activity raises threats of harm to human health or the environment, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically.” (Gilbert, 2005b; Myers and Raffensperger, 2006; Raffensperger and Tickner, 1999). The precautionary principle incorporates elements of science and ethical philosophy into a single statement, acknowledging that ethics and values are part of the decision making process. Although the conceptual value of the precautionary principle to public health protection is obvious, the actual implementation of it in toxicological risk assessment is not straightforward, and remains a point of considerable debate (Marchant, 2003; Goldstein, 2006; Peterson, 2006). With the increased relevance of toxicological data and evaluation in issues fundamental to society there has been increased awareness of the possibility of conflicts of interest influencing the decision-making process (Maurissen et al., 2005). The disclosure of conflicts of interest as well as the development of appropriate guidelines continues to be a challenge (NAS, 2003; Goozner, 2004; Krimsky and Rothenberg, 2001). These issues go to the core of one’s individual values and integrity in the interpretation and communication of research results. Many professional societies, including the Society of Toxicology (http://www.toxicology.org/ai/asot/ ethics.asp), have developed codes of ethics for their members. As the field of toxicology has matured and its influence on societal issues has increased so has the need for the profession to make a commitment to examine the ethical, legal, and social implications of research and practice of toxicology.

General Characteristics of the Toxic Response One could define a poison as any agent capable of producing a deleterious response in a biological system, seriously injuring function or producing death. This is not, however, a useful working definition for the very simple reason that virtually every known chemical has the potential to produce injury or death if it is present in a sufficient amount. Paracelsus (1493–1541), a Swiss/German/Austrian physician, scientist, and philosopher, phrased this well when he noted, “What is there that is not poison? All things are poison and nothing

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Table 2.1 Approximate Acute LD50 s of Some Representative Chemical Agents agent

ld50 , mg/kg∗

Ethyl alcohol Sodium chloride Ferrous sulfate Morphine sulfate Phenobarbital sodium Picrotoxin Strychnine sulfate Nicotine d−Tubocurarine Hemicholinium-3 Tetrodotoxin Dioxin (TCDD) Botulinum toxin

10000 4000 1500 900 150 5 2 1 0.5 0.2 0.10 0.001 0.00001

∗

LD50 is the dosage (mg/kg body weight) causing death in 50% of exposed animals.

[is] without poison. Solely the dose determines that a thing is not a poison.” Among chemicals there is a wide spectrum of doses needed to produce deleterious effects, serious injury, or death. This is demonstrated in Table 2-1, which shows the dosage of chemicals needed to produce death in 50% of treated animals (LD50 ). Some chemicals produce death in microgram doses and are commonly thought of as being extremely poisonous. Other chemicals may be relatively harmless after doses in excess of several grams. It should be noted, however, that measures of acute lethality such as LD50 may not accurately reflect the full spectrum of toxicity, or hazard, associated with exposure to a chemical. For example, some chemicals with low acute toxicity may have carcinogenic, teratogenic, or neurobehavioral effects at doses that produce no evidence of acute toxicity. In addition, there is growing recognition that genetic factors can account for individual susceptibility to a range of responses.

CLASSIFICATION OF TOXIC AGENTS Toxic agents are classified in a variety of ways, depending on the interests and needs of the classifier. In this textbook, for example, toxic agents are discussed in terms of their target organs (liver, kidney, hematopoietic system, etc.), use (pesticide, solvent, food additive, etc.), source (animal and plant toxins), and effects (cancer, mutation, liver injury, etc.). The term toxin generally refers to toxic substances that are produced by biological systems such as plants, animals, fungi, or bacteria. The term toxicant is used in speaking of toxic substances that are produced by or are a by-product of anthropogenic (human-made) activities. Thus, zeralanone, produced by a mold, is a toxin, whereas “dioxin” [2,3,7,8-tetrachlorodibenzo- pdioxin (TCDD)], produced during the production and/or combustion of certain chlorinated organic chemicals, is a toxicant. Some toxicants can be produced by both natural and anthropogenic activities. For example, polyaromatic hydrocarbons are produced by the combustion of organic matter, which may occur both through natural processes (e.g., forest fires) and through anthropogenic activities (e.g., combustion of coal for energy production; cigarette smoking). Arsenic, a toxic metalloid, may occur as a natural contaminant of groundwater or may contaminate groundwater secondary to indus-

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trial activities. Generally, such toxic substances are referred to as toxicants, rather than toxins, because, although they are naturally produced, they are not produced by biological systems. Toxic agents may also be classified in terms of their physical state (gas, dust, liquid), their chemical stability or reactivity (explosive, flammable, oxidizer), general chemical structure (aromatic amine, halogenated hydrocarbon, etc.), or poisoning potential (extremely toxic, very toxic, slightly toxic, etc.). Classification of toxic agents on the basis of their biochemical mechanisms of action (e.g., alkylating agent, cholinesterase inhibitor, methemoglobin producer) is usually more informative than classification by general terms such as irritants and corrosives. But more general classifications such as air pollutants, occupation-related agents, and acute and chronic poisons can provide a useful focus on a specific problem. It is evident from this discussion that no single classification is applicable to the entire spectrum of toxic agents and that combinations of classification systems or a classification based on other factors may be needed to provide the best rating system for a special purpose. Nevertheless, classification systems that take into consideration both the chemical and the biological properties of an agent and the exposure characteristics are most likely to be useful for regulatory or control purposes and for toxicology in general.

SPECTRUM OF UNDESIRED EFFECTS The spectrum of undesired effects of chemicals is broad. Some effects are deleterious and others are not. In therapeutics, for example, each drug produces a number of effects, but usually only one effect is associated with the primary objective of the therapy; all the other effects are referred to as undesirable or side effects of that drug for that therapeutic indication. However, some of these side effects may be desired for another therapeutic indication. For example, the “first-generation” antihistamine diphenhydramine (Benadryl) is effective in reducing histamine responses associated with allergies, but it readily enters the brain and causes mild central nervous system (CNS) depression (drowsiness, delayed reaction time). With the advent of newer and selective histamine receptor antagonists that do not cross the blood–brain barrier and thus do not have this CNSdepressant side effect, diphenhydramine is used less commonly today as an antihistamine. However, it is widely used as an “over the counter” sleep remedy, often in combination with analgesics (e.g., Tylenol PM, Excedrin PM, etc), taking advantage of the CNSdepressant effects. Some side effects of drugs are never desirable and are always deleterious to the well-being of humans. These are referred to as the adverse, deleterious, or toxic effects of the drug.

Allergic Reactions Chemical allergy is an immunologically mediated adverse reaction to a chemical resulting from previous sensitization to that chemical or to a structurally similar one. The term hypersensitivity is most often used to describe this allergic state, but allergic reaction and sensitization reaction are also used to describe this situation when pre-exposure of the chemical is required to produce the toxic effect (see Chap. 12). Once sensitization has occurred, allergic reactions may result from exposure to relatively very low doses of chemicals; therefore population-based dose–response curves for allergic reactions have seldom been obtained. Because of this omission, some people assumed that allergic reactions are not dose-related. Thus, they do not consider the allergic reaction to be a true toxic response. However, for a given allergic individual, allergic reactions are

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dose-related. For example, it is well known that the allergic response to pollen in sensitized individuals is related to the concentration of pollen in the air. In addition, because the allergic response is an undesirable, adverse, deleterious effect, it obviously is also a toxic response. Sensitization reactions are sometimes very severe and may be fatal. Most chemicals and their metabolic products are not sufficiently large to be recognized by the immune system as a foreign substance and thus must first combine with an endogenous protein to form an antigen (or immunogen). A molecule that must combine with an endogenous protein to elicit an allergic reaction is called a hapten. The hapten-protein complex (antigen) is then capable of eliciting the formation of antibodies, and usually at least 1 or 2 weeks is required for the synthesis of significant amounts of antibodies. Subsequent exposure to the chemical results in an antigen–antibody interaction, which provokes the typical manifestations of allergy. The manifestations of allergy are numerous. They may involve various organ systems and range in severity from minor skin disturbance to fatal anaphylactic shock. The pattern of allergic response differs in various species. In humans, involvement of the skin (e.g., dermatitis, urticaria, and itching) and involvement of the eyes (e.g., conjunctivitis) are most common, whereas in guinea pigs, bronchiolar constriction leading to asphyxia is the most common. However, chemically induced asthma (characterized by bronchiolar constriction) certainly does occur in some humans, and the incidence of allergic asthma has increased substantially in recent years. Hypersensitivity reactions are discussed in more detail in Chap. 12.

serious lack of oxygen delivery to tissues after exposure to doses of methemoglobin-producing chemicals that would be harmless to individuals with normal NADH-cytochrome b5 reductase activity.

Immediate versus Delayed Toxicity Immediate toxic effects can be defined as those that occur or develop rapidly after a single administration of a substance, whereas delayed toxic effects are those that occur after the lapse of some time. Carcinogenic effects of chemicals usually have a long latency period, often 20 to 30 years after the initial exposure, before tumors are observed in humans. For example, daughters of mothers who took diethylstilbestrol (DES) during pregnancy have a greatly increased risk of developing vaginal cancer, but not other types of cancer, in young adulthood, some 20 to 30 years after their in utero exposure to DES (Hatch et al., 1998). Also, delayed neurotoxicity is observed after exposure to some organophosphorus insecticides that act by covalent modification of an enzyme referred to as neuropathy target esterase (NTE), a neuronal protein with serine esterase activity (Glynn et al., 1999). Binding of certain organophosphates (OP) to this protein initiates degeneration of long axons in the peripheral and central nervous system. The most notorious of the compounds that produce this type of neurotoxic effect is triorthocresylphosphate (TOCP). The effect is not observed until at least several days after exposure to the toxic compound. In contrast, most substances produce immediate toxic effects but do not produce delayed effects.

Idiosyncratic Reactions

Reversible versus Irreversible Toxic Effects

Chemical idiosyncrasy refers to a genetically determined abnormal reactivity to a chemical (Goldstein et al., 1974; Levine, 1978). The response observed is usually qualitatively similar to that observed in all individuals but may take the form of extreme sensitivity to low doses or extreme insensitivity to high doses of the chemical. However, while some people use the term idiosyncratic as a catchall to refer to all reactions that occur with low frequency, it should not be used in that manner (Goldstein et al., 1974). A classic example of an idiosyncratic reaction is provided by patients who exhibit prolonged muscular relaxation and apnea (inability to breathe) lasting several hours after a standard dose of succinylcholine. Succinylcholine usually produces skeletal muscle relaxation of only short duration because of its very rapid metabolic degradation by an enzyme that is present normally in the bloodstream called plasma butyrylcholinesterase (also referred to as pseudocholinesterase). Patients exhibiting this idiosyncratic reaction have a genetic polymorphism in the gene for the enzyme butyrylcholinesterase, which is less active in breaking down succinylcholine. Family pedigree and molecular genetic analyses have demonstrated that the presence of low plasma butyrylcholinesterase activity is due to the presence of one or more single nucleotide polymorphisms in this gene (Bartels et al., 1992). Similarly, there is a group of people who are abnormally sensitive to nitrites and certain other chemicals that have in common the ability to oxidize the iron in hemoglobin to produce methemoglobin, which is incapable of carrying oxygen to the tissues. The unusual phenotype is inherited as an autosomal recessive trait and is characterized by a deficiency in NADH-cytochrome b5 reductase activity. The genetic basis for this idiosyncratic response has been identified as a single nucleotide change in codon 127, which results in replacement of serine with proline (Kobayashi et al., 1990). The consequence of this genetic deficiency is that these individuals may suffer from a

Some toxic effects of chemicals are reversible, and others are irreversible. If a chemical produces pathological injury to a tissue, the ability of that tissue to regenerate largely determines whether the effect is reversible or irreversible. Thus, for a tissue such as liver, which has a high ability to regenerate, most injuries are reversible, whereas injury to the CNS is largely irreversible because differentiated cells of the CNS cannot divide and be replaced. Carcinogenic and teratogenic effects of chemicals, once they occur, are usually considered irreversible toxic effects.

Local versus Systemic Toxicity Another distinction between types of effects is made on the basis of the general site of action. Local effects are those that occur at the site of first contact between the biological system and the toxicant. Such effects are produced by the ingestion of caustic substances or the inhalation of irritant materials. For example, chlorine gas reacts with lung tissue at the site of contact, causing damage and swelling of the tissue, with possibly fatal consequences, even though very little of the chemical is absorbed into the bloodstream. The alternative to local effects is systemic effects. Systemic effects require absorption and distribution of a toxicant from its entry point to a distant site, at which deleterious effects are produced. Most substances except highly reactive materials produce systemic effects. For some materials, both effects can be demonstrated. For example, tetraethyl lead produces effects on skin at the site of absorption and then is transported systemically to produce its typical effects on the CNS and other organs. If the local effect is marked, there may also be indirect systemic effects. For example, kidney damage after a severe acid burn is an indirect systemic effect because the toxicant does not reach the kidney.

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Most chemicals that produce systemic toxicity do not cause a similar degree of toxicity in all organs; instead, they usually elicit their major toxicity in only one or two organs. These sites are referred to as the target organs of toxicity of a particular chemical. The target organ of toxicity is often not the site of the highest concentration of the chemical. For example, lead is concentrated in bone, but its toxicity is due to its effects in soft tissues, particularly the brain. DDT is concentrated in adipose tissue but produces no known toxic effects in that tissue. The target organ of toxicity most frequently involved in systemic toxicity is the CNS (brain and spinal cord). Even with many compounds having a prominent effect elsewhere, damage to the CNS can be demonstrated by the use of appropriate and sensitive methods. Next in order of frequency of involvement in systemic toxicity are the circulatory system; the blood and hematopoietic system; visceral organs such as the liver, kidney, and lung; and the skin. Muscle and bone are least often the target tissues for systemic effects. With substances that have a predominantly local effect, the frequency with which tissues react depends largely on the portal of entry (skin, gastrointestinal tract, or respiratory tract).

Interaction of Chemicals Because of the large number of different chemicals an individual may come in contact with at any given time (workplace, drugs, diet, hobbies, etc.), it is necessary, in assessing the spectrum of responses, to consider how different chemicals may interact with each other. Interactions can occur in a variety of ways. Chemical interactions are known to occur by a number of mechanisms, such as alterations in absorption, protein binding, and the biotransformation and excretion of one or both of the interacting toxicants. In addition to these modes of interaction, the response of the organism to combinations of toxicants may be increased or decreased because of toxicologic responses at the site of action. The effects of two chemicals given simultaneously produce a response that may simply be additive of their individual responses or may be greater or less than that expected by addition of their individual responses. The study of these interactions often leads to a better understanding of the mechanism of toxicity of the chemicals involved. A number of terms have been used to describe pharmacologic and toxicologic interactions. An additive effect occurs when the combined effect of two chemicals is equal to the sum of the effects of each agent given alone (example: 2 + 3 = 5). The effect most commonly observed when two chemicals are given together is an additive effect. For example, when two organophosphate insecticides are given together, the cholinesterase inhibition is usually additive. A synergistic effect occurs when the combined effects of two chemicals are much greater than the sum of the effects of each agent given alone (example: 2 + 2 = 20). For example, both carbon tetrachloride and ethanol are hepatotoxic compounds, but together they produce much more liver injury than the mathematical sum of their individual effects on liver at a given dose would suggest. Potentiation occurs when one substance does not have a toxic effect on a certain organ or system but when added to another chemical makes that chemical much more toxic (example: 0 + 2 = 10). Isopropanol, for example, is not hepatotoxic, but when it is administered in addition to carbon tetrachloride, the hepatotoxicity of carbon tetrachloride is much greater than when it is given alone. Antagonism occurs when two chemicals administered together interfere with each other’s actions or one interferes with the action of the other (example: 4 + 6 = 8; 4 + (−4) = 0; 4 + 0 = 1). Antag-

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onistic effects of chemicals are often very desirable in toxicology and are the basis of many antidotes. There are four major types of antagonism: functional, chemical, dispositional, and receptor. Functional antagonism occurs when two chemicals counterbalance each other by producing opposite effects on the same physiologic function. Advantage is taken of this principle in that the blood pressure can markedly fall during severe barbiturate intoxication, which can be effectively antagonized by the intravenous administration of a vasopressor agent such as norepinephrine or metaraminol. Similarly, many chemicals, when given at toxic dose levels, produce convulsions, and the convulsions often can be controlled by giving anticonvulsants such as the benzodiazepines (e.g., diazepam). Chemical antagonism or inactivation is simply a chemical reaction between two compounds that produces a less toxic product. For example, dimercaprol (British antilewisite, or BAL) chelates with metal ions such as arsenic, mercury, and lead and decreases their toxicity. The use of antitoxins in the treatment of various animal toxins is also an example of chemical antagonism. The use of the strongly basic low-molecular-weight protein protamine sulfate to form a stable complex with heparin, which abolishes its anticoagulant activity, is another example. Dispositional antagonism occurs when the disposition—that is, the absorption, distribution, biotransformation, or excretion of a chemical—is altered so that the concentration and/or duration of the chemical at the target organ are diminished. Thus, the prevention of absorption of a toxicant by ipecac or charcoal and the increased excretion of a chemical by administration of an osmotic diuretic or alteration of the pH of the urine are examples of dispositional antagonism. If the parent compound is responsible for the toxicity of the chemical (such as the anticoagulant warfarin) and its metabolic breakdown products are less toxic than the parent compound, increasing the compound’s metabolism (biotransformation) by administering a drug that increases the activity of the metabolizing enzymes (e.g., a “microsomal enzyme inducer” such as phenobarbital) will decrease its toxicity. However, if the chemical’s toxicity is largely due to a metabolic product (as in the case of the organophosphate insecticide parathion), inhibiting its biotransformation by an inhibitor of microsomal enzyme activity (SKF-525A or piperonyl butoxide) will decrease its toxicity. Receptor antagonism occurs when two chemicals that bind to the same receptor produce less of an effect when given together than the addition of their separate effects (example: 4 + 6 = 8) or when one chemical antagonizes the effect of the second chemical (example: 0 + 4 = 1). Receptor antagonists are often termed blockers. This concept is used to advantage in the clinical treatment of poisoning. For example, the receptor antagonist naloxone is used to treat the respiratory depressive effects of morphine and other morphine-like narcotics by competitive binding to the same receptor. Another example of receptor antagonism is the use of the antiestrogen drug tamoxifen to lower breast cancer risk among women at high risk for this estrogen-related cancer. Tamoxifen competitively block estradiol from binding to its receptor. Treatment of organophosphate insecticide poisoning with atropine is an example not of the antidote competing with the poison for the receptor (cholinesterase) but involves blocking the receptor (cholinergic receptor) for the excess acetylcholine that accumulates by poisoning of the cholinesterase by the organophosphate (see Chap. 22).

Tolerance Tolerance is a state of decreased responsiveness to a toxic effect of a chemical resulting from prior exposure to that chemical or to a

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structurally related chemical. Two major mechanisms are responsible for tolerance: one is due to a decreased amount of toxicant reaching the site where the toxic effect is produced (dispositional tolerance), and the other is due to a reduced responsiveness of a tissue to the chemical. Comparatively less is known about the cellular mechanisms responsible for altering the responsiveness of a tissue to a toxic chemical than is known about dispositional tolerance. Two chemicals known to produce dispositional tolerance are carbon tetrachloride and cadmium. Carbon tetrachloride produces tolerance to itself by decreasing the formation of the reactive metabolite (trichloromethyl radical) that produces liver injury (see Chap. 13). The mechanism of cadmium tolerance is explained by induction of metallothionein, a metal-binding protein. Subsequent binding of cadmium to metallothionein rather than to critical macromolecules decreases its toxicity.

CHARACTERISTICS OF EXPOSURE Toxic effects in a biological system are not produced by a chemical agent unless that agent or its metabolic breakdown (biotransformation) products reach appropriate sites in the body at a concentration and for a length of time sufficient to produce a toxic manifestation. Many chemicals are of relatively low toxicity in the “native” form but, when acted on by enzymes in the body, are converted to intermediate forms that interfere with normal cellular biochemistry and physiology. Thus, whether a toxic response occurs is dependent on the chemical and physical properties of the agent, the exposure situation, how the agent is metabolized by the system, the concentration of the active form at the particular target site(s), and the overall susceptibility of the biological system or subject. Thus, to characterize fully the potential hazard of a specific chemical agent, we need to know not only what type of effect it produces and the dose required to produce that effect but also information about the agent, the exposure, and its disposition by the subject. Two major factors that influence toxicity as it relates to the exposure situation for a specific chemical are the route of exposure, and the duration, and frequency of exposure.

Route and Site of Exposure The major routes (pathways) by which toxic agents gain access to the body are the gastrointestinal tract (ingestion), lungs (inhalation), skin (topical, percutaneous, or dermal), and other parenteral (other than intestinal canal) routes. Toxic agents generally produce the greatest effect and the most rapid response when given directly into the bloodstream (the intravenous route). An approximate descending order of effectiveness for the other routes would be inhalation, intraperitoneal, subcutaneous, intramuscular, intradermal, oral, and dermal. The “vehicle” (the material in which the chemical is dissolved) and other formulation factors can markedly alter absorption after ingestion, inhalation, or topical exposure. In addition, the route of administration can influence the toxicity of agents. For example, an agent that acts on the CNS, but is efficiently detoxified in the liver, would be expected to be less toxic when given orally than when given via inhalation, because the oral route requires that nearly all of the dose pass through the liver before reaching the systemic circulation and then the CNS. Occupational exposure to toxic agents most frequently results from breathing contaminated air (inhalation) and/or direct and prolonged contact of the skin with the substance (dermal exposure),

whereas accidental and suicidal poisoning occurs most frequently by oral ingestion. Comparison of the lethal dose of a toxic substance by different routes of exposure often provides useful information about its extent of absorption. In instances when the toxic dose after oral or dermal administration is similar to the toxic dose after intravenous administration, the assumption is that the toxic agent is absorbed readily and rapidly. Conversely, in cases where the toxic dose by the dermal route is several orders of magnitude higher than the oral toxic dose, it is likely that the skin provides an effective barrier to absorption of the agent. Toxic effects by any route of exposure can also be influenced by the concentration of the agent in its vehicle, the total volume of the vehicle and the properties of the vehicle to which the biological system is exposed, and the rate at which exposure occurs. Studies in which the concentration of a chemical in the blood is determined at various times after exposure are often needed to clarify the role of these and other factors in the toxicity of a compound. For more details on the absorption of toxicants, see Chap. 5.

Duration and Frequency of Exposure Toxicologists usually divide the exposure of experimental animals to chemicals into four categories: acute, subacute, subchronic, and chronic. Acute exposure is defined as exposure to a chemical for less than 24 hours, and examples of exposure routes are intraperitoneal, intravenous, and subcutaneous injection; oral intubation; and dermal application. Whereas acute exposure usually refers to a single administration, repeated exposures may be given within a 24-hours period for some slightly toxic or practically nontoxic chemicals. Acute exposure by inhalation refers to continuous exposure for less than 24 hours, most frequently for 4 hours. Repeated exposure is divided into three categories: subacute, subchronic, and chronic. Subacute exposure refers to repeated exposure to a chemical for 1 month or less, subchronic for 1 to 3 months, and chronic for more than 3 months, although usually this refers to studies with at least 1 year of repeated dosing. These three categories of repeated exposure can be by any route, but most often they occur by the oral route, with the chemical added directly to the diet. In human exposure situations, the frequency and duration of exposure are usually not as clearly defined as in controlled animal studies, but many of the same terms are used to describe general exposure situations. Thus, workplace or environmental exposures may be described as acute (occurring from a single incident or episode), subchronic (occurring repeatedly over several weeks or months), or chronic (occurring repeatedly for many months or years). For many chemicals, the toxic effects that follow a single exposure are quite different from those produced by repeated exposure. For example, the primary acute toxic manifestation of benzene is central nervous system (CNS) depression, but repeated exposures can result in bone marrow toxicity and an increased risk for leukemia. Acute exposure to chemicals that are rapidly absorbed is likely to produce immediate toxic effects but also can produce delayed toxicity that may or may not be similar to the toxic effects of chronic exposure. Conversely, chronic exposure to a toxic chemical may produce some immediate (acute) effects after each administration in addition to the long-term, low-level, or chronic effects of the toxic substance. In characterizing the toxicity of a specific chemical, it is evident that information is needed not only for the single-dose (acute) and long-term (chronic) effects but also for exposures of intermediate duration. The other time-related factor that is important in the temporal characterization of repeated

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Figure 2-2. Diagrammatic view of the relationship between dose and concentration at the target site under different conditions of dose frequency and elimination rate. Line A. A chemical with very slow elimination (e.g., half-life of 1 year). Line B. A chemical with a rate of elimination equal to frequency of dosing (e.g., 1 day). Line C. Rate of elimination faster than the dosing frequency (e.g., 5 h). Blue-shaded area is representative of the concentration of chemical at the target site necessary to elicit a toxic response.

exposures is the frequency of exposure. The relationship between elimination rate and frequency of exposure is shown in Fig. 2-2. A chemical that produces severe effects with a single dose may have no effect if the same total dose is given in several intervals. For the chemical depicted by line B in Fig. 2-2, in which the half-life for elimination (time necessary for 50% of the chemical to be removed from the bloodstream) is approximately equal to the dosing frequency, a theoretical toxic concentration (shown conceptually as two Concentration Units in Fig. 2-2) is not reached until the fourth dose, whereas that concentration is reached with only two doses for chemical A, which has an elimination rate much slower than the dosing interval (time between each repeated dose). Conversely, for chemical C, where the elimination rate is much shorter than the dosing interval, a toxic concentration at the site of toxic effect will never be reached regardless of how many doses are administered. Of course, it is possible that residual cell or tissue damage occurs with each dose even though the chemical itself is not accumulating. The important consideration, then, is whether the interval between doses is sufficient to allow for complete repair of tissue damage. It is evident that with any type of repeated exposure, the production of a toxic effect is influenced not only by the frequency of exposure but may, in fact, be totally dependent on the frequency rather than the duration of exposure. Chronic toxic effects may occur, therefore, if the chemical accumulates in the biological system (rate of absorption exceeds the rate of biotransformation and/or excretion), if it produces irreversible toxic effects, or if there is insufficient time for the system to recover from the toxic damage within the exposure frequency interval. For additional discussion of these relationships, see Chaps. 5 and 7.

DOSE–RESPONSE RELATIONSHIP The characteristics of exposure and the spectrum of toxic effects come together in a correlative relationship customarily referred to as the dose–response relationship. Whatever response is selected for measurement, the relationship between the degree of response of the biological system and the amount of toxicant administered assumes a form that occurs so consistently as to be considered the most fundamental and pervasive concept in toxicology. From a practical perspective, there are two types of dose– response relationships: (1) the individual dose–response relationship, which describes the response of an individual organism to varying doses of a chemical, often referred to as a “graded” response because the measured effect is continuous over a range of doses, and (2) a quantal dose–response relationship, which characterizes the distribution of individual responses to different doses in a population of individual organisms.

Individual, or Graded, Dose–Response Relationships Individual dose–response relationships are characterized by a doserelated increase in the severity of the response. The dose relatedness of the response often results from an alteration of a specific biochemical process. For example, Fig. 2-3 shows the dose–response relationship between different dietary doses of the organophosphate insecticide chlorpyrifos and the extent of inhibition of two

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Quantal Dose–Response Relationships In contrast to the “graded” or continuous-scale dose–response relationship that occurs in individuals, the dose–response relationships in a population are by definition quantal—or “all or none”—in nature; that is, at any given dose, an individual in the population is classified as either a “responder” or a “nonresponder.” Although these distinctions of “quantal population” and “graded individual” dose–response relationships are useful, the two types of responses are conceptually identical. The ordinate in both cases is simply labeled the response, which may be the degree of response in an individual or system or the fraction of a population responding, and the abscissa is the range in administered doses. A widely used statistical approach for estimating the response of a population to a toxic exposure is the “Effective Dose” or ED. Generally, the mid-point, or 50%, response level is used, giving rise to the “ED50 ” value. However, any response level, such as an ED01 , ED10 or ED30 could be chosen. A graphical representation of an approximate ED50 is shown in Fig. 2-4. Note that these data are

Figure 2-3. Dose–response relationship between different doses of the organophosphate insecticide chlorpyrifos and esterase enzyme inhibition in the brain. Open circles and blue lines represent acetylcholinesterase activity and closed circles represent carboxylesterase activity in the brains of pregnant female Long-Evans rats given 5 daily doses of chlorpyrifos. A. Dose–response curve plotted on an arithmetic scale. B. Same data plotted on a semi-log scale. (From Lassiter et al., Gestational exposure to chloryprifos: Dose response profiles for cholinesterase and carboxylesterase activity. Toxicol Sci 52:92– 100, 1999, with permission.)

different enzymes in the brain and liver: acetylcholinesterase and carboxylesterase. In the brain, the degree of inhibition of both enzymes is clearly dose-related and spans a wide range, although the amount of inhibition per unit dose is different for the two enzymes. From the shapes of these two dose–response curves it is evident that, in the brain, cholinesterase is more easily inhibited than carboxylesterase. The toxicologic response that results is directly related to the degree of cholinesterase enzyme inhibition in the brain. Thus, clinical signs and symptoms for chlorpyrifos would follow a dose–response relationship similar to that for brain cholinesterase. However, for many chemicals, more than one effect may result because of multiple different target sites in different tissues. Thus, the observed response to varying doses of a chemical in the whole organism is often complicated by the fact that most toxic substances have multiple sites or mechanisms of toxicity, each with its own “dose–response” relationship and subsequent adverse effect. Note that when these dose–response data are plotted using the base 10 log of the dose on the abscissa (Fig. 2.3B), a better “fit” of the data to a straight line usually occurs. This is typical of many graded as well as quantal dose–response relationships.

Figure 2-4. Diagram of quantal dose–response relationship. The abscissa is a log dosage of the chemical. In the top panel the ordinate is response frequency, in the middle panel the ordinate is percent response, and in the bottom panel the response is in probit units (see text).

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“quantal”. Where death is the measured end-point, the ED50 would be referred to as the Lethal Dose 50 (LD50 ). Historically, determination of the LD50 was often the first experiment performed with a new chemical. Today, it is widely recognized that the LD50 is of marginal value as a measure of hazard, although it does provide a useful “ball park” indication of the relative hazard of a compound to cause serious, life-threatening poisoning from a single exposure. Although death is an obvious quantal end-point to measure, it should be noted that any quantal response could be used. For example, the LD50 of lead or DDT is not a relevant endpoint when characterizing hazards of the agents to children or wildlife, respectively. Even continuous variables can be converted to quantal responses if desired. For example, an antihypertensive drug that lowers blood pressure might be evaluated in a population by assigning a “responder” as an individual who’s blood pressure was lowered by 10 mm Hg or more. Note that, in this example, an individual that responded to a change in blood pressure of 50 mm Hg would classified the same as an individual with a change in only 10 mm Hg, yet an individual with a change in 8 mm Hg would be classified as a “non-responder”. The top panel of Fig. 2-4 shows that quantal dose responses typically exhibit a normal or Gaussian distribution. The frequency histogram in this panel also shows the relationship between dose and effect. The bars represent the percentage of animals that responded at each dose minus the percentage that responded at the immediately lower dose. One can clearly see that only a few animals responded to the lowest dose and the highest dose. Larger numbers of animals responded to doses intermediate between these two extremes, and the maximum frequency of response occurred in the middle portion of the dose range. Thus, we have a bell-shaped curve known as a normal frequency distribution. The reason for this normal distribution is that there are differences in susceptibility to chemicals among individuals; this is known as biological variation. Animals responding at the left end of the curve are referred to as hypersusceptible, and those at the right end of the curve are called resistant. If the numbers of individuals responding at each consecutive dose are added together, a cumulative, quantal dose– response relationship is obtained. When a sufficiently large number of doses is used with a large number of animals per dose, a sigmoid dose–response curve is observed, as depicted in the middle panel of Fig. 2-4. With the lowest dose (6 mg/kg), 1% of the animals respond. A normally distributed sigmoid curve such as this one approaches a response of 0% as the dose is decreased and approaches 100% as the dose is increased; but—theoretically—it never passes through 0 and 100%. However, the minimally effective dose of any chemical that evokes a stated all-or-none response is called the threshold dose even though it cannot be determined experimentally. For a normally distributed population response, the sigmoid curve has a relatively linear portion between 16 and 84%. These values represent the limits of 1 standard deviation (SD) of the mean (and the median) in a population with truly normal or Gaussian distribution. However, it is usually not practical to describe the dose–response curve from this type of plot because one does not usually have large enough sample sizes to define the sigmoid curve adequately. In a normally distributed population, the mean ±1 SD represents 68.3% of the population, the mean ±2 SD represents 95.5% of the population, and the mean ±3 SD equals 99.7% of the population. Because quantal dose–response phenomena are usually normally distributed, one can convert the percent response to units of deviation from the mean or normal equivalent deviations (NEDs). Thus, the NED for a 50% response is 0; an NED of +1 is equated

21

with an 84.1% response. Traditionally, units of NED are converted by the addition of 5 to the value to avoid negative numbers; these converted units are called probit units (Bliss, 1957). The probit (from the contraction of probability unit), then, is an NED plus 5. In this transformation, a 50% response becomes a probit of 5, a +1 deviation becomes a probit of 6, and a –1 deviation is a probit of 4. The data given in the top two panels of Fig. 2-4 are replotted in the bottom panel with the response plotted in probit units. The data in the middle panel (which was in the form of a sigmoid curve) and the top panel (a bell-shaped curve) form a straight line when transformed into probit units. In essence, what is accomplished in a probit transformation is an adjustment of quantal data to an assumed normal population distribution, resulting in a straight line. The ED50 is obtained by drawing a horizontal line from the probit unit 5, which is the 50% response point, to the dose–effect line. At the point of intersection, a vertical line is drawn, and this line intersects the abscissa at the ED50 point. It is evident from the line that information with respect to the effective dose for 90% or for 10% of the population also may be derived by a similar procedure. Mathematically, it can be demonstrated that the range of values encompassed by the confidence limits is narrowest at the midpoint of the line (ED50 ) and widest at both extremes (ED10 and ED90 ) of the dose–response curve (dotted lines in Fig. 2-5). In addition to the ED50 , the slope of the dose–response curve can also be obtained. Figure 2-5 demonstrates the dose–response curves for the response of two compounds. Compound A exhibits a “flat” dose–response curve, showing that a large change in dosage is required before a significant change in response will be observed. However, compound B exhibits a “steep” dose–response curve, where a relatively small change in dosage will cause a large change in response. It is evident that the ED50 for both compounds is the same (8 mg/kg). However, the slopes of the dose– response curves are quite different. At one-half of ED50 of the compounds (4 mg/kg), less than 1% of the animals exposed to compound B would respond but 20% of the animals given compound A would respond. In Figs. 2-4 and 2-5 the dosage has been given on a log basis. Although the use of the log of the dosage is empiric, log-dosage plots for normally distributed quantal data provide a more nearly linear representation of the data. It must be remembered, however,

Figure 2-5. Comparison of dose–response relationship for two different chemicals, plotted on a log dose-probit scale. Note that the slope of the dose–response is steeper for chemical B than chemical A. Dotted lines represents the confidence limits for chemical A.

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Table 2.2 Allometric Scaling of Dose Across Different Species fold difference, relative to humans, normalized by body weight species Mouse Rat Guinea pig Rabbit Cat Monkey Dog Human ∗

weight (kg)

surface area (cm2 )∗

mg/kg

(bw)2/3

(bw)3/4

0.30 0.2 0.4 1.5 2 4 12 70

103 365 582 1410 1710 2720 5680 18500

1 1 1 1 1 1 1 1

13.0 6.9 5.5 3.5 3.2 2.6 1.8 1.0

7.0 4.3 3.6 2.6 2.4 2.0 1.5 1.0

Surface area of animals is closely approximated by the formula: SA = 10.5 × (Body wt, in grams)2/3 .

that this is not universally the case. Some radiation effects, for example, give a better probit fit when the dose is expressed arithmetically rather than logarithmically. There are other situations in which other functions (e.g., exponentials) of dosage provide a better fit to the data than does the log function. It is also conventional to express the dosage in milligrams per kilogram. It might be argued that expression of dosage on a mole-per-kilogram basis would be better, particularly for making comparisons among a series of compounds. Although such an argument has considerable merit, dosage is usually expressed in milligrams per kilogram. One might also view dosage on the basis of body weight as being less appropriate than other bases, such as surface area. The term Allometry refers to the field of study that examines the relationships between body weight and other biological and physical parameters such as rate of basal metabolism (caloric consumption), heart rate, blood flow, etc. Allometric studies revealed that the relationship between body weight and various other physiological parameters can be closely estimated by the formula, Y = aW b , where Y is the biological parameter of interest, a and b are constants that relate Y to body weight (Rodricks et al., 2001). In general, organ sizes between species seem to scale best when b is equal to 1, whereas metabolically derived parameters scale better when b is 0.67–0.75. The relationship between body surface area and body weight across most mammalian species is closely described by the formula SA = 10.5 × (body weight, in grams)0.67 (Harkness and Wagner, 1995). Empirical comparisons of toxicity data across species confirm that this relationship is appropriate for toxicological scaling. For example, Travis and White (1988) analyzed a number of toxicity testing data sets for 27 different chemotherapeutic drugs for which toxicity data were available in mouse, rat, hamster, dog, monkey, and human. They found that the exponent of body weight that gave the best correlation with toxicity was 0.73, with 95% confidence bounds of 0.69–0.77 (Rodricks et al., 2001). Table 2-2 illustrates the differences in comparative doses when scaling is done by body weight (mg/kg) versus an allometric approach that uses an exponent of either 0.67 or 0.75. Thus, if a scaling factor of (BW)2/3 is used, a mouse would need to receive a dose 13 times greater than humans for an equivalent toxic response, where as a it would be seven times greater if a scaling factor of (BW)3/4 was used. However, not all toxic responses will necessarily scale across species in the same way. For example, acute lethality seemed to correlate better across species when body weight, rather than body surface area, was used (Rhomberg and Wolf, 1998).

Shape of the Dose–Response Curve Essential Nutrients The shape of the dose–response relationship has many important implications in toxicity assessment. For example, for substances that are required for normal physiologic function and survival (e.g., vitamins and essential trace elements such as chromium, cobalt, and selenium), the shape of the “graded” dose– response relationship in an individual over the entire dose range is actually U-shaped (Fig. 2-6). That is, at very low doses, there is a high level of adverse effect, which decreases with an increasing dose. This region of the dose–response relationship for essential nutrients is commonly referred to as a deficiency. As the dose is increased to a point where the deficiency no longer exists, no adverse response is detected and the organism is in a state of homeostasis. However, as the dose is increased to abnormally high levels, an adverse response (usually qualitatively different from that observed at deficient doses) appears and increases in magnitude with increasing dose, just as with other toxic substances. Thus, it is recognized that high doses of vitamin A can cause liver toxicity and birth defects,

Figure 2-6. Individual dose–response relationship for an essential substance such as a vitamin or trace element. It is generally recognized that, for most types of toxic responses, a threshold exists such that at doses below the threshold, no toxicity is evident. For essential substances, doses below the minimum daily requirement, as well as those above the threshold for safety, may be associated with toxic effects. The blue-shaded region represents the “region of homeostasis”—the dose range that results in neither deficiency or toxicity.

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(curve A, Fig. 2-7). However, there is also substantial clinical and epidemiologic evidence that low to moderate consumption of alcohol reduces the incidence of coronary heart disease and stroke (curve B, Fig. 2-7) (Hanna et al., 1997). Thus, when all responses are plotted on the ordinate, a “U-shaped” dose–response curve is obtained (curve C, Fig. 2-7). U-shaped dose–response relationships have obvious implications for the process of low dose extrapolation in risk assessment.

Figure 2-7. Hypothetical dose–response relationship depicting characteristics of hormesis. Hormetic effects of a substance are hypothesized to occur when relatively low doses result in the stimulation of a beneficial or protective response (B), such as induction of enzymatic pathways that protect against oxidative stress. Although low doses provide a potential beneficial effect, a threshold is exceeded as the dose increases and the net effects will be detrimental (A), resulting in a typical dose-related increase in toxicity. The complete dose– response curve (C) is conceptually similar to the individual dose–response relationship for essential nutrients shown in Fig. 2-6.

high doses of selenium can affect the brain, and high doses of estrogens may increase the risk of breast cancer, even though low doses of all these substances are essential for life. Hormesis There is considerable evidence to suggest that some nonnutritional toxic substances may also impart beneficial or stimulatory effects at low doses but that, at higher doses, they produce adverse effects. This concept of “hormesis” was first described for radiation effects but may also pertain to most chemical responses (Calabrese and Blaine, 2005). Thus, in plotting dose versus response over a wide range of doses, the effects of hormesis may also result in a “U-shaped” dose–response curve. In its original development, the concept of hormesis pertained to the ability of substances to stimulate biological systems at low doses but to inhibit them at high doses. The application of the concept of hormesis to whole-animal toxicologic dose–response relationships may also be relevant but requires that the “response” on the ordinate be variant with dose. For example, chronic alcohol consumption is well recognized to increase the risk of esophageal cancer, liver cancer, and cirrhosis of the liver at relatively high doses, and this response is dose-related

Threshold Another important aspect of the dose–response relationship at low doses is the concept of the threshold. It has long been recognized that acute toxicologic responses are associated with thresholds; that is, there is some dose below which the probability of an individual responding is zero. Obviously, the identification of a threshold depends on the particular response that is measured, the sensitivity of the measurement, and the number of subjects studied. For the individual dose–response relationship, thresholds for most toxic effects certainly exist, although interindividual variability in response and qualitative changes in response pattern with dose make it difficult to establish a true “no effects” threshold for any chemical. The biological basis of thresholds for acute responses is well established and frequently can be demonstrated on the basis of mechanistic information (Aldridge, 1986). The traditional approaches to establishing acceptable levels of exposure to chemicals are inherently different for threshold versus nonthreshold responses. The existence of thresholds for chronic responses is less well defined, especially in the area of chemical carcinogenesis. It is, of course, impossible to scientifically prove the absence of a threshold, as one can never prove a negative. Nevertheless, for the identification of “safe” levels of exposure to a substance, the absence or presence of a threshold is important for practical reasons (see Chap. 4). A classic example of the difficulty of establishing thresholds experimentally is provided by the “ED01” study, where over 24,000 mice and 81 different treatment groups were used to determine the shape of the dose–response relationship for the prototypical carcinogen 2acetylaminofluorene (2-AAF). The study was designed to identify a statistically significant response of 1% (0.01 probability). The mice were exposed to 2-AAF at one of seven different doses in the dose range of 30 to 150 ppm (plus 0 dose control) (Littlefield et al., 1979). Eight “sacrifice intervals” were used to determine how quickly tumors developed. The dose–response relationship between 2-AAF exposure and liver and bladder cancer at 24 and 33 months of exposure are shown in Fig. 2-8. Both types of tumors demonstrated increasing incidence with increasing dose, but the shapes of the two curves are dramatically different. For liver tumors, no clear threshold was evident, whereas for bladder tumors, an apparent threshold was evident. However, the apparent threshold, or “no observable adverse effect level” (NOAEL), for bladder cancer was lower at 33 months (45 ppm) than at 24 months (75 ppm). Of course, the ability to detect a low incidence of tumors depends on the number of animals used in the study. Thus, although a threshold (a dose below which no response occurs) appears evident for bladder tumors in Fig. 2-8, one cannot say for certain that tumors would not occur if more animals had been included in the lower-dose groups. (See Chap. 4 for more discussion on statistical issues related to extrapolation of dose–response curves and the determination of NOAELs.) In evaluating the shape of the dose–response relationship in populations, it is realistic to consider inflections in the shape of the dose–response curve rather than absolute thresholds. That is, the slope of the dose–response relationship at high doses may be

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m

although the risk decreases proportionately with a decrease in the dose. The existence or lack of existence of a threshold dose for carcinogens has many regulatory implications and is a point of considerable controversy and research in the field of quantitative risk assessment for chemical carcinogens (see Chap. 4).

Assumptions in Deriving the Dose–Response Relationship

m

Figure 2-8. Dose–response relationship for carcinogens.

A number of assumptions must be considered before dose–response relationships can be used appropriately. The first is that the response is due to the chemical administered. To describe the relationship between a toxic material and an observed effect or response, one must know with reasonable certainty that the relationship is indeed a causal one. For some data, it is not always apparent that the response is a result of chemical exposure. For example, an epidemiologic study might result in the discovery of an “association” between a response (e.g., disease) and one or more variables. Frequently, the data are presented similarly to the presentation of “dose response” in pharmacology and toxicology. Use of the dose response in this context is suspect unless other convincing evidence supports a causal connection between the estimated dose and the measured endpoint (response). Unfortunately, in nearly all retrospective and case-control studies and even in many prospective studies, the dose, duration, frequency, and routes of exposure are seldom quantified, and other potential etiologic factors are frequently present. In its most strict usage, then, the dose–response relationship is based on the knowledge that the effect is a result of a known toxic agent or agents. A second assumption seems simple and obvious: The magnitude of the response is in fact related to the dose. Perhaps because of its apparent simplicity, this assumption is often a source of misunderstanding. It is really a composite of three other assumptions that recur frequently:

Eight groups of male mice were administered 2-acetylaminofluorine (2AAF) in the diet from weaning. The percent of animals with liver (blue line) or bladder (black line) tumors at 24 months (A) or 33 months (B) are shown. Most of the animals in the high-dose group (150 ppm) did not survive to 33 months; thus, those data are not shown in B.

1. There is a molecular target site (or sites) with which the chemical interacts to initiate the response. 2. The production of a response and the degree of response are related to the concentration of the chemical at the target site. 3. The concentration at the site is, in turn, related to the dose administered.

substantially different from the slope at low doses, usually because of dispositional differences in the chemical. Saturation of biotransformation pathways, protein-binding sites or receptors, and depletion of intracellular cofactors represent some reasons why sharp inflections in the dose–response relationship may occur. For example, the widely used analgesic acetaminophen has a very low rate of liver toxicity at normal therapeutic doses. Even though a toxic metabolite [N -acetyl- p-benzoquinoneimine (NAPQI)] is produced in the liver at therapeutic doses, it is rapidly detoxified through conjugation with the intracellular antioxidant glutathione. However, at very high doses, the level of intracellular glutathione in the liver is depleted and NAPQI accumulates, causing serious and potentially fatal liver toxicity. This effect is analogous to the rapid change in pH of a buffered solution that occurs when the buffer capacity is exceeded. Some toxic responses, most notably the development of cancer after the administration of genotoxic carcinogens, are often considered to be linear at low doses and thus do not exhibit a threshold. In such circumstances, there is no dose with “zero” risk,

The third assumption in using the dose–response relationship is that there exists both a quantifiable method of measuring and a precise means of expressing the toxicity. For any given dose–response relationship, a great variety of criteria or endpoints of toxicity could be used. The ideal criterion would be one closely associated with the molecular events resulting from exposure to the toxicant. It follows from this that a given chemical may have a family of dose–response relationships, one for each toxic endpoint. For example, a chemical that produces cancer through genotoxic effects, liver damage through inhibition of a specific enzyme, and CNS effects via a different mechanism may have three distinct dose–response relationships, one for each endpoint. Early in the assessment of toxicity, little mechanistic information is usually available; thus establishing a dose–response relationship based on the molecular mechanism of action is usually impossible. Indeed, it might not be approachable even for well-known toxicants. In the absence of a mechanistic, molecular ideal criterion of toxicity, one looks to a measure of toxicity that is unequivocal and clearly relevant to the toxic effect. Such measures are often referred to as “effects-related biomarkers.” For

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example, with a new compound chemically related to the class of organophosphate insecticides, one might approach the measurement of toxicity by measuring the inhibition of cholinesterase in blood. In this way, one would be measuring, in a readily accessible system and using a technique that is convenient and reasonably precise, a prominent effect of the chemical and one that is usually pertinent to the mechanism by which toxicity is produced. The selection of a toxic endpoint for measurement is not always so straightforward. Even the example cited above may be misleading, as an organophosphate may produce a decrease in blood cholinesterase, but this change may not be directly related to its toxicity. As additional data are gathered to suggest a mechanism of toxicity for any substance, other measures of toxicity may be selected. Although many endpoints are quantitative and precise, they are often indirect measures of toxicity. Changes in enzyme levels in blood can be indicative of tissue damage. For example, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are used to detect liver damage. Use of these enzymes in serum is yet another example of an effects-related biomarker because the change in enzyme activity in the blood is directly related to damage to liver cells. Much of clinical diagnostic medicine relies on effects-related biomarkers, but to be useful the relationship between the biomarker and the disease must be carefully established. Patterns of isozymes and their alteration may provide insight into the organ or system that is the site of toxic effects. As discussed later in this chapter, the new tools of toxicogenomics provide an unprecedented opportunity to discover new “effects-related biomarkers” in toxicology. Many direct measures of effects are also not necessarily related to the mechanism by which a substance produces harm to an organism but have the advantage of permitting a causal relation to be drawn between the chemical and its action. For example, measurement of the alteration of the tone of smooth or skeletal muscle for substances acting on muscles represents a fundamental approach to toxicological assessment. Similarly, measures of heart rate, blood pressure, and electrical activity of heart muscle, nerve, and brain are examples of the use of physiologic functions as indices of toxicity. Measurement can also take the form of a still higher level of integration, such as the degree of motor activity or behavioral change. The measurements used as examples in the preceding discussion all assume prior information about the toxicant, such as its target organ or site of action or a fundamental effect. However, such information is usually available only after toxicological screening and testing based on other measures of toxicity. With a new substance, the customary starting point is a single dose acute toxicity test designed to provide preliminary identification of target organ toxicity. Studies specifically designed with lethality as an end-point are no longer recommended by United States or international agencies. Data from acute studies provides essential information for choosing doses for repeated dosing studies as well as choosing specific toxicological endpoints for further study. Key elements of the study design must be a careful, disciplined, detailed observation of the intact animal extending from the time of administration of the toxicant to any clinical signs of distress, which may include detailed behavioral observations or physiological measures. It is recommended that these observations be taken over a 14-day period. From properly conducted observations, immensely informative data can be gathered by a trained toxicologist. Second, an acute toxicity study ordinarily is supported by histological examination of major tissues and organs for abnormalities. From these observations, one can usually obtain more specific information about the events leading to the various

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Figure 2-9. Comparison of effective dose (ED), toxic dose (TD), and lethal dose (LD). The plot is of log dosage versus percentage of population responding in probit units.

endpoints, the target organs involved, and often a suggestion about the possible mechanism of toxicity at a relatively fundamental level.

Evaluating the Dose–Response Relationship Comparison of Dose Responses Figure 2-9 illustrates a hypothetical quantal dose–response curve for a desirable effect of a chemical effective dose (ED) such as anesthesia, a toxic dose (TD) effect such as liver injury, and the lethal dose (LD). As depicted in Fig. 2-9, a parallelism is apparent between the ED curve and the curve depicting mortality (LD). It is tempting to view the parallel dose–response curves as indicative of identity of mechanism—that is, to conclude that the lethality is a simple extension of the therapeutic effect. Whereas this conclusion may ultimately prove to be correct in any particular case, it is not warranted solely on the basis of the two parallel lines. The same admonition applies to any pair of parallel “effect” curves or any other pair of toxicity or lethality curves. Therapeutic Index The hypothetical curves in Fig. 2-9 illustrate two other interrelated points: the importance of the selection of the toxic criterion and the interpretation of comparative effect. The concept of the “therapeutic index,” which was introduced by Paul Ehrlich in 1913, can be used to illustrate this relationship. Although the therapeutic index is directed toward a comparison of the therapeutically effective dose to the toxic dose of a chemical, it is equally applicable to considerations of comparative toxicity. The therapeutic index (TI) in its broadest sense is defined as the ratio of the dose required to produce a toxic effect and the dose needed to elicit the desired therapeutic response. Similarly, an index of comparative toxicity is obtained by the ratio of doses of two different materials to produce an identical response or the ratio of doses of the same material necessary to yield different toxic effects. The most commonly used index of effect, whether beneficial or toxic, is the median effect dose (ED50 ). The therapeutic index of a drug is an approximate statement about the relative safety of a drug expressed as the ratio of the adverse endpoint or toxic dose (historically the lethal dose) to the therapeutic dose: Therapeutic Index = TD50 /ED50

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Figure 2-10. Schematic representation of the difference in the dose–response curves for four chemicals (A–D), illustrating the difference between potency and efficacy (see text).

From Fig. 2-9 one can approximate a therapeutic index by using these median doses. The larger the ratio, the greater the relative safety. The ED50 is approximately 20, and the TD50 is about 60; thus, the therapeutic index is 3, a number indicating that reasonable care in exposure to the drug is necessary to avoid toxicity. However, the use of the median effective and median toxic doses is not without disadvantages, because median doses tell nothing about the slopes of the dose–response curves for therapeutic and toxic effects. Margins of Safety and Exposure One way to overcome this deficiency is to use the ED99 for the desired effect and the TD1 for the undesired effect. These parameters are used in the calculation of the margin of safety (MOS): Margin of safety = TD1 /ED99 The quantitative comparisons described above have been used mainly after a single administration of chemicals. However, for chemicals for which there is no beneficial or effective dose and exposures are likely to occur repeatedly, the ratio of TD1 to ED99 has little relevance. Thus, for non-drug chemicals, the term margin of safety has found use in risk-assessment procedures as an indicator of the magnitude of the difference between an estimated “exposed dose” to a human population and the NOAEL or other benchmark dose determined in experimental animals. A measure of the degree of accumulation of a chemical and/or its toxic effects can also be estimated from quantal toxicity data. The chronicity index of a chemical is a unitless value obtained by dividing its 1-dose TD50 by its 90-dose (90-day) TD50 , with both expressed in milligrams per kilogram per day. Theoretically, if no cumulative effect occurs over the doses, the chronicity index will be 1. If a compound were absolutely cumulative, the chronicity index would be 90. Historically, statistical procedures similar to those used to calculate the LD50 can also be used to determine the lethal time 50 (LT50 ), or the time required for half the animals to die (Litchfield, 1949). The LT50 value for a chemical indicates the time course of the toxic effects but does not indicate whether one chemical is more toxic than another. Frequently, dose–response curves from repeated-dose experimental animal studies (subacute, subchronic, or chronic) are used

to estimate the NOAEL, or some other “benchmark” measure of minimal toxic response, such as the dose estimated to produce toxic effects in 10% of the population (TD10 ) (see also Chap. 4). These estimates of minimal toxic dose, derived from quantal dose–response curves, can be used in risk assessment to derive a “margin of exposure” (MOE) index. This index compares the estimated daily exposure, in milligrams per kilogram per day, that might occur under a given set of circumstances to some estimated value from the quantal dose–response relationship (e.g., NOAEL or TD10 ). Like the MOS, the MOE is often expressed as a ratio of these two values. Thus, for example, if an estimate of human exposure to a pesticide residue yielded a value of 0.001 mg/kg/day, and a TD10 of 1 mg/kg/day was determined for that same pesticide, the MOE would be 1000. This value indicates that the estimate of daily exposure under the described set of conditions is 1/1000 the estimated daily dose that would cause evident toxicity in 10% of exposed animals. (See Chap. 4 for a more complete discussion of benchmark doses, NOAELs, and MOE.) Potency versus Efficacy To compare the toxic effects of two or more chemicals, the dose response to the toxic effects of each chemical must be established. One can then compare the potency and maximal efficacy of the two chemicals to produce a toxic effect. These two important terms can be explained by reference to Fig. 2-10, which depicts dose–response curves to four different chemicals for the frequency of a particular toxic effect, such as the production of tumors. Chemical A is said to be more potent than chemical B because of their relative positions along the dosage axis. Potency thus refers to the range of doses over which a chemical produces increasing responses. Thus, A is more potent than B and C is more potent than D. Maximal efficacy reflects the limit of the dose–response relationship on the response axis to a certain chemical. Chemicals A and B have equal maximal efficacy, whereas the maximal efficacy of C is less than that of D.

VARIATION IN TOXIC RESPONSES Selective Toxicity Selective toxicity means that a chemical produces injury to one kind of living matter without harming another form of life even though

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the two may exist in intimate contact (Albert, 1965, 1973). The living matter that is injured is termed the uneconomic form (or undesirable), and the matter protected is called the economic form (or desirable). They may be related to each other as parasite and host or may be two tissues in one organism. This biological diversity interferes with the ability of ecotoxicologists to predict the toxic effects of a chemical in one species (humans) from experiments performed in another species (laboratory animals). However, by taking advantage of the biological diversity, it is possible to develop chemicals that are lethal for an undesired species and harmless for other species. In agriculture, for example, there are fungi, insects, and even competitive plants that injure the crop, and thus selective pesticides are needed. Similarly, animal husbandry and human medicine require chemicals, such as antibiotics, that are selectively toxic to the undesirable form but do not produce damage to the desirable form. Drugs and other chemicals used for selective toxic purposes are selective for one of two reasons. Either (1) the chemical is equally toxic to both economic and uneconomic cells but is accumulated mainly by uneconomic cells or (2) it reacts fairly specifically with a cytological or a biochemical feature that is absent from or does not play an important role in the economic form (Albert, 1965, 1973). Selectivity resulting from differences in distribution usually is caused by differences in the absorption, biotransformation, or excretion of the toxicant. The selective toxicity of an insecticide spray may be partly due to a larger surface area per unit weight that causes the insect to absorb a proportionally larger dose than does the mammal being sprayed. The effectiveness of radioactive iodine in the treatment of hyperthyroidism (as well as its thyroid carcinogenicity) is due to the selective ability of the thyroid gland to accumulate iodine. A major reason why chemicals are toxic to one, but not to another, type of tissue is that there are differences in accumulation of the ultimate toxic compound in various tissues. This, in turn, may be due to differences in the ability of various tissues to transport or biotransform the chemical into the ultimate toxic product. Selective toxicity caused by differences in comparative cytology is exemplified by a comparison of plant and animal cells. Plants differ from animals in many ways—for example, absence of a nervous system, an efficient circulatory system, and muscles as well as the presence of a photosynthetic mechanism and cell walls. The fact that bacteria contain cell walls and humans do not has been utilized in developing selective toxic chemotherapeutic agents, such as penicillin and cephalosporins, that kill bacteria but are relatively nontoxic to mammalian cells. Selective toxicity can also be a result of a difference in biochemistry in the two types of cells. For example, bacteria do not absorb folic acid but synthesize it from p-aminobenzoic acid, glutamic acid, and pteridine, whereas mammals cannot synthesize folic acid but have to absorb it from the diet. Thus, sulfonamide drugs are selectively toxic to bacteria because the sulfonamides, which resemble p-aminobenzoic acid in both charge and dimensions, antagonize the incorporation of p-aminobenzoic acid into the folic acid molecule—a reaction that humans do not carry out.

Species Differences Although a basic tenet of toxicology is that “experimental results in animals, when properly qualified, are applicable to humans,” it is important to recognize that both quantitative and qualitative differences in response to toxic substances may occur among different species. As discussed above, there are many reasons for selective

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toxicity among different species. Even among phylogenetically similar species (e.g., rats, mice, guinea pigs, and hamsters), large differences in response may occur. For example, the LD50 for the highly toxic dioxin, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), differs by more than 1000-fold between guinea pigs and hamsters. Not only does the lethal dose for TCDD vary widely among species, so do the particular target organs affected. Species differences in response to carcinogenic chemicals represent an important issue in regulatory risk assessment. As discussed in Chap. 4, extrapolation of laboratory animal data to infer human cancer risk is currently a key component of regulatory decision making. The validity of this approach of course depends on the relevance of the experimental animal model to humans. Large differences in carcinogenic response between experimental animal species are not unusual. For example, mice are highly resistant to the hepatocarcinogenic effects of the fungal toxin aflatoxin B1 . Dietary doses as high as 10,000 parts per billion (ppb) failed to produce liver cancer in mice, whereas in rats dietary doses as low as 15 ppb produced a significant increase in liver tumors (Wogan et al., 1974). The mechanistic basis for this dramatic difference in response appears to be entirely related to species differences in the expression of a particular form of glutathione Stransferase (mGSTA3-3) that has unusually high catalytic activity toward the carcinogenic epoxide of aflatoxin (Eaton and Gallagher, 1994). Mice express this enzyme constitutively, whereas rats normally express a closely related form with much less detoxifying activity toward aflatoxin epoxide. Interestingly, rats do possess the gene for a form of glutathione S-transferase with high catalytic activity toward aflatoxin epoxide (rGSTA5-5) that is inducible by certain dietary antioxidants and drugs. Thus, dietary treatment can dramatically change the sensitivity of a species to a carcinogen. Other examples in which large species differences in response to carcinogens have been observed include the development of renal tumors from 2,3,5-trimethylpentane and d-limonene in male rats (Lehman-McKeeman and Caudill, 1992), the production of liver tumors from “peroxisomal proliferators” such as the antilipidemic drug clofibrate and the common solvent trichloroethylene (Roberts, 1999), and the induction of nasal carcinomas in rats after inhalation exposure to formaldehyde (Monticello and Morgan, 1997). Identifying the mechanistic basis for species differences in response to chemicals is an important part of toxicology because only through a thorough understanding of these differences can the relevance of animal data to human response be verified.

Individual Differences in Response Even within a species, large interindividual differences in response to a chemical can occur because of subtle genetic differences. Hereditary differences in a single gene that occur in more than 1% of the population are referred to as genetic polymorphism and may be responsible for idiosyncratic reactions to chemicals, as discussed earlier in this chapter. However, genetic polymorphism may have other important but less dramatic effects than those described for acute idiosyncratic responses (such as that occurring in pseudocholinesterase-deficient individuals after succinylcholine exposure). For example, it is recognized that approximately 50% of the Caucasian population has a gene deletion for the enzyme glutathione S-transferase M1. This enzyme has no apparent significant physiologic function, and thus homozygotes for the gene deletion (e.g., those who lack both copies of the normal gene) are functionally and physiologically normal. However, epidemiologic studies have indicated that smokers who are homozygous for the null allele

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may be at slightly increased risk of developing lung cancer compared with smokers who have one or both copies of the normal gene (Mohr et al., 2003). Chapter 6 provides additional examples of genetic differences in biotransformation enzymes that may be important determinants of variability in individual susceptibility to chemical exposures. Genetic polymorphism in physiologically important genes may also be responsible for interindividual differences in toxic responses. For example, studies in transgenic mice have shown that mice possessing one copy of a mutated p53 gene (a so-called tumor suppressor gene; see Chap. 8) are much more susceptible to some chemical carcinogens than are mice with two normal copies of the gene (Tennant et al., 1999). In humans, there is evidence that possessing one mutated copy of a tumor suppressor gene greatly increases the risk of developing certain cancers. For example, retinoblastoma is a largely inherited form of cancer that arises because of the presence of two copies of a defective tumor suppressor gene (the Rb gene) (Wiman, 1993). Individuals with one mutated copy of the Rb gene and one normal copy are not destined to acquire the disease (as are those with two copies of the mutated gene), although their chance of acquiring it is much greater than that of persons with two normal Rb genes. This is the case because both copies of the gene must be nonfunctional for the disease to develop. With one mutated copy present genetically, the probability of acquiring a mutation of the second gene (potentially from exposure to environmental mutagens) is much greater than the probability of acquiring independent mutations in both copies of the gene as would be necessary in people with two normal Rb alleles. (See Chap. 8 for additional discussion of tumor suppressor genes.) As our understanding of the human genome increases, more “susceptibility” genes will be discovered, and it is likely that the etiology of many chronic diseases will be shown to be related to a combination of genetics and environment. Simple blood tests may ultimately be developed that allow an individual to learn whether he or she may be particularly susceptible to specific drugs or environmental pollutants. Although the public health significance of this type of information could be immense, the disclosure of such information raises many important ethical and legal issues that must be addressed before wide use of such tests. The study of “gene-environment” interactions, or “Ecogenetics” (Costa and Eaton, 2006) is a rapidly developing field of substantial relevance to toxicology. It is likely that the majority of chronic diseases develop as a result of the complex interplay between multiple genes and the myriad of environmental factors, including diet, lifestyle, and occupational and/or environmental exposures to toxic substances.

DESCRIPTIVE ANIMAL TOXICITY TESTS Two main principles underlie all descriptive animal toxicity testing. The first is that the effects produced by a compound in laboratory animals, when properly qualified, are applicable to humans. This premise applies to all of experimental biology and medicine. On the basis of dose per unit of body surface, toxic effects in humans are usually in the same range as those in experimental animals. On a body weight basis, humans are generally more vulnerable than are experimental animals. When one has an awareness of these quantitative differences, appropriate safety factors can be applied to calculate relatively safe doses for humans. All known chemical carcinogens in humans, with the possible exception of arsenic, are carcinogenic in some species but not in all laboratory animals. It has become increas-

ingly evident that the converse—that all chemicals carcinogenic in animals are also carcinogenic in humans—is not true (Dybing and Sanner, 1999; Grisham, 1997; Hengstler et al., 1999). However, for regulatory and risk assessment purposes, positive carcinogenicity tests in animals are usually interpreted as indicative of potential human carcinogenicity. If a clear understanding of the mechanism of action of the carcinogen indicates that a positive response in animals is not relevant to humans, a positive animal bioassay may be considered irrelevant for human risk assessment (see Chap. 4). This species variation in carcinogenic response appears to be due in many instances to differences in biotransformation of the procarcinogen to the ultimate carcinogen (see Chap. 6). The second principle is that exposure of experimental animals to chemicals in high doses is a necessary and valid method of discovering possible hazards in humans. This principle is based on the quantal dose–response concept that the incidence of an effect in a population is greater as the dose or exposure increases. Practical considerations in the design of experimental model systems require that the number of animals used in toxicology experiments always be small compared with the size of human populations at risk. Obtaining statistically valid results from such small groups of animals requires the use of relatively large doses so that the effect will occur frequently enough to be detected. However, the use of high doses can create problems in interpretation if the response(s) obtained at high doses does not occur at low doses. Thus, for example, it has been shown that bladder tumors observed in rats fed very high doses of saccharin will not occur at the much lower doses of saccharin encountered in the human diet. At the high concentrations fed to rats, saccharin forms an insoluble precipitate in the bladder that subsequently results in chronic irritation of bladder epithelium, enhanced cell proliferation, and ultimately bladder tumors (Cohen, 1998, 1999). In vitro studies have shown that precipitation of saccharin in human urine will not occur at the concentrations that could be obtained from even extraordinary consumption of this artificial sweetener. Examples such as this illustrate the importance of considering the molecular, biochemical, and cellular mechanisms responsible for toxicological responses when extrapolating from high to low dose and across species. Toxicity tests are not designed to demonstrate that a chemical is safe but to characterize the toxic effects a chemical can produce. Although there are no set toxicology tests that have to be performed on every chemical intended for commerce, a tiered approach typical of many hazard assessment programs is shown illustrated in Fig. 2-11. Depending on the eventual use of the chemical, the toxic effects produced by structural analogs of the chemical, as well as the toxic effects produced by the chemical itself, contribute to the determination of the toxicology tests that should be performed. The FDA, EPA, and Organization for Economic Cooperation and Development (OECD) have written good laboratory practice (GLP) standards that stipulate that procedure must be defined and accountability documented. These guidelines are expected to be followed when toxicity tests are conducted in support of the introduction of a chemical to the market. The following sections provide an overview of basic toxicity testing procedures in use today. For a detailed description of these tests, the reader is referred to several authoritative texts on this subject (Williams and Hottendorf, 1999; Hayes, 2001; Jacobson-Kram and Keller, 2001). Although different countries have often had different testing requirements for toxicity testing/product safety evaluation, efforts to “harmonize” such testing protocols have resulted in more

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Test Material Identification

Chemical Characterization

Literature Review

Structure/Activity Assessment

Short-Term Animal Studies (Acute/Short-Term Repeated Dose)

In Vitro Genetic Toxicology

Metabolism/Pharmacokinetics

Subchronic Toxicity

Reproductive/Teratology

Chronic Toxicity

Oncogenicity

Figure 2-11. Typical Tiered Testing Scheme for the Toxicological Evaluation of New Chemicals (From: Wilson et al., 2001. In: Hayes, 2001; Principles and Methods in Toxicology, 4th ed, Fig. 19-1, p. 918.)

standardized approaches. The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) includes regulatory authorities from Europe, Japan, and the United States (primarily the FDA), as well as experts from the pharmaceutical industry in the three regions, who worked together to develop internationally recognized scientific and technical approaches to pharmaceutical product registration. ICH has adopted guidelines for most areas of toxicity testing (Table 2-3). In addition to safety assessment (ICH Guidelines designated with an “S”), ICH has also established guidelines on Quality (Q), Efficacy (E) and Multidisciplinary (M) topics. [see: http://www.ich.org/cache/compo/276-254-1.html for a description of current ICH guidelines and (Pugsley and Curtiss, 2006) for a detailed discussion of in vitro and in vivo pharmacological methods development that has been informed by the ICH regulatory guidance document for pre-clinical safety testing of drugs]. Typically, a tiered approach is used, with subsequent tests dependent on results of initial studies. A general framework for how new chemicals are evaluated for toxicity is shown in Fig 2-11. Early studies require careful chemical evaluation of the compound or mixture to assess purity, stability, solubility, and other physicochemical factors that could impact the ability of the test compound to be delivered effectively to animals. Once this information is obtained, the chemical structure of the test compound is compared with similar chemicals for which toxicological information is already available. Structure-activity relationships may be derived from a

review of existing toxicological literature, and can provide additional guidance on design of acute and repeated dose experiments, and what specialized tests need to be completed. Once such basic information has been compiled and evaluated, the test compound is then administered to animals in acute and repeated dose studies.

Acute Toxicity Testing Generally, the first toxicity test performed on a new chemical is acute toxicity, determined from the administration of a single exposure. The objectives of acute toxicity testing are to: (1) provide an estimate of the intrinsic toxicity of the substance, often times expressed as an approximate lethal dose (e.g., LD50 ), (2) provide information on target organs and other clinical manifestations of toxicity, (3) identify species differences and susceptible species, (4) establish the reversibility of the toxic response, and (5) provide information that will assist in the design and dose selection for longer term (sub-chronic, chronic) studies. It should be noted that the ICH recommended in 1991 (D’Arcy and Harron, 1992) the elimination of LD50 determinations for pharmaceuticals, although other regulatory requirements, e.g., pesticide registration, may still require determinations of LD50 s. The LD50 and other acute toxic effects are determined after one or more routes of administration (one route being oral or the intended route of exposure) in one or more species. The species most often used are the mouse and rat. Studies are performed in both adult

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Table 2.3 International Conference on Harmonization (ICH) Codification of “Safety” Protocols. Titles and abbreviations adopted November, 2005 S1A S1B S1C(R1)

Carcinogenicity studies Need for Carcinogenicity Studies of Pharmaceuticals Testing for Carcinogenicity of Pharmaceuticals Dose Selection for Carcinogenicity Studies of Pharmaceuticals & Limit Dose

S2A S2B

Genotoxicity studies Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals

S3A S3B

Toxicokinetics and pharmacokinetics Note for Guidance on Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies Pharmacokinetics: Guidance for Repeated Dose Tissue Distribution Studies

S4

Toxicity testing Single Dose Toxicity Tests Duration of Chronic Toxicity Testing in Animals (Rodent and Non Rodent Toxicity Testing)

S5(R2)

Reproductive toxicology Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility

S6

Biotechnological products Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

S7A S7B

Pharmacology studies Safety Pharmacology Studies for Human Pharmaceuticals The Non-Clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals

S8

Immunotoxicology studies Immunotoxicity Studies for Human Pharmaceuticals

M3(R1)

Joint safety/efficacy (multidisciplinary) topic Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals

Data from: http://www.ich.org/cache/compo/276-254-1.html.

male and female animals. Food is often withheld the night before dosing. The number of animals that die in a 14-day period after a single dosage is tabulated. In addition to mortality and weight, daily examination of test animals should be conducted for signs of intoxication, lethargy, behavioral modifications, morbidity, food consumption, and so on. Determination of the LD50 has become a public issue because of increasing concern for the welfare and protection of laboratory animals. The LD50 is not a biological constant. Many factors influence toxicity and thus may alter the estimation of the LD50 in any particular study. Factors such as animal strain, age, and weight, type of feed, caging, pretrial fasting time, method of administration, volume and type of suspension medium, and duration of observation have all been shown to influence adverse responses to toxic substances. These and other factors have been discussed in detail in earlier editions of this textbook (Doull, 1980). Because of this inherent variability in LD50 estimates, it is now recognized that for most purposes it is only necessary to characterize the LD50 within an order of magnitude range such as 5–50 mg/kg, 50–500 mg/kg, and so on. There are several traditional approaches to determining the LD50 and its 95% confidence limit as well as the slope of the probit line. The reader is referred to the classic works of Litchfield and Wilcoxon (1949), Bliss (1957), and Finney (1971) for a description of the mechanics of these procedures. Other statistical tech-

niques that require fewer animals, such as the “moving averages” method of Thompson and Weill (Weil, 1952), are available but do not provide confidence limits for the LD50 and the slope of the probit line. Finney (1985) has succinctly summarized the advantages and deficiencies of many of the traditional methods. For most circumstances, an adequate estimate of the LD50 and an approximation of the 95% confidence intervals can be obtained with as few as 6 to 9 animals, using the “up-and-down” method as modified by Bruce (1985). When this method was compared with traditional methods that typically utilize 40 to 50 animals, excellent agreement was obtained for all 10 compounds tested (Bruce, 1987). In mice and rats the LD50 is usually determined as described above, but in the larger species only an approximation of the LD50 is obtained by increasing the dose in the same animal until serious toxic effects are evident. If there is a reasonable likelihood of substantial exposure to the material by dermal or inhalation exposure, acute dermal and acute inhalation studies are performed. When animals are exposed acutely to chemicals in the air they breathe or the water they (fish) live in, the dose the animals receive is usually not known. For these situations, the lethal concentration 50 (LC50 ) is usually determined; that is, the concentration of chemical in the air or water that causes death to 50% of the animals. In reporting an LC50 , it is imperative that the time of exposure be indicated. The acute dermal toxicity test is usually performed in rabbits. The site of application is shaved. The test substance is kept in contact with the skin for 24 hours by

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wrapping the skin with an impervious plastic material. At the end of the exposure period, the wrapping is removed and the skin is wiped to remove any test substance still remaining. Animals are observed at various intervals for 14 days, and the LD50 is calculated. If no toxicity is evident at 2 g/kg, further acute dermal toxicity testing is usually not performed. Acute inhalation studies are performed that are similar to other acute toxicity studies except that the route of exposure is inhalation. Most often, the length of exposure is 4 hours. Although by themselves LD50 and LC50 values are of limited significance given the growing sophistication of target organ toxicity endpoints and mechanistic analysis. The most meaningful scientific information derived from acute toxicity tests comes from clinical observations and postmortem examination of animals rather than from the specific LD50 value.

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area may be occluded. Some 2 to 3 weeks after the last treatment, the animals are challenged with a nonirritating concentration of the test substance and the development of erythema is evaluated.

Subacute (Repeated-Dose Study) Subacute toxicity tests are performed to obtain information on the toxicity of a chemical after repeated administration and as an aid to establish doses for subchronic studies. A typical protocol is to give three to four different dosages of the chemicals to the animals by mixing it in their feed. For rats, 10 animals per sex per dose are often used; for dogs, three dosages and 3 to 4 animals per sex are used. Clinical chemistry and histopathology are performed after 14 days of exposure, as described below in the section on subchronic toxicity testing.

Skin and Eye Irritations

Subchronic

The ability of a chemical to irritate the skin and eye after an acute exposure is usually determined in rabbits. For the dermal irritation test (Draize test), rabbits are prepared by removal of fur on a section of the back by electric clippers. The chemical is applied to the skin (0.5 mL of liquid or 0.5 g of solid) under four covered gauze patches (1 in. square; one intact and two abraded skin sites on each animal) and usually kept in contact for 4 hours. The nature of the covering patches depends on whether occlusive, semiocclusive, or nonocclusive tests are desired. For occlusive testing, the test material is covered with an impervious plastic sheet; for semiocclusive tests, a gauze dressing may be used. Occasionally, studies may require that the material be applied to abraded skin. The degree of skin irritation is scored for erythema (redness), eschar (scab), and edema (swelling) formation, and corrosive action. These dermal irritation observations are repeated at various intervals after the covered patch has been removed. To determine the degree of ocular irritation, the chemical is instilled into one eye (0.1 mL of liquid or 100 mg of solid) of each test rabbit. The contralateral eye is used as the control. The eyes of the rabbits are then examined at various times after application. Controversy over this test has led to the development of alternative in vitro models for evaluating cutaneous and ocular toxicity of substances. The various in vitro methods that have been evaluated for this purpose include epidermal keratinocyte and corneal epithelial cell culture models. These and other in vitro tests have been reviewed recently (Davila et al., 1998).

The toxicity of a chemical after subchronic exposure is then determined. Subchronic exposure can last for different periods of time, but 90 days is the most common test duration. The principal goals of the subchronic study are to establish a NOAEL and to further identify and characterize the specific organ or organs affected by the test compound after repeated administration. One may also obtain a “lowest observed adverse effect level” (LOAEL) as well as the NOAEL for the species tested. The numbers obtained for NOAEL and LOAEL will depend on how closely the dosages are spaced and the number of animals examined. Determinations of NOAELs and LOAELs have numerous regulatory implications. For example, the EPA utilizes the NOAEL to calculate the reference dose (RfD), which may be used to establish regulatory values for “acceptable” pollutant levels (Barnes and Dourson, 1988) (see Chap. 4). An alternative to the NOAEL approach referred to as the benchmark dose uses all the experimental data to fit one or more dose–response curves (Crump, 1984). These curves are then used to estimate a benchmark dose that is defined as “the statistical lower bound on a dose corresponding to a specified level of risk” (Allen et al., 1994a). Although subchronic studies are frequently the primary or sole source of experimental data to determine both the NOAEL and the benchmark dose, these concepts can be applied to other types of toxicity testing protocols, such as that for chronic toxicity or developmental toxicity (Allen et al., 1994a, 1994b; Faustman et al., 1994) (see also Chap. 4 for a complete discussion of the derivation and use of NOAELs, RfDs, and benchmark doses). If chronic studies have been completed, these data are generally used for NOAEL and LOAEL estimates in preference to data from subchronic studies. A subchronic study is usually conducted in two species (usually rat and dog for FDA; and mouse for EPA) by the route of intended exposure (usually oral). At least three doses are employed (a high dose that produces toxicity but does not cause more than 10% fatalities, a low dose that produces no apparent toxic effects, and an intermediate dose) with 10 to 20 rodents and 4 to 6 dogs of each sex per dose. Each animal should be uniquely identified with permanent markings such as ear tags, tattoos, or electronically coded microchip implants. Only healthy animals should be used, and each animal should be housed individually in an adequately controlled environment. When the test compound is administered in the diet over a prolonged period of time (sub-chronic and chronic studies), the concentration in the diet should be adjusted periodically (weekly for the first 12–14 weeks) to maintain a constant intake of material based on food consumption and rate of change in body weight (Wilson et al., 2001). Animals should be observed

Sensitization Information about the potential of a chemical to sensitize skin is needed in addition to irritation testing for all materials that may repeatedly come into contact with the skin. Numerous procedures have been developed to determine the potential of substances to induce a sensitization reaction in humans (delayed hypersensitivity reaction), including the Draize test, the open epicutaneous test, the Buehler test, Freund’s complete adjuvant test, the optimization test, the split adjuvant test, and the guinea pig maximization test (Maibach and Patrick, 2001; Rush et al., 1995). Although they differ in regard to route and frequency of duration, they all utilize the guinea pig as the preferred test species. In general, the test chemical is administered to the shaved skin topically, intradermally, or both and may include the use of adjuvant to enhance the sensitivity of the assay. Multiple administrations of the test substance are generally given over a period of 2 to 4 weeks. Depending on the specific protocol, the treated

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once or twice daily for signs of toxicity, including changes in body weight, diet consumption, changes in fur color or texture, respiratory or cardiovascular distress, motor and behavioral abnormalities, and palpable masses. All premature deaths should be recorded and necropsied as soon as possible. Severely moribund animals should be terminated immediately to preserve tissues and reduce unnecessary suffering. At the end of the 90-day study, all the remaining animals should be terminated and blood and tissues should be collected for further analysis. The gross and microscopic condition of the organs and tissues (about 15 to 20) and the weight of the major organs (about 12) are recorded and evaluated. Hematology and blood chemistry measurements are usually done before, in the middle of, and at the termination of exposure. Hematology measurements usually include hemoglobin concentration, hematocrit, erythrocyte counts, total and differential leukocyte counts, platelet count, clotting time, and prothrombin time. Clinical chemistry determinations commonly made include glucose, calcium, potassium, urea nitrogen, alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (GGT), sorbitol dehydrogenase, lactic dehydrogenase, alkaline phosphatase, creatinine, bilirubin, triglycerides, cholesterol, albumin, globulin, and total protein. Urinalysis is usually performed in the middle of and at the termination of the testing period and often includes determination of specific gravity or osmolarity, pH, proteins, glucose, ketones, bilirubin, and urobilinogen as well as microscopic examination of formed elements. If humans are likely to have significant exposure to the chemical by dermal contact or inhalation, subchronic dermal and/or inhalation experiments may also be required. Subchronic toxicity studies not only characterize the dose–response relationship of a test substance after repeated administration but also provide data for a more reasonable prediction of appropriate doses for chronic exposure studies. For chemicals that are to be registered as drugs, acute and subchronic studies (and potentially additional special tests if a chemical has unusual toxic effects or therapeutic purposes) must be completed before the company can file an Investigational New Drug (IND) application with the FDA. If the application is approved, clinical trials can commence. At the same time phase I, phase II, and phase III clinical trials are performed, chronic exposure of the animals to the test compound can be carried out in laboratory animals, along with additional specialized tests.

Chronic Long-term or chronic exposure studies are performed similarly to subchronic studies except that the period of exposure is longer than 3 months. In rodents, chronic exposures are usually for 6 months to 2 years. Chronic studies in nonrodent species are usually for 1 year but may be longer. The length of exposure is somewhat dependent on the intended period of exposure in humans. For example, for pharmaceuticals, the ICH S4 guidance calls for studies of 6 months in duration in rodents, and 9 months in non-rodents. However, if the chemical is a food additive with the potential for lifetime exposure in humans, a chronic study up to 2 years in duration is likely to be required. Dose selection is critical in these studies to ensure that premature mortality from chronic toxicity does not limit the number of animals that survive to a normal life expectancy. Most regulatory guidelines require that the highest dose administered be the estimated maximum tolerable dose (MTD). This is generally derived from subchronic studies, but additional longer studies (e.g.,

6 months) may be necessary if delayed effects or extensive cumulative toxicity are indicated in the 90-day subchronic study. The MTD has had various definitions (Haseman, 1985). The MTD has been defined by some regulatory agencies as the dose that suppresses body weight gain slightly (i.e., 10%) in a 90-day subchronic study (Reno, 1997). However, regulatory agencies may also consider the use of parameters other than weight gain, such as physiological and pharmacokinetic considerations and urinary metabolite profiles, as indicators of an appropriate MTD (Reno, 1997). Generally, one or two additional doses, usually fractions of the MTD (e.g., one-half and one-quarter MTD), and a control group are tested. Chronic toxicity tests may include a consideration of the carcinogenic potential of chemicals so that a separate lifetime feeding study that addresses carcinogenicity does not have to be performed. However, specific chronic studies designed to assess the carcinogenic potential of a substance may be required (see below).

Developmental and Reproductive Toxicity The effects of chemicals on reproduction and development also need to be determined. Developmental toxicology is the study of adverse effects on the developing organism occurring anytime during the life span of the organism that may result from exposure to chemical or physical agents before conception (either parent), during prenatal development, or postnatally until the time of puberty. Teratology is the study of defects induced during development between conception and birth (see Chap. 10). Reproductive toxicology is the study of the occurrence of adverse effects on the male or female reproductive system that may result from exposure to chemical or physical agents (see Chap. 20). Several types of animal tests are utilized to examine the potential of an agent to alter development and reproduction. General fertility and reproductive performance (segment I) tests are usually performed in rats with two or three doses (20 rats per sex per dose) of the test chemical (neither produces maternal toxicity). Males are given the chemical 60 days and females 14 days before mating. The animals are given the chemical throughout gestation and lactation. Typical observations made include the percentage of females that become pregnant, the number of stillborn and live offspring, and the weight, growth, survival, and general condition of the offspring during the first 3 weeks of life. The potential of chemicals to disrupt normal embryonic and/or fetal development (teratogenic effects) is also determined in laboratory animals. Current guidelines for these segment II studies call for the use of two species, including one nonrodent species (usually rabbits). Teratogens are most effective when administered during the first trimester, the period of organogenesis. Thus, the animals (usually 12 rabbits and 24 rats or mice per group) are usually exposed to one of three dosages during organogenesis (day 7 to 17 in rodents and days 7 to 19 in rabbits), and the fetuses are removed by cesarean section a day before the estimated time of delivery (gestational days 29 for rabbit, 20 for rat, and 18 for mouse). The uterus is excised and weighed and then examined for the number of live, dead, and resorbed fetuses. Live fetuses are weighed; half of each litter is examined for skeletal abnormalities and the remaining half for soft tissue anomalies. The perinatal and postnatal toxicities of chemicals also are often examined (segment III). This test is performed by administering the test compound to rats from the 15th day of gestation throughout delivery and lactation and determining its effect on the birthweight, survival, and growth of the offspring during the first 3 weeks of life.

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In some instances a multigenerational study may be chosen, often in place of segment III studies, to determine the effects of chemicals on the reproductive system. At least three dosage levels are given to groups of 25 female and 25 male rats shortly after weaning (30 to 40 days of age). These rats are referred to as the F0 generation. Dosing continues throughout breeding (about 140 days of age), gestation, and lactation. The offspring (F1 generation) have thus been exposed to the chemical in utero, via lactation, and in the feed thereafter. When the F1 generation is about 140 days old, about 25 females and 25 males are bred to produce the F2 generation, and administration of the chemical is continued. The F2 generation is thus also exposed to the chemical in utero and via lactation. The F1 and F2 litters are examined as soon as possible after delivery. The percentage of F0 and F1 females that get pregnant, the number of pregnancies that go to full term, the litter size, the number of stillborn, and the number of live births are recorded. Viability counts and pup weights are recorded at birth and at 4, 7, 14, and 21 days of age. The fertility index (percentage of mating resulting in pregnancy), gestation index (percentage of pregnancies resulting in live litters), viability index (percentage of animals that survive 4 days or longer), and lactation index (percentage of animals alive at 4 days that survived the 21-day lactation period) are then calculated. Gross necropsy and histopathology are performed on some of the parents (F0 and F1 ), with the greatest attention being paid to the reproductive organs, and gross necropsy is performed on all weanlings. The International Commission on Harmonization (ICH) guidelines provide for flexible guidelines that address six “ICH stages” of development: premating and conception (stage A), conception to implantation (stage B), implantation to closure of the hard palate (stage C), closure of the hard palate to end of pregnancy (stage D), birth and weaning (stage E), and weaning to sexual maturity (stage F). All of these stages are covered in the segment I to segment III studies described above (Christian, 1997). Numerous short-term tests for teratogenicity have been developed (Faustman, 1988). These tests utilize whole-embryo culture, organ culture, and primary and established cell cultures to examine developmental processes and estimate the potential teratogenic risks of chemicals. Many of these in utero test systems are under evaluation for use in screening new chemicals for teratogenic effects. These systems vary in their ability to identify specific teratogenic events and alterations in cell growth and differentiation. In general, the available assays cannot identify functional or behavioral teratogens (Faustman, 1988).

Mutagenicity Mutagenesis is the ability of chemicals to cause changes in the genetic material in the nucleus of cells in ways that allow the changes to be transmitted during cell division. Mutations can occur in either of two cell types, with substantially different consequences. Germinal mutations damage DNA in sperm and ova, which can undergo meiotic division and therefore have the potential for transmission of the mutations to future generations. If mutations are present at the time of fertilization in either the egg or the sperm, the resulting combination of genetic material may not be viable, and the death may occur in the early stages of embryonic cell division. Alternatively, the mutation in the genetic material may not affect early embryogenesis but may result in the death of the fetus at a later developmental period, resulting in abortion. Congenital abnormalities may also result from mutations. Somatic mutations refer to mutations in all

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other cell types and are not heritable but may result in cell death or transmission of a genetic defect to other cells in the same tissue through mitotic division. Because the initiating event of chemical carcinogenesis is thought to be a mutagenic one, mutagenic tests are often used to screen for potential carcinogens. Numerous in vivo and in vitro procedures have been devised to test chemicals for their ability to cause mutations. Some genetic alterations are visible with the light microscope. In this case, cytogenetic analysis of bone marrow smears is used after the animals have been exposed to the test agent. Because some mutations are incompatible with normal development, the mutagenic potential of a chemical can also be evaluated by the dominant lethal test. This test is usually performed in rodents. The male is exposed to a single dose of the test compound and then is mated with two untreated females weekly for 8 weeks. The females are killed before term, and the number of live embryos and the number of corpora lutea are determined. The test for mutagens that has received the widest attention is the Salmonella/microsome test developed by Ames and colleagues (Ames et al., 1975). This test uses several mutant strains of Salmonella typhimurium that lack the enzyme phosphoribosyl ATP synthetase, which is required for histidine synthesis. These strains are unable to grow in a histidine-deficient medium unless a reverse or back-mutation to the wild type has occurred. Other mutations in these bacteria have been introduced to enhance the sensitivity of the strains to mutagenesis. The two most significant additional mutations enhance penetration of substances into the bacteria and decrease the ability of the bacteria to repair DNA damage. Because many chemicals are not mutagenic or carcinogenic unless they are biotransformed to a toxic product by enzymes in the endoplasmic reticulum (microsomes), rat liver microsomes are usually added to the medium containing the mutant strain and the test chemical. The number of reverse mutations is then quantified by the number of bacterial colonies that grow in a histidine-deficient medium. Strains of yeast have recently been developed that detect genetic alterations arising during cell division after exposure to nongenotoxic carcinogens as well as mutations that arise directly from genotoxic carcinogens. This test identifies deletions of genetic material that occur during recombination events in cell division that may result from oxidative damage to DNA, direct mutagenic effects, alterations in fidelity of DNA repair, and/or changes in cell cycle regulation (Galli and Schiestl, 1999). Mutagenicity is discussed in detail in Chap. 9. With the advent of techniques that readily allow manipulation of the mouse genome, transgenic animals have been developed that allow for in vivo assessment of mutagenicity of compounds. For example, two commercially available mouse strains, the “MutaMouse”, and “BigBlue” contain the lac operon of E. coli that has been inserted into genomic DNA using a lambda phage to DNA to produce a recoverable shuttle vector. Stable, homozygous strains of these transgenic animals (both mice and rats have been engineered) can be exposed to potential mutagenic agents. Following in vivo exposure, the target lac genes can be recovered from virtually any cell type or organ and analyzed for mutations (Brusick, 2001).

Oncogenicity Bioassays Oncogenicity studies are both time consuming and expensive, and are usually only done when there is reason to suspect that a chemcial may be carcinogenic, or when there may be wide spread, long term exposures to humans (e.g., widely used food additives, drinking

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p

u

water contaminants, or pharmaceuticals that are likely to be administered repeatedly for long periods of time). Chemicals that test positive in several mutagenicity assays are likely to be carcinogenic, and thus are frequent candidates for oncogenicity bioassay assessment. In the United States, the National Toxicology Program (NTP) has the primary responsibility for evaluating non-drug chemicals for carcinogenic potential. For pharmaceuticals, the FDA may require the manufacturer to conduct oncogenicity studies as part of the preclinical assessment, depending on the intended use of the drug, and the results of mutagenicity assays and other toxicological data. Studies to evaluate the oncogenic (carcinogenic) potential of chemicals are usually performed in rats and mice and extend over the average lifetime of the species (18 months to 2 years for mice; 2 to 2.5 years for rats). To ensure that 30 rats per dose survive the 2-year study, 60 rats per group per sex are often started in the study. Both gross and microscopic pathological examinations are made not only on animals that survive the chronic exposure but also on those that die prematurely. The use of the MTD in carcinogenicity has been the subject of controversy. The premise that high doses are necessary for testing the carcinogenic potential of chemicals is derived from the statistical and experimental design limitations of chronic bioassays. Consider that a 0.5% increase in cancer incidence in the United States would result in over 1 million additional cancer deaths each year—clearly an unacceptably high risk. However, identifying with statistical confidence a 0.5% incidence of cancer in a group of experimental animals would require a minimum of 1000 test animals, and this assumes that no tumors were present in the absence of exposure (zero background incidence). Figure 2-12 shows the statistical relationship between minimum detectable tumor incidence and the number of test animals per group. This curve shows that in a chronic bioassay with 50 animals per test group, a tumor incidence of about 8% could exist even though no animals in the test group had tumors. This example assumes that there are no tumors in the control group. These statistical considerations illustrate why animals are tested at doses higher than those that occur in human exposure. Because it is impractical to use the large number of animals that would be required to test the potential carcinogenicity of a chemical at the doses usually encountered by people, the alternative is to assume that there is a relationship

Figure 2-12. Statistical limitations in the power of experimental animal studies to detect tumorigenic effects.

between the administered dose and the tumorigenic response and give animals doses of the chemical that are high enough to produce a measurable tumor response in a reasonable size test group, such as 40 to 50 animals per dose. The limitations of this approach are discussed in Chap. 4. For non-mutagenic pharmaceutical agents, ICH S1C provides the following guidance on dose selection for oncogenicity studies: “The doses selected for rodent bioassays for non-genotoxic pharmaceuticals should provide an exposure to the agent that (1) allow an adequate margin of safety over the human therapeutic exposure, (2) are tolerated without significant chronic physiological dysfunction and are compatible with good survival, (3) are guided by a comprehensive set of animal and human data that focus broadly on the properties of the agent and the suitability of the animal (4) and permit data interpretation in the context of clinical use.” Another approach for establishing maximum doses for use in chronic animal toxicity testing of drugs is often used for substances for which basic human pharmacokinetic data are available (for example, new pharmaceutical agents which have completed phase I clinical trials). For chronic animal studies performed on drugs where single-dose human pharmacokinetic data are available, a daily dose that would provide an area under the curve (AUC) in laboratory animals equivalent to 25 times the AUC in humans given the highest (single) daily dose to be used therapeutically may be used, rather than the MTD. Based on a series of assumptions regarding allometric scaling between rodents and humans (Table 2-2), the ICH noted that it may not be necessary to exceed a dose of 1500 mg/kg/day where there is no evidence of genotoxicity, and where the maximum recommended human dose does not exceed 500 mg/day. Most regulatory guidelines require that both benign and malignant tumors be reported in oncogenicity bioassays. Statistical increases above the control incidence of tumors (either all tumors or specific tumor types) in the treatment groups are considered indicative of carcinogenic potential of the chemical unless there are qualifying factors that suggest otherwise (lack of a dose response, unusually low incidence of tumors in the control group compared with “historic” controls, etc.; Huff, 1999). Thus, the conclusion as to whether a given chronic bioassay is positive or negative for carcinogenic potential of the test substance requires careful consideration of background tumor incidence. Properly designed chronic oncogenicity studies require that a concurrent control group matched for variables such as age, diet, housing conditions be used. For some tumor types, the “background” incidence of tumors is surprisingly high. Figure 2-13 shows the background tumor incidence for various tumors in male and female F-344 rats used in 27 National Toxicology Program 2-year rodent carcinogenicity studies. The data shown represent the percent of animals in control (nonexposed) groups that developed the specified tumor type by the end of the 2-year study. These studies involved more than 1300 rats of each sex. Figure 214 shows similar data for control (nonexposed) male and female B6C3F1 mice from 30 recent NTP 2-year carcinogenicity studies and includes data from over 1400 mice of each sex. There are several key points that can be derived from these summary data:

1. Tumors, both benign and malignant, are not uncommon events in animals even in the absence of exposure to any known carcinogen. 2. There are numerous different tumor types that develop “spontaneously” in both sexes of both rats and mice, but at different rates.

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Taken together, these data demonstrate the importance of including concurrent control animals in such studies. In addition, comparisons of the concurrent control results to “historic” controls accumulated over years of study may be important in identifying potentially spurious “false-positive” results. The relatively high variability in background tumor incidence among groups of healthy, highly inbred strains of animals maintained on nutritionally balanced and consistent diets in rather sterile environments highlights the dilemma in interpreting the significance of both positive and negative results in regard to the human population, which is genetically diverse, has tremendous variability in diet, nutritional status, and overall health; and lives in an environment full of potentially carcinogenic substances, both natural and human-made. Finally, it should be noted that both inbred and outbred strains have distinct background tumor patterns and the NTP and most other testing programs select strains based on the particular needs of the agent under study. For example, the NTP used the Wistar rat for chemicals that may have the testis as a target organ, based on acute, sub-chronic or other bioassay results. Similarly, the NTP used the Sprague-Dawley strain of rat in studies of estrogenic agents such as genistein because its mammary tumors are responsive to estrogenic stimulation, as are humans.

Figure 2-13. Most frequently occurring tumors in untreated control rats from recent NTP 2-year rodent carcinogenicity studies. The values shown represent the mean ±SD of the percentage of animals developing the specified tumor type at the end of the 2-year study. The values were obtained from 27 different studies involving a combined total of between 1319 and 1353 animals per tumor type.

3. Background tumors that are common in one species may be uncommon in another (for example, testicular interstitial cell adenomas are very common in male rats but rare in male mice; liver adenomas/carcinomas are about 10 times more prevalent in male mice than in male rats). 4. Even within the same species and strain, large gender differences in background tumor incidence are sometimes observed (for example, adrenal gland pheochromocytomas are about seven times more prevalent in male F344 rats than in female F344 rats; lung and liver tumors are twice as prevalent in male B6C3F1 mice as in female B6C3F1 mice). 5. Even when the general protocols, diets, environment, strain and source of animals, and other variables are relatively constant, background tumor incidence can vary widely, as shown by the relatively large standard deviations for some tumor types in the NTP bioassay program. For example, the range in liver adenoma/carcinoma incidence in 30 different groups of unexposed (control) male B6C3F1 mice went from a low of 10% to a high of 68%. Pituitary gland adenomas/carcinomas ranged from 12 to 60% and 30 to 76% in unexposed male and female F344 rats, respectively, and from 0 to 36% in unexposed female B6C3F1 mice.

Figure 2-14. Most frequently occurring tumors in untreated control mice from recent NTP 2-year rodent carcinogenicity studies. The values shown represent the mean ±SD of the percentage of animals developing the specified tumor type at the end of the 2-year study. The values were obtained from 30 different studies involving a total of between 1447 and 1474 animals per tumor type.

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Neurotoxicity Assessment Neurotoxicity or a neurotoxic effect is defined as an adverse change in the chemistry, structure or function of the nervous system following exposure to a chemical or physical agent. The structure, function, and development of the nervous system and its vulnerability to chemicals, is examined in Chap. 16. When evaluating the potential neurological effects of a compound, effects may be on the central or peripheral nervous system or related to exposure that occurred during development or as an adult. The developing nervous system is particularly sensitive to chemical exposures (see Chap. 10). In vitro systems often using cell culture techniques is a rapidly developing area of neurotoxicity assessment. Specific cell lines are available to examine effects on neuron or glial cells such as proliferation, migration, apoptosis, synpatogenesis and other endpoints. In vitro assays have a number of potential advantages including minimizing the use of animal, lower costs, and adaptable to high through put screening. It is also possible to use an in vitro model to examine the interaction of chemicals, such as food additives, on neuronal cells (Lau et al., 2006). The principle and challenges on in vitro neurotoxicity testing are well described (Tiffany-Castiglioni, 2004). Procedures for the neurobehavioral evaluation of animals were initially developed as part of the scientific investigation of behavioral motivation. Some of these procedures were then used to evaluate the neuropharmacological properties of new drugs. Now animals are commonly used to evaluate the neurotoxic properties of chemicals. A wide range of adult and developmental animal tests are used to access neurobehavioral function. In addition, neuropathological assessment is an important part of the neurotoxicity evaluation and best practices have been developed for developmental neurotoxicity (Bolon et al., 2006). Irwin developed a basic screen for behavioral function in mice (Irwin, 1968), which was subsequently refined to the functional observational battery (FOB) (Moser, 2000). The FOB can also be used in the evaluation of drug safety (Redfern et al., 2005). The U.S. EPA established a protocol for the evaluation of developmental neurotoxicity (DNT) in laboratory animals (U.S. EPA 870.6300 and OECD 426) (EPA, 1998; OECD 2004). These protocols include tests of neurobehavioral function, such as auditory startle, learning and memory function, changes in motor activity, and neuropathologic examination and morphometric analysis. Methods and procedures for developmental neurotoxicity evaluation are well established (Claudio et al., 2000; Cory-Slechta et al., 2001; Dorman et al., 2001; Garman et al., 2001; Mileson and Ferenc, 2001). Recent studies examine the neurotoxicity of multiple chemical exposures in animals (Moser et al. 2006). Methods are also available to examine cognitive measures on weanling rodents in DNT studies (Ehman and Moser 2006). Non-human primates have been invaluable in evaluating the effects of neurotoxoicants and the risk assessment process (Burbacher and Grant 2000). Sophisticated assessment of operant behavior, and learning and memory assessment of rodents has been used to evaluate the effects of lead (Cory-Slechta, 1995, 1996, 2003). Monkeys can also be used to evaluate the low level effects of neurotoxicants such as mercury on vision, auditory function and vibration sensitivity (Burbacher et al., 2005; Rice and Gilbert, 1982, 1992, 1995). There is remarkable concordance between human and animal neurotoxicity assessment, for example, in lead, mercury, and PCBs (Rice, 1995). Human testing for the neurological effects of occupational exposures to chemicals is advancing rapidly (Anger, 2003; Farahat et al., 2003; Kamel et al., 2003; McCauley et al., 2006) and even the neurotoxic effects of war (Binder et al., 1999, 2001). These methods

have also been applied to Hispanic workers (Rohlman et al., 2001b) and populations with limited education or literacy (Rohlman et al., 2003). The WHO has also recommended a test battery for humans (Anger et al., 2000). There are also neurobehavioral test batteries for assessing children (Rohlman et al., 2001a). Evaluation of the childhood neurological effects of lead (Lanphear et al., 2005; Needleman and Bellinger, 1991) and mercury (Myers et al., 2000) have added enormously to our understanding of the health effects of these chemicals and to the methodology of human neurobehavioral testing. In summary, the neurontoxicological evaluation is an important aspect of developing a hazard and risk assessment of environmental chemicals and drugs.

Immunotoxicity Assessment Under normal conditions, the immune system is responsible for host defense against pathogenic infections and certain cancers. However, environmental exposures can alter immune system development and/or function and lead to hypersensitivity, autoimmunity, or immunosuppression, the outcome of which may be expressed as a pathology in most any organ or tissue (see Chap. 12). Our understanding of the biological processes underlying immune system dysfunction remains incomplete. However, advances in molecular biology (including use of transgenic/knockout mice), analytical methods (including gene expression arrays and multiparameter flow cytometry), animal models (including adoptive transfers in immunocompromised mice and host resistance to viral, bacterial, or tumor cell challenge), and other methods are greatly advancing our knowledge. From a toxicologist’s perspective, evaluation of immune system toxicity represents special challenges. Development of hypersensitivity can take various forms, depending on the mechanism underlying the associated immune response, and standard assumptions regarding dose-response relationships may not necessarily apply. For example, a single or incidental exposure to beryllium has been associated with chronic beryllium disease in some individuals. We are only just beginning to understand the biological basis underlying such individual susceptibility. In the case of chronic beryllium disease, a genetic polymorphism in a gene involved in antigen recognition may be associated with increased susceptibility (see Bartell et al., 2000). Although our ability to predict immunogenicity remains poor, research efforts are continuing to identify aspects of the chemical and the individual that confer immunogenicity and underlie hypersensitivity. For example, the increasing incidence of allergic asthma among preschool-age children in the United States since the 1980s, may be associated with exposure to allergens (e.g., dust mites, molds, and animal dander), genetic factors, and other factors in the in utero and post-natal environment (see Donovan and Finn, 1999; Armstrong et al., 2005). Immunosuppression is another form of immune system toxicity, which can result in a failure to respond to pathogenic infection, a prolonged infection period, or expression of a latent infection or cancer. Various chemicals have been associated with immunosuppression. Broad spectrum and targeted immunosuppressive chemicals are designed and used therapeutically to reduce organ transplant rejection or suppress inflammation. However, a large number of chemicals have been associated with immunosuppression, including organochlorine pesticides, diethylstilbesterol, lead, and halogenated aromatic hydrocarbons (including TCDD), and exposures that occur during critical stages may present special risk to development (Holladay, 2005).

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Autoimmunity is a specific immune system disorder in which components of the immune system attack normal (self) tissues. Cases of autoimmunity have been reported for a wide range of chemicals including therapeutic drugs, metals, pesticides, and solvents. As with other forms of immune system toxicity, autoimmunity can present in most any tissue. Finally, new forms of immunotoxicity are appearing based on novel forms of clinical therapy and immunomodulation. These include the variously classified “tumor lysis syndromes” and “cytokine storms” that arise from massive cytokine dysregulation. A recent example involved six healthy volunteers who had enrolled in a Phase 1 clinical trial in the UK who developed a severe cytokine response to an anti-CD28 monoclonal antibody leading to systemic organ failures (Bhogal and Combes, 2006). Such cases are stark reminders of the challenges we face in understanding how the immune system is regulated, developing reliable test systems for identifying such risks prior to human use, and safe means for testing these agents in humans. As described in Chapter 12, current practice for evaluating potential toxic effects of xenobiotic exposures on the immune system involves a tiered approach to immunotoxicity screening (Luster et al., 2003). This tiered approach is generally accepted world-wide in the registration of novel chemical and therapeutic products. Most recently, final guidance to the pharmaceutical industry was published in April, 2006 by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (Table 2-3). This guidance, which applies to the nonclinical (animal) testing of human pharmaceuticals is the accepted standard in the U.S., EU, and Japan, and demonstrates the continued commitment by these regulatory bodies to understand the potential risks posed by novel therapeutics. Tiered testing relies on the concept that standard toxicity studies can provide good evidence for immunotoxicity when considered with known biological properties of the chemical, including structural similarities to known immunomodulators, disposition, and other clinical information, such as increased occurrence of infections or tumors. Evaluation of hematological changes, including differential effects on white blood cells and immunoglobulin changes, and alterations in lymphoid organ weights or histology, can provide strong evidence of potential effects to the immune system. Should such evaluations indicate a potential effect on immune system function, more detailed evaluations may be considered, including the evaluation of functional effects (e.g., T-cell dependent antibody response or Natural Killer cell activity), flow cytometric immunophenotyping, or host resistance studies. Thus, as with other areas of toxicology, the evaluation of immune system toxicity requires the toxicologist to be vigilant in observing early indications from a variety of sources in developing a weight-of-evidence assessment regarding potential injury/dysfunction.

Other Descriptive Toxicity Tests Most of the tests described above will be included in a “standard” toxicity testing protocol because they are required by the various regulatory agencies. Additional tests may be required or included in the protocol to provide information relating a special route of exposure, such as inhalation. Inhalation toxicity tests in animals usually are carried out in a dynamic (flowing) chamber rather than in static chambers to avoid particulate settling and exhaled gas complications. Such studies usually require special dispersing and analytic methodologies, depending on whether the agent to be tested is a gas, vapor, or aerosol; additional information on methods, concepts,

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and problems associated with inhalation toxicology is provided in Chaps. 15 and 28. The duration of exposure for inhalation toxicity tests can be acute, subchronic, or chronic, but acute studies are more common with inhalation toxicology. Other special types of animal toxicity tests include toxicokinetics (absorption, distribution, biotransformation, and excretion), the development of appropriate antidotes and treatment regimens for poisoning, and the development of analytic techniques to detect residues of chemicals in tissues and other biological materials.

TOXICOGENOMICS In the past decade, numerous new genome-based technologies have become available that allow for the large-scale analysis of biological responses to external stimuli. Traditional scientific approaches to elucidate the biochemical and molecular effects of toxic substances focused largely on examining biochemical pathways that were logically connected to observed responses identified through gross pathology, histology, blood chemistry, or behavioral observations. Such “hypothesis driven” research into understanding mechanism of action remains a mainstay of current scientific investigations in toxicology. However, technologies now available allow one to examine the entire “universe” of biological responses to a toxic substance (Fig. 2-15). These new “hypothesis generating” technologies include: genomics (characterization of much or all of the genome of an organism), transcriptomics (characterization of most or all of the mRNAs, or transcriptome, expressed in a given cell/tissue), proteomics (characterization of most or all of the proteins expressed in a given cell/tissue), and metabonomics (characterization of most or all of the small molecules in a cell or tissue, including substrates, products, and co-factors of enzyme reactions). Other “omics” approaches (e.g., “lipidomics”, “nutrigenomics”) are being devised to look broadly at the biological response of an organism to change. The integration of all of these levels of molecular function (genomics, transcriptomics, proteomics, metabonomics, etc.) to the understanding of how a living organism functions at the cellular level is sometimes referred to as “Systems Biology” (Weston and Hood, 2004). Because each level of analysis generates a very large quantity of data, the collection, organization, evaluation and statistical analysis is in itself an enormous undertaking. The field of “Bioinformatics” has been developed to address the many computational and statistical challenges of “omics” data. In the field of toxicology, the term “toxicogenomics” is used to define the area of research that “combines transcript, protein and metabolite profiling with conventional toxicology to investigate the interaction between genes and environmental stress in disease causation” (Waters and Fostel, 2004). A conceptual model for how the various new “omics” technologies can be incorporated into toxicological evaluation is shown in Fig. 2-15. Genomics: The genome of an organism represents the full complement of genes that are determined at fertilization by the combination of the parental DNA. Thus, each cell of an organism has the same genome, characterized by the nucleotide sequences inherited from its parents. The human genome consists of approximately 3 billion base pairs of deoxyribonucleotides. Within the human genome, there is, on average, about 0.1% variability in DNA sequence between any two individuals, and it is these differences that contribute to the uniqueness of each person. Most of this variability exists as “single nucleotide polymorphism”, or SNPs, although larger segments of DNA may be variable between individuals, including the duplication or loss of entire genes. The identification of particular genetic variants, such as the GSTM1 polymorphism, that might

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0100 010110 10101001 0101011010 101001010110 10101110111001 001010010010100

Treatment

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Gene, protein, or metabolite-expression profiles Histopathology

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Identify gene/protein functional groups, pathways, and networks

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Weight, physiology

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Multi-domain multi-genome knowledgebase

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Nature Reviews Genetics

Figure 2-15. Conceptual approach for incorporating “omics” technologies and resulting large databases into toxicological evaluation. Data from experiments that evaluate the effects of a chemical on global patterns of gene expression (transcriptomics), protein content (proteomics), and small molecules/metabolites (metabonomics/metabolomics), combined with genomic information from both the test species (e.g. rats, mice) and the target species of interest (e.g., humans), are analyzed by computational tools (bioinformatics) for unique or potentially predictive patterns of toxicity. Essential to the use of omics data for predictive toxicology/safety assessment is the ability to reliably tie observed omics patterns to traditional measures of toxicity, such as histopathology and clinical chemistry (phenotypic anchoring). (From: Waters MD, Fostel JM: Toxicogenomics and systems toxicology: Aims and prospects. Nat Rev Genet 5(12):936–948, 2004, with permission.)

contribute to interindividual differences in susceptibility to chemicals or other environmental factors discussed previously, represents a relatively new and growing area of study that aims to understand the complex interactions between the human genome and the environment (Costa and Eaton, 2006). Although the genome provides the blueprint for biological function, in order for the genomic information to be utilized in a cell, it must be expressed. Expression of the genome occurs when the coding sequence of DNA is converted to messenger RNA (mRNA). For any given cell, transcription of the genomic information contained in that cell is only partial. It is the differential expression of genes in a given cell that is largely responsible for the diverse function of the thousands of different cells, tissues, and organs that constitute an individual organism. Thus, understanding which genes are expressed

in a given tissue, at what level, and how toxicants perturb the “transcriptome” is of great relevance to toxicology. In addition to coding for mRNAs that provide the blueprint for protein synthesis, genomic DNA also generates small interfering RNAs (siRNA, microRNAs) which are biologically active and can participate in the regulation of gene expression. Furthermore, methylation of DNA is an important determinant of gene expression in cells and tissues, and exogenous chemicals can interfere with transcriptional function via alterating DNA methylation (Watson and Goodman, 2002). Importantly, although such epigenetic changes do not result in the alteration of the genomic sequence, they can result in heritable phenotypic changes. Thus, genomic analyses in toxicology may also include techniques to identify toxicant-induced changes in DNA methylation patterns (Watson and Goodman, 2002).

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Transcriptomics: Among the first changes that a cell will exhibit following exposure to a toxic substance is a change in gene expression. The transcriptome (all of the mature mRNA species present in a cell at a given point in time) is dynamic, and represents the steady-state between the rate of synthesis (transcription) and degradation of mRNAs in a cell. Toxicologists have utilized the so-called “Northern blot” analysis to assess the level of expression of individual genes in cells or tissues for decades. The “Reverse Transcriptase Polymerase Chain Reaction” (RT-PCR) allows one to quantitatively measure the relative number of mRNA species in a sample for specific genes. Using general primers, it is also possible to amplify the entire transcriptome quantitatively to make many complete copies of the transcriptome in a test tube. Thus, large amounts of material for analysis can be obtained from a relatively small number of cells. Finally, using microarray technologies, where tens of thousands of unique oligonucleotides (or cDNAs) are anchored on a solid matrix, toxicologists can now quantitatively assess the expression of thousands of unique mRNAs in a single sample, thus capturing an “expression profile” of the entire transcriptome in one analysis. There is great promise that gene expression profiles may be used to provide signatures of specific types of toxic responses, such as a cellular response to DNA damage or oxidative stress. There is also hope that such signature changes in gene expression could be used to facilitate more accurate cross-species extrapolation, allowing comparison of, for example, toxicant-induced changes in gene expression in rat hepatocytes with that of human hepatocytes under identical experimental conditions. However, one of the major challenges in toxicogenomics is the recognition that transcriptional regulation is highly dynamic, and that gene expression profiles can change dramatically with both dose and time. Because microarray experiments are relatively expensive and highly data intensive, it becomes both costly and challenging to conduct and analyze experiments with extensive dose and time course data (although costs are declining). Although changes in gene expression often contribute to, or are reflective of, phenotypic changes that occur in response to a toxic substance, the transcriptome is still somewhat far removed from the ultimate biochemical functions that dictate the actual biological function of the cell. Because the functional expression of a gene generally requires the translation of the mRNA to a protein, there is also great interest in looking at the “proteome”–the entire compliment of proteins that are present in a cell or tissue at a given point in time. Proteomics: Analysis of the proteome of a cell or tissue is much more difficult than analysis of the transcriptome, primarily because it is not yet possible to “amplify” the number of copies of proteins in a cell. Furthermore, unambiguous identification of specific proteins is much more difficult than that for individual mRNAs. Identification of specific proteins is generally done using a combination of separation techniques (e.g., 2D-gel electrophoresis, high performance liquid chromatography), followed by tandem mass spectrometry for identification (Aebersold and Mann, 2003). Because of size limitations for accurate mass spectrometry, protein mixtures are usually digested to smaller peptide fragments. The mixture of peptide fragments is resolved into individual components, and the identity of the specific peptides is determined based on high resolution mass analysis and sequential degradation (sequential loss of single amino acids) of the peptides by various means (Aebersold and Mann, 2003). The large and complex set of peptide mass fragments is then analyzed by computers and compared with a large database of mass fragments of known peptides/proteins. Because as few as 5 amino acid

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sequences may provide unique identification of a specific protein, the presence and relative abundance of specific proteins in a sample can then be reconstructed through bioinformatic analyses. As with transcriptomics, it is hoped that changes in protein expression can be used as specific biomarkers for particular types of toxic responses. Of course, such conceptual approaches have been used for years, e.g., use of serum transaminase proteins as indicators of liver damage, or the presence of prostate specific antigen (PSA) in serum as a potential biomarker of early stage prostate hyperplasia or cancer. The potential power of proteomics lies in the ability to identify unique patterns of protein expression, or identification of unique proteins or peptides, that are predictive of early toxic response or later development of disease. Metabonomics/metabolomics: These two terms are often used interchangeably to describe the analysis of the “universe” of small molecules that serve as substrates, products, and co-factors of the milieu of enzymatic reactions and other metabolic processes that define living cells, and thus the organism. Metabonomics has been defined as “the comprehensive and simultaneous systematic profiling of metabolite levels and their systematic and temporal change through such effects on diet, lifestyle, environment, genetic and pharmaceuticals, both beneficial and adverse, in whole organisms” (Lindon et al., 2003, 2006). The term “metabolomics” has been used principally in studies in plants and in vitro or single cell systems (Fiehn, 2002). Regardless of the specific term used (metabonomics will be used here), the concept of quantitatively analyzing toxicantinduced changes in the “metabolic profile” (the “metabonome”) of a cell, tissue or body fluid in some ways represents the “Holy Grail” of toxicogenomics, because the changes in these small molecules must represent a biologically relevant integration of all of the molecular, biochemical and cellular perturbations that lead to the development of toxicity (Fig. 2-15). In other words, changes in the metabonome should reflect the biologically relevant changes in gene transcription, translation, protein function, and other cellular processes, including temporal and adaptive responses, while ignoring biologically irrelevant changes in these factors. Although conceptually superior to either transcriptomics or proteomics for predictive toxicology, metabonomics lags significantly in technological development of readily accessible tools for thorough analysis of the metabonome. Two approaches for identifying and measuring hundreds, or even thousands, of small molecules in biological samples have emerged – Nuclear Magnetic Resonance (NMR), and mass spectrometry (Lindon et al., 2003, 2006). Both have their advantages and limitations, and it is likely that the most successful approaches to applying metabonomics to toxicological problems will utilize both techniques. Bioinformatics: One feature in common among all of the various “omics” technologies is the ability to generate very large volumes of data (literally millions of data points from a single experiment). Both the data management and statistical evaluation of toxicogenomics studies represents an enormous challenge. The emerging field of bioinformatics has developed to address these challenges. Numerous commercial platforms for conducting microarray analysis of the transcriptome are available, and sophisticated software is available for both data management and analysis. One of the major challenges in statistical analysis of large data sets is the large number of “false positives” that will result from multiple comparisons. In a typical gene array experiment, it is not uncommon for an investigator to make >20,000 different comparisons. At the typical “95%” statistical confidence limit, one would expect

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more than 1000 of the noted differences to occur just by chance alone. Thus, more rigorous statistical methods have been developed to reduce the so-called “false discovery rate” in such experiments (Storey et al., 2005; Gao, 2006). Challenges in using “omics” technologies for predictive toxicology and risk assessment: A conceptual framework for incorporating these new technologies into toxicology, sometimes referred to as “Systems Toxicology” is shown in Fig. 2-15. Several key components of such an approach include: (1) large databases of treatmentspecific information, such as results of transcriptomic, proteomic and metabonomic analyses from target tissues and/or body fluids derived from toxicant-treated animals, (2) genomic databases that describe the DNA sequence information from the species of interest, (3) computational tools that extract information from these and other databases and the published literature to identify critical pathways and networks that are altered by the toxicant treatment, and (4) comparison with traditional toxicological endpoints to ensure that the observed “omics responses are closely aligned with the toxicant-related pathophysiology in the animal (histopathology, clinical chemistry, etc) – a process called “phenotypic anchoring” (Waters and Fostel, 2004). Toxicogenomics tools are becoming indespensible for research aimed at identifying the mechanisms and mode of action of toxic substances. However, the incorporation of such approaches into routine toxicity assessment presents numerous challenges. Numerous working group reports and publications have addressed the challenges of incorporating toxicogenomics data into predictive

toxicology and risk assessment (Bammler, 2006; Maggioli et al., 2006; Boverhof and Zacharewski, 2006). One of the major challenges to incorporating toxicogenomic data into risk assessment is related to the highly dynamic processes that preceded an observed toxic response. Traditional measure of toxicity, such as histopathological changes in a tissue, tend to be stable or even irreversible, whereas the myriad of molecular, biochemical, and cellular changes that give rise to the toxic response(s) are highly dynamic, frequently changing by the hour. Thus, the profiles of mRNAs, proteins and/or metabolites captured at a single point in time may be dramatically different, depending on the specific point in time the sample was collected. Many of the observed changes may be the result of direct effects of the toxicant on specific targets, whereas others will be compensatory or feedback mechanisms invoked in response to the initial damage. Nevertheless, patterns of change in transcript, protein and/or metabolite profiles are likely to provide informative “signatures” of toxic response that will be of great value in predictive toxicology. Such approaches may be particularly useful in pharmaceutical development, where toxicogenomic profiles may help to accelerate preclinical evaluation of drug candidates by identifying “class prediction” profiles indicative of certain types of desirable (pharmacological efficacy) as well as adverse (e.g., DNA damage, oxidative stress) responses. Finally, it is likely that the introduction of omics technologies to toxicity testing will eventually contribute to the reduction, refinement and replacement (the “3Rs”) of animals in toxicity testing and product safety evaluations (Kroeger, 2006).

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CHAPTER 3

MECHANISMS OF TOXICITY Zolt´an Gregus STEP 3—CELLULAR DYSFUNCTION AND RESULTANT TOXICITIES

STEP 1—DELIVERY: FROM THE SITE OF EXPOSURE TO THE TARGET

Toxicant-Induced Cellular Dysregulation Dysregulation of Gene Expression Dysregulation of Ongoing Cellular Activity Toxic Alteration of Cellular Maintenance Impairment of Internal Cellular Maintenance: Mechanisms of Toxic Cell Death Impairment of External Cellular Maintenance

Absorption versus Presystemic Elimination Absorption Presystemic Elimination Distribution to and Away from the Target Mechanisms Facilitating Distribution to a Target Mechanisms Opposing Distribution to a Target Excretion versus Reabsorption Excretion Reabsorption Toxication versus Detoxication Toxication Detoxication

STEP 4—INAPPROPRIATE REPAIR AND ADAPTATION Mechanisms of Repair Molecular Repair Cellular Repair: A Strategy in Peripheral Neurons Tissue Repair Mechanisms of Adaptation Adaptation by Decreasing Delivery to the Target Adaptation by Decreasing the Target Density or Responsiveness Adaptation by Increasing Repair Adaptation by Compensating Dysfunction When Repair and Adaptation Fail When Repair Fails When Adaptation Fails Toxicity Resulting from Inappropriate Repair and Adaptation Tissue Necrosis Fibrosis Carcinogenesis

STEP 2—REACTION OF THE ULTIMATE TOXICANT WITH THE TARGET MOLECULE Attributes of Target Molecules Types of Reactions Noncovalent Binding Covalent Binding Hydrogen Abstraction Electron Transfer Enzymatic Reactions Effects of Toxicants on Target Molecules Dysfunction of Target Molecules Destruction of Target Molecules Neoantigen Formation Toxicity Not Initiated by Reaction with Target Molecules

CONCLUSIONS

a better understanding of fundamental physiologic and biochemical processes ranging from neurotransmission (e.g., curare-type arrow poisons) through deoxyribonucleic acid (DNA) repair (e.g., alkylating agents) to transcription, translation, and signal transduction pathways (e.g., chemicals acting through transcription factors, such as the aryl hydrocarbon receptor). Pathologic conditions such as cancer and Parkinson’s disease are better understood because of studies on the mechanism of toxicity of chemical carcinogens and 1,2,3,6tetrahydro-1-methyl-4-phenylpyridine (MPTP), respectively. Continued research on mechanisms of toxicity will undoubtedly continue to provide such insights. This chapter reviews the cellular mechanisms that contribute to the manifestation of toxicities. Although such mechanisms are dealt with elsewhere in this volume, they are discussed in detail in this chapter in an integrated and comprehensive manner. We provide an overview of the mechanisms of chemical toxicity by relating a

Depending primarily on the degree and route of exposure, chemicals may adversely affect the function and/or structure of living organisms. The qualitative and quantitative characterization of these harmful or toxic effects is essential for an evaluation of the potential hazard posed by a particular chemical. It is also valuable to understand the mechanisms responsible for the manifestation of toxicity—that is, how a toxicant enters an organism, how it interacts with target molecules, and how the organism deals with the insult. An understanding of the mechanisms of toxicity is of both practical and theoretical importance. Such information provides a rational basis for interpreting descriptive toxicity data, estimating the probability that a chemical will cause harmful effects, establishing procedures to prevent or antagonize the toxic effects, designing drugs and industrial chemicals that are less hazardous, and developing pesticides that are more selectively toxic for their target organisms. Elucidation of the mechanisms of chemical toxicity has led to 45

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Toxicant

1 Delivery

2a

2b

Interaction with target molecule

Alteration of biological environment

3 Cellular dysfunction, injury

4 Inappropriate repair and adaptation

T O X I C I T Y

Figure 3-1. Potential stages in the development of toxicity after chemical exposure.

series of events that begins with exposure, involves a multitude of interactions between the invading toxicant and the organism, and culminates in a toxic effect. This chapter focuses on mechanisms that have been identified definitively or tentatively in humans or animals. As a result of the huge number of potential toxicants and the multitude of biological structures and processes that can be impaired, there are a tremendous number of possible toxic effects. Correspondingly, there are various pathways that may lead to toxicity (Fig. 3-1). A common course is when a toxicant delivered to its target reacts with it, and the resultant cellular dysfunction manifests itself in toxicity. An example of this route to toxicity is that taken by the puffer fish poison, tetrodotoxin. After ingestion, this poison reaches the voltage-gated Na+ channels of motoneurons (step 1). Interaction of tetrodotoxin with this target (step 2a) results in blockade

of Na+ channels, inhibition of the activity of motor neurons (step 3), and ultimately skeletal muscle paralysis. No repair mechanisms can prevent the onset of such toxicity. Sometimes a xenobiotic does not react with a specific target molecule but rather adversely influences the biological (micro) environment, causing molecular, organellar, cellular, or organ dysfunction leading to deleterious effects. For example, 2,4-dinitrophenol, after entering the mitochondrial matrix space (step 1), collapses the outwardly directed proton gradient across the inner membrane by its mere presence there (step 2b), causing mitochondrial dysfunction (step 3), which is manifest in toxic effects such as hyperthermia and seizures. Chemicals that precipitate in renal tubules and block urine formation represent another example for such a course (step 2b). The most complex path to toxicity involves more steps (Fig. 3-1). First, the toxicant is delivered to its target or targets (step 1), after which the ultimate toxicant interacts with endogenous target molecules (step 2a), triggering perturbations in cell function and/or structure (step 3), which initiate repair mechanisms at the molecular, cellular, and/or tissue levels as well as adaptive mechanisms to diminish delivery, boost repair capacity and/or compensate for dysfunction (step 4). When the perturbations induced by the toxicant exceed repair and adaptive capacity or when repair and adaptation becomes malfunctional, toxicity occurs. Tissue necrosis, cancer, and fibrosis are examples of chemically induced toxicities whose development follow this four-step course.

STEP 1—DELIVERY: FROM THE SITE OF EXPOSURE TO THE TARGET Theoretically, the intensity of a toxic effect depends primarily on the concentration and persistence of the ultimate toxicant at its site of action. The ultimate toxicant is the chemical species that reacts with the endogenous target molecule (e.g., receptor, enzyme, DNA, microfilamental protein, lipid) or critically alters the biological (micro) environment, initiating structural and/or functional alterations that result is toxicity. Often the ultimate toxicant is the original chemical to which the organism is exposed (parent compound). In other cases, the ultimate toxicant is a metabolite of the parent compound or a reactive oxygen or nitrogen species (ROS or RNS) generated during the biotransformation of the toxicant. Occasionally, the ultimate toxicant is an unchanged or altered endogenous molecule (Table 3-1). The concentration of the ultimate toxicant at the target molecule depends on the relative effectiveness of the processes that increase or decrease it’s concentration at the target site (Fig. 3-2). The accumulation of the ultimate toxicant at its target is facilitated by its absorption, distribution to the site of action, reabsorption, and toxication (metabolic activation). Conversely, presystemic elimination, distribution away from the site of action, excretion, and detoxication oppose these processes and work against the accumulation of the ultimate toxicant at the target molecule.

Absorption versus Presystemic Elimination Absorption Absorption is the transfer of a chemical from the site of exposure, usually an external or internal body surface (e.g., skin, mucosa of the alimentary and respiratory tracts), into the systemic circulation. Whereas transporters may contribute to the gastrointestinal absorption of some chemicals (e.g., salicylate and valproate by monocarboxylate transporters, some β-lactam antibiotics and ACE inhibitor drugs by peptide transporters, Fe2+ , Cd2+ , as well

CHAPTER 3

MECHANISMS OF TOXICITY

47

Table 3.1 Types of Ultimate Toxicants and Their Sources Parent xenobiotics as ultimate toxicants Pb ions Tetrodotoxin TCDD Methylisocyanate HCN CO Xenobiotic metabolites as ultimate toxicants Amygdalin → HCN Arsenate → Arsenite Fluoroacetate → Fluorocitrate Ethylene glycol → Oxalic acid Hexane → 2,5-Hexanedione Acetaminophen → N -Acetyl- p-benzoquinoneimine CCl4 → CCl3 OO• Benzo[a]pyrene (BP) → BP-7,8-diol-9,10-epoxide Benzo[a]pyrene (BP) → BP-Radical cation Reactive oxygen or nitrogen species as ultimate toxicants ⎫ Hydrogen peroxide ⎬ → Hydroxyl radical (HO• ) Diquat, doxorubicin, nitrofurantoin ⎭ Cr(V), Fe(II), Mn(II), Ni(II) Paraquat → O•2¯ + NO• → Peroxynitrite (ONOO– ) Endogenous compounds as ultimate toxicants Sulfonamides → albumin-bound bilirubin → Bilirubin CCl3 OO• → unsaturated fatty acids → Lipid peroxyl radicals CCl3 OO• → unsaturated fatty acids → Lipid alkoxyl radicals CCl3 OO• → unsaturated fatty acids → 4-Hydroxynon-2-enal HO• → proteins → Protein carbonyls

as some other divalent metal ions by the divalent metal-ion transporter, and arsenate by phosphate transporters), the vast majority of toxicants traverse epithelial barriers and reach the blood capillaries by diffusing through cells. The rate of absorption is related to the concentration of the chemical at the absorbing surface, which depends on the rate of exposure and the dissolution of the chemical. It is also related to the area of the exposed site, the characteristics of the epithelial layer through which absorption takes place (e.g., the thickness of the stratum corneum in the skin), the intensity of the subepithelial microcirculation, and the physicochemical properties of the toxicant. Lipid solubility is usually the most important property influencing absorption. In general, lipid-soluble chemicals are absorbed more readily than are water-soluble substances. Presystemic Elimination During transfer from the site of exposure to the systemic circulation, toxicants may be eliminated. This is not unusual for chemicals absorbed from the gastrointestinal (GI) tract because they must first pass through the GI mucosal cells, liver, and lung before being distributed to the rest of the body by the systemic circulation. The GI mucosa and the liver may eliminate a significant fraction of a toxicant during its passage through these tissues, decreasing its systemic availability. For example, ethanol is oxidized by alcohol dehydrogenase in the gastric mucosa (Lim et al., 1993), cyclosporine is returned from the enterocyte into the intestinal lumen by P-glycoprotein (an ATP-dependent xenobiotic transporter) and is also hydroxylated by cytochrome P450 (CYP3A4) in these cells (Lin et al., 1999), morphine is glucuronidated in intestinal mu-

cosa and liver, and manganese is taken up from the portal blood into liver and excreted into bile. Such processes may prevent a considerable quantity of chemicals from reaching the systemic blood. Thus, presystemic or first-pass elimination reduces the toxic effects of chemicals that reach their target sites by way of the systemic circulation. In contrast, the processes involved in presystemic elimination may contribute to injury of the digestive mucosa, liver, and lungs by chemicals such as ethanol, iron salts, α-amanitin, and paraquat because these processes promote their delivery to those sites.

Distribution to and Away from the Target Toxicants exit the blood during the distribution phase, enter the extracellular space, and may penetrate into cells. Chemicals dissolved in plasma water may diffuse through the capillary endothelium via aqueous intercellular spaces and transcellular pores called fenestrae and/or across the cell membrane. Lipid-soluble compounds move readily into cells by diffusion. In contrast, highly ionized and hydrophilic xenobiotics (e.g., tubocurarine and aminoglycosides) are largely restricted to the extracellular space unless specialized membrane carrier systems are available to transport them. During distribution, toxicants reach their site or sites of action, usually a macromolecule on either the surface or the interior of a particular type of cell. Chemicals also may be distributed to the site or sites of toxication, usually an intracellular enzyme, where the ultimate toxicant is formed. Some mechanisms facilitate whereas others delay the distribution of toxicants to their targets.

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UNIT 1

GENERAL PRINCIPLES OF TOXICOLOGY

EXPOSURE SITE Skin, GI tract, respiratory tract, injection/bite site, placenta TOXICANT

Absorption Distribution Toward Target Reabsorption Toxication

D E L I V E R Y

Presystemic Elimination Distribution Away from Target Excretion Detoxication

ULTIMATE TOXICANT Target Molecule (Protein, lipid, nucleic acid macromolecular complex) TARGET SITE

Figure 3-2. The process of toxicant delivery is the first step in the development of toxicity.

Mechanisms Facilitating Distribution to a Target Distribution of toxicants to specific target sites may be enhanced by (1) the porosity of the capillary endothelium, (2) specialized membrane transport, (3) accumulation in cell organelles, and (4) reversible intracellular binding. Porosity of the Capillary Endothelium Endothelial cells in the hepatic sinusoids and in the renal peritubular capillaries have larger fenestrae (50 to 150 nm in diameter) that permit passage of even protein-bound xenobiotics. This favors the accumulation of chemicals in the liver and kidneys. Specialized Transport Across the Plasma Membrane Specialized ion channels and membrane transporters can contribute to the delivery of toxicants to intracellular targets. For example, aquaglyceroporin channels may mediate influx of arsenite, which is present at physiological pH as uncharged As(OH)3 , voltage-gated Ca2+ channels permit the entry of cations such as lead or barium ions into excitable cells, and Na+ , K+ -ATPase promotes intracellular accumulation of thallous ion. Paraquat enters into pneumocytes via hitherto unspecified transporters, hepatocellular uptake of α-amanitin is mediated by the Na-dependent bile acid transporter (NTCP) and an organic anion transporting polypeptide (OATP1B3) and that of microcystin by OATP1B1 and OATP1B3, organic anion transporters such as human OAT1 and OAT3 mediate renal tubular uptake of ochratoxin and mercuric ion (the latter as the di-cysteine conjugate Cys-Hg-Cys), whereas both OAT1 and amino acid transporters can carry methylmercury as its cysteine conjugate CH3 -Hg-Cys, and an MPTP metabolite (MPP+ ) enters into extrapyramidal dopamin-

ergic neurons by means of the dopamine transporter. Endocytosis of some toxicant-protein complexes, such as Cd-metallothionein or hydrocarbons bound to the male rat-specific α2u -globulin, by renal proximal tubular cells can also occur. In addition, lipoprotein receptor–mediated endocytosis contributes to entry of lipoproteinbound toxicants into cells equipped with such transporters. Membrane recycling can internalize cationic aminoglycosides associated with anionic phospholipids in the brush border membrane of renal tubular cells (Laurent et al., 1990). This process may also contribute to cellular uptake of heavy metal ions. Such uptake mechanisms facilitate the entry of toxicants into specific cells, rendering those cells targets. Thus, carrier-mediated uptake of paraquat by pneumocytes and internalization of aminoglycosides by renal proximal tubular cells expose those cells to toxic concentrations of those chemicals. Accumulation in Cell Organelles Amphipathic xenobiotics with a protonable amine group and lipophilic character accumulate in lysosomes as well as mitochondria and cause adverse effects there. Lysosomal accumulation occurs by pH trapping, i.e., diffusion of the amine (e.g., amiodarone, amitriptyline, fluoxetine) in unprotonated form into the acidic interior of the organelle, where the amine is protonated, preventing its efflux. The entrapped amine inhibits lysosomal phospholipases, impairing degradation of lysosomal phospholipids, and causing phospholipidosis. Mitochondrial accumulation takes place electrophoretically. The amine is protonated in the intermembrane space (to where the mitochondria eject protons). The cation thus formed will then be sucked into the matrix space by the strong negative potential there (–220 mV), where it may impair β-oxidation and oxidative phosphorylation. By such mechanisms, the valued antiarrhytmic drug amiodarone is entrapped in the hepatic lysosomes and mitochondria, causing phospholipidosis (Kodavanti and Mehendale, 1990) and microvesiculas steatosis with other liver lesions (Fromenty and Pessayre, 1997), respectively. The cationic metabolite of MPTP (MPP+ ) also electrophoretically accumulates in the mitochondria of dopaminergic neurons, causing mitochondrial dysfunction and cell death, whereas highly lipophilic local anesthetics (e.g., tetracaine, bupivacaine), when overdosed or inadvertently injected into a blood vessel, accumulate in cardiac mitochondria, compromising mitochondrial energy production and causing cardiac failure. Human equilibrative nucleoside transporter 1 (ENT1) in the mitochondrial inner membrane appears responsible for targeting fialuridine (an already withdrawn thymidine nucleoside analogue antiviral drug) into human mitochondria, where it inhibits mitochondrial DNA synthesis, thereby inducing hepatotoxicity. The fact that ENT1 is not localized in rodent mitochondria may account for the dramatic difference in mitochondrial toxicity of fialuridine between humans and rodents (Lee et al., 2006). Reversible Intracellular Binding Binding to the pigment melanin, an intracellular polyanionic aromatic polymer, is a mechanism by which chemicals such as organic and inorganic cations and polycyclic aromatic hydrocarbons can accumulate in melanincontaining cells in the retina, the substantia nigra, and the skin (Larsson, 1993). The release of melanin-bound toxicants is thought to contribute to the retinal toxicity associated with chlorpromazine and chloroquine, injury to substantia nigra neurons by MPTP and manganese, and the induction of melanoma by polycyclic aromatics. Keratins are the major structural proteins in the epidermis and its appendages (nail and hair), constituting up to 85% of fully differentiated keratinocytes (skin cells). As keratins are abundant in cysteine residues, they can sequester thiol-reactive metal ions and metalloid compounds, whose nail and hair contents are indicative

CHAPTER 3

MECHANISMS OF TOXICITY

of exposure. Release of keratin-bound arsenic in keratinocytes may adversely affect these cells, leading to dermal lesions common in arsenicism.

Mechanisms Opposing Distribution to a Target Distribution of toxicants to specific sites may be hindered by several processes. The processes include (1) binding to plasma proteins, (2) specialized barriers, (3) distribution to storage sites such as adipose tissue, (4) association with intracellular binding proteins, and (5) export from cells. Binding to Plasma Proteins As long as xenobiotics such as DDT and TCDD are bound to high-molecular-weight proteins or lipoproteins in plasma, they cannot leave the capillaries by diffusion. Even if they exit the bloodstream through fenestrae, they have difficulty permeating cell membranes. Dissociation from proteins is required for most xenobiotics to leave the blood and enter cells. Therefore, strong binding to plasma proteins delays and prolongs the effects and elimination of toxicants. Specialized Barriers Brain capillaries have very low aqueous porosity because their endothelial cells lack fenestrae and are joined by extremely tight junctions. This blood–brain barrier prevents the access of hydrophilic chemicals to the brain except for those that can be actively transported. In the choroid plexus, where the capillaries are fenestrated, the choroidal epithelial cells are sealed together by tight junctions, forming the blood-cerebrospinal fluid barrier. Watersoluble toxicants also have restricted access to reproductive cells, which are separated from capillaries by other cells. The oocyte in the ovary is surrounded by multiple layers of granulosa cells, and the spermatogenic cells are supported by Sertoli cells that are tightly joined in the seminiferous tubules to form the blood-testis barrier (see Chap. 20). Transfer of hydrophilic toxicants across the placenta is also restricted. However, none of these barriers are effective against lipophilic substances. Distribution to Storage Sites Some chemicals accumulate in tissues (i.e., storage sites) where they do not exert significant effects. For example, highly lipophilic substances such as chlorinated hydrocarbon insecticides concentrate in adipocytes, whereas lead is deposited in bone by substituting for Ca2+ in hydroxyapatite. Such storage decreases the availability of these toxicants for their target sites and acts as a temporary protective mechanism. However, insecticides may return to the circulation and be distributed to their target site, the nervous tissue, when there is a rapid lipid loss as a result of fasting. This contributes to the lethality of pesticide-exposed birds during migration or during the winter months, when food is restricted. The possibility that lead is mobilized from the bone during pregnancy is of concern. Association with Intracellular Binding Proteins Binding to nontarget intracellular sites also reduces the concentration of toxicants at the target site, at least temporarily. Metallothionein, a cysteinerich cytoplasmic protein, serves such a function in acute cadmium intoxication (Klaassen et al., 1999). Export from Cells Intracellular toxicants may be transported back into the extracellular space. This occurs in brain capillary endothelial cells. In their luminal membrane, these cells contain ATP-dependent membrane transporters (ATP-binding cassette or ABC transporters) such as the multidrug-resistance protein (MDR1), or P-glycoprotein, which extrudes chemicals and contributes to the blood-brain barrier (Schinkel, 1999). Compared to normal mice, mice with disrupted mdr1a gene exhibit 100-fold higher brain levels of and sensitivity to ivermectin, a neurotoxic pesticide and human antihelmintic drug

49

that is one of many P-glycoprotein substrates (Schinkel, 1999). The ooctye is also equipped with the P-glycoprotein that provides protection against chemicals that are substrates for this efflux pump (Elbling et al., 1993). Hematopoietic stem cells (and perhaps other stem cells) are also protected by ABC transporters, such as MDR1, MRP1 and BCRP (breast cancer resistance protein), of which the latter confers these cells resistance to mitoxantrone. ABC transporters that export drugs were first identified in tumor cells which often overexpress them, thereby making these cells resistant to antitumor drugs these transporters pump out.

Excretion versus Reabsorption Excretion Excretion is the removal of xenobiotics from the blood and their return to the external environment. Excretion is a physical mechanism whereas biotransformation is a chemical mechanism for eliminating the toxicant. For nonvolatile chemicals, the major excretory structures in the body are the renal glomeruli, which hydrostatically filter small molecules ( ultrarapid metabolizers (UMs). Exposure to metabolites (especially the maximum concentration, Cmax ) follows the opposite rank order. The difference in exposure between PMs and EMs (i.e., the fold increase in AUC associated with the PM phenotype versus the EM phenotype) depends on fractional metabolism (fm), a measure of how much of the drug’s clearance is determined by CYP2D6 (or whatever enzyme is responsible for metabolizing the drug in question). The fold increase in AUC (AUCPM /AUCEM ) is equal to 1/(1 − f m); hence, the impact of the polymorphism increases dramatically as fm approaches unity. If CYP2D6 accounted for half of a drug’s clearance ( f m = 0.5), exposure to that drug would increase twofold in CYP2D6 PMs. However, if CYP2D6 accounted for 90% of a drug’s clearance (fm = 0.9), then exposure to that drug would increase tenfold in CYP2D6 PMs. This is why PMs are often at increased risk of adverse drug events either due to an exaggerated

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Table 6-3 Drugs That Have Been Withdrawn or Carry a Black Box Warning for Idiosyncratic Hepatotoxicitya

drug name

typical dose (mg)

indication

reactive metabolite and evidence of mechanism-based enzyme inhibition

Drugs withdrawn Benoxaprofen

300–600

NSAID

Acyl glucuronide

Bromfenac Iproniazid

25–50 25–150

NSAID Depression

Acyl glucuronide Multiple. MBI of MAO-B

Nefazodone

200

Depression

Tienilic acid

250–500

Diuretic

Troglitazone

400

Diabetes

Quinoneimine. MBI of CYP3A4 Thiophene epoxide, sulfoxide. MBI of CYP2C9 Quinone methide, sulfenic acid, α-ketoisocyanate

25–50

Psoriasis

Bosentan

125–250

PAH

Dacarbazine

140–315

Melanoma, lymphoma

Dantrolene

300–400

Muscle relaxant

Felbamate

1200

Epilepsy

Flutamide

750

Prostate cancer

Gemtuzumab

9 mg/m2

AML

Isoniazid

300

Tuberculosis

Ketoconazole

200

Fungal infections

Naltrexone Nevirapine Pemoline Tolcapone

50 200–400 37.5–112.5 300

Alcoholism, addiction AIDS Hyperactivity Parkinsonism

Trovafloxacin

100–500

Antibiotic

Valproic acid (VPA)

1000–4200

Epilepsy

Black box warnings Acitretin

a

No. Acitretin, a retinoid, is esterified to etretinate No Methyl diazohydroxide and methyl cation (CH+ 3) Nitro-reduction intermediates bind to GSH 2-Phenylpropenal (atropaldehyde) Unknown metabolites bind to protein and GSH Reductive activation to a di-radical Multiple. MBI of CYP1A1, 2A6, 2C19, and 3A4. Di-aldehyde No No No Nitro reduction and then quinoneimine formation No, but it has a lipophilic difluorinated side chain present in a toxic fluoroquinolone—now withdrawn CoA esterification and epoxidation of 4-ene-VPA

inductionb , immunological and other mechanistic aspects

Inducer (rat AhR and PPARα agonist) Anti-MAO-B and antimitochondria

Anti-LKM2 (anti-CYP2C9)

Inducer (CAR/PXR agonist)

Etretinate was withdrawn in 2002 Inhibits BSEP. Inducer (PXR agonist) Hepatotoxic to laboratory animals Inducer (AhR agonist)

Inducer (PXR agonist) Inhibits mitochondrial respiration Designed to intercalate and bind DNA Radicals formed by myeloperoxidase Auto-inhibitor (inhibits CYP3A4) Inducer (PXR agonist) Uncouples mitochondrial respiration Eosinophilia suggests an immune-mediated hepatitis

Disrupts mitochondrial β-oxidation

Data Adapted from Walgren JL, Mitchell MD, Thompson DC: Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit Rev Toxicol 35:325–361, 2005. The receptor agonist assignment is based on the CYP-induced enzyme (CYP1A for AhR; CYP2B, CYP2C, and/or CYP3A for CAR/PXR; CYP4A for PPARα). AhR, aryl hydrocarbon receptor; AIDS, acquired immunodeficiency syndrome; AML, acute myeloid leukemia; anti-LKM2 , auto-antibodies against liver and kidney microsomes (anti-LKM1 contains antibodies against CYP2D6, whereas anti-LKM2 contains antibodies against CYP2C9); BSEP, bile salt export pump (a bile canalicular transporter); CYP, cytochrome P450; GSH, glutathione; MAO, monoamine oxidase; MBI, mechanism-based inhibitor (a.k.a. suicide inactivator); NSAID, nonsteroidal anti-inflammatory drug; PAH, pulmonary arterial hypertension; PPARα, peroxisome proliferator activated receptor-alpha; PXR, pregnane X receptor; VPA, valproic acid. b

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

171

Table 6-4 The Relationship Between Genotype and Phenotype for a Polymorphically Expressed Enzyme with Active (wt), Partially Active (∗ x), and Inactive (∗ y) Alleles genotype

alleles

conventional phenotypea

activity scoreb

activity score phenotypec

Duplication of active alleles (n = 2 or more) Two fully active wild-type (wt) alleles One fully active + one partially active allele One fully active + one inactive allele Two partially active alleles One partially active + one inactive allele Two inactive alleles

(wt/wt)n wt/wt wt/∗ x wt/∗ y ∗ ∗ x/ x ∗ ∗ x/ y ∗ ∗ y/ y

UM EM EM EM EM or IM IM PM

2×n 1+1=2 1 + 0.5 = 1.5 1+0=1 0.5 + 0.5 = 1 0.5 + 0 = 0.5 0 + 0 = zero

UM High EM Medium EM Low EM Low EM IM PM

a

The phenotypes are ultrarapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM), based on the particular combination of alleles that are fully active (wt), partially active (∗ x), or inactive (∗ y). b In the case of CYP2D6 (Data from Zineh et al., 2004), various activity scores have been determined experimentally as follows: Activity score = 1.0 for each ∗ 1 (wt), ∗ 2, ∗ 35, and ∗ 41 [2988G]. Activity score = 0.75 for each ∗ 9, ∗ 29, ∗ 45, and ∗ 46. Activity score = 0.5 for each ∗ 10, ∗ 17, and ∗ 41 [2988A]. Activity score = 0 for each ∗ 3, ∗ 4, ∗ 5, ∗ 6, ∗ 7, ∗ 8, ∗ 11, ∗ 12, ∗ 15, ∗ 36, ∗ 40, and ∗ 42. c Activity scores are classified as follows (from Zineh et al., 2004): UM activity score => 2.0 (e.g., [∗ 1/∗ 1]n where n is 2 or more gene duplications). High EM activity score = 1.75 to 2.0 (e.g., ∗ 1/∗ 1). Medium EM activity score = 1.5 (e.g., ∗ 1/∗ 17 or ∗ 9/∗ 9). Low EM activity score = 1.0 to 1.25 (e.g., ∗ 1/∗ 4, ∗ 17/∗ 17, or ∗ 9/∗ 17). IM activity score = 0.5 to 0.75 (e.g., ∗ 4/∗ 9 or ∗ 4/∗ 17). PM activity score = 0 (e.g., ∗ 4/∗ 4).

pharmacological response to the drug or due to its toxic side effects. An exception is omeprazole and other proton pump inhibitors, which actually provide improved control over gastroesophageal reflux disease (GERD) in CYP2C19 PMs compared with EMs (Furuta et al., 2005). There are a minority of cases where the UMs, not the PMs, are at increased risk, especially in cases where the polymorphic enzyme catalyzes the formation of a pharmacologically active or toxic metabolite. For example, CYP2D6 converts the prodrug codeine into the active metabolite morphine, and there are reports of morphine intoxication in CYP2D6 UMs; one case being the death of a baby being breast fed by a CYP2D6 UM mother who was prescribed codeine (Gasche et al., 2004; Koren et al., 2006). Many cases have now been described where the PM phenotype requires dosage adjustment to prevent drug toxicity or an exaggerated pharmacological response. For example, CYP2D6 PMs are at increased risk for perhexiline hepatotoxicity and at increased risk for an exaggerated pharmacological response to debrisoquine and sparteine, three drugs that were not approved in the USA because of the high incidence of adverse events in the PMs. On the other hand, when drugs are converted to active metabolites by CYP2D6, then PMs derive inadequate therapeutic effect. For example, CYP2D6 converts codeine to morphine; hence, codeine is a less-effective analgesic in PMs. Similarly, CYP2D6 converts the breast cancer drug tamoxifen to endoxifen (which is 30- to 100-fold more potent than tamoxifen in suppressing estrogen-dependent cell proliferation), hence, CYP2D6 PMs are at increased risk for breast cancer recurrence following tamoxifen adjuvant therapy (Goetz et al., 2005). Other examples of genetic polymorphisms that affect drug disposition in humans include CYP2C9 (warfarin), CYP2C19 (omeprazole), CYP3A4 (clopidogrel, irinotecan, thioridazine), CYP3A5 (tacrolimus), UGT1A1 (irinotecan), UGT2B7 (morphine), TPMT (6-mercaptopurine), NAT2 (isoniazid), COMT (levodopa), DPD (5-

fluorouracil), and the efflux transporter P-glycoprotein (digoxin) (Robert et al., 2005; Relling and Giacomini, 2006). Most of these drugs identified in parentheses all have a narrow therapeutic target. Details of these genetic polymorphisms are given later in the chapter. Genetic polymorphisms in xenobiotic-biotransforming enzymes have an impact on the incidence of certain environmental diseases. For example, ethnic differences in the incidence of polymorphisms in alcohol dehydrogenase and aldehdye dehydrogenase impact the incidence of alcoholism (Li, 2000). Genetic polymorphisms in CYP2A6 impact the incidence of cigarette-smoking-induced lung cancer. Individuals lacking CYP2A6 are PMs of nicotine (so they tend to smoke less than CYP2A6 EMs) and are poor activators of tobacco-specific mutagens (so they form fewer DNA-reactive metabolites than do CYP2A6 EMs) (Kamataki et al., 2005). Genetic polymorphisms can be the underlying cause of a disease. For example, the severe and mild hyperbilirubinemia associated with Crigler–Najjar syndrome and Gilbert’s syndrome, respectively, reflect a complete and partial loss of the UDP-glucuronosyltransferase responsible for conjugating bilirubin, namely, UGT1A1. The hyperbilirubinemic Gunn rat is the rodent equivalent of Crigler–Najjar syndrome. Genetic polymorphisms have been described in laboratory animals. For example, laboratory-bred rabbits have about 50:50 chance of being a poor or rapid acetylator of certain drugs a (such as isoniazid) because N -acetyltransferase 2 (NAT2) is polymorphically expressed in rabbits. Point 24 Environmental factors can introduce as much variation in drug metabolism as can genetic factors, and this is especially true of drug–drug interactions. To take CYP3A4 as an example, PMs can be created pharmacologically with inhibitors (such as ketoconazole and erythromycin), whereas UMs can be created pharmacologically with inducers (such as rifampin and the herbal agent

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DISPOSITION OF TOXICANTS

St. John’s wort). Whereas the impact of genetic polymorphisms on drug disposition and safety is often identified during clinical trials, the impact of drug–drug interactions may not be identified until after the drug has been approved and is under postmarketing surveillance. Drugs (and other xenobiotics) can be viewed from a victim and perpetrator perspective. A drug whose clearance is largely determined by a single route of elimination, such as metabolism by a single CYP enzyme, is considered a victim drug (also known as a sensitive or object drug). Such drugs have a high victim potential because a diminution or loss of that elimination pathway, either due to a genetic deficiency in the relevant CYP enzyme or due to its inhibition by another, concomitantly administered drug, will result in a large decrease in victim drug clearance and a correspondingly large increase in exposure (the magnitude of which will depend on fractional metabolism, as indicated in Point 23). Some victim drugs have a high therapeutic index, hence, they are therapeutically effective in PMs to UMs and cause roughly the same incidence of adverse effects regardless of genotype. However, in the case of victim drugs with a narrow therapeutic index, dosage must be increased in UMs to achieve a therapeutic effect and/or it must be decreased in PMs to prevent adverse drug events. Perpetrators are those drugs (or other environmental factors) that inhibit or induce the enzyme that is otherwise responsible for clearing a victim drug. In other words, perpetrators are drugs that cause drug–drug interactions. Perpetrators are also known as precipitants. Genetic polymorphisms that result in the partial or complete loss of enzyme activity can also be viewed as perpetrators inasmuch they cause a decrease in the clearance of—and an increase in exposure to—victim drugs. Terfenadine (Seldane), cisapride (Propulsid), astemizole (Hismanal), and cerivastatin (Baycol) are all victim drugs, so much so that they have all been withdrawn from the market (Fung et al., 2001). The first three are all victim drugs because they are extensively metabolized by CYP3A4. Inhibition of CYP3A4 by various antimycotic drugs, such as ketoconazole, and antibiotic drugs, such as erythromycin, decrease the clearance of terfenadine, cisapride, and astemizole and increase their plasma concentrations to levels that, in some individuals, cause ventricular arrhythmias (QT prolongation) which can result in fatal heart attacks. Cerivastatin is extensively metabolized by CYP2C8. Its metabolism is inhibited by gemfibrozil (actually by gemfibrozil glucuronide), and the combination of cerivastatin (Baycol) and gemfibrozil (Lopid) was associated with a high incidence of fatal, cerivastatin-induced rhabdomyolysis (Ogilvie et al., 2006). Mibefradil (Posicor) is the only drug withdrawn from the U.S. market largely because of its perpetrator potential. This calcium channel blocker not only causes extensive inhibition of CYP3A4, it causes prolonged inhibition of the enzyme by virtue of being a metabolism-dependent inhibitor of CYP3A4. By inactivating CYP3A4 in an irreversible manner—such that restoration of normal CYP3A4 activity required the synthesis of new enzyme— mibefradil inhibits CYP3A4 long after treatment with the drug is discontinued. The in vitro technique of reaction phenotyping (also known as enzyme mapping) is the process of identifying which enzyme or enzymes are largely responsible for metabolizing a drug candidate (Williams et al., 2003b; Ogilvie et al., 2007). Reaction phenotyping allows an assessment of the victim potential of a drug candidate or other xenobiotic. Drug candidates can also be evaluated in vitro for their potential to inhibit or induce cytochrome P450, which allows

an assessment of their perpetrator potential (Tucker et al., 2001; Bjornsson et al., 2003; U.S. Food and Drug Administration, 2006). Herbal remedies can also interact with drugs. For example, St. John’s wort contains hyperforin, which is a potent PXR agonist and, as such, is an inducer of CYP3A4 (along with several other xenobiotic-metabolizing enzymes) and P-glycoprotein (MDR1 or ABCB1). To prevent a loss of therapeutic efficacy, the use of St. John’s wort is not recommended for patients on antirejection drugs (such as cyclosporine and tacrolimus), anti-HIV drugs (such as indinavir and nevirapine), anticoagulants (such as warfarin and phenprocoumon), or oral contraceptive steroids (Pal and Mitra, 2006). Food can affect drug disposition in a number of ways. It can affect absorption, which is why some drugs are taken with meals and others are taken between meals. Large quantities of cruciferous vegetables (e.g., broccoli and Brussels sprouts) can induce hepatic CYP1A2 (Conney, 1982), whereas grapefruit juice can inhibit intestinal CYP3A4 (Paine et al., 2006). Point 25 Although drug–drug interactions can cause an increase in the incidence of adverse events or, in the case of induction, a loss of therapeutic efficacy, not all drug–drug interactions are undesirable. For example, some anti-HIV drugs, like ritonavir, inhibit CYP3A4 and thereby improve the pharmacokinetic profile of other anti-HIV drugs, like saquinavir and lopinavir. Tumors often over-express various transporters and glutathione transferases, which limit the effectiveness of several anticancer drugs. Drugs that inhibit transporters and glutathione transferase are being developed to enhance the efficacy of anticancer drugs. Point 26 Although the amount of functional enzyme determines whether an individual is a poor or rapid metabolizer, there are other factors that can impact the rate of xenobiotic metabolism. Not surprisingly, severe liver disease decreases the rate of metabolism of a large number of drugs whose clearance is determined by hepatic metabolism. For certain xenobiotic-metabolizing enzymes, particularly the conjugating enzymes, cofactor availability can impact the rate of xenobiotic biotransformation. The liver usually has sufficient NADPH to saturate reactions catalyzed by cytochrome P450, but it does not have sufficient levels of UDP-glucuronic acid (UDPGA) or 3 -phosphoadenosine-5 -phosphosulfate (PAPS) to saturate the enzymes responsible for glucuronidating or sulfonating xenobiotics. Consequently, alterations in the levels of these cofactors can impact the rate or extent of glucuronidation and sulfonation. For example, fasting lowers hepatic levels of these cofactors, and fasting decreases the extent to which acetaminophen is conjugated. Consequently, fasting increases the extent to which acetaminophen is metabolized to a toxic metabolite by cytochrome P450 (including CYP2E1, which is induced by fasting), for which reason fasting is suspected of being one of the risk factors for acetaminophen hepatotoxicity (Whitcomb, 1994). Point 27 Stereochemical aspects can play an important role in the interaction between a xenobiotic and its biotransforming enzyme (both from a substrate and inhibitor perspective), and xenobioticbiotransforming enzymes can play a key role in converting one stereoisomer to another, a process known as mutarotation or inversion of configuration. A xenobiotic that contains a chiral center can exist in two mirror-image forms called stereoisomers or enantiomers. The biotransformation of some chiral xenobiotics occurs stereoselectively; meaning that one enantiomer (stereoisomer) is biotransformed faster than its antipode. For example, the antiepileptic drug Mesantoin® , which is a racemic mixture of R- and S-mephenytoin, is biotransformed stereoselectively in humans, such that the S-enantiomer

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

is rapidly hydroxylated (by CYP2C19) and eliminated faster than the R-enantiomer. The ability of some chiral xenobiotics to inhibit xenobiotic-biotransforming enzymes can also occur stereoselectively. For example, quinidine is a potent inhibitor of CYP2D6, where as quinine, its antipode, has relatively little inhibitory effect on this enzyme. In some cases, achiral molecules (or achiral centers) are converted to a mixture of enantiomeric metabolites, and this conversion may proceed stereoselectively such that one enantiomer is formed preferentially over its antipode. For example, several cytochrome P450 enzymes catalyze the 6-hydroxylation of steroid hormones. Some P450 enzymes (such as the rat enzyme CYP2A1) preferentially catalyze the 6α-hydroxylation reaction, whereas other P450 enzymes (such as the CYP3A enzymes in all mammalian species) preferentially catalyze the 6β-hydroxylation reaction (which is a major route of hepatic steroid biotransformation). Inversion of configuration is the process whereby one enantiomer is converted to its antipode via an achiral intermediate. This interconversion can occur nonenzymatically, as in the case of thalidomide, or it can be catalyzed by a xenobiotic-metabolizing enzyme, as in the case of lisofylline, or by an endobiotic-metabolizing enzyme, as in the case of simvastatin (Fig. 6-2). In the case of thalidomide, inversion of configuration (as shown in Fig. 6-2) occurs spontaneously, although the process is facilitated by albumin (Eriksson et al., 1998). In the mid-to-late 1950s, mainly in Europe, thalidomide was prescribed to pregnant women in the first trimester to treat morning sickness. Unfortunately, whereas (R)-thalidomide is an effective sedative, the (S)-enantiomer is a teratogen that produces phocomelia (limb shortening) and other congenital defects in the offspring largely as a result of its ability to inhibit angiogenesis (vasculogenesis). The teratogenic effect of thalidomide cannot be circumvented by administering only the (R)-enantiomer because spontaneous or albumin-facilitated racemization quickly produces the teratogenic (S)-enantiomer. Thalidomide blocks the release of tissue necrosis factor alpha (TNFα) and it has been approved for the treatment of erythema nodosum leprosum (an acute inflammatory reaction associated with lepromatous leprosy), and is useful in the treatment of several other inflammatory conditions and immune-mediated diseases. Because of its ability to inhibit angiogenesis (the process that supplies tumors with new blood vessels), thalidomide is also under investigation as an anticancer drug. It is ironic that a drug with antivascular side effects that was originally prescribed as a sedative is now in clinical trials for vascular diseases with sedation as a side effect (Franks et al., 2004). Ketones can be reduced by carbonyl reductases to a mixture of enantiomeric secondary alcohols, and this can occur with a high degree of stereoselectivity. For example, pentoxifylline is reduced by carbonyl reductases in blood and liver to a mixture of secondary alcohols with the major metabolite having an S-configuration, as shown in Fig. 6-2. Interestingly, the minor metabolite, a secondary alcohol with the R-configuration, has pharmacological properties distinct from those of its S-antipode and its ketone precursor, pentoxifylline. This minor metabolite is known as lisofylline, which is under clinical investigation for the treatment of various diseases. Through the action of carbonyl reductase, lisofylline is oxidized to pentoxifylline and then reduced to its antipode (i.e., R-alcohol → ketone → S-alcohol), the net result being an inversion of configuration (Lillibridge et al., 1996). The same type of interconversion explains why the administration of pure R-albuterol to human volunteers results in the formation of S-albuterol, just as the admin-

173

istration of pure S-albuterol leads to the formation of R-albuterol (Boulton and Fawcett, 1997). In the case of simvastatin (which contains a β-hydroxylactone), the interconversion of two secondary alcohols (β-hydroxylactone ↔ α-hydroxy-lactone) involves hydrolysis of the lactone ring (by paraoxonase 3 [hPON3] in humans) followed by formation of a thioether with coenzyme A, the first step in the β-oxidation of fatty acids, followed by dehydration to an achiral intermediate. Reversal of the steps by a combination of hydrolysis and lactonization (condensation) restores the lactone ring with the hydroxyl group in the original β-configuration or in the opposing α-configuration. In humans, the initial hydrolysis of the lactone ring and the final lactonization (condensation) reaction are both catalyzed by paraoxonase-3 (hPON3) (Draganov and La Du, 2004). Point 28 Mass spectrometry is widely used to characterize the structure of metabolites, and many instruments now come equipped with software to assist in this process, based on the fact that certain xenobiotic reactions are associated with discrete changes in mass. For example, the loss of 2 atomic mass units (amu) signifies dehydrogenation, whereas the loss of 14 amu usually signifies demethylation (–CH2 ). Several reactions result in an increase in mass, including reduction (+2 amu = 2H), methylation (+14 amu = CH2 ), oxidation (+16 amu = O), hydration (+18 amu = H2 O), acetylation (+42 amu = C2 H2 O), glucosylation (+162 = C6 H10 O5 ), sulfonation (+80 amu = SO3 ), glucuronidation (+176 amu = C6 H8 O6 ), and conjugation with glutathione (+305 amu = C10 H15 N3 O6 S), glycine (+74 amu = C2 H4 NO2 ), and taurine (+42 amu = C2 H6 NO3 S). Occasionally, routine changes in mass can arise from unexpected reactions. For example, ziprasidone is converted to two metabolites, each of which involves an increase of 16 amu, which normally indicates addition of oxygen (e.g., hydroxylation, sulfoxidation, N-oxygenation). One of the metabolites is indeed formed by addition of oxygen to ziprasidone (sulfoxidation), as shown in Fig. 6-3 (Beedham et al., 2003). However, the other metabolite is formed by a combination of reduction (+2 amu) and methylation (+14). Therefore, care must be exercised in interpreting routes of metabolism based on changes in mass. Mass spectrometry can typically provide information on which region of a molecule has undergone biotransformation, but it can seldom distinguish between several closely related possibilities. For example, based on mass spectrometry alone, it might be possible to ascertain that a certain phenyl group has been hydroxylated. However, analysis by nuclear magnetic resonance (NMR) is required to ascertain whether the hydroxylation occurred at the ortho, meta, or para position. Point 29 As mentioned in Point 18, xenobioticbiotransforming enzymes are widely distributed throughout the body. The advent of high-throughput assays based on quantitative real-time reverse-transcription polymerase chain reaction (QPCR) has facilitated comprehensive measurements of the levels of mRNA encoding numerous xenobiotic-biotransforming enzymes, transporters, and xenosensors in a wide range of human tissues (Nishimura et al., 2003, 2004; Furukawa et al., 2004; Nishimura and Naito, 2005, 2006). These data are very informative as far as mRNA expression is concerned, but it can be difficult to draw conclusions about the activity of a given enzyme in a given tissue based on mRNA levels alone. For instance, based on mRNA levels in human liver, the levels of the top-ten CYP enzymes follow the rank order: CYP2E1 2A6 > 2C8 > 2C18 ≈ 1A2 ≈ 4A11 ≈ 8B1 ≈ 2C9 ≈ 3A4 > CYP51A1 (Nishimura et al., 2003). This contrasts substantially from the levels of CYP

CH 3 N

Carbonyl reductase

O

N

NH

HO

O

H

N O

N CH 3

O

O

OH

CH 3 NH

O

NH

O

N O

O O

O

O

Secondary alcohol (S-configuration)

N

N

H

H

O

O

O

N

O

CH 3 Pentoxifylline (ketone)

Thalidomide intermediate

CH 3

R(+)-Thalidomide

N

Carbonyl reductase

S(+)-Thalidomide

N

H

O

OH

N N

O

CH 3 Secondary alcohol (R-configuration) Lisofylline hPON3

Simvastatin β-hydroxy lactone HO

-H

β-hydroxy ring opened

2O

O O

2O

hP O N 3

"Simvastatin" α-hydroxy lactone HO

H 2O

CoASH, ATP

O

-H

α-hydroxy ring opened

SCo A

O

SCo A

O

O

SCo A

O

O HO

O

H -H

H

HO +H

2O

OH

H

OH +H

R

O

2O

OH -H

2O

R

2O

R

O

β-oxidation

Figure 6-2. Stereochemical aspects of xenobiotic biotransformation: Inversion of configuration by nonenzymatic means (thalidomide), by carbonyl reductase (lisofylline), and by hydrolysis and condensation (lactonization) of a lactone ring (simvastatin).

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

175

Cl S

N

Cl Reduction

N

HS

H

N

N

N

N

H

N

(Aldehyde oxidase)

N

H O

O

Ziprasidone

Dihydroziprasidone

S-Oxidation (CYP3A4)

S-Methylation (TMT)

Cl S O

N N

Cl

N

H

H3C

S

N

N

N

N

H N

H O Ziprasidone sulfoxide (mass increases by 16 amu relative to ziprasidone)

O S-Methyldihydroziprasidone (mass increases by 16 amu relative to ziprasidone)

Figure 6-3. Conversion of ziprasidone to two different metabolites both involving a mass increase of 16 amu (relative to ziprasidone).

protein, which follow the rank order: CYP3A4 > 2C9 ≈ 3A5 ≈ 2E1 > 1A2 > 2A6 > 2C8 > 2C19 > 2B6 > 2D6 (RowlandYeo et al., 2004; Howgate et al., 2006). In the case of CYP2E1, hepatic mRNA levels are more than 17 times higher than the levels of CYP3A4 mRNA, but CYP2E1 protein levels are less than half those of CYP3A4. This discrepancy is due to the fact that, under normal conditions, most of the mRNA encoding CYP2E1 is sequestered in the cytoplasm and is not available for translation (Gonzalez, 2007). The mRNA for FMO2 is present at very high levels in human lung, but is not translated into functional enzyme due to the presence of a truncation mutation in Caucasians and Asians. However, about 26% of African Americans, 7% of Puerto Ricans, and 2% of Mexicans have one normal allele and express a functional protein (Cashman and Zhang, 2006).

HYDROLYSIS, REDUCTION, AND OXIDATION Hydrolysis Mammals contain a variety of enzymes that hydrolyze xenobiotics containing such functional groups as a carboxylic acid ester (delapril and procaine), amide (procainamide), thioester (spironolactone), phosphoric acid ester (paraoxon), acid anhydride (diisopropylfluorophosphate [DFP]), and lactone (spironolactone), as shown in Fig. 6-4. The major hydrolytic enzymes are the carboxylesterases, cholinesterases, and paraoxonases (for which lactonase is a more encompassing name), but they are by no means the only hydrolytic enzymes involved in xenobiotic biotransformation. The first two classes of hydrolytic enzymes, the carboxylesterases and cholinesterases, are known as serine esterases because their catalytic site contains a nucleophilic serine residue that participates in the hydrolysis of various xenobiotic and endobiotic substrates. This serine residue becomes phosphorylated by organophosphorus (OP) compounds, such as those used as insecticides, herbicides, fungi-

cides, nematicides, and plant growth regulators. However, following completion of the human genome project, it was determined that, of the estimated 22,500 human genes, about 5% (1227 genes) can be classified as serine hydrolases based on structural features (motifs) predicted from their DNA sequence, of which about 150 can be classified as serine proteases (Casida and Quistad, 2005). Based on the large number of potential serine hydrolases, it is not surprising that enzymes other than those described in this chapter (namely, the carboxylesterases, cholinesterases, and paraoxonases) may participate in xenobiotic metabolism. Aldehyde dehydrogenases, carbonic anhydrases, carboxypeptidases, lipases, proteases, and even albumin have all been shown to have hydrolytic (esteratic) activity toward various xenobiotics. Cytochrome P450 can catalyze the cleavage of certain xenobiotics containing a carboxylic acid ester (see section “Cytochrome P450” for examples). Based on the large number of potential serine hydrolases, it is not surprising that enzymes other than carboxylesterases and cholinesterases represent additional targets for phosphorylation and inactivation by OP compounds. Some of these OP targets have been identified; they include enzymes involved in the hydrolysis of endobiotics and certain receptors (reviewed in Casida and Quistad, 2005). Differential binding to these numerous sites may account for some of the differences observed among OP compounds. However, in insects, nematodes, and mammals, the critical site is acetylcholinesterase, the cholinesterase that hydrolyzes acetylcholine and thereby terminates its neurotransmitter activity. Phosphorylation of acetylcholinesterase is the principal mechanism of OP toxicity, with 70–90% inhibition usually proving lethal, and reversal of this phosphorylation event is one of the strategies to treat OP poisoning (e.g., with pralidoxime). In the presence of an alcohol, carboxylesterases and certain other hydrolytic enzymes can catalyze the transesterification of xenobiotics, which accounts for the conversion of cocaine (a methyl ester) to ethylcocaine (the corresponding ethyl ester) (Fig. 6-4).

(A2) Carboxylic acid ester (procaine)

(A1) Carboxylic acid ester (delapril) O

NH2

CH2

2

O

OC2H5

C CH

NH

CH

NH2

O

C

N

H 2O

CH2COOH

C

H2O

+

OH

hCE1

CH3

C2H5OH

N

+

HO

hCE2 N

R C

COOH

O

O (C) Thioester (spironolactone)

(B) Amide (procainamide) NH2

O

O

H 2O

+ N C

O

O

NH2 H2O

N

+

H 2N

CH3COOH

NH COOH

O

SCOCH3

O

SH

O

(E) Acid anhydride (diisopropylfluorophosphate)

(D) Phosphoric acid ester (paraoxon) O C2H5

O

O

P

C2H5

O

O

OH

CH3

O

H 2O

+

C2H5

O

hPON1

P

O

CH

O

CH3

C2H5

P

O

CH

O

H2O

CH3

CH3

CH3

F

CH

CH3

O

P

O

CH

CH3 CH3

OH

+

OH

NO2

NO2

(F) Transesterification (cocaine)

(G) Lactone (spironolactone) O methyl ester

O H3C

C N

ethyl ester

ethanol CH3CH2OH H 3C

OCH3

C N

O O

COOH

O

OH

O

OCH2CH3

H 2O

O

hCE1

C

O

CH3OH

hPON3

O

methanol SCOCH3

O

(H) Phosphate prodrugs (fosamprenavir)

SCOCH3

O

OH OH O

P OH

O H N

O

H 2O N

O

NH2

S

O O

O

H N

O Alkaline Phosphatase

N

O

NH2

S

O O

+

H3PO4

O

Figure 6-4. Examples of reactions catalyzed by carboxylesterases, cholinesterases, organophosphatases, and alkaline phosphatase. hCE1 and hCE2, human carboxylesterases 1 and 2; hPON1 and hPON3 (human) paraoxonase 1 and 3.

HF

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

Transesterification occurs when ethanol, not water, cleaves the catalytic transition state, i.e., the esteratic bond between the active serine residue on the enzyme and the carbonyl group on the xenobiotic:

177

O

O CH 3O

C

CH 2CH 2

C

OCH 3

Dimethylester of succinic acid

Enzyme−O−CO−R + CH3 CH2 OH → Enzyme−OH + CH3 CH2 −O−CO−R

Carboxylesterase

Carboxylesterases in serum, liver, intestine, and other tissues and the cholinesterases in blood (and muscles depending on the route of xenobiotic exposure) collectively determine the duration and site of action of certain drugs. For example, procaine—a carboxylic acid ester—is rapidly hydrolyzed, which is why this drug is used mainly as a local anesthetic. In contrast, procainamide, the amide analog of procaine, is hydrolyzed much more slowly; hence, this drug reaches the systematic circulation, where it is useful in the treatment of cardiac arrhythmia. In general, enzymatic hydrolysis of amides occurs more slowly than esters, although electronic factors can influence the rate of hydrolysis. The presence of electron-withdrawing substituents weakens an amide bond, making it more susceptible to enzymatic hydrolysis. The hydrolysis of xenobiotics by carboxylesterases and other hydrolytic enzymes is not always a detoxication process. Figure 6-5 shows some examples in which carboxylesterases convert xenobiotics to toxic and tumorigenic metabolites. In 1953, Aldridge classified hydrolytic enzymes on the basis of their interaction with OP compounds, classifying those that hydrolyze OP compounds as A-esterases, those that are inhibited by OP compounds as B-esterases, and those that do not interact with OP compounds as C-esterases. Although the terms are still widely used, the classification system of Aldridge can be somewhat confusing because it divides the paraoxonases into the A- and Cesterase class (the human paraoxonase hPNO1 hydrolyzes OP compounds and so can be classified as an A-esterase, whereas hPON2 and hPON3 can be classified as C-esterases because they do not hydrolyze OP compounds, nor are they inhibited by them). Furthermore, carboxylesterases and cholinesterases, two distinct classes of hydrolytic enzymes, are both B-esterases according to Aldridge because both are inhibited by OP compounds. Carboxylesterases Carboxylesterases are ∼60 kDa glycoproteins that are present in a wide variety of tissues, including serum. Most of the carboxylesterase activity in liver is associated with the endoplasmic reticulum, although considerable carboxylesterase activity is present in lysosomes and cytosol. Although by some estimates human liver and brain may express 5 and 30 carboxylesterases, respectively (Liederer and Borchardt, 2006), the hydrolysis of xenobiotic esters and amides in humans is largely catalyzed by just two carboxylesterases called hCE1 and hCE2. hCE1 mRNA is detected at very high levels in the liver, followed by the trachea and lung (at less than 10% of the level detected in the liver) (Nishimura and Naito, 2006). It is encoded by two genes, namely, CES1A1 and CES1A2, that differ only in the amino acid sequence of the encoded signal peptide, for which reason both genes are considered to encode the same enzyme, hCE1. The second human carboxylesterase, hCE2, is encoded by CES2A1, and is expressed to approximately the same extent in the liver and small intestine, followed by the colon and kidney (at less than half of the level detected in the liver) (Nishimura and Naito, 2006). In various mammalian species, carboxylesterases are found in the plasma, whereas human plasma does not contain carboxylesterases, meaning that

2

HOOC

CH3OH

Methanol

CH 2CH 2

COOH

Succinic acid

Nasal epithelium

Epithelium degeneration O H 3C

C

O

CH

CH 3

Vinyl acetate

Carboxylesterase

O

O

CH 3

CH

Acetaldehyde H 3C

C

OH

Acetate

Covalent binding to DNA and proteins

Nasal tumors O N

C

CH 3

N

CH 3

NO

1,3-Dimethyl-3-phenyl-1-nitrosourea Carboxylesterase

[HO

O N

C

OH

N

N

CH 3]

Methyldiazonium hydroxide

CH 3 N-methyl-N-phenyl1-nitro-formic acid

Covalent binding to DNA

Skin tumors

Figure 6-5. Activation of xenobiotics to toxic and tumorigenic metabolites by carboxylesterases.

178

UNIT 2

DISPOSITION OF TOXICANTS

butyrylcholinesterases and paraoxonases, not carboxylesterases, are responsible for the hydrolysis of amide and ester-containing compounds in the plasma (Satoh and Hosokawa, 2006). In rats, hydrolases A and B appear to be the counterparts of hCE1 and hCE2, respectively. The HUGO Gene Nomenclature Committee has officially approved gene names for CES1, 2, 3, 4, and 7 (http://www.gene.ucl.ac.uk/nomenclature/index.html), with CES1 and CES2 corresponding to hCE1 and hCE2. However, Satoh and Hosokawa (2006) have proposed that in all mammalian species, the carboxylesterases can be assigned to one of five gene families (designated CES 1–5) with three human genes in the first gene family (namely, CES1A1, 1A2, and 1A3), and a single member of the remaining families (i.e., CES2A1, 3A1, 4C1, and 5A1). As illustrated in Fig. 6-4, hCE1 generally catalyzes the hydrolysis of xenobiotics with a small alcoholic leaving group (such as ethanol, as in the case of delapril) whereas hCE2 generally catalyzes the hydrolysis of xenobiotics with a small acidic or large alcoholic leaving group (as in the case of procaine), although there are xenobiotics that are hydrolyzed by both enzymes. hCE1 is more active than hCE2 at catalyzing the hydrolysis of numerous xenobiotics such as oseltamivir, benazepril, cilazepril, quinapril, temocapril, imidapril, meperidine, delapril, and clopidogrel, as well as the transesterification of the methyl esters of cocaine (Fig. 6-4), whereas hCE2 is more active than hCE1 at hydrolyzing aspirin, heroin, cocaine benzoyl ester, 6-acetylmorphine, oxybutynin, and the anticancer drug irinotecan (7-ethyl-10-[4-(1piperidino)-1-piperidino]carbonyoxycamptothecin, also known as CPT-11) (Satoh and Hosokawa, 2006; Shi et al., 2006; Tang et al., 2006). The hydrolysis of irinotecan by hCE2 (and, to a much lesser extent, by hCE1) produces SN-38, a topoisomerase inhibitor that is responsible for both the anticancer effects of irinotecan and many of its side effects, especially diarrhea, which can be understood, at least in part, by the high levels of hCE2 in the intestine (Satoh et al., 2002). Genetic polymorphisms (single nucleotide polymorphisms or SNPs) have been described for hCE2, some of which appear to lower the rate of hydrolysis of irinotecan to SN-38 (Kubo et al., 2005). Genetic polymorphisms have also been described for hCE1, but these are less likely to impair overall hCE1 because the enzyme is encoded by two genes. Therefore, a true hCE1 PM would only arise if genetic polymorphisms affected both alleles of both hCES1A1 and hCES1A2. A phenotype for hCE1 that might be classified as high extensive metabolizer has been described. It arises from a single nucleotide polymorphism in the promoter region of hCES1A2 (but not hCES1A1) that increases the expression of hCE1 and thereby increases the rate of hydrolysis of imidapril to its active metabolite imidaprilat, an angiotensin-converting enzyme (ACE) inhibitor, which increases its antihypertensive effect (Geshi et al., 2005). In addition to hydrolyzing xenobiotics, carboxylesterases hydrolyze numerous endogenous compounds, such as palmitoyl-CoA, monoacylglycerol, diacylglycerol, retinyl ester, platelet-activating factor, and other esterified lipids. Carboxylesterases can also catalyze the synthesis of fatty acid ethyl esters, which represents a nonoxidative pathway of ethanol metabolism in adipose and certain other tissues. In the case of platelet-activating factor, carboxylesterases catalyze both the deacetylation of PAF and its subsequent esterification with fatty acids to form phosphatidylcholine (Satoh and Hosokawa, 1998). Certain carboxylesterases also have a physiologic function in anchoring other proteins to the endoplasmic reticulum. For example, the lysosomal enzyme β-glucuronidase is also present in the endoplasmic reticulum, where it is anchored in the lumen by egasyn,

a microsomal carboxylesterase related to hCE1. Egasyn binds to β-glucuronidase at its active site serine residue, which effectively abolishes the carboxylesterase activity of egasyn, although there is no corresponding loss of β-glucuronidase activity. Binding of OP compounds to egasyn causes the release of β-glucuronidase into plasma, which serves as the basis for a test for OP exposure (Fujikawa et al., 2005). The retention of β-glucuronidase in the lumen of the ER is thought to be physiologically significant. Glucuronidation by microsomal UGTs is a major pathway in the clearance of many of the endogenous aglycones (such as bilirubin) and xenobiotics (such as drugs). However, hydrolysis of glucuronides by β-glucuronidase complexed with egasyn in the lumen of the ER appears to be an important mechanism for recycling endogenous compounds, such as steroid hormones (Dwivedi et al., 1987). The acute-phase response protein, C-reactive protein, is similarly anchored in the endoplasmic reticulum by egasyn. The mechanism of catalysis by carboxylesterases is analogous to the mechanism of catalysis by serine-proteases. In the case of carboxylesterases, it involves charge relay among a catalytic triad comprising an acidic amino acid residue (glutamate [Glu335 ]), a basic residue (histidine [His448 ]), and a nucleophilic residue (serine [Ser203 ]) (Yan et al., 1994; Satoh and Hosokawa, 1998). (These amino acid residues, numbered for a rat carboxylesterase, differ slightly in other species, but the overall location and function of these residues are the same in all mammalian carboxylesterases.) The mechanism of catalysis of carboxylesterases is shown in Fig. 6-6, and is discussed in more detail in the section on Epoxide Hydrolase. Organophosphorus compounds bind to the nucleophilic OH-group on the active site serine residue to form a phosphorus– oxygen bond, which is not readily cleaved by water. Therefore, OP compounds bind stoichiometrically to carboxylesterases and inhibit their enzymatic activity, for which reason they are also classified as B-esterases (Aldridge, 1953). Surprisingly, the stoichiometric binding of OP compounds to carboxylesterases is an important determinant of OP toxicity, as outlined in the following section on cholinesterases.

Cholinesterases (AChE and BChE) Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE, also known as pseudocholinesterase) are related enzymes. As the names imply, AChE and BChE have high activity toward acetylcholine and butyrylcholine (and propionylcholine), respectively. BChE can also hydrolyze bambuterol, chlorpropaine, cocaine, methylprednisolone acetate, heroin, isosorbide diaspirinate, mivacurium, procaine, succinylcholine, tetracaine, and other drugs. Eserine is an inhibitor of both enzymes, whereas BW284C51 is a selective inhibitor of AChE, and iso-OMPA is a selective inhibitor of BChE (Liederer and Borchardt, 2006). Drugs that selectively inhibit brain AChE and BChE activity, such as rivastigmine (Exelon® ), have been used to treat Alzheimer’s disease. Other drugs that inhibit AChE and are used to treat Alzheimer’s disease include tacrine (Cognex® ), gelantamine (Reminyl® ), and donepezil (Aricept® ). Both enzymes exist in six different forms with differing solubility: monomer (G1), dimer (G2), tetramer (G4), tailed tetramers (A4), double tetramers (A8), and triple tetramers (A12). G1, G2, and G4 contain 1, 2, and 4 subunits, each with a catalytic site. These various forms can each exist in three states: soluble (hydrophilic), immobilized (asymmetric), and amphiphilic globular (membrane-bound through attachment to the phospholipid bilayer) (Nigg and Knaak, 2000). All forms are expressed in muscle. In the case of AChE, the

CHAPTER 6

Carboxylesterase

Epoxide hydrolase

H+

O

O

Glu335

O R1

BIOTRANSFORMATION OF XENOBIOTICS

C

O

O

O

R2

O

H

H

N H

O

O

N

Ser203

N

O

N

C His448

Asp226

His431

Enzyme-substrate complex

Enzyme-substrate complex

O

O Glu335

HO

O R2

O

R1

O

C H O

O

H

Glu376 and 404

H

N

N

O

O

N

Ser203

Glu376 and 404

N

C His448

Acylated enzyme intermediate

Asp226

His431

Alkylated enzyme intermediate

H2O

H2O

R2OH

O

O Glu335

HO

O R1

O C

H

H

O H

O

O

Glu376 and 404

H

N N

N

O

O C

H N O H

Ser203

His448

Nucleophilic attack by water

Asp226

His431

Nucleophilic attack by water HO

R1COOH OH

Enzyme ready to bind substrate

Enzyme ready to bind substrate

Figure 6-6. Catalytic cycle of microsomal carboxylesterase (left) and microsomal epoxide hydrolase (right), two α/β-hydrolase fold enzymes.

major form in brain is the tetramer G4 (anchored with a 20-kDa side chain containing fatty acids), but the major form in erythrocytes is the dimer G2 (anchored with a glycolipid-phosphatidylinositol side chain). In the case of BChE, the major form in serum is the tetramer G4 (a glycoprotein with Mr 342 kDa). In both AChE and BChE, the esteratic site (containing the active site serine residue) is adjacent to an anionic (negatively charged) site that interacts with the positively charged nitrogen on acetylcholine and butyrylcholine. Genetic variants of AChE that severely impair its activity have not been described, which is not surprising given the key role that AChE plays in terminating neurotransmission by acetylcholine.

179

Based on measurements of erythrocyte AChE activity, familial reductions of 30% have been reported, and a reduction of 50% has been linked to paroxysmal nocturnal hemoglobinuria (Nigg and Knaak, 2000). At least 10 genetic variants of BChE have been described following the discovery of PMs of succinylcholine. Succinylcholine is a muscle relaxant whose duration of action is determined by serum BChE. In some individuals (∼2% of Caucasians), succinylcholine causes prolonged muscular relaxation and apnea, which led to the discovery of a genetic variant of BChE (Asp70 → Gly70 ) (La Du, 1992; Lockridge, 1992). Although this allelozyme has markedly diminished activity toward succinylcholine (which is the genetic basis for the exaggerated response to this muscle relaxant in affected individuals), it nevertheless has appreciable activity toward other substrates, such as acetylcholine and benzoylcholine. The wild type and variant form of BChE are equally sensitive to the inhibitory effect of OP compounds, but the allelic variant is relatively resistant to the inhibitory effect of dibucaine, a local anesthetic, which forms the basis of a diagnostic test for its presence (frequently called a test for atypical pseudocholinesterase). The discovery of the BChE allelozyme (the so-called atypical pseudocholinesterase) is of historical interest because it ushered in the new field of pharmacogenetics. Since its initial discovery in the late 1950s, several allelic variants of BChE and many other xenobiotic-biotransforming enzymes have been identified (see Point 23). Carboxylesterases and cholinesterases in the blood and tissues play an important role in limiting the amount of OP compounds that reaches AChE in the brain, inhibition of which is the mechanism of toxicity of OP pesticides (and carbamate insecticides), such that a 70–90% loss of AChE activity is lethal to mammals, insects, and nematodes. The symptoms of OP toxicity resemble those caused by excessive stimulation of cholinergic nerves. The covalent interaction between OP compounds and brain AChE is analogous to their binding to the active site serine residue in all serine esterases (B-esterases). As previously mentioned, certain OP compounds are hydrolyzed by A-esterases (the paraoxonases) but bind stoichiometrically and, for the most part, irreversibly to B-esterases (carboxylesterases and cholinesterases). Surprisingly, stoichiometric binding of OP compounds to carboxylesterase and cholinesterase (and perhaps to numerous other enzymes and receptors that have structural features common to serine esterases) plays an important role in limiting the toxicity of OP compounds. Numerous studies have shown an inverse relationship between serine esterase activity and susceptibility to the toxic effect of OP compounds. Factors that decrease serine esterase activity potentiate the toxic effects of OP compounds, whereas factors that increase serine esterase activity have a protective effect. For example, the susceptibility of animals to the toxicity of parathion, malathion, and diisopropylfluorophosphate (DFP) is inversely related to the level of serum esterase activity (which reflects both carboxylesterase and BChE activity). Differences in the susceptibility of several mammalian species to OP toxicity can be abolished by pretreatment with selective serine esterase inhibitors such as cresylbenzodioxaphosphorin oxide, the active metabolite of tri-ortho-tolylphosphate (which is also known as tri-ortho-cresylphosphate or TOCP). Esterases are not the only enzymes involved in the detoxication of OP pesticides. Certain OP compounds are detoxified by cytochrome P450, flavin monooxygenases, and glutathione transferases. However, paraoxonases, enzymes that catalyze the hydrolysis of certain OP compounds, appear to play only a minor role in determining susceptibility to OP toxicity, as outlined in the following section.

180

UNIT 2

DISPOSITION OF TOXICANTS

Paraoxonases (Lactonases) Paraoxonases catalyze the hydrolysis of a broad range of organophosphates, organophosphinites, aromatic carboxylic acid esters, cyclic carbonates, and lactones. They are calcium-dependent enzymes containing a critical sulfhydryl (-SH) group; as such they are inhibited by EDTA, metal ions (Cu and Ba), and various mercurials such as phenylmercuric acetate (PMA) and para-chloromercuribenzoate (PCMB). Based on the observation that A-esterases are inhibited by PCMB but not OP compounds, Augustinsson (1966) postulated that, in the case of paraoxonases, OP compounds bind to a nucleophilic SH-group on an active site cysteine residue and form a phosphorus–sulfur bond, which is readily cleaved by water. A strong argument against this postulate is the fact that there is no loss of activity when the only potential active site cysteine residue in human paraoxonase (Cys283) is substituted with serine or alanine (Sorenson et al., 1995). Paraoxonase requires Ca2+ , both for stability and catalytic activity, which raises the possibility that the hydrolysis of OP compounds by paraoxonase involves metal-catalyzed hydrolysis, analogous to that proposed for calciumdependent phospholipase A2 or zinc-dependent phosphotriesterase activity (Sorenson et al., 1995). Humans express three paraoxonases designated hPON1, hPON2, and hPON3. hPON1 is present in liver microsomes and plasma, where it is associated exclusively with high-density lipoprotein (HDL). hPON2 is not present in plasma but it is expressed in several tissues. hPON3 is expressed in serum and liver and kidney microsomes. In addition to differences in tissue distribution, the three paraoxonases in mice respond differently to oxidative stress, which decreases PON1, increases PON3, and has no effect on PON2. Only hPON1 has appreciable arylesterase activity and the ability to hydrolyze the toxic oxon metabolites of OP insecticides such as parathion, diazinon, and chlorpyrifos (Draganov and La Du, 2004). However, all three enzymes can catalyze the hydrolysis of various lactones, for which reason the name “lactonase” is more encompassing. They also catalyze the reverse reaction, i.e., the lactonization of hydroxy-carboxylic acids, as illustrated in Fig. 6-2. Hydrolysis and lactonization of statins, such a lovastatin and simvastatin, is catalyzed only by hPON3. Reports of the same reaction being catalyzed by hPON1 appear to be attributable to trace contamination with hPON3 (Draganov and La Du, 2004). The lactone dihydrocoumarin is the only substrate reported for hPON2. There is evidence to suggest that hPON1 protects against atherosclerosis by hydrolyzing specific derivatives of oxidized cholesterol and/or phospholipids in atherosclerotic lesions and in oxidized low-density lipoprotein (LDL). For example, mice lacking PON1 (knockout mice or PON1 null mice) are predisposed to atherogenesis (Draganov and La Du, 2004). It has been suggested that PON1 has two active sites, one involved in hydrolytic reactions and the other involved in protecting LDL against oxidation. The ability of PON1 to protect LDL from copper-induced oxidation requires a cysteine residue at position 284 (which is not required for esteratic activity) but which does not require calcium (which is required for catalytic activity). Based on its ability to hydrolyze OP compounds, it seems reasonable to assume that PON1 would play an important role in determining susceptibility to OP toxicity, but this may not be the case. PON1 knockout mice are no more susceptible than wildtype mice to the toxic effects of paraoxon (the active metabolite of parathion). However, it is possible that PON1 plays a larger role in limiting the toxicity of other OP compounds such as diazoxon and chlorpyrifos oxon because it hydrolyzes these oxons 10–20

times faster than it hydrolyzes paraoxon (Draganov and La Du, 2004). hPON1 hydrolyzes the unsaturated cyclic carbonate prodrug prulifloxacin to its active metabolite NM394, and the rate of hydrolysis is influenced by a genetic polymorphism at amino acid 192. Whether this position is a glutamine or arginine residue affects the hydrolytic activity of hPON1 in a substrate-dependent manner, with some substrates being hydrolyzed faster by hPON1192R , such as paraoxon, and others being hydrolyzed faster by hPON1192Q , such as certain lactones. There is clinical evidence supporting—but also some excluding—a link between the hPON1192R genetic polymorphism and the development of cardiovascular disease, which is consistent with the decreased ability of this allelozyme to metabolize oxidized lipids (Draganov and La Du, 2004). Diisopropylfluorophosphatase (DFPase), which catalyzes the release of fluoride from DFP, is a hydrolytic enzyme related to the paraoxonases. It hydrolyzes the nerve gas agents sarin and soman (Liederer and Borchardt, 2006).

Prodrugs and Alkaline Phosphatase Many prodrugs are designed to be hydrolyzed by hydrolytic enzymes (reviewed by Liederer and Borchardt, 2006). Some prodrugs, such as propranolol ester, are hydrolyzed by both carboxylesterases and cholinesterases (both AChE and BChE), whereas others are preferentially or specifically hydrolyzed by carboxylesterases (capecitabine, irinotecan), BChE (bambuterol, methylprednisolone acetate), hPNO1 (prulifloxacin) or hPON3 (lovastatin, simvastatin). In some cases the prodrug is hydrolyzed by an enzyme that has yet to be fully characterized. For example, the hydrolysis of valacyclovir to the antiviral drug acyclovir is catalyzed by a human enzyme named valacyclovirase (genesymbol: BPHL). Some prodrugs are hydrolyzed with a high degree of stereoselectivity. For example, in the case of prodrugs of ibuprofen and flurbiprofen, the R-enantiomer is hydrolyzed about 50 times faster than the S-enantiomer. Some prodrugs, such as fosphenytoin (Cerebyx® ) and fosamprenavir (Lexiva® ), are designed to be hydrolyzed by alkaline phosphatase, high concentrations of which are present on the luminal surface of the enterocytes lining the wall of the small intestine. Hydrolysis of these prodrugs by alkaline phosphatase releases the active drug at the surface of the enterocytes, where it can be readily absorbed. As a result of their ability to hydrolyze prodrugs, hydrolytic enzymes may have clinical applications in the treatment of certain cancers. They might be used, for example, to activate prodrugs in vivo and thereby generate potent anticancer agents in highly selected target sites (e.g., at the surface of tumor cells, or inside the tumor cells themselves). For example, carboxylesterases might be targeted to tumor sites with hybrid monoclonal antibodies (i.e., bifunctional antibodies that recognize the carboxylesterase and the tumor cell), or the cDNA encoding a carboxylesterase might be targeted to the tumor cells via a viral vector. In the case of irinotecan, this therapeutic strategy would release the anticancer drug SN-38 in the vicinity of the tumor cells, which would reduce the systemic levels and side effects of this otherwise highly toxic drug (Senter et al., 1996). Some prodrugs, such as capecitabine, are activated by hydrolytic enzymes in the tumors themselves. There is evidence to suggest that, by hydrolyzing SN-38-glucuronide (a metabolite of the anticancer drug irinotecan), β-glucuronidase in colonic tumor cells may play a role in exposing such tumors to the topoisomerase inhibitor SN-38 (Tobin et al., 2006).

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

Peptidases With the advent of recombinant DNA technology, numerous human peptides have been mass-produced for use as therapeutic agents, and several recombinant peptide hormones, growth factors, cytokines, soluble receptors, and humanized monoclonal antibodies currently are used clinically. To avoid acid-precipitation and proteolytic degradation in the gastrointestinal tract, peptides are administered parenterally. Nevertheless, peptides are hydrolyzed in the blood and tissues by a variety of peptidases, including aminopeptidases and carboxypeptidases, which hydrolyze amino acids at the N - and C-terminus, respectively, and endopeptidases, which cleave peptides at specific internal sites (trypsin, for example, cleaves peptides on the C-terminal side of arginine or lysine residues) (Humphrey and Ringrose, 1986). Peptidases cleave the amide linkage between adjacent amino acids, hence, they function as amidases. As in the case of carboxylesterases, the active site of peptidases contains either a serine or cysteine residue, which initiates a nucleophilic attack on the carbonyl moiety of the amide bond. As previously noted, the mechanism of catalysis by serine proteases, such as chymotrypsin, is similar to that by serine esterases (B-esterases). Epoxide Hydrolases Epoxide hydrolases catalyze the transaddition of water to alkene epoxides and arene oxides (oxiranes), which can form during the cytochrome P450-dependent oxidation of aliphatic alkenes and aromatic hydrocarbons, respectively. As shown in Fig. 6-7, the products of this hydrolysis are vicinal diols with a trans-configuration (i.e., trans-1,2-dihydrodiols); a notable exception being the conversion of leukotriene A4 (LTA4) to leukotriene B4 (LTB4), in which case the two hydroxyl groups that result from epoxide hydrolysis appear on nonadjacent carbon atoms. Epoxide hydrolases play an important role in detoxifying electrophilic epoxides that might otherwise bind to proteins and nucleic acids and cause cellular toxicity and genetic mutations. Although the levels vary from one tissue to the next, epoxide hydrolase has been found in the microsomal fraction of virtually all tissues, including the liver, testis, ovary, lung, kidney, skin, intestine, colon, spleen, thymus, brain, and heart. There are five distinct forms of epoxide hydrolase in mammals: microsomal epoxide hydrolase (mEH, which is the product of the gene EPHX1), soluble epoxide hydrolase (the gene product of EPHX2), cholesterol epoxide hydrolase, leukotriene A4 (LTA4 , the gene product of LTA4H) hydrolase, and hepoxilin hydrolase (Fretland and Omiecinski, 2000; Morisseau and Hammock, 2005).

181

As their names imply, the latter three enzymes appear to hydrolyze endogenous epoxides exclusively, and have virtually no capacity to detoxify xenobiotic oxides. LTA4 hydrolase is distinct from the other epoxide hydrolases because it is a bifunctional zinc metalloenzyme that has both epoxide hydrolase and peptidase activity, and because the two hydroxyl groups introduced during the conversion of LTA4 to LTB4 are eight carbon atoms apart. In contrast to the high degree of substrate specificity displayed by the cholesterol, LTA4 and hepoxilin epoxide hydrolases, mEH hydrolyzes a wide variety of xenobiotics with an alkene epoxide or arene oxide. sEH hydrolyzes some xenobiotic epoxides and oxides, such a trans-stilbene oxide, but it also plays an important role in the hydrolysis of endogenous fatty acid epoxides, such as the epoxyeicosatrienoic acids (EETs) that are formed by epoxidation of arachidonic acid by cytochrome P450 and the leukotoxins that are formed by the epoxidation of linoleic acid by leukocytes (Fretland and Omiecinski, 2000; Morisseau and Hammock, 2005). In the case of EETs, hydrolysis by sEH terminates their vasodilatory effects. Male sEH knockout mice (sEH null mice) have significantly lower blood pressure than wild-type mice, supporting the important role of sEH in EET hydrolysis (and the important vasodilatory effects of EETs), and supporting the notion that inhibitors of sEH might be useful in the treatment of hypertension. In the case of leukotoxins, hydrolysis by sEH can be considered an activation process because it produces diols that perturb membrane permeability and calcium homeostasis, which results in vascular inflammation and cardio-pulmonary toxicity, including acute respiratory distress syndrome (ARDS) and multiple organ failure (Morisseau and Hammock, 2005). In general, mEH prefers mono-substituted epoxides and disubstituted epoxides with a cis configuration, such as cis-stilbene oxide, whereas sEH prefers tetra- and tri-substituted epoxides and di-substituted epoxides with a gem configuration (both substituents on the same carbon atom) or the trans configuration, such as transstilbene oxide, as shown in Fig. 6-8. In rodents, sEH and mEH are both inducible enzymes; sEH is under the control of PPARα, so it is induced following treatment of rats and mice with peroxisome

H

Microsomal epoxide hydrolase (pH 9.0)

O H

H2 O

Alkene epoxide OH

O C

CH 2

H2O

C

H

cis-stilbene oxide

H

Styrene 7,8-epoxide

OH H

CH2OH

C

C

H

OH

Styrene 7,8-glycol 1,2-diphenyl-1,2-ethanediol

Arene oxide H

HO

O H

+ H2 O

H

H

H

O H2O

OH H

Naphthalene 1,2-oxide

Naphthalene 1,2-dihydrodiol

Figure 6-7. Examples of the hydrolyation of an alkene epoxide (top) and an arene oxide (bottom) by epoxide hydrolase.

Soluble (cytosolic) epoxide hydrolase (pH 7.4)

trans-stilbene oxide

Figure 6-8. Stereoselective hydrolyation of stilbene oxide by microsomal and soluble epoxide hydrolase.

182

UNIT 2

DISPOSITION OF TOXICANTS

proliferators, whereas mEH is under the control of Nrf2, so it is induced in response to oxidative stress or exposure to electrophiles and glutathione depletors (see section “Quinone Reduction— NQO1 and NQO2”). Treatment of mice with the CAR agonist phenobarbital induces mEH about two- to threefold, whereas treatment with Nrf2 activators such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and ethoxyquin induces mEH by an order of magnitude or more. Many epoxides and oxides are intermediary metabolites formed during the cytochrome P450-dependent oxidation of unsaturated aliphatic and aromatic xenobiotics. These electrophilic metabolites might otherwise bind to proteins and nucleic acids and cause cellular toxicity and genetic mutations. In general, sEH and mEH are found in the same tissues and cell types that contain cytochrome P450. For example, the distribution of epoxide hydrolase parallels that of cytochrome P450 in liver, lung, and testis. In other words, both enzymes are located in the centrilobular region of the liver (zone 3), in Clara and type II cells in the lung, and in Leydig cells in the testis. The co-localization of epoxide hydrolase and cytochrome P450 presumably ensures the rapid detoxication of alkene epoxides and arene oxides generated during the oxidative metabolism of xenobiotics. Electrophilic epoxides and arene oxides are constantly produced during the cytochrome P450-dependent oxidation of unsaturated aliphatic and aromatic xenobiotics, and are highly reactive to cellular macromolecules such as DNA and protein. Epoxide hydrolase can rapidly convert these potentially toxic metabolites to the corresponding dihydrodiols, which are less reactive and easier to excrete. Thus, epoxide hydrolases are widely considered as a group of detoxication enzymes. In some cases, however, further oxidation of a dihydrodiol can lead to the formation of diol epoxide derivatives that are no longer substrates for epoxide hydrolase because the oxirane ring is protected by bulky substituents that sterically hinder interaction with the enzyme. This point proved to be extremely important in elucidating the mechanism by which polycyclic aromatic hydrocarbons cause tumors in laboratory animals (Conney, 1982). Tumorigenic polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are converted by cytochrome P450 to a variety of arene oxides that bind covalently to DNA, making them highly mutagenic to bacteria. One of the major arene oxides formed from benzo[a]pyrene, namely, the 4,5-oxide, is highly mutagenic to bacteria but weakly mutagenic to mammalian cells. This discrepancy reflects the rapid inactivation of benzo[a]pyrene 4,5-oxide by epoxide hydrolase in mammalian cells. However, one of the arene oxides formed from benzo[a]pyrene, namely, benzo[a]pyrene 7,8dihydrodiol-9,10-oxide, is not a substrate for epoxide hydrolase and is highly mutagenic to mammalian cells and considerably more potent than benzo[a]pyrene as a lung tumorigen in mice. Benzo[a]pyrene 7,8-dihydrodiol-9,10-oxide is known as a bayregion diolepoxide, and analogous bay-region diolepoxides are now recognized as tumorigenic metabolites of numerous polycyclic aromatic hydrocarbons. A feature common to all bay-region epoxides is their resistance to hydrolyation by epoxide hydrolase, which results from steric hindrance from the nearby dihydrodiol group. As shown in Fig. 6-9, benzo[a]pyrene 7,8-dihydrodiol-9,10-oxide is formed in three steps: Benzo[a]pyrene is converted to the 7,8-oxide, which is converted to the 7,8-dihydrodiol, which is converted to the corresponding 9,10-epoxide. The first and third steps are epoxidation reactions catalyzed by cytochrome P450 or prostaglandin H synthase, but the second step is catalyzed by epoxide hydrolase. Consequently, even though epoxide hydrolase plays a major role in detoxifying sev-

eral benzo[a]pyrene oxides, such as the 4,5-oxide, it nevertheless plays a role in converting benzo[a]pyrene to its ultimate tumorigenic metabolite, benzo[a]pyrene 7,8-dihydrodiol-9,10-oxide. The importance of mEH in the conversion of polycyclic aromatic hydrocarbons to their ultimate carcinogenic metabolites, namely, diol-epoxides, is illustrated by the observation that mEH knockout mice (mEH-null mice) are less sensitive than wild-type mice to the tumorigenic effects of 7,12-dimethylbenz[a]anthracene (DMBA) (Fretland and Omiecinski, 2000; Morisseau and Hammock, 2005). Not all epoxides are highly reactive and toxic to the cells that produce them. The major metabolite of carbamazepine is an epoxide, which is so stable that carbamazepine 10,11-epoxide is a major circulating metabolite in patients treated with this antiepileptic drug. (Carbamazepine is converted to a second epoxide, which is less stable and more cytotoxic, as shown in the section on Cytochrome P450.) Vitamin K epoxide is also a nontoxic epoxide, which is formed and consumed during the vitamin K-dependent γ carboxylation of prothrombin and other clotting factors in the liver. Vitamin K epoxide is not hydrated by epoxide hydrolase but is reduced by vitamin K epoxide reductase. This enzyme is inhibited by warfarin and related coumarin anticoagulants, which interrupts the synthesis of several clotting factors. Some drugs actually contain an epoxide, such as scopolamine and tiotropium. Epoxide hydrolase is one of several proteins (so-called preneoplastic antigens) that are overexpressed in chemically induced foci and nodules that eventually develop into liver tumors. Several alcohols, ketones, and imidazoles stimulate microsomal epoxide hydrolase activity in vitro. Epoxide hydrolase cannot be inhibited by antibodies raised against the purified enzyme, but it can be inhibited by certain epoxides, such as 1,1,1-trichloropropene oxide and cyclohexene oxide, and certain drugs, such as valpromide (the amide analog of valproic acid) and progabide, a γ -aminobutyric acid (GABA) agonist. These latter two drugs potentiate the neurotoxicity of carbamazepine by inhibiting epoxide hydrolase, leading to increased plasma levels of carbamazepine 10,11-epoxide and presumably the more toxic 2,3-epoxide (Kroetz et al., 1993). Several genetic polymorphisms have been identified in the coding region and the 5 region (i.e., the regulatory region) of the gene encoding human mEH (Daly, 1999). Two variants involve substitutions at amino acid 113 (Tyr → His) or amino acid 139 (His → Arg), which are encoded by exons 3 and 4, respectively. Although these allelic variant forms of mEH have near-normal enzymatic activity (at least 65% of normal), they appear to be less stable than the wild-type enzyme. The possibility that these amino acid substitutions might predispose individuals to the adverse effects of antiepileptic drugs has been examined, but no such association was found (Daly, 1999). More recently, a case was reported of a man who had a defect in mEH expression and suffered acute and severe phenytoin toxicity (Morisseau and Hammock, 2005). The microsomal and soluble forms of epoxide hydrolase show no evident sequence identity and, accordingly, are immunochemically distinct proteins (Beetham et al., 1995). Nevertheless, mEH and sEH catalyze reactions by the same mechanism, and similar amino acids are involved in catalysis, namely, a nucleophilic acid (Asp226 in mEH and Asp334 in sEH), a basic histidine (His431 in mEH and His523 in sEH), an orienting acid (Glu404 in mEH and Asp495 in sEH) and polarizing tyrosine residues (Tyr299 and Tyr374 in mEH and Tyr382 and Tyr465 in mEH) (Morisseau and Hammock, 2005). The mechanism of catalysis by epoxide hydrolase is similar to that of carboxylesterase, in that the catalytic site comprises

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

183

Bay region 12 10

1 2

11

3

9 8 6

P450 PHS

4 7

O

Epoxide hydrolase

P4 50

5

O

HO

HO OH

OH benzo[a]pyrene

(+) benzo[a]pyrene 7,8-oxide

(–) benzo[a]pyrene 7,8-dihydrodiol

P450 AKRs

Dihydrodiol dehydrogenase

Resistant to hydrolyation by epoxide hydrolase

Covalent binding to DNA

HO

O

(+) benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide

OH

benzo[a]pyrene 4,5-oxide

catechol Mutation of the 12th codon of the Hras oncogene

Epoxide hydrolase

oxidation

reduction

Lung and skin tumors

OH OH benzo[a]pyrene 4,5-dihydrodiol

O O ortho-quinone

Figure 6-9. Role of epoxide hydrolase in the inactivation of benzo[a]pyrene 4,5-oxide and in the conversion of benzo[a]pyrene to its tumorigenic bayregion diolepoxide. Also shown is the role of dihydrodiol dehydrogenase, a member of the aldo–keto reductase (AKR) superfamily, in the formation of reactive catechol and ortho-quinone metabolites of benzo[a]pyrene.

three amino acid residues that form a catalytic triad, as shown in Fig. 6-6. The attack of the nucleophile Asp226 on the carbon of the oxirane ring initiates enzymatic activity, leading to the formation of an α-hydroxyester–enzyme intermediate, with the negative charge developing on the oxygen atom stabilized by a putative oxyanion hole. The His431 residue (which is activated by Glu376 and Glu404 ) activates a water molecule by abstracting a proton (H+ ). The activated (nucleophilic) water then attacks the Cγ atom of Asp226 , resulting in the hydrolysis of the ester bond in the acyl–enzyme intermediate, which restores the active enzyme and results in formation of a vicinal diol with a trans-configuration (Armstrong, 1999). The second step, namely, cleavage of the ester bond in the acyl–enzyme intermediate, resembles the cleavage of the ester or amide bond in substrates for serine esterases and proteases. Although epoxide hydrolase and carboxylesterase both have a catalytic triad comprising a nucleophilic, basic, and acidic amino

acid residue, there are striking differences in their catalytic machinery, which account for the fact that carboxylesterases primarily hydrolyze esters and amides, whereas epoxide hydrolases primarily hydrolyze epoxides and oxides. In the triad, both enzymes have histidine as the base and either glutamate or aspartate as the acid, but they differ in the type of amino acids for the nucleophile. Even during catalysis, there is a major difference. In carboxylesterases, the same carbonyl carbon atom of the substrate is attacked initially by the nucleophile Ser203 to form an α-hydroxyester-enzyme ester that is subsequently attacked by the activated water to release the alcohol product. In contrast, two different atoms in epoxide hydrolase are targets of nucleophilic attacks. First the less hindered carbon atom of the oxirane ring is attacked by the nucleophile Asp226 to form a covalently bound ester, and next this ester is hydrolyzed by an activated water molecule that attacks the Cγ atom of the Asp226 residue, as illustrated in Fig. 6-6. Therefore, in carboxylesterase,

184

UNIT 2

DISPOSITION OF TOXICANTS

Azo-reduction

[4H] H2N

N

N

SO2NH2

H 2N

NH2

NH2

+

H2N

SO2NH2

NH2 Prontosil

1,2,4-Triaminobenzene

Sulfanilamide

Nitro-reduction

O

OH O2N

CH

CH

NH

C

CHCl2

[6H]

H2N

CH

CH2OH

Nitrobenzene

NHOH [2H]

Nitrosobenzene

NH

C

CHCl2

Arylamine metabolite

NO [2H]

CH

CH2OH

Chloramphenicol

NO2

O

OH

NH2 [2 H ]

Phenylhydroxylamine

Aniline

Figure 6-10. Examples of drugs that undergo azo reduction (prontosil) and nitro reduction (chloramphenicol and nitrobenzene).

the oxygen introduced to the product is derived from the activated water molecule. In contrast, in epoxide hydrolase, the oxygen introduced to the product is derived from the nucleophile Asp226 (Fig. 6-6). Carboxylesterases and epoxide hydrolases exhibit no primary sequence identity, but they share surprising similarities in the topology of the structure and sequential arrangement of the catalytic triad. Both are members of the α/β-hydrolase fold enzymes, a superfamily of proteins that include lipases, esterases, and haloalkane dehydrogenases (Beetham et al., 1995). Functionally, proteins in this superfamily all catalyze hydrolytic reactions; structurally, they all contain a similar core segment that is composed of eight β-sheets connected by α-helices. They all have a catalytic triad and the arrangement of the amino acid residues in the triad (i.e., the order of the nucleophile, the acid, and the base in the primary sequence) is the mirror image of the arrangement in other hydrolytic enzymes such as trypsin. All three active-site residues are located on loops that are the best conserved structural features in the fold, which likely provides catalysis with certain flexibility to hydrolyze numerous structurally distinct substrates.

Reduction Certain metals (e.g., pentavalent arsenic) and xenobiotics containing an aldehyde, ketone, alkene, disulfide, sulfoxide, quinone, N oxide, hydroxamic acid, amidoxime, isoxazole, isothiazole, azo, or nitro group are often reduced in vivo, although it is sometimes difficult to ascertain whether the reaction proceeds enzymatically or

nonenzymatically by interaction with reducing agents (such as the reduced forms of glutathione, FAD, FMN, and NAD[P]). Some of these functional groups can be either reduced or oxidized. For example, aldehydes (R–CHO) can be reduced to an alcohol (R–CH2 OH) or oxidized to a carboxylic acid (R–COOH), whereas sulfoxides (R1 –SO–R2 ) can be reduced to a sulfide (R1 –S–R2 ) or oxidized to a sulfone (R1 –SO2 –R2 ). Likewise, some enzymes, such as alcohol dehydrogenase, aldehyde oxidase, and cytochrome P450, can catalyze both reductive and oxidative reactions depending on the substrate or conditions. In the case of halogenated hydrocarbons, such as halothane, dehalogenation can proceed by an oxidative or reductive pathway, both of which are catalyzed by the same enzyme (namely, cytochrome P450). In some cases, such as azo-reduction, nitro-reduction, and the reduction of certain alkenes, the reaction is largely catalyzed by intestinal microflora.

Azo- and Nitro-Reduction Prontosil and chloramphenicol are examples of drugs that undergo azo- and nitro-reduction, respectively, as shown in Fig. 6-10. Reduction of prontosil is of historical interest. Treatment of streptococcal and pneumococcal infections with prontosil marked the beginning of specific antibacterial chemotherapy. Subsequently, it was discovered that the active drug was not prontosil but its metabolite, sulfanilamide (para-aminobenzene sulfonamide), a product of azo-reduction. During azo-reduction, the nitrogen–nitrogen double bond is sequentially reduced and cleaved to produce two primary amines, a reaction requiring four reducing equivalents. Nitro-reduction requires six reducing equivalents,

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

CH2OH

LIVER

CH3 O2N

O2N

NO2

INTESTINE

O2N

NO2

CH2OH O2N

NO2

UGT

2,6-Dinitrobenzylalcohol

CH2O-glucuronide NH2

O2N

NO2

nitroreductase

-Glucuronidase

2,6-Dinitrobenzylalcohol glucuronide

CH2OH

CH2OH

LIVER

CH2O-glucuronide

P450

2,6-Dinitrotoluene

185

O2N

O2 N

NH2

CH2OH NHOH

O2N

NHOX

P450 Acetylation sulfation 2-Amino-6-nitrobenzylalcohol

– XO

Covalent binding to protein and DNA

X = COCH3 SO3H

Figure 6-11. Role of nitro reduction by intestinal microflora in the activation of the rat liver tumorigen, 2,6-dinitrotoluene.

which are consumed in three sequential reactions, as shown in Fig. 6-10 for the conversion of nitrobenzene to aniline. Azo- and nitro-reduction reactions are generally catalyzed by intestinal microflora. However, under certain conditions, such as low oxygen tension, the reactions can be catalyzed by liver microsomal cytochrome P450 and NAD(P)H-quinone oxidoreductase (NQO1, a cytosolic flavoprotein that is also known as DT-diaphorase) and, in the case of nitroaromatics, by cytosolic aldehyde oxidase. The anaerobic environment of the lower gastrointestinal tract is well suited for azo- and nitro-reduction, which is why intestinal microflora contribute significantly to these reactions. The reduction of quinic acid to benzoic acid is another example of a reductive reaction catalyzed by gut microflora, as shown in Fig. 6-1. Nitro-reduction by intestinal microflora is thought to play an important role in the toxicity of several nitroaromatic compounds including 2,6-dinitrotoluene, which is hepatotumorigenic to male rats. The role of nitro-reduction in the metabolic activation of 2,6dinitrotoluene is shown in Fig. 6-11 (Long and Rickert, 1982; Mirsalis and Butterworth, 1982). The biotransformation of 2,6dinitrotoluene begins in the liver, where it is oxidized by cytochrome P450 and conjugated with glucuronic acid. This glucuronide is excreted in bile and undergoes biotransformation by intestinal microflora. One or both of the nitro groups are reduced to amines by nitroreductase, and the glucuronide is hydrolyzed by β-glucuronidase. The deconjugated metabolites are absorbed and transported to the liver, where the newly formed amine group is

N -hydroxylated by cytochrome P450 and conjugated with acetate or sulfonate. These conjugates form good leaving groups, which renders the nitrogen highly susceptible to nucleophilic attack from proteins and DNA; this ostensibly leads to mutations and the formation of liver tumors. The complexity of the metabolic scheme shown in Fig. 6-11 underscores an important principle, namely, that the activation of some chemical tumorigens to DNA-reactive metabolites involves several different biotransforming enzymes and may take place in more than one tissue. Consequently, the ability of 2,6dinitrotoluene to bind to DNA and cause mutations is not revealed in most of the short-term assays for assessing the genotoxic potential of chemical agents. These in vitro assays for genotoxicity do not make allowance for biotransformation by intestinal microflora or, in some cases, the conjugating enzymes. Nitro-reduction by intestinal microflora also plays an important role in the biotransformation of musk xylene (1,3,5-trinitro-2tbutyl-4,6-dimethylbenzene). Reduction of one or both of the nitro groups is required for musk xylene to induce (as well as markedly inhibit) liver microsomal cytochrome P450 (namely, CYP2B) in rodents (Lehman-McKeeman et al., 1999).

Carbonyl Reduction—SDRs and AKRs A variety of xenobiotics contain a carbonyl function (R–CHO and R1 –CO–R2 ) that undergoes reduction in vivo. The reduction of aldehydes to primary alcohols and of ketones to secondary alcohols is generally

186

UNIT 2

DISPOSITION OF TOXICANTS

A

Cl

B OH Cl3C

O F

(CH2)3

C

CH OH

N OH

Chloral hydrate

Haloperidol – H 2O

O

Carbonyl reductase Cl

Cl3C

C H

OH F

C

Alcohol dehydrogenase (CH2)3

N OH

H Cl3C

CH2OH

Reduced haloperidol Trichloroethanol

Figure 6-12. Reduction of xenobiotics by carbonyl reductase (A) and alcohol dehydrogenase (B).

catalyzed in mammals by NAD(P)H-dependent reductases belonging to one of two superfamilies, the aldo–keto reductases (AKRs), and the short-chain dehydrogenases/reductases (SDRs), as shown in Table 6-5 (Jez and Penning, 2001; Oppermann et al., 2001; Matsunaga et al., 2006). Humans contain 39 SDR members, two of which, namely, cytosolic and microsomal carbonyl reductase, play a role in the reduction of a wide variety of carbonyl-containing xenobiotics (other species express more than two carbonyl reductases). Erythrocytes also contain carbonyl reductase, which contributes significantly to the reduction of haloperidol, as shown in Fig. 6-12. From the alternative names given in Table 6-5, it is apparent that the cytosolic and microsomal carbonyl reductases have both been studied for their role in endobiotic metabolism, namely, the reduction of prostaglandin derivatives and 11β-hydroxysteroids, respectively. The AKRs are members of a superfamily of cytosolic enzymes that reduce both xenobiotic and endobiotic compounds, as their alternative names imply (Table 6-5). Various members of the AKR superfamily can function as dihydrodiol dehydrogenases and oxidize the trans-dihydrodiols of various polycyclic aromatic hydrocarbon oxiranes (formed by epoxide hydrolase) to the corresponding ortho-quinones, as shown previously in Fig. 6-9. The role of AKR as an oxidizing enzyme is discussed in the section on oxidative enzymes (see section “Dihydrodiol Dehydrogenase”). As mentioned earlier (see Point 8), one of the AKRs, namely, AKR7A (also known as aflatoxin aldehyde reductase) is one of the many enzymes induced following activation of Nrf2 by oxidative stress, exposure to electrophiles, or depletion of glutathione. In certain cases, the reduction of aldehydes to alcohols can be catalyzed by alcohol dehydrogenase, as shown in Fig. 6-12

for the conversion of the sedative-hypnotic, chloral hydrate, to trichloroethanol. As shown in Table 6-5, alcohol dehydrogenases belong to the medium chain dehydrogenases/reductases (MDRs). They typically convert alcohols to aldehydes, for which reason they are discussed later in the section on oxidative reactions (see section “Alcohol Dehydrogenases”). In the case of chloral hydrate, the reverse reaction is favored by the presence of the trichloromethyl group, which is a strong electron-withdrawing group. The SDR carbonyl reductases are monomeric, NADPHdependent enzymes present in erythrocytes and both the cytosolic and microsomal fraction of the liver, kidney, brain, and many other tissues. The major circulating metabolite of the antipsychotic drug, haloperidol, is a secondary alcohol formed by carbonyl reductases in the blood and liver, as shown in Fig. 6-12. Other xenobiotics that are reduced by carbonyl reductases include pentoxifylline (see Fig. 6-2), acetohexamide, daunorubicin, doxorubicin, loxoprofen, menadione, 4-nitroacetophenone, timiperone, and R-warfarin (Rosemond and Walsh, 2004). As shown in Fig. 6-2, the reduction of ketones to secondary alcohols by carbonyl reductases may proceed with a high degree of stereoselectivity, as in the case of pentoxifylline (Lillibridge et al., 1996). Liver cytosol and microsomes contain different forms of carbonyl reductase, and these can differ in the degree to which they stereoselectively reduce ketones to secondary alcohols. For example, keto-reduction of pentoxifylline produces two enantiomeric secondary alcohols: one with the R-configuration (which is known as lisofylline) and one with the S-configuration, as shown in Fig. 6-2. Reduction of pentoxifylline by cytosolic carbonyl reductase results in the stereospecific formation of the optical antipode of lisofylline, whereas the same reaction catalyzed by microsomal

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

187

Table 6-5 Human Aldo–Keto Reductases (AKRs), Short-Chain Dehydrogenases/Reductases (SDRs), Medium-Chain Dehydrogenases/Reductases (MDRs), and Quinone Reductases (NQOs) enzyme superfamily

example enzymes

alternative names

subcellular localization

Aldo–keto reductase (AKR) 13 enzymes

AKR1A1 AKR1B1 AKR1B10 AKR1C1 AKR1C1, 1C2, 1C4 AKR1C1, 1C2, 1C3, 1C4 AKR1C3 AKR1C4 AKR1D1 AKR6A3, 6A5, 6A9 AKR7A2 AKR7A3

Aldehyde reductase Aldose reductase Small intestine reductase 20α-Hydroxysteroid dehydrogenase Dihydrodiol dehydrogenases (DD1, DD2, DD4) 3α-Hydroxysteroid dehydrogenase 17β-Hydroxysteroid dehydrogenase type V Chlordecone reductase 4 -3-Ketosteroid-5β-reductase Shaker-channel subunit Kvb1, Kvb2, Kvb3 Aflatoxin B1 aldehyde reductase 2 Aflatoxin B1 aldehyde reductase 3

Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Golgi Cytosol

Short chain dehydrogenase/ reductases (SDR) 39 enzymes

Cytosolic carbonyl reductase

Cytosol

Microsomal carbonyl reductase ADH1A ADH1B ADH1C ADH4 ADH5 ADH6 ADH7

Xenobiotic ketone reductase with pH 6.0 activity; Prostaglandin 9-ketoreductase; Human placental NADP-linked 15-hydroprostaglandin dehydrogenase 11β-Hydroxysteroid dehydrogenase; 11β-reductase, 11-oxidoreductase Class I ADH; ADH1, α, β, γ ; hADH1,2,3 Class I ADH; ADH2, β, γ ; ADHII; hADH4 Class I ADH; ADH3, γ Class II ADH; π , hADH7 Class III ADH; χ Class V ADH Class IV ADH; μ or σ

Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol

NQO1 NQO2

DT diaphorase, menadione reductase N -Ribosyldihydronicotinamide dehydrogenase

Cytosol Cytosol

Medium chain dehydrogenase/ reductases (MDR) 7 ADH enzymes

NQO 2 enzymes

Microsomes

NQO, NADPH-Quinone oxidoreducta2e.

carbonyl reductase produces both lisofylline and its optical antipode in a ratio of about 1 to 5 (Lillibridge et al., 1996). In rat liver cytosol, the reduction of quinones is primarily catalyzed by NQO1 and NQO2 (see section “Quinone Reduction— NQO1 and NQO2”), whereas in human liver cytosol, quinone reduction is catalyzed by both NQO and carbonyl reductases. Various members of the AKR superfamily have been implicated in the reduction of such carbonyl-containing xenobiotics as the tobacco-specific nitrosamine NNK, acetohexamide, daunorubicin, naloxone, naltrexone, befunolol, ethacrynic acid, ketoprofen, ketotifen, haloperidol, loxoprofen, metyrapone, oxo-nortyptyline, and numerous aromatic and aliphatic aldehydes (Rosemond and Walsh, 2004). Many of the xenobiotics reduced by AKRs and also reduced by SDRs, and in most cases the relative contribution of individual carbonyl-reducing enzymes is not known. Genetic polymorphisms of AKRs or SDRs have not been shown to impact the disposition or safety of carbonyl-containing drugs, and there appear to be no reports of drug–drug interactions involving the inhibition or induction of AKRs or SDRs (Rosemond and Walsh, 2004).

Disulfide Reduction Some disulfides are reduced and cleaved to their sulfhydryl components, as shown in Fig. 6-13 for the alcohol

deterrent, disulfiram (Antabuse). As shown in Fig. 6-13, disulfide reduction by glutathione is a three-step process, the last of which is catalyzed by glutathione reductase. The first steps can be catalyzed by glutathione transferase, or they can occur nonenzymatically.

Sulfoxide and N-Oxide Reduction Thioredoxin-dependent enzymes in liver and kidney cytosol have been reported to reduce sulfoxides, which themselves may be formed by cytochrome P450 or flavin monooxygenases (Anders et al., 1981). It has been suggested that recycling through these counteracting enzyme systems may prolong the half-life of certain xenobiotics. Sulindac is a sulfoxide that undergoes reduction to a sulfide, which is excreted in bile and reabsorbed from the intestine (Ratnayake et al., 1981). This enterohepatic cycling prolongs the duration of action of the drug such that this nonsteroidal anti-inflammatory drug (NSAID) need only be taken twice daily. Sulfoxide reduction may also occur nonenzymatically at an appreciable rate, as in the case of the proton pump inhibitor rabeprazole (Miura et al., 2006). Diethyldithiocarbamate methyl ester, a metabolite of disulfiram, is oxidized to a sulfine, which is reduced to the parent methyl ester by glutathione. In the latter reaction, two molecules of glutathione (GSH) are oxidized with reduction

188

UNIT 2

A

S

H5C2 N

S

C

S

S

DISPOSITION OF TOXICANTS

C2H5

C

N

H5C2

C2H5 Disulfiram (Antabuse)

S

H5C2 2×

N

C

SH

H5C2 Diethyldithiocarbamate

B GSH

XSH

XSSG

XSSX

GSH NADP

2GSH

NADPH + H+

XSH

GSSG

Figure 6-13. Biotransformation of disulfiram by disulfide reduction (A) and the general mechanism of glutathione-dependent disulfide reduction of xenobiotics (B). GSH, glutathione; XSSX, xenobiotic disulfide; GSSG, reduced glutathione. The last reaction in (B) is catalyzed by glutathione reductase.

of the sulfine oxygen to water (Madan et al., 1994), as shown below: R1 R2 C = S+ –O− + 2GSH → R1 R2 C = S + GSSG + H2 O Just as sulfoxide reduction can reverse the effect of sulfoxidation, so the reduction of N -oxides can reverse the N -oxygenation of amines, which is catalyzed by flavin monooxygenases and cytochrome P450. Under reduced oxygen tension, reduction of the N oxides of imipramine, tiaramide, indicine, and N ,N -dimethylaniline can be catalyzed by mitochondrial and/or microsomal enzymes in the presence of NADH or NADPH (Sugiura and Kato, 1977). The NADPH-dependent reduction of N -oxides in liver microsomes appears to be catalyzed by cytochrome P450 (Sugiura et al., 1976), although in some cases NADPH-cytochrome P450 reductase may play an important role. As a class, N -oxides are not inherently toxic compounds. However, certain aromatic and aliphatic N -oxides have been exploited as bioreductive drugs (also known as DNA affinic drugs) for the treat-

ment of certain cancers and infectious diseases (Wardman et al., 1995). In these cases, N -oxides have been used as prodrugs that are converted to cytotoxic or DNA-binding drugs under hypoxic conditions. The fact that N -oxides of certain drugs are converted to toxic metabolites under hypoxic conditions is the basis for their selective toxicity to certain solid tumors (namely, those that are hypoxic and, hence, resistant to radiotherapy) and anaerobic bacteria. For example, tirapazamine (SR 4233) is a benzotriazine di-N -oxide that is preferentially toxic to hypoxic cells, such as those present in solid tumors, apparently due to its rapid activation by one-electron reduction of the N -oxide to an oxidizing nitroxide radical, as shown in Fig. 6-14 (Walton et al., 1992). This reaction is catalyzed by cytochrome P450 and NADPH-cytochrome P450 reductase (Saunders et al., 2000). Two-electron reduction of the di-N -oxide, SR 4233, produces a mono-N -oxide, SR 4317, which undergoes a second N oxide reduction to SR 4330. Like SR 4233, the antibacterial agent, quindoxin, is a di-N -oxide whose cytotoxicity is dependent on reductive activation, which is favored by anaerobic conditions. Bioreductive alkylating agents, which include such drugs as mitomycins, anthracyclins, and aziridinylbenzoquinones represent another class of anticancer agents that require activation by reduction. However, for this class of agents, bioactivation also involves a two-electron reduction reaction, which is largely catalyzed by NQO, which is described in the next section. Quinone Reduction—NQO1 and NQO2 Quinones can be reduced to hydroquinones by two closely related, cytosolic flavoproteins, namely, NQO1 and NQO2. The former enzyme, NAD(P)Hquinone oxidoreductase-1, is also known as DT-diaphorase. The latter enzyme, NAD(P)H-quinone oxidoreductase-2, is also known as NRH-quinone oxidoreductase because it prefers the unusual electron donor dihydronicotinamide riboside (NMR) over NAD(P)H. Although they are closely related enzymes (both contain two 27kDa subunits each with an FAD prosthetic group), NQO1 and NQO2 have different substrate specificities, and they can be distinguished on the basis of their differential inhibition by dicoumarol and quercetin (which are selective inhibitors of NQO1 and NQO2, respectively). NQO2 may have a physiological role in the metabolism of vitamin K hydroquinone (Chen et al., 2000). An example of the type of reaction catalyzed by NQO is shown in Fig. 6-15. Formation of the hydroquinone involves a two-electron reduction of the quinone with stoichiometric oxidation of NAD[P]H without oxygen consumption. (The two-electron reduction of certain quinones can also be catalyzed by carbonyl reductase, especially in humans.) In contrast, NADPH-cytochrome P450 reductase, a microsomal flavoprotein, catalyzes the one-electron reduction of quinones to semiquinone radicals that, in addition to being reactive metabolites themselves, cause oxidative stress by reacting with oxygen to form reactive oxygen species (ROS), which leads to nonstoichiometric oxidation of NADPH and oxygen consumption, as shown in Fig. 6-15. The two-electron reduction of quinones is a nontoxic reaction—one that is not associated with semiquinone formation and oxidative stress—provided the resultant hydroquinone is sufficiently stable to undergo glucuronidation or sulfonation. However, there are quinone-containing xenobiotics that, despite undergoing two-electron reduction by NQO, produce semiquinone free radicals, oxidative stress, DNA damage, and cytotoxicity. Many of these xenobiotics are being developed as anticancer drugs because NQO1 is often overexpressed in tumor cells. The properties of the hydroquinone determine whether, during the metabolism of quinonecontaining xenobiotics, NQO functions as a protective antioxidant

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

189

Sulfoxide reduction O

2H

H 2O

S CH3

CH

CH

CH3

S CH3 CH3

F

F CH2COOH

CH2COOH

[O ]

Sulindac sulfide

Sulindac Note: The sulfoxide is a chiral center, hence, sulindac is a racemic mixture

N-oxide reduction O N

O

N

2e –

N

N

NH2

O Tirapazamine (SR 4233) 1e –

N

2e –

N

NH2

SR 4317

N

N

N

NH2

SR 4330

Disproportionation

Nitroxide radical

Toxicity to hypoxic cells, such as those in solid tumors

Figure 6-14. Examples of sulfoxide and N-oxide reduction. Note that tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a representative of a class of agents that are activated by reduction, which may be clinically useful in the treatment of certain tumors.

or a pro-oxidant activator leading to the formation of reactive oxygen species and reactive semiquinone free radicals. The latter are thought to form not from the one-electron reduction of the quinone but from the two-electron reduction of the quinone (Q) to the hydroquinone (QH2 ), which then undergoes one-electron oxidation or perhaps disproportionation to form the reactive semiquinone (QH): QH2 + Q ↔ 2QH Drugs or drug candidates that are activated by NQO to anticancer agents include the aziridinylbenzoquinone diaziquone, the anthraquinone mitoxantrone, the indolquinones mitomycin C and EO9 (an analog of mitomycin C that is more rapidly reduced by NQO1), and the anthracycline antibiotics daunorubicin and doxorubicin (Gutierrez, 2000). These so-called bioreductive alkylating agents are reduced by NQO1 to generate semiquinone free radicals and other reactive intermediates that undergo nucleophilic additions with DNA, resulting in single-strand DNA breaks. The reason such drugs are preferentially toxic to tumor cells is that tumor cells, especially those in solid tumors, are hypoxic, and hypoxia induces the synthesis of NQO1 (by a mechanism that involves the Activator Protein 1 [AP-1] and Nuclear Factor-κB [NF-κB] response elements in the 5 -promoter region of the NQO1 gene). Therefore, tumor cells often express high levels of NQO1, which predisposes them to the toxic effects of quinone-reductive anticancer drugs like mitomycin C. Interestingly, mitomycin C also upregulates the expression of NQO1, which may enable this anticancer

drug to stimulate its own metabolic activation in tumor cells (Yao et al., 1997). Some cancer chemotherapeutic agents, such as the N oxide SR 4233, are inactivated, not activated, by NQO, as shown in Fig. 6-14. NQO can activate certain nitroaromatic compounds (R–NO2 ) to the corresponding hydroxylamine (R–NHOH), which can be activated by acetylation or sulfonation (by pathways analogous to those shown in Fig. 6-11). Dinitropyrenes and the nitroaromatic compound CB 1954 are activated by NQO. The latter compound was under consideration as an anticancer agent. However, although it is activated by reduction by rat NQO, the nitroaromatic compound CB 1954 is not activated by human NQO. Oxidative stress appears to be an important component to the mechanism of toxicity of several xenobiotics that either contain a quinone or can be biotransformed to a quinone (Anders, 1985). The production of superoxide anion radicals and oxidative stress are responsible, at least in part, for the cardiotoxic effects of doxorubicin (adriamycin) and daunorubicin (daunomycin), the pulmonary toxicity of paraquat and nitrofurantoin, and the neurotoxic effects of 6-hydroxydopamine. Oxidative stress also plays an important role in the destruction of pancreatic beta cells by alloxan and dialuric acid. Tissues low in superoxide dismutase activity, such as the heart, are especially susceptible to the oxidative stress associated with the redox cycling of quinones. This accounts, at least in part, for the cardiotoxic effects of adriamycin and related anticancer agents, although other susceptibility factors have been proposed (Mordente et al., 2001).

190

UNIT 2

DISPOSITION OF TOXICANTS

2H , 2e

O

OH

CH 3

CH3 DT-diaphorase (NADPH-quinone oxidoreductase)

O

OH

Menadione

Hydroquinone

H,e NADPHcytochrome P450 reductase

O2

Superoxide anion

O CH3

HO 2 Perhydroxyl radical

H 2 O 2 Hydrogen peroxide OH Semiquinone radical HO

Damage to proteins and DNA

Reactive oxygen species

O2

Hydroxyl radical

Lipid peroxidation

Figure 6-15. Two-electron reduction of menadione to a hydroquinone, and production of reactive oxygen species during its one-electron reduction to a semiquinone radical.

As already mentioned in this section, it is now apparent that the structure of the hydroquinones produced by NQO determines whether the two-electron reduction of quinones results in xenobiotic detoxication or activation. Hydroquinones formed by two-electron reduction of unsubstituted or methyl-substituted 1,4naphthoquinones (such as menadione) or the corresponding quinone epoxides are relatively stable to autoxidation, whereas the methoxyl, glutathionyl, and hydroxyl derivatives of these compounds undergo autoxidation with production of semiquinones and reactive oxygen species. The ability of glutathionyl derivatives to undergo redox cycling indicates that conjugation with glutathione does not prevent quinones from serving as substrates for NQO. The glutathione conjugates of quinones can also be reduced to hydroquinones by carbonyl reductases, which actually have a binding site for glutathione. In human carbonyl reductase, this binding site is Cys227 , which is involved in binding both substrate and glutathione (Tinguely and Wermuth, 1999). Although oxidative stress is an important mechanism by which quinones cause cellular damage (through the intermediacy of semiquinone radicals and the generation of reactive oxygen species), it should be noted that quinones are Michael acceptors, and cellular damage can occur through direct alkylation of critical cellular proteins and/or DNA (Bolton et al., 2000). NQO1 is inducible up to tenfold by two classes of inducers, which have been categorized as bifunctional and monofunctional inducers (Prochaska and Talalay, 1988). The bifunctional inducers include compounds like β-naphthoflavone, benzo[a]pyrene, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD or dioxin), which induce both oxidative enzymes (such

as the cytochrome P450 enzyme CYP1A1) and conjugating enzymes (such as glutathione transferase and UDP-glucuronosyltransferase). The monofunctional inducers tend to induce conjugating and other non-CYP enzymes (although in mice, monofunctional inducers can induce CYP2C55 and 2U1, as well as aldehyde oxidase). These inducers signal through two distinct mechanisms, one involving the XRE (xenobiotic-response element) and one involving the ARE (antioxidant response element), which is also known as the EpRE (electrophilic response element). (Response elements are short sequences of DNA, often located in the 5 -promoter region of a gene, that bind the transcription factors that control gene expression.) Some enzymes, such as CYP1A1, are largely regulated by the XRE, whereas others are largely regulated by ARE, such as glutathione transferase (see Point 8). Some enzymes, such as NQO1, are regulated by both. The monofunctional inducers can be subdivided into two chemical classes: those that cause oxidative stress through redox cycling (e.g., the quinone, menadione, and the phenolic antioxidants tert-butylhydroquinone and 3,5-di-tert-butylcatechol), and those that cause oxidative stress by depleting glutathione (e.g., fumarates, maleates, acrylates, isothiocyanates, and other Michael acceptors that react with glutathione). The flavonoid β-naphthoflavone and the polycyclic aromatic hydrocarbon benzo[a]pyrene induce NQO1 by both mechanisms; the parent compound binds to the Ah receptor and is responsible for inducing CYP1A1, as well as NQO1, via the XRE, whereas electrophilic and/or redox active metabolites of β-naphthoflavone and benzo[a]pyrene are responsible for inducing glutathione

O

O

O

Br

Br Br

HN HOCH2 O

HN

Gut flora N

O

HN N H

O HO

O NADPH + H+

N H

NADP +

BVU

Dihydro-BVU O

OH

DPD

HBr

Sorivudine

Dihydropyrimidine dehydrogenase (DPD)

HN O

N H

Inactivated DPD O

O F

HN O

F

O

in liver

O NADP +

O

N H

5-Fluorouracil (5-FU)

HOOC

F HN

HN

P450 N

NADP H + H+

O

N H

5-Fluorodihydrouracil

H 2N

α-Fluoro-β-alanine

Tegafur

Increase in 5-FU levels when DPD is inactivated

F

– NH3 – CO2

Toxicity to bone marrow and intestines. May be fatal

Figure 6-16. Reduction of 5-fluorouracil by dihydropyrimidine dehydrogenase and its inhibition (suicide inactivation) by Sorivudine.

Note: Inhibition of dihydropyrimidine dehydrogenase is the mechanism of fatal interactions between Sorivudine and the 5-fluorouracil prodrug, tegafur.

192

UNIT 2

DISPOSITION OF TOXICANTS

transferase, as well as NQO1, via the ARE. The situation with benzo[a]pyrene is quite intriguing. This polycyclic aromatic hydrocarbon binds directly to the Ah receptor, which binds to the XRE and induces the synthesis of CYP1A1, which in turn converts benzo[a]pyrene to electrophilic metabolites (such as arene oxides and diolepoxides) and redox active metabolites (such as catechols), as shown in Fig. 6-9. These electrophilic and redox active metabolites then induce enzymes that are regulated by the ARE. However, the catechol metabolites of benzo[a]pyrene are further converted by dihydrodiol dehydrogenase to ortho-quinones (Fig. 6-9), and are thereby converted back into planar, hydrophobic compounds that are highly effective ligands for the Ah receptor (Burczynski and Penning, 2000). This may be toxicologically important, because the Ah receptor may translocate ortho-quinone metabolites of benzo[a]pyrene into the nucleus, where they might damage DNA (Bolton et al., 2000). Among the monofunctional inducers that apparently increase NQO1 via ARE is sulforaphane, an ingredient of broccoli that may be responsible for the anticarcinogenic effects of this cruciferous vegetable (Zhang et al., 1992). Isothiocyanates (which are also present at high levels in cruciferous vegetables) likely exert their chemopreventive effects largely through induction of the detoxifying enzymes under the control of ARE, namely, glutathione transferase (GSTA1), microsomal epoxide hydrolase, aldo– keto reductase (AKR7A, also known as aflatoxin aldehyde reductase), NAD(P)H-quinone oxidoreductase (NQO1, also known as DT-diaphorase), glutamate-cysteine ligase (GCL), as well as genes involved in apoptosis. One isothiocyanate in particular, phenethyl isothiocyanate, has been found to activate Nrf2 and activate numerous genes in addition to those encoding xenobioticbiotransforming enzymes and oxidant defense systems. Microarray studies carried out in wild-type and Nrf2 knockout mice treated with phenethylisothiocyanate showed that the most highly inducible genes include the very low density lipoprotein (VLDL) receptor, Gprotein signaling modulator 2, early growth response 1, pancreatic lipase-related protein 2, histocompatibility 2 (K region), general transcription factor IIB, myoglobin, potassium voltage-gated channel Q2, and SLC39A10 (Hu et al., 2006). As with other xenosensors, activation of Nrf2 results in a pleiotypic response in which a large number of genes are activated (or repressed). As mentioned above (this section), hypoxia and the anticancer agent mitomycin C are also inducers of NQO1, which has implications for cancer chemotherapy. NQO1 and NQO2 are polymorphically expressed enzymes, and several lines of evidence suggest that NQO1 and/or NQO2 plays a key role in protecting bone marrow from the hematotoxic effects of benzene or other environmental factors (Iskander and Jaiswal, 2005). In humans, a high percentage of individuals with myeloid and other types of leukemia are homo- or heterozygous for a null mutant allele of NQO1. This polymorphism, NQO1*2, is a SNP (C609 T) that changes Pro187 to Ser187 , which destabilizes the protein and targets it for rapid degradation by the ubiquitin proteasomal pathway (Ross, 2005). Mice lacking NQO1 or NQO2 (knockout or null mice) have no developmental abnormalities but have increased granulocytes in the blood and myelogenous hyperplasia of the bone marrow (due to decreased apoptosis). Mice lacking NQO1 are substantially more susceptible than wild-type mice to benzeneinduced hematotoxicity (Iskander and Jaiswal, 2005; Ross, 2005). The hematotoxicity of benzene is thought to involve its conversion to hydroquinone in the liver and its subsequent oxidation to benzoquinone by myeloperoxidase in the bone marrow (discussed later in

the section on Peroxidase-dependent cooxidation). NQO would be expected to play a role in detoxifying benzoquinone, and the loss of this protective mechanism may be the mechanism by which loss of NQO potentiates benzene hematotoxicity. However, loss of NQO also impairs apoptosis, which also represents a plausible explanation for the association between loss of NQO and increased susceptibility to benzene hematotoxicity. This latter mechanism (i.e., impaired apoptosis) likely accounts for the observation that NQO1 and NQO2 null mice are more susceptible than wild-type mice to skin carcinogenesis by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, an effect attributable to the diol-epoxides, not the quinone metabolites, of these polycyclic aromatic hydrocarbons (Iskander and Jaiswal, 2005). Dihydropyrimidine Dehydrogenase (DPD) In 1993, 15 Japanese patients died as a result of an interaction between two oral medications: Sorivudine—a new antiviral drug for herpes zoster— and Tegafur—a prodrug that is converted in the liver to the anticancer agent, 5-fluorouracil. The deaths occurred within 40 days of the Japanese government’s approval of Sorivudine for clinical

CCl4

e

Reductive dehalogenation

Cl

CCl3

O2

O

e

O

CCl3

RH Cl R

CCl2

HOOC

CHCl3

H2O

CCl3

Lipid peroxidation

P450 [O ]

2HCl HOCCl3 GSSG + H2O

CO Carbon monoxide

2GSH

HCl H2O

2HCl

O Cl

C Phosgene

Cl

CO2 Carbon dioxide

Figure 6-17. Reductive dehalogenation of carbon tetrachloride to a trichloromethyl free radical that initiates lipid peroxidation. RH, unsaturated lipid; R·, lipid dienyl radical; GSH, reduced glutathione; GSSG, oxidized glutathione.

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

Reductive dehalogenation

F

e

Br H,e

F

F

Cl

C

C

F

H

F

F

Cl

C

C

F

H

F

Cl

C

C

F

H

F C

e

F

P450

F

[O ]

H

F

F

Cl

C

C

F

O

Br

H

C H

HBr

2-chloro-1,1-difluoroethylene F C

F

OH F

C

F

O

trifluoroacetic acid

Liver damage in rats

Br

Cl

F

Lipid peroxidation Binding to protein

Oxidative dehalogenation

2-chloro-1,1,1-trifluoroethane

carbon-centered radical

193

C F

OH

Cl

Cl C O

trifluoroacetylchloride

Binding to protein (Neo-antigen formation)

Immune hepatitis in humans

Figure 6-18. Activation of halothane by reductive and oxidative dehalogenation and their role in liver toxicity in rats and humans.

use. The mechanism of the lethal interaction between Sorivudine and Tegafur is illustrated in Fig. 6-16, and involves inhibition of dihydropyrimidine dehydrogenase (DPD), an NADPH-requiring, homodimeric protein (Mr ∼210 kDa) containing FMN/FAD and an iron–sulfur cluster in each subunit. The enzyme is located mainly in liver cytosol, where it catalyzes the reduction of 5-fluorouracil and related pyrimidines. Sorivudine is converted in part by gut flora to (E)-5-(2-bromovinyl) uracil (BVU), which lacks antiviral activity but which is converted by DPD to a metabolite that binds covalently to the enzyme. The irreversible inactivation (a.k.a. suicidal inactivation) of DPD by Sorivudine causes a marked inhibition of 5-fluorouracil metabolism, which increases blood levels of 5fluorouracil to toxic and, in some cases, lethal levels (Ogura et al., 1998; Kanamitsu et al., 2000). Several genetic polymorphisms that result in a partial or complete loss of DPD activity have been described (van Kuilenburg et al., 2004; Robert et al., 2005). Severe 5-fluorouracil toxicity has also been documented in individuals who are heterozygous for allelic variants of DPD, and 5-fluorouracil lethality has been documented in rare individuals who are completely deficient in DPD (one individual in about 10,000). 5-Fluorouracil is one of the most frequently prescribed anticancer drugs, for which reason assessing an individual’s DPD genotype (by analyzing DNA for allelic variants) or phenotyping (by measuring DPD activity in peripheral blood

mononuclear cells or PBMCs) is advocated prior to 5-fluorouracil therapy so that the dosage of this anticancer drug can be adjusted on an individual basis.

Dehalogenation There are three major mechanisms for removing halogens (F, Cl, Br, and I) from aliphatic xenobiotics (Anders, 1985). The first, known as reductive dehalogenation, involves replacement of a halogen with hydrogen, as shown below: X

X

X C C X X

H

+2H – HX

Pentahaloethane

X X X C C H X H Tetrahaloethane

In the second mechanism, known as oxidative dehalogenation, a halogen and hydrogen on the same carbon atom are replaced with oxygen. Depending on the structure of the haloalkane, oxidative dehalogenation leads to the formation of an acylhalide or aldehyde, as shown below: X

X

X C C X X

H

Pentahaloethane

+[O] – HX

X

X

X C C O X Tetrahaloacetylhalide

194

UNIT 2

DISPOSITION OF TOXICANTS

A third mechanism of dehalogenation involves the elimination of two halogens on adjacent carbon atoms to form a carbon–carbon double bond, as shown below: X X X C C X X H Pentahaloethane

+2H – 2HX

X

X C C

X

H

Trihaloethylene

A variation on this third mechanism is dehydrohalogenation, in which a halogen and hydrogen on adjacent carbon atoms are eliminated to form a carbon–carbon double bond. Reductive and oxidative dehalogenation are both catalyzed by cytochrome P450. (The ability of cytochrome P450 to catalyze both reductive and oxidative reactions is explained later in the section on Cytochrome P450.) Dehalogenation reactions leading to double bond formation are catalyzed by cytochrome P450 and glutathione transferase. These reactions play an important role in the biotransformation and metabolic activation of several halogenated alkanes, as the following examples illustrate. The hepatotoxicity of carbon tetrachloride (CCl4 ) and several related halogenated alkanes is dependent on their biotransformation by reductive dehalogenation (Plaa, 2000). The first step in reductive dehalogenation is a one-electron reduction catalyzed by cytochrome P450, which produces a potentially toxic, carbon-centered radical and inorganic halide. In the case of CCl4 , reductive dechlorination produces a trichloromethyl radical ( rCCl3 ), which initiates lipid peroxidation and produces a variety of other metabolites, as shown in Fig. 6-17. Halothane can also be converted by reductive dehalogenation to a carbon-centered radical, as shown in Fig. 6-18. The mechanism is identical to that described for carbon tetrachloride, although in the case of halothane the radical is generated through loss of bromine, which is a better leaving group than chlorine. Figure 6-18 also shows that halothane can undergo oxidative dehalogenation, which involves oxygen insertion at the C–H bond to generate an unstable halohydrin (CF3 COHClBr) that decomposes to a reactive acylhalide (CF3 COCl), which can bind to cellular proteins (particularly to amine groups) or further decompose to trifluoroacetic acid (CF3 COOH). Both the oxidative and reductive pathways of halothane metabolism generate reactive intermediates capable of binding to proteins and other cellular macromolecules. The relative importance of these two pathways to halothane-induced hepatotoxicity appears to be species dependent. In rats, halothane-induced hepatotoxicity is promoted by those conditions favoring the reductive dehalogenation of halothane, such as moderate hypoxia (10–14% oxygen) plus treatment with the cytochrome P450 inducers, phenobarbital, and pregnenolone-16α-carbonitrile. In contrast to the situation in rats, halothane-induced hepatotoxicity in guinea pigs is largely the result of oxidative dehalogenation of halothane (Lunam et al., 1989). In guinea pigs, halothane hepatotoxicity is not enhanced by moderate hypoxia and is diminished by the use of deuterated halothane, which impedes the oxidative dehalogenation of halothane because the P450-dependent insertion of oxygen into a carbon–deuterium bond is energetically less favorable (and therefore slower) than inserting oxygen into a carbon–hydrogen bond. Halothane hepatitis in humans is a rare but severe form of liver necrosis associated with repeated exposure to this volatile anesthetic. In humans, as in guinea pigs, halothane hepatotoxicity

appears to result from the oxidative dehalogenation of halothane, as shown in Fig. 6-18. Serum samples from patients suffering from halothane hepatitis contain antibodies directed against neoantigens formed by the trifluoroacetylation of proteins. These antibodies have been used to identify which specific proteins in the endoplasmic reticulum are targets for trifluoroacetylation during the oxidative dehalogenation of halothane (Pohl et al., 1989). The concept that halothane is activated by cytochrome P450 to trifluoroacetylhalide, which binds covalently to proteins and elicits an immune response, has been extended to other volatile anesthetics, such as enflurane, methoxyflurane, and isoflurane. In other words, these halogenated aliphatic hydrocarbons, like halothane, may be converted to acylhalides that form immunogens by binding covalently to proteins. In addition to accounting for rare instances of enflurane hepatitis, this mechanism of hepatotoxicity can also account for reports of a cross-sensitization between enflurane and halothane, in which enflurane causes liver damage in patients previously exposed to halothane. One of the metabolites generated from the reductive dehalogenation of halothane is 2-chloro-1,1-difluoroethylene (Fig. 6-18). The formation of this metabolite involves the loss of two halogens

A X

X

CH2

CH2

X

GS

GS

CH2

CH2

X

RS

CH 2

CH2 + GSSR

X

B X

X

CH 2

CH2

X

GS

GS

X + CH 2

CH 2

RS

CH 2

CH 2 + GSSR

X

Figure 6-19. Glutathione-dependent dehalogenation of 1,2-dihaloethane to ethylene. (A) Nucleophilic attack on carbon and (B) nucleophilic attack on halide.

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

195

H Cl

C

Cl

Cl

C

Cl

CCl 2

CCl 3 HCl

DDT

DDE

Figure 6-20. Dehydrochlorination of the pesticide DDT to DDE, a glutathione-dependent reaction.

NH 2

H 2N

O

N

H 3C

O

O

N

CH 3

DB289

O HO

O-Demethylation (CYP4F Enzymes)

O N

S

F

N O H

CP-544,439 NH 2

H 2N

O

N

H 3C

O

Dehydroxylation (Aldehyde oxidase)

OH

N

Amidoxime metabolite O

O N

Dehydroxylation (Cytochrome b5)

H 2N

S

F

O

Deoxy CP-544,439 NH 2

H 2N

O H 3C

N

O

NH

Amidine metabolite

Figure 6-21. Dehydroxylation by cytochrome b5 and aldehyde oxidase.

from adjacent carbon atoms with formation of a carbon–carbon double bond. This type of dehalogenation reaction can also be catalyzed by glutathione transferases. Glutathione initiates the reaction with a nucleophilic attack either on the electrophilic carbon to which the halogen is attached (mechanism A) or on the halide itself (mechanism B), as shown in Fig. 6-19 for the dehalogenation of 1,2dihaloethane to ethylene. The insecticide DDT is detoxified by dehydrochlorination to DDE by DDT-dehydrochlorinase, as shown in

Fig. 6-20. The activity of this glutathione-dependent reaction correlates well with resistance to DDT in houseflies. Dehydroxylation—Cytochrome b5 and Aldehyde Oxidase In the presence of NADH and NADH-cytochrome b5 reductase (CYB5R3), the microsomal hemoprotein cytochrome b5 (CYB5A) can catalyze the N -dehydroxylation of various amidoximes, as shown in Fig. 6-21 for the amidoxime metabolite of

196

UNIT 2

DISPOSITION OF TOXICANTS

Oxidation

Reduction

O

R

H

R

Aldehydes

H N

NO2

O

OH

OH

Carboxylic acids H N

N

N

Nitroaromatics O

Hydroxylamines

R

R

N

NH

N O

H N

N

OH

O

Isoxazoles

Imino alcohols R

R N

NH

N N

N

NH

S

SH

O

Azaheterocyclic aromatic compounds

Isothiazoles

Lactams

O N

Alicyclic imines

N

O

Lactams

OH N

Hydroxamic acids

Imino thiols

O

H N

Amides

Figure 6-22. Examples of oxidation and reduction catalyzed by aldehyde oxidase.

the antimicrobial prodrug DB289 (Saulter et al., 2005). The complex of NADH-cytochrome b5 reductase and cytochrome b5 , in the presence of NADH, can also efficiently reduce arylhydroxylamine metabolites of drugs such as sulfamethoxazole and dapsone, as well as carcinogens that are generated through the oxidation of compounds such as 4-aminobiphenyl and PhIP by CYP1A1, 1A2, 1B1, lactoperoxidase, and myeloperoxidase (see sections “Cytochrome P450” and “Peroxidase-Dependent Cooxidation”) (Kurian et al., 2006). Because these carcinogenic arylhydroxylamines can be further activated by glucuronidation, sulfonation, or acetylation in various tissues (see sections “Glucuronidation,” “Sulfonation,” and “Acetylation”), reduction by NADH-cytochrome b5 reductase and cytochrome b5 represents a competing detoxication pathway. Figure 6-21 also shows a similar dehydroxylation reaction catalyzed by aldehyde oxidase, an enzyme that can catalyze both reductive and oxidative reaction, as discussed in the next section.

Aldehyde Oxidase—Reductive Reactions Aldehyde oxidase is a cytosolic molybdozyme that catalyzes the oxidation of some xenobiotics and the reduction of others. The types of oxidative and reductive reactions catalyzed by aldehyde oxidase are shown in Fig. 6-22. In contrast to the large number of drugs that are known to be (or suspected of being) oxidized by aldehyde oxidase in vivo, only a few drugs are known to be (or suspected of being) reduced by aldehyde oxidase in vivo, including nitrofurazone, zonisamide, and ziprasidone. The reductive metabolism of ziprasidone by aldehyde

oxidase is shown in Fig. 6-3, and the reductive dehydroxylation of CP-544,439 is shown in Fig. 6-21. The features of aldehyde oxidase and the oxidative reactions it catalyzes are discussed later in the section on “Aldehyde Oxidase.”

Oxidation Alcohol, Aldehyde, Ketone Oxidation–Reduction Systems Alcohols, aldehydes, and ketones are oxidized by a number of enzymes, including alcohol dehydrogenase, aldehyde dehydrogenase, AKRs (such as those with dihydrodiol dehydrogenase activity), the molybdenum-containing enzymes, aldehyde oxidase, and xanthine dehydrogenase/oxidase, and cytochrome P450. For example, simple alcohols (such as methanol and ethanol) are oxidized to aldehydes (namely, formaldehyde and acetaldehyde) by alcohol dehydrogenase. These aldehydes are further oxidized to carboxylic acids (formic acid and acetic acid) by aldehyde dehydrogenase, as shown in Fig. 6-23. Many of the aforementioned enzymes can also catalyze the reduction of xenobiotics, as discussed in the previous section on “Reduction.” Alcohol Dehydrogenase Alcohol dehydrogenases (ADHs) belong to the medium-chain dehydrogenases/reductases (MDRs), as shown in Table 6-5. ADHs are zinc-containing, cytosolic enzymes present in several tissues including liver, which has the highest levels, kidney, lung, and gastric mucosa (Agarwal, 1992). Human ADHs are dimeric proteins consisting of two 40-kDa subunits designated

CHAPTER 6

R

CH 2 OH

BIOTRANSFORMATION OF XENOBIOTICS

ADH

R

C

O

ALDH

H N AD

N AD H + H

R

197

C

O OH

N A D + H 2O

N AD H + H

Figure 6-23. Oxidation of alcohols to aldehydes and carboxylic acids by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).

α, β, γ , π, χ, and either σ or μ. As shown in Table 6-5, there are seven human ADHs, and these are categorized into five classes (I– V). Class I comprises three enzymes: ADH1A, which contains at least one alpha subunit (αα, αβ, or αγ ), ADH1B, which contains at least one beta subunit (ββ or βγ ), and ADH1C, which contains two gamma subunits (γ γ ). ADH1A, -1B, and 1C were formerly known as ADH1, -2, and -3. Class II contains ADH4, which is composed of two pi subunits (ππ ). Class III contains ADH5, which is composed of two chi subunits (χ χ). Class IV contains ADH7, which is composed of two subunits designated mu (μμ) or sigma (σ σ ). Class V contains ADH6 (for which there is no subunit designation) (Brennan et al., 2004); see also www.gene,ucl.ac.uk/ nomenclature/genefamily/ADH.shtml. Of the seven ADHs, only ADH1B and ADH1C are polymorphic. There are three allelic variants of the beta subunit (β1 , β2 , and β3 ), which differ by a single amino acid, and two allelic variants of the gamma subunit (γ1 and γ2 ), which differ by two amino acids. Consequently, the human ADH enzymes comprise nine subunits, all of which can combine as homodimers. In addition, the α, β, and γ subunits (and their allelic variants) can form heterodimers with each other (but not with the π, χ , or σ /μ subunits, which only form homodimers). ADH1B enzymes that differ in the type of β subunits are known as allelozymes, as are ADH1C enzymes that differ in the type of γ subunit. The class I ADH isozymes (ADH1A or α-ADH, ADH1B or β-ADH, and ADH1C or γ -ADH) are responsible for the oxidation of ethanol and other small, aliphatic alcohols, and they are strongly inhibited by pyrazole and its 4-alkyl derivatives (e.g., 4methylpyrazole). High levels of class I ADH isozymes are expressed in liver and adrenals, with lower levels in kidney, lung, blood vessels (in the case of ADH1B) and other tissues, but not brain. The class II enzyme ADH4 (π -ADH) is primarily expressed in liver (with lower levels in stomach), where it preferentially oxidizes larger aliphatic and aromatic alcohols. ADH4 differs from the ADH1 isozymes in that it plays little or no role in ethanol and methanol oxidation, and it is not inhibited by pyrazole. The class III enzyme ADH5 (χ-ADH) preferentially oxidizes long-chain alcohols (pentanol and larger) and aromatic alcohols (such as cinnamyl alcohol). Like ADH4, ADH5 is not inhibited by pyrazole. However, in contrast to ADH4, which is largely confined to the liver, ADH5 is ubiquitous, being present in virtually all tissues (including brain), where it appears to play an important role in detoxifying formaldehyde. In fact, ADH5 and formaldehyde dehydrogenase are identical enzymes (Koivusalo et al., 1989). The class IV enzyme ADH7 (μ- or σ -ADH) is a low-affinity (high K m ), high-capacity (high Vmax ) enzyme, and is the most active of the medium-chain ADHs in oxidizing retinol. It is the major ADH expressed in human stomach and other areas of the gas-

trointestinal tract (esophagus, gingiva, mouth, and tongue). In contrast the other ADHs, ADH7 is not expressed in adult human liver. Inasmuch as ADH7 is expressed in the upper gastrointestinal tract, where chronic alcohol consumption leads to cancer development, there is considerable interest in the role of ADH7 in the conversion of ethanol to acetaldehyde (a suspected upper GI tract carcinogen or co-carcinogen) and in its role in the metabolism of retinol (a vitamin required for epithelial cell growth and differentiation), which might be inhibited by alcohol consumption (Seitz and Oneta, 1998). The class I ADH isozymes differ in their capacity to oxidize ethanol. Even the allelozymes, which differ in a single amino acid, differ markedly in the affinity (K m ) and/or capacity (Vmax ) for oxidizing ethanol to acetaldehyde. The homodimer, β2 β2 and heterodimers containing at least one β2 subunit (i.e., the ADH1B*2 allelozymes) are especially active in oxidizing ethanol at physiological pH. ADH1B*2 (formerly known as ADH2*2) is known as atypical ADH, and is responsible for the unusually rapid conversion of ethanol to acetaldehyde in 90% of the Pacific Rim Asian population (Japanese, Chinese, Korean). The atypical ADH is expressed to a much lesser degree in Caucasians (70% similar (Mackenzie et al., 2005). UGT2A1 and 2A2 share five exons with a variable first exon, similar to the UGT1A enzymes, whereas UGT2A3 is made up of six unique exons. A summary of the current understanding of the tissue distribution and substrate specificity of the human UGT1 and UGT2 enzymes can be found in Table 6-16. Suffice it to say that these enzymes are expressed in a wide variety of tissues, and some enzymes— including UGT1A7, 1A8, 1A10, and 2A1—are expressed only in extrahepatic tissues, which has implications for the common practice of using human liver microsomes to investigate the role of glucuronidation in drug metabolism. Of the hepatically expressed UGT enzymes, UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 are considered to be the UGT enzymes most important for hepatic drug metabolism because UGT1A5, 2B4, 2B10, 2B11, 2B17, and 2B28 are reported to have low or negligible activity toward xenobiotics (Miners et al., 2006). UGT1A7, 1A8, and 1A10 expressed in the gastrointestinal tract may also be important for prehepatic elimination of various orally administered drugs. Numerous UGT1 and UGT2 enzymes are expressed throughout the gastrointestinal tract, where they contribute significantly to the first pass elimination of numerous xenobiotics. Several UGT2B enzymes are expressed in steroidsensitive tissues, such as prostate and mammary gland, where they presumably terminate the effects of steroid hormones. Probe drugs have been identified for some but not all of the human UGTs, including UGT1A1 (17β-estradiol 3-glucuronidation and bilirubin), UGT1A3 (hexafluoro-1α,25-trihydroxyvitamin D3), UGT1A4 (trifluoperazine), UGT1A6 (serotonin and 1-naphthol), UGT1A9 (propofol), UGT2B7 (morphine 6-glucuronidation and zidovudine [AZT]), and UGT2B15 (S-oxazepam) (Miners et al., 2006). The glucuronidation of morphine by UGT2B7 involves conjugation of the phenolic 3-hydroxyl and the alcoholic 6-hydroxyl group in a 7:1 ratio. The 6-O-glucuronide is 600 times more potent an analgesic than the parent drug, whereas the 3-O-glucuronide is devoid of analgesic activity. UGT2B7 is present in the brain, where it might facilitate the analgesic effect of morphine through formation of the 6-O-glucuronide, which presumably does not readily cross the blood–brain barrier and may be retained in the brain longer than morphine (Tukey and Strassburg, 2000). Only UGT2B7 catalyzes the 6-glucuronidation of morphine, whereas several UGTs including UGT1A1, 1A3, 1A6, 1A8, 1A9, 1A10 as well as 2B7 can catalyze the 3-glucuronidation (Stone et al., 2003). Selective probe inhibitors have only been characterized for UGT1A4 (hecogenin) and 2B7 (fluconazole) (Miners et al., 2006). Drug–drug interactions that are at least partly due to inhibition of UGTs have been reported. For instance, plasma levels of indomethacin are increased about twofold upon coadministration of diflunisal, and in vitro studies indicate that this interaction is due in part to inhibition of indomethacin glucuronidation in the intestine (Gidal et al., 2003; Mano et al., 2006). Valproic acid coadministration increases the AUC of lorazepam and lamotrigine by 20% and 160%, respectively (Williams et al., 2004). In contrast to the situation with CYP enzymes, there are fewer inhibitory drug– drug interactions caused by the inhibition of UGT enzymes, and AUC increases are rarely greater than twofold (Williams et al., 2004), whereas dramatic AUC increases have been reported for CYP enzymes, such as the 190-fold increase in AUC reported for the

CYP1A2 substrate ramelteon (RozeremTM ) upon coadministration of fluvoxamine (RozeremTM prescribing information, 2005). The low magnitude of UGT-based inhibitory interactions is partly due to the fact that most drugs that are primarily cleared by glucuronidation are metabolized by several UGTs. Drug–drug interactions due to induction of UGT enzymes have also been observed. Rifampin coadministration increases mycophenolic acid clearance by 30%, and increases the AUC of its acyl glucuronide (formed by UGT2B7) and its 7-O-glucuronide (formed by various UGT1 enzymes) by more than 100% and 20%, respectively (Naesens et al., 2006). As mentioned above and as shown in Fig. 6-52, UGTs can form unusual conjugates including bisglucuronides, diglucuronides, N -carbamoyl glucuronides, N -glucosides, and other glycoside conjugates. Bisglucuronides (i.e., a glucuronide in which two separate functional groups on the aglycone are glucuronidated) are more common than diglucuronides, and include the bisglucuronides of bilirubin, morphine, octylgallate, diosmetin, phenolphthalein, and hydroxylated polycyclic aromatic hydrocarbons (such as hydroxylated chrysene and benzo[a]pyrene) (Murai et al., 2006). A diglucuronide is a glucuronide in which a single functional group on the aglycone is conjugated twice resulting in two glucuronosyl groups in tandem (Murai et al., 2006). Diglucuronides of the xenobiotics nalmefene and 4-hydroxybiphenyl, and of the endogenous steroids androsterone, 5α-dihydrotestosterone, 17β-estradiol, estriol, estrone, and testosterone have previously been detected in dogs. Rat liver microsomes do not form diglucuronides of these steroids, whereas monkey liver microsomes form detectable levels of the 5α-dihydrotestosterone, testosterone, and 17β-estradiol diglucuronides, with human liver microsomes forming only the diglucuronide of 5α-dihydrotestosterone (see Fig. 6-52) (Murai et al., 2005). In all cases, it is the 2 -hydroxyl group of the first glucuronide moiety that is subject to additional glucuronidation. In the case of 5α-dihydrotestosterone, only human UGT1A8 (an intestinal UGT) has been found to produce the diglucuronide from 5α-dihydrotestosterone itself, although UGT1A4, 2B15, and 2B17 can produce the monoglucuronide, and UGT1A1 and 1A9 can produce the diglucuronide when the monoglucuronide is the substrate (Murai et al., 2006). Human intestinal microsomes form the diglucuronide more efficiently than human liver microsomes, reflecting the fact that UGT1A8, an extrahepatic UGT, is the predominant enzyme involved in the diglucuronidation of 5α-dihydrotestosterone. N -Carbamoyl glucuronidation has been reported for relatively few primary amines, or the demethylated metabolites of secondary and tertiary amines, and includes drugs such as sertraline (Fig. 6-52), carvedilol, varenicline, mofegiline, garenoxacin, tocainide, and sibutramine (Gipple et al., 1982; Tremaine et al., 1989; Beconi et al., 2003; Hayakawa et al., 2003; Link et al., 2006; Obach et al., 2006). Marked species difference have been found in the formation of N -carbamoyl glucuronides, and humans have only been found to produce these conjugates from even fewer drugs, including sertraline, varenicline, and mofegiline. To form this type of conjugate in vitro, the incubation must be performed under a CO2 atmosphere, in a carbonate buffer. Although not directly demonstrated, it has been hypothesized that a transient carbamic acid intermediate is formed by the interaction of the amine with the dissolved CO2 , followed by glucuronidation (Obach et al., 2005). Because the intermediate is not stable, the hypothesis that UGT also catalyzes the formation of the carbamic acid cannot be disproved. However, in the case of sertraline, and varenicline it is predominantly UGT2B7 that forms the N -carbamoyl glucuronide, which also conjugates various carboxylic acids (Obach et al., 2005, 2006). Given that the in vitro

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

265

Figure 6-53. Structure of the human UGT1 locus which encodes multiple forms of UDP–glucuronosyltransferase. Note that these microsomal enzymes face the lumen of the endoplasmic reticulum.

formation of N -carbamoyl glucuronides occurs only under special incubation conditions that are not typically employed, it is possible that many other primary and secondary amines or their oxidative metabolites can be converted to such conjugates but have not been detected because of the unusual incubation conditions required to support the formation of N -carbamoyl glucuronides. Finally, although human UGTs typically are highly selective in the use of UDPGA as a sugar donor, they can accommodate the use of other sugar donors such as UDP-glucose, UDP-galactose, and UDP-xylose in an aglycone-dependent manner, as mentioned previously (Tang and Ma, 2005). For instance, recombinant human UGT1A1 can utilize UDPGA, UDP-xylose, or UDP-glucose to form glycosides, but only with bilirubin (Tang and Ma, 2005). It has also been demonstrated that recombinant human UGT2B7 can glycosidate an endothelin ETA receptor antagonist with UDPGA, UDP-glucose, and UDP-galactose, but can glycosidate diclofenac with only UDPGA (Tang and Ma, 2005). In the case of an aldosereductase inhibitor (AS-3201), Toide and colleagues (2004) found that UGT2B4, 2B7, and especially 2B15 all preferentially utilize UDP-glucose over UDPGA. Several other compounds have been found to be glucosidated in mammals, including 5-aminosalicylic acid, bromfenac, pranoprofen, pantothenic acid, hyodeoxycholic acid, mycophenolic acid, sulphadimidine, sulphamerazine, sulphamethoxazole, and various barbiturates (e.g., phenobarbital and amobarbital) (Tang and Carro-Ciampi, 1980; Nakano et al., 1986; Arima, 1990; Kirkman et al., 1998; Toide et al., 2004; Picard et al., 2005). In the case of the carboxyl-containing amine, bromfenac, the aglycone was observed in rat bile after base hydrolysis, and it was concluded that it was formed by hydrolysis of an acyl glucuronide (Kirkman et al., 1998). In later studies to characterize the stability of the putative acyl glucuronide, an N -glucoside was detected (Kirkman et al., 1998). 5-Aminosalicylic acid is structurally similar to bromfenac in that both NSAIDs contain a primary amine near the carboxyl group, and both are converted to N -glucosides (Kirkman et al., 1998). In the human metabolism of barbiturates, N -glucosides are the major metabolites found in urine, and glucuronides have not been detected. Accordingly, it was theorized that enzymes other

than UGTs may be involved in the conjugation of barbiturates in humans (Toide et al., 2004). This latter theory is interesting because liver homogenates from cats, which lack UGT activity, can N -glucosidate amobarbital (however, it should be noted that glucose conjugates can sometimes form nonenzymatically) (Carro-Ciampi et al., 1985). However, Toide and colleagues (2004) have demonstrated that the N-glucosidation of amobarbital in human liver microsomes correlates highly with the N-glucosidation of AS-3201, but not its N-glucuronidation, indicating that UGT2B15 is probably the predominant human UGT responsible for the glucosidation of amobarbital. In humans, Crigler–Najjar syndrome (type I and II) and Gilbert disease are congenital defects in bilirubin conjugation. The diseases have historically been differentiated largely on the basis of the severity of symptoms and total plasma concentrations of bilirubin (e.g., Crigler–Najjar Type I: 310–855 μM, Crigler–Najjar Type II: 100–430 μM, and Gilbert disease: 20–100 μM). The major bilirubin-conjugating enzyme in humans is UGT1A1. Genetic polymorphisms in exons 2-5 (which affect all enzymes encoded by the UGT1A locus), in exon 1 (which affect only UGT1A1), in the promoter regions, and in introns 1 and 3 have been identified in patients with Crigler–Najjar syndrome or Gilbert disease. More than 60 genetic polymorphisms are associated with these diseases. A current list of all UGT polymorphisms and phenotypes (when known) can be found at http://galien.pha.ulaval.ca/alleles/alleles.html. Some polymorphisms cause the introduction of a premature stop codon in one of the exons 2-5 (which causes a loss of all UGT1A enzymes, analogous to the Gunn rat) and are associated with type I Crigler–Najjar syndrome, a severe form of the disease characterized by a complete loss of bilirubin-conjugating activity and marked hyperbilirubinemia. Type I Crigler–Najjar syndrome is also associated with various frameshifts and deletions in exons 1-5, and in at least three cases, with changes in introns 1 and 3 that affect splice donor or acceptor sites. Other polymorphisms are associated with the less severe type II Crigler–Najjar syndrome (i.e., UGT1A1*7–9, 12, 26, 30, 33–38, 42, 48, 51, 52, 59, and 64). Individuals with Gilbert disease have an occasionally transient, and generally mild hyperbilirubinemia which

266

UNIT 2

DISPOSITION OF TOXICANTS

is often caused by the addition of one “TA” segment in the TATA promoter region (i.e., UGT1A1∗ 28: A(TA)6 TAA → A(TA)7 TAA). There is some overlap between Crigler–Najjar Type II and Gilbert disease, not only in terms of the plasma concentrations of bilirubin, but also in the type of polymorphism that underlies the disease. In addition to the ∗ 28 allele, the ∗ 6, ∗ 27, ∗ 29, ∗ 60, and ∗ 62 alleles are associated with Gilbert disease, and some of these polymorphisms affect coding regions of the UGT1A1 gene. In addition, the UGT1A1∗ 37 allele produces a A(TA)8 TAA promoter defect which results in Crigler–Najjar Type II. A Korean individual heterozygous for three UGT1A1 alleles associated with Gilbert disease (i.e., likely ∗ 6, ∗ 28, and ∗ 60), was found to have total bilirubin concentrations as high as 193 μM (Seok Seo et al., 2007), which is a concentration typically associated with Crigler–Najjar Type II. Crigler–Najjar Type II and Gilbert disease (in contrast to Crigler–Najjar Type I) typically respond to some extent to phenobarbital treatment, which stimulates bilirubin conjugation presumably by inducing UGT1A1. Type I Crigler–Najjar syndrome is also associated with impaired glucuronidation of propofol, ethinylestradiol, and various phenolic substrates for UGT1A enzymes. Polymorphisms that might affect the other UGT1A enzymes have not been thoroughly characterized in vivo, but there are data to suggest that polymorphisms in these enzymes may modify the risk of developing certain types of cancer (Nagar and Remmel, 2006). The UGT1A1*28 allele has received widespread attention in recent years due to the impact this variant has on the toxicity of the topoisomerase I inhibitor, irinotecan, that is used primarily to treat colorectal cancer. The disposition of irinotecan is complex, with conversion to the active metabolite, SN-38, occurring mainly in the liver by hydrolysis by carboxylesterase. The active metabolite SN-38 is subsequently glucuronidated primarily by UGT1A1, with potential contribution from UGT1A6, 1A7, 1A9, and 1A10 (Nagar and Blanchard, 2006). The UGT1A1*28 variant has now been referenced in the Camptosar® prescribing information, which notes that patients with reduced UGT1A1 activity have a higher exposure to SN-38 (which is 50–100 times more toxic than the glucuronide), and that the dose of irinotecan should be adjusted downward accordingly (Nagar and Blanchard, 2006). The toxicity of SN-38 primarily manifests as severe diarrhea and myelosuppression (in the form of leucopenia, severe thrombocytopenia, severe anemia, or grade 3– 4 neutropenia) (Nagar and Blanchard, 2006). Several studies have demonstrated grade 3–4 neutropenia and/or grade 3–4 diarrhea upon irinotecan administration in patients with at least one UGT1A1*28 or *27 allele, and one study implicated high activity variants of UGT1A7 and 1A9 with diarrhea (Nagar and Blanchard, 2006). In at least one study, the UGT1A1*28 allele has also been reported to increase the risk of invasive breast cancer in premenopausal women by 1.8-fold, which was attributed to decreased glucuronidation of estradiol, but other studies appear to contradict this finding (Nagar and Remmel, 2006). Polymorphisms have been identified in UGT2B4, 2B7, 2B15, and 2B28. For example, oxazepam is glucuronidated by UGT2B15, which preferentially glucuronidates S-oxazepam over its Renantiomer. Ten percent (10%) of the population appear to be poor glucuronidators of S-oxazepam, and one study has implicated the low activity UGT2B15*2 allele as a possible determinant of such variation (Nagar and Remmel, 2006). Such polymorphisms also appear to be the underlying cause of alterations in hyodeoxycholate glucuronidation in gastric mucosa (Tukey and Strassburg, 2000). Human UGT1A6 glucuronidates acetaminophen and the glucuronidation of acetaminophen in humans is enhanced by cigarette

smoking and dietary cabbage and Brussels sprouts, which suggests that human UGT1A6 is inducible by polycyclic aromatic hydrocarbons and derivatives of indole 3-carbinol (Bock et al., 1994). Ligands for the Ah receptor, such as those present in cigarette smoke, induce CYP1A2, which would be expected to enhance the hepatotoxicity of acetaminophen. Increased acetaminophen glucuronidation may explain why cigarette smoking does not enhance the hepatotoxicity of acetaminophen. Conversely, decreased glucuronidation may explain why some individuals with Gilbert’s syndrome are predisposed to the hepatotoxic effects of acetaminophen (de Morais et al., 1992). Low rates of glucuronidation predispose newborns to jaundice and to the toxic effects of chloramphenicol; the latter was once used prophylactically to prevent opportunistic infections in newborns until it was found to cause severe cyanosis and even death (gray baby syndrome). Glucuronidation generally detoxifies xenobiotics and potentially toxic endobiotics, such as bilirubin, for which reason glucuronidation is generally considered a beneficial process. However, steroid hormones glucuronidated on the D-ring (but not the A-ring) cause cholestasis, and induction of UGT activity has been implicated as an epigenetic mechanism of thyroid tumor formation in rodents (McClain, 1989; Curran and DeGroot, 1991). Inducers of UGTs cause a decrease in serum thyroid hormone levels, which triggers a compensatory increase in thyroid stimulating hormone (TSH). During sustained exposure to the enzyme-inducing agent, prolonged stimulation of the thyroid gland by TSH (>6 months) results in the development of thyroid follicular cell neoplasia. Glucuronidation followed by biliary excretion is a major pathway of thyroxine biotransformation in rodents whereas deiodination is the major pathway (up to 85%) of thyroxine metabolism in humans. In contrast to the situation in rodents, prolonged stimulation of the thyroid gland by TSH in humans will result in malignant tumors only in exceptional circumstances and possibly only in conjunction with some thyroid abnormality. Therefore, chemicals that cause thyroid tumors in rats or mice by inducing UGT activity are unlikely to cause such tumors in humans. In support of this conclusion, extensive epidemiological data in epileptic patients suggest that phenobarbital and other anticonvulsants do not function as thyroid (or liver) tumor promoters in humans (Parkinson et al., 2006). In some cases, glucuronidation represents an important event in the toxicity of xenobiotics. For example, the aromatic amines that cause bladder cancer (such as benzidine, 2-aminonaphthalene, and 4-aminobiphenyl) undergo N-hydroxylation in the liver followed by N-glucuronidation of the resultant N -hydroxyaromatic amine, although direct N-glucuronidation also occurs, and is a competing pathway of hepatic metabolism. In the case of 4-aminobiphenyl, the competing pathways of ring hydroxylation and O-esterification (i.e., O-glucuronidation, O-sulfonation, or O-acetylation) are detoxication pathways, whereas N-esterification catalyzed by UGTs, SULTs, or NATs represent activating pathways (see section entitled Acetylation) (Cohen et al., 2006). Benzidine and 2-aminonaphthalene are particularly tumorigenic; with accumulating data which demonstrates that the risk of bladder cancer may increase by up to 100fold in workers exposed to these substances in the course of their occupation in various manufacturing processes (Al-Zoughool et al., 2006). The N -glucuronides of such carcinogens, which accumulate in the urine of the bladder, are unstable in acidic pH and thus are hydrolyzed to the corresponding unstable, tumorigenic N hydroxyaromatic amine, as shown in Fig. 6-54. N -Hydroxyaromatic amines can give rise to highly electrophilic aromatic nitrenium ions that can bind to DNA and other macromolecules, or they can be

R- COOH (e.g., diclofenac and related NSAIDs)

NH2

Acyl-glucuronidation 2-Aminonaphthalene (2-Naphthylamine)

Acyl migration

Nucleophic displacement

O

COOH O O

C

R

N-hydroxylation (CYP1A2) OH H N

HO

COOH O OH

OH

Acylglucuronide

OCOR N-Hydroxy-2-naphthylamine

N-glucuronidation glucuronide

glucuronic acid

PROTEIN

OH

O

HO R

OH

C

Acylated protein

COOH

The 2-O-β-, 3-O-β-, and 4-O-βisomers also form and undergo ring opening and protein binding

PROTEIN

OH OCOR CH2

N

NH

L ys

PROTEIN

HO

OH

O Toxicity/Immune hepatitis

ring opening H2 O Amadori rearrangement

Acidic pH of urine β-Glucuronidase in intestine

H2N

Lys

PROTEIN

glucuronic acid

H2 O

COOH

OCOR

OH HC

OCOR CH

O

HO Activation of DNA-reactive metabolites that cause bladder or colon tumors

COOH

OH

N-Hydroxy-2-naphthylamine

Imine formation OH

Figure 6-54. Role of glucuronidation in the activation of xenobiotics to toxic metabolites.

NH

Lys PROTEIN

HO OH Protein adducts containing a rearranged glucuronic acid

268

UNIT 2

DISPOSITION OF TOXICANTS

converted to reactive acetoxy metabolites directly in the bladder epithelium via NAT-mediated O-acetylation, which also leads to the formation of aromatic nitrenium ions (Al-Zoughool et al., 2006). Because N-glucuronidation of aromatic amines can also occur directly without a prior oxidation, this reaction competes with oxidation, and therefore a decrease in UGT activity in the liver would favor N-hydroxylation, with subsequent O-acetylation by NAT and spontaneous formation of aromatic nitrenium ions in the liver, rather than the bladder. In contrast, decreased acetylation with normal UGT activity would lead to a greater accumulation of N -hydroxyaromatic amines in the bladder with increased bladder tumor formation. Concordant with this scenario, benzidine has been shown to induce predominantly liver tumors in rats (fast acetylators) but bladder cancer in dogs (poor acetylators) (Al-Zoughool et al., 2006). In humans, there are sex differences in aromatic amine carcinogenicity. Irrespective of ethnicity or race, men are 2.5–5 times more likely to develop bladder cancer than women in general, and in particular male smokers, hairdressers, dye and textile workers who are exposed to aromatic amines have several times increased risk relative to their female counterparts. Male mice, which N -glucuronidate 4aminobiphenyl faster than females, were found during the treatment with this carcinogen to have a 2.2-fold higher rate of DNA-adduct formation, and increased rates of bladder tumor formation relative to females, and female mice were found to have tumors only in the liver (Al-Zoughool et al., 2006). In mice, coadministration of 4-aminobiphenyl with hecogenin, which in humans has been found to inhibit hepatic UGT1A4 (Uchaipichat et al., 2006), was found to increase DNA-adduct formation in a statistically significant manner in the livers of male mice, and to slightly decrease adduct formation in the bladders of both male and female mice (Al-Zoughool et al., 2006). The available literature regarding sex differences in human UGT activity is conflicting. On the one hand, glucuronidation of temazepam, oxazepam, propranolol, and salicylic acid were 20–60% higher in men than in women (Al-Zoughool et al., 2006), whereas 4-methyumbelliferone glucuronidation in cryopreserved human hepatocytes was found to be an average of 40% higher in female samples (n = 33), than in male samples (n = 31). The carcinogenicity of aromatic amines is multifactorial, and involves not only hepatic N-glucuronidation but also hepatic oxidation, hepatic and bladder acetylation, and possibly peroxidation of N -hydroxyN -acetyl aromatic amines in the bladder (Al-Zoughool et al., 2006). Therefore, sex differences in UGT activity alone may not fully explain the sex differences observed in the carcinogenicity of aromatic amines. A similar mechanism may be involved in colon tumor formation by aromatic amines, although in this case hydrolysis of the N -glucuronide is probably catalyzed by β-glucuronidase in intestinal microflora. Some acylglucuronides are reactive intermediates that bind covalently to protein by mechanisms that may or may not result in cleavage of the glucuronic acid moiety, as shown in Fig. 6-54. Several drugs, including the NSAIDs benoxaprofen, bromfenac, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indoprofen, ketoprofen, ketorolac, loxoprofen, sulindac, suprofen (very similar to the diuretic, tienilic acid), tiaprofenic acid, tolmetin, and zomepirac, contain a carboxylic acid moiety that can be glucuronidated to form a reactive acylglucuronide that can form covalent adducts with proteins. Acyl glucuronides vary widely in their reactivity, from the highly reactive zomepirac and tolmetin acyl glucuronides to the less reactive acyl glucuronides of ibuprofen and salicylic acid (Shipkova et al., 2003). A relationship between the reactivity of acyl glucuronides and the substitution near the carboxylic acid has been found. In general, α-unsubstituted acetic acid

derivatives such as zomepirac, tolmetin, and diclofenac exhibit the highest degree of covalent binding, while mono-α-substituted acetic acids such as fenoprofen show intermediate levels, and fully substituted α-acetic acids such as furosemide, ketoprofen, ibuprofen, and suprofen exhibit lower levels of covalent binding (Bolze et al., 2002). However, a direct correlation between the ability of acyl glucuronides to give rise to covalent adducts with proteins such as albumin and their ability to cause drug-related toxicity has not been firmly established, and other mechanisms may come into play. For instance, diclofenac is still a widely used drug in spite of the fact that its acyl glucuronide is very reactive, whereas zomepirac, which is less reactive than these other drugs, was withdrawn from the market in 1983 (Chen et al., 2006). Neoantigens formed by binding of acylglucuronides to protein might be the cause of rare cases of NSAID-induced immune hepatitis. Covalent adducts with proteins in the liver, kidneys, colon, small intestine, skeletal muscle, and bladder were detected in rats administered diflunisal, and in the liver, lungs, and spleen of rats administered diclofenac of UGT activity (Shipkova et al., 2003). Covalent binding of acyl glucuronides appears to be selective, with diclofenac acyl glucuronide forming adducts with dipeptidyl peptidase in rat liver, and with aminopeptidase N and sucrase-isomaltase in rat intestine (Shipkova et al., 2003). Human and rat liver UGTs are themselves targets of adducts formed by ketoprofen acyl glucuronide, which may cause nonspecific irreversible inhibition (Shipkova et al., 2003). Binding of acyl glucuronides to protein can involve isomerization reactions that lead to the retention of a rearranged glucuronide moiety (Fig. 6-54). Formation of a common neoantigen (i.e., one that contains a rearranged glucuronic acid moiety) might explain the allergic cross reactivities (cross sensitization) observed among different NSAIDs (Spahn-Langguth and Benet, 1992; KretzRommel and Boelsterli, 1994). There are reports of the detection of antibodies to acyl glucuronide–protein adducts in rats administered diflunisal, and in humans treated with valproic acid (Shipkova et al., 2003). The inherent reactivity of acyl glucuronides (covered above) can be investigated by determining the rate of disappearance of the acyl glucuronide in aqueous buffers at various pH levels (determination of hydrolysis and intramolecular rearrangement), by including human serum albumin to assess the formation of covalently bound adducts, or by the use of a lysine–phenylalanine dipeptide (Lys-Phe) to assess adduct formation. The use of the lysine–phenylalanine dipeptide allows for the rapid quantitative LC/MS/MS analysis of adducts produced by Schiff’s base formation (Wang et al., 2004). The rank order of the reactivity of the acyl glucuronides of some carboxylic acid-containing drugs determined by this approach is as follows: tolmetin > zomepirac > diclofenac > ketoprofen > fenoprofen > ibuprofen > furosemide (Wang et al., 2004). Some glucuronide conjugates have been found to act as substrates for further biotransformation by oxidation or even by further conjugation. For instance, in male Wag/Rij rats, estradiol 17β-glucuronide can be sulfonated by one or more SULTs to estradiol 3-sulfate-17β-glucuronide (Sun et al., 2006). In addition, the acyl glucuronide of 4 -hydroxydiclofenac can be formed either by glucuronidation of the oxidative metabolite of diclofenac, 4 hydroxydiclofenac (an example of conjugation following oxidation, as historically conceptualized), or the acyl glucuronide of the parent can be 4 -hydroxylated by CYP2C8 (Kumar et al., 2002), (an example of oxidative metabolism occurring after conjugation). Glucuronidation has been shown to convert several other CYP2C9 substrates into CYP2C8 substrates or inhibitors. For instance, CYP2C8 has been shown to catalyze the oxidation of several glucuronides, the aglycones of which are CYP2C9 substrates, including

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

Safrole

2-Acetylaminofluorene (2-AAF)

7-12-Dimethylbenz[a]anthracene (DMBA)

1´-hydroxylation (P450)

N-hydroxylation (P450)

269

7-Methyhydroxylation (P450)

OH N C

CH 3

CH 3

O

O CH 2

N-Hydroxy-2-AAF

CH

O

CH OH

CH 2

PAPS 1´-Hydroxysafrole PAP OSO 3

OH PAPS

PAPS

PAP

PAP

N C

CH 3

O

CH 3

O H 2C

CH

O

CH OSO 3

SO 4

CH 2

1´-Sulfoxysafrole

OSO 3

N C

CH 3

SO 4

SO 4

O O CH 3

Nitrenium ion H 2C

CH

O

CH Carbonium ion

CH 2

N C

Carbonium ion

CH 3

O

Carbonium ion

DNA binding and tumor formation

Figure 6-55. Role of sulfonation in the generation of tumorigenic metabolites (nitrenium or carbonium ions) of 2-acetylaminofluorene, safrole, and 7,12-dimethylbenz[a]anthracene (DMBA).

estradiol 17β-glucuronide and the acyl glucuronides of naproxen, the PPAR-α/agonist MRL-C, and gemfibrozil (Delaforge et al., 2005; Kochansky et al., 2005; Ogilvie et al., 2006). In the case of gemfibrozil, the CYP2C8-mediated hydroxylation of its 1-Oβ-glucuronide can apparently lead to the formation of a reactive product that causes irreversible inhibition of this enzyme (Ogilvie et al., 2006). The formation of a hydroxylated gemfibrozil acyl glucuronide by CYP2C8 leads to the irreversible inactivation of this enzyme, which leads to significant drug–drug interactions with drugs such as repaglinide and cerivastatin (Ogilvie et al., 2006). Kumar and colleagues demonstrated that formation of the acyl glucuronide of 4 -hydroxydiclofenac leads to an underestimation of

hepatic clearance of diclofenac when it is not accounted for. It may also be that oxidation of glucuronide metabolites can lead to toxicity. A case report described the formation of an IgM antibody that bound erythrocytes, but only in the presence of the 4 -hydroxydiclofenac acyl glucuronide in a patient that developed hemolysis during diclofenac treatment (Shipkova et al., 2003). A determination of the absolute amount of diclofenac acyl glucuronide formed in vivo relative to the amount of 4 -hydroxydiclofenac formed in vivo would be confounded by the rapid hydrolysis of the glucuronide to the aglycone, and it would therefore be likely that detection of 4 hydroxydiclofenac acyl glucuronide would be attributed to oxidative metabolism occurring prior to conjugation. Two recent reports

270

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suggest that direct glucuronidation with subsequent oxidation (by a combination of UGT2B7 and CYP2C8 in humans) may be the major determinants of diclofenac clearance in humans (possibly as high as 75%) and monkeys (>90%) (Kumar et al., 2002; Prueksaritanont et al., 2006), as opposed to earlier in vivo data that suggested oxidative metabolism by CYP2C9 alone is the major determinant of clearance (Stierlin and Faigle, 1979; Stierlin et al., 1979). Prueksaritanont and colleagues (2006) further note that there are no clinical reports that implicate pharmacokinetic interactions between diclofenac and potent CYP2C9 inhibitors or inducers. Taken together, these observations suggest that the CYP-mediated oxidation of glucuronide metabolites has implications not only for the prediction of in vivo drug–drug interactions from in vitro data (i.e., gemfibrozil), but also for the prediction of in vivo clearance (i.e., diclofenac), and possibly also toxicity, as in the case of immune-mediated toxicity of diclofenac.

Sulfonation Many of the xenobiotics and endogenous substrates that undergo Oglucuronidation also undergo sulfonation, as illustrated in Fig. 6-34 for acetaminophen (Mulder, 1981; Paulson et al., 1986). Sulfonation generally produces a highly water-soluble sulfuric acid ester. The reaction is catalyzed by sulfotransferases (SULT), a large multigene family of enzymes found primarily in the liver, kidney, intestinal tract, lung, platelets, and brain. In mammals, there are two major classes of SULTs: (1) membrane-bound SULTs in the Golgi apparatus, and (2) soluble SULTs in the cytoplasm (Gamage et al., 2006). The membrane-bound SULTs are responsible for the sulfonation of glycosaminoglycans, proteins, and peptides such as cholecystokinin, factors V and VIII, α-2-glycoprotein, gastrin, and p-selective glycoprotein ligand-1, thereby modulating their structure and function. At least five different Golgi-resident N -acetylglucosamine 6-O-sulfotransferases have been identified in humans. They are important for many biological processes including cell–cell adhesion, axon function, T-cell response, cell proliferation, and modulation of viral and bacterial infection (reviewed by Grunwell and Bertozzi, 2002), but they have no activity toward xenobiotics (Wang and James, 2006). Brachymorphic mice are undersized because the defect in PAPS synthesis prevents the normal sulfonation of glycosaminoglycans and proteoglycans, such as heparin and chondroitin, which are important components of cartilage. These particular sulfonation reactions are catalyzed by membrane-bound sulfotransferase, which are thought not to play a role in xenobiotic sulfonation. This section will focus on the cytosolic SULTs, which are known for the sulfonation of various drugs, mutagens, flavonoids, and other xenobiotics, as well as endogenous substrates such as bile acids, thyroid hormones, catecholamine neurotransmitters, and steroids. The cofactor for the sulfonation reaction is 3 phosphoadenosine-5 -phosphosulfate (PAPS), the structure of which is shown in Fig. 6-49. The sulfonation of aliphatic alcohols and phenols, R-OH, proceeds as follows: O R OH + phosphoadenosine

O

O P O S O O

O

O R O S O + phosphoadenosine O

O O P

O

+ H

O

Sulfonation involves the transfer of sulfonate not sulfate (i.e., − SO− 3 not SO4 ) from PAPS to the xenobiotic. SULTs are single α/β

globular proteins that contain a PAPS-binding site which is present on a characteristic five-stranded β-sheet along with the core of the catalytic site. The central β-sheet is surrounded by α-helices (Wang and James, 2006). The sulfonate donor PAPS is synthesized from inorganic sulfate (SO2− 4 ) and ATP in a two-step reaction. The first reaction is catalyzed by ATP sulfurylase, which converts ATP and SO2− to adenosine-5 -phosphosulfate (APS) and pyrophosphate. 4 The second reaction is catalyzed by APS kinase, which transfers a phosphate group from ATP to the 3 -position of APS. The major source of sulfate required for the synthesis of PAPS appears to be derived from cysteine through a complex oxidation sequence. Because the concentration of free cysteine is limited, the cellular concentrations of PAPS (4–80 μM) are considerably lower than those of UDP-glucuronic acid (200–350 μM) and glutathione (5– 10 mM). This topic has been thoroughly reviewed, and is outside the scope of this chapter (Klaassen and Boles, 1997). The relatively low concentration of PAPS limits the capacity for xenobiotic sulfonation. In general, sulfonation is a high-affinity but low-capacity pathway of xenobiotic conjugation, whereas glucuronidation is a low-affinity but high-capacity pathway. Acetaminophen is one of the several xenobiotics that are substrates for both sulfotransferases and UDP-glucuronosyltransferases (see Fig. 6-34). The relative amount of sulfonate and glucuronide conjugates of acetaminophen is dependent on dose. At low doses, acetaminophen sulfonate is the main conjugate formed due to the high affinity of sulfotransferases. As the dose increases, the proportion of acetaminophen conjugated with sulfonate decreases, whereas the proportion conjugated with glucuronic acid increases. In some cases, even the absolute amount of xenobiotic conjugated with sulfonate can decrease at high doses apparently because of substrate inhibition of sulfotransferase. Sulfonation is not limited to phenols and aliphatic alcohols (which are often the products of oxidative or hydrolytic biotransformation), although these represent the largest groups of substrates for sulfotransferases. Certain aromatic amines, such as aniline and 2-aminonaphthalene, can undergo sulfonation to the corresponding sulfamates. The primary amines in cisapride and DPC423 can also be directly N -sulfonated. The N -oxide group in minoxidil and the N -hydroxy group in N -hydroxy-2-aminonaphthalene and N hydroxy-2-acetylaminofluorene can also be sulfonated. In all cases, the conjugation reaction involves nucleophilic attack of oxygen or nitrogen on the electrophilic sulfur atom in PAPS with cleavage of the phosphosulfate bond. Table 6-17 lists some examples of xenobiotics and endogenous compounds that are sulfonated without prior biotransformation by oxidative enzymes. An even greater number of xenobiotics are sulfonated after a hydroxyl group is exposed or introduced during oxidative or hydrolytic biotransformation. Carboxylic acids can be conjugated with glucuronic acid but not with sulfonate. However, a number of carboxylic acids, such as benzoic acid, naphthoic acid, naphthylacetic acid, salicylic acid, and naproxen, are competitive inhibitors of sulfotransferases (Rao and Duffel, 1991). Pentachlorophenol and 2,6-dichloro-4-nitrophenol are potent sulfotransferase inhibitors because they bind to the enzyme but cannot initiate a nucleophilic attack on PAPS due to the presence of electron-withdrawing substituents in the ortho- and para-positions on the aromatic ring. Sulfonate conjugates of xenobiotics are excreted mainly in urine. Those excreted in bile may be hydrolyzed by aryl sulfatases present in gut microflora, which contributes to the enterohepatic circulation of certain xenobiotics. Sulfatases are also present in the endoplasmic reticulum and lysosomes, where they primarily hydrolyze sulfonates of endogenous compounds presumably in a

CHAPTER 6

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271

Table 6-17 Properties of the Human Cytosolic Sulfotransferases (SULTs) human sult

major tissue distribution

major substratesa

SULT1A1

Liver (very high), platelets. placenta, adrenals, endometrium, colon, jejunum, brain leukocytes

SULT1A2

Liver, bladder tumors

SULT1A3

Jejunum and colon mucosa (very high), platelets, placenta, brain, leukocytes Liver, pancreas, colon, brainb Colon (highest), liver, leukocytes Fetal lung and kidney, kidney, stomach, thyroid gland Kidney, ovary, spinal cord, fetal kidney, fetal lung Liver (highest), endometrium, jejunum, adrenals, mammary epithelial cells, fetal liver

4-Nitrophenol, 4-ethylphenol, 4-cresol, 2-naphthol, other phenols, acetaminophen, minoxidil, N -hydroxy-PhIP, T2, T3, 17β-estradiol (and other phenolic steroids), dopamine, benzylic alcohols, 2-nitropropane, aromatic amines, hydroxylamines, hydroxamic acids, apomorphine, troglitazone, 4-Nitrophenol, N -hydroxy-2-acetylaminofluorene, 2-naphthol, aromatic hydroxylamines, hydroxamic acids Dopamine, 4-nitrophenol, 1-hydroxymethylpyrene, norepinephrine, salbutamol, dobutamine Not characterized. Likely similar to SULT1A3 4-Nitrophenol, T2, T3, r-T3, T4, dopamine, benzylic alcohols 4-Nitrophenol, N -hydroxy-2-AAF, aryl hydroxylamines, thyroid hormones

SULT1A4 SULT1B1 SULT1C2 SULT1C4 SULT1E1

SULT2A1

Liver (highest), adrenals, jejunum, brain

SULT2B1 v1

Placenta (highest), prostate, trachea, skin

SULT2B1 v2 SULT4A1 v1

Brain: cortex, globus pallidus, islands of Calleja, septum, thalamus, red nucleus, substantia nigra and pituitary

4-Nitrophenol, N -hydroxy-2-AAF, 17β-estrone, bisphenol-A, 4-octylphenol, nonylphenol, diethylstilbestrol, 1-hydroxymethylpyrene 17β-Estradiol, estrone, ethinyl estradiol, 17β-estrone, equilenin, 2-hydroxy-estrone, 2-hydroxy-estradiol, 4-hydroxy-estrone, 4-hydroxy-estradiol, diethylstilbestrol, tamoxifen, thyroid hormones, 4-hydroxylonazolac, pregnenolone, dehydroepiandrosterone, 1-naphthol, naringenin Dehydroepiandrosterone (DHEA), 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, hycanthone, bile acids, pregnenolone, testosterone, androgens, estrone, 17β-estradiol, other hydroxysteroids, budenoside Dehydroepiandrosterone, pregnenolone, oxysterols, other hydroxysteroids Cholesterol, dehydroepiandrosterone, other hydroxysteroids Endogenous: 4 unidentified compounds from mouse brain homogenate.c Other: T3, T4, estrone, 4-nitrophenol, 2-naphthylamine, 2-naphthold

SULT4A1 v2 Substrates in bold are reported to be selective probe substrates for the SULT listed (Coughtrie and Fisher, 2005). T4 is thyroxine. T2 and T3 are di- and tri-iodothyronine. b Data from Bradley, Benner (2005). c Sakakibara et al.: Reported that recombinant human SULT4A1 expressed in E. coli (and subsequently purified) sulfonated four distinct endogenous substances from mouse brain homogenate (2002). d Sakakibara et al.: Reported that recombinant human SULT4A1 expressed in E. coli (and subsequently purified) sulfonated these prototypical SULT substrates (2002). Adapted from Gamage N, Barnett A, Hempel N, et al.: Human sulfotransferases and their role in chemical metabolism. Toxicol Sci 90:5–22, 2006; Wang LQ, James MO: Inhibition of sulfotransferases by xenobiotics. Curr Drug Metab 7:83–104, 2006; Blanchard RL, Freimuth RR, Buck J, Weinshilboum RM, Coughtrie MW: A proposed nomenclature system for the cytosolic sulfotransferase (SULT) superfamily. Pharmacogenetics 14:199–211, 2004. a

manner analogous to that described for microsomal β-glucuronidase (Dwivedi et al., 1987) (see comments on egasyn in section “Carboxylesterases”). Sulfonation facilitates the deiodination of thyroxine and triiodothyronine and can determine the rate of elimination of thyroid hormones in some species. Inhibition of SULTs can occur with exposure to drugs such as mefenamic acid, salicylic acid, clomiphene, danazol, environmental chemicals, such as hydroxylated PCBs, hydroxylated PAHs, pentachlorophenol, triclosan, and bisphenol A, and dietary constituents, such as catechins, colorants, phytoestrogens, and flavonoids. Adverse effects on human health can potentially result from SULT inhibition, such as the thyroid hormone disruption that occurs with exposure to hydroxylated PCBs (Wang and James, 2006). In contrast, given that some sulfonate conjugates are chemically reactive, inhibition of their formation may be protective. There are a few reports of drug–drug interactions due to SULT inhibition. It has been reported that the sulfonation rates of both acetaminophen and salicylamide are decreased when these

drugs are coadministered, and dapsone and lamivudine have been found to decrease acetaminophen sulfonation (Wang and James, 2006). Coadministration of acetaminophen with ethinyl estradiol increases its AUC by up to 54% and decreases the AUC of ethinyl estradiol sulfate by ∼40%, indicating that acetaminophen may directly inhibit one or more SULTs (Rogers et al., 1987). Drug–drug interactions involving induction of SULT are detailed later in this section. Sulfonation may represent a benign metabolic pathway compared with competing pathways that can lead to the activation of promutagens and procarcinogens. For instance, sulfonation of hydroquinones, phenols, and aminophenols can prevent or reduce the formation of reactive quinones, semiquinones, and quinnone amines. Sulfonation of aromatic amines such as 2-amino-3,8dimethylimidazo-[4,5-f]quinoxaline (MelQx), which leads to sulfamate formation can compete with activation by N-O-acetylation or sulfonation (Wang and James, 2006).

272

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Like glucuronide conjugates, some sulfonate conjugates are substrates for further biotransformation. For instance, the 7- and 4sulfates of daidzein and genistein can be sulfonated by SULT1E1 to disulfates (Nakano et al., 2004). Other examples include the oxidation of sulfonate conjugates of testosterone and estrogens. Dehydroepiandrosterone-3-sulfate is 16α-hydroxylated by CYP3A7, the major CYP enzyme expressed in human fetal liver (Ingelman-Sundberg et al., 1975; Kitada et al., 1987; Ohmori et al., 1998). CYP2C12, which is expressed in female but not male rats, catalyzes the oxidation of a steroid di-sulfate (namely, 5αandrostane-3α,17β-diol-3,17-disulfate) (Ryan et al., 1984). Multiple sulfotransferases have been identified in all mammalian species examined. An international workshop approved the abbreviation “SULT” for sulfotransferase (although ST remains a common abbreviation) and developed a nomenclature system based on amino acid sequences (and, to some extent, function). SULT nomenclature is available on line at: http://www.fccc.edu/research/ labs/blanchard/sult/accessions.html. The SULTs are arranged into gene families that share at least 45% amino acid sequence identity. The nine gene families identified to date (vertebrate: SULT1– SULT6; insect: SULT101; and plant: SULT201–SULT202) are subdivided into subfamilies that are at least 60% identical (Blanchard et al., 2004; Gamage et al., 2006). For example, SULT1 is divided into five subfamilies designated SULT1A–SULT1E. Two SULTs that share more than 60% similarity are considered individual members of the same subfamily. For example, SULT1A1, SULT1A2, SULT1A3, and SULT1A4 are four individual members of the human SULT1A subfamily. In general, the first published sequence in a subfamily is designated as enzyme 1 and subsequent enzymes within that subfamily are assigned on the basis of percentage amino acid identity relative to the “1” enzyme (Blanchard et al., 2004). Exceptions to this rule have been made to maintain historical use of a name (e.g., SULT2A1). Variant forms with different amino acid sequences encoded by the same gene are designated by “vn” at the end. For instance, the SULTs initially referenced as SULT2B1a and SULT2B1b are now called SULT2B1 v1 and SULT2B1 v2. Although nine SULT gene families have been identified, these have not been identified in all mammalian species. Currently, SULT1 and SULT2 are the only gene families subdivided into multiple subfamilies (five in the case of SULT1 [SULT1A–1E]; two in the case of SULT2 [SULT2A and SULT2B]). Most of the SULTs cloned belong to one of the two families, SULT1 and SULT2. These two families are functionally different; the SULT1 enzymes catalyze the sulfonation of phenols, isoflavones, the procarcinogen N -OH-2-acetylaminofluorene, endogenous compounds such as 17β-estradiol (including its glucuronide conjugate; see section “Glucuronidation”), and other steroids, iodothyronines, endogenous catecholamines, and eicosanoids. The SULT2 enzymes catalyze the sulfonation of the 3β-hydroxy groups of steroids with unsaturated A rings, bile acids, benzylic alcohols of polycyclic aromatic hydrocarbons, and other primary and secondary alcohols. A sulfotransferase that catalyzes the sulfonation of heterocyclic amines such as 2-naphthylamine, desipramine, and aniline (to form sulfamates) has been cloned from rabbit and mouse (SULT3A1). SULT4A1 has been identified in rat, mouse, and human. These enzymes are expressed in the cerebral cortex, cerebellum, pituitary, and brainstem and do not sulfonate typical SULT substrates (Blanchard et al., 2004; Gamage et al., 2006). These SULTs share ∼97% amino acid sequence identity across species, which suggests that SULT4A1 likely serves a critical endogenous function. The SULT4A1 sequence has also been identified as SULT5A1,

but the 4A1 nomenclature has been retained. A separate gene, SULT5A1, has been cloned from mouse, but no information on its function is available. SULT6A1 has been cloned from chicken liver, and the recombinant enzyme was found to sulfonate 17β-estradiol and corticosterone (Blanchard et al., 2004). SULT201 and 202 represent two families of plant SULTs. SULTT101A1 is an insect SULT cloned from Spodoptera frugiperda and converts retinol to anhydroretinol via a retinyl sulfate intermediate (Blanchard et al., 2004). SULT101A also exhibits sulfotransferase activity toward ethanol, dopamine, vanillin, 4-nitrophenol, serotonin, and hydroxybenzylhydrazine (Blanchard et al., 2004). Thirteen cytosolic SULTs have been cloned from rat, and they belong either to the SULT1, SULT2, or SULT4 gene families. The individual rat enzymes are SULT1A1, 1B1, 1C1, 1C2, 1C3, 1D1, 1E1, 1E2, 2A1, 2A2, 2A3, 2A4, and 4A1. Eleven genes encoding 13 cytosolic SULTs have been identified in humans, and they belong either to the SULT1, SULT2, or SULT4 gene families. The individual human enzymes are listed in Table 6-17. Various single-nucleotide polymorphisms have been reported in most of the SULT genes with rare single-base deletions in SULT1A2 and 4A1 (Glatt and Meinl, 2004). With a few exceptions, the functional consequences of most of these polymorphisms remain unknown. Several of the human SULT genes have multiple initiation sites for transcription, which produces different mRNA transcripts. Consequently, in some cases, different versions of the same human SULT gene have been cloned several times. For example, there are three alternative first exons (exons 1a, 1b, and 1c) in the human SULT1A3 gene (none of which contains a coding region), and five SULT1A3 cDNAs have been cloned from various human tissues, each with a unique 5 -region (Nagata and Yamazoe, 2000). Historically, human liver cytosol was found to contain two phenol sulfotransferase activities (PST) that could be distinguished by their thermal stability; hence, they were known as TS-PST (thermally stable) and TL-PST (thermally labile) (Weinshilboum, 1992a; Weinshilboum et al., 1997). It is now known that TS-PST actually reflects the activity of two SULTs, namely, SULT1A1 and SULT1A2 which share 93% identity, whereas TL-PST reflects the activity of SULT1A3 (and likely SULT1A4), which is 60% similar to both SULT1A1 and 2 (Gamage et al., 2006). Hence, the four members of the SULT1A gene subfamily in human were represented functionally by TS-PST and TL-PST activity. SULT1A1 and SULT1A2 function as homo- and heterodimers, and are coregulated. Although these two individual SULTs are not catalytically identical, they are sufficiently similar to consider them as the single activity traditionally known as TS-PST. Because of differences in their substrate specificity, SULT1A1/2 and 1A3 were also known as phenol-PST and monoamine-PST, respectively. SULT1A3 preferentially catalyzes the sulfonation of dopamine, epinephrine, and levodopa, whereas SULT1A1 and 1A2 preferentially catalyze the sulfonation of simple phenols, such as phenol, 4-nitrophenol, minoxidil, and acetaminophen. SULT1A1 and 1A2 also catalyze the N -sulfonation of 2-aminonaphthalene. SULT1A1/2 and SULT1A3 can also be distinguished by differences in their sensitivity to the inhibitory effects of 2,6-dichloro-4-nitrophenol. The expression of SULT1A1 and 1A2 in human liver is largely determined by genetic factors, which also determines the corresponding sulfotransferase activity in blood platelets. Inherited variation in platelet SULT1A1 and 1A2 largely reflects genetic polymorphisms in these enzymes. One allelic variant of SULT1A1 known as SULT1A1*2 (Arg213 → His213 ) is associated with decreased activity

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

in platelets but not liver, and decreased thermal stability (Glatt and Meinl, 2004). This particular genetic polymorphism is common in both Caucasians and Nigerians (with an allele frequency of 0.31 and 0.37, respectively), and is correlated with interindividual variation in the sulfonation of acetaminophen. Low SULT1A1 and 1A2 activity predisposes individuals to diet-induced migraine headaches, possibly due to impaired sulfonation of unidentified phenolic compounds in the diet that cause such headaches. A fourth member of the human SULT1A subfamily, SULT1A4, has been recently described, which appears to be a duplication of SULT1A3, and these two enzymes share >99% sequence identity (Bradley and Benner, 2005; Gamage et al., 2006). The genes for both of these SULTs lie on chromosome 16p, which contains a segmental duplication that results in two nearly identical, transcriptionally active copies of SULT1A3 and SULT1A4. Each copy shares exons with an adjacent copy of SULT1A1. Four nonsynonymous SNPs were reported for these genes, which show different enzyme activities (Gamage et al., 2006). Human SULT1B1, like the corresponding enzyme in other species, catalyzes the sulfonation of thyroid hormones, 2-naphthol, and dopamine. SULT1B1 levels in human liver cytosol vary widely, possibly due to polymorphisms (e.g., Glu186 → Gly186 and Glu204 → Asp204 ) (Glatt and Meinl, 2004). SULT1B1 is also expressed in human colon, small intestine, and blood leukocytes. Humans have two SULT1C enzymes (SULT1C2 and SULT1C4). Their function has not been determined, although the corresponding rat enzyme (SULT1C1) catalyzes the sulfonation of N -hydroxy-2-acetylaminofluorene (see Fig. 6-55). SULT1C2 is expressed at high levels in the thyroid, stomach, and kidneys (Blanchard et al., 2004). High levels of SULT1C4 are expressed in fetal liver and kidney, with hepatic levels declining in adulthood, but it is also present in adult ovary and brain. Human SULT1E1 has been identified as a high affinity estrogen sulfotransferase. SULT1A1 also catalyzes the sulfonation of estrogens, such as 17β-estradiol, but it does so with a much lower affinity than does SULT1E1. The sulfonation of ethinyl estradiol in human hepatocytes is inducible by rifampin (Li et al., 1999), which raises the possibility that SULT1E1 is an inducible enzyme. In addition to human liver, SULT1E1 is expressed in placenta, breast, brain, testes, adrenal glands, and uterine tissue. SULT1E1 has been studied in SULT1E1-deficient mice, and it was shown that these mice had spontaneous fetal loss caused by placental thrombosis, which was reversible by administration of antiestrogens (Gamage et al., 2006). SULT2A1 is the human alcohol sulfotransferase, long known as DHEA-ST (for its ability to sulfonate dehydroepiandrosterone). In addition to DHEA, substrates for SULT2A1 include steroid hormones, bile acids, and cholesterol. Furthermore, SULT2A1 converts several procarcinogens to electrophilic metabolites, including hydroxymethyl polycyclic aromatic hydrocarbons, N -hydroxy-2acetylaminofluorene, and 1 -hydoxysafrole, as shown in Fig. 6-55. The thermal stability of SULT2A1 is intermediate between that of the four phenol SULTs (SULT1A1/2 and 1A3/4), and the enzyme is resistant to the inhibitory effects of 2,6-dichloro-4-nitrophenol. SULT2A1 is not expressed in blood platelets, but the activity of this enzyme has been measured in human liver cytosol. SULT2A1 is also expressed in adrenal cortex, brain, and intestine (Blanchard et al., 2004). SULT2A1 is bimodally distributed, possibly due to a genetic polymorphism that apparently lies outside of the coding region, and perhaps outside of the SULT2A1 gene itself, with a high activity group composed of ∼25% of the population (Glatt and Meinl,

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2004). Several SULT2A1 SNPs have been identified, but the underlying basis for the high activity group remains to be determined as these polymorphisms appear to be too rare to explain the bimodal distribution. Human SULT2B1 is also a DHEA-sulfotransferase. It is expressed in placenta, prostate, and trachea. The SULT2B1 gene can be transcribed from one of the two exons, both of which contain coding sequences, hence, two forms of SULT2B1 (known as 2B1 v1 and 2B1 v2) with different N-terminal amino acid sequences can be transcribed by alternate splicing of precursor mRNA. This situation is analogous to the alternative splicing of multiple exons 1 in the UGT1 gene family (see Fig. 6-53). SULT2B1 v1 appears to catalyze the sulfonation of pregnenolone, and SULT2B1 v2 can catalyze the sulfonation of both pregnenolone and cholesterol (Blanchard et al., 2004). The SULT enzymes were previously categorized into five classes based on their catalytic activity. These five functional classes were: Arylsulfotransferase, which sulfonates numerous phenolic xenobiotics; alcohol sulfotransferase, which sulfonates primary and secondary alcohols including nonaromatic hydroxysteroids (for which reason these enzymes are also known as hydroxysteroid SULTs); estrogen sulfotransferase, which sulfonates estrone and other aromatic hydroxysteroids; tyrosine ester sulfotransferase, which sulfonates tyrosine methyl ester and 2cyanoethyl-N -hydroxythioacetamide, and bile salt sulfotransferase, which sulfonates conjugated and unconjugated bile acids. The arylsulfotransferase and estrogen sulfotransferase are composed largely of SULT1 enzymes, which catalyze the sulfonation of phenolic xenobiotics, catechols, and aromatic (phenolic) steroids. The alcohol sulfotransferase and bile salt sulfotransferase are composed largely of SULT2 enzymes, which catalyze the sulfonation of a variety of primary and secondary alcohols, bile acids, and hydroxysteroids (such as dehydroepiandrosterone or DHEA). In rats, sulfotransferase activity varies considerably with the sex and age. In mature rats, phenol sulfotransferase activity (SULT1A activity) is higher in males, whereas alcohol sulfotransferase and bile acid sulfotransferase activities (SULT2 activities) are higher in females. Sex differences in the developmental expression of individual sulfotransferases are the result of a complex interplay between gonadal, thyroidal, and pituitary hormones, which similarly determine sex differences in CYP enzyme expression. However, compared with CYP enzymes, the SULTs are refractory or only marginally responsive to the enzyme-inducing effects of 3methylcholanthrene and phenobarbital, although one or more individual SULT2 enzymes are inducible by PCN. Likewise, SULT1A1, 2A1, or 2E1 expressed in Caco-2 cells are refractory to various polycyclic aromatic hydrocarbons, and 3-methylcholanthrene has no effect on SULT1A1 and 1A3 mRNA levels in primary human hepatocytes (Gamage et al., 2006). From rodent studies, it is generally held that AhR agonists have suppressive effects on SULT regulation. 2-AAF, TCDD, 3-methylcholanthrene, and β-naphthoflavone markedly suppress SULT1A1 and 2A activities and mRNA levels in rat livers (Gamage et al., 2006). In contrast, there is evidence that the rat liver hydroxysteroid SULTs (SULT2s) may be inducible by tamoxifen and estrogens (Gamage et al., 2006). For mouse SULT2A2, a functional nuclear response element responsive for CAR has been reported (Gamage et al., 2006). Human SULT2A1 has been reported to be regulated by FXR, PXR, VDR, and PPARα, whereas SULT1A1, 2A1, and 2A9 genes in mice are regulated by CAR (Tirona and Kim, 2005; Gamage et al., 2006). There are conflicting data regarding the influence of CAR ligands on

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SULT1A1 and 1A3. One study reports an 11-fold increase in SULT1A1 mRNA in primary human hepatocytes by the CAR ligand, CITCO, whereas another study could not reproduce these results for either SULT1A1 or 1A3 (Gamage et al., 2006). The glucocorticoid, dexamethasone, has been reported to induce both murine and human SULT2A1 through PXR and GR activation (Gamage et al., 2006). Human SULT2A1 mRNA and protein have also been reported to be induced by rifampin, vitamin D3 , phenobarbital, TCPOBOP, and the PPARα-agonist, ciprofibrate (Runge-Morris and Kocarek, 2005). Induction of SULTs by rifampin, on the other hand, may be clinically relevant. Rifampin (600 mg q.d.) has been reported to cause up to a 190% increase in the clearance of ethinyl estradiol (35 μg q.d.) (Barditch-Crovo et al., 1999). The interaction between ethinyl estradiol-containing oral contraceptives and antibiotics such as rifampin is often attributed to the induction of CYP3A4, which is the major CYP involved in the oxidative metabolism of ethinyl estradiol (e.g., Ortho-Evra® prescribing information, 2005). Several lines of evidence suggest that induction of CYP3A4 is not the predominant mechanism by which rifampin increases the clearance of ethinyl estradiol. First, Li and colleagues (1999) reported that treatment of primary cultures of human hepatocytes with rifampin (33.3 μM) caused up to a 3.3-fold increase in ethinyl estradiol 3-Osulfate formation. Second, SULTs 1A1, 1A2, 1A3, 1E1, and 2A1 catalyze the 3-O-sulfonation of ethinyl estradiol with K m values ranging from 6.7 to 4500 nM, nearer the pharmacologically relevant concentrations (Schrag et al., 2004). Finally, it is known that ethinyl estradiol is predominantly excreted in bile and urine as the 3-sulfate and, to a lesser extent, the 3-glucuronide (Li et al., 1999), which suggests that 3-sulfonation is the major pathway of ethinyl estradiol metabolism. Taken together, these data suggest that induction of SULTs can be clinically relevant at least for low-dose drugs that can be sulfonated with high affinity. Extrapolation of animal data with regard to biotransformation by SULTs is confounded by the number of SULTs, the expression pattern of SULTs and pronounced sexual dimorphisms in many rodents (Gamage et al., 2006). For instance, expression of SULTs in humans appears to have a largely extrahepatic pattern, whereas rodent SULT expression is predominantly hepatic. For instance, based on RT-PCR measurements of mRNA levels in human tissues, SULT1A3 is expressed at the greatest level in the small intestine, 1B1 in the colon, 2B1 in the placenta, and 4A1 in the brain (Nishimura and Naito, 2006). Of the SULTs that have been characterized by RT-PCR in various human tissues, only SULT1A1, 1E1, and 2A1 are predominantly expressed in the liver (Nishimura and Naito, 2006). Additionally, humans have four members of the SULT1A subfamily whereas rodents have only one. In contrast, rats have four members of the SULT2A subfamily, whereas humans have only a single SULT2A gene. Human equivalents of mouse SULT3A1 and 5A1 have not yet been identified in humans (Gamage et al., 2006). There are also significant differences between other mammalian species. For instance, sulfotransferase activity is low in pigs but high in cats. The high sulfotransferase activity in cats offsets their low capacity to conjugate xenobiotics with glucuronic acid. In general, sulfonation is an effective means of decreasing the pharmacological and toxicological activity of xenobiotics. There are cases, however, in which sulfonation increases the toxicity of foreign chemicals because certain sulfonate conjugates are chemically unstable and degrade to form potent electrophilic species. As shown in Fig. 6-55, sulfonation plays an important role in the acti-

vation of aromatic amines, methyl-substituted polycyclic aromatic hydrocarbons, and safrole to tumorigenic metabolites. To exert its tumorigenic effect in rodents, safrole must be hydroxylated by CYP to 1 -hydroxysafrole, which is then sulfonated to the electrophilic and tumor-initiating metabolite, 1 -sulfooxysafrole (Boberg et al., 1983). 1 -Hydroxysafrole is a more potent hepatotumorigen than safrole. Two lines of evidence support a major role for sulfonation in the hepatotumorigenic effect of 1 -hydroxysafrole. First, the hepatotumorigenic effect of 1 -hydroxysafrole can be inhibited by treating mice with the sulfotransferase inhibitor, pentachlorophenol. Second, the hepatotumorigenic effect of 1 -hydroxysafrole is markedly reduced in brachymorphic mice, which have a diminished capacity to sulfonate xenobiotics because of a genetic defect in PAPS synthesis. The sulfo-conjugates of benzylic and allylic alcohols, aromatic hydroxylamines, and hydroxamic acids (including those in cooked meat) are short-lived electrophiles capable of reacting with nucleophilic substances including proteins and DNA (Wang and James, 2006). Sulfonation can also convert procarcinogens and promutagens to electrophilic nitrenium or carbocation intermediates such as N -hydroxy-2-acetylaminofluorene (NOH-AAF), 1-hydroxymethylpyrene (1-HMP), 1 -hydroxysafrole, and the cooked food mutagen N -hydroxy-2-amino-1-methyl-6phenylimidazo(4,5-b)pyridine (N-OH-PhIP) (Wang and James, 2006). Some drugs must be converted to a sulfonate conjugate to exert their desired effect, including triamterene, cicletanine, and minoxidil (Wang and James, 2006). Sulfonation (as well as glucuronidation) converts morphine to more potent analgesics than the parent, with morphine 6-sulfate being 30 times more potent and with morphine-6-glucuronide being 45–800 times more potent than morphine itself in rats (Wang and James, 2006). Similarly, several sulfonated steroids such as pregnenolone sulfate and DHEA sulfate interact directly with neurotransmitter receptors. It has been found that pregnenolone sulfate and DHEA sulfate enhance memory in mice. Prevention of hydrolysis of these sulfates by the steroid sulfatase inhibitor, (para-O-sulfamoyl)-N -tetradecanoyl tyramine, increases the memory enhancement caused by DHEA sulfate in rats, which suggests there is an important role of these sulfates in the central nervous system (Wang and James, 2006). Polymorphisms with consequences for the bioactivation of xenobiotics have been reported. For instance, the human SULT1A*Arg (*1) allelozyme expressed in Salmonella typhinurium is 12- to 350-fold more active in the sulfonation of 2-acetylamino-4hydroxyaminotoluene, 2-nitropropoane, 2,4-dinitrobenzylalcohol, (–)-1-(α-hydroxyethyl) pyrene, and 1-hydroxymethylpyrene to mutagens than are cells expressing SULT1A1*His (Gamage et al., 2006). Enantioselective sulfonation of promutagens has been reported, as in the case of 1-(α-hydroxyethyl) pyrene, for which SULT2A1 exhibits a 15-fold preference for the (+)-enantiomer, and SULT1E1 exhibits a 160-fold preference for the (–)-enantiomer (Gamage et al., 2006).

Methylation Methylation is a common but generally minor pathway of xenobiotic biotransformation. Methylation differs from most other conjugation reactions because it generally decreases the water solubility of xenobiotics and masks functional groups that might otherwise be metabolized by other conjugating enzymes. One exception to this rule is the N-methylation of pyridine-containing xenobiotics, such as nicotine, which produces quaternary ammonium ions that

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are water soluble and readily excreted. Another exception is the S-methylation of thioethers to form positively charged sulfonium ions, a reaction catalyzed by thioether methyltransferase (TEMT), which has only been identified in mice (Weinshilboum et al., 1999). The cofactor for methylation is S-adenosylmethionine (SAM), the structure of which is shown in Fig. 6-49. The methyl group bound to the sulfonium ion in SAM has the characteristics of a carbonium ion and is transferred to xenobiotics and endogenous substrates by nucleophilic attack from an electron-rich heteroatom (O, N , or S). Consequently, the functional groups involved in methylation reactions are phenols, catechols, aliphatic and aromatic amines, N -heterocyclics, and sulfhydryl-containing compounds. The conversion of benzo[a]pyrene to 6-methylbenzo[a]pyrene is a rare example of C-methylation. Another reaction that appears to involve C-methylation, the conversion of cocaine to ethylcocaine, is actually a transesterification reaction, as shown in Fig. 6-4. Metals can also be methylated. Inorganic mercury and arsenic can both be dimethylated, and inorganic selenium can be trimethylated. The selenium atom in ebselen is methylated following the ring opening of this antiinflammatory drug. Some examples of xenobiotics and endogenous substrates that undergo O-, N-, or S-methylation are shown in Fig. 6-56. During these methylation reactions, SAM is converted to S-adenosyl-l-homocysteine (SAH). This section will cover the following methyltransferases: AS3MT, COMT, GNMT, HNMT, INMT, NNMT, PNMT, POMT (TMT), TEMT, and TPMT. Other methyltransferases that have been implicated as drug metabolizing enzymes include GAMT and PEMT (Nishimura and Naito, 2006). The O-methylation of phenols and catechols is catalyzed by two different enzymes known as catechol-O-methyltransferase (COMT) and the enzyme historically termed phenol Omethyltransferase (POMT) (Weinshilboum, 1989, 1992b). POMT is a microsomal enzyme that methylates phenols but not catechols, and COMT is both a cytosolic and microsomal enzyme with the converse substrate specificity, i.e., an absolute requirement for catechol substrates (Weinshilboum, 2006). COMT plays a greater role in the biotransformation of catechols than POMT plays in the biotransformation of phenols. It should be noted that there is strong evidence to suggest that the membrane-bound POMT is the same enzyme as TMT (Weinshilboum, 2006). COMT was originally described as a cytosolic, Mg2+ -requiring, monomeric enzyme (Mr 25,000). However, in rats and humans, the enzyme is encoded by the COMT gene (on chromosome 22 in humans) with two different promoters and transcription initiation sites. Transcription at one site produces a cytosolic form of COMT, whereas transcription from the other site produces a membrane-bound form by adding 50 hydrophobic amino acids to the N-terminal of the microsomal COMT, which targets this form to the endoplasmic reticulum (Weinshilboum et al., 1999; Weinshilboum, 2006). The microsomal COMT is expressed at high levels in the brain and lymphocytes (Weinshilboum, 2006). The cytosolic form of COMT is present in virtually all tissues, including erythrocytes, but the highest concentrations are found in liver and kidney. The membrane-bound form is more highly expressed in brain. Substrates for COMT include several catecholamine neurotransmitters, such as epinephrine, norepinephrine, and dopamine; and catechol drugs, such as the anti-Parkinson’s disease agent ldopa (3,4-dihydroxyphenylalanine) and the antihypertensive drug methyldopa (α-methyl-3,4-dihydroxyphenylalanine). Catechol estrogens, which are formed by 2- or 4-hydroxylation of the steroid A-ring, are substrates for COMT, as are drugs that are converted

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to catechols either by two consecutive hydroxylation reactions (as in the case of phenobarbital and diclofenac), by ring opening of a methylenedioxy group (as in the case of stiripentol and 3,4methylenedioxymethamphetamine), or by hydrolysis of vicinal esters (as in the case of ibopamine). Formation of catechol estrogens, particularly 4-hydroxyestradiol, has been suggested to play an important role in estrogen-induced tumor formation in hamster kidney, rat pituitary, and mouse uterus (Zhu and Liehr, 1993) (see section “CYP1B1”). These tissues contain high levels of epinephrine or dopamine, which inhibit the O-methylation of 4-hydroxyestradiol by COMT. Nontarget tissues do not contain high levels of catecholamines, which suggests that 4-hydroxyestradiol induces tumor formation in those tissues that fail to methylate and detoxify this catechol estrogen. These observations in animals are especially intriguing in view of subsequent epidemiological evidence demonstrating that low COMT activity appears to increase the risk of breast cancer, with odds ratios ranging from 1.7 to 3.0 (Weinshilboum, 2006). In the 1970s, when COMT levels in erythrocytes (predominantly the cytosolic form) were measured in human subjects, it was apparent that there was a subpopulation that displayed low levels of this enzyme. Segregation analysis indicated that erythrocyte COMT activity was an autosomal codominant trait, and that erythrocyte levels correlated with relative COMT levels in liver and lymphocyte cytosol (Weinshilboum, 2006). It was subsequently found that COMT is encoded by a single gene with alleles for a low activity form (COMTL ) and high activity form (COMTH ) (Weinshilboum, 1989, 1992b, 2006; Weinshilboum et al., 1999). This polymorphism results from a single G → A transition in exon 4 that results in the substitution Val108 Met in cytosolic COMT and Val158 Met in microsomal COMT (Weinshilboum et al., 1999; Weinshilboum, 2006). The presence of methionine at position 108 in the cytosolic enzyme not only decreases the catalytic activity of COMT, but it decreases the thermal stability of the enzyme, which has long been used to differentiate COMTL (thermolabile) from COMTH (thermostable). In Caucasians, these allelic variants are expressed with equal frequency, so that 25% of the population is homozygous for either the low or high activity enzyme, and 50% is heterozygous and have intermediate COMT activity. COMT activity is generally higher in Asians and African Americans due to a higher frequency of the COMTH allele (∼0.75 for Asians and African Americans vs. ∼0.5 for Caucasians [McLeod et al., 1994]). Subsequent resequencing of the COMT gene has revealed numerous single-nucleotide polymorphisms, with at least eight that occur with a frequency >10% in Caucasians, and 11 such SNPs in African Americans. Several of these SNPs are found in the intronic regions. A list of current COMT (and many other) alleles can be found at http://alfred.med.yale.edu/alfred/index.asp. The genetically determined levels of COMT in erythrocytes correlate with individual differences in the proportion of l-dopa converted to 3-O-methyldopa and the proportion of methyldopa converted to its 3-O-methyl metabolite. O-Methylation is normally a minor pathway of l-dopa biotransformation, but 3-O-methyldopa is the major metabolite when l-dopa is administered with a dopa decarboxylase inhibitor, such as carbidopa or benserazide, which is common clinical practice. High COMT activity, resulting in extensive O-methylation of l-dopa to 3-O-methyldopa, has been associated with poor therapeutic management of Parkinson’s disease and an increased incidence of drug-induced toxicity (dyskinesia). A large number of epidemiological studies have been performed to examine the effects of the COMT Val108/158 Met polymorphism, and there is no evidence that the genetic polymorphism in COMT represents a risk modifier for the development of Parkinson’s disease

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DISPOSITION OF TOXICANTS

O-Methylation

COO

COO

CH

CH2

CH2

NH2

CH NH2

SAM HO

CH3O OH

OH

L-Dopa

3-O-Methyl-L-dopa

N-Methylation CH2CH2NH2 SAM

N

HN

CH2CH2NH2

Histamine

CH3

N

N-Methylhistamine

N

N

SAM

CH3

N

N

CH3

N CH3

Nicotine

N-Methylnicotinium ion

S-Methylation S

SH N

N

SAM N

N H

6-Mercaptopurine

CH3 N

N

N

N H

6-Methylmercaptopurine

Figure 6-56. Examples of compounds that undergo O-, N-, or S-methylation.

(Weinshilboum et al., 1999). However, Egan and colleagues have demonstrated that COMT genotype was related in an allele-dosage manner to cognitive performance, with individuals homozygous for Met108 (COMTL phenotype) demonstrating increased executive cognition, as measured by the Wisconsin Card Sorting Test (Egan et al., 2001). The impact of the Met108 allele was attributed to decreased dopamine catabolism in the prefrontal cortex, which results in enhanced neuronal function. Conversely, those individuals who are homozygous for Val108 (COMTH phenotype) appear to have decreased executive cognition, and may be at a slightly increased risk of developing schizophrenia (Egan et al., 2001). Several N -methyltransferases have been described in humans and other mammals, including phenylethanolamine N methyltransferase (PNMT), which catalyzes the N-methylation of the neurotransmitter norepinephrine to form epinephrine; histamine N -methyltransferase (HNMT), which specifically methylates the imidazole ring of histamine and closely related compounds (Fig. 6-56); and nicotinamide N -methyltransferase (NNMT), which methylates compounds containing a pyridine ring, such as nicotinamide, or an indole ring, such as tryptophan and serotonin (Weinshilboum, 1989, 1992b; Weinshilboum et al., 1999). PNMT is a cytosolic enzyme expressed at high levels in adrenal medullary chromafin cells, and in neurons of the medulla oblongata, hypothalamus, as well as in sensory nuclei of the vagus nerve and the retina,

and is not thought to play a significant role in the biotransformation of xenobiotics (Ji et al., 2005). Histamine N -methyltransferase is a cytosolic enzyme (Mr 33,000) which is highly expressed in kidney, liver, colon, prostate, ovary, and spinal cord cells (Horton et al., 2005). Its activity (which can be measured in erythrocytes) varies sixfold among individuals due to a genetic polymorphism (C → T) that results in a point mutation, namely Thr115 Ile. The latter allele (Ile115 ) is quite common in Caucasians and Han Chinese (10% frequency) and encodes a variant of HNMT with decreased catalytic activity and thermal stability. HNMT may influence efficacy of some drugs by a mechanism that is not yet fully understood. For instance, individuals who are heterozygous for the Ile115 allele have been found to exhibit significantly decreased methylprednisolone-induced cortisol suppression relative to (Thr115 )-homozygous individuals (Hon et al., 2006). Several other polymorphisms in the noncoding region of the HNMT gene have also been identified. HNMT can be inhibited by several antihistamines, quinacrine, amodiaquine, metoprine, and tacrine (Horton et al., 2005). NNMT is a monomeric, cytosolic enzyme (Mr ∼30,000) that appears to be a member of a family of methyltransferases that includes PNMT and TEMT (the thioether S-methyltransferase present in mouse lung). NNMT catalyzes the N-methylation of nicotinamide and structurally related pyridine compounds (including pyridine

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itself) to form positively charged pyridinium ions. Nicotinic acid (niacin), a commonly used lipid-lowering agent, is converted to nicotinamide in vivo, which is then methylated by NNMT (or it is incorporated into nicotinamide adenine dinucleotide, NAD). In contrast to many other methyltransferases, NNMT is not expressed in erythrocytes. Nicotinamide N -methyltransferase activity in human liver, like HNMT activity in erythrocytes, varies considerably from one individual to the next, and has a trimodal distribution dependent on variations in mRNA and protein levels, and up to 25% of the general population have high NNMT levels (Souto et al., 2005; Williams et al., 2005). It is not known to what extent genetic polymorphisms account for this variation. However, 10 SNPs in the untranslated regions of the gene have been detected in a Spanish population (Souto et al., 2005). A genome-wide scan for genes associated with plasma homocysteine levels determined that there was a statistically significant association with the NNMT gene, and, moreover that one SNP (dbSNP ID#: rs694539), has a greater statistically significant association with homocysteine levels ( p = 0.017) (Souto et al., 2005). Homocysteine plasma levels are an independent intermediate risk marker for osteoporotic fractures, congestive heart failure, venous thrombosis, myocardial infarction, stroke, and Alzheimer’s disease (Souto et al., 2005). In humans, the only source of homocysteine is from the demethylation of methionine in a multistep pathway that involves SAM-dependent methyltransferases to form S-adenosylhomocysteine, the immediate precursor to homocysteine. Taken together, these data suggest that high methyltransferase activity could contribute to hyperhomocysteinemia. Of the many SAM-dependent methyltransferase genes examined for an association with homocysteine levels, only NNMT (which is highly expressed in the liver) was found to show a significant association (Souto et al., 2005). NNMT is reported to be expressed in the brain and has been implicated as a component of the etiology of idiopathic Parkinson disease because it can convert 4-phenylpyridine to MPP+ , which is known to cause Parkinson’s disease symptoms due to its toxic effect on neuronal mitochondria (see section “Monoamine Oxidase, Diamine Oxidase, and Polyamine Oxidase” and Fig. 6-28) (Williams et al., 2005). There are numerous other human N -methyltransferases (as well as O-, S-, and C-methyltransferases) that appear to play relatively specific roles in the methylation of endogenous compounds, and most have not been well-characterized with regard to their capability to methylate xenobiotics (there are at least 39 SAM-dependent methyltransferases in humans [Souto et al., 2005]). For instance, indolethylamine N -methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds (Thompson et al., 1999). Other such enzymes that were initially thought to play a role only in the N-methylation of endogenous compounds were later found to play a role, albeit a minor one, in the N-methylation of one or more xenobiotics. Amine N -methyltransferase (AMNT, also called arylamine N -methyltransferase or nicotine N -methyltransferase), which is highly expressed in human thyroid and is also found in adrenal gland and lung, exhibits some activity toward tryptamine and has been also found to preferentially methylate the pyridine nitrogen of Rnicotine, which gives rise to nicotine isomethonium ions (Hukkanen et al., 2005). Glycine N -methyltransferase (GNMT) is thought to play an important role in the regulation of methyl group metabolism in the liver and pancreas through regulation of the ratio between Sadenosyl-l-methionine and S-adenosyl-l-homocysteine. Rat data show that the tetrameric form of the GNMT has catalytic activity,

277

and the dimeric form binds polycyclic aromatic hydrocarbons. There is also evidence that the dimeric form of human GNMT sequesters benzo[a]pyrene, and thereby decreases its cytotoxic effects (Chen et al., 2004b; Lee et al., 2006). The system that is used to classify human N methyltransferases may not be appropriate for other species. In guinea pigs, for example, nicotine and histamine are both methylated by a common N -methyltransferase. Guinea pigs have an unusually high capacity to methylate histamine and xenobiotics. The major route of nicotine biotransformation in the guinea pig is methylation, although R-nicotine is preferentially methylated over its S-enantiomer (Cundy et al., 1985). Guinea pigs also methylate the imidazole ring of cimetidine. S-Methylation is an important pathway in the biotransformation of sulfhydryl-containing xenobiotics, such as the antihypertensive drug captopril, the antirheumatic agent d-penicillamine, the antineoplastic and immunosuppressive drugs 6-mercaptopurine, 6-thioguanine, and azathioprine, metabolites of the alcohol deterrent disulfiram, and the deacetylated metabolite of the antidiuretic, spironolactone. In humans, S-methylation is catalyzed by at least two enzymes, thiopurine methyltransferase (TPMT) and thiol methyltransferase (TMT, which may be the same enzyme as POMT). TPMT is a cytoplasmic enzyme that preferentially methylates aromatic and heterocyclic compounds such as the thiopurine drugs 6-mercaptopurine, 6-thioguanine, and azathioprine. TMT is a microsomal enzyme that preferentially methylates aliphatic sulfhydryl compounds such as captopril, d-penicillamine, and disulfiram derivatives. TMT has also been found to methylate the heterocyclic thiol-containing leaving groups of some cephalosporins (Wood et al., 2002), the thiazolidinedione drug, MK-0767, a dual α/γ peroxisome proliferator-activated receptor (PPAR) agonist (Karanam et al., 2004; Kochansky et al., 2006), dithiothreitol (Weinshilboum, 2006), and some thiofuran flavoring agents (Lake et al., 2003). Although a gene that encodes TMT has not yet been definitively identified, there is strong evidence to suggest that the membrane-bound POMT is the same enzyme as TMT, which means that TMT could also catalyze the O-methylation of phenols (Weinshilboum, 2006). Both TMT and TPMT are present in erythrocytes at levels that reflect the expression of TPMT and TMT in liver and other tissues. Although TPMT and TMT are independently regulated, their expression in erythrocytes is largely determined by genetic factors. TPMT is encoded by a single gene with alleles for a low activity form (TPMTL ) and for a high activity form (TPMTH ). The allele frequency of TPMTL and TPMTH are 0.06 and 0.94, respectively, which produces a tri-modal distribution of TPMT activity with low, intermediate, and high activity expressed in 0.3, 11.1, and 88.6% of Caucasians, respectively. At least 21 separate genetic polymorphisms are associated with low TPMT activity, with the *2, *3A, and *3C alleles accounting for greater than 95% of the TPMTL phenotype (Weinshilboum, 2006). In Caucasians, the allele that is most commonly associated with the TPMTL phenotype is TPMT*3A (5%), which contains two nonsynonymous single-nucleotide polymorphisms: Ala154 Thr and Tyr240 Cys (Weinshilboum, 2006). These amino acid changes (and those in the TPMT*3B and *3C variants) lead to aggregation and rapid degradation of expressed TPMT by a ubiquitin/proteasome-dependent mechanism (Wang et al., 2005a). Cancer patients with low TPMT activity are at increased risk for thiopurine-induced myelotoxicity, in contrast to the potential need for higher-than-normal doses to achieve therapeutic levels of

278

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DISPOSITION OF TOXICANTS

thiopurines in patients with high TPMT activity (Weinshilboum, 1989, 1992b). The thiopurine drugs metabolized by TPMT have a relatively narrow therapeutic index, and are used to treat lifethreatening illnesses such as acute lymphoblastic leukemia or organtransplant patients. The thiopurines are also oxidized by xanthine oxidase, but since there is extensive variation in TPMT activity and xanthine oxidase is not present in hematopoietic tissues, TPMT activity in these tissues is more important in the avoidance of lifethreatening myelosuppression at standard doses (Weinshilboum, 2006). Phenotyping for the TPMT genetic polymorphism represents one of the first examples in which testing for a genetic variant has entered standard clinical practice (Weinshilboum et al., 1999). The clinical relevance of TPMT polymorphisms is reflected by the inclusion of TPMT as a “valid biomarker” for pharmacogenomics, along with CYP2D6 polymorphisms, in the FDA’s 2005 “Guidance for Pharmacogenomic Data Submission.” TPMT can be inhibited by benzoic acid derivatives, which also complicates therapy with drugs that are metabolized by TPMT. Patients with inflammatory bowel disorders such as Crohn disease are often treated with thiopurine drugs, which are metabolized by TPMT, and with sulfasalazine or olsalazine, which are potent TPMT inhibitors. The combination of these drugs can lead to thiopurine-induced myelosuppression. A genetic polymorphism for TMT also has been described, but its pharmacological and toxicological significance remain to be determined. The molecular basis for the polymorphism has not been determined, but studies have shown that 98% of the fivefold individual variation in erythrocyte TMT activity is due to inheritance, with an allele for high TMT activity having a frequency of 0.12. TMT is relatively specific for aliphatic sulfhydryl compounds such as 2-mercatoethanol, captopril, d-penicillamine, and N -acetylcysteine. TMT also rapidly methylates a dihydrometabolite of ziprasidone that is formed by aldehyde oxidase as shown in Fig. 6-3 (Obach and Walsky, 2005). TMT is present at high levels in the colonic mucosa and is also expressed in liver microsomes and erythrocyte membranes. TMT is not inhibited by benzoic acid derivatives, but it is inhibited by the cytochrome P450 inhibitor proadifen (a.k.a. SKE-525A) (Weinshilboum et al., 1999). Some of the hydrogen sulfide produced by anaerobic bacteria in the intestinal tract is converted by S-methyltransferases to methane thiol and then to dimethylsulfide. Another source of substrates for S-methyltransferases is the thioethers of glutathione conjugates. Glutathione conjugates are hydrolyzed to cysteine conjugates, which can either be acetylated to form mercapturic acids or cleaved by cysteine-conjugate β-lyase (CCBL1). This β-lyase pathway converts the cysteine conjugate to pyruvate, ammonia, and a sulfhydryl-containing xenobiotic, which is a potential substrate for S-methylation. Methylation can also lead to increased toxicity. A recently characterized methyltransferase, AS3MT (previously called Cyt19), methylates inorganic arsenic to form methylarsonic and dimethylarsonic acids, which are more cytotoxic and genotoxic than arsenate and arsenite (Wood et al., 2006). As many as 27 polymorphisms have been identified in this gene, with two rare alleles that cause markedly decreased activity and immunoreactive protein levels, and one frequent allele (i.e., ∼10% in both African Americans and Caucasians) that causes increased activity and immunoreactive protein levels (Wood et al., 2006). Up to 1% of African Americans and Caucasians would be expected to be homozygous for the allele that encodes the high activity AS3MT, and this may potentially lead to increased arsenic toxicity in such individuals.

Acetylation N-Acetylation is a major route of biotransformation for xenobiotics containing an aromatic amine (R–NH2 ) or a hydrazine group (R–NH–NH2 ), which are converted to aromatic amides (R–NH–COCH3 ) and hydrazides (R–NH–NH–COCH3 ), respectively (Evans, 1992). Xenobiotics containing primary aliphatic amines are rarely substrates for N-acetylation, a notable exception being cysteine conjugates, which are formed from glutathione conjugates and converted to mercapturic acids by N-acetylation in the kidney (see section “Glutathione Conjugation”). Like methylation, N-acetylation masks an amine with a nonionizable group, so that many N -acetylated metabolites are less water soluble than the parent compound. Nevertheless, N-acetylation of certain xenobiotics, such as isoniazid, facilitates their urinary excretion. The N-acetylation of xenobiotics is catalyzed by N acetyltransferases (NATs) and requires the cofactor acetylcoenzyme A (acetyl-CoA), the structure of which is shown in Fig. 6-49. The reaction occurs in two sequential steps according to a ping-pong Bi–Bi mechanism (Hein, 1988). In the first step, the acetyl group from acetyl-CoA is transferred to a cysteine residue in the NAT active site with release of coenzyme A (E-SH + CoA-SCOCH3 → E-S-COCH3 + CoA-SH). In the second step, the acetyl group is transferred from the acylated enzyme to the amino group of the substrate with regeneration of the enzyme. For strongly basic amines, the rate of N-acetylation is determined by the first step (acetylation of the enzyme), whereas the rate of N-acetylation of weakly basic amines is determined by the second step (transfer of the acetyl group from the acylated enzyme to the acceptor amine). In certain cases (discussed below), N -acetyltransferases can catalyze the O-acetylation of xenobiotics. N -Acetyltransferases are cytosolic enzymes found in liver and many other tissues of most mammalian species, with the notable exception of the dog and fox, which are unable to acetylate xenobiotics. In contrast to other xenobiotic-biotransforming enzymes, the number of N -acetyltransferases known to play a role in xenobiotic metabolism is limited (Vatsis et al., 1995; Boukouvala and Fakis, 2005). NAT activities are distinguishable from other N acetyltransferases such as those involved in melatonin synthesis and serotonin metabolism (arylalkylamine N -acetyltransferases) but are indistinguishable from the group of bacterial enzymes termed N -hydroxyarylamine O-acetyltransferases (Boukouvala and Fakis, 2005). Rabbits and hamsters express only two N -acetyltransferases, known as NAT1 and NAT2, whereas mice and rats express three enzymes, namely, NAT1, NAT2, and NAT3. The Human Genome Organisation (HUGO) Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature) has designated NAT as the official symbol for arylamine N -acetyltransferases. The two well-known and characterized xenobiotic-acetylating human enzymes are NAT1 and NAT2, which are encoded by two highly polymorphic genes located on chromosome 8. Other HUGO-approved human NAT gene symbols include NAT5–6 and NAT8–14, located on other chromosomes. The activities and expression pattern of these enzymes have not yet been definitively characterized, although some of these genes have been associated with atopic dermatitis or psoriasis (NAT9) and nasopharyngeal cancer (NAT6) (Helms et al., 2003; Bowcock and Cookson, 2004; Duh et al., 2004; Yamada and Ymamoto, 2005; Morar et al., 2006). Individual NATs and their allelic variants were named in the order of their description in the literature, which makes for a somewhat confusing nomenclature system (Vatsis et al., 1995). For example, in humans, the

CHAPTER 6

BIOTRANSFORMATION OF XENOBIOTICS

“wild-type” NAT1 and NAT2 alleles are designated NAT1*4 and NAT2*4, respectively, because they are the most common alleles in some but not all ethnic groups (Hein, 2006).For NAT enzymes, the term “wild-type” may be somewhat arbitrary because it depends on the particular ethnic group that is studied. The official website for maintaining and updating NAT allele nomenclature is http://www.louisville.edu/medschool/pharmacology/NAT.html. The frequency of some SNPs and alleles in various ethnic groups is available online from the National Cancer Institute’s SNP500Cancer database (http://snp500cancer.nci.nih.gov). In each species examined, NAT1 and NAT2 are closely related proteins (79–95% identical in amino acid sequence) with an active site cysteine residue (Cys68 ) in the N-terminal region (Grant et al., 1992; Vatsis et al., 1995). Human NAT1 and NAT2 genes are composed of intronless open reading frames of 870 bp on the same chromosome with a NAT pseudogene (NATP1) between them, and encode proteins of 290 amino acids that share 87% homology in the coding region (Boukouvala and Fakis, 2005; Hein, 2006). In spite of this apparently simple structure, NAT genes are fairly complex. For instance, comparisons of genomic and cDNA clones of the human NAT2 gene performed in the early 1990s revealed that the 5 untranslated region is contained in a “noncoding exon,” 8 kb upstream of the coding region (Boukouvala and Fakis, 2005). A similar type of unusual structure was later revealed for NAT2 genes in rabbit, hamster, mouse, and rat. More recent sequence alignments of expressed sequence tags with genomic sequences reveal that the presence of one or more upstream “noncoding exons” is typical for all vertebrate NAT genes, with the contiguous coding region contained in a single exon in the 3 untranslated region (Boukouvala and Fakis, 2005). Furthermore, the splice site nearest the coding region appears to be universally conserved at position-6, relative to the first codon. The primary transcript of both NAT1 and 2 genes are also subject to alternative intron splicing in human, rat, and possibly chicken, whereas alternative splicing for NAT2 has been observed in rabbit and hamster. Alternative splicing in the case of NATs generates mRNAs with variable 5 untranslated regions. The presence of “noncoding exons” lying upstream of higher eukaryotic genes (especially intronless genes) is common, and their transcription is likely required for transport of the entire transcript to the cytoplasm (Boukouvala and Fakis, 2005). The differential transcription of upstream noncoding exons has been frequently associated with cell-specific regulation of transcription and translation, and recent studies show that certain noncoding NAT exons are present to different extents in different tissues (Boukouvala and Fakis, 2005). There is also evidence of differential utilization of multiple tandem poly-adenosine repeats in the 3 untranslated regions in different species, but the precise effect of polyadenylation of NATs remains unknown (Boukouvala and Fakis, 2005). Despite their coexistence on the same chromosome, NAT1 and NAT2 are independently regulated proteins. For instance, human NAT1 protein and/or mRNA has been detected in every tissue examined (e.g., liver, gastrointestinal tract, leukocytes, erythrocytes, bladder, uroepithelial cells, mammary, lung, placenta, kidney, pineal gland, skeletal muscle, heart, brain, and pancreas) and are present from the blastocyst stage, whereas NAT2 is thought to be mainly expressed in liver and intestine. However, most (but not all) of the tissues that express NAT1 also appear to express low levels of NAT2, at least at the level of mRNA (Debiec-Rychter et al., 1999). Hein has also challenged the hypothesis that NAT2 is expressed mainly in the liver and intestine, and noted that the O-acetylation of N -hydroxy4-aminobiphenyl in human urinary bladder cytosol did not correlate

279

with NAT1 activity (4-aminobenzoic acid N-acetylation) consistent with acetylation by both enzymes (Hein, 2006). Regulation of human NAT1 is complex, and involves a promoter region composed of an AP1-box flanked by two TCATT boxes (Boukouvala and Fakis, 2005). The 3 TCATT box is required for expression whereas the 5 -box attenuates promoter activity. Transcription factors such as c-Fos/Fra, c-Jun, and YY1 bind to the NAT1 promoter (Boukouvala and Fakis, 2005). In addition, because transcription of human NAT1 can begin with different upstream “noncoding exons,” it is likely that there are additional promoters that regulate expression. NAT1 and NAT2 also have different but overlapping substrate specificities, although no substrate is exclusively N acetylated by one enzyme or the other. Substrates preferentially N acetylated by human NAT1 include 4-aminobenzoic acid (PABA), 4aminosalicylic acid, sulfamethoxazole, and sulfanilamide, whereas substrates preferentially N -acetylated by human NAT2 include isoniazid, hydralazine, procainamide, dapsone, aminoglutethimide, and sulfamethazine. Some investigators have used 4-aminobenzoic acid as a selective probe substrate for wild-type NAT1, and either sulfamethazine or sulfadiazine as selective probe substrates for wild-type NAT2 (Winter and Unadkat, 2005). Some xenobiotics, such as the carcinogenic aromatic amine, 2-aminofluorene, are N acetylated equally well by NAT1 and NAT2. Other drugs that are substrates for either NAT1 or NAT2 include acebutolol, amantadine, amonafide, amrinone, benzocaine, declopramide, metamizole, and phenelzine (Gonzalez and Tukey, 2006; Sirot et al., 2006). Several drugs are N -acetylated following their biotransformation by hydrolysis, reduction, or oxidation. For example, caffeine is N 3-demethylated by CYP1A2 to paraxanthine (Fig. 6-44), which is then N -demethylated to 1-methylxanthine and N -acetylated to 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by NAT2. Other drugs converted to metabolites that are N -acetylated by NAT2 include sulfasalazine, nitrazepam, and clonazepam. Examples of drugs that are N -acetylated by NAT1 and NAT2 are shown in Fig. 6-57. It should be noted, however, that there are species differences in the substrate specificity of N -acetyltransferases. For example, 4-aminobenzoic acid is preferentially N -acetylated by NAT1 in humans and rabbits but by NAT2 in mice and hamsters. Genetic polymorphisms for N-acetylation have been documented in humans, hamsters, rabbits, and mice (Evans, 1992; Grant et al., 1992; Vatsis et al., 1995; Hirvonen, 1999; Hein et al., 2000). A series of clinical observations in the 1950s established the existence of slow and fast acetylators of the antitubercular drug isoniazid. In general, ∼50% of patients treated with isoniazid have adverse events such as peripheral neuropathy and hepatotoxicity. Slow acetylators also exhibit a higher incidence of adverse events with clonazepam, hydralazine, procainamide, and sulfonamides (Sirot et al., 2006). The incidence of the slow acetylator phenotype is high in Middle Eastern populations (e.g., ∼92% in Egyptians), intermediate in Caucasian and African populations (e.g., ∼50–59% in Caucasian Americans, Australians, and Europeans; ∼41% in African Americans; ∼50–60% in black Africans), and low in Asian populations (e.g., 50% in the U.K.) but is rarely observed in Koreans (i.e., 90% (exactly 93.8%) of the dose to be eliminated, and about seven half-lives for >99% (exactly 99.2%) elimination. Thus, given the elimination T1/2 of a toxicant, the length of time it takes for near complete washout of a toxicant after discontinuation of its exposure can easily be estimated. As will be seen in next section, the concept of T1/2 is applicable to toxicants that exhibit multi-exponential kinetics. We can infer from the mono-exponential decline of blood or plasma concentration that the toxicant equilibrates very rapidly between blood and the various tissues relative to the rate of elimination, such that extravascular equilibration is achieved nearly instantaneously and maintained thereafter. Depiction of the body system by a one-compartment model does not mean that the concentration of the toxicant is the same throughout the body, but it does assume that the changes that occur in the plasma concentration reflect proportional changes in tissue toxicant concentrations (Fig. 7-2 upper, right panel). In other words, toxicant concentrations in tissues are expected to decline with the same elimination rate constant or T1/2 as in plasma.

Two-Compartment Model After rapid iv administration of some toxicants, the semi-logarithmic plot of plasma concentration versus time does not yield a straight line but a curve that implies more than one dispositional phase (Fig. 7-2). In these instances, it takes some time for the toxicant to be taken up into certain tissue groupings, and to then reach an equilibration with the concentration in plasma; hence, a multi-compartmental model is needed for the description of its kinetics in the body (Fig. 7-1). The concept of tissue groupings with distinct uptake and equilibration rates of toxicant becomes apparent when we consider the factors that govern the uptake of a lipid-soluble, organic toxicant. Table 7-2 presents data on the volume and blood perfusion rate of various organs and tissues in a standard size human. From these data and assuming reasonable partitioning ratios of a typical lipidsoluble, organic compound in the various tissue types, we can estimate the uptake equilibration half-times of the toxicant in each organ or tissue region during constant, continuous exposure. The results suggest that the tissues can be grouped into rapid-equilibrating visceral organs, moderately slow-equilibrating lean body tissues (mainly skeletal muscle), and very slow-equilibrating body fat; these

308

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DISPOSITION OF TOXICANTS

Table 7-1 Elimination of a Toxicant that Follows First-Order Kinetics (kel = 0.3 h−1 ) by 1 Hour After IV Administration at Four Different Dose Levels dose (mg)

toxicant remaining (mg)

toxicant eliminated (mg)

toxicant eliminated (% of dose)

10 30 90 250

7.4 22 67 185

2.6 8 23 65

26 26 26 26

groupings give rise to three distinct uptake phases; that is, halftimes of 1 L/kg). The mechanisms of tissue sequestration include partitioning of a toxicant into tissue fat, high affinity binding to tissue proteins, trapping in specialized organelles (e.g., pH trapping of amine compounds in acidic lysozomes), and concentrative uptake by active transporters. In fact, the equation below is an alternate form of Equation (7-7), which features the interplay of binding to plasma and tissue proteins in determining the partitioning of a toxicant in that only free or unbound drug can freely diffuse across membrane and cellular barriers. Vd = Vp +

f up × Vt,i f ut,i

(7-8)

where f up is the unbound fraction of toxicant in plasma (Table 7-3) and f ut,i is the effective unbound fraction in a tissue region. Here, Pt,i is governed by the ratio f up / f ut,i . Thus, a toxicant that has high affinity for plasma proteins (e.g., albumin and/or α1 -acid glycoprotein) relative to tissue proteins has a restricted distribution volume; for example, the anticoagulant warfarin with a plasma-bound fraction of 0.995 or an f up of 0.005 (Table 7-3). On the other hand, a toxciant that has a high affinity for tissue proteins and lesser affinity for plasma proteins can have a very high Vd . For example, the tricyclic antidepressant nortriptyline has a good affinity for plasma proteins with a bound fraction of 0.92 or an f up of 0.08; however, binding of nortriptyline to tissues constituents is so much higher such that it has a Vd of 18 times the body weight in adult humans.

Table 7-3 Volume of Distribution (Vd ) and Unbound or Free Fraction in Plasma (fup ) for Several Drugs that are of Clinical Toxicological Interest.a Volumes of Body Fluid Compartments in Healthy Human Adults are Included for Comparison. chemical Chloroquine Nortriptyline Oxycodone Acetaminophen Phenytoin Warfarin Epoetin alfa a

Vd (L/kg) ∼200 18 2.0 Body size = 1.0 0.95 Total body water = 0.60 0.64 Extracellular fluid = 0.27 0.14 0.05 Plasma volume = 0.045

Vd and f up values taken from Thummel et al. (2006).

f up ∼0.45 0.080 0.55 ka2 > ka3 Same Bioavailability

Plasma Conc

1

1

313

2 3

2 3

Time

Time

Figure 7-5. Influence of absorption rate on the time to peak (Tp ) and maximum plasma concentration (Cmax ) of a toxicant that exhibit one-compartment kinetics. The left panel illustrates the change in plasma concentration-time curves as the first-order absorption rate constant (ka ) decreases, while keep the extent of absorption or bioavailability (F), hence the AUC, constant. The right panel displays the same curves in a semi-logarithmic plot. Time to peak plasma concentration shows a progressive delay as ka decreases, along with a decrease in Cmax . In case 1 and 2, the terminal decline in plasma concentration is governed by elimination half-life; hence, the parallel decline in the semi-logarithmic plot. In case 3 where ka  kel , the absorption becomes so slow that decline in plasma concentration in the terminal phase reflects the absorption half-life; that is, washout of toxicant is rate-limited by absorption. Accordingly, the terminal decline is slower than in case 1 and 2.

Parent Vp

Metabolite Vm

Km

Kp

Parent Plasma Conc

Metabolite Metabolite

km >> kp

Parent

km fourfold) in Cmax and AUC∞ 0 as the methanol vapor concentration is raised from 1200 ppm to 4800 ppm. It should be noted that a constant T1/2 or kel does not exist during the saturation regime; it varies depending upon the saturating methanol dose. In addition to the complication of dose-dependent kinetics, there are chemicals whose clearance kinetics changes over time (i.e., time-dependent kinetics). A common cause of time-dependent kinetics is auto-induction of xenobiotic metabolizing enzymes; that is, the substrate is capable of inducing its own metabolism through activation of gene transcription. The classic example of auto-induction is with the antiepileptic drug, carbamazepine. Daily administration of carbamazepine leads to a continual increase in clearance and shortening in elimination half-life over the first few weeks of therapy (Bertilsson et al., 1986).

CHAPTER 7

TOXICOKINETICS

100

120

Blood Methanol (μg/ml)

315

1200 ppm

100

10

4800 ppm 80

1

60 40

0.1 20 0.01

0 0

120

240

360

Time (min)

480

0

120

240

360

480

Time (min)

Figure 7-8. Predicted time course of blood methanol concentration following a 120-minute exposure to 1200 and 4800 ppm of methanol vapor in the female monkey based on the toxicokinetic model reported by Burbacher et al. (2004) that features a saturable (Michaelis–Menten type) metabolic clearance. The left panel is a rectilinear plot of the simulated blood methanol concentration–time curves at the two exposure levels. The right panel shows the same simulation in a semi-logarithmic plot. The washout of blood methanol following the 120-min inhalation exposure at 1200 ppm follows a typical concave or exponential pattern in the rectilinear plot (left panel) and is linear in a semi-logarithmic plot (right panel). The post exposure profile at 4800 ppm shows a linear segment during the first 120 min of washout and becomes exponential thereafter in the rectilinear plot (left panel). The linear segment reflects saturation of alcohol dehydrogenase, which is the principal enzyme responsible for the metabolism of methanol. The in vivo K M for this simulation was set at 32.7 μg/mL. At concentrations above K M , the kinetics approach zero-order kinetics. At concentrations below K M , washout kinetics become first-order with a half-life of about 60 min. The right panel shows a characteristic convex semilogarithmic plot for the initial phase of zero-order kinetics and becomes linear as expected for first-order kinetics when the concentration falls below K M . It should also be noted that the maximum blood methanol following 4800 ppm exposure is predicted to be 5.9-times higher than that following 1200 ppm. Also, the area under the blood concentration-time curve (AUC) from time zero to 480 min at 4800 ppm exposure is eightfold higher than at 1200 ppm. Under linear kinetics, the increase in maximum blood methanol and AUC ought to be proportionate to the dose increase, i.e., a fourfold increment. Here, we observe a more than proportionate increase in blood methanol concentration in relation to the dose, which is another hallmark of saturation kinetics.

Accumulation During Continuous or Intermittent Exposure It stands to reason that continual or chronic exposure to a chemical leads to its cumulative intake and accumulation in the body. For a chemical that follows first-order elimination kinetics, the elimination rate increases as the body burden increases. Therefore, at a fixed level of continuous exposure, accumulation of a toxicant in the body eventually reaches a point when the intake rate of the toxicant equals its elimination rate, from thereon the body burden stays constant. This is referred to as the steady state. Figure 7-9 illustrates the rise of toxicant concentration in plasma over time during continuous exposure and the eventual attainment of a plateau or the steady-state. Steady-state concentration of a toxicant in plasma (Css ) is related to the intake rate (Rin ) and clearance of the toxicant. Css =

Rin Cl

(7-16)

For a one-compartment model, an exponential rise in plasma concentration is expected during continuous exposure and the time it takes for a toxicant to reach steady state is governed by its elimination half-life. It takes one half-life to reach 50%, four half-lives to reach 93.8%, and seven half-lives to reach 99.2% of steady state.

Time to attainment of steady state is not dependent on the intake rate of the toxicant. The left panel of Fig. 7-9 shows the same time to 50% steady state at three different rates of intake; on the other hand, the steady-state concentration is strictly proportional to the intake rate. Change in clearance of a toxicant often leads to a corresponding change in elimination half-life (see right panel of Fig. 7-9), in which case both the time to reach and magnitude of steady-state concentration are altered. The same steady-state principle applies to toxicants that exhibit multi-compartmental kinetics; except that, accumulation occurs in a multi-phasic fashion reflective of the multiple exponential half-lives for inter-compartmental distribution and elimination. Typically, the rise in plasma concentration is relatively rapid at the beginning, being governed by the early (distribution) half-life, and becomes slower at later times when the terminal (elimination) half-life takes hold. The concept of accumulation applies to intermittent exposure as well. Figure 7-10 shows a typical occupational exposure scenario to volatile chemicals at the workplace over the course of a week. Whether accumulation occurs from day to day and further from week to week depends on the intervals between exposure and the elimination half-life of the chemicals involved. For a chemical with relatively short half-life compared to the interval between work shifts and the “exposure holiday” over the weekend, little accumulation is expected. In contrast, for a chemical with elimination

316

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DISPOSITION OF TOXICANTS

4

Plasma Conc.

2

1

1 5

3

Time

Time

Figure 7-9. Accumulation of plasma toxicant concentration over time during constant, continuous exposure as a function of exposure level (left panel) and elimination half-life (right panel). These simulations are based on a one-compartment model at a constant apparent volume of distribution. Case 1 serves as the reference with an elimination half-life set equal to one arbitrary time unit. In the left panel, which illustrates accumulation of toxicant as a function of exposure level, exposure level is raised by twofold in Case 2 and lowered by 50% in Case 3. The changes in eventual steady state concentration are proportional to the changes in exposure level, i.e., increased by twofold in Case 2 and decreased by 50% in Case 3. During continuous exposure, 50% of steady state is achieved in one half-life. Near plateau or steady-state (>90%) is reached after four half-lives. Since the elimination half-life is constant across Cases 1–3 in the left panel, the time it takes to attain 50% of steady-state concentration (see arrows) is the same, i.e., one time unit. Right panel illustrates the influence of elimination half-life and clearance on accumulation at a fixed constant rate of exposure. Case 4 represents a 50% decrease in clearance and a corresponding twofold increase in elimination half-life compared to Case 1. Case 5 represents a twofold increase in clearance and a corresponding 50% decrease in elimination half-life. Changes in both the time to attain steady-state and the steady-state concentration are evident. In Case 4, the steady-state concentration increased by twofold as a result of a 50% reduction in clearance, and the time to achieved 50% of steady state increased by twofold as a result of the prolonged elimination half-life. In Case 5, the steady-state concentration is reduced by 50%, while the time to reach 50% steady state is shortened by 50%.

T1/2 = 24 hrs

Plasma Conc.

half-life approaching or exceeding the between-shift intervals (>12 to 24 hours), progressive accumulation is expected over the successive workdays. Washout of the chemical may not be complete over the weekend and result in a significant carry forward of body burden into the next week. It should also be noted that the overall exposure as measured by the AUC over the cycle of a week is dependent on the toxicant clearance.

T1/2 = 8 hrs

Conclusion For many chemicals, blood or plasma chemical concentration versus time data can be adequately described by a one- or twocompartment, classic pharmacokinetic model when basic assumptions are made (e.g., instantaneous mixing within compartments and first-order kinetics). In some instances, more sophisticated models with increased numbers of compartments will be needed to describe blood or plasma toxicokinetic data; for example, if the chemical is preferentially sequestered and turns over slowly in select tissues. The parameters of the classic compartmental models are usually estimated by statistical fitting of data to the model equations using nonlinear regression methods. A number of software packages are available for both data fitting and simulations with classic compartmental models; examples include WinNonlin (Pharsight Corp., Palo Alto, CA), SAAM II (SAAM Institute, University of Washington, Seattle, WA), ADAPT II (University of Southern California, Los Angeles, CA), and PK Solutions (Summit Research Services, Montrose, CO).

Mon

Tue

Wed

Thur

Fri

Sat

Sun

Mon

Figure 7-10. Simulated accumulation of plasma concentration from occupational exposure over the cycle of a work week compared between two industrial chemicals with short and long elimination half-lives. Shading represents the exposure period during the 8-hour workday, Monday through Friday. Intake of the chemical into the systemic circulation is assumed to occur at a constant rate during exposure. Exposure is negligible over the weekend. For the chemical with the short elimination half-life of 8 hours, minimal accumulation occurs from day to day over the work days. Near complete washout of the chemical is observed when work resumes on Monday (see arrow). For the chemical with the long elimination half-life of 24 hours, progressive accumulation is observed over the five work days. Washout of the longer half-life chemical over the weekend is incomplete; a significant residual is carried into the next work week. Because of its lower clearance, the overall AUC of the long half-life chemical over the cycle of a week is higher by threefold.

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Knowledge of toxicokinetic data and compartmental modeling are useful in deciding what dose or dosing regimen of chemical to use in the planning of toxicology studies (e.g., targeting a toxic level of exposure), in choosing appropriate sampling times for biological monitoring, and in seeking an understanding of the dynamics of a toxic event (e.g., what blood or plasma concentrations are achieved to produce a specific response, how accumulation of a chemical controls the onset and degree of toxicity, and the persistence of toxic effects following termination of exposure).

317

Intravenous Injection

Blood

Brain

PHYSIOLOGIC TOXICOKINETICS The primary difference between physiologic compartmental models and classic compartmental models lies in the basis for assigning the rate constants that describe the transport of chemicals into and out of the compartments (Andersen, 1991). In classic kinetics, the rate constants are defined by the data; thus, these models are often referred to as data-based models. In physiologic models, the rate constants represent known or hypothesized biological processes, and these models are commonly referred to as physiologically based models. The concept of incorporating biological realism into the analysis of drug or xenobiotic distribution and elimination is not new. For example, one of the first physiologic models was proposed by Teorell (1937). This model contained all the important determinants in chemical disposition that are considered valid today. Unfortunately, the computational tools required to solve the underlying equations were not available at that time. With advances in computer science, the software and hardware needed to implement physiological models are now well within the reach of toxicologists. The advantages of physiologically based models compared with classic models are that (1) these models can describe the time course of distribution of toxicants to any organ or tissue, (2) they allow estimation of the effects of changing physiologic parameters on tissue concentrations, (3) the same model can predict the toxicokinetics of chemicals across species by allometric scaling, and (4) complex dosing regimes and saturable processes such as metabolism and binding are easily accommodated (Gargas and Andersen, 1988). The disadvantages are that (1) much more information is needed to implement these models compared with classic models, (2) the mathematics can be difficult for many toxicologists to handle, and (3) values for parameters are often ill defined in various species, strains, and disease states. Nevertheless, physiologically based toxicokinetic models are conceptually sound and are potentially useful tools for gaining rich insight into the kinetics of toxicants beyond what classic toxicokinetic models can provide.

Basic Model Structure Physiologic models are fundamentally complex compartmental models; it generally consists of a system of tissue or organ compartments that are interconnected by the circulatory network. If necessary, each tissue or organ compartments can further be divided into extracellular and intracellular compartments to describe movement of toxicant at the cellular level. The exact model structure, or how the compartments are organized and linked together, depends on both the chemical and the organism being studied. For example, a physiologic model describing the disposition of a chemical in fish would require a description of the gills (Nichols et al., 1994), whereas a model for the same chemical in mammals would require a lung compartment (Ramsey and Andersen, 1984). Model structures can also vary with the chemicals being studied. For

Other Tissues

Kidney Kr

Km

Liver Kb Kec Intestine Kf Figure 7-11. Physiologic model for a hypothetical toxicant that is soluble in water, has a low vapor pressure (not volatile), and has a relatively large molecular weight (MW > 100). This hypothetical chemical is eliminated through metabolism in the liver (K m ), biliary excretion (K b ), renal excretion (K r ) into the urine, and fecal excretion (K f ). The chemical can also undergo enterohepatic circulation (K ec ). Perfusion-limited compartments are noted in blue and diffusionlimited compartments are noted in white.

example, a model for a non-volatile, water-soluble chemical, which might be administered by intravenous injection (Fig. 7-11), has a structure different from that of a model for a volatile organic chemical for which inhalation is the likely route of exposure (Fig. 7-12). The route of administration is not the only difference between these two models. For example, the first model has a compartment for the intestines, because biliary excretion, fecal elimination, and enterohepatic circulation are presumed important in the disposition of this chemical. The second model has a compartment for fat because fat is an important storage organ for organics. However, the models are not completely different. Both contain a liver compartment because the hepatic metabolism of each chemical is an important element of its disposition. It is important to realize that there is no generic physiologic model. Models are simplifications of reality and should contain elements believed to represent the essential disposition features of a chemical. In view of the fact that physiologic modeling requires more effort than does classic compartmental modeling, what then accounts for the popularity of this approach among toxicologists? The answer lies in the potential predictive power of physiologic models. Toxicologists are constantly faced with the issue of extrapolation in risk assessments—from laboratory animals to humans, from high to low doses, from occasional to continuous exposure, and from single chemicals to mixtures. Because the kinetic constants in physiologic models represent measurable biological or chemical processes, the resultant physiologic models have the potential for extrapolation from observed data to predicted scenarios.

318

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(Ramsey and Andersen, 1984; Andersen et al., 1984; Travis et al., 1990). The conclusion is that the same model structure is capable of describing the chemicals’ kinetics in two different species. Because the parameters underlying the model structure represent measurable biological and chemical determinants, the appropriate values for those parameters can be chosen for each species, forming the basis for successful interspecies extrapolation. Even though the same model structure is used for both rodents and humans, the simulated and the observed kinetics of both chemicals differ between rats and humans. The terminal half-life of both organics is longer in the human compared with the rat. This longer half-life for humans is due to the fact that clearance rates for smaller species are faster than those for larger ones. Even though the larger species breathes more air or pumps more blood per unit of time than does the smaller species, blood flows and ventilation rates per unit of body mass are greater for the smaller species. The smaller species has more breaths per minute or heartbeats per minute than does the larger species, even though each breath or stroke volume is smaller. The faster flows per unit mass result in a more efficient delivery of a chemical to organs responsible for elimination. Thus, a smaller species can eliminate the chemical faster than a larger one can. Because the parameters in physiologic models represent real, measurable values, such as blood flows and ventilation rates; the same model structure can resolve such disparate kinetic behaviors among species.

Figure 7-12. Physiological model for a typical volatile organic chemical. Chemicals for which this model would be appropriate have low molecular weights (MW < 100), are soluble in organic solvents, and have significant vapor pressures (volatile). Transport of chemical throughout the body by blood is depicted by the black arrows. Elimination of chemical as depicted by the model includes metabolism (dashed arrow) and exhalation (black arrow). All compartments are perfusion-limited.

One of the best illustrations of the predictive power of physiologic models is their ability to extrapolate kinetic behavior from laboratory animals to humans. For example, physiologic models developed for styrene and benzene correctly simulate the concentration of each chemical in the blood of both rodents and humans (Ramsey and Andersen, 1984; Travis et al., 1990). Simulations are the outcomes or results (such as a chemical’s concentration in blood or tissue) of numerically solving the model equations over a time period of concern, using a set of initial conditions (such as intravenous dose) and parameter values appropriate for the species (such as organ weights and blood flow). Both styrene and benzene are volatile organic chemicals; thus, the model structures for the kinetics of both chemicals in rodents and humans are identical to that shown in Fig. 7-12. However, the parameter values for rodents and humans are different. Humans have larger body weights than rodents, and thus weights of organs such as the liver are larger. Because humans are larger, they also breathe more air per unit of time than do rodents, and a human heart pumps a larger volume of blood per unit of time than does that of a rodent, although the rodent’s heart beats more times in the same period. The parameters that describe the chemical behavior of styrene and benzene, such as solubility in tissues, are similar in the rodents and human models. This is often the case because the composition of tissues in different species is similar. For both styrene and benzene, there are experimental data for humans and rodents and the model simulations can be compared with the actual data to see how well the model has performed

Compartments The basic unit of the physiologic model is the lumped compartment, which is often depicted as a box in a graphical scheme (Fig. 7-13). A compartment represents a definable anatomical site or tissue type in the body that acts as a unit in effecting a measurable kinetic process (Rowland, 1984, 1985). A compartment may represent a particular structure or functional portion of an organ, a segment of blood vessel with surrounding tissue, an entire discrete organ such as the liver or kidney, or a widely distributed tissue type such as fat or skin. Compartments usually consist of three individual well-mixed regions, or sub-compartments, that correspond to specific physiologic spaces or regions of the organ or tissue. These sub-compartments are: (1) the vascular space through which the compartment is perfused with blood, (2) the interstitial space that forms the matrix for the cells, and (3) the intracellular space consisting of the cells in the tissue (Gerlowski and Jain, 1983). As shown in Fig. 7-13, the toxicant enters the vascular subcompartment at a certain rate in mass per unit of time (e.g., mg/h). The rate of entry is a product of the blood flow rate to the tissue (Q t in L/h) and the concentration of the toxicant in the blood entering the tissue (Cin in mg/L). Within the compartment, the toxicant moves from the vascular space to the interstitial space at a certain net rate (Flux1 ) and moves from the interstitial space to the intracellular space at different net rate (Flux2 ). Some toxicants can bind to cell components; thus, within a compartment there may be both free and bound toxicants. The toxicant leaves the vascular space at a certain venous concentration (Cout ). Cout is equal to the concentration of the toxicant in the vascular space assuming a well-mixed compartment.

Parameters The most common types of parameters, or information required, in physiologic models are anatomic, physiologic, thermodynamic, and transport.

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319

been examples of remarkable success with quantitative prediction of in vivo metabolic clearance based on in vitro biochemical data, there are also notable failures. Unfortunately, estimation of metabolic parameters from in vivo studies is also fraught with difficulties, especially when multiple metabolic pathways and enzymes are present. Estimation of metabolic parameters remains a challenging aspect of physiologically based toxicokinetic modeling.

Figure 7-13. Schematic representation of a lumped tissue compartment in a physiologic model. The blood capillary and cell membranes separating the vascular, interstitial, and intracellular sub-compartments are depicted in heavy black lines. The vascular and interstitial sub-compartments are often combined into a single extracellular sub-compartment. Q t is blood flow, Cin is chemical concentration into the compartment, and Cout is chemical concentration out of the compartment.

Anatomic Anatomic parameters are used to describe the physical size of various compartments. The size is generally specified as a volume (milliliters or liters), because a unit density is assumed even though weights of organs and tissues are most frequently obtained experimentally. If a compartment contains sub-compartments such as those in Fig. 7-13, those volumes also must be known. Volumes of compartments often can be obtained from the literature or from specific toxicokinetic experiments. For example, kidney, liver, brain, and lung can be weighed. Obtaining precise data for volumes of compartments representing widely distributed tissues such as fat or muscle is more difficult. If necessary, these tissues can be removed by dissection and weighed. Among the numerous sources of general information on organ and tissue volumes across species, Brown et al. (1997) is a good starting point. Physiologic Physiologic parameters encompass a wide variety of processes in biological systems. The most commonly used physiologic parameters are blood flows and lung ventilation. The blood flow rate (Q t in volume per unit time, such as mL/min or L/h) to individual compartments must be known. Additionally, information on the total blood flow rate or cardiac output (Q c ) is necessary. If inhalation is the route for exposure to the chemical or is a route of elimination, the alveolar ventilation rate (Q p ) also must be known. Blood flow rates and ventilation rates can be taken from the literature or can be obtained experimentally. Parameters for renal excretion and hepatic metabolism are another subset of physiologic parameters, and are required, if these processes are important in describing the elimination of a chemical. For example, glomerular filtration rate and renal tubular transport parameters are required to describe renal clearance. If a chemical is known to be metabolized via a saturable process, both Vmax (the maximum rate of metabolism) and K M (the concentration of chemical at one-half Vmax ) for each of the enzymes involved must be obtained so that elimination of the chemical by metabolism can be described in the model. In principle, these parameters can be determined from in vitro metabolism studies with cultured cells, tissue homogenates, or cellular fractions containing the metabolic enzymes (e.g., microsomes for cytochrome P450 enzymes and UDP-GTs), along with appropriate in vitro-in vivo scaling techniques (Iwatsubo et al., 1997; MacGregor et al., 2001; Miners et al., 2006). Although there have

Thermodynamic Thermodynamic parameters relate the total concentration of a chemical in a tissue (Ct ) to the concentration of free chemical in that tissue (Cf ). Two important assumptions are that (1) total and free concentrations are in equilibrium with each other, and (2) only free chemical can be exchanged between the tissue sub-compartments (Lutz et al., 1980). Most often, total concentration is measured experimentally; however, it is the free concentration that is available for passage across membrane barriers, binding to proteins, metabolism, or carrier-mediated transport. Various mathematical expressions describe the relationship between these two entities. In the simplest situation, the toxicant is a freely diffusible water-soluble chemical that does not bind to any molecules. In this case, free concentration of the chemical is equal to the total concentration of the chemical in the tissue: total = free, or Ct = Cf . The affinity of many chemicals for tissues of different composition varies. The extent to which a chemical partitions into a tissue is directly dependent on the composition of the tissue and independent of the concentration of the chemical. Thus, the relationship between free and total concentration becomes one of proportionality: total = free × partition coefficient, or Ct = Cf × Pt . In this case, Pt is called a tissue partition coefficient, which is most often determined from tissue distribution studies in animals, ideally at steady-state during continuous intravenous infusion of the chemical. Estimation of Pt based on in vitro binding studies with human or animal tissues or tissue fractions has proven successful in some cases (Lin et al., 1982; MacGregor et al., 2001). Knowledge of the value of Pt permits an indirect calculation of the free concentration of toxicant in the tissue or Cf = Ct /Pt . Table 7-4 compares the partition coefficients for a number of toxic volatile organic chemicals. The larger values for the fat/blood partition coefficients compared with those for other tissues suggests that these chemicals distribute into fat to a greater extent than they distribute into other tissues. This has been observed experimentally. Fat and fatty tissues, such as bone marrow, contain higher concentrations of benzene than do tissues such as liver and blood. Similarly, styrene concentrations in fatty tissue are higher than styrene concentrations in other tissues. It should be noted that lipophilic organic compounds often can bind to plasma proteins and/or blood cell constituents, in which case the observed tissue/blood partition coefficients will be a function of both the tissue and blood partition coefficient (i.e., Pt /Pb ). Hence, partitioning or binding to blood Table 7-4 Partition Coefficients for Four Volatile Organic Chemicals in Several Tissues chemical

blood/air

muscle/blood

fat/blood

Isoprene Benzene Styrene Methanol

3 18 40 1350

0.67 0.61 1 3

24 28 50 11

320

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DISPOSITION OF TOXICANTS

constituents (Pb ) must be known in order to estimate the true thermodynamic partitioning coefficient for a tissue or the free toxicant concentration at equilibrium. Pb can be determined from in vitro studies of blood cells to plasma distribution and plasma protein binding of the chemical. A more complex relationship between the free concentration and the total concentration of a chemical in tissues is also possible. For example, the chemical may bind to saturable binding sites on tissue components. In these cases, nonlinear functions relating the free concentration in the tissue to the total concentration are necessary. Examples in which more complex binding has been used are physiologic models for dioxin and tertiary-amyl methyl ether (Andersen et al., 1993; Collins et al., 1999). Transport Passage of a chemical across a biological membrane may occur by passive diffusion, carrier-mediated transport involving either facilitated or active transporters, or a combination thereof (Himmelstein and Lutz, 1979). The simplest of these processes— passive diffusion is a first-order process described by Fick’s law of diffusion. Diffusion of a chemical occurs during its passage across the blood capillary membrane (Flux1 in Fig. 7-13) and across cell membrane (Flux2 in Fig. 7-13). Flux refers to the rate of transfer of a chemical across a boundary. For simple diffusion, the net flux (mg/h) from one side of a membrane to the other is governed by the barrier permeability and the chemical concentration gradient. Flux = PA × (C1 − C2 ) = PA × C1 − PA × C2

(7-17)

The term PA is often called the permeability-area product for the membrane or cellular barrier in flow units (e.g., L/h), and is a product of the barrier permeability coefficient (P in velocity units, e.g., μm/h) for the chemical and the total barrier surface area (A, in μm2 ). The permeability coefficient takes into account the diffusivity of the specific chemical and the thickness of the cell membrane. C1 and C2 are the respective free concentrations of the chemical in the originating and receiving compartments. Diffusional flux is enhanced when the barrier thickness is small, the barrier surface area is large, and a large concentration gradient exists. Membrane transporters offer an additional route of entry into cells, and allow more effective tissue penetration for chemicals that have limited passive permeability. Alternately, the presence of efflux transporters at epithelial or endothelial barriers can limit toxicant penetration into critical organs, even for highly permeable toxicant (e.g., P-glycoprotein mediated efflux functions as part of the blood–brain barrier). For both transporter-mediated influx and efflux processes, the kinetics is saturable and can be characterized by Tmax (the maximum transport rate) and K T (the concentration of toxicant at one-half Tmax ) for each of the transporters involved. In principle, kinetic parameters for passive permeability or carrier-mediated transport can be estimated from in vitro studies with cultured cell systems. However, the predictability and applicability of such in vitro approaches for physiologic modeling has not been systematically evaluated (MacGregor et al., 2001). At this time, the transport parameters have to be estimated from in vivo data, which are at times difficult and carry some degree of uncertainty. There are two limiting conditions for the uptake of a toxicant into tissues: perfusion-limited and diffusion-limited. An understanding of the assumptions underlying the limiting conditions is critical because the assumptions change the way in which the model equations are written to describe the movement of a toxicant into and out of the compartment.

Perfusion-Limited Compartments A perfusion-limited compartment, alternately referred to as blood flow-limited or simply flow-limited compartment, describes the situation when permeability across the cellular or membrane barriers (PA) for a particular toxicant is much greater than the blood flow rate to the tissue (Q t ), i.e., PA Q t . In this case, uptake of toxicant by tissue sub-compartments is limited by the rate at which the toxicant is presented to the tissue via the arterial inflow, and not by the rate at which the toxicant penetrates through the vascular endothelium, which is fairly porous in most tissues, or gains passage across the cell membranes. As a result, equilibration of a toxicant between the blood in the tissue vasculature and the interstitial sub-compartment is maintained at all times, and the two subcompartments are usually lumped together as a single extracellular compartment. An important exception to this vascular-interstitial equilibrium relationship is in the brain, where the capillary endothelium with its tight junctions poses a diffusional barrier between the vascular space and the brain interstitium. Furthermore, as indicated in Fig. 7-13, the cell membrane separates the extracellular compartment from the intracellular compartment. The cell membrane is the most crucial diffusional barrier in a tissue. Nonetheless, for molecules that are very small (molecular weight < 100) or lipophilic (log P > 2), cellular permeability generally does not limit the rate at which a molecule moves across cell membranes. For these molecules, flux across the cell membrane is fast compared with the tissue perfusion rate (PA Q t ), and the molecules rapidly distribute throughout the sub-compartments. In this case, free toxicant in the intracellular compartment is always in equilibrium with the extracellular compartment, and these tissue subcompartments can be lumped as a single compartment. Such a flowlimited tissue compartment is shown in Fig. 7-14. Movement into and out of the entire tissue compartment can be described by a single equation. Vt ×

dCt = Q t × (Cin − Cout ) dt

(7-18)

where Vt is the volume of the tissue compartment, Ct is the toxicant concentration in the compartment (Vt × C equals the amount of toxicant in the compartment), Vt × dCt /dt is the change in the amount of toxicant in the compartment with time, expressed as mass per unit of time, Q t is blood flow to the tissue, Cin is the toxicant concentration entering the compartment, and Cout is the toxicant

Figure 7-14. Schematic representation of a tissue compartment that features blood flow–limited uptake kinetics. Rapid exchange of toxicant between the extracellular space (blue) and intracellular space (light blue), unhindered by a significant diffusional barrier as symbolized by the dashed line, allows equilibrium to be maintained between the two sub-compartments at all times. In effect, a single compartment represents the tissue distribution of the toxicant. Q t is blood flow, Cin is the chemical concentration entering the compartment via the arterial inflow, and Cout is the chemical concentration leaving the compartment in the venous outflow.

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concentration leaving the compartment. Equations of this type are called mass-balance differential equations. Differential refers to the term dCt /dt. Mass balance refers to the requirement that the rate of change in the amount of toxicant in a compartment (left-hand side of Equation (7-18)) equals the difference in the rate of entry via arterial inflow and the rate of departure via venous outflow (right-hand side of Equation (7-18)). In the perfusion-limited case, the concentration of chemical in the venous drainage from the tissue is equal to the free concentration of chemical in the tissue (i.e., Cout = Cf ) when the chemical is not bound to blood constituents. As was noted previously, Cf (or Cout ) can be related to the total concentration of chemical in the tissue through a simple linear partition coefficient, Cout = Cf = Ct /Pt . In this case, the differential equation describing the rate of change in the amount of a chemical in a tissue becomes Vt × dCt /dt = Q t × [Cin − Ct /Pt ]

(7-19)

In the event the chemical does bind to blood constituents, blood partitioning coefficient needs to be recognized in the mass balance equation. Vt × dCt /dt = Q t × [Cin − Ct /(Pt /Pb )]

(7-20)

321

Figure 7-15. Schematic representation of a tissue compartment that features membrane-limited uptake kinetics. Perfusion of blood into and out of the extracellular compartment is depicted by thick arrows. Transmembrane transport (flux) from the extracellular to the intracellular sub-compartment is depicted by thin double arrows. Q t is blood flow, Cin is chemical concentration entering the compartment, and Cout is chemical concentration leaving the compartment.

describe the events in these two sub-compartments: Extracellular: Vt1 × dCt1 /dt = Q t × (Cin − Cout ) − PAt × (Ct1 /Pt1 )+PAt × (Ct2 /Pt2 ) (7-21) Intracellular: Vt2 × dCt2 /dt = PAt × (Ct1 /Pt1 ) − PAt × (Ct2 /Pt2 )

The physiologic model shown in Fig. 7-12, which was developed for volatile organic chemicals such as styrene and benzene, is a good example of a model in which all the compartments are described as flow-limited. Distribution of a toxicant in all the compartments is described by using equations of the type noted above. In a flow-limited compartment, the assumption is that the concentrations of a toxicant in all parts of the tissue are in equilibrium. For this reason, the compartments are generally drawn as simple boxes (Fig. 7-12) or boxes with dashed lines that symbolize the equilibrium between the intracellular and extracellular sub-compartments (Fig. 7-14). Additionally, with a flow-limited model, estimates of fluxes between sub-compartments are not required to develop the mass balance differential equation for the compartment. Given the challenges in measuring flux across the vascular endothelium and cell membrane, this is a simplifying assumption that significantly reduces the number of parameters required in the physiologic model.

Diffusion-Limited Compartments When uptake of a toxicant into a compartment is governed by its diffusion or transport across cell membrane barriers, the model is said to be diffusion-limited or barrier-limited. Diffusion-limited uptake or release occurs when the flux, or the transport of a toxicant across cell membranes, is slow compared with blood flow to the tissue. In this case, the permeability-area product is small compared with blood flow, i.e., PA  Q t . The distribution of large polar molecules into tissue cells is likely to be limited by the rate at which the molecules pass through cell membranes. In contrast, entry into the interstitial space of the tissue through the leaky capillaries of the vascular space is usually rapid even for large molecules. Figure 7-15 shows the structure of such a compartment. The toxicant concentrations in the vascular and interstitial spaces are in equilibrium and make up the extracellular sub-compartment, where uptake from the incoming blood is flow-limited. The rate of toxicant uptake across the cell membrane from the extracellular space into the intracellular space is limited by membrane permeability, and is thus diffusionlimited. Two mass balance differential equations are necessary to

(7-22)

Q t is blood flow, and C is the chemical concentration in entering blood (in), exiting blood (out), tissue extracellular space (t1), or tissue intracellular space (t2). The subscript (t) for the PA term acknowledges the fact that PA, reflecting either via passive diffusion or carrier-mediated processes, can differ between tissues. Both equations feature fluxes or transfers across the cell membrane that are driven by free concentration. Hence, partition coefficients are needed to convert extracellular and intracellular tissue concentration to their corresponding free concentration. The physiologic model in Fig. 7-11 is composed of two diffusion-limited compartments each of which contain two sub-compartments—extracellular and intracellular space, and several perfusion-limited compartments.

Specialized Compartments Lung The inclusion of a lung compartment in a physiologic model is an important consideration because inhalation is a common route of exposure to many volatile toxic chemicals. Additionally, the lung compartment serves as an instructive example of the assumptions and simplifications that can be incorporated into physiologic models while maintaining the overall objective of describing processes and compartments in biologically relevant terms. For example, although lung physiology and anatomy are complex, Haggard (1924) developed a simple approximation that sufficiently describes the uptake of many volatile chemicals by the lungs. A diagram of this simplified lung compartment is shown in Fig. 7-16. The assumptions inherent in this compartment description are as follows: (1) ventilation is continuous, not cyclic; (2) conducting airways (nasal passages, larynx, trachea, bronchi, and bronchioles) function as inert tubes, carrying the vapor to the pulmonary or gas exchange region; (3) diffusion of vapor across the lung cell and capillary walls is rapid compared with blood flow through the lung; (4) all chemicals disappearing from the inspired air appears in the arterial blood (i.e., there is no hold-up of chemical in the lung tissue and insignificant lung mass); and (5) vapor in the alveolar air and arterial blood within the lung compartment are in rapid equilibrium and are related by Pb/a , the

322

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chemical in the blood increases. Additionally, the time to reach the steady-state concentration and the time to clear the chemical also increase with increasing Pb/a . Fortunately, Pb/a is readily measured by using in vitro techniques in which a volatile chemical in air is equilibrated with blood in a closed system, such as a sealed vial (Gargas and Andersen, 1988). Figure 7-16. Simple model for exchange of volatile chemicals in the alveolar region of the respiratory tract. Rapid exchange in the lumped lung compartment between the alveolar gas (blue) and the pulmonary blood (light blue) maintains the equilibrium between them as symbolized by the dashed line. Q p is alveolar ventilation (L/h); Q c is cardiac output (L/h); Cinh is inhaled vapor concentration (mg/L); Cart is concentration of vapor in the arterial blood; Cven is concentration of vapor in the mixed venous blood. The equilibrium relationship between the chemical in the alveolar air (Calv ) and the chemical in the arterial blood (Cart ) is determined by the blood/air partition coefficient Pb/a , i.e., Calv = Cart /Pb/a .

blood/air partition coefficient (e.g., Calv = Cart /Pb/a ). Pb/a is a thermodynamic parameter that quantifies the equilibrium partitioning of a volatile chemical between blood and air. In the lung compartment depicted in Fig. 7-16, the rate of inhalation of a volatile chemical is controlled by the ventilation rate (Q p ) and the inhaled concentration (Cinh ). The rate of exhalation of the chemical is a product of the ventilation rate and the chemical’s concentration in the alveoli (Calv ). Chemical also can enter the lung compartment via venous blood returning from the heart, at a rate represented by the product of cardiac output (Q c ) and the concentration of chemical in venous blood (Cven ). Chemical leaving the lungs via the blood at a rate determined by both cardiac output and the concentration of chemical in arterial blood (Cart ). Putting these four processes together, a mass balance differential equation can be written for the rate of change in the amount of chemical in the lung compartment (L): dL = Q p × (Cinh − Calv ) + Q c × (Cven − Cart ) dt

(7-23)

Because of these assumptions, at steady-state the rate of change in the amount of chemical in the lung compartment becomes zero (dL/dt = 0). Calv can be replaced by Cart /Pb/a , and the differential equation can be solved for the arterial blood concentration: Cart = (Q p × Cinh + Q c × Cven )/(Q c + Q p /Pb/a )

Liver The liver is almost always featured as a distinct compartment in physiologic models because biotransformation is an important route of elimination for many toxicants and the liver is considered the major organ for biotransformation of xenobiotics. A simple compartmental structure for the liver is depicted in Fig. 7-17, where uptake into the liver compartment is assumed to be flow-limited. This liver compartment is similar to the general tissue compartment in Fig. 7-14, except that the liver compartment contains an additional process for metabolic elimination. Under first-order elimination, the rate of hepatic metabolism (R) by the liver can be presented as: R = Cll × Cf

(7-25)

where Cf is the free concentration of toxicant in the liver (mg/L), and Cll is the clearance of free toxicant within the liver (L/h). The latter parameter is conceptually the same as the intrinsic hepatic clearance term (Clint,h ) in Equation (7-12). In the case of a single enzyme mediating the biotransformation and Michaelis–Menten kinetics are obeyed, Cll is related to the maximum rate of metabolism, Vmax (in mg/h) and the Michaelis constant, K M (in mg/L) (Andersen, 1981). As a result, the rate of hepatic metabolism can be expressed in terms of the Michaelis parameters. R = [Vmax /(K M + Cf )] × Cf

(7-26)

Under non-saturating or first-order condition (i.e., Cf  K M ), Cll becomes equal to the ratio of Vmax /K M . Because many chemicals at toxic levels display saturable metabolism, the above equation is a key factor in the success of physiologic models for simulation of chemical disposition across a wide range of doses. Other, more complex expressions for metabolism also can be incorporated into physiologic models. Bi-substrate second-order

(7-24)

This algebraic equation is incorporated into physiologic models for many volatile organics. Because the lung is viewed here as a portal of entry and not as a target organ, the concentration of a chemical delivered to other organs by the arterial blood is of primary interest. The assumptions of continuous ventilation, dead space, rapid equilibration with arterial blood, and no hold-up of vapor in the lung tissues have worked extremely well with many volatile organics, especially relatively lipophilic volatile solvents. Indeed, the use of these assumptions simplifies and speeds model calculations and may be entirely adequate for describing the chemical behavior of relatively inert vapors with low water solubility. Inspection of the equation for calculating the arterial concentration of the inhaled organic vapor indicates that the term Pb/a , the blood/air partition coefficient of the chemical, becomes an important term for simulating the uptake of various volatile organics. As the value for Pb/a increases, the maximum concentration of the

Figure 7-17. Schematic representation of a flow-limited liver compartment in which metabolic elimination occurs. R, in milligrams per hour, is the rate of metabolism. Q l is hepatic blood flow, Cin is chemical concentration entering the liver compartment, and Cout is chemical concentration out of the liver compartment. It should be noted that the liver receives blood from two sources, arterial inflow via hepatic artery and outflow from the upper mesentery via portal vein. This dual inflow is featured in the physiologic model featuring enterohepatic circulation in Fig. 7-11. Inflow via hepatic artery is often ignored, as in this case and in the physiologic model shown in Fig. 7-12, because of its relatively small flow rate compared to portal flow.

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Figure 7-18. Model simulations compared with experimental data obtained in growing adult rats dosed orally with 203Hg-labelled methylmercury chloride. Panel A shows the physiologic model for methylmercury and its demethylated product, inorganic mercury. Primed symbols denote the parameters for inorganic mercury. Panels B and C show the respective time course of methylmercury and inorganic mercury concentration in blood, liver, kidneys and the brain. Panel C shows the slow accumulation of total mercury in hair and skin, or the integument (skin plus hair). Reprinted from Farris FF, Dedrick RL, Allen P, et al.: Physiological model for the pharmacokientics of methyl mercury in the growing rat. Toxciol Appl Pharmacol 119:74–90, 1993. With permission from Elsevier.

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reactions, reactions involving the destruction of enzymes, inhibition of enzymes, or the depletion of cofactors, have been simulated using physiologic models. Metabolism can be also included in other compartments in much the same way as described for the liver. Blood In a physiologic model, the tissue compartments are linked together by the circulatory network. Figures 7-11 and 7-12 represent different approaches toward describing the circulating blood in physiologic models. In general, the arterial system delivers a chemical into various tissues. Exceptions are the liver, which receives arterial and portal blood, and the lungs, which receive mixed venous blood from the right cardiac ventricle. In the body, the venous blood supplies draining from tissue compartments eventually merge in the vena cava and heart chambers to form mixed venous blood. In Fig. 7-11, a blood compartment is created in which the input is the sum of the toxicant outflow from each compartment (Q t × Cvt ). Outflow from the blood compartment is a product of the blood concentration in the compartment and the total cardiac output (Q c × Cbl ). The mass-balance differential equation for the blood compartment in Fig. 7-11 is as follows: Vbl × dCbl /dt = (Q br × Cvbr + Q ot × Cvot + Q k × Cvk + Q l Cvl ) − Q c × Cbl ,

(7-27)

where Vbl is the volume of the blood compartment; C is concentration; Q is blood flow; bl, br, ot, k, and l represent the blood, brain, other tissues, kidney, and liver compartments, respectively; and vbr, vot, vk, and vl represent the venous blood leaving the organs. Q c is the total blood flow equal to the sum of the venous blood flows from each organ. In contrast, the physiologic model in Fig. 7-12 does not feature an explicit blood compartment. For simplicity, the blood volumes of the heart and the major blood vessels that are not within organs are assumed to be negligible. The venous concentration of a chemical returning to the lungs is simply the weighted average of the concentrations in the venous blood emerging from the tissues. Cv = (Q l × Cvl × Q rp × Cvrp + Q pp × Cvpp + Q f × Cvf )/Q c (7-28) where C is concentration; Q is blood flow; v, l, rp, pp, and f represent the venous blood entering the lungs, liver, richly perfused, poorly perfused, and fat tissue compartments, respectively; and vl, vrp, vpp, and vf represent the venous blood leaving the corresponding organs. Q c is the total blood flow equal to the sum of the blood flows exiting each organ. In the physiologic model in Fig. 7-12, the blood concentration entering each tissue compartment is the arterial concentration (Cart ) that was calculated above for the lung compartment (Equation (7-24)). The decision to use one formulation as opposed to another to describe blood in a physiologic model depends on the role the blood plays in disposition and the type of application. If the toxicokinetics after intravenous injection is to be simulated or if binding to or metabolism by blood components is suspected, a separate compartment for the blood that incorporates these additional processes is required. A blood compartment is obviously needed if the model were developed to explain a set of blood concentration–time data for a toxicant. On the other hand, if blood is simply a conduit to the other compartments, as in the case for

inhaled volatile organics shown in Fig. 7-12, the algebraic solution is acceptable. Figure 7-18 shows the application of physiologic modeling in elucidating the disposition fate of methylmercury and its demethylated product, inorganic mercury following a single peroral administration in the growing adult rat (Farris et al., 1993). The model scheme presented in Panel A for both the organic and inorganic forms of mercury has the following unique features: (1) increase in compartment size due to growth over the long duration of the tissue washout experiment; (2) mercury enters the gut lumen via biliary excretion and secretory transport from gut tissue to lumen, some of which is reabsorbed; (3) uptake of methylmercury from blood into tissues, except for the brain, is assumed to be rate-limited by plasma flow because of sequestration of mercury in rat erythrocytes and its slow release; (4) uptake of methylmercury across the blood brain barrier is rate-limited by transport; (5) uptake of inorganic mercury into all tissues, except the liver, is limited by permeability/transport; (6) mercury is transferred from skin into hair, where it is irreversibly bound; and (7) mercury in hair is either shed or ingested by the animal during grooming. The model simulations displayed in Panels B, C, and D show two prominent features of methylmercurcy disposition: (1) methylmercury is rapidly demethylated to inorganic mercury, which is slowly eliminated from the brain and the kidneys, two major sites of methylmercury toxicities; and (2) a significant portion of mercury is sequestered in hair and the ingestion of hair by the animal contributes to the remarkable persistence of mercury in the rat. This example illustrates the capability of physiologic models to deal with the varied and complex disposition kinetics of toxicants from a wide range of sources under a multitude of experimental and environmental exposure scenarios.

Conclusions The second section provides an introduction to the simpler elements of physiologic models and the important assumptions that underlie model structures. For more detailed aspects of physiologic modeling, the readers can consult several in-depth and well-annotated reviews on physiologically based toxicokinetic models (Clewell and Anderson, 1994, 1996; O’Flaherty, 1998; Krishnan and Anderson, 2001; Krishnan et al., 2002; Anderson, 2003). Computer softwares are available for numerically integrating the system of differential equations that form the models. Investigators have successfully used Advanced Continuous Simulation Language (Pharsight Corp., Palo Alto, CA), Simulation Control Program (Simulation Resources, Inc., Berrien Springs, MI), MATLAB (The MathWorks, Inc., Natick, MA), and SAS software applications to name a few. Choice of software depends on prior experience, familiarity with the computer language used, and cost of the software package. The field of physiologic modeling is evolving as toxicologists and pharmacologists develop increasingly more sophisticated applications. Three-dimensional visualizations of xenobiotic transport in fish and vapor transport in the rodent nose, physiologic models of a parent chemical linked in series with one or more active metabolites, models describing biochemical interactions among xenobiotics, and more biologically realistic descriptions of tissues previously viewed as simple lumped compartments are just a few of the more sophisticated applications. Finally, physiologically based toxicokinetic models are beginning to be linked to biologically based toxicodynamic models to simulate the entire exposure → dose → response paradigm that is basic to the science of toxicology.

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REFERENCES Andersen ME: A physiologically based toxicokinetic description of the metabolism of inhaled gases and vapors: Analysis at steady state. Toxicol Appl Pharmacol 60:509–526, 1981. Andersen ME: Physiological modeling of organic compounds. Ann Occup Hyg 35:309–321, 1991. Andersen ME: Toxicokinetic modeling and its applications in chemical risk assessment. Toxicol Lett 138:9–27, 2003. Andersen ME, Gargas ML, Ramsey JC: Inhalation pharmacokinetics: Evaluating systemic extraction, total in vivo metabolism, and the time course of enzyme induction for inhaled styrene in rats based on arterial blood:inhaled air concentration ratios. Toxicol Appl Pharmacol 73:176–187, 1984. Andersen ME, Mills JJ, Gargas ML, et al.: Modeling receptor-mediated processes with dioxin: Implications for pharmacokinetics and risk assessment. Risk Anal 13:25–36, 1993. Bertilsson L, Tomson T, Tybring G: Pharmacokinetics: time-dependent changes—autoinduction of carbamazepine epoxidation. J Clin Pharmacol 26:459–462, 1986. Brown RP, Delp MD, Lindstedt SL, et al.: Physiological parameter values for physiologically based pharmacokinetic models. Toxicol Ind Health 13:407–484, 1997. Burbacher TM, Shen DD, Lalovic B, et al.: Chronic maternal methanol inhalation in nonhuman primates (Macaca fascicularis): Exposure and toxicokinetics prior to and during pregnancy. Neurotoxicol Teratol 26:201–221, 2004. Clewell HJ IIIrd, Andersen ME. Physiologically-based pharmacokinetic modeling and bioactivation of xenobiotics. Toxicol Ind Health 10:1– 24, 1994. Clewell HJ IIIrd, Andersen ME. Use of physiologically based pharmacokinetic modeling to investigate individual versus population risk. Toxicology 111:315–329, 1996. Collins AS, Sumner SCJ, Borghoff SJ, et al.: A physiological model for tertamyl methyl ether and tert-amyl alcohol: Hypothesis testing of model structures. Toxicol Sci 49:15–28, 1999. Farris FF, Dedrick RL, Allen P, et al.: Physiological model for the pharmacokientics of methyl mercury in the growing rat. Toxciol Appl Pharmacol 119:74–90, 1993. Gargas ML, Andersen ME: Physiologically based approaches for examining the pharmacokinetics of inhaled vapors, in Gardner DE, Crapo JD, Massaro EJ (eds): Toxicology of the Lung. New York: Raven Press, 1988, pp. 449–476. Gerloski LE, Jain RK: Physiologically based pharmacokinetic modeling: Principles and applications. J Pharm Sci 72:1103–1127, 1983. Gibaldi M, Perrier D: Pharmacokinetics, 2nd ed. New York: Marcel Dekker, 1982. Haggard HW: The absorption, distribution, and elimination of ethyl ether: II. Analysis of the mechanism of the absorption and elimination of such a gas or vapor as ethyl ether. J Biol Chem 49:753–770, 1924. Himmelstein KJ, Lutz RJ: A review of the applications of physiologically based pharmacokinetic modeling. J Pharmacokinet Biopharm 7:127– 145, 1979. Iwatsubo T, Hirota N, Ooie T, et al.: Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol Ther 73:147–171, 1997.

Krishnan K, Anderson ME: Physiologically based pharmacokinetic modeling in toxicology, in Hayes AW (ed): Principles and Methods of Toxicology. Philadelphia: Taylor & Francis, 2001, pp. 193– 241. Krishnan K, Haddad S, Beliveau M, et al.: Physiological modeling and extrapolation of pharmacokinetic interactions from binary to more complex chemical mixtures. Environ Health Perspect 110 (Suppl 6):989– 994, 2002. Lin JH, Sugiyama Y, Awazu S, et al.: In vitro and in vivo evaluation of the tissue-to-blood partition coefficient for physiological pharmacokinetic models. J Pharmacokinet Biopharm 10:637–647, 1982. Lutz RJ, Dedrick RL, Zaharko DS: Physiological pharmacokinetics: An in vivo approach to membrane transport. Pharmacol Ther 11:559–592, 1980. MacGregor JT, Collins JM, Sugiyama Y, et al.: In vitro human tissue models in risk assessment: report of a consensus-building workshop. Toxicol Sci 59:17–36, 2001. Miners JO, Knights KM, Houston JB, et al.: In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises. Biochem Pharmacol 71:1531–1539, 2006. Nichols J, Rheingans P, Lothenbach D, et al.: Three-dimensional visualization of physiologically based kinetic model outputs. Environ Health Perspect 102:952–956, 1994. O’Flaherty EJ. Physiologically based models of metal kinetics. Crit Rev Toxicol 28:271–317, 1998. Ramsey JC, Andersen ME: A physiologically based description of the inhalation pharmacokinetics of styrene in rats and humans. Toxicol Appl Pharmacol 73:159–175, 1984. Rowland M: Physiologic pharmacokinetic models and interanimal species scaling. Pharmacol Ther 29:49–68, 1985. Rowland M: Physiologic pharmacokinetic models: Relevance, experience, and future trends. Drug Metab Rev 15:55–74, 1984. Rowland M, Tozer TN: Clinical Pharmacokinetics. Baltimore: Williams & Wilkins, 1995. Schentag JJ, Jusko WJ: Renal clearance and tissue accumulation of gentamicin. Clin Pharmacol Ther 22:364-70, 1977. Teorell T: Kinetics of distribution of substances administered to the body: I. The extravascular modes of administration. Arch Int Pharmacodyn Ther 57:205–225, 1937. Thummel KE, Shen DD, Isoherranen N, Smith HE: Appendix II. Design and optimization of dosage regimens: Pharmacokinetic data. In: Brunton LL, Parker KL, Buxton IOL, Blumenthal DK (eds.): Goodman & Gilman’s: The Pharmacological Basis of Therapeutics, 11th ed. The McGraw-Hill Companies, 2006. Tozer NT, Rowland M: Introduction to Pharmacokinetics and Pharmacodynamics: The Quantitative Basis of Drug Therapy. Lippincott Williams & Wilkins, 2006, p. 75. Travis CC, Quillen JL, Arms AD: Pharmacokinetics of benzene. Toxicol Appl Pharmacol 102:400–420, 1990. Wilkinson GR. Clearance approaches in Pharmacology. Pharmacol Rev 39:1–47, 1987.

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CHAPTER 8

CHEMICAL CARCINOGENESIS James E. Klaunig and Lisa M. Kamendulis Receptor Mediated Hormonal Mode of Action DNA Methylation and Carcinogenesis Oxidative Stress and Chemical Carcinogenesis Gap Junctional Intercellular Communication and Carcinogenesis Modifiers of Chemical Carcinogenic Effects Polymorphisms in Carcinogen Metabolism and DNA Repair Proto-Oncogenes and Tumor-Suppressor Genes Retroviruses DNA Viruses Proto-Oncogenes Tumor Suppressor Genes Hormesis and Carcinogenesis Chemoprevention

OVERVIEW HISTORICAL BACKGROUND Definitions MULTISTAGE CARCINOGENESIS Initiation Promotion Progression MECHANISMS OF ACTION OF CHEMICAL CARCINOGENS Genotoxic Carcinogens Direct-Acting (Activation-Independent) Carcinogens Indirect-Acting Genotoxic Carcinogens Mutagenesis Damage by Alkylating Electrophiles DNA Repair DNA Repair Mechanisms Mismatch Repair of Single-Base Mispairs Excision Repair End-Joining Repair of Nonhomologous DNA Classes of Genotoxic Carcinogens Polyaromatic Hydrocarbons Alkylating Agents Aromatic Amines and Amides Inorganic Carcinogens Arsenic Beryllium Cadmium Chromium Nickel Lead Nongenotoxic (Epigenetic) Carcinogens Cytotoxicity α2u -Globulin-Binding Chemicals

TEST SYSTEMS FOR CARCINOGENICITY ASSESSMENT Short-term Tests for Mutagenicity In Vitro Gene Mutation Assays In Vivo Gene Mutation Assays Chromosomal Alterations DNA Damage Transformation Assays Chronic Testing for Carcinogenicity Chronic (2-Year) Bioassay Organ-Specific Bioassays and MultiStage Animal Models Transgenic Animals in Carcinogenicity Assessment CHEMICAL CARCINOGENESIS IN HUMANS Classification Evaluation of Carcinogenicity in Humans SUMMARY

OVERVIEW

decades, many aspects of the causes, prevention, and treatment of human cancers remain unresolved.

Cancer is a disease of cellular mutation, proliferation, and aberrant cell growth. It ranks as one of the leading causes of death in the world. In the United States, cancer ranks as the second leading cause of death, with over one million new cases of cancer diagnosed and more than one half million Americans die from cancer annually. Multiple causes of cancer have been either firmly established or suggested, including infectious agents, radiation, and chemicals. Estimates suggest that 70–90% of all human cancers have a linkage to environmental, dietary, and behavioral factors (Fig. 8-1). While our understanding of the biology of the progression from a normal cell to a malignant one has advanced considerably in the past several

HISTORICAL BACKGROUND A strong historical foundation for the linkage of the induction of cancer by chemicals has been documented (Table 8-1). In 1775, Percival Pott described a linkage between the increased occurrence of scrotal and nasal cancer among chimney sweepers and their profession. Pott (1775) concluded that chimney soot was the causative agent for cancer induction in these individuals. Other investigators also recognized an association between exposure to chemicals and the 329

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Causes of Human Cancers

35 30

% of Human Cancers

25 20 15 10 5 0 Salt/other food additives/contaminants

Ionizing/ultraviolet radiation

Environmental pollution

Prescription drugs/medicine procedures

Alcohol

Socioeconomic status

Reproductive factors

Perinatal factors/growth

Viruses/other biologic agents

Family history of cancer

Occupational factors

Sedentary lifestyle

Adult diet/obesity

Tobacco

Figure 8-1. Major causes of Human Cancers. Adapted from Harvard Reports on Cancer Prevention volume 1 (1996).

induction of human cancer. Thiersch (1875) described a relationship between sunlight exposure and skin cancer in humans. A linkage between an increased incidence of lung cancer and uranium mining was noted by Harting and Hesse (1879). Butlin, in a follow-up of Potts’ observations, noted that scrotal cancer in chimney sweepers in the European continent was relatively rare compared to that seen in England (Butlin, 1892). He attributed this difference to better hygiene practices by the European sweepers as well as to the use of younger boys as sweeps in England, suggesting that the age of exposure and the duration of exposure influence the formation of the cancer. Rehn (1895) reported a linkage between the manufacturer of aniline dyes and the induction of bladder cancer in dye workers. Due to the increase in demand for synthetic dyes in the 19th century, production of the aniline-based dyes increased, as did the development of skin and bladder cancer in exposed workers. The specific chemicals in the dyes related to the cause of skin and bladder cancer were later determined to be the aromatic compounds 2-napthylamine and benzidine (Hueper et al., 1938). Exposure of workers to metals such as chromium (Alwens and Jonas, 1932), nickel (Stephens, 1932), as well as asbestos (Wood and Gloyne, 1934) was shown to be associated with an increased incidence of lung cancers in workers.

Based on these human observations, further investigations in the first half of the 20th century were performed to examine the role of these chemical mixtures and individual chemical compounds in the mixtures using animal models. Initial studies by Yamagiwa and Ichikawa (1915) examined the linkage between exposure to coal tar and its derivatives on cancer induction in humans by painting coal tar on rabbit ears. This resulted in skin cancer in the rabbits. Subsequently, Kennaway and Hieger (1930) purified the compound dibenz(a,h)anthracene from the coal tar extract, and determined that dibenz(a,h)anthracene was at least one compound in the coal tar mixture that was responsible for inducing skin cancer in mice. Cook et al. (1933) similarly isolated another polyaromatic hydrocarbon, benzo(a)pyrene, from the coal tar and showed this to also function as a carcinogen in rodents. In similar approaches with aromatic amines, Hueper et al. (1937) reported that 2-naphthylamine induced bladder tumors in dogs, and Yoshida and Kinosita (1936) found that feeding aminoazotoluene or aminoazobenzene to rodents induced liver cancer. Studies with the aromatic amine compounds in animal models correlated with the human epidemiological studies that had revealed an association between bladder cancer in humans and exposure to aniline dyes, and helped to define the specific chemicals in the dyes responsible for the cancer induction. These

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Table 8-1 Historical Events in Chemically Induced Cancer date

investigator(s)

causative agent(s)

1775 1822 1875 1876 1879 1892 1895 1902 1915 1915 1920 1928 1930 1932 1932 1933 1936 1934 1934 1936 1938 1941

Pott Ayrton Thiersch Manourriez Harting and Hesse Butlin Rehn Frieben Davis Yamagiwa, Ichikawa, and Tsusui Leitch and Seguina Delore and Bergamo Kennaway and Hieger Stephens Alwens Cook, Hewett, and Hieger Yoshida and Kinosita Wood and Gloyne Neitzel Kawahata Hueper, Wiley, and Wolfe Berenblum, Rous, MacKenzie, and Kidd

1951

Miller and Miller

Soot and chimney sweeps Arsenic-containing metal Sunlight Coal tar Lung cancer and uranium Soot and chimney sweeps Manufacture of aniline dyes X-rays Pipe smokers and betel nut chewers Induction of skin cancer in rabbits and mice by coal tar Radium radiation Benzene Tumor induction by dibenz[a,h] anthracene Nickel Chromium compounds Isolation of the carcinogen benzo[a]pyrene from coal tar Induction of liver cancer in rats by o-aminoazotoluene Arsenicals, beryllium, and asbestos Mineral oil mists and radiation Coal tar fumes Induction of urinary cancer in dogs by 2-naphthylamine Initiation and promotion stages in skin carcinogenesis with benzo[a]pyrene Carcinogen binding to cellular macromolecule

epidemiological and experimental studies have shown a clear relationship between the induction of cancer in humans and rodents by a specific chemical or chemical mixture. In addition, this experimental work, demonstrated that animal models could be used as surrogates for humans in the study of chemical carcinogenesis.

Definitions An understanding of the cellular and molecular aspects of the cancer process requires an understanding of the pathology and scien-

tific terms involved in defining neoplasia (Table 8-2). Neoplasia is often defined as new growth or autonomous growth of tissue. The resulting neoplastic lesion is referred to as a neoplasm. Both benign and malignant neoplasms can be induced by chemical carcinogens. Benign neoplasms are lesions characterized by expansive growth, frequently exhibiting slow rates of proliferation that do not invade surrounding tissue or other organs. In contrast, a malignant neoplasm demonstrates invasive growth characteristics, capable of spreading throughout the organ of origin, and through metastasis to other tissues and organs. Metastases are

Table 8-2 Terminology Neoplasia Neoplasm Benign

Malignant Metastases Tumor Cancer Carcinogen Genotoxic Nongenotoxic

New growth or autonomous growth of tissue The lesion resulting from the neoplasia Lesions characterized by expansive growth, frequently exhibiting slow rates of proliferation that do not invade surrounding tissues Lesions demonstrating invasive growth, capable of metastases to other tissues and organs Secondary growths derived from a primary malignant neoplasm Lesion characterized by swelling or increase in size, may or may not be neoplastic Malignant neoplasm A physical or chemical agent that causes or induces neoplasia Carcinogens that interact with DNA resulting in mutation Carcinogens that modify gene expression but do not damage DNA

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Table 8-3 Neoplasm Nomenclature tissue of origin Connective tissue Bone Fibrous Fat Lipid Blood cells and related cells Hematopoietic cells Lymphoid tissue Muscle Smooth Striated Endothelium Mesothelium Epithelial Squamous Respiratory Renal epithelium Liver cells Urinary epithelium Testicular epithelium Melanocytes

benign neoplasm

malignant neoplasm

Osteoma Fibroma Lipoma

Osteosarcoma Fibrosarcoma Liposarcoma Leukemias Lymphomas

Leiomyoma Rhabdomyoma Hemangioma

Leiomyosarcoma Rhabdomyosarcoma Angiosarcoma Mesothelioma

Squamous cell papilloma Bronchial adenoma Renal tubular adenoma Liver cell adenoma Transitional cell papilloma

Squamous cell or carcinoma Bronchogenic carcinoma Renal cell carcinoma Hepatocellular carcinoma Transitional cell carcinoma Seminoma Malignant melanoma

secondary growths derived from the cells of the primary malignant neoplasm. The term “tumor” describes a lesion that may or may not be neoplastic, and is characterized by swelling or an increase in size. In classifying neoplasms, the nomenclature reflects both the tissue or cell of origin, and the characteristics of the type of tissue involved (Table 8-3). For benign neoplasms, the tissue of origin is frequently followed by the suffix “oma”; for example, a benign fibrous neoplasm would be termed fibroma, and a benign glandular epithelium termed an adenoma. Malignant neoplasms from epithelial origin are called carcinomas while those derived from mesenchymal origin are referred to as sarcoma. Thus, a malignant neoplasm of fibrous tissue would be a fibrosarcoma, whereas that derived from bone would be an ostoesarcoma. Similarly, a malignant neoplasm from the liver would be a hepatocellular carcinoma, whereas that derived from skin would be referred to as a squamous cell carcinoma. Preneoplastic lesions have also been observed in a number of target organs in both animal models and humans, and reflect an early reversible lesion in neoplasm progression. The characterization and study of preneoplastic cells has led to a further understanding of the process of cancer formation. The term “cancer” describes the subset of neoplasia that represents malignant neoplasms. A carcinogen is an agent, chemical or physical, that causes or induces neoplasia. This definition has been more thoroughly defined as an agent whose administration to previously untreated animals leads to a statistically significant increased incidence of neoplasia of one or more histogenetic types, as compared with the incidence of the appropriate untreated control animals (Pitot, 1986). Thus, the induction of both benign and malignant neoplasms is included in this definition. Carcinogens can be chemicals, viruses, hormones, radiation, or solid materials. Carcinogens either produce new neoplastic growth in a tissue or organ or increase the incidence and/or multiplicity of background spontaneous neoplastic formation in the target tissue.

Table 8-4 Features of Genotoxic and Nongenotoxic Carcinogens Genotoxic carcinogens Mutagenic Can be complete carcinogens Tumorigenicity is dose responsive No theoretical threshold Nongenotoxic carcinogens Nonmutagenic Threshold, reversible Tumorigenicity is dose responsive May function at tumor promotion stage No direct DNA damage Species, strain, tissue specificity

Carcinogens may be genotoxic, meaning that they interact physically with DNA to damage or change its structure. Other carcinogens may change how DNA expresses information without modifying or directly damaging its structure, or may create a situation in a cell or tissue that makes it more susceptible to DNA damage from other sources. Chemicals belonging to this latter category are referred to as nongenotoxic carcinogens. Common features of genotoxic and nongenotoxic carcinogens are shown in Table 8-4. This knowledge has led to continued efforts, using both epidemiological information and experimental animal models, to assess chemical carcinogenicity of occupational, industrial, and environmental agents, to gain an understanding of the mechanisms of action for these agents, and to determine the relevance of human exposure to cancer risk.

CHAPTER 8

Normal Cell Repair

DNA Damage

CHEMICAL CARCINOGENESIS

Initiated Cell

Focal Lesion Apoptosis

Cancer Apoptosis

Proliferation

Initiation

333

Proliferation

Promotion

Progression

Figure 8-2. Multistage model of carcinogenesis.

MULTISTAGE CARCINOGENESIS Through extensive experimental studies with animal models and evaluation of human cancers, it has been shown that the carcinogenesis process involves a series of definable and reproducible stages. Operationally, these stages have been defined as initiation, promotion, and progression (Fig. 8-2). These steps follow a temporal sequence of events that have been observed in a wide variety of target tissues. The defining characteristics of each of these stages are outlined in Table 8-5.

Initiation The first stage of the cancer process involves initiation, a process that is defined as a stable, heritable change. This stage is a rapid, irreversible process that results in a carcinogen-induced mutational event. Chemical and physical agents that function at this stage are Table 8-5 Characteristics of the Stages of the Carcinogenesis Process Initiation DNA modification Mutation Genotoxic One cell division necessary to lock in mutation Modification is not enough to produce cancer Nonreversible Single treatment can induce mutation Promotion No direct DNA modification Nongenotoxic No direct mutation Multiple cell divisions necessary Clonal expansion of the initiated cell population Increase in cell proliferation or decrease in cell death (apoptosis) Reversible Multiple treatments (prolonged treatment) necessary Threshold Progression DNA modification Genotoxic event?? Mutation, chromosome disarrangement Changes from preneoplasia to neoplasia benign/malignant Irreversible Number of treatments needed with compound unknown (may require only single treatment)

referred to as initiators or initiating agents. Initiating agents lead to genetic changes including mutations and deletions. Chemical carcinogens that covalently bind to DNA and form adducts that result in mutations are initiating agents. Included among chemicals classified as initiating carcinogens are compounds such as polycyclic hydrocarbons and nitrosamines, biological agents such as viruses, and physical agents such as X-rays and UV light. Most chemical carcinogens that function at the initiation stage of the cancer process are indirect-acting genotoxic compounds that require metabolic activation in the target cell to produce the DNA-damaging event. For indirect-acting genotoric compounds, the chemical must be taken into the target site and metabolized. The ultimate form of the carcinogen is then able to bind to nuclear DNA, resulting in adduct formation. The initiating event becomes “fixed” when the DNA adducts or other damage to DNA are not correctly repaired or are incompletely repaired prior to DNA synthesis. This event can lead to inappropriate base pairing and/or formation of a mutation. Initiation by itself does not appear to be sufficient for neoplastic formation. Once initiated cells are formed, their fate has multiple potential outcomes: (1) the initiated cell can remain in a static nondividing state through influences by growth control via either normal surrounding cells or through endocrine influence; (2) the initiated cell may possess mutations incompatible with viability or normal function and be deleted through apoptotic mechanisms; or (3) the cell, through stimuli such as intrinsic factors or from chemical exposure, may undergo cell division resulting in the growth in the proliferation of the initiated cell. In some instances, typically following relatively high doses and/or repeated exposure to the genotoxic carcinogen, a chemical carcinogen may function as a complete carcinogen, i.e., it is capable of progressing through all stages of the cancer process.

Promotion Derived from either endogenous or exogenous stimuli of cell growth, the second stage of the carcinogenesis process involves the selective clonal expansion of initiated cells to produce a preneoplastic lesion. This is referred to as the promotion stage of the carcinogenesis process. Both exogenous and endogenous agents that function at this stage are referred to as tumor promoters. Tumor promoters are not mutagenic and generally are not able to induce tumors by themselves; rather they act through several mechanisms involving gene expression changes that result in sustained cell proliferation, either through increases in cell proliferation and/or the inhibition of apoptosis. Nongenotoxic carcinogens frequently function at the tumor promotion stage. The growth of preneoplastic lesions requires repeated applications of or continuous exposure to tumor-promoting

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NON-ORGAN-DIRECTED TOXICITY

compounds. While initial exposure to tumor promoters may result in an increase in cell proliferation and/or DNA synthesis in all tissues of the organ, this is usually a transient effect and with repeated applications of the tumor promoter only the initiated cells continue to clonally expand and divide (Fig. 8-2). Promotion is a reversible phenomenon whereby upon removal of the promoting agent, the focal cells may return to single initiated cell thresholds. In addition, these agents demonstrate a well-documented threshold for their effects, below a certain dose or frequency of application, tumor promoters are unable to induce cell proliferation. Multiple chemical compounds as well as physical agents have been linked to the tumor promotion stage of the cancer process. Tumor promoters in general show organ-specific effects, e.g., a tumor promoter of the liver, such as phenobarbital will not function as a tumor promoter in the skin or other tissues.

Progression The final stage of the carcinogenesis process, progression, involves the conversion of benign preneoplastic lesions into neoplastic cancer. In this stage, due to increase in DNA synthesis cell proliferation in the preneoplastic lesions, additional genotoxic events may occur resulting in additional DNA damage including chromosomal aberrations and translocations. These events result in the transfer from preneoplastic, clonally derived cell populations into neoplastic cell populations. Chemicals that impact on the progression stage are usually genotoxic agents. By definition, the progression stage is an irreversible stage in that neoplasm formation, whether benign or malignant, occurs. With the formation of neoplasia, an autonomous growth and/or lack of growth control is achieved. Spontaneous progression can occur from spontaneous karyotypic changes that occur in mitotically active initiated cells during promotion. An accumulation of nonrandom chromosomal aberrations and karyotypic instability are hallmarks of progression. As such, chemicals that function as progressor agents are usually clastogenic and are capable of causing chromosomal abnormalities. Complete carcinogens have the ability to function at the initiation, promotion, and progression stages and hence by definition have genotoxic properties. In addition, a number of model systems are available to test for carcinogenicity and/or to study the multistep mechanisms involved in chemical carcinogenesis. The models are described in a later section of this chapter.

MECHANISMS OF ACTION OF CHEMICAL CARCINOGENS The development of neoplasia requires two major events: the formation of an initiated, mutated cell and the selective proliferation of the mutated cell to form a neoplasm. Both these events can be induced or acted upon by chemical carcinogens. Chemicals that induce cancer have been broadly classified into one of two categories—genotoxic or DNA reactive, and nongenotoxic or epigenetic carcinogens— based on their relative abilities to interact with genomic DNA.

Genotoxic Carcinogens Genotoxic carcinogens initiate tumors by producing DNA damage. Experimental and epidemiological observations made in the middle of the 20th century identified a number of chemicals that could cause cancer in humans or experimental animals. Coal tar carcinogens including benzo(a)pyrene, pesticides such as 2-acetylaminofluorine,

Table 8-6 Examples of Genotoxic Carcinogens Direct-acting carcinogens Nitrogen or sulfur mustards Propane sulfone Methyl methane sulfonate Ethyleneimine B-Propiolactone 1,2,3,4-Diepoxybutane Dimethyl sulfate Bis-(Chloromethyl) ether Dimethylcarbamyl chloride Chemicals requiring activation (indirect-acting carcinogens) Polycyclic aromatic hydrocarbons and heterocyclic aromatics Aromatic amines N -Nitrosoamines Azo dyes Hydrazines Cycasin Safrole Chlorinated hydrocarbons Aflatoxin Mycotoxin Pyrrolizidine alkaloids Bracken fern Carbamates

and azo dyes such as diaminobenzamide were among the first chemical carcinogens to be studied. DNA reactive carcinogens can be further subdivided according to whether they are active in their parent form (i.e., direct-acting carcinogens—agents that can directly bind to DNA without being metabolized) and those that require metabolic activation (i.e., indirect-acting carcinogens—compounds that require metabolism in order to react with DNA). Examples of direct-acting and indirect-acting carcinogens are listed in Table 8-6. Direct-Acting (Activation-Independent) Carcinogens A variety of carcinogens do not require metabolic activation or chemical modification to induce cancer, and are termed direct acting or activationindependent carcinogens. These chemicals are also defined as ultimate carcinogens. Examination of the chemical structure of these agents reveals that they are highly reactive electrophilic molecules that can interact with and bind to nucleophiles, such as cellular macromolecules including DNA. Common electrophilic species are shown in Fig. 8-3. Generally, chemicals containing these moieties are highly reactive and frequently result in tumor formation at the site of chemical exposure. Direct-acting carcinogens include epoxides, imines, alkyl and sulfate esters, and mustard gases (Fox and Scott, 1980; Sontag, 1981). Direct-acting electrophilic carcinogenic chemicals typically test positive in the Ames test without additional bioactivation with a liver metabolic fraction. The relative carcinogenic strength of directacting carcinogens depends in part on the relative rates of interaction between the chemical and genomic DNA, as well as competing reactions with the chemical and other cellular nucleophiles. The relative carcinogenic activity of direct-acting carcinogens is dependent upon such competing reactions and also on detoxification reactions. Chemical stability, transport, and membrane permeability determine the carcinogenic activity of the chemical. Direct-acting

CHAPTER 8

1. Carbonium ions H R

2. Nitrenium ions

3. Free radicals H

N+

R

C+

R

H

R

4. Diazonium ions

CHEMICAL CARCINOGENESIS

C R

5. Epoxides

+ – R N N OH

6. Aziridinium ions

R

R N + R

O R

335

reactive form, detoxification pathways may also occur resulting in inactivation of the carcinogen. Indirect-acting genotoxic carcinogens usually produce their neoplastic effects, not at the site of exposure (as seen with directacting genotoxic carcinogens) but at the target tissue where the metabolic activation of the chemical occurs. Indirect-acting genotoxic carcinogens include the polycyclic aromatic hydrocarbons (PAHs), nitrosamines, aromatic amines, and aflatoxin B1. Figure 8-4 shows the parent (procarcinogen) and metabolites for several representative indirect-acting genotoxic carcinogens.

Mutagenesis 7. Episultonium ions + S—R

10. Halo ethers

8. Strained Lactones

9. Sulfonates

O

O

RSO2OCH3

11. Enals O

CICH2OCH2CI

RCH

CHC H

Figure 8-3. Structures of reactive carcinogenic electrophiles.

carcinogens are typically carcinogenic at multiple sites and in all species examined. A number of direct-acting alkylating agents, including a number of chemotherapeutic chemicals, are carcinogenic in humans (Vainio et al., 1991). Indirect-Acting Genotoxic Carcinogens An important discovery in the understanding of chemical carcinogenesis came from the investigations of the Millers who established that many carcinogens are not intrinsically carcinogenic, but require metabolic activation to be carcinogenic. They demonstrated that azo dyes covalently bind to proteins in liver, leading to the conclusion that carcinogens may bind to proteins that are critical for cell growth control (Miller and Miller, 1947). Subsequent work with benzo(a)pyrene showed covalent binding of benzo(a)pyrene or the metabolites of benzo(a)pyrene in rodents (Miller, 1951). Additional investigations with other indirect-acting genotoxic carcinogens confirmed that metabolism of the parent compound was necessary to produce a metabolite (activation) that was able to interact with DNA (Miller, 1970). It has since been shown that the majority of DNA reactive carcinogens are found as parent compounds, or procarcinogens. Procarcinogens are stable chemicals that require subsequent metabolism to be carcinogenic (Miller and Miller, 1981; Weisburger and Wiliams, in Becker, 1981; Conney, 1982; Miller et al., 1983). The terms procarcinogen, proximate carcinogen, and ultimate carcinogen have been coined to define the parent compound (procarcinogen) and its metabolite form, either intermediate (proximate carcinogen) or final (ultimate carcinogen) that reacts with DNA (Fig. 8-4). The ultimate form of the carcinogen is most likely the chemical species that results in mutation and neoplastic transformation. The ultimate form of certain carcinogenic chemicals is not known, whereas for other chemicals there may be more than one ultimate carcinogenic metabolite depending on the metabolic pathway followed. It is important to note that besides activation of the procarcinogen to a DNA

The reaction of a carcinogen with genomic DNA, either directly or indirectly, may result in DNA adduct formation or DNA damage, and frequently produces a mutation. Several mechanisms of mutagenesis are known to occur. Modification of DNA by electrophilic carcinogens can lead to a number of products. Modified DNA are dependent upon when in the cell cycle the adducts are formed; where the adducts are formed; and, the type of repair process used in response to the damage. Transitions are a substitution of one pyrimidine by the other, or one purine by the other (changes within a chemical class), whereas a transversion occurs when a purine is replaced by a pyrimidine, or a pyrimidine is replaced by a purine (changes across a chemical class). Carcinogens can induce transitions and transversions several ways. In one scenario, when adducts (or apurinic or apyrmidinic sites) are encountered by the DNA replication processes, they may be misread. The polymerase may preferentially insert an adenine (A) in response to a noninformative site. Thus, the daughter strand of an A, C, G, or T adduct will have an adenine (A) and this change is fixed (mutation) and resistant to subsequent DNA repair. A second outcome, a shift in the reading frame (resulting in a frame-shift mutation) may also result from carcinogen–DNA adducts formation. Most frame-shift mutations are deletions and occur more frequently when the carcinogen–DNA adduct is formed on a nucleotide. In a third scenario, DNA strand breaks can also result from carcinogen DNA adducts. These may arise either as a result of excision–repair mechanisms that are incomplete during DNA replication or via direct alkylation of the phosphodiester backbone leading to backbone cleavage. Strand scission can lead to double-strand breaks, recombination, or loss of heterozygosity.

Damage by Alkylating Electrophiles As noted above, most chemical carcinogens require metabolic activation to exert a carcinogenic effect. The ultimate carcinogenic forms of these chemicals are frequently strong electrophiles (Fig. 8-3) that can readily form covalent adducts with nucleophilic targets. Because of their unpaired electrons, S:, O:, and N: atoms are nucleophilic targets of many electrophiles. The extent of adducts formed is limited by the structure of DNA, where bulky electrophilic chemicals can bind, and size of the ultimate carcinogenic form. In general, the stronger electrophiles display a greater range of nucleophilic targets (i.e., they can attack weak and strong nucleophiles), whereas weak electrophiles are only capable of alkylating strong nucleophiles (e.g., S: atoms in amino acids). In addition, the metabolic capability of a target cell will dictate the extent and types of electrophiles generated from the procarcinogenic parent. An important and abundant source of nucleophiles is contained not only in the DNA bases, but also in the phosphodiester

336

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

Procarcinogen

Ul ti mate (Ut) Carci nogen

P roximate (Px) Carcinogen

Direct epoxidation

O

O

O

O

C

C

C

C

O

O O

O

O

O

OCH3

Aflatoxin B1 (P r )

O

OCH3

Af latoxin B1 2,3 epoxide (Ut)

N-Hydroxylation

NH2

H2 N Benzidine (Pr)

O

H

C H3C

N

O H NH2 N-hydroxy diacetyl Benzidine (Px)

C N

NH

H3C

H

N-Acetyl benzidine Nitrenium ion (Ut)

Two-step epoxidation

O

HO

O

OH Benzo(a)pyrene (Pr)

Benzo(a)pyrene 7,8 diol-9,10 epoxide (Ut)

Benzo(a)pyrene 7,8 epoxide (Px)

H2C

C

O

OH

CH2

CH2

HC C

ester

HC

c

CH2

O O H2C

O

O H2C

Safrole

H3C N

O

H2C

l' hydroxy Safrole (Px)

CH3

N O Dimethynitrosamine (Pr)

H3C N

CH2OH

O

Safrole l' O-ester (Ut)

H3C N

N OH HCOH CH3+ + N2 + H2O

N O Hydroxymethyl, methyl nitrosamin (Px)

Methyl carbonium ion (Ut)

Figure 8-4. Structures of representative indirect-acting carcinogens and their metabolic derivatives. The proximate (Px) and ultimate (Ut) carcinogenic forms result from the metabolism of the procarcinogenic form (Pr).

backbone (Fig. 8-5). Whereas carcinogen DNA adducts may be formed at all sites in DNA, the most common sites of alkylation include the N7 of guanine, the N3 of adenine, the N1 of adenine, the N3 of guanine, and the O6 of guanine. Alkylations of phosphate may also occur at a high frequency. Selective examples of carcinogen

interactions with proteins and nucleic acids are shown in Fig. 8-6. Different electrophilic carcinogens will often display different preferences for nucleophilic sites in DNA and different spectra of damage. Dimethylnitrosamine and diethylnitrosamine, for example, are metabolized by P450 oxidation to yield a methyl carbonium ion

CHAPTER 8

CHEMICAL CARCINOGENESIS

Base Adenine

337

Positions Alkylated 7

NH2

1

N

N

1-,3-,7-

N

N

3

Guanine

6

O

7

OH N

N

H2N

3-,7-,O6-

N

N

3

Cytosine

NH2

3 N

3-, O2-

N

HO O2

Thymine

O

4

OH

3

CH3

N HO

3-, O2- O4-

N

O2

Phosphate

O O

P

O

OH

Figure 8-5. Examples of cellular nucleophiles and sites of possible adduct formation. + (CH+ 3 ) and an ethyl carbonium ion (CH3 CH3 ), respectively. Despite the structural similarities of the ultimate electrophiles, they display significant differences in alkylation profiles (Pegg, 1984). The relative proportions of methylated bases present in DNA following reaction with carcinogen-methylating agents are shown in Table 8-7 (Pegg, 1984). The predominant adduct formed following exposure to methylating chemicals such as methylmethane sulfonate is 7-methylguanine. In contrast, ethylating agents produce adducts predominately in the phosphate backbone of DNA. The carcinogenic potential of the type of adducts formed is often debated; some believe that O6 -alkylguanine is the most carcinogenic adduct, while others report that O4 -alkylthymine is more important in the carcinogenic process, due to its persistence relative to other adducts (Pegg, 1984; Swenberg et al., 1984). Another common modification to DNA is the hydroxylation of DNA bases. Oxidative DNA adducts have been identified in all four DNA bases (Fig. 8-7); however, 8-hydroxyguanine is among the most prevalent oxidative DNA adduct (Floyd, 1990). The source of oxidative DNA damage is typically formed from free radical reactions that occur endogenously in the cell or from exogenous sources (Floyd, 1990; Ames and Shigenaga, 1993; Klaunig and Kamendulis, 2004). Although a relatively large amount of oxidative DNA adducts have been proposed to be formed per day, repair mechanisms exist that maintain the cellular level at a low rate and keep endogenous mutations to a relatively low level. The role of oxidative

damage and oxidative stress is discussed in greater detail later in this chapter. Methylation of deoxycytidine residues is a well-studied DNA adduct. This reaction occurs by the transfer of a methyl group from S-adenosylmethionine by DNA methyltransferases (Holliday, 1990). Methylation of DNA results in heritable expression or repression of genes, with hypomethylation associated with active transcription of genes, while hypermethylated genes tend to be rarely transcribed. Chemical carcinogens may inhibit DNA methylation by several mechanisms including forming covalent adducts, single strand breaks in the DNA, alteration of methionine pools, and inactivation of the DNA methyltransferase responsible for methylation (Riggs and Jones, 1983). The importance of DNA methylation in chemical carcinogenesis is discussed later in this chapter. Although a large number of adducts can be formed following exposure to chemicals, whether a particular DNA adduct will result in mutation and participate in the carcinogenesis process is dependent in part on the persistence of the adduct through the process of DNA replication, which is also in part dependent upon DNA repair.

DNA Repair Following the formation of a carcinogen DNA adduct, the persistence of the adduct is a major determinant of the outcome. This

338

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

CH3 C O N

C

N

C H2

N

DNA

N-(deoxyguanosine-8-yl)-acetylaminofluorene in DNA

O

O

O

OH

C

H

H N

H

O

H

O

NH2

deoxyribose

H3CO

O

C

C

N

O

H N

C

C

N

H

C

C

N

N

H

CH N

H

N

N

N

deoxyribose

N C H2

N

C

C

DNA

CH3

O

NH2

3-(deoxyguanosine N2-yl)-acetylaminofluorene in DNA

7

Aflatoxin B1 N- guanine-adduct

H

N

O

CH2CH2OH

N

N

H

N

N

N N

N N deoxyribose DNA

O

H2N

N

N

N deoxyribose

deoxyribose DNA

1, N 6 -ethenoadenine in DNA

DNA

3,N 4-ethenocytosine in DNA

N-7-(2-hydroxyethyl) guanosine in DNA

Figure 8-6. Select structures of protein and nucleic adducts of certain chemical carcinogens.

persistence depends on the ability of the cell to repair the altered DNA. The detection of unique DNA adducts has proven to be important in understanding the mechanism of action of specific carcinogens and has been correlated with carcinogenesis (Goth and Rajewsky, 1974; Kadlubar et al., 1981; Becker and Shank, 1985; Swenberg et al., 1985). However, the presence of a DNA adduct is not sufficient for the carcinogenesis process to proceed. The relative rates or persistence of particular DNA adducts may be an important determinant of carcinogenicity, for example, O4 -ethylthymine is relatively stable in DNA while O6 -ethylguanine does not persist in DNA after continuous exposure to diethylnitrosamine (Swenberg et al., 1984). The persistence of DNA adducts of trans-4-aminostilbene does not correlate with organ carcinogenicity and/or tissue susceptibility. While the liver and kidney exhibited the greatest burden and persistence of the adduct and the Zymbals gland showed the least amount of DNA adducts, the latter tissue was more susceptible to carcinogenesis by this chemical (Neumann, 1983). As such, differences in susceptibility to carcinogenesis are likely the result of a number of factors, including DNA replication within a tissue and repair of a DNA adduct.

The quantification of covalent DNA adducts in tissues has been used to demonstrate exposure of humans to carcinogenic chemicals and to assess the relative risk from exposure to carcinogenic chemicals. For example, DNA adducts of carcinogenic polyaromatic hydrocarbons have been demonstrated at relatively high levels in tissues and blood of smokers and foundry workers compared with nonexposed individuals (Perera et al., 1991). In addition, DNA adducts of aflatoxin B1 were seen in samples of human placenta and cord blood in individuals in Taiwan, an area that has a high incidence of liver cancer (Hsieh and Hsieh, 1993). The presence of macromolecular carcinogen adducts may be important to their mechanism of carcinogenicity, the presence and persistence of the adducts is only one factor in the process of cancer development. Experimental and epidemiological evidence indicates that the development of cancer following exposure to chemical carcinogens is a relatively rare event. This can be explained by the ability of a cell to recognize and repair damaged DNA. During the DNA repair, the DNA region containing the adduct is removed and a new patch of DNA is synthesized, using the opposite intact strand as a template. The new DNA segment is then spliced into the DNA molecule in

CHAPTER 8

CHEMICAL CARCINOGENESIS

339

Table 8-7 Relative Proportions of Methylated Bases Present in DNA after Reaction with Carcinogenic Alkylating Agents percent of total alkylation by

1-Alkyladenine 3-Alkyladenine 7-Alkyladenine 3-Alkylguanine 7-Alkylguanine O6 -Alkylguanine 3-Alkylcytosine O2 -Alkylcytosine 3-Alkylthymine O2 -Alkylthymine O4 -Alkylthymine Alkylphosphates

OH

HN O

N-ethyl-N -nitrosourea

0.7 8 1.5 0.8 68 7.5 0.5 0.1 0.3 0.1 0.1–0.7 12

0.3 4 0.4 0.6 12 8 0.2 3 0.8 7 1–4 53

NH2

O

O OH

N

5-hydroxy-dU

thymine glycol

O

NH2 OH

HN

H N

N

OH N O H uracil glycol

N H

8-oxo-dA

O N H

N

H2N

8-oxo-dG

O

NH2 H N

N

H N

HN

O N

OH OH

N H

O

5-hydroxy-dC

O

CH3

HN

N H

O

N H

O N

NH2

Fapy-dA

H N

HN H2N

diethyl-nitrosamine or

dimethyl-nitrosamine, N -methyl-N-nitrosourea, or 1,2-dimethyl-hydrazine

O N

NH2

Fapy-dG

Figure 8-7. Structures of selected oxidative DNA bases.

place of the defective one. To be effective in restoring a cell to normal, repair of DNA must occur prior to cell division; if repair is not complete prior to replication, the presence of the adducts can give rise to mispairing of bases and other genetic effects such as rearrangements and translocations of DNA segments. Thus, a chemical that alters the repair process or the rate of cell division can itself affect the frequency of neoplastic transformation.

DNA Repair Mechanisms Although cells possess mechanisms to repair many types of DNA damage, these are not always completely effective, and residual

DNA damage can lead to the insertion of an incorrect base during DNA replication, followed by transcription and translation of the mutated templates, ultimately leading to the synthesis of altered protein. Mutations in an oncogene, tumor-suppressor gene, or gene that controls the cell cycle can result in a clonal cell population with a survival advantage. The development of a cancer requires many such events, occurring over a long period of time, and for this reason human cancer induction often takes place within the context of chronic exposure to chemical carcinogens. A number of structural alterations may occur in DNA as a result of interaction with reactive chemicals or radiation, and the more frequent types of damage are depicted in Fig. 8-8. The reaction of chemical species with DNA can produce adducts within the bases, sugar, and phosphate backbone of DNA. In addition, bifunctional alkylating agents (such as mustards) may cause DNA crosslinking between two opposing bases. Other structural changes such as pyrimidine dimer formation are specific for exposure to UV light, while double-stranded breaks in DNA are more commonly associated with ionizing radiation. A variety of mechanisms have evolved to effectively remediate and repair DNA damage. The more common types of DNA repair mechanisms seen in mammalians are listed in Table 8-8. In addition to the proofreading activity of DNA polymerases that can correct miscopied bases during replication, cells have several mechanisms for repairing DNA damage. Repair of DNA Table 8-8 DNA Repair Pathways 1. Direct reversal of DNA damage 2. Excision repair systems Base excision repair Nucleotide excision repair Mismatch repair 3. Postreplicational repair (recombination repair) 4. Nonhomologous-end-jointing (NHEJ): double-strand break repair

340

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

Figure 8-8. Common Forms of DNA Damage.

damage does not always occur prior to cell replication, and in addition, repair of DNA damage by some chemicals is relatively inefficient, as such, exposure to chemicals that cause DNA damage can increase the probability of acquiring mutations that ultimately lead to cancer development. Mismatch Repair of Single-Base Mispairs Spontaneous mutations may occur through normal cellular DNA replication errors. Many spontaneous mutations are point mutations, a change in a single base pair in the DNA sequence. The issue for mismatch repair is determining which is the normal DNA and which is the damaged DNA strand, and therefore repairing the mutated strand such that the correct base pairs are restored. Depurination is a fairly common occurrence and a spontaneous event in mammals, and results in the formation of apurinic sites. If these lesions are left unrepaired, mutations are generated during DNA replication since the DNA synthetic machinery is unable to determine the appropriate base with which to pair. All mammalian cells possess apurinic endonucleases that function to cut DNA near apurinic sites. The cut is then extended by exonucleases, and the resulting gap repaired by DNA polymerases and ligases. Excision Repair DNA regions containing chemically modified bases, or DNA chemical adducts, are typically repaired by excision repair processes. DNA adducts cause a distortion in the normal shape of DNA. Proteins that slide along the surface of a doublestranded DNA molecule recognize the irregularities in the shape of the double helix, and affect the repair of the lesion. DNA lesions that are repaired by excision repair processes include thymine–thymine dimers, produced following exposure to UV light; dimers that interfere with both replication and transcription of DNA. The repair

of DNA regions containing bases altered by the attachment of large chemical adducts such as benzo(a)pyrene are also effectively repaired by excision repair processes (Fig. 8-9). End-Joining Repair of Nonhomologous DNA A cell that has double-strand breaks can be repaired by joining the free DNA ends. The joining of broken ends from different chromosomes, however, will lead to the translocation of DNA pieces from one chromosome to another, translocations that have the potential to enable abnormal cell growth by placing a proto-oncogene next to, and therefore under the control of, another gene promoter. Double-strand breaks can be caused by ionizing radiation, and drugs such as anticancer drugs. Double-strand breaks are correctly repaired only when the free ends of DNA rejoin exactly. The repair of double-stranded DNA is therefore confounded by the absence of single-stranded regions that can signal the correct base pairing during the rejoining process. Homologous recombination is one of two mechanisms responsible for the repair of double-strand breaks. In this process, the doublestrand break on one chromosome is repaired using the information on the homologous, intact chromosome. The predominant mechanism for double-stranded DNA repair in multicellular organisms is nonhomologous repair, and involves the rejoining of the ends of the two DNA molecules. Although this process yields a continuous double-stranded molecule, several base pairs are lost at the joining point. This type of deletion may produce a possibly mutagenic coding change.

Classes of Genotoxic Carcinogens Polyaromatic Hydrocarbons Polyaromatic hydrocarbons are found at high levels in charcoal broiled foods, cigarette smoke,

CHAPTER 8

CHEMICAL CARCINOGENESIS

341

Damaged base 5’

3’

3’

5’ AP site

DNA glycosylase

5’

3’

3’ 5’

5’ AP endonuclease

3’

5’

3’ DNA polymerase I DNA ligase

3’

5’

5’

3’

(a) Damaged nucleotide 5’

3’

5’

3’

Uvr-A, Uvr-B, Uvr-C

5’

3’

3’

5’

5’

3’

3’

5’ DNA polymerase I DNA ligase

5’

3’

3’

5’

(b) Figure 8-9. (A) DNA repair by base excision. (B) DNA repair by nucleotide excision.

and in diesel exhaust. Representative chemicals belonging to this class are shown in Fig. 8-10a. Benzo(a)pyrene is a representative polycyclic hydrocarbon that has been studied extensively. The metabolism and pathways that lead to tumor formation have been characterized through the work of a number of laboratories (Conney, 1982). The ultimate carcinogen is a diol epoxide of benzo(a)pyrene,

formed following three separate enzymatic reactions (Sims et al., 1974). Benzo[a]pyrene is first oxidized by cytochrome P4501A1 to form benzo[a]pyrene 7,8-oxide, further metabolized by epoxide hydrolase, yielding the 7,8-dihydrodiol. And then further metabolism by cytochrome P4501A1 to yield the ultimate carcinogen, the 7,8-dihydrodiol-9,10-epoxide (Yang et al., 1976; Lowe and

342

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

H3C

Dibenz(a,c)anthracene

Dibenz(a,h)anthracene

3-Methylcholanthrene

CH3

Benzo(a)pyrene

Chrysene

CH3

7,12-dimethylbenz(a)anthracene

N

Perylene

7H-Dibenzo(c,q)carbazole

Benzo(e)pyrene

(a) Bay region 12

1

11

2

10 9

O

Epoxide hydrolase

3 P-450

P-450 PHS

8

4 7

6

5

HO

HO

O

OH benzo(a)pyrene

(+) benzo(a)pyrene 7,8-oxide

(-) benzo(a)pyrene 7,8-dihydrodiol

OH (+) benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide Resistant to hydrolyation by epoxide hydrolase

P450

Covalent binding to DNA

Epoxide hydrolase

O

benzo(a)pyrene 4,5-oxide

Mutation of the 12th codon of the Hras oncogene

OH

Lung and skin tumors

OH benzo(a)pyrene 4,5-dihydrodiol

(b) Figure 8-10. (A) Chemical structures of selected carcinogenic polycyclic hydrocarbons. (B) Role of epoxide hydrolase in the activation of benzo[a]pyrene 4,5-oxide and in the conversion of benzo[a]pyrene to its tumorigenic bay-region diole poxide.

Silverman, 1994). The Bay region or K region of the benzo(a)pyrene molecule is the site of metabolic targeting and DNA interaction (Fig. 8-10b). The importance of the bay region (K region) of the benzo(a) pyrene molecule was demonstrated from the understanding of the metabolism of the benzo(a)pyrene to its ultimate DNA reactive form. Similar regions (to the Bay region) have been iden-

tified in other carcinogenic polycyclic hydrocarbons and used to access and predict carcinogenic potential of other PAHs. Alkylating Agents Alkylating chemicals represent an important class of chemical carcinogens. Whereas some alkylating chemicals are direct-acting genotoxic agents, many require

CHAPTER 8

CHEMICAL CARCINOGENESIS

O R

343

R

S

O

O

R

N

N R

O

Diakyl nitrosamines

Alkyl alkanesulfonates

R O

N

N

R NH2

O

N

H

N

N NO2

O

NH2

N-Nitrosoureas

N-Alkyl-N '-nitro-N-nitrosoguanidine

Figure 8-11. Structures of representative methylating and ethylating agents.

Cl

Cl

O

HOOC N H

Cl

O Cl

Chlorambucil

Cyclophosphamide

DNA

Cl R

N

N

P

R

N

R

N+

N Cl

Cl

Cl

Monoadduct

DNA R

H2O

DNA N+

R

N OH

DNA

DNA R

N DNA

Interstrand DNA Crosslink Figure 8-12. Nitrogen mustards and proposed mechanism for the reaction of nitrogen mustards with DNA.

metabolic activation to produce electrophilic metabolites that can react with DNA. Alkylating agents can be classified into several groups including the direct-acting alkylalkanesulfonates (methyl- and ethyl methanesulfonate) and nitrosamides (N -methyl-N -nitrosourea,

N -ethyl-N -nitrosourea, N -methyl-N -nitro-N -nitrosoguanidine, and the indirect-acting nitrosamides (dimethyl- and diethylnitrosamines) (Fig. 8-11). The alkylating compounds (or in the case of diethylnitrosamine and dimethylnitrosamine; their metabolites)

344

UNIT 3

H

O C

NON-ORGAN-DIRECTED TOXICITY

H C

H

H

Ethylene oxide O H

H C

H

C C

H H

H

Propylene oxide Figure 8-13. Chemical structures for ethylene and propylene oxide.

readily react with DNA at more than 12 sites. The N7 position of guanine and the N3 position of adenine are the most reactive sites in DNA for alkylating chemicals. DNA methylation reactions occur more readily and thus exhibit >20 more adducts than with ethylation reactions. However, ethylation reactions have a greater affinity for oxygen centers, an event that appears to correlate with the mutagenicity and carcinogenicity of these compounds. Nitrosamines were initially used as solvents in chemistry. Their toxic effects were identified when workers using the nitrosamines solvents developed jaundice and liver damage. Subsequent studies in animal models revealed that dimethylnitrosamine and diethylnitrosamine were highly hepatotoxic and hepatocarcinogenic.

Other alkylating chemicals including the nitrogen mustards (e.g., chlorambucil, cyclophosphamide) have been used in cancer chemotherapy. They produce DNA adducts as well as induce the formation of DNA strand breaks. The alkylation of DNA by nitrogen mustards requires the formation of highly reactive N alkylazirdinium ions (Fig. 8-12). Nitrogen mustards can produce a wide spectrum of mutations including base pair substitutions (AT and GC) and deletions. In addition, nitrogen mustards are potent clastogens causing chromosomal aberrations and sister chromatid exchanges (SCEs), predominantly in GC-rich regions. Ethylene oxide and propylene oxide are other examples of mutagenic and carcinogenic alkylating agents (Fig. 8-13). Ethylene oxide is a direct-acting alkylating carcinogen in rodents, and perhaps of human concern (Hogstedt et al., 1986). Ethylene oxide is mutagenic in short term in vitro assays, and produces chromosomal aberrations and SCEs in eukaryotic cells (Ehrenberg and Hussain, 1981). Propylene oxide is also a mutagenic rodent carcinogen inducing nasal tumors in rodents following inhalation exposure (NTP, 1985). Alkylation of DNA by ethylene and propylene oxide occurs predominantly at the N7 position of guanine, yielding 7(2-hydroxyethyl)guanine and 7-(2-hydroxypropyl)guanine adducts, respectively. These adducts represent the major adducts formed following either in vitro or in vivo exposure (Walker et al., 1992). Vinyl chloride is another known rodent and human carcinogen, producing angiosarcomas in the liver and tumors in the lung and hematopoetic system in humans (Doll, 1985). Limited evidence also suggests that vinyl chloride exposure results in brain tumors. Vinyl chloride is mutagenic and is metabolized by cytochrome P450 to form chloroethylene oxide, which rearranges nonenzymatically to produce chloroacetaldehyde, both of which can alkylate DNA. Vinyl chloride and metabolites form several DNA adducts including

NH2

NH2

N

4-Aminobiphenyl (ABP)

NH2

4-Aminoazobenzene

Aniline

H 2N

N

NH 2

NH

NH2

Diphenylamine

Benzidine 2-Aminophenanthrene NH2

Cl

O CH3

C H2 N

CH2

NH2

N

2-Naphthylamine

H

Cl

4,4'-Methylene-bis-2-chloroaniline Figure 8-14. Chemical structures of selected carcinogenic aromatic amines.

2-Acetylaminofluorene (2-AAF)

CHAPTER 8

CHEMICAL CARCINOGENESIS

345

Table 8-9 Carcinogenicity of Metals animal

human

metal

species

tumor site

tumor type

exposure

tumor type

Arsenic

Mice, dogs, rats

None observed

None observed

Beryllium

Mice, rats, monkeys Mice, rats, chickens Mice, rats, rabbits

Bone Lung Injection site Testes Injection site Lung

Osteosarcoma Carcinoma Sarcoma Teratoma Sarcoma Carcinoma

Cu refinery As pesticides Chemical plants Drinking water (oral) None observed

Pulmonary carcinoma Lymphoma, leukemia Dermal carcinoma Hepatic angiosarcoma None observed

CD refinery

Pulmonary carcinoma Pulmonary carcinoma Gastrointestinal carcinoma

Rats, rabbits Hamsters, mice, rats, rabbits Mice, rats Mice, rats, cats, hamsters, rabbits Guinea pigs, rats Rats Chickens, rats, hamsters

Injection site Injection site

Sarcoma Sarcoma

CR refinery Chrome plating Chromate pigments None observed None observed

Kidney Injection site Lung Kidney

Carcinoma Carcinoma Carcinoma Carcinoma

None observed Ni refinery

Injection site Testes Testes

Sarcoma Carcinoma Teratoma

None observed None observed

Cadmium Chromium

Cobalt Iron Lead Nickel

Titanium Zinc

7-(2 -oxoethyl)guanine, N 2 , 3-ethenoguaine, 3,N 4 -ethenocytosine, and 1,N 6 -ethenoadenine (Guengerich, 1994). Aromatic Amines and Amides Aromatic amines and amides encompass a class of chemicals with varied structures (aromatic amines, e.g. aniline dyes, 2-naphthylamine, benzidine, 2acetylaminofluorene) (Fig. 8-14). Because of their use in the dye industry and other industrial processes their carcinogen potential in humans was realized as early as the late 19th century. While proper industrial hygiene processes have considerably reduced the human exposure to aromatic amines and amides in the workplace, exposure to these chemicals still occurs through cigarette smoke and environmental sources. The aromatic amines undergo both phase-I and phase-II metabolism. Phase-I reactions occur mainly by cytochrome P450-mediated reactions, yielding hydroxylated metabolites that are often associated with adduct formation in proteins and DNA, and produce liver and bladder carcinogenicity (Miller et al., 1964). For example, metabolism of 2-acetylaminofluorene (AAF) results in the formation of N -hydroxy-AAF, which is a metabolite responsible for the liver tumorigenicity. Similarly, 1-napthylamine exhibits carcinogenic activity only in test systems capable of producing the N -hydroxy metabolite of naphthylamine. Aromatic amines are capable of forming adducts with several DNA bases.

Inorganic Carcinogens Several metals exhibit carcinogenicity in experimental animals and/or exposed humans. Table 8-9 provides a listing of some common metals and their corresponding carcinogenicity in animals and humans. Additional details are provided below.

None observed None observed None observed Pulmonary carcinoma Nasolaryngeal carcinoma Gastric and renal carcinoma Sarcoma (?) None observed None observed

Arsenic Arsenic compounds are poorly mutagenic in both bacterial and mammalian cell assays (Lofroth and Ames, 1978). Metallic arsenic, arsenic trioxide, sodium arsenite, sodium arsenate, potassium arsenite, lead arsenate, calcium arsenate, and pesticide mixtures containing arsenic have been tested for carcinogenicity in experimental animals (IARC, 1980, 1987). In the majority of studies in experimental animals—including oral exposure studies in mice, rats, and dogs; dermal exposure studies in mice; inhalation exposure studies in mice; injection studies in mice and rats; and intramedullary injection studies in rats and rabbits—no tumors were observed or the results were inconclusive, and thus it has previously been concluded that limited evidence exists for the carcinogenicity of inorganic arsenic compounds in experimental animals (IARC, 1987). In contrast, inorganic arsenic compounds are known human carcinogens, based on sufficient evidence of carcinogenicity in humans. Epidemiological studies of humans exposed to arsenic compounds demonstrated that exposure to inorganic arsenic compounds increases the risk of cancer in the skin, lung, digestive tract, liver, bladder, kidney, and lymphatic and hematopoietic systems (IARC, 1973, 1980). Several of the epidemiological studies have reported dose–response relationships between arsenic in drinking water and several types of cancer, including bladder, kidney, lung, and skin cancer (Cantor, 1997; Ferreccio et al., 2000). The mechanisms for cancer formation are unclear but possibly involve the induction of oxidative stress, altered cell signaling, modulation of apoptosis, and/or altered cell cycle (Harris and Shi, 2003; Quian and Shi, 2003; Hughes and Kitchin, 2006). The latency period in humans of arsenic-related carcinogenesis is considered to be 30–50 years. The first signs of chronic exposure, frequently seen in water supplies contaminated with arsenic, are skin pigmentation, depigmentation, hyperkeratosis of palms and soles, and skin lesions. A unique

346

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

peripheral vascular disease associated with chronic arsenic exposure is black foot disease, starting with numbness and ulceration of extremities and ending in gangrene and spontaneous amputations (Chen et al., 1988). Beryllium Beryllium and its salts are not mutagenic and do not appear to induce cellular transformation (IARC, 1993). Mechanistically, beryllium salts bind to nucleoproteins and inhibit enyzmes involved in DNA synthesis, resulting in infidelity of DNA synthesis and also induce gene mutations in cultured cells (Leonard and Lauwerys, 1987). Studies in animal models have consistently reported increases in lung tumors in rodents and nonhuman primates exposed to beryllium or beryllium compounds (IARC, 1993; Finch et al., 1996; NTP, 1998). Beryllium metal and several beryllium compounds (e.g., beryllium–aluminum alloy, beryllium ore, beryllium chloride, beryllium hydroxide, beryllium sulfate tetrahydrate, and beryllium oxide) induced lung tumors in rats. Beryllium oxide and beryllium sulfate produced lung cancer (anaplastic carcinoma) in monkeys after intrabronchial implantation or inhalation. In rabbits, osteosarcomas were reported after exposure to beryllium metal, beryllium carbonate, beryllium oxide, beryllium phosphate, beryllium silicate, or zinc beryllium silicate (IARC, 1993). Beryllium and beryllium compounds have been classified as human carcinogens based on animal studies and evidence of carcinogenicity in humans. Epidemiological studies indicate an increased risk of lung cancer in occupational groups exposed to beryllium or beryllium compounds (Steenland and Ward, 1991; Ward et al., 1992). Further, an association with lung cancer has consistently been observed in occupational populations exposed to beryllium or beryllium compounds. Acute beryllium pneumonitis, a marker for exposure to beryllium has been shown to be associated with higher lung cancer rates (Steenland and Ward, 1991). Cadmium Animal studies have shown that cadmium and cadmium compounds induce tumor formation at various sites in multiple species of experimental animals, following multiple exposure routes, including the induction of prostate tumors in rats, testicular tumors in rats and mice, lymphoma in mice, adrenal-gland tumors in hamsters and mice, and lung and liver tumors in mice (IARC, 1993; Waalkes et al., 1994, 1999). It has been suggested that ionic cadmium, or compounds that release ionic cadmium, is the cause of genetic damage and thus the carcinogenic species. Increased frequencies of chromosomal aberrations (changes in chromosome structure or number) have been observed in lymphocytes of workers occupationally exposed to cadmium. Many studies of cultured mammalian cells have shown that cadmium compounds cause genetic damage, including gene mutations, DNA strand breaks, chromosomal damage, cell transformation, and disrupted DNA repair (IARC, 1993). Cadmium and cadmium compounds have been classified as known human carcinogens based on evidence of carcinogenicity in humans, including epidemiological and mechanistic information that indicate a causal relationship between exposure to cadmium and cadmium compounds and human cancer (IARC, 1993). Epidemiological studies of cadmium workers found that exposure to various cadmium compounds increased the risk of death from lung cancer (IARC, 1993). Follow-up analysis of some of these cohorts has confirmed that cadmium exposure is associated with elevated lung cancer risk under some industrial circumstances (Sorahan et al., 1995; Sorahan and Lancashire, 1997). Some epidemiological evidence has also suggested an association between cadmium exposure and prostate cancer (Shigematsu et al., 1982; van der Gulden et al.,

1995), kidney (Mandel et al., 1995), and bladder (Siemiatycki et al., 1994). Chromium Chromium has multiple oxidation states: from −2 to +6; however, the most common forms are the trivalent (III) and hexavalent (VI) forms. With regard to carcinogenicity, chromium III does not exhibit carcinogenicity in laboratory animals whereas chromium VI has been tested to be positive for genotoxicity and carcinogenicity in a variety of bioassays (Langard, 1988; IARC, 1990). Chromium VI compounds cause genetic damage including gene mutations and DNA damage in bacteria. Several chromium VI compounds also caused mutations in yeast and insects. Many chromium VI compounds caused genetic damage in cultured human and other animal cells and in experimental animals exposed in vivo, including SCE, chromosomal aberrations, and cell transformation. Chromosomal aberrations, SCE, and aneuploidy were observed in workers exposed to chromium VI compounds (IARC, 1990). Chromium VI (calcium chromate, chromium trioxide, sodium dichromate, lead chromates, strontium chromate, or zinc chromates) exposure in rats following inhalation, intrabronchial, intrapleural, intratracheal, intramuscular, or subcutaneous administration resulted in benign and malignant lung tumors in rats in a number of studies. In mice, calcium chromate caused benign lung tumors and chromium trioxide caused malignant lung tumors. Exposure of hamsters, guinea pigs, and rabbits to chromium VI compounds by intratracheal instillation did not cause lung tumors (IARC, 1980, 1990). While the mechanisms for chromium VI carcinogenicity remain unresolved, it has been speculated that the reduction of chromium VI by glutathione is involved (Connett and Whtterhahn, 1985; Kortenkamp and O’Brien, 1994). Hexavalent chromium (chromium VI) compounds have been classified as known human carcinogens based on data from animal studies and human epidemiological studies. Human epidemiological studies have consistently reported increased risks of lung cancer among chromate workers. Chromate workers are exposed to a variety of chromium compounds, including chromium VI and trivalent (III) compounds. In addition, an increased risk of a rare cancer of the sinonasal cavity was observed in these workers (IARC, 1990). Some studies suggested that exposure to chromium among workers, such as chromium-exposed arc welders, chromate pigment workers, chrome platers, and chromium tanning workers, may be associated with leukemia and bone cancer (Costa, 1997). Nickel Many studies in cultured rodent and human cells have shown that a variety of nickel compounds, including both soluble and insoluble forms of nickel, exhibit genotoxicity, producing DNA strand breaks, mutations, chromosomal damage, cell transformation, and modulation of DNA repair. Soluble nickel salts can be complete carcinogens and/or initiators of carcinogenesis (Kasprzak et al., 1990; Diwan et al., 1992). In rats and mice, inhalation or intratracheal instillation of nickel subsulfide or nickel oxide produced dose-related increases of benign and malignant lung tumors (IARC, 1990; NTP, 1996). Inhalation of nickel compounds also caused malignant and benign pheochromocytoma in rats (NTP, 1996). Shortterm intraperitoneal exposure during gestation to soluble nickel salt induced malignant pituitary tumors in the offspring. Additionally, exposure to nickel acetate through the placenta followed by exposure of the offspring to barbital (a known tumor promoter) produces kidney tumors (renal cortical and pelvic tumors) (Diwan et al., 1992). In adult rats, injection of soluble nickel salts followed by exposure to a promoting carcinogen resulted in kidney cancer (renal cortical

CHAPTER 8

CHEMICAL CARCINOGENESIS

adenocarcinomas) that frequently metastasized to the lung, liver, and spleen (Kasprzak et al., 1990). The carcinogenic properties of metallic nickel are believed to be due to ionic nickel, which can slowly dissolve in the body from nickel compounds. Nickel compounds are classified as known human carcinogens (IARC, 1990) based on animal data and sufficient evidence of carcinogenicity from human studies. The IARC (1990) evaluation of nickel and nickel compounds concluded that nickel compounds are carcinogenic to humans based on sufficient evidence in the nickel refining industry and very strong evidence of carcinogenicity of a variety of nickel compounds in experimental studies in rodents. Several cohort studies of workers exposed to various nickel compounds showed an elevated risk of death from lung and nasal cancers (IARC, 1990). An excess risk of lung and nasal cancer was seen in nickel refinery workers exposed primarily to soluble nickel compounds (Anderson et al., 1996). Lead Lead compounds do not appear to cause genetic damage directly, but may do so through several indirect mechanisms, including inhibition of DNA synthesis and repair, oxidative damage, and interaction with DNA-binding proteins and tumor-suppressor proteins (NTP, 2003). Lead has exhibited conflicting results concerning its genotoxicity; it does not cause mutations in bacteria, but does cause chromosomal aberrations in vitro and in vivo, and causes DNA damage in vivo and in cell-free systems, whereas in mammalian systems, conflicting results were observed. Lead also inhibits the activity of DNA and RNA polymerase in cell-free systems and in mammalian cell cultures. Conflicting results were observed for SCE and micronucleus formation in mammalian test systems. In studies with laboratory animals, carcinogenicity has been observed for soluble (lead acetate and lead subacetate) and insoluble (lead phosphate, lead chromate) inorganic lead compounds as well as for tetraethyl lead (an organic lead compound), following exposure via oral, injection, and in offspring exposed via the placenta or lactation. Although kidney tumors (including adenomas, carcinomas, and adenocarcinomas) were most frequently associated with lead exposure, tumors of the brain, hematopoietic system, and lung were reported in some studies (IARC, 1980, 1987; Waalkes et al., 1995). Lead also appears to function as a tumor promoter, leading to increased incidence in kidney tumors initiated by N -ethyl-N -hydroxyethylnitrosamine and N -(4 -fluoro-4biphenyl)acetamide (IARC, 1980, 1987). The mechanisms by which lead causes cancer are not understood. Lead and lead compounds are classified as reasonably anticipated to be human carcinogens based on limited evidence from studies in humans and sufficient evidence from studies in experimental animals. Lead exposure has been associated with increased risk of lung, stomach, and bladder cancer in diverse human populations (Fu and Boffetta, 1995; Steenland and Boffetta, 2000; NTP, 2003). Epidemiological studies link lead exposure to increased risk for lung and stomach cancer. However, most studies of lead exposure and cancer reviewed had limitations, including poor exposure assessment and failure to control for confounding factors.

organ and tissue targets induced by nongenotoxic carcinogens are many times in tissues where a significant incidence of background, spontaneous tumors is seen in the animal model. Prolonged exposure to relatively high levels of chemicals is usually necessary for the production of tumors. In addition, with nongenotoxic carcinogens, tumors are not theoretically expected to occur at exposures below a threshold at which relevant cellular effects are not observed. In contrast to DNA-reactive genotoxic effects, non-DNA reactive mechanisms may be unique to the rodent species used for testing. Certain chemical carcinogens have been well studied and provide examples for the use of mechanistic information in risk assessment. Further, the biochemical modes of action for non-DNA reactive carcinogens are diverse. Examples include agents that function via sustained cytotoxicity, receptor-mediated (e.g., CAR, PPARα, AhR) effects, hormonal perturbation, as well as the induction of oxidative stress and modulation of methylation status (Table 8-10). Each of these potential mechanisms is discussed in greater detail in the following sections. Cytotoxicity Cytotoxicity and consequent regenerative hyperplasia is a well-documented mode of action for a variety of nonDNA reactive chemical carcinogens (Dietrich and Swenberg, 1991). Chloroform-induced liver and kidney tumors and melamine-induced bladder tumors are classic examples of chemical carcinogens that are classified as functioning via a cytolethal mode of action (Bull et al., 1986; Butterworth, 1990; Larson et al., 1994; Pereira, 1994; Andersen et al., 1998). Chemicals that function through this mechanism produce sustained cell death, often related to metabolism of Table 8-10 Proposed Modes of Action for Selected Nongenotoxic Chemical Carcinogens mode of action

example

Cytotoxicity

Chloroform Melamine d-limonene, 1,4-dichlorobenzene Phenobarbital Trichloroethylene Perchloroethylene Diethylhexylphthalate Fibrates (e.g., clofibrate) TCDD Polychlorinated biphenyls (PCBs) Polybrominated biphenyls (PBBs) Biogenic amines Steroid and peptide hormones DES Phytoestrogens (bisphenol-A) Tamoxifen Phenobarbital Phenobarbital Choline deficiency Diethanolamine Ethanol TCDD Lindane Dieldrin Acrylonitrile

α2u -Globulin-binding Receptor mediated CAR PPARα

AhR

Hormonal

Altered methylation

Nongenotoxic (Epigenetic) Carcinogens A number of chemicals that produce tumors in experimental animals following chronic treatment appear to act via mechanisms not involving direct binding, damage, or interaction of the chemical or its metabolites with DNA (Williams and Whysner, 1996). Based on the lack of genotoxicity, yet their ability to induce tumors in rodent models, these agents have been labeled nongenotoxic carcinogens. The

347

Oxidative stress inducers

348

UNIT 3

NON-ORGAN-DIRECTED TOXICITY

CH3 Cl

CH3 CH3 H

CH3

CH3

C

CH

CH2

CH3

CH3

C

2,2,4-trimethylpentane C8H18

CH2 Cl D-limonene

1,4-dichlorobenzene

Figure 8-15. Examples of selective α2u-globulin-binding chemicals.

the chemical, that is accompanied by persistent regenerative growth, resulting in the potential for the acquisition of “spontaneous” DNA mutations and allowing mutated cells to accumulate and proliferate. This process then gives rise to preneoplastic focal lesions that upon further expansion can lead to tumor formation. Chloroform has been shown to induce mouse liver tumors only at doses that produce liver necrosis, thus demonstrating an association between necrosis with compensatory hyperplasia and the resulting tumorigenicity, and also supports that a threshold for the induction of tumors is likely at doses that do not produce toxicity. It is important to note that the induction of cytotoxicity may be observed with many carcinogens both genotoxic and nongenotoxic when high toxic exposures occur. Thus, the induction of cytotoxicity with compensatory hyperplasia may contribute to the observed tumorigenicity of many carcinogenic chemicals at high doses. α2u -Globulin-Binding Chemicals The carcinogens d-limonene, 1,4-dichlorobenzene, and trimethylpentane (Fig. 8-15) induce renal tumors selectively in the male rat, and provide excellent examples of species, sex, and tissue specificity of non-DNA reactive carcinogens. The mechanism for the species and sex specificity is related to the ability of these compounds to bind to α2u -globulin, a protein synthesized by the male rat liver at the onset of puberty, as the mechanism of tumorigenesis. α2u -Globulin is filtered through the glomerulus, and only partially excreted (∼50%) in the urine. The reabsorbed fraction accumulates in the lysosomes of the P2 segment of the proximal tubules, where it is hydrolyzed to amino acids (Melnick et al., 1996). Chemicals with the ability to bind to α2u globulin prevent the digestion of α2u -globulin and result in the accumulation in lysosomes, dysfunction of this organelle, and subsequent release of digestive enzymes and cell necrosis. The greater loss of tubule cells leads to increased cell proliferation in the P2 segment, which may be responsible for the tumor development and malignant transformation (Dietrich and Swenberg, 1991). Receptor Mediated P450 Inducers: Phenobarbital-like Carcinogens Phenobarbital is a commonly studied non-DNA reactive compound that is known to cause tumors by a nongenotoxic mechanism involving liver hyperplasia (Williams and Whysner, 1996). One feature seen following phenobarbital exposure is the induction of P450 enzymes, particularly CYP2B (Nims and Lubet, 1996). Because a number of diverse chemicals are known to induce various members of the P450 system (e.g., dieldrin, ethanol, TCDD), the specificity of this effect to carcinogenesis has been questioned. Recent evidence has shown that

the induction of CYP2B is mediated by activation of the constitutive androstane receptor (CAR), a member of the nuclear receptor family (Honkakoski et al., 1998; Ueda et al., 2002; Kodama et al., 2004). CAR-null mice show no induction of CYP2B following phenobarbital exposure (Wei et al., 2000). Other phenobarbital responses that are critical for tumor formation include increased cell proliferation, inhibition of apoptosis, inhibition of gap junctional communication, hypertrophy, and development of preneoplastic focal lesions in the liver (Whysner et al., 1996), effects that have all been shown to be CAR-dependent (Wei et al., 2000; Kodama et al., 2004) (Fig. 8-16). Peroxisome Proliferator Activated Receptor α (PPARα) A wide array of chemicals are capable of increasing the number and volume of peroxisomes in the cytoplasm of cells. These chemicals, termed peroxisome proliferators, include chemicals such as herbicides, chlorinated solvents (e.g., trichloroethylene and perchloroethylene), plasticizers (e.g., diethylhexylphthalate and other phthalates), lipid lowering fibrate drugs (e.g., ciprofibrate, clofibrate), and natural products. In addition, many of these chemicals produce liver enlargement and hepatocellular carcinoma in rats and mice through non-DNA reactive mechanisms (Lake, 1995; Reddy and Rao, 1997). Two additional tumor types are also associated with exposure to peroxisome proliferating compounds: Leydig cell tumors and pancreatic acinar-cell tumors in the rat. Studies conducted either in vivo or in vitro in primary hepatocyte cultures have shown important interspecies differences in the hepatic peroxisome proliferation responses to chemicals within the class of compounds. The rat and mouse were clearly responsive species, whereas primates and the guinea pig proved to be nonresponders. The Syrian hamster exhibits an intermediate response (Bentley et al., 1993; Lake, 1995). Due to the wide structural diversity of this chemical class, the mechanism(s) involved in peroxisome proliferation and tumorigenesis went unrecognized for years. The currently accepted mode of action for this class of chemicals involves agonist binding to the nuclear hormone receptor, peroxisome proliferator-activated receptor alpha (PPARα). Largely through the use of PPARα knockout mice, the activation of PPARα by agonists is needed for these chemicals to induce peroxisome proliferation and tumorigenesis in rodents (Issemann and Green, 1990; Lee et al., 1995; Peters et al., 1998, reviewed in Klaunig et al., 2003). PPARα is highly expressed in cells that have active fatty acid oxidation capacity (e.g., hepatocytes, cardiomyocytes, enterocytes). It is well documented that PPARα plays a central role in lipid metabolism and acts as a transcription factor to modulate gene expression following ligand activation. This latter effect arises through the heterodimerization of PPAR and RXRα, which results in binding

CHAPTER 8

CHEMICAL CARCINOGENESIS

349

CYP Induction Cell Proliferation

P

CAR PP

CAR

RXR

Increased Transcription

CamK

P

Phenobarbital

CAR

P

CAR RXR PBREM

CYP2B6, Cell 2 Proliferation Genes

Figure 8-16. Proposed mechanism for the involvement of the constitutive androstane receptor (CAR) in phenobarbital-induced gene expression changes. PP, protein phosphatase; CamK, CaM Kinase; RXR, retinoic acid receptor; PBREM, phenobarbital response element. Following dephosphorylation of CAR by protein phosphatase, CAR crosses the cell membrane and becomes phosphorylated by CaM kinase. CAR then forms a dimer with RXR and binds to PBREMs, resulting in increased gene expression.

9-cis-Retinoic Acid

RXR

PPARα Agonist

PPA R

TCACCT n T CACCT (PPRE - PPAR Response Element)

Enzyme Induction Peroxisome Proliferation Altered Lipid Metabolism

Altered Cell Growth DNA Synthesis Tumor Promotion

Figure 8-17. Mechanism for altered gene expression by peroxisome proliferator activated receptor α (PPARα) agonist binding. Following agonist binding to PPARα, the receptor dimerizes with the retinoic acid receptor (RXR). This complex then binds to PPREs, resulting in enhanced gene transcription.

to response elements (PPREs) and subsequent modulation of target gene transcription (Fig. 8-17). Following this event is the induction of cell proliferation and suppression of apoptosis (Marsman et al., 1988; James and Roberts, 1996; Burkhardt et al., 2001). Both these events would then be expected to affect tumor development as these effects would enhance the rate of fixation of DNA damage in the genome, leading to changes in gene expression such as the silencing of tumor suppressor genes or increased expression of oncogenes,

or suppress apoptosis that may normally remove DNA-damaged, potentially tumorigenic, cells. Because humans are exposed to a number of chemicals that are PPARα ligands, the relevance of this mode of action to humans has been evaluated (Klaunig et al., 2003). Although the same events would be expected to occur in exposed humans, several species differences have been noted, including a lack of induction of cell proliferation in nonhuman primates (Pugh et al., 2000), and the finding that the amount of PPARα in human liver is at least tenfold lower compared with the rat or mouse (Palmer et al., 1998; Tugwood et al., 1998). Based on these kinetic and dynamic differences between species, it has been concluded that tumors are not likely to occur in humans (Klaunig et al., 2003). Aryl Hydrocarbon Receptor (AhR) Agonists of the AhR including TCDD and selective members of the polychlorinatedand brominated-biphenyl (PCBs and PBBs) class of compounds (Fig. 8-18) have been linked to tumor development, and appear to function as hepatic tumor promoters (Pitot et al., 1982; IARC, 1997). The tumor response has been determined to be AhR dependent (Knutson and Poland, 1982) (Fig. 8-19). Upon ligand binding to the AhR, the ligand-bound AhR translocates to the nucleus, dimerizes with the Ah receptor nuclear translocator (ARNT), and binds to aryl hydrocarbon response elements [AREs also known as dioxin response elements (DRE) and xenobiotic response elements (XRE)] (for review see Nebert et al., 2000). AhRARNT-dependent genes include cytochrome P450 family members, NAD(P)H:quinine oxidoreductase, a cytosolic aldehyde dehydrogenase 3, a UDP-glucuronosyltransferase, and a glutathione transferase (Nebert et al., 2000), genes that are involved in metabolic

350

UNIT 3

CI

CI

NON-ORGAN-DIRECTED TOXICITY

O

CI

O

CI

other promoting chemicals, TCDD does inhibit apoptosis in hepatic foci (Stinchcombe et al., 1995).

2, 3, 7, 8-p-TCDD

3’

2’

2

3

4’ para

4

5’ meta

6’ ortho

6

5

PCB* *Prototypical structure for PCBs or PBBs. PCBs and PBB exhibit varying degrees of Chlorination or Bromination at all position of the biphenyl molecular

Figure 8-18. Selective examples of aryl hydrocarbon receptor-binding chemicals. Prototypical structure for PCBs or PBBs. PCBs and PBBs exhibit varying degrees of chlorination or bromination at positions 2-6 and 2 -6 of the biphenyl molecule.

activation as well as detoxification of chemicals. It has been hypothesized that there are additional AhR-ARNT-dependent genes (Nebert et al., 2000). AhR knockout mice showed a diminished response to tumor induction by AhR ligands (Nakatsuru et al., 2004), conversely, constitutively overexpressed AhR resulted in an increased incidence of liver tumors (Moennikes et al., 2004). As seen with

Hormonal Mode of Action Hormonally active chemicals include biogenic amines, steroids, and peptide hormones that cause tissuespecific changes through interaction with a receptor. In addition, a number of non-DNA reactive chemicals can induce neoplasms in rodents through receptor-mediated mechanisms, and/or perturbation of hormonal balance. Trophic hormones are known to induce cell proliferation at their target organs. This action may lead to the development of tumors when the mechanisms of hormonal control are disrupted and some or other hormone shows persistently increased levels. Several well-studied examples include the induction of ovarian neoplasms via decreased estradiol and increased LH levels (Capen et al., 1995) and the induction of thyroid tumors in rats by phenobarbital-type P450 inducers (McClain, 1995; Williams, 1995). Estrogenic agents can induce tumors in estrogen-dependent tissue. Selective estrogenic chemicals (agonists and antagonists) are shown in Fig. 8-20. Oral administration of 17β-estradiol to female mice increases the incidence of mammary tumors (Highman et al., 1977; Welsch et al., 1977), whereas subcutaneous administration of estradiol to young female mice produced tumors of the cervix and vagina (Pan and Gardner, 1948). Evidence that estrogenic chemicals are carcinogenic to humans comes from epidemiological data on breast and ovarian cancer, which indicates that individuals with higher circulating estrogen levels and those with exposure to the potent estrogenic agent diethylstilbesterol (DES) are at increased risk of cancer development. DES was first shown to induce mammary tumors in male mice following subcutaneous administration of the hormone. DES has been causally associated to the higher incidence of adenocarcinomas of the vagina and cervix in daughters of women treated with the hormone during pregnancy (Herbst and Scully, 1970; Herbst et al., 1972; Noller et al., 1972). The mechanism of action for DES is believed to function through its ability to induce

Metabolism of ligand by CYP1 forms CYP 1

AHR AHR

AHR ligand

ARNT

HSP 90

Increased

HSP 90 AHR

ARNT

AHR AIP

XRE

CYP1A1, CYP1A2, CYP1B1

transcription

Figure 8-19. Proposed mechanism of aryl hydrocarbon receptor (AhR) mediated gene expression. ARNT, AhR nuclear transporter; XRE, xenobiotic response element. Non-ligand bound AhR is maintained in the cytoplasm via association with chaperone proteins (e.g., AIP, Hsp90). Following ligand binding, chaperone proteins dissociate and AhR translocates to the nucleus where it binds with ARNT. The heterodimer binds to XREs resulting in an increase in gene transcription.

CHAPTER 8

CHEMICAL CARCINOGENESIS

351

OH

H

H

H

HO

(Agonist) 17β-estradiol (Agonist)

CH3 HO

C

OH

CH3

CH3

Bisphenol A

OCH2 CH2 N CH3

(Agonist)

C C CH3CH2 Tamoxifen

Tamoxifen (Antagonist) Figure 8-20. Chemical examples of estrogenic agonists and antagonists.

aneuploidy (Li et al., 1997; Tsutsui et al., 1997). The effects of steroidal chemicals on the cell cycle (Sutherland et al., 1995) and on microtubule assembly (Metzler et al., 1996) may be important in the aneuploidy inducing effects of some hormonal agents (Li et al., 1996). Chronic exogenous administration of hormonally active chemicals, including synthetic estrogens and anabolic steroids, can increase hepatic adenoma incidence in rats (Li et al., 1992) and in humans (IARC, 1997). Women of child-bearing age are sensitive to hepatic adenoma formation, which can be exacerbated by oral contraceptive use. These adenomas will regress upon hormone cessation (Edmondson et al., 1977) and can progress with continued administration (Christopherson et al., 1978). Chronic administration (>8 years) is required to detect this increased liver tumor risk from oral contraceptives (Tavani et al., 1993). Many substances in plants (phytoestrogens) have been described. These include compounds such as genestein, daidzein, glycetein, equol, and their metabolites found in soy products and various lignan derivatives (Adlercreutz and Mazur, 1997). In addition, a number of environmental nonsteroidal synthetic compounds have been identified that demonstrate apparent estrogenic activity (e.g., nonyl-phenol, bisphenol-A, chlorinated hydrocarbons) (Soto et al., 1997). The potential for these chemicals to induce cancer in humans is an area of current investigation. Species and tissue specificity in response to receptor and hormone-mediated carcinogenesis is often observed. As an example, tamoxifen is antiestrogenic in the chick oviduct, estrogenic in the mouse uterus with acute administration, but antiestrogenic with chronic administration in the same tissue, while it is estrogenic in the rat uterus. Whereas this may be in part due to tissue and species differences in coactivator and corepressor levels and availability,

many other pharmacokinetic and pharmacodynamic properties are likely to participate (Carthew et al., 1995). To further exemplify the complexity of the role of estrogen in cancer development, estrogens have also been shown to be protective in prostate cancer. Induction of ovarian tumors by dietary administration of nitrofurantoin in mice is an example of a tumorigenic effect secondary to drug-induced hormonal disturbance. Nitrofurantoin treatment resulted in ovarian atrophy with absence of graafian follicles and sterility. The reduction of follicles induced a reduction of sex steroids by the ovary, resulting in increased production of gonadotrophins, notably LH, presumably due to the decreased negative feedback on the hypothalamus–pituitary axis by estrogens. Persistent stimulation by LH of the ovary cells resulted in development of tumors (Capen et al., 1995). A similar effect is known to occur with dopamine agonists. The induction of hypoprolactinemia changes the number of LH receptors and leads to enhanced sensitivity to LH and a higher stimulation by LH, similar to nitrofurantoin. A number of chemicals that reduce thyroid hormone concentrations (T4 and/or T3) and increase thyroid-stimulating hormone (TSH) have been shown to induce neoplasia in the rodent thyroid. TSH demonstrates proliferative activity in the thyroid, with chronic drug-induced TSH increases leading to progression of follicular cell hypertrophy, hyperplasia, and eventually neoplasia. Inducers of metabolic enzymes in the liver, a classic and well-studied example being phenobarbital (Hood et al., 1999), result in increased thyroid hormone metabolism and as such lead to increases in TSH levels (Fig. 8-21). It is this latter event that is associated with the development of thyroid tumors in rodents. Recent evidence suggests that the enhancement of metabolism and excretion of thyroid hormones by xenobiotics (via induction of Phase II enzymes) is a consequence of CAR (and presumably PXR) activation. Qualitatively, thyroid gland

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Brain Pituitary TSH

+

Thyroid ACTH

Phenobarbital (Enhanced Metabolism)

T3

Liver Adrenal Glands Figure 8-21. Mechanism for phenobarbital-induced thyroid tumors. Through alteration of gene expression, phenobarbital enhances metabolism of thyroid hormone (T3) resulting in enhanced TSH production. Increased TSH then leads to increased cell proliferation in the thyroid.

function in rodents is much more susceptible to disturbances of the thyroid hormone levels than it is in humans. In contrast to humans, rodents lack the thyroid-binding globulin, which is the predominant plasma protein responsible for thyroxin binding and transport in humans (McClain, 1995). A relatively higher level of functional activity is present in rat thyroid compared to humans.

DNA Methylation and Carcinogenesis Post-DNA synthetic methylation of the 5-position on cytosine (5-methylcytosine, 5mC) is a naturally occurring modification to DNA in higher eukaryotes that influences gene expression (Holliday, 1990) (Fig. 8-22). Under normal conditions, DNA is methylated symmetrically on both strands. Immediately following DNA replication, the newly synthesized double-stranded DNA contains hemimethylated sites that signal for DNA maintenance methylases to transfer methyl groups from S-adenosylmethionine to cytosine residues on the new DNA strand (Hergersberg, 1991). The degree of methylation within a gene inversely correlates with the expression of that gene; hypermethylation of genes is associated with gene silencing, whereas hypermethylation results in an enhanced expression of genes. Several chemical carcinogens are known to modify DNA methylation, methyltransferase activity, and chromosomal structure. During carcinogenesis, both hypomethylation and hypermethylation of the genome have been observed (Counts and Goodman, 1995; Baylin, 1997). Increased methylation of CpG islands have been observed in bladder cancer and tumor suppressor genes such as the retinoblastoma gene, p16ink4a , and p14 A R F have been reported to be hypermethylated in tumors (Stirzaker et al., 1997; Belinsky et al., 1998; My¨oh¨anen et al., 1998; Salem et al., 2000; Esteller et al., 2001). Hypomethylation has been associated with increased mutation rates. Most metastatic neoplasms in humans have significantly lower 5MeC than normal tissue (Gama-Sosa et al., 1983); furthermore, many oncogenes are hypomethylated and their expression amplified. Choline and methionine, which can be derived from dietary sources, provide a source of methyl groups used in methylation reactions. Rats exposed to choline and/or methionine-deficient

NH2 N

Substrates

O

N 5´

CH2

C H

H

H H

O

N

CH2

C H

CH3

N

Products

O 5´

NH2

H H

H O

H 3´

H 3´

Deoxycytidine

+ DNMT

5-Methyl-cytidine

NH2 N H HO O

NH2

CI S

NH2 N N

N O

H HO

CH3

N

N NH2

N

N O

S O

HO

OH

S-adenosyl-L-methionine (SAM)

HO

OH

S-adenosyl homocysteine (SAH)

Figure 8-22. Substrates and products involved in DNA methylation. Methylation of DNA occurs through an enzymatic reaction catalyzed by DNA methyltransferases (DNMTs). DNA is methylated at the 5-position of cytosine and requires the cofactor S-adenosyl methionine.

CHAPTER 8

CHEMICAL CARCINOGENESIS

353

Table 8-11 Evidence for Choline Depletion in Hepatocarcinogenesis choline

methionine

folate

hepatocellular carcinoma

duration (months)

reference

– – – –

+ + Low –

+ + – +

100% 73% 100% 51%

12 12 15 13

Nakae et al., 1992 Lombardi et al., 1988 Henning et al., 1996 Ghoshal et al., 1984

diets resulted in hepatocellular proliferation and neoplasia (Abanobi et al., 1982; Wainfan and Poirier, 1992) (Table 8-11). In rodents fed choline or methyl donor groups deficient diets, the hepatic neoplasia is thought to arise from hypomethylation of c-myc, c-fos, and c-H-ras proto-oncogenes, due to the decreased availability of Sadenosyl-methionine (Newberne et al., 1982; Wainfan and Poirier, 1992). Chemicals such as diethanolamine result in hepatic neoplasia, in part via mechanisms involving choline depletion, altered methylation, and modulation of gene expression (Kamendulis and Klaunig, 2005; Carnell et al., 2006). Reactive oxygen species have also been shown to modify DNA methylation, resulting in changes in DNA methylation profiles through interfering with the ability of methyltransferases to interact with DNA leading to hypomethylation of CpG sites (Weitzman et al., 1994; Turk et al., 1995). Also, the presence of 8-OHdG in DNA can lead to hypomethylation of DNA, because this adduct if present in CpCpGpGp sequences will inhibit the methylation of adjacent C residues. Thus, oxidative DNA damage may be an important contributor to the carcinogenesis process brought about by the loss of DNA methylation, allowing the expression of normally quiescent genes. Also, the abnormal methylation pattern observed in cells transformed by chemical oxidants may contribute to an overall aberrant gene expression and promote the tumor process.

Oxidative Stress and Chemical Carcinogenesis Experimental evidence has shown that increases in reactive oxygen in the cell, through either physiological modification or through chemical carcinogen exposure, contribute to the carcinogenesis processes (Vuillaume, 1987; Trush and Kensler, 1991; Witz, 1991; Guyton and Kensler, 1993). Reactive oxygen species encompass a series of reactive compounds including the superoxide anion (• O2 -), hydroperoxyl radical (HO2 • .), hydrogen peroxide (H2 O2 ), and the hydroxyl radical (• OH), all derived through the reduction of molecular oxygen (Table 8-12). Oxygen radicals can be produced by both endogenous and exogenous sources and are typically counterbalanced by antioxidants (Table 8-13). Antioxidant defenses are both enzymatic (e.g., superoxide dismutase, glutathione peroxidase, and catalase) and nonenzymatic (e.g., vitamin E, vitamin C, β-carotene, glutathione (Betteridge, 2000; Abuja and Albertini, 2001). Importantly, many of these antioxidants are provided through dietary intake (Clarkson and Thompson, 2000). Endogenous sources of reactive oxygen species include oxidative phosphorylation, P450 metabolism, peroxisomes, and inflammatory cell activation (Table 8-13). Within the mitochondria, a small percentage of oxygen is converted into the superoxide anion via oneelectron reduction of molecular oxygen. Superoxide can be dismutated by superoxide dismutase to yield hydrogen peroxide (Barber

Table 8-12 Pathways for Intercellular Oxidant Generation Generation of reactive oxygen species via reduction of molecular oxygen O2 + e− → O−• 2 (superoxide anion) • O−• 2 + H2 O → HO2 (hydroperoxyl radical) HO•2 + e− + H → H2 O2 (hydrogen peroxide) H2 O2 + e− → OH− + • OH (hydroxyl radical) A series of oxygen radicals are produced by the reduction of molecular oxygen. Of the radicals produced, the hydroxyl radical, hydroperoxyl radical, and the superoxide anion are sufficiently reactive and may interact with biomolecules.

and Harris, 1994). In the presence of partially reduced metal ions, hydrogen peroxide is converted to the highly reactive hydroxyl radical through Fenton and Haber-Weiss reactions (Betteridge, 2000). Neutrophils, eosinophils, and macrophages represent another intracellular source of reactive oxygen species. Activated macrophages, through “respiratory burst” elicit a rapid increase in oxygen uptake that gives rise to a variety of reactive oxygen species including superoxide anion, hydrogen peroxide, and nitric oxide. The release of cytokines and reactive oxygen intermediates from activated Kupffer cells (the resident macrophage of the liver) has been implicated in hepatotoxicological and hepatocarcinogenic events (Rose et al., 1999; Rusyn et al., 1999); in particular, recent studies show that Kupffer cells may function at the promotion stage of carcinogenesis. Reactive oxygen species can also be produced by cytochrome P450mediated mechanisms including: (1) redox cycling in the presence of molecular oxygen, (2) peroxidase-catalyzed single-electron drug oxidations, and (3) “futile cycling” of cytochromes P450 (Parke, 1994; Parke and Sapota, 1996). Ethanol, phenobarbital, and a number of chlorinated and nonchlorinated compounds such as dieldrin, TCDD, and lindane are among the xenobiotics shown to increase in reactive oxygen species through P450-mediated mechanisms (Eksrom and Ingleman-Sundberg, 1989; Rice et al., 1994; Klaunig et al., 1997). Chemicals classified as peroxisome proliferators activated receptor α (PPARα) agonists (e.g., clofibrate, phthalate esters) represent chemicals that induce cytochrome P4504A, and increase the formation of peroxisomes. As such, an increase in H2 O2 production often accompanies exposure to chemicals that stimulate the number and activity of peroxisomes (Rao and Reddy, 1991; Wade et al., 1992). Through these or other currently unknown mechanisms, a number of chemicals that induce cancer (e.g., chlorinated compounds, radiation, metal ions, barbiturates, and some PPARα agonists) induce reactive oxygen species formation and/or oxidative stress (Rice-Evans and Burdon, 1993; Klaunig et al., 1997; Kamendulis and Klaunig, 2005).

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Table 8-13 Reactive Oxygen Species Generation and Removal in the Cell Cellular oxidants Endogenous Mitochondria • O−• 2 , H2 O2 , OH Cytochrome P450 O−• 2 , H2 O 2 Macrophage/inflammatory cells • − O−• 2 , NO, H2 O2 ,OCl Peroxisomes H 2 O2 Cellular antioxidants Enzymatic Superoxide dismutase Catalase Glutathione peroxidase Glutaredoxin Thioredoxin Oxidants > Antioxidants

Exogenous Redox cycling compounds O−• 2 Metals (Fenton reaction) H2 O2 + Fe2+ → OH− + • OH + Fe3+ Radiation • OH

Non-enzymatic Vitamin E Glutathione Vitamin C Catechins Oxidative Damage (DNA, RNA, Lipid, Protein)

Oxidants can be produced via both endogenous and exogenous sources. Antioxidants function to maintain the cellular redox balancing. However, excess production of oxidants and or inadequate supplies of antioxidants result in damage to cellular biomolecules and may impact on neoplastic development.

CHEMICAL CARCINOGEN

Map Kinase Cascades (ERK, P38, JNK)

P

Nucleus

Transcription Factors

P

P

Target Genes (Cell proliferation Differentiation Apoptosis)

Transcription Figure 8-23. Role of reactive oxygen species (ROS) on altered gene expression. Many chemical carcinogens increase ROS and activate signaling cascades, such as the mitogen activated (MAP) kinase family. These serene/threonine kinases activate transcription factors, leading to DNA binding and gene expression changes.

Oxidative Damage and Carcinogenesis Reactive oxygen species left unbalanced by antioxidants can result in damage to cellular macromolecules. In DNA, reactive oxygen species can produce single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA crosslinks (von Sonntag, 1987; Dizdaroglu, 1992; Demple and Harrison, 1994). Persistent DNA damage can result in either arrest or induction of transcription, induction of signal transduction pathways, replication errors, and genomic instability, events that are potentially involved in carcinogenesis. Oxidation of guanine at the C8 position results in the formation of 8-hydroxydeoxyguanosine (8-OHdG; see Fig. 8-7). This oxidative DNA adduct is mutagenic in bacterial and mammalian cells, produces dose-related increases in cellular transformation, and causes G → T transversions which are commonly observed in mutated oncogenes and tumor suppressor genes (Shibutani et al., 1991; Moriya, 1993; Hussain and Haris, 1998; Zhang et al., 2000). During DNA replication, 8-OHdG in the nucleotide pool can incorporate into the DNA template strand opposite dC or dA resulting in A:T to C:G transversions (Cheng et al., 1992; Demple and Harrison, 1994). Oxidative damage to mitochondrial DNA and mutations in mitochondrial DNA have been identified in a number of cancers (Schumacher et al., 1973; Horton et al., 1996; Cavalli and Liang, 1998; Tamura et al., 1999). Compared to nuclear DNA, mitochondrial genome is relatively susceptible to oxidative base damage due to (1) close proximity to the electron transport system, a major source of reactive oxygen species (Barber and Harris, 1994); (2) mitochondrial DNA is not protected by histones; and (3) DNA repair capacity is limited in the mitochondria (Bohr and Dianov, 1999; Sawyer and Van Houten, 1999). Although many pathways exist that enable the formation of oxidative DNA damage, mammalian cells also possess specific repair pathways for the remediation of oxidative DNA damage (Tchou and Grollman, 1993; Fortini et al., 1999). Apart from oxidized nucleic acids, oxygen radicals can damage cellular biomembranes resulting in lipid peroxidation. Peroxidation of biomembranes generates a variety of products, including reactive electrophiles, such as epoxides and aldehydes, including

CHAPTER 8

CHEMICAL CARCINOGENESIS

355

CHEMICAL CARCINOGENS

NF- B I B kinase

P

P

I B

I B

(Inactive)

NF-

I B

(Active)

NF-

Nucleus

NF- B

Transcription of Target Genes (Cytokines, Cell proliferation genes, Adhesion molecules)

Figure 8-24. Interaction between reactive oxygen species (ROS) and NFkB-induced gene expression. A number of chemicals that increase ROS have been shown to activate NFkB. It is proposed that ROS enhance the dissociation of IkB from the NFkB/IkB complex leading to active NFkB. NFkB can then translocate into the nucleus where it binds DNA and increases gene transcription.

malondialdehyde (MDA) (Janero, 1990). MDA is a highly reactive aldehyde and exhibits reactivity toward nucleophiles and can form MDA–MDA dimers. Both MDA and the MDA–MDA dimers are mutagenic in bacterial assays and the mouse lymphoma assay (Riggins and Marnett, 2001).

Oxidative Stress and Cell Growth Regulation Reactive oxygen species production and oxidative stress can affect both cell proliferation and apoptosis (Cerutti, 1985; Burdon, 1995; Slater et al., 1995). H2 O2 and superoxide anion can induce cell proliferation in several mammalian cell types (D’Souza et al., 1993). Conversely, high concentrations of reactive oxygen species can initiate apoptosis (Dypbukt et al., 1994). It has clearly been demonstrated that low levels of reactive oxygen species influence signal transduction pathways and alter gene expression (Fiorani et al., 1995). Many xenobiotics, by increasing cellular levels of oxidants, alter gene expression through activation of signaling pathways including cAMP-mediated cascades, calcium-calmodulin pathways, and transcription factors such as AP-1 and NF-κB as well as signaling through mitogenactivated protein (MAP) kinases, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and the p38 kinases (Kerr, 1992; Muller et al., 1997; Timblin et al., 1997). Activation of these signaling cascades by reactive oxygen species induced by chemical carcinogens ultimately leads to altered gene expression for a number of genes including those affecting proliferation, dif-

ferentiation, and apoptosis (Hollander and Fornace, 1989; Amstad et al., 1992; Pinkus, 1993; Zawaski, 1993; Brown et al., 1998; Chang and Karin, 2001; Martindale and Holbrook, 2002) (Fig. 8-23). Activation of NFκB, a ubiquitously expressed transcription factor, is regulated, in part, by reactive oxygen species and the cellular redox status, and has been observed to occur following a wide variety of extracellular stimuli, including exposure to chemical carcinogens such as PPARα agonists (Rusyn et al., 2003) and PCBs (Beauerle et al., 1988; Li and Karin, 1998; Schulze-Oshoff et al., 1998; Pahl, 1999; Nebreda and Porrai, 2000; Molina et al., 2001; Glauert et al., 2002) (Fig. 8-24).

Gap Junctional Intercellular Communication and Carcinogenesis Cells within an organism communicate in a variety of ways including through gap junctions, which are aggregates of connexin proteins that form a conduit between two adjacent cells (Lowenstein et al., 1981) (Fig. 8-25). Gap junctional intercellular communication appears to play an important role in the regulation of cell growth and cell death, in part through the ability to exchange small molecules (85% concordance with the 2-year rodent bioassay; (2) the stages involved in the clonal transformation of SHE cells (e.g., morphological transformation, immortalization, and tumorigenicity) closely resemble those associated with the classic defined stages of carcinogenesis (e.g., initiation, promotion, and progression); and (3) nongenotoxic/non-DNA reactive carcinogens elicit a positive response on morphological transformation in the SHE cell assay (Barrett and Lamb, 1985; LeBeouf et al., 1990; Isfort et al., 1994, 1996). The SHE assay offers an alternative, regulatory approved, means to check the validity of the positive result before embarking on potentially unnecessary and expensive chronic or subchronic testing protocols.

Chronic Testing for Carcinogenicity The majority of in vivo carcinogenicity testing is performed in rodent models. The administration of chemicals in the diet, often for extended periods, for assessment of their safety and/or toxic-

Donehower, 1996 Spalding et al., 1993

ity began in the 1930s (Sasaki and Yoshida, 1935). Animal testing today remains a standard approach for determining the potential carcinogenic activity of xenobiotics. In addition to the lifetime exposure rodent models, organ-specific model systems, multistage models, and transgenic models are being developed and used in carcinogen testing (Table 8-21). Chronic (2-Year) Bioassay Two-year studies in laboratory rodents remain the primary method by which chemicals or physical agents are identified as having the potential to be hazardous to humans. The most common rodents used are the rat and mouse. Typically the bioassays are conducted over the lifespan of the rodents (2 years). Historically, selective rodent strains have been used in the chronic bioassay; however, each strain has both advantages and disadvantages. For example, the National Toxicology Program (NTP) typically uses Fisher 344 (F344) rats and B6C3F1 mice. The F344 rat has a high incidence of testicular tumors and leukemias, whereas the B6C3F1 mouse is associated with a high background of liver tumors (Table 8-22). In the chronic bioassay, two or three dose levels of a test chemical and a vehicle control are administered to 50 males and 50 females (mice and rats), beginning at 8 weeks of age, continuing throughout their lifespan. The route of administration can be via oral (gavage), dietary (mixed in feed), or inhalation (via inhalation chambers) exposure. Typically a number of short-term in vivo tests are conducted prior to the chronic bioassay to determine acute toxicity profiles, appropriate route of administration, and the maximum tolerated dose (MTD). Generally, the MTD is used to set the high dose in the chronic study. The use of the MTD as the upper dose level has been questioned by many investigators, as it is recognized that the doses selected often represent doses that are considered unrealistically high for human exposure. Pharmacokinetics and metabolism at high dose are frequently unrepresentative of those at lower doses; in

CHAPTER 8

CHEMICAL CARCINOGENESIS

Table 8-22 Spontaneous Tumor Incidence (Combined Benign and Malignant) in Selected Sites of the Two Species, B6C3F1 Mice and F344 Rats, Used in the NCI/NTP Bioassay B6C3F1 MICE

F344 RATS

site

male

female

male

female

Liver Adenoma Carcinoma Pituitary Adrenal Thyroid Hematopoietic Mammary gland Lung

10.3 21.3 0.7 3.8 1.3 12.7 0 17.1

4.0 4.1 8.3 1.0 2.1 27.2 1.9 7.5

3.4 0.8 24.7 19.4 10.7 30.1 2.5 2.4

3.0 0.2 47.5 8.0 9.3 18.9 26.1 1.2

addition, a general relationship between toxicity and carcinogenicity cannot be drawn for all classes of chemicals. During the study, food consumption and bodyweight gain should be monitored, and the animals observed clinically on a regular basis, and at necropsy the tumor number, location, and pathological diagnosis for each animal is thoroughly assessed. Organ-Specific Bioassays and MultiStage Animal Models Many tissue-specific bioassays have been developed with the underlying goal being to produce a sensitive and reliable assay that could be conduced in a time frame shorter in duration than the 2-year chronic bioassay. These assays are commonly used to detect carcinogenic activity of chemicals in various target organs (Weisburger and Williams, 1984). Of the many models available, three models, the liver, skin, and lung models are more widely used. Carcinogenicity Testing in the Liver The liver represents a major target organ for chemical carcinogens. It has been estimated that nearly half of the chemicals tested in the 2-year chronic bioassay by the National Toxicology Program showed an increased incidence of liver cancer. The rodent liver has been used as an animal model for carcinogenesis since the 1930s. Early pioneering work by Peraino and Pitot as well as Farber showed the multistaged process that occurs in the liver. The multistage nature of carcinogenesis is paralleled in the animal models; the system is characterized by welldefined changes including the formation of initiated cells by genotoxic agents that then progress to preneoplastic focal lesions, some of which subsequently convert into neoplasms. The use of preneoplastic lesions as endpoints in carcinogenicity testing may shorten the experiment from 2 years to a few months. Several rodent liver focus assays have been developed to assess the ability of a chemical to induce liver cancer, and study the mechanisms involved in tumor development (Williams, 1982; Bannasch, 1986a; Ito et al., 1989). Liver carcinogenesis assays have been developed to study and distinguish chemicals that affect the initiation or promotion stage of hepatocarcinogenesis. During the assay for initiating activity of a chemical, the test substance is given prior to exposure to a promoting chemical. Although a single initiating dose can result in the induction of focal lesions, exposure over several weeks is often used to increase

367

the sensitivity of the model (Williams, 1982; Parnell et al., 1988). Phenobarbital is a commonly used tumor promoter; however, a wide range of chemicals have also been used as promoting agents (Solt and Farber, 1976; Oesterle and Deml, 1988). To assess the promoting activity of a chemical, the liver is initiated with a genotoxic chemical, often diethylnitrosamine. The test chemical is then administered for a duration of weeks to several months, and chemical substances with promoting activity may stimulate the proliferation of initiated cells or may inhibit the proliferation of the surrounding putatively normal cells. The dose of the initiating carcinogen should represent a dose that will not itself induce liver tumors during the course of the experiment. Another method commonly used was developed in Japan by Ito and coworkers (Ogiso et al., 1990; Ito et al., 1994; Shirai et al., 1999). The entire assay takes only 8 weeks to perform. Rats are initiated with a single dose of diethylnitrosamine, followed by a 2-week recovery period, after which point the animals are exposed to the test compound for 8 weeks. After 1 week of exposure to the test substance, the animals are given a 2/3 partial hepatectomy. The control group receives the same initiation and partial hepatectomy, but is not exposed to the test chemical. Hepatic focal lesions, while individually are clonal in nature express a number of phenotypic alterations in various enzyme markers. A common endpoint assessed is the formation of liver lesions that stain for glutathione-S-transferase pi (GST-P), a marker that stains many focal lesions in the rat. Using this assay, these investigators have demonstrated a significant correlation between the results obtained using this assay and the mediumand long-term study results (Ogiso et al., 1990). This group has also modified the original procedure to enable the detection of promoting agents. In this protocol, carcinogens are given over a 4-week period to initiate the formation of focal lesions, after which, test chemicals are administered for an additional 24- to 36-week period (Ito et al., 1996). In this manner, the ability of the test chemical to promote the growth of preneoplastic lesions can be assessed. The newborn mouse model originally described by Shubik and coworkers (Pietra et al., 1959) has also been used as a model for hepatocarcinogenesis (Vesselinovitch et al., 1978; Fujii, 1991). In this model, a single dose of diethylnitrosamine is administered to infant mice to initiate focal lesions. This step is then followed by exposure to test chemicals for several weeks to assess their potential to promote focal lesion development in the liver. Identification of hepatic foci in H&E-stained sections is regarded as the most reliable approach for the diagnosis and quantification of preneoplastic liver lesions in rodents. Preneoplastic lesions are obligatory precursor lesions that can lead to liver tumors and will progress to benign and malignant liver cell tumors without further chemical exposure, and are used as endpoints in carcinogenicity testing (Pitot et al., 1987; Pereira and HerrenFreund, 1988; Ito et al., 1989; Maronpot et al., 1989; Williams, 1989). In addition to the sensitive detection of these preneoplastic lesions in conventional H&E staining, a number of histochemically detectable phenotypic alterations have been used for their quantification; however, these markers are only useful in the rat model, as focal lesions in mice to not exhibit these same phenotypic markers. Carcinogenicity Testing in the Skin The Mouse Skin model is one of the most extensively studied and used models for understanding multistage carcinogenesis. This model of carcinogenesis is an assay that has been used to dissect mechanisms of carcinogenesis and also is purported to be a useful intermediate-term cancer bioassay. The skin was the target organ of the first experimental induction

368

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Figure 8-30. Experiments demonstrating the initiation and promotion phases of carcinogens in mice. Group 2: Application of promoter repeated at twice-weekly intervals for several months. Group 3: Application of promoter delayed for several months and then applied twice weekly. Group 6: Promoter applied at monthly intervals.

of chemical carcinogenesis (Yamagiwa and Ichikawa, 1915). The early studies by Friedwald and Rous (1944) and Berenblum and Shubik (1947) introduced the two-stage concept of carcinogenesis in the skin (Fig. 8-30). This model exploits many of the unique properties of the mouse skin; one major being that the development of neoplasia can be followed visually. In addition, the number and relative size of papillomas and carcinomas can be quantified as the tumors progress. Both initiating and promoting activities of chemical carcinogens can be assessed using this model. In the promotion assay, a number of chemical carcinogens have been used to initiate cells in the mouse skin including urethane, UV light, benzo(a)pyrene, and dimethylbenzanthracene, with the latter of more common usage. The requirement for all initiating agents is to induce a genotoxic event that upon failure to repair DNA damage results in the formation of a mutated cell. Grossly, initiated cells of the skin appear identical to normal skin. Initiation in skin is frequently linked with the mutation of the CHr gene. Since the terminally differentiated cells in the skin are no longer capable of undergoing cell division, only initiated cells retain their proliferative capacity and thus represent the cell populations that give rise to tumors. To assess promotion by a chemical, an initiating chemical is applied first and is followed by the administration of a test substance for several weeks on the shaved skin of mice (Slaga, 1984). The promotion of initiated karatinocytes is commonly assessed using the phorbol ester TPA, which is routinely included as a positive control in this assay. The current hypothesis is that during the initiation stage the expansion of initiated cells occurs as a result of inflammation and hyperplasia from either TPA or through mechanical wound healing mechanisms. Upon repeated application of tumor promoters, selective clonal expansion of initiated keratinocytes occurs, resulting in skin papillomas, which over time can progress to carcinomas. In the standard two-stage protocol for mouse skin, malignant progression is relatively rare with approximately 5% of the papillomas progressing to the carcinoma stage. For the detection of initiating activity,

the test substance is applied to skin prior to promotion with phorbol esters. Several mouse models are available, including hairless mice, SENCAR mice, both of which have enhanced sensitivity to induction of skin cancer (Brown and Balmain, 1995; Sundberg et al., 1997).

Carcinogenicity Testing in the Lung Strain A mice are genetically susceptible to the development of lung tumors, with lung tumors being observed in control animals as early as 3–4 weeks of age, with a steady increase to nearly 100% by 24 months of age (Shimkin and Stoner, 1975). Chemically induced tumors appear to be derived from hyperplastic lesions that progress to adenoma, carcinoma within adenoma, and ultimately, to carcinomas (Stoner et al., 1993). In this model, carcinogenicity is typically assessed as an acceleration of tumor development, rather than an increase in the tumor incidence. The protocol currently used is that the chemical is administered for a period of 8 weeks, after which the animals remain on test for 4 additional months without chemical exposure. The strain A mouse lung tumor assay is sensitive to particular classes of chemicals, such as polycyclic aromatic hydrocarbons, nitrosamines, nitrosoureas, carbamates, aflatoxin, certain metals, and hydrazines (Stoner and Shimkin, 1985; Maronpot et al., 1986; Stoner, 1991).

Carcinogenicity Testing in Other Organs Test systems to examine the ability of a chemical to promote neoplastic development at organ sites other than liver, skin, and lung have also been developed. The available systems include animal models directed at examining carcinogenicity in the kidney, bladder, pancreas, stomach, colon, small intestine, and oral cavity. These models vary in the initiating carcinogen used, and frequency, duration, and site of application, as well as the duration of promoting chemical exposure. Table 8-23 provides an overview of the animal models available for these target organs.

CHAPTER 8

CHEMICAL CARCINOGENESIS

369

Table 8-23 organ

species

initiating carcinogen

initiator duration

carcinogen (promoter) duration

references

Kidney

Rat1

Single exposure

20 wks

Hiasa et al., 1991

Bladder

Rat

4 wks

32 wks

Fukushima et al., 1983

Pancreas

Rat, Syrian Hamsters

Stomach (forestomach) Stomach (glandular)

Rat

N -ethyl-N hydroxyethylnitrosamine N -nitrosobutyl(4hydroxybutyl)amine N -nitrosobis(2oxopropyl)amine, N -nitroso(2hydroxypropyl)(2oxopropyl)amine Benzo(a)pyrene

Rat

N -methyl-N  -nitro-N nitronitrosoguanidine

Colon Small intestine

Rat Rat Mice

2 exposures 2–9 wks 4 wks

Oral cavity (lip, oral, nasal) Oral cavity (tongue) Buccal cells (squamous cell carcinoma) Buccal cells (squamous cell carcinoma)

Rat

Azoxymethane 1,2-Dimethylhydrazine N -ethyl-N  -nitro-N nitrosoguanidine 4-Nitroquinoline N -oxide

Rat

Single exposure

40 wks

Silva et al., 1995

40 wks

Takahashi et al., 1986

12 wks 16–20 wks

4 wks2

Yamashita et al., 1994 Lindenschmidt et al., 1987; Jagadeesan et al., 1994 Johansson et al., 1989

4-Nitroquinoline N -oxide

8 wks3

Tanaka et al., 1995

Syrian Hamster

—

—

Repeated application4 10–16 wks

Solt et al., 1987

Syrian Hamster

dimethylbenz[a]anthracene

Single exposure

45 wks

Gimenez-Conti and Slaga, 1993

Transgenic Animals in Carcinogenicity Assessment Due to the development of animal models with genetic alterations that invoke a susceptibility to carcinogenesis by chemical agents, the use of transgenic and knockout animals in carcinogenicity assessment is gaining more popularity. The common models that have been used include the Tg.AC and rasH2 transgenic mice, and p53+/− and XPA−/− knockout mice (Gulezian et al., 2000). Recently, the feasibility of the use of these animal models as alternative assays for the 2-year chronic bioassay was assessed by the Health and Environmental Sciences Institute (HESI) branch of the International Life Sciences Institute (ILSI). In this assessment, 21 chemicals were evaluated, encompassing genotoxic, nongenotoxic, and noncarcinogenic chemicals. The conclusions drawn from the scientific review suggested that these models appear to have usefulness as screening models for assessment of chemical carcinogenicity; however, they do not provide definitive proof of potential human carcinogenicity. Further the scientific panel suggested that these models could be used in place of the mouse 2-year bioassay (Tennant et al., 1998; Cohen et al., 2001). Coupled with information on genotoxicity, particularly DNA reactivity, structure–activity relationships, results from other bioassays, and the results of other mechanistic investi-

1–2x/week, 4 wks Single exposure

Longnecker et al., 1984, 1985

gations including toxicokinetics, metabolism, and mechanistic information, these alternate mouse models for carcinogenicity appear to be useful models for assessing the carcinogenicity of chemical agents.

CHEMICAL CARCINOGENESIS IN HUMANS A number of factors have been implicated in the induction of cancer in humans. Infectious agents, lifestyle, medical treatments, environmental and occupational exposure account either directly or indirectly for the majority of cancers seen in humans. Of these, the component that contributes the most to human cancer induction and progression is lifestyle: tobacco use, alcohol use, and poor diet (Fig. 8-1 and Table 8-24). Tobacco usage either through smoking tobacco, chewing tobacco, or tobacco snuff-type products is estimated to be responsible for 25–40% of all human cancers. In particular a strong correlation between tobacco usage and mouth, larynx, lung, esophageal, and bladder cancer exists. It has been estimated (Doll and Peto, 1981) that 85–90% of all lung cancer cases in the United States are a direct result of tobacco use. The induction of pancreatic cancer also appears to have a linkage to tobacco use. Alcohol

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Table 8-24 Carcinogenic Factors Associated with Lifestyle chemical(s)

neoplasm(s)

Alcohol beverage Aflatoxins Betel chewing Dietary intake (fat, protein, calories) Tobacco smoking

Esophagus, liver, oropharynx, and larynx Liver Mouth Breast, colon, endometrium, gallbladder Mouth, pharynx, larynx, lung, esophagus, bladder

Table 8-25 Occupational Human Carcinogens agent

industrial process

neoplasms

Asbestos Arsenic Alkylating agents (mechloro-ethamine hydrochloride and bis[chloromethyl]ether) Benzene Benzidine, beta-naphthylamine Chromium and chromates Nickel Polynuclear aromatic hydrocarbons Vinyl chloride monomer Wood dust Beryllium Cadmium Ethylene oxide Formaldehyde Polychlorinated biphenyls

Construction, asbestos mining Mining and smelting Chemical manufacturing

Peritoneum, bronchus Sking, bronchus, liver Bronchus

Chemical manufacturing Dye and textile Tanning, pigment making Nickel refining Steel making, roofing, chimney cleaning Chemical manufacturing Cabinet making Aircraft manufacturing, electronics Smelting Production of hospital supplies Plastic, textile, and chemical Electrical-equipment production and maintenance

Bone marrow Urinary bladder Nasal sinus, bronchus Nasal sinus, bronchus Skin, scrotum, bronchus Liver Nasal sinus Bronchus Bronchus Bone marrow Nasal sinus, bronchus Liver

Table 8-26 Human Carcinogenic Chemicals Associated with Medical Therapy and Diagnosis chemical or drug

associated neoplasms

Alkylating agents (cyclophospamide, melphalan) Azathioprine Chloramphenicol Diethylstilbestrol Estrogens Phenacetin Phenytoin Thorotrast

Bladder, leukemia Lymphoma, reticulum cell sarcoma, skin, Kaposi’s sarcoma (?) Leukemia Vagina (clear cell carcinoma) Liver cell adenoma, endometrium, skin Renal pelvis (carcinoma) Lymphoma, neuroblastoma Liver (angiosarcoma)

consumption contributes anywhere from 2 to 4% of cancers of the esophagus, liver, and larynx. Poor diets whether high fat, low protein, high calories, or diets lacking in needed antioxidants and minerals account for anywhere from 10 to 70% of human cancers. Diet contaminated by molds such as Aspergillus flavis (which produces aflatoxin B1) have been linked epidemiologically to a higher incidence of liver cancer. It also appears that aflatoxin B1 exposure coupled with hepatitis B virus infection produces an increased incidence of liver cancer compared to aflatoxin B1 or hepatitis B exposure individually. Mold contaminated food stuffs have also been shown to produce nitroso compounds. There is substantial evidence that over nutrition either through excess calories and/or high fat diets contribute to a number of human

cancers (Doll and Peto, 1981). In particular high fat and high calorie diets have been linked to breast, colon, and gall bladder cancer in humans. Diets poor in antioxidants and/or vitamins such as vitamin A and vitamin E probably also contribute to the onset of cancer. The method of cooking may also influence the production of carcinogens produced in the cooking process. Carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons are formed during broiling and grilling of meat. Acrylamide, a suspected human carcinogen has been found in fried foods at low concentrations. A number of occupations have been associated with the development of specific cancers (Table 8-25). As noted earlier, the linkage between chimney sweepers who as young boys in England were exposed to polyaromatic hydrocarbons through constant exposure to soot, developed scrotal cancer. The linkage between occupational

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371

Table 8-27 IARC Classification of the Evaluation of Carcinogenicity for Human Beings group

evidence

1. Agent is carcinogenic to humans

Human data strong Animal data strong Human epidemiology data suggestive Animal data positive Human epidemiology data weak Animal data positive Human and animal data inadequate Human and animal data negative

2A. Agent is probably carcinogenic to humans 2B. Agent is possibly carcinogenic to humans 3. Agent is not classifiable as to carcinogenicity to humans 4. Agent is probably not carcinogenic to humans

exposure to asbestos and the development of bronchiogenic carcinoma and as well as malignant mesothelioma has been clearly established. The appearance of bronchiogenic carcinoma was much higher in shipyard workers who were exposed to both asbestos as well as cigarette smoking. Muscat and Wynder (1991) noted no association between cigarette smoking and mesothelioma formation. Similarly, asbestos exposure by itself (without smoking) does not seem to increase the risk of bronchiogenic carcinoma. Aromatic amines used in the chemical and dye industries have been shown to produce or induce bladder cancer in humans. Prolonged high exposure to benzene in an occupational setting has been linked to the formation of acute myelogenous leukemia in humans. A number of medical therapy and medical diagnostic tools have also been linked to the induction of human cancer (Table 8-26). Cancer chemotherapeutic drugs, such as the alkylating agent cyclophos-

Table 8-28 USEPA Cancer Guidelines Descriptors Carcinogenic to humans – strong evidence of human carcinogenicity, including convincing epidemiologic evidence of a causal association between human exposure and cancer. – the mode(s) of carcinogenic action and associated key precursor events have been identified in animals, and there is strong evidence that the key precursor events in animals are anticipated to occur in humans. Likely to be carcinogenic to humans – weight of the evidence is adequate to demonstrate carcinogenic potential to an agent in animal experiments in more than one species, sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans Suggestive evidence of carcinogenic potential – the weight of evidence is suggestive of carcinogenicity; a concern for potential carcinogenic effects in humans is raised, but the data are judged not sufficient for a stronger conclusion. Inadequate information to assess carcinogenic potential – available data are judged inadequate for applying one of the other descriptors. Not likely to be carcinogenic to humans – This descriptor is appropriate when the available data are considered robust; there is no basis for human hazard concern, evidence in both humans and animals that the agent is not carcinogenic.

phamide, have been associated with bladder tumors and leukemia in patients receiving these treatments. The administration of the synthetic estrogenic compound diethylstilbestrol to pregnant women, in order to improve embryo implantation and prevent spontaneous abortion, has been shown to result in the formation of clear cell carcinomas of the vagina in the female offspring of mothers treated with diethylstilbestrol during pregnancy. The use of oral contraceptives containing synthetic estrogens as their major or only component has been implicated in the induction of liver cell adenomas. In addition, an association exists between prolonged use of estrogenic oral contraceptives and an increased incidence of premenopausal breast cancer. Androgenic steroids and synthetic testosterone compounds have been implicated in hepatocellular carcinoma induction. Therapeutic immunosuppression given to transplant patients or arising secondary to selective diseases such as acquired immune deficiency syndrome (AIDS) result in an increase in a variety of different neoplasms. These results further support the role of the immune system in identifying and removing early preneoplastic cells from the body. In addition, the previously used diagnostic tracer Thorotrast has been sufficiently linked to the formation of hemangiosarcomas in the liver.

Classification Evaluation of Carcinogenicity in Humans The assessment and designation of a chemical or a mixture of chemicals as carcinogenic in humans is evaluated by various agencies worldwide. The evaluation usually encompasses both epidemiological and experimental animal and in vitro data utilizing assays as described earlier in this chapter. One of the first devised schemes for the classification of an agent’s carcinogenicity was devised by the International Agency for Research on Cancer (IARC) (Table 8-27). The IARC approach assigns the chemical or mixture to one of five groupings based upon strength of evidence for the Table 8-29 USEPA Mode of Action Definitions Mode of action: Key events and processes, starting with the interaction of an agent with a cell, through functional and anatomical changes, resulting in cancer or other health endpoints. Key event: Empirically observable precursor step that is itself a necessary element of the mode of action or is a biologically based marker for such an element.

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Table 8-30 US EPA Mode of Action Framework Mode of action criteria Summary description of the hypothesized mode of action Identification of key events Strength, consistency, specificity of association Dose–response concordance Temporal relationship Biological plausibility and coherence Consideration of the possibility of other MOAs Is the mode of action sufficiently supported in the test animals? Is the mode of action relevant to humans? Which populations or life stages can be particularly susceptible to the mode or action?

agent’s possible, probable, or definite carcinogenicity to humans. In Group 1 classification, the agent or mixture is classified as definitely carcinogenic to humans. The second grouping is Group 2A in which the agent is probably carcinogenic to humans. In Group 2B, the agent is classified as possibly carcinogenic to humans. In Group 3, the agent is not classifiable. In the last group, Group 4, the agent is not carcinogenic to humans. The IARC produces a series of monographs that describe the methodology for the evaluation of specific chemicals and the rationale for their final classification. Currently, more than 100 chemical agents or mixtures or exposure circumstances have been classified by IARC as falling in Group 1, which shows sufficient evidence for carcinogenicity to humans. Similar classifications exist for the U.S. EPA, the Food & Drug Administration, and the European Community (EC). The

classification of agents with regard to human carcinogenicity can many times be very difficult, in particular, when animal data and/or epidemiological data in humans are inconclusive or confounded. New U.S. EPA Guidelines for Cancer Risk Assessment (2005) uses descriptors for defining the relative carcinogenic risk to humans (Table 8-28). These descriptors include: carcinogenic to humans; likely to be carcinogenic to humans; suggestive evidence of carcinogenic potential; inadequate information to access carcinogenic potential; and, not likely to be carcinogenic in humans. The EPA Guidelines take into account the understanding of the mode of carcinogenic action and associated key precursor events needed for the cancer to form (Table 8-29). Central to the U.S. EPA Guidelines for Cancer Risk Assessment is the utilization of the mode of action framework to define the key events in rodents, and assessment of whether those same key events and mode of action can occur in humans (Table 8-30). This approach is similar to that developed by the International Program on Chemical Safety and by panels in the International Life Sciences Institute.

SUMMARY The induction of cancer by chemicals is well established in animal models as well as in humans. Linkages between chemicals found in human lifestyle, occupational exposure, and environmental exposure provides strong evidence for the induction or contribution of environmental occupational lifestyle carcinogens to human cancer. Cancer is a multistage process that involves a mutational event followed by the selected clonal proliferation of the mutated cell. The multistage nature of the process has been extensively examined with regard to molecular, cellular, tissue, and organ events.

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CHAPTER 9

GENETIC TOXICOLOGY R. Julian Preston and George R. Hoffmann ASSAYS FOR DETECTING GENETIC ALTERATIONS

WHAT IS GENETIC TOXICOLOGY?

Introduction to Assay Design DNA Damage and Repair Assays Gene Mutations in Prokaryotes Genetic Alterations in Nonmammalian Eukaryotes Gene Mutations and Chromosome Aberrations Mitotic Recombination Gene Mutations in Mammals Gene Mutations In Vitro Gene Mutations In Vivo Transgenic Assays Mammalian Cytogenetic Assays Chromosome Aberrations Micronuclei Sister Chromatid Exchanges Aneuploidy Germ Cell Mutagenesis Gene Mutations Chromosomal Alterations Dominant Lethal Mutations Development of Testing Strategies

HISTORY OF GENETIC TOXICOLOGY HEALTH IMPACT OF GENETIC ALTERATIONS Somatic Cells Germ Cells CANCER AND GENETIC RISK ASSESSMENTS Cancer Risk Assessment Genetic Risk Assessment MECHANISMS OF INDUCTION OF GENETIC ALTERATIONS DNA Damage Ionizing Radiations Ultraviolet Light Chemicals Endogenous Agents DNA Repair Base Excision Repair Nucleotide Excision Repair Double-Strand Break Repair Mismatch Repair O 6 -Methylguanine-DNA Methyltransferase Repair Formation of Gene Mutations Somatic Cells Germ Cells Formation of Chromosomal Alterations Somatic Cells Germ Cells

HUMAN POPULATION MONITORING NEW APPROACHES FOR GENETIC TOXICOLOGY Advances in Cytogenetics Molecular Analysis of Mutations and Gene Expression CONCLUSIONS ACKNOWLEDGMENTS

WHAT IS GENETIC TOXICOLOGY?

cer and genetic risk assessments, the mechanisms underlying genetic toxicology assays, the assays that can be used for detecting genotoxic endpoints, the use of the same assays for better understanding mechanisms of mutagenesis, and new methods for the assessment of genetic alterations. The field is evolving rapidly, and the present snapshot will set the stage for considering this evolution.

Genetic toxicology is a branch of the field of toxicology that assesses the effects of chemical and physical agents on the hereditary material (DNA) and on the genetic processes of living cells. Such effects can be assessed directly by measuring the interaction of agents with DNA or more indirectly through the assessment of DNA repair or the production of gene mutations or chromosome alterations. Given the risk assessment framework of this chapter, it is important at the outset to distinguish between genotoxicity and mutagenicity. Genotoxicity covers a broader spectrum of endpoints than mutagenicity. For example, unscheduled DNA synthesis, sister chromatid exchanges, and DNA strand breaks are measures of genotoxicity, not mutagenicity, because they are not themselves transmissible from cell to cell or generation to generation. Mutagenicity on the other hand refers to the production of transmissible genetic alterations. This chapter discusses the history of the development of the field of genetic toxicology, the use of genetic toxicology data in can-

HISTORY OF GENETIC TOXICOLOGY The field of genetic toxicology can be considered to have its roots in the pioneering work of H.J. Muller (1927), who showed that X-rays could induce mutations in the fruit fly, Drosophila. In his studies he showed not only that radiation exposure could increase the overall frequencies of mutations but also that the types of mutations induced were exactly the same in effect, or phenotype, as those observed in the absence of radiation exposure. Thus, the induced mutagenic responses must be assessed in relation to background 381

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mutations. As a conclusion to this study of radiation-induced mutations, Muller predicted the utility of mutagenesis studies not only for the study of mutations themselves but also for gene mapping approaches. Karl Sax (1938) built upon Muller’s original studies of radiation-induced mutations by showing that X-rays could also induce structural alterations to chromosomes in Tradescantia pollen grains. Sax and his colleagues, notably in the absence of a knowledge of DNA structure and chromosomal organization, showed that at least two critical lesions in a nuclear target are required for the production of an exchange within (intrachromosome) or between (interchromosome) chromosomes. We know now that the lesions identified by Sax are DNA double-strand breaks, base damages, or multiply damaged sites (reviewed by Ward, 1988). In addition, Sax and colleagues (Sax, 1939; Sax and Luippold, 1952) showed that the yield of chromosome aberrations was reduced if the total dose of X-rays was delivered over extended periods of time or split into two fractions separated by several hours. These observations led to the concept of restitution of radiation-induced damage, which was later recognized as involving specific DNA repair processes (see below). Consideration of the genetic effects of exogenous agents on cells was expanded to include chemicals in 1946, when Charlotte Auerbach and colleagues reported that mustard gas could induce mutations in Drosophila and that these mutations were phenotypically similar to those induced by X-rays (Auerbach and Robson, 1946). Thus, the field of chemical mutagenesis was initiated to run in parallel with studies of radiation mutagenesis. These original studies of Auerbach (actually conducted in 1941) are placed in a historical and biological perspective by the delightful review of Geoffrey Beale (1993). Although the scientific value of the analysis of mutations in Drosophila was clear, there was an impression that the extrapolation to predict similar effects in human populations was too wide a step. Thus, a research effort of great magnitude was initiated to attempt to assess radiation-induced mutations in mice. This effort resulted in the publication by William Russell (1951) of data on X-ray-induced mutations using a mouse specific-locus mutation assay. These data clearly showed that the type of results obtained with Drosophila could be replicated in a mammalian system. The mouse tester strain developed for the specific-locus assay has recessive mutations at seven loci coding for visible mutations, such as coat color, eye color, and ear shape. This homozygous recessive tester strain can be used for identifying recessive mutations induced in wild-type genes at the same loci in mice treated with radiation or chemical mutagens. It was noteworthy that the mutation rate for X-ray-induced mutations in germ cells was similar in mouse and Drosophila. Subsequent studies by Liane Russell and colleagues showed that chemicals could induce mutations at the same seven loci (Russell et al., 1981). Over the next 20 years, genetic toxicologists investigated the induction of mutations and chromosomal alterations in somatic and germ cells largely following exposures to radiation. The ability to grow cells in vitro, either as primary cultures or as transformed cell lines, enhanced these quantitative studies. The in vitro culture of human lymphocytes, stimulated to reenter the cell cycle by phytohemagglutinin, greatly expanded the information on the assessment of chromosomal alterations in human cells [an excellent review by Hsu (1979) is recommended]. It also became feasible to use cytogenetic alterations in human lymphocytes as a biodosimeter for assessing human exposures to ionizing radiations (Bender and Gooch, 1962).

Two events during the 1970s served to expand the utility of mutagenicity data into the realm of risk assessment. The Millers and their colleagues (Miller and Miller, 1977) showed that chemical carcinogens could react to form stable, covalent derivatives with DNA, RNA, and proteins both in vitro and in vivo. In addition, they reported that these derivatives could require the metabolism of the parent chemical to form reactive metabolites. This metabolism is required for some chemicals to become mutagens and carcinogens. Metabolic capability is endogenous in vivo, but most cell lines in vitro have lost this capacity. To overcome this for in vitro mutagenicity studies, Heinrich Malling and colleagues developed an exogenous metabolizing system based upon a rodent liver homogenate (S9) (Malling and Frantz, 1973). Although this exogenous metabolism system has had utility, it does have drawbacks related to species and tissue specificity and loss of cellular compartmentalization. The development of transgenic cell lines containing inducible P450 genes has overcome this drawback to some extent (Crespi and Miller, 1999). The second development in the 1970s that changed the field of genetic toxicology was the development by Bruce Ames et al. (1975) of a simple, inexpensive mutation assay with the bacterium Salmonella typhimurium. This assay can be used to detect chemically induced reverse mutations at the histidine locus and can include the exogenous metabolizing S9 system described above. The Ames assay, as it is generally called, has been expanded and modified to enhance its specificity as discussed below (under section “Gene Mutations in Prokaryotes”). The assay has been used extensively, especially for hazard identification, as part of the cancer risk assessment process. This use was based on the assumption that carcinogens were mutagens, given that cancer required mutation induction. This latter dogma proved to be somewhat inhibitory, in some ways, to the field of genetic toxicology because it provided a framework that was too rigid. Nonetheless, over the decade of the mid-1970s to mid-1980s somewhere on the order of 200 short-term genotoxicity and mutagenicity assays were developed for screening potentially carcinogenic chemicals. The screens included mutation induction, DNA damage, DNA repair, and cell killing or other genotoxic activities. Several international collaborative studies were organized to establish the sensitivity and specificity of a select group of assays as well as to assess interlaboratory variation (IPCS, 1988). In summary, most assays were able to detect carcinogens or noncarcinogens with an efficiency of about 70% as compared with the outcome of 2-year cancer bioassays. There are a number of possible reasons for the imperfect correspondence, the most likely being that there is a group of chemical carcinogens that do not induce cancer by a direct mutagenic action. The latter point was addressed to some extent by Tennant et al. (1987), who compared the effectiveness of a small standard battery of well-characterized short-term assays to identify carcinogens. Again, this battery predicted about 70% of known carcinogens. Subsequently, the lack of a tight correlation between carcinogenicity and mutagenicity (and the converse, noncarcinogenicity and nonmutagenicity) was found to be due to the fact that some chemicals were not directly mutagenic but instead induced the damage necessary for tumor development indirectly by, for example, clonally expanding preexisting mutant cells (i.e., tumor promotion) or through the production of reactive oxygen species. This class of chemicals has been given the rather unfortunate name of nongenotoxic to contrast them with genotoxic ones; the classification as not directly mutagenic is more appropriate. In the context of the mechanism of their mutagenicity, it is even more preferable to distinguish between DNA-reactivity and its correlate

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non-DNA-reactivity. In the past 10 years or so, emphasis has been placed on identifying mechanisms whereby nondirectly mutagenic chemicals can be involved in tumor production. Those identified include cytotoxicity with regenerative cell proliferation, mitogenicity, receptor-mediated processes, changes in methylation status, and alterations in cell–cell communication. In the past 10 years or so, the field of genetic toxicology has moved away from the short-term assay approach for assessing carcinogenicity to a much more mechanistic approach, fueled to quite an extent by the advances in molecular biology. The ability to manipulate and characterize DNA, RNA, and proteins and to understand basic cellular processes and how they can be perturbed has advanced enormously over this period. Knowing how to take advantage of these technical developments is paramount. This chapter addresses current genetic toxicology: the assays for qualitative and quantitative assessment of cellular changes induced by chemical and physical agents, the underlying molecular mechanisms for these changes, and how such information can be incorporated into cancer and genetic risk assessments. In addition, the way forward for the field is addressed in the form of an epilogue. Thus, the preceding historical overview sets the stage for the rest of the chapter.

HEALTH IMPACT OF GENETIC ALTERATIONS The importance of mutations and chromosomal alterations for human health is evident from their roles in genetic disorders, including birth defects and cancer. Therefore, mutations in both germ cells and somatic cells need to be considered when an overall risk resulting from mutations is concerned.

Somatic Cells An association between mutation and cancer has long been evident, such as through the correlation between the mutagenicity and carcinogenicity of chemicals, especially in biological systems that have the requisite metabolic activation capabilities. Moreover, human chromosome instability syndromes and DNA repair deficiencies are associated with increased cancer risk (Friedberg, 1985). Cancer cytogenetics has greatly strengthened the association in that specific chromosomal alterations, including deletions, translocations, inversions, and amplifications, have been implicated in many human leukemias and lymphomas as well as in some solid tumors (Rabbitts, 1994). Critical evidence that mutation plays a central role in cancer has come from molecular studies of oncogenes and tumor suppressor genes. Oncogenes are genes that stimulate the transformation of normal cells into cancer cells (Bishop, 1991). They originate when genes called proto-oncogenes, involved in normal cellular growth and development, are genetically altered. Normal regulation of cellular proliferation requires a balance between factors that promote growth and those that restrict it. Mutational alteration of protooncogenes can lead to overexpression of their growth-stimulating activity, whereas mutations that inactivate tumor suppressor genes, which normally restrain cellular proliferation, free cells from their inhibitory influence (Hanahan and Weinberg, 2000). The action of oncogenes is genetically dominant in that a single active oncogene is expressed even though its normal allele is present in the same cell. Proto-oncogenes can be converted into

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active oncogenes by point mutations or chromosomal alterations. Base-pair substitutions in ras proto-oncogenes are found in many human tumors (Bishop, 1991; Barrett, 1993). Among chromosomal alterations that activate proto-oncogenes, translocations are especially prevalent (Rabbitts, 1994). For example, Burkitt’s lymphoma involves a translocation between the long arm of chromosome 8, which is the site of the c-MYC oncogene, and chromosome 14 (about 90% of cases), 22, or 2. A translocation can activate a protooncogene by moving it to a new chromosomal location, typically the site of a T-cell receptor or immunoglobulin gene, where its expression is enhanced. A similar translocation-based mechanism also applies to various other hematopoietic cancers. Alternatively, the translocation may join two genes, resulting in a protein fusion that contributes to cancer development. Fusions have been implicated in other hematopoietic cancers and some solid tumors (Rabbitts, 1994). Like translocations, other chromosomal alterations can activate proto-oncogenes, and genetic amplification of oncogenes can magnify their expression (Bishop, 1991). Mutational inactivation or deletion of tumor suppressor genes has been implicated in many cancers. Unlike oncogenes, the cancercausing alleles that arise from tumor suppressor genes are typically recessive in that they are not expressed when they are heterozygous (Evans and Prosser, 1992). However, several genetic mechanisms, including mutation, deletion, chromosome loss, and mitotic recombination, can inactivate or eliminate the normal dominant allele, leading to the expression of the recessive cancer gene in a formerly heterozygous cell (Cavenee et al., 1983). The inactivation of tumor suppressor genes has been associated with various cancers, including those of the eye, kidney, colon, brain, breast, lung, and bladder (Fearon and Vogelstein, 1990; Marshall, 1991). Gene mutations in a tumor suppressor gene called P53, located on chromosome 17, occur in many different human cancers, and molecular characterization of P53 mutations has linked specific human cancers to mutagen exposures (Harris, 1993; Aguilar et al., 1994; Royds and Iacopetta, 2006). In the simplest model for the action of tumor suppressor genes, two events are considered to be required for the development of retinoblastoma, a tumor of the eye, because both normal alleles must be inactivated or lost (Knudson, 1997). In sporadic forms of the cancer (i.e., no family history), the two genetic events occur independently, but in familial forms (e.g., familial retinoblastoma), the first mutation is inherited, leaving the need for only a single additional event for expression. The strong predisposition to cancer in the inherited disease stems from the high likelihood that a loss of heterozygosity will occur by mutation, recombination, or aneuploidy in at least one or a few cells in the development of the affected organ. The simple model involving two events and a single pair of alleles cannot explain all observations concerning tumor suppressor genes because many cancers involve more than one tumor suppressor gene. For example, the childhood kidney tumor called Wilms’ tumor can be caused by damage in at least three different genes (Marshall, 1991), and colorectal carcinomas are often found to have lost not only the wild-type P53 tumor suppressor gene but also other tumor suppressor genes (Fearon and Vogelstein, 1990; Stoler et al., 1999). Moreover, a single mutation in a tumor suppressor gene, even though not fully expressed, may contribute to carcinogenesis. For example, a single P53 mutation in a developing colorectal tumor may confer a growth advantage that contributes to the development of the disease (Venkatachalam et al., 1998). Subsequent loss of heterozygosity will increase the growth advantage as the tumor progresses from benign to malignant (Fearon and

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Vogelstein, 1990). In this regard (mutation and selection), carcinogenesis has been likened to an evolutionary process, with genomic instability providing the substrate and with growth advantage as the selection pressure. Many cancers involve both activation of oncogenes and inactivation of tumor suppressor genes (Fearon and Vogelstein, 1990; Bishop, 1991). The observation of multiple genetic changes supports the view that cancer results from an accumulation of genetic alterations and that carcinogenesis is a multistep process (Kinzler et al., 1996; Hahn et al., 1999; Stoler et al., 1999). At least three stages have been defined in carcinogenesis: initiation, promotion, and progression (Barrett, 1993). Initiation involves the induction of a genetic alteration, such as the mutational activation of a ras proto-oncogene by a mutagen. It is an irreversible step that starts the process toward cancer. Promotion involves cellular proliferation in an initiated cell population. Promotion can lead to the development of benign tumors such as papillomas. Agents called promoters stimulate this process. Promoters may be mutagenic but are not necessarily so. Progression involves the continuation of cell proliferation and the accumulation of additional irreversible genetic changes; it is marked by increasing genetic instability and malignancy. More recent studies are beginning to change this view, leading to the concept of acquired capabilities (Hanahan and Weinberg, 2000). In their Hallmarks of Cancer, Hanahan and Weinberg describe a set of six acquired characteristics that are essential for the formation of all tumors irrespective of tumor type and species. These characteristics are broadly described as follows: self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. It seems probable that there is no specific order for obtaining these characteristics. Gene mutations, chromosome aberrations, and aneuploidy are all implicated in the development of cancer. Mutagens and clastogens (chromosome breaking agents) contribute to carcinogenesis as initiators. Their role does not have to be restricted to initiation, however, in that mutagens, clastogens, and aneugens (agents that induce aneuploidy) may contribute to the multiple genetic alterations that characterize progression or the development of acquired capabilities. Other agents that contribute to carcinogenesis, such as promoters, need not be mutagens. However, the role of mutations is critical, and analyzing mutations and mutagenic effects is essential for understanding and predicting chemical carcinogenesis.

Germ Cells The relevance of gene mutations to health is evident from the many disorders that are inherited as simple Mendelian characteristics (Mohrenweiser, 1991). About 1.3% of newborns suffer from autosomal dominant (1%), autosomal recessive (0.25%), or sex-linked (0.05%) genetic diseases (NRC, 1990; Sankaranarayanan, 1998). Molecular analysis of the mutations responsible for Mendelian diseases has revealed that almost half these mutations are basepair substitutions; of the remainder, most are small deletions (Sankaranarayanan, 1998). Many genetic disorders (e.g., cystic fibrosis, phenylketonuria, Tay-Sachs disease) are caused by the expression of recessive mutations. These mutations are mainly inherited from previous generations and are expressed when an individual inherits the mutant gene from both parents. New mutations make a larger contribution to the incidence of dominant diseases than to that of recessive diseases because only a single dominant mutation is required for expression.

Thus, new dominant mutations are expressed in the first generation. If a dominant disorder is severe, its transmission between generations is unlikely because of reduced fitness. For dominants with a mild effect, reduced penetrance, or a late age of onset, the contribution from previous generations is apt to be greater than that from new mutations. Estimating the proportion of all Mendelian genetic diseases that can be ascribed to new mutations is not straightforward; a rough estimate is 20% (Wyrobek, 1993; Shelby, 1994). Besides causing diseases that exhibit Mendelian inheritance, gene mutations undoubtedly contribute to human disease through the genetic component of disorders with a complex etiology (Sankaranarayanan et al., 1999). Some 3% (UNSCEAR, 2001) or 5–6% (Sankaranarayanan, 1998) of infants are affected by congenital abnormalities; if one includes multifactorial disorders that often have a late onset, such as heart disease, hypertension, and diabetes, the proportion of the population affected increases to more than 60% (Sankaranarayanan, 1998; UNSCEAR, 2001). Such frequencies are necessarily approximate because of differences among surveys in the reporting and classification of disorders. A higher prevalence would be found if less severe disorders were included in the tabulation. Nevertheless, such estimates provide a sense of the large impact of genetic disease. Sensitive cytogenetic methods have led to the discovery of minor variations in chromosome structure that have no apparent effect. On the other hand, other relatively minor structural chromosome aberrations cause fetal death or serious abnormalities. Aneuploidy (gain or loss of one or more chromosomes) also contributes to fetal deaths and causes disorders such as Down syndrome. About 4 infants per 1000 live births have syndromes associated with chromosomal abnormalities, including translocations and aneuploidy. The majority of these syndromes (about 85%) result from trisomies (NRC, 1990). The majority of the adverse effects of chromosomal abnormalities occur prenatally. It has been estimated that 5% of all recognized pregnancies involve chromosomal abnormalities, as do about 6% of infant deaths and 30% of all spontaneous embryonic and fetal deaths (Mohrenweiser, 1991). Among the abnormalities that have been observed, aneuploidy is the most common, followed by polyploidy. Structural aberrations constitute about 5% of the total. Unlike gene mutations, many of which are inherited from the previous generation, about 85% of the chromosomal anomalies observed in newborns arose de novo in the germ cells of the parents (Mohrenweiser, 1991). The frequency of aneuploidy assessed directly in human sperm, initially by standard karyotyping and more recently by fluorescence in situ hybridization (FISH), is 3–4%; about 0.4% are sex chromosome aneuploidies (Martin et al., 1991, 1996). The frequency of aneuploidy in human oocytes is about 18% (Martin et al., 1991).

CANCER AND GENETIC RISK ASSESSMENTS Cancer Risk Assessment The formalized process for conducting a cancer risk assessment has many variations based upon national requirements and regulations. A summary of some of the different approaches can be found in Moolenaar (1994). There are ongoing attempts, for example, by the International Program on Chemical Safety (IPCS), to develop a harmonized approach to cancer (and genetic) risk assessments. However, no unified approach is currently available. Thus, for the purpose of this chapter, the formalized approach developed by the U.S. Environmental Protection Agency (EPA) based upon

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the paradigm presented by the National Research Council (NRC, 1983) is described here for depicting the use of genetic toxicology in the risk assessment process. Genetic toxicology data have been used until recently solely for hazard identification. Namely, if a chemical is DNA-reactive, then tumors are considered to be produced by this chemical via direct mutagenicity. This has led, in turn, to the use of the default linear extrapolation from the rodent bioassay tumor data to exposure levels consistent with human environmental or occupational exposures (EPA, 1986). The assessment of risk requires the application of a series of default options, e.g., from laboratory animals to humans, from high to low exposures, from intermittent to chronic lifetime exposures, and from route to route of exposure. Default options are “generic approaches, based on general scientific knowledge and policy judgement that are applied to various elements of the risk assessment process when specific scientific information is not available” (NRC, 1994). The default options have been, in some ways, the Achilles’ heel of the cancer risk-assessment process because they have a very significant impact on low exposure risk but are based on an uncertain database. This concern led the EPA (1996) to develop a very different approach, initially described in the Proposed Guidelines for Carcinogen Risk Assessment, now released as the Guidelines for Carcinogen Risk Assessment (EPA, 2005). In these guidelines, the emphasis is on using mechanistic data, when available, to inform the risk assessment process, particularly for dose–response assessment. The goal is to develop biologically based dose–response models for estimating cancer risk at low environmental exposures. This does, in general, bring the EPA approach into some harmony with those in other countries (Moolenaar, 1994), where a more narrative approach to risk assessment is preferred to a strictly quantitative one. The outcome of a more mechanistically based cancer risk assessment process is that there is a greater impetus to developing a database for mechanisms in addition to the yes/no output from genotoxicity assays. The same group of genotoxicity assays can be used for the collection of both types of information. The advent of molecular biology techniques has certainly aided in the pursuit of mechanisms of mutagenicity and carcinogenicity. It is anticipated that the cancer risk assessment process will evolve as new types of data are obtained. In this regard, some of the issues that remain to be more firmly elucidated are (1) the relative sensitivities of different species (particularly rodent and human) to the induction of organ-specific mutations and tumors by chemicals and radiation; (2) the shape of the dose response for genetic alterations and tumors at low (environmental) exposure levels, especially for genotoxic chemicals; and (3) the relative sensitivity of susceptible subpopulations of all types. A better understanding of these major issues will greatly reduce the uncertainty in cancer risk assessments by, in part, replacing default options with biological data. The recently released EPA (2005) Guidelines provide a framework for cancer risk assessment that utilizes a mode-of-action (MoA) as the means of describing the “necessary but not sufficient” steps required for a chemical to produce a tumor. A particular MoA can further be defined by a set of key events that are required for tumor development (Preston and Williams, 2005). In addition, the key events can be used to establish whether or not a particular MoA described for a rodent model is plausible in humans (the so-called Human Relevance Framework, Meek et al., 2003). This more defined approach based on the use of the best available science can possibly be extended to include noncancer health effects (Seed et al., 2005).

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Figure 9-1. Parallelogram approach for genetic risk assessment. Data obtained for genetic alterations in rodent somatic and germ cells and human somatic cells are used to estimate the frequency of the same genetic alterations in human germ cells. The final step is to estimate the frequency of these genetic alterations that are transmitted to offspring.

Genetic Risk Assessment The approach for conducting a genetic risk assessment is less well defined than that for cancer risk. In fact, only a handful of genetic risk assessments have been conducted. An in-depth discussion of the topic can be found in the book Methods for Genetic Risk Assessment (Brusick, 1994). The reader is also referred to the genetic risk for ethylene oxide developed by the EPA (Rhomberg et al., 1990) and the discussion of this and a recalculation presented by Preston et al. (1995). These two articles serve to highlight the difficulties with and uncertainties in genetic risk assessments. The general approach is to use rodent germ cell and somatic cell data for induced genetic alterations and human data for induced genetic alterations in somatic cells (when available) to estimate the frequency of genetic alterations in human germ cells. This is the “parallelogram approach” (Fig. 9-1) first used by Brewen and Preston (1974) for X-irradiation and subsequently more fully developed for chemical exposures by Sobels (1982). The aim of this approach is to develop two sensitivity factors: (1) somatic to germ cell in the rodent and (2) rodent to human using somatic cells. These factors can then be used to estimate genetic alterations in human germ cells. Of course, for a complete estimate of genetic risk, it is necessary to obtain an estimate of the frequency of genetic alterations transmitted to the offspring (UNSCEAR, 2001). In addition, separate genetic risk assessments need to be conducted for males and females, given the considerable difference in germ cell development and observed and predicted sensitivity differences.

MECHANISMS OF INDUCTION OF GENETIC ALTERATIONS DNA Damage The types of DNA damage produced by ionizing radiations, nonionizing radiations, and chemicals are many and varied, including single- and double-strand breaks in the DNA backbone, crosslinks between DNA bases or between DNA bases and proteins, and chemical addition to the DNA bases (adducts) (Fig. 9-2). The aim of this section is to introduce the topic of DNA damage because such

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Apurinic Site (monofunctional alkylating agents)

T CH3 Alkylation (monofunctional alkylating agents)

Intercalations (acridines)

A Apyridmidinic Site (monofunctional alkylating agents)

A

Radical Formation (BrdU + light, X-rays) Single-Strand Breaks (X-rays, UV, etc.) Adduct of a Bulky Molecule (e.g., benzo(a)pyrene)

T CH3

T

Pyrimidine Dimers (UV)

Phosphotriesters (monofunctional alkylating agents)

Base Damage (X-rays) Double-Strand Breaks (ionizing radiation)

G

Interstrand Cross Links (bifunctional alkylating agents)

G

Intrastrand Cross Links (polyfunctional alkylating agents)

DNA-Protein Cross Links (X-rays, polyfunctional alkylating agents)

Figure 9-2. Spectrum of DNA damage induced by physical and chemical agents.

damage is the substrate for the formation of genetic alterations and genotoxicity in general. However, much greater detail can be found in recent reviews that are referenced at the appropriate places within each section. It should be noted that endogenous processes and exogenous agents can produce DNA damage, but mutations themselves are produced by errors in DNA repair or replication that are a consequence of the induced DNA damage.

Ionizing Radiations Ionizing radiations such as X-rays, gamma rays, and alpha particles produce DNA single- and double-strand breaks and a broad range of base damages (Goodhead, 1994; Wallace, 1994; Ward, 1994). In addition, it has been reported more recently that multiply damaged sites or clustered lesions can be formed that appear to be more difficult to repair. Such lesions consist of multi single lesions, including oxidized purine or pyrimidine bases, sites of base loss, and single-strand breaks. These multiple lesions can be formed in DNA from the same radiation energy depo-

sition event (Blaisdell et al., 2001). The relative proportions of these different classes of DNA damage vary with type of radiation. For example, single-strand breaks and base damages predominate with X-rays, for which ionization density is sparse, whereas the frequencies of single- and double-strand breaks are more similar with alpha particles, for which ionization density is dense. The frequencies of individual base damages have been assessed using monoclonal antibodies, for example (Le et al., 1998), but only a very few of the total spectrum have so far been studied. More recently, it has been demonstrated that the modified histone gamma-H2AX can be used as a sensitive marker of DNA double-strand breaks (Nakamura et al., 2006).

Ultraviolet Light Ultraviolet light (a nonionizing radiation) induces two predominant lesions, cyclobutane pyrimidine dimers and 6,4-photoproducts. These lesions have been studied extensively because they can both be quantitated by chemical and immunological

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methods (Friedberg et al., 1995). In part because of this feature, the repair of cyclobutane dimers and 6,4-photoproducts has been extremely well characterized, as discussed below. Chemicals Chemicals can produce DNA alterations either directly (DNA-reactive) as adducts or indirectly by intercalation of a chemical between the base pairs (e.g., 9-aminoacridine) (see Heflich, 1991, for a review). Many electrophilic chemicals react with DNA, forming covalent addition products (adducts). The DNA base involved and the positions on DNA bases can be specific for a given chemical. Such specificity of DNA damage can result in a spectrum of mutations that is chemical specific, i.e., a fingerprint of sorts (Dogliotti et al., 1998). Some alkylated bases can mispair, causing mutations when DNA is replicated. Alkylated bases can also lead to secondary alterations in DNA. For example, the alkyl group of an N7-alkylguanine adduct, which is a major adduct formed by many alkylating agents, labilizes the bond that connects the base to deoxyribose, thereby stimulating base loss. Base loss from DNA leaves an apurinic or apyrimidinic site, commonly called an AP site. The insertion of incorrect bases into AP sites causes mutations (Laval et al., 1990). Bulky DNA adducts formed, for example, by metabolites of benzo(a)pyrene or N -2-acetylaminofluorene are recognized by the cell in a similar way to UV damages and are repaired similarly (see below). Such adducts can also hinder polymerases and cause mutation as a consequence of errors that they trigger in replication. Endogenous Agents Endogenous agents are responsible for several hundred DNA damages per cell per day (Lindahl, 2000). The majority of these damages are altered DNA bases (e.g., 8oxoguanine and thymine glycol) and AP sites. The cellular processes that can lead to DNA damage are oxygen consumption that results in the formation of reactive oxygen species (e.g., superoxide • O2 , hydroxyl free radicals • OH, and hydrogen peroxide) and deamination of cytosines and 5-methylcytosines leading to uracils and thymines, respectively. The process of DNA replication itself is somewhat error-prone, and an incorrect base can be added by replication polymerases. The frequencies of these endogenously produced DNA damages can be increased by exogenous (genotoxic) agents.

DNA Repair The cell is faced with the problem of how to cope with the quite extensive DNA damage that it sustains. In a general sense, two processes are present to achieve this. If the damage is extensive, the cell can undergo apoptosis (programmed cell death), effectively releasing it from becoming a mutant cell (Evan and Littlewood, 1998). If the damage is less severe, it can be repaired by a range of processes that are part of a generalized cellular DNA damage response network that returns the DNA to its undamaged state (error-free repair) or to an improved but still altered state (error-prone repair). As a feature of this error-prone repair, it has recently been demonstrated that a family of polymerases, the eukaryotic translesion synthesis polymerases (e.g., human Y-family polymerases eta, iota, kappa, and Rev1) can bypass lesions that otherwise would block replication by the normal processive polymerases (Rattray and Strathern, 2003; Prakash et al., 2005). These polymerases have the ability to bypass specific DNA lesions or groups of lesions. The result of the bypass can be an incorrect DNA sequence or a correct one depending on the induced lesion and the particular bypass polymerase. The basic

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principles underlying most repair processes (other than translesion synthesis) are damage recognition, removal of damage (except for strand breaks or cleavage of pyrimidine dimers), repair DNA synthesis, and ligation. In order to achieve this for different types of DNA lesions, cells have modified the protein complexes used for other housekeeping processes (e.g., transcription, replication, and recombination). This chapter presents a brief outline of the major classes of DNA repair; much greater detail can be found in the reviews provided for each section and general reviews by Van Houten and Albertini (1995), Friedberg (2000), and Wood et al. (2005). Base Excision Repair The major pathways by which DNA base damages are repaired involve a glycosylase that removes the damaged base, causing the production of an apurinic or apyrimidinic site that can be filled by the appropriate base or processed further (Demple and Harrison, 1994; Seeberg et al., 1995; Wood, 1996; McCullough et al., 1999; Sung and Demple, 2006). The resulting gap from this further processing can be filled by a DNA polymerase, followed by ligation to the parental DNA. The size of the gap is dependent upon the particular polymerase involved in the repair (i.e., polymerase β for short patches; polymerase δ or ∈ for longer patches). Oxidative damage, either background or induced, are important substrates for base excision repair (Lindahl, 2000). The role of translesion bypass polymerases in the repair of DNA base alterations is discussed above. Nucleotide Excision Repair The nucleotide excision repair (NER) system provides the cell’s ability to remove bulky lesions from DNA. In the past decade the NER process has been studied extensively, and a complete characterization of the genes and proteins involved has been obtained (Reardon and Sancar, 2006). NER uses about 30 proteins to remove a damage-containing oligonucleotide from DNA. The basic steps are damage recognition, incision, excision, repair synthesis, and ligation. The characterization of these steps has been enhanced by the use of rodent mutant cell lines and cells from individuals with the UV-sensitivity, skin cancer-prone syndrome xeroderma pigmentosum (XP, for which there are at least seven distinct genetic complementation groups). Of particular interest is the link between NER and transcription, for which the DNA damage in actively transcribing genes, and specifically the transcribed strand, is preferentially and thus more rapidly repaired than the DNA damage in the rest of the genome (Lommel et al., 1995; Jiang and Sancar, 2006). Thus, the cell protects the integrity of the transcription process. This link between transcription and repair appears to be provided by two factors: (1) when a bulky lesion is located on the transcribed strand of an active gene, RNA polymerase II is blocked, thus providing a signal for recruiting the NER complex, and (2) a major component of the NER complex is the TFII H basal transcription factor. The involvement of TFII H in repair also provides some specificity to the incisions in the DNA required to remove the damaged nucleotide. An incision on the 3 side of the damage is made first by the XPG protein followed by one on the 5 side by the XPF-ERRC1 complex. The lesion is removed in the 27–30 nucleotide segment formed by the two incisions. The gap is filled by polymerase δ or ∈ in the presence of replication factor C and proliferating cell nuclear antigen (PCNA). Ligation by DNA ligase I completes the process. This NER process has been reconstituted in vitro, allowing for complete characterization, kinetic studies, and estimates of fidelity (Aboussekhra et al., 1995).

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Double-Strand Break Repair Cell survival is seriously compromised by the presence in the cell of broken chromosomes. Unrepaired double-strand breaks trigger one or more DNA damage response systems to either check cell-cycle progression or induce apoptosis. In order to reduce the probability of persistent DNA double-strand breaks, cells have developed an array of specific repair pathways. These pathways are largely similar across a broad range of species from yeast to humans, although the most frequently used one is different among species. There are two general pathways for repair of DNA double-strand breaks: homologous recombination and nonhomologous end-joining. These two can be considered as being in competition for the double-strand break substrate (Haber, 2000; Sonoda et al., 2006). Homologous Recombination Eukaryotes undergo homologous recombination as part of their normal activities both in germ cells (meiotic recombination) and somatic cells (mitotic recombination). The repair of double-strand breaks (and single-strand gaps) basically uses the same process and complex of proteins, although some different protein–protein interactions are involved (Shinohara and Ogawa, 1995). In eukaryotes, the process has been characterized most extensively for yeast, but evidence is accumulating that a very similar process occurs in mammalian cells, including human (Johnson et al., 1999; Cahill et al., 2006). The basic steps in double-strand break repair are as follows. The initial step is the production of a 3 -ended single-stranded tail by exonucleases or helicase activity. Through a process of strand invasion, whereby the single-stranded tail invades an undamaged homologous DNA molecule, together with DNA synthesis, a so-called Holliday junction DNA complex is formed. By cleavage of this junction, two DNA molecules are produced (with or without a structural crossover), neither of which now contain a strand break. Additional models have been proposed but probably play a minor role in mammalian cells (Haber, 2000). A detailed description of the specific enzymes known to be involved can be found in Shinohara and Ogawa (1995) and Cahill et al. (2006). Nonhomologous End-Joining (NHEJ) The characterization of NHEJ in mammalian cells was greatly enhanced by the observation that mammalian cell lines that are hypersensitive to ionizing radiation are also defective in the V(D)J recombination process, which is the means by which the huge range of an antibody’s antigen-binding sites and T-cell receptor proteins are generated during mammalian lymphoid cell development. V(D)J recombination requires the production of double-strand breaks, recombination of DNA pieces, and subsequent religation. A major component of the NHEJ repair complex is a DNA-dependent protein kinase (DNA-PK). This protein, a serine/threonine kinase, consists of a catalytic subunit (DNA-PKcs ) and a DNA-end-binding protein consisting of KU70 and KU80 subunits. The specific role of DNA-PK in the repair of double-strand breaks is unclear in mammalian cells; a detailed discussion of what is known and some possible models of NHEJ are presented in the reviews by Critchlow and Jackson (1998) and Burma et al. (2006). Perhaps the most viable role of DNA-PK is to align the broken DNA ends to facilitate their ligation. In addition, DNA-PK might serve as a signal molecule for recruiting other repair proteins known to be involved in yeast and to some extent in mammalian cells. The final ligation step is performed by DNA ligase IV in human cells.

Mismatch Repair The study of DNA mismatch repair systems has received considerable attention over the past few years, in part

because an association has been demonstrated between genetic defects in mismatch repair genes and the genomic instability associated with cancer susceptibility syndromes and sporadic cancers. In general, DNA mismatch repair systems operate to repair mismatched bases formed during DNA replication, genetic recombination, and as a result of DNA damage induced by chemical and physical agents. Detailed reviews can be found in Kolodner (1995), Jiricny (1998), Modrich and Lahue (1996), and Jun et al. (2006). The principal steps in all cells from prokaryotes to human are damage recognition by a specific protein that binds to the mismatch, stabilizing of the binding by the addition of one or more proteins, cutting the DNA at a distance from the mismatch, excision past the mismatch, resynthesis, and ligation. In some prokaryotes, the cutting of the DNA (for DNA replication mismatches) is directed to the strand that contains the incorrect base by using the fact that recently replicated DNA is unmethylated at N 6 -methyladenine at a GATC sequence. The question of whether or not strand-specific mismatch repair occurs in mammalian cells has not been resolved, although some evidence does point to its occurrence (Modrich, 1997). Strand specificity for DNA mismatches resulting from induced DNA damage has not been identified. O 6 -Methylguanine-DNA Methyltransferase Repair The main role for O 6 -methylguanine-DNA methyltransferase (MGMT) is to protect cells against the toxic effects of simple alkylating agents. The methyl group is transferred from O 6 -methylguanine in DNA to a cysteine residue in MGMT. The adducted base is reverted to a normal one by the enzyme, which is itself inactivated by the reaction. Details of the MGMT enzyme properties and the gene isolation and characterization can be found in Tano et al. (1990), Grombacher et al. (1996), and Margison et al. (2003). The probability that induced DNA damage can be converted into a genetic alteration is influenced by the particular repair pathway(s) recruited, the rate of repair of the damage, and the fidelity and completeness of the repair. The mechanisms of induction of gene mutations and chromosome alterations discussed in the following sections build upon the assessment of the probability of repair versus misrepair versus nonrepair that can be derived from a knowledge of the mechanism of action of the different DNA repair mechanisms. The preceding sections, together with the references provided, should assist in this assessment.

Formation of Gene Mutations Somatic Cells Gene mutations are considered to be small DNA sequence changes confined to a single gene; larger genomic changes are considered below, under section “Formation of Chromosomal Alterations.” The general classes of gene mutations are base substitutions and small additions or deletions. More detailed classifications can be found in the review by Ripley (1991). Base substitutions are the replacement of the correct nucleotide by an incorrect one; they can be further subdivided as transitions where the change is purine for purine or pyrimidine for pyrimidine; and transversions where the change is purine for pyrimidine and vice versa. Frameshift mutations are strictly the addition or deletion of one or a few base pairs (not in multiples of three) in protein-coding regions. The definition is more generally expanded to include such additions and deletions in any DNA region. For the discussion of the mechanism of induction of gene mutations and chromosomal alterations, it is necessary to distinguish chemicals by their general mode of action. Chemicals that can produce genetic alterations

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with similar effectiveness in all stages of the cell cycle are called radiomimetic because they act like radiation in this regard. Chemicals that produce genetic alterations far more effectively in the S phase of the cell cycle are described as nonradiomimetic. The great majority of chemicals are nonradiomimetic; the radiomimetic group includes bleomycin, streptonigrin, neocarzinostatin, and 8-ethoxycaffeine. Gene mutations can arise in the absence of specific exogenous exposures to radiations and chemicals. The great majority of socalled spontaneous (background) mutations arise from replication of an altered template. The DNA alterations that arise are either the result of oxidative damage or are produced from the deamination of 5-methylcytosine to thymine at CpG sites resulting in G:C → A:T transitions. Mutations induced by ionizing radiations tend to be deletions ranging in size from a few bases to multilocus events (Thacker, 1992). The rapid rate of repair of the majority of radiation-induced DNA damages greatly reduces the probability of DNA lesions being present at the time of DNA replication. Thus, mutations induced by ionizing radiations are generally the result of errors of DNA repair (Preston, 1992). The low frequencies of gene mutations are produced from any unrepaired DNA base damage present during DNA replication. Gene mutations produced by a majority of chemicals and nonionizing radiations are base substitutions, frameshifts, and small deletions. Of these mutations, a very high proportion is produced by errors of DNA replication on a damaged template. Thus, the probability of a DNA adduct, for example, being converted into a mutation is determined, to a significant extent by the number of induced DNA adducts that remain in the DNA at the time that it is replicated. Thus, relative mutation frequency will be the outcome of the race between repair and replication, i.e., the more repair that takes place prior to replication, the lower the mutation frequency for a given amount of induced DNA damage. Significant regulators

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of the race are cell cycle checkpoint genes (e.g., P53) because if the cell is checked from entering the S phase at a G1 /S checkpoint, more repair can take place prior to the cell starting to replicate its DNA (Mercer, 1998). The proportion of chemically induced gene mutations that result from DNA repair errors is low, given that the DNA repair processes involved are error-free, and that, as a generalization, repair of chemically induced DNA damage is slower than for ionizing radiation damage, leading to the balance tipping toward replication prior to repair, especially for cells in the S phase at the time of exposure. However, in the case of translesion bypass, discussed above, gene mutations can be produced at relatively high frequencies. Germ Cells The mechanism of production of gene mutations in germ cells is basically the same as in somatic cells. Ionizing radiations produce mainly deletions via errors of DNA repair; the majority of chemicals induce base substitutions, frameshifts, and small deletions by errors of DNA replication (Favor, 1999). An important consideration for assessing gene mutations induced by chemicals in germ cells is the relationship between exposure and the timing of DNA replication. Figure 9-3 depicts the stages in oogenesis and spermatogenesis when DNA replicates. A few features are worthy of note. The spermatogonial stem cell in humans and rodents has a long cell cycle time, 8 days or longer, with only a small fraction being occupied by the S phase. Thus, the probability of DNA repair taking place prior to DNA replication is high, for both acute and chronic treatments. However, for considerations of genetic risk, it is the spermatogonial stem cell that is the major contributor because it is present, in general, throughout the reproductive lifetime of an individual. Each time a spermatogonial stem cell divides it produces a differentiating spermatogonium and a stem cell. Thus, the stem cell can accumulate genetic damage from chronic exposures. Differentiating spermatogonia, as far as the

Figure 9-3. The stages of spermatogenesis (A) and oogenesis (B) indicating the periods of cell division and DNA replication (S phase).

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induction of gene mutation is concerned, are the same as mitotically dividing somatic cells. The first S phase after gametogenesis occurs in the zygote, formed following fertilization. This fact needs to be balanced by the lack of DNA repair in late spermatids and sperm. Thus, DNA damage induced in these stages will remain until the zygote. Postmeiotic germ cells are particularly sensitive to mutation induction by nonradiomimetic chemicals, especially following acute exposures (Russell, 2004). The fairly short duration of this stage (approximately 21 days in the mouse) means that their contribution to genetic risk following chronic exposures is quite small. For oogenesis (Fig. 9-3) similar observations on gene mutation induction and timing of S phase can be made. In this case the primary oocyte arrests prior to birth, and there is no further S phase until the zygote. For this reason, the oocyte is resistant to the induction of gene mutations by nonradiomimetic chemicals but not to radiation, for which DNA repair is the mode of formation of mutations, and DNA repair occurs in oocytes (Brewen and Preston, 1982). These mechanistic aspects of the production of gene mutations (and chromosome alterations described in the following two sections) by chemicals and radiations in somatic and germ cells are most important for considerations of the design of genetic toxicology assays, the interpretation of the data generated, and the incorporation of the data into cancer and genetic risk assessments.

Formation of Chromosomal Alterations Somatic Cells Structural Chromosome Aberrations There are components of the formation of chromosome aberrations, sister chromatid exchanges (the apparently reciprocal exchange between the sister chromatids of a single chromosome), and gene mutations that are similar. In particular, damaged DNA serves as the substrate leading to all these events. However, chromosome aberrations induced by ionizing radiations are generally formed by errors of DNA repair, whereas those produced by nonradiomimetic chemicals are generally formed by errors of DNA replication on a damaged DNA template. The DNA repair errors that lead to the formation of chromosome aberrations following ionizing radiation (and radiomimetic chemical) exposure arise from misligation of double-strand breaks or interaction of coincidentally repairing regions during nucleotide excision repair of damaged bases. The details of the DNA damage types and their repair are described above. Thus, the overall kinetics and fidelity of DNA repair influence the sensitivity of cells to the induction of chromosomal aberrations produced by misrepair. The broad outcomes of misrepair are that incorrect rejoining of chromosomal pieces during repair leads to chromosomal exchanges within (e.g., inversions and interstitial deletions) and between (e.g., dicentrics and reciprocal translocations) chromosomes. In fact, using fluorescence in situ hybridization, it can be shown that very complex rearrangements take place (Anderson et al., 2000). Failure to rejoin double-strand breaks or to complete repair of other types of DNA damage leads to terminal deletions. Acentric fragments arise from interstitial deletions, terminal deletions, and the formation of dicentric chromosomes and rings. The failure to incorporate an acentric fragment into a daughter nucleus at anaphase/telophase, or the failure of a whole chromosome to segregate at anaphase to the cellular poles, can result in the formation of a membrane-bounded micronucleus that resides in the cytoplasm.

Errors of DNA replication on a damaged template can lead to a variety of chromosomal alterations. The majority of these involve deletion or exchange of individual chromatids (chromatid-type aberrations). Thus, nonradiomimetic chemicals induce only chromatidtype aberrations, whereas radiations and radiomimetic chemicals induce chromatid-type aberrations in the S and G2 phases of the cell cycle, but chromosome-type aberrations affecting both chromatids in G1 . The reason for this latter observation is that the G1 (or G0 ) chromosome behaves as a single DNA molecule and aberrations formed in it will be replicated in the S phase and will involve both chromatids. This distinction is important for considerations of outcome of the aberrations and the probability of an effect on cells because for chromatid-type aberrations, one chromatid remains intact and genetically unaltered, in contrast to chromosome-type aberrations in which both chromatids are damaged (Preston et al., 1995). Numerical Chromosome Changes Numerical changes (e.g., monosomies, trisomies, and ploidy changes) can arise from errors in chromosomal segregation. The complexity of the control and the mechanics of the mitotic process means that alteration of various cellular components can result in failure to segregate the sister chromatids to separate daughter cells or in failure to segregate a chromosome to either pole (Bickel and Orr-Weaver, 1996; Preston, 1996; Hunt, 2006). The mechanisms underlying chromosomal loss are pertinent to those involved in the formation of micronuclei. A limited set of chemicals has been demonstrated to cause aneuploidy through interaction with components of the structures that facilitate chromosome movement (Preston, 1996; Aardema et al., 1998). These include benomyl, griseofulvin, nocodazole, colchicine, colecemid, vinblastine, and paclitaxel. These chemicals affect tubulin polymerization or spindle microtubule stability. To date, other mechanisms of aneuploidy induction by chemicals have not been firmly identified. Sister Chromatid Exchanges Sister chromatid exchanges (SCEs) are produced during the S phase and are presumed to be a consequence of errors in the replication process, perhaps at the sites of stalled replication complexes (Painter, 1980; Heartlein et al., 1983; Preston, 1991). Because SCEs are apparently reciprocal exchanges, it is quite possible that they result from a recombination process occurring at the site of the stalled replication fork. It is, in fact, this mode of action that makes assays for SCE less than ideal for detecting effects due directly to a chemical exposure. The creation of intracellular conditions that slow the progress of DNA replication, for example, could lead to the formation of SCE.

Germ Cells The formation of chromosomal alterations in germ cells is basically the same as that for somatic cells, namely, via misrepair for ionizing radiations and radiomimetic chemicals for treatments in G1 and G2 , and by errors of replication for all radiations and chemicals for DNA damage present during the S phase. Also, the restrictions on the timing of formation of chromosomal alterations induced by nonradiomimetic chemicals in germ cells is as described above for gene mutations, namely at the specific stages where DNA synthesis occurs, as depicted in Fig. 9-3. The types of aberrations formed in germ cells are the same as those formed in somatic cells (e.g., deletions, inversions, translocations), although their appearance in diplotene/diakinesis of meiosis I, where analysis is frequently conducted, is rather different because of the homologous chromosome pairing that takes place in meiotic cells (see the review by Leonard, 1973). The specific segregation of

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chromosomes during meiosis influences the probability of recovery of an aberration, particularly a reciprocal translocation, in the offspring of a treated parent. This is discussed in detail in Preston et al. (1995).

ASSAYS FOR DETECTING GENETIC ALTERATIONS Introduction to Assay Design Genetic toxicology assays are used to identify germ-cell mutagens, somatic-cell mutagens, and potential carcinogens. They detect diverse kinds of genetic alterations that are relevant for human health, including gene mutations, chromosome aberrations, and aneuploidy. Over the last three decades, hundreds of chemicals and complex mixtures have been evaluated for genotoxic effects. Genetic toxicology assays serve two interrelated but distinct purposes in the toxicologic evaluation of chemicals: (1) identifying mutagens for purposes of hazard identification and (2) characterizing dose–response relationships and mutagenic mechanisms, both of which contribute to an understanding of genetic and carcinogenic risks. A common experience in surveying the mutagenicity literature is encountering a bewildering array of assays in viruses, bacteria, fungi, cultured mammalian cells, plants, insects, and mammals. More than 200 assays for mutagens have been proposed, and useful information has been obtained from many of them. Although most genetic toxicology testing and evaluation relies on relatively few assays, data from relatively obscure assays can sometimes contribute to a judgment about the genetic activity of a compound. Table 9-1 lists key assays that have a prominent place in genetic toxicology. Table 9-2 is a more comprehensive list that provides literature citations to many of the assays that one might encounter in the genetic toxicology literature. Even this extensive table is not exhaustive, in that it emphasizes methods in applied genetic toxicology and not those assays whose use has been largely restricted to studies of mutational mechanisms. The commonly used assays rely on phenotypic effects as indicators of gene mutations or small deletions and on cytological methods for observing gross chromosomal damage. Detailed information on assay design, testing data, controls, sample sizes, and other factors in effective testing is found in the references cited. Some assays for gene mutations detect forward mutations whereas others detect reversion. Forward mutations are genetic alterations in a wild-type gene and are detected by a change in phenotype caused by the alteration or loss of gene function. In contrast, a back mutation or reversion is a mutation that restores gene function in a mutant, bringing about a return to the wild-type phenotype. In principle, forward-mutation assays should respond to a broad spectrum of mutagens because any mutation that interferes with gene expression should confer the detectable phenotype. In contrast, a reversion assay might be expected to have a more restricted mutational response because only mutations that correct or compensate for the specific mutation in a particular mutant will be detected. In fact, some reversion assays respond to a broader spectrum of mutational changes than one might expect because mutations at a site other than that of the original mutation, either within the test gene or in a different gene (i.e., a suppressor mutation), can sometimes confer the selected phenotype. Both forward mutation assays and reversion assays are used extensively in genetic toxicology. The simplest gene mutation assays rely on selection techniques to detect mutations. A selection technique is a means of imposing

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Table 9-1 Principal Assays in Genetic Toxicology I. Pivotal assays A. A well-characterized assay for gene mutations The Salmonella/mammalian microsome assay (Ames test) B. A mammalian assay for chromosome damage in vivo Metaphase analysis or micronucleus assay in rodent bone marrow II. Other assays offering an extensive database or unique genetic endpoint A. Assays for gene mutations E. coli WP2 tryptophan reversion assay TK or HPRT forward mutation assays in cultured mammalian cells Drosophila sex-linked recessive lethal assay B. Cytogenetic analysis in cultured Chinese hamster or human cells Assays for chromosome aberrations and micronuclei Assays for aneuploidy C. Other indicators of genetic damage Mammalian DNA damage and repair assays Mitotic recombination assays in yeast and Drosophila D. Mammalian germ-cell assays Mouse specific-locus tests Assays for skeletal or cataract mutations in mice Cytogenetic analysis and heritable translocation assays DNA damage and repair in rodent germ cells Mutation analysis in tandem-repeat loci in mice

experimental conditions under which only cells or organisms that have undergone mutation can grow. Selection techniques greatly facilitate the identification of rare cells that have experienced mutation among the many cells that have not. Forward mutations and reversions can both be detected by selection techniques in microorganisms and cultured mammalian cells. Because of their speed, low cost, and ease of detecting events that occur at low frequency (i.e., mutation), assays in microorganisms and cell cultures have figured prominently in genetic toxicology. Studying mutagenesis in intact animals requires assays of more complex design than the simple selection methods used in microorganisms and cultured cells. Genetic toxicology assays therefore range from inexpensive short-term tests that can be performed in a few days to complicated assays for mutations in mammalian germ cells. Even in multicellular organisms, there has been an emphasis on designing assays that detect mutations with great efficiency. Nevertheless, there remains a gradation in which an increase in relevance for human risk entails more elaborate and costly tests. The most expensive mammalian tests are typically reserved for agents of special importance in basic research or risk assessment, whereas the simpler assays can be applied more broadly. Cytogenetic assays differ in design from typical gene mutation assays because of their reliance on cytological rather than genetic methods. The goal in cytogenetic methods is the unequivocal visual recognition of cells that have experienced genetic damage. The alterations measured include chromosome aberrations, micronuclei, SCEs, and changes in chromosome numbers (aneuploidy). In all mutagenicity testing, one must be aware of possible sources of error. Factors to consider in the application of mutagenicity assays are the choice of suitable organisms and growth

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Table 9-2 Overview of Genetic Toxicology Assays assays I. DNA Damage and Repair Assays A. Direct detection of DNA damage: Alkaline elution assays for DNA strand breakage Comet assay for DNA strand breakage Assays for chemical adducts in DNA B. Bacterial assays for DNA damage: Differential killing of repair-deficient and wild-type strains Induction of the SOS system by DNA damage C. Assays for repairable DNA damage in mammalian cells: Unscheduled DNA synthesis (UDS) in rat hepatocytes UDS in rodent hepatocytes in vivo II. Prokaryote Gene Mutation Assays A. Bacterial reverse mutation assays: Salmonella/mammalian microsome assay (Ames test) E. coli WP2 tryptophan reversion assay Salmonella specific base-pair substitution assay (Ames-II assay) E. coli lacZ specific reversion assay B. Bacterial forward mutation assays: E. coli lacI assay Resistance to toxic metabolites or analogs in Salmonella III. Assays in Nonmammalian Eukaryotes: A. Fungal assays: Forward mutations, reversion, and small deletions Mitotic crossing over and gene conversion in yeast Mitotic aneuploidy: chromosome loss or gain in yeast Meiotic nondisjunction in yeast or Neurospora B. Plant assays: Gene mutations affecting chlorophyll in seedlings or waxy in pollen Tradescantia stamen hair color mutations Chromosome aberrations or micronuclei in mitotic or meiotic cells Aneuploidy detected by pigmentation or cytogenetics C. Drosophila assays: Sex-linked recessive lethal test in germ cells Heritable translocation assays Sex chromosome loss tests for aneuploidy Induction of mitotic recombination in eyes or wings IV. Mammalian Gene Mutation Assays A. In vitro assays for forward mutations: tk mutations in mouse lymphoma or human cells hprt or xprt mutations in Chinese hamster or human cells B. In vivo assays for gene mutations in somatic cells: Mouse spot test (somatic cell specific locus test) hprt mutations (6-thioguanine-resistance) in rodent lymphocytes C. Transgenic assays: r ” mice Mutations in the bacterial lacI gene in “Big Blue and rats Mutations in the bacterial lacZ gene in the “MutaTM Mouse” Mutations in the phage cII gene in lacI or lacZ transgenic mice Point mutations and deletions in the lacZ plasmid mouse Point mutations and deletions in delta gpt mice and rats

selected literature citations

Elia et al., 1994 Fairbairn et al., 1995; Singh, 2000; Tice et al., 2000 Chang et al., 1994; Kriek et al., 1998; Phillips et al., 2000 Hamasaki et al., 1992 Quillardet and Hofnung, 1993; Yasunaga et al., 2004 Madle et al., 1994 Madle et al., 1994

Ames et al., 1975; Kier et al., 1986; Kirkland et al., 1990; Maron and Ames, 1983; Mortelmans and Zeiger, 2000 Kirkland et al., 1990; Mortelmans and Riccio, 2000 Gee et al., 1994, 1998 Cupples and Miller, 1989; Cupples et al., 1990; Josephy, 2000 Calos and Miller, 1981; Halliday and Glickman, 1991 Jurado et al., 1994; Vlasakova et al., 2005

Zimmermann et al., 1984; Crouse, 2000 Zimmermann et al., 1984; Zimmermann, 1992 Zimmermann et al., 1984; Parry, 1993; Aardema et al., 1998 Zimmermann et al., 1984 Grant, 1994 Grant, 1994 Grant, 1994 Parry, 1993; Grant, 1994; Aardema et al., 1998 Lee et al., 1983; Mason et al., 1987 Mason et al., 1987 Aardema et al. 1998; Osgood and Cyr, 1998 Vogel et al., 1999

Kirkland et al., 1990; Clements, 2000; Moore et al., 2003 DeMarini et al., 1989 Styles and Penman, 1985; Lambert et al., 2005 Cariello and Skopek, 1993; Lambert et al., 2005

Mirsalis et al., 1994; Lambert et al., 2005 Mirsalis et al., 1994; Lambert et al., 2005 Swiger, 2001; Lambert et al., 2005 Lambert et al., 2005 Okada et al., 1999; Lambert et al., 2005

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Table 9-2 Overview of Genetic Toxicology Assays assays V. Mammalian Cytogenetic Assays A. Chromosome aberrations: Metaphase analysis in cultured Chinese hamster or human cells Metaphase analysis of rodent bone marrow or lymphocytes in vivo B. Micronuclei: Cytokinesis-block micronucleus assay in human lymphocytes Micronucleus assay in mammalian cell lines In vivo micronucleus assay in rodent bone marrow or blood C. Sister chromatid exchange: SCE in human cells or Chinese hamster cells SCE in rodent tissues, especially bone marrow D. Aneuploidy in mitotic cells: Mitotic disturbance seen by staining spindles and chromosomes Hyperploidy detected by chromosome counting Chromosome gain or loss in cells with intact cytoplasm Micronucleus assay with centromere labeling Hyperploid cells in vivo in mouse bone marrow Mouse bone marrow micronucleus assay with centromere labeling VI. Germ Cell Mutagenesis A. Measurement of DNA damage Molecular dosimetry based on mutagen adducts UDS in rodent germ cells Alkaline elution assays for DNA strand breaks in rodent testes B. Gene mutations Mouse specific locus test for gene mutations and deletions Mouse electrophoretic specific-locus test Dominant mutations causing mouse skeletal defects or cataracts Mouse tandem-repeat loci analysis C. Chromosomal aberrations Cytogenetic analysis in oocytes, spermatogonia, or spermatocytes Micronuclei in mouse spermatids Mouse heritable translocation test D. Dominant lethal mutations Mouse or rat dominant lethal assay E. Aneuploidy Cytogenetic analysis for aneuploidy arising by nondisjunction Sex chromosome loss test for nondisjunction or breakage Micronucleus assay in spermatids with centromere labeling FISH with probes for specific chromosomes in sperm

selected literature citations

Ishidate et al., 1988; Kirkland et al., 1990; Galloway et al., 1994 Preston et al., 1981; Kirkland et al., 1990; Tice et al., 1994

Fenech, 2000; Fenech et al., 2003 Kirsch-Volders et al., 2000, 2003 Heddle et al., 1991; Hayashi et al., 2000; Krishna and Hayashi, 2000; Hamada et al., 2001 Tucker et al., 1993a Tucker et al., 1993a Parry, 1998 Galloway and Ivett, 1986; Aardema et al., 1998 Natarajan, 1993 Lynch and Parry, 1993; Natarajan, 1993; Aardema et al., 1998; Fenech, 2000 Aardema et al., 1998 Heddle et al., 1991; Adler et al., 1994; Aardema et al., 1998

Russell and Shelby, 1985 Bentley et al., 1994; Sotomayor and Sega, 2000 Bentley et al., 1994

Russell et al., 1981; Kirkland et al., 1990; Ehling, 1991; Russell and Russell, 1992 Lewis, 1991 Ehling, 1991 Yauk, 2004 Kirkland et al., 1990; Tease, 1992; Russo, 2000 Hayashi et al., 2000; Russo, 2000 Russell and Shelby, 1985 Adler et al., 1994 Allen et al., 1986; Adler, 1993; Aardema et al., 1998 Russell and Shelby, 1985; Adler, 1993 Aardema et al., 1998 Russo, 2000; Wyrobek et al., 2005

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conditions, appropriate monitoring of genotypes and phenotypes, effective experimental design and treatment conditions, inclusion of proper positive and negative controls, and sound methods of data analysis (Kirkland et al., 1990). Many compounds that are not themselves mutagenic or carcinogenic can be activated into mutagens and carcinogens by metabolism. Such compounds are called promutagens and procarcinogens. Because microorganisms and mammalian cell cultures lack many of the metabolic capabilities of intact mammals, provision must be made for metabolic activation in order to detect promutagens in many genetic assays. Incorporating an in vitro metabolic activation system derived from a mammalian tissue homogenate is the most common means of adding metabolic activation to microbial or cell culture assays. For example, the promutagens dimethylnitrosamine and benzo[a]pyrene are not themselves mutagenic in bacteria, but they are mutagenic in bacterial assays if the bacteria are treated with the promutagen in the presence of a homogenate from mammalian liver. The most widely used metabolic activation system in microbial and cell culture assays is a postmitochondrial supernatant from a rat liver homogenate, along with appropriate buffers and cofactors (Maron and Ames, 1983; Kirkland et al., 1990). The standard liver metabolic activation system is called an S9 mixture, designating a supernatant from centrifugation at 9000g (Malling and Frantz, 1973). Most of the short-term assays in Table 9-2 require exogenous metabolic activation to detect promutagens. Exceptions are assays in intact mammals and a few simpler assays that have a high level of endogenous cytochrome P450 metabolism, such as the detection of unscheduled DNA synthesis (UDS) in cultured hepatocytes (Madle et al., 1994). Rat liver S9 provides a broad assemblage of metabolic reactions, but they are not necessarily the same as those of hepatic metabolism in an intact rat. Metabolic activation systems based on homogenates from other species or organs have found some use, but they may similarly differ from the species or organs of their origin. Therefore, alternative metabolic activation systems tend to be more useful if chosen for mechanistic reasons rather than simply testing another species or organ. For example, metabolism by intact hepatocytes (Langenbach and Oglesby, 1983) can preserve elements of the cellular compartmentalization of reactions that would be altered when tissues are made into homogenates. Likewise, a system that includes a reductive reaction not encompassed by standard S9 is required for detecting the mutagenicity of some azo dyes and nitro compounds (Dellarco and Prival, 1989). Despite their usefulness, in vitro metabolic activation systems, however well refined, cannot mimic mammalian metabolism perfectly. There are differences among tissues in reactions that activate or inactivate foreign compounds, and organisms of the normal flora of the gut can contribute to metabolism in intact mammals. Agents that induce enzyme systems or otherwise alter the physiological state can also modify the metabolism of toxicants, and the balance between activation and detoxication reactions in vitro may differ from that in vivo. An interesting development with respect to metabolic activation is the incorporation of genes encoding human enzymes into microorganisms or cell cultures. For example, the expression of human cytochrome P4501A2 in Salmonella tester strains from the Ames assay permits the activation of such promutagens as 2-aminoanthracene and 2-aminofluorene without an S9 mixture (Josephy et al., 1995). Mammalian cell lines have also been genetically engineered to express human Phase-I and Phase-II en-

zymes, including those catalyzing reactions of metabolic activation (Sawada and Kamataki, 1998). Many cell lines stably expressing a single form of P450 have been established. Mutagenesis can be measured through such endpoints as HPRT mutations and cytogenetic alterations, and the cells are well suited to analyzing the contribution of different enzymes to the activation of promutagens. Metabolic activation is so central to genetic toxicology that all mutagenicity testing programs must provide for it in the choice of assays and procedures. In special circumstances, other forms of activation may be relevant, and assays are adapted accordingly. Some chemicals are subject to photochemical activation, such that genotoxic effects depend both on the chemical and on its being irradiated with ultraviolet or visible light. Many of the assays listed in Table 9-2, including gene-mutation assays in bacteria and cultured mammalian cells, cytogenetic assays, and the comet assay, have been adapted so that they can measure photogenotoxic effects (BrendlerSchwaab et al., 2004).

DNA Damage and Repair Assays Some assays measure DNA damage itself, rather than mutational consequences of DNA damage. They may do so directly, through such indicators as chemical adducts or strand breaks in DNA, or indirectly, through the measurement of biological repair processes. Adducts in DNA are detected by 32 P-postlabeling, immunological methods using antibodies against specific adducts, or fluorometric methods in the case of such fluorescent compounds as polynuclear aromatic hydrocarbons and aflatoxins (Chang et al., 1994; Kriek et al., 1998; Phillips et al., 2000). The 32 P-postlabeling method is highly sensitive and applicable to diverse mutagens. The measurement of adducts after human chemical exposures has proven useful in human monitoring and molecular dosimetry (Chang et al., 1994; Kriek et al., 1998; Phillips et al., 2000). DNA strand breakage can be measured by alkaline elution and electrophoretic methods (Elia et al., 1994). The applicability of DNA damage assays to rodent testes (Bentley et al., 1994) makes these methods helpful in interpreting risks to germ cells. Single-cell gel electrophoresis, also called the comet assay, is a widely used, rapid method of measuring DNA damage (Fairbairn et al., 1995; Singh, 2000; Tice et al., 2000). In this assay cells are incorporated into agarose on slides, lysed so as to liberate their DNA, and subjected to electrophoresis. The DNA is stained with a fluorescent dye for observation and image analysis. Because broken DNA fragments migrate more quickly than larger pieces of DNA, a blur of fragments (a “comet”) is observed when the DNA is extensively damaged. The extent of DNA damage can be estimated from the length and other attributes of the comet tail. Variations in the procedure permit the general detection of DNA strand breakage under alkaline conditions (Fairbairn et al., 1995; Singh, 2000; Tice et al., 2000) or the preferential detection of double-strand breaks under neutral conditions (Fairbairn et al., 1995). Although the comet assay is relatively new and needs further evaluation, it appears to be a sensitive indicator of DNA damage with broad applicability. It has been used most commonly with human lymphocytes (Fairbairn et al., 1995; Singh, 2000) and other mammalian cells (Tice et al., 2000), but it can be adapted to diverse species, including plants, worms, mollusks, fish, and amphibians (Cotelle and F´erard, 1999). This adaptability suggests that it will find diverse uses in environmental genetic toxicology. The occurrence of DNA repair can serve as an easily measured indicator of DNA damage. Repair assays have been developed in

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microorganisms, cultured mammalian cells, and intact mammals (Table 9-2). Greater toxicity of a chemical in DNA-repair-deficient strains than in their repair-proficient counterparts has served as an indicator of DNA damage in bacteria (e.g., polA− and polA+ in E. coli or rec− and rec+ in Bacillus subtilis) (Hamasaki et al., 1992). The induction of bacterial SOS functions, indicated by phage induction or by colorimetry in the SOS chromotest, can similarly serve as a general indicator of genetic damage (Quillardet and Hofnung, 1993). The most common repair assay in mammalian cells is the measurement of unscheduled DNA synthesis (UDS). UDS is a measure of excision repair, and its occurrence indicates that DNA had been damaged (Madle et al., 1994). The absence of UDS, however, does not provide clear evidence that DNA has not been damaged, because some classes of damage are not readily excised, and some excisable damage may not be detected as a consequence of assay insensitivity. Though bacterial repair assays have declined in usage over the years, UDS assays continue to be used because of their applicability to cultured hepatocytes with endogenous cytochrome P450 enzyme activities and to tissues of intact animals, including hepatocytes (Madle et al., 1994) and germinal tissue (Bentley et al., 1994; Sotomayor and Sega, 2000).

Gene Mutations in Prokaryotes The most common means of detecting mutations in microorganisms is selecting for reversion in strains that have a specific nutritional requirement differing from wild-type members of the species; such strains are called auxotrophs. For example, the widely used assay developed by Bruce Ames and his colleagues is based on measuring reversion in several histidine auxotrophs in Salmonella enterica Serovar typhimurium, commonly called Salmonella typhimurium. In the Ames assay one measures the frequency of histidineindependent bacteria that arise in a histidine-requiring strain in the presence or absence of the chemical being tested. Auxotrophic bacteria are treated with the chemical of interest by one of several procedures (e.g., the standard plate-incorporation assay) and plated on medium that is deficient in histidine (Ames et al., 1975; Maron and Ames, 1983; Kirkland et al., 1990; Mortelmans and Zeiger, 2000). The assay is conducted using genetically different strains so that reversion by base-pair substitutions and frameshift mutations in several DNA sequence contexts can be detected and distinguished. Because Salmonella does not metabolize promutagens in the same way as mammalian tissues, the assay is generally performed in the presence and absence of a rat liver S9 metabolic activation system. Hence, the Ames assay is also called the Salmonella/microsome assay. The principal strains of the Ames test and their characteristics are summarized in Table 9-3. In addition to the histidine alleles that provide the target for measuring mutagenesis, the Ames tester strains contain other genes and plasmids that enhance the assay. Part I of the table gives genotypes, and Part II explains the rationale for including specific genetic characteristics in the strains. Part III summarizes the principal DNA target in each strain and their mechanisms of reversion. Taken together, the Ames strains detect a broad array of mutations, and they complement one another. For example, strains TA102 and TA104, which are sensitive to agents that cause oxidative damage in DNA, detect the A:T → G:C base-pair substitutions that are not detected by hisG46 strains (Mortelmans and Zeiger, 2000). TA102 also detects agents that cause DNA crosslinks because it has

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an intact excision repair system whereas the other common tester strains do not. The most common version of the Ames assay is the plateincorporation test (Ames et al., 1975; Maron and Ames, 1983; Mortelmans and Zeiger, 2000). In this procedure, the bacterial tester strain, the test compound (or solvent control), and the S9 metabolic activation system (or buffer for samples without S9) are added to 2 mL of molten agar containing biotin and a trace of histidine to allow a few cell divisions, mixed, and immediately poured onto the surface of a petri dish selective for histidine-independent revertants. For general testing it is recommended to use at least 3 plates per dose, 5 doses, samples with and without S9, and appropriate concurrent positive and negative controls (Mortelmans and Zeiger, 2000). Variations on the standard plate-incorporation assay confer advantages for some applications. These include a preincubation assay that facilitates the detection of unstable compounds and short-lived metabolites; a desiccator assay for testing volatile chemicals and gases; a microsuspension assay for working with small quantities of test agent; assays incorporating reductive metabolism rather than the conventional S9 system; and assays under hypoxic conditions (Mortelmans and Zeiger, 2000). Whereas simplicity is a great merit of microbial assays, it can also be deceptive. Even assays that are simple in design and application can be performed incorrectly. For example, in the Ames assay one may see very small colonies in the petri dishes at highly toxic doses (Maron and Ames, 1983; Kirkland et al., 1990; Mortelmans and Zeiger, 2000). Counting such colonies as revertants would be an error because they may actually be nonrevertant survivors that grew on the low concentration of histidine in the plates. Were there millions of survivors, the amount of histidine would have been insufficient to allow any of them (except real revertants) to form colonies. This artifact is easily avoided by checking that there is a faint lawn of bacterial growth in the plates; one can also confirm that colonies are revertants by streaking them on medium without histidine to be sure that they grow in its absence. Such pitfalls exist in all mutagenicity tests. Therefore, anyone performing mutagenicity tests must have detailed familiarity with the laboratory application and literature of the assay and be observant about the responsiveness of the assay. Although information from the Ames assay has become a standard in genetic toxicology testing, equivalent information can be obtained from other bacterial assays. Like the Ames assay, the WP2 tryptophan reversion assay in Escherichia coli (Kirkland et al., 1990; Mortelmans and Riccio, 2000) incorporates genetic features that enhance assay sensitivity, can accommodate S9 metabolic activation, and performs well in many laboratories. Mutations are detected by selecting for reversion of a trpE allele from Trp− to Trp+ . Its responsiveness to mutagens most closely resembles TA102 among the Ames strains (Mortelmans and Riccio, 2000). Bacterial reversion assays are commonly used for testing purposes, but they also provide information on molecular mechanisms of mutagenesis. The broader understanding of mutational mechanisms that comes from refined genetic assays and molecular analysis of mutations can contribute to the interpretation of mutational hazards. The primary reversion mechanisms of the Ames strains, summarized in Table 9-3, were initially determined by genetic and biochemical means (Maron and Ames, 1983). An ingenious method called allele-specific colony hybridization greatly facilitated the molecular analysis of revertants in the Ames assay (Koch et al., 1994), and many spontaneous and induced revertants have been cloned or amplified by the polymerase chain reaction (PCR) and sequenced (Levine et al., 1994; DeMarini, 2000).

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Table 9-3 The Ames Assay: Tester Strains and Their Characteristics i. standard tester strains of SALMONELLA TYPHIMURIUM strain

target allele

chromosomal genotype

plasmids

TA1535 TA100 TA1538 TA98 TA1537 TA97 TA102 TA104

hisG46 hisG46 hisD3052 hisD3052 hisC3076 hisD6610 hisG428 hisG428

hisG46 rfa uvrB hisG46 rfa uvrB hisD3052 rfa uvrB hisD3052 rfa uvrB hisC3076 rfa uvrB hisD6610 hisO1242 rfa uvrB his (G)8476 rfa hisG428 rfa uvrB

None pKM101 (mucAB Apr ) None pKM101 (mucAB Apr ) None pKM101 (mucAB Apr ) pKM101 (mucAB Apr ) and pAQ1 (hisG428 Tcr ) pKM101 (mucAB Apr )

characteristic

ii. genetic characteristics of the ames tester strains rationale for inclusion in the tester strain

rfa uvrB mucAB Apr hisO1242 his (G)8476 Tcr strain TA1535, TA100

TA1538, TA98 TA1537 TA97 TA102, TA104

∗

Alters the lipopolysaccharide wall, conferring greater permeability to mutagens. Deletes the excision repair system, increasing sensitivity to many mutagens; retention of excision in TA102 permits the detection of DNA-crosslinking agents. Enhances sensitivity to some mutagens whose activity depends on the SOS system. Permits selection for the presence of pKM101 by ampicillin resistance. Affects regulation of histidine genes, enhancing revertibility of hisD6610 in TA97. Eliminates the chromosomal hisG gene, allowing detection of the reversion of hisG428 on pAQ1 in TA102. Permits selection for the presence of pAQ1 in TA102 by tetracycline resistance. iii. mechanisms of reversion detected by the ames tester strains primary target∗ mutations detected GGG/CCC Base-pair substitutions, principally those beginning at G:C base pairs (G:C → A:T; G:C → T:A; G:C → C:G), but also A:T → C:G. These strains do not detect A:T → G:C. CGCGCGCG/GCGCGCGC Frameshift mutations, especially −2 frameshifts ( GC or CG), +1 frameshifts, other small deletions, and some complex mutations. GGGGG/CCCCC Frameshift mutations, mainly −1 ( G or C; less frequently T), but also some +CG frameshifts. GGGGGG/CCCCCC Frameshift mutations, combining the specificity of TA1537 at the primary target and with some characteristics of TA98. TAA/ATT Base-pair substitutions, principally those beginning at A:T base pairs (A:T → G:C; A:T → T:A; A:T → C:G), but also G:C → T:A and G:C → A:T.

The sequences before and after the backslash represent the two complementary strands of DNA.

Part III of Table 9-3 is by necessity a simplification with respect both to targets and mechanisms of reversion of the Ames strains. Some mutations that bring about reversion to histidine independence fall outside the primary target, and the full target has been found to be as much as 76 base pairs in hisD3052 (DeMarini et al., 1998). Other revertants can arise by suppressor mutations in other genes. It has been shown that hisG46, hisG428, hisC3076, hisD6610, and hisD3052 all revert by multiple mechanisms and that the spectrum of classes of revertants may vary depending on the mutagen, experimental conditions, and other elements of the genotype (Cebula and Koch, 1990; Prival and Cebula, 1992; DeMarini et al., 1998; Mortelmans and Zeiger, 2000). The development of Salmonella strains that are highly specific with respect to mechanisms of reversion has made the identification of particular base-pair substitutions more straightforward. These strains (TA7001–TA7006) each revert from his− to his+ by a single kind of mutation (e.g., G:C to T:A), and collectively they permit the specific detection of all six possible base-pair substitutions (Gee et al., 1994, 1998; Mortelmans and Zeiger, 2000).

Specific reversion assays are also available in E. coli. A versatile system based on reversion of lacZ mutations in E. coli permits the specific detection of all six possible base-pair substitutions (Cupples and Miller, 1989; Josephy, 2000) and frameshift mutations for which one or two bases have been added or deleted in various sequence contexts (Cupples et al., 1990; Josephy, 2000). The versatility of the lacZ assay has been expanded through the introduction of useful characteristics into the strains parallel to those incorporated into the Ames strains. Among the features added to the lacZ assay are DNA repair deficiencies, permeability alterations, plasmid-enhanced mutagenesis, and enzymes of mutagen metabolism (Josephy, 2000). Bacterial forward mutation assays, such as selections for resistance to arabinose or to purine or pyrimidine analogs in Salmonella (Jurado et al., 1994; Vlasakova et al., 2005), are also used in research and testing, though less extensively than reversion assays. A versatile forward mutation assay that has contributed greatly to an understanding of mechanisms of mutagenesis is the lacI system in E. coli (Calos and Miller, 1981; Halliday and Glickman, 1991).

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Mutations in the lacI gene, which encodes the repressor of the lactose operon, are easily identified by phenotype, cloned or amplified by PCR, and sequenced. The lacI gene is widely used as a target for mutagenesis both in E. coli and in transgenic mice, and more than 30,000 lacI mutants had been sequenced by the mid-1990s (Mirsalis et al., 1994).

Genetic Alterations in Nonmammalian Eukaryotes Gene Mutations and Chromosome Aberrations Many early studies of mutagenesis used yeasts, mycelial fungi, plants, and insects as experimental organisms. Even though well-characterized genetic systems permit the analysis of a diverse array of genetic alterations in these organisms (Table 9-2), they have been largely supplanted in genetic toxicology by bacterial and mammalian systems. Exceptions are to be found where the assays in nonmammalian eukaryotes permit the study of genetic endpoints that are not readily analyzed in mammals or where the organism has special attributes that fit a particular application. The fruit fly, Drosophila, has long occupied a prominent place in genetic research. In fact, the first unequivocal evidence of chemical mutagenesis was obtained in Scotland in 1941 when Charlotte Auerbach and J.M. Robson demonstrated that mustard gas is mutagenic in Drosophila. Drosophila continues to be used in modern mutation research (Potter and Turenchalk, 2000) but its role in genetic toxicology is now more limited. The Drosophila assay of greatest historical importance is the sex-linked recessive lethal (SLRL) test. A strength of the SLRL test is that it permits the detection of recessive lethal mutations at 600–800 different loci on the X chromosome by screening for the presence or absence of wild-type males in the offspring of specifically designed crosses (Mason et al., 1987). The genetic alterations include gene mutations and small deletions. The spontaneous frequency of SLRL is about 0.2% , and a significant increase over this frequency in the lineages derived from treated males indicates mutagenesis. Although it requires screening large numbers of fruit fly vials, the SLRL test yields information about mutagenesis in germ cells, which is lacking in all microbial and cell culture systems. However, means of exposure, measurement of doses, metabolism, and gametogenesis in insects differ from those in mammalian toxicology, thereby introducing doubt about the relevance of Drosophila assays to human genetic risk. Drosophila assays are also available for studying the induction of chromosome abnormalities in germ cells, specifically heritable translocations (Mason et al., 1987) and sex-chromosome loss (Osgood and Cyr, 1998). Genetic and cytogenetic assays in plants (Grant, 1994) also occupy a more restricted niche in modern genetic toxicology than they did years ago. However, plant assays continue to find use in special applications, such as in situ monitoring for mutagens and exploration of the metabolism of promutagens by agricultural plants. In situ monitoring entails looking for evidence of mutagenesis in organisms that are grown in the environment of interest. Natural populations of organisms can also be examined for evidence of genetic damage (Klekowski et al., 1994; Jha, 2004). For example, frequencies of chlorophyll mutations in red mangroves have been correlated with concentrations of polycyclic hydrocarbons in the sediments in which they were growing (Klekowski et al., 1994). Although studies of natural populations are of obvious interest, they require utmost precaution when characterizing the environments and defining appropriate control populations.

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Mitotic Recombination Assays in nonmammalian eukaryotes continue to be important in the study of induced recombination. Recombinogenic effects in yeast have long been used as a general indicator of genetic damage (Zimmermann et al., 1984), and interest in the induction of recombination has increased as recombinational events have been implicated in the etiology of cancer (Sengstag, 1994; Bishop and Schiestl, 2003). The widely used assays for recombinogens are those that detect mitotic crossing over and mitotic gene conversion in the yeast Saccharomyces cerevisiae (Zimmermann, 1992). Hundreds of chemicals have been tested for recombinogenic effects in straightforward yeast assays. In yeast strain D7, for example, mitotic crossing over involving the ade2 locus is detected on the basis of pink and red colony color, and mitotic gene conversion at the trp5 locus is detected by selection for growth without tryptophan. Strategies have also been devised to detect recombinogenic effects in human lymphocytes (Turner et al., 2003), other mammalian cells, mice, plants, and mycelial fungi (Hoffmann, 1994). At least 350 chemicals have been evaluated in Drosophila somatic cell assays in which recombinogenic effects are detected by examining wings or eyes for regions in which recessive alleles are expressed in heterozygotes (Vogel et al., 1999).

Gene Mutations in Mammals Gene Mutations In Vitro Mutagenicity assays in cultured mammalian cells have some of the same advantages as microbial assays with respect to speed and cost, and they use similar approaches. The most widely used assays for gene mutations in mammalian cells detect forward mutations that confer resistance to a toxic chemical. For example, mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT enzyme; HPRT gene) confer resistance to the purine analogue 6-thioguanine (Walker et al., 1999), and thymidine kinase mutations (TK enzyme; TK gene) confer resistance to the pyrimidine analogue trifluorothymidine (Clements, 2000). HPRT and TK mutations may therefore be detected by attempting to grow cells in the presence of purine analogues and pyrimidine analogues, respectively. For historical reasons, HPRT assays have most commonly been conducted in Chinese hamster cells or human cells, while TK assays have used mouse lymphoma cells or human cells. Forward-mutation assays typically respond to diverse mechanisms of mutagenesis, but there are exceptions. For example, a specific alteration in the target gene confers resistance to ouabain, but alterations that eliminate the gene function are lethal (DeMarini et al., 1989). Because the ability to detect various kinds of mutations is desirable, assays that do not do so, such as ouabain resistance, are not useful for general mutagenicity testing. Gene Mutations In Vivo In vivo assays involve treating intact animals and analyzing appropriate tissues for genetic effects. The choice of suitable doses, treatment procedures, controls, and sample sizes is critical in the conduct of in vivo tests. Mutations may be detected either in somatic cells or in germ cells. The latter are of special interest with respect to risk for future generations. The mouse spot test is a traditional genetic assay for gene mutations in somatic cells (Styles and Penman, 1985; Lambert et al., 2005). Visible spots of altered phenotype in mice heterozygous for coat color genes indicate mutations in the progenitor cells of the altered regions. Although straightforward in design, the spot test is less used today than other somatic cell assays or than its germ-cell counterpart, the mouse specific-locus test.

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Cells that are amenable to positive selection for mutants when collected from intact animals form the basis for efficient in vivo mutation-detection assays analogous to those in mammalian cell cultures. Lymphocytes with mutations in the HPRT gene are readily detected by selection for resistance to 6-thioguanine. The hprt assay in mice and rats, and HPRT in monkeys (Casciano et al., 1999; Walker et al., 1999) is of special interest because it permits comparisons to the measurement of HPRT mutations in humans, an important assay in human mutational monitoring (Cole and Skopek, 1994; Albertini and Hayes, 1997). Besides determining whether agents are mutagenic, mutation assays provide information on mechanisms of mutagenesis that contributes to an understanding of mutational hazards. Base-pair substitutions and large deletions, which may be indistinguishable on the basis of phenotype, can be differentiated through the use of probes for the target gene and Southern blotting, in that base substitutions are too subtle to be detectable on the blots, whereas gross structural alterations are visible (Cole and Skopek, 1994; Albertini and Hayes, 1997). Molecular analysis has been used to determine proportions of mutations ascribable to deletions and other structural alterations in several assays, including the specific-locus test for germ-cell mutations in mice (Favor, 1999) and the human HPRT assay (Cole and Skopek, 1994). Gene mutations have been characterized at the molecular level by DNA sequence analysis both in transgenic rodents (Mirsalis et al., 1994; Lambert et al., 2005) and in endogenous mammalian genes (Cariello and Skopek, 1993). Many HPRT mutations from human cells in vitro and in vivo have been analyzed at the molecular level and classified with respect to base-pair substitutions, frameshifts, small deletions, large deletions, and other alterations (Cole and Skopek, 1994).

Transgenic Assays Transgenic animals are products of DNA technology in which the animal contains foreign DNA sequences that have been added to the genome. The foreign DNA is represented in all the somatic cells of the animal and is transmitted in the germ line to progeny. Mutagenicity assays in transgenic animals combine in vivo metabolic activation and pharmacodynamics with simple microbial detection systems, and they permit the analysis of mutations induced in diverse mammalian tissues (Mirsalis et al., 1994; Lambert et al., 2005). The transgenic animals that have figured most heavily in genetic toxicology are rodents that carry lac genes from E. coli. The bacterial genes were introduced into mice or rats by injecting a vector carrying the genes into fertilized oocytes (Mirsalis et al., 1994; Lambert et al., 2005). The strains are commonly referred to by their r mouse” and “Big Blue r rat,” commercial names—the “Big Blue TM which use lacI as a target for mutagenesis, and the “Muta Mouse,” which uses lacZ (Lambert et al., 2005). After mutagenic treatment of the transgenic animals, the lac genes are recovered from the animal, packaged in phage λ, and transferred to E. coli for mutational analysis. Mutant plaques are identified on the basis of phenotype, and mutant frequencies can be calculated for different tissues of the treated animals (Mirsalis et al., 1994). The cII locus may be used as a second target gene in both the lacZ and lacI transgenic assays (Swiger, 2001; Lambert et al., 2005). Its use offers technical advantages as a small, easily sequenced target in which mutations are detected by positive selection, and it permits interesting comparisons both within and between assays. A lacZ transgenic mouse, which uses a plasmid-based system rather than a phage vector is available and has the advantage

that deletion mutants are more readily recovered than in the phagebased lac systems (Lambert et al., 2005). Deletions may also be detected in the gpt delta mouse and rat using a phage vector system. These transgenic animals detect two kinds of genetic events in two targets—point mutations in gpt detected by resistance to 6thioguanine and spi deletions that permit growth on P2 lysogens (Okada et al., 1999; Lambert et al., 2005). Other transgenic assays are under development and offer the prospect of expanding the versatility of such assays (Lambert et al., 2005). Various mutagens, including alkylating agents, nitrosamines, procarbazine, cyclophosphamide, and polycyclic aromatic hydrocarbons have been studied in transgenic mouse assays, and mutant frequencies have been analyzed in such diverse tissues as liver, skin, spleen, kidney, bladder, small intestine, bone marrow, and testis (Lambert et al., 2005). Tissue-specific mutant frequencies can be compared to the distribution of adducts among tissues and to the site specificity of carcinogenesis (Mirsalis et al., 1994). An important issue that remains to be resolved is the extent to which transgenes resemble endogenous genes. Although their mutational responses tend to be comparable (Lambert et al., 2005), some differences have been noted (Burkhart and Malling, 1993; Lambert et al., 2005), and questions have been raised about the relevance of mutations that might be recovered from dying or dead animal tissues (Burkhart and Malling, 1994). Therefore, transgenic animals offer promising models for the study of chemical mutagenesis, but they must be further characterized before their ultimate place in hazard assessment is clear.

Mammalian Cytogenetic Assays Chromosome Aberrations Cytogenetic assays rely on the use of microscopy for the direct observation of the effects of interest. This approach differs sharply from the indirectness of traditional genetic assays in which one observes a phenotype and reaches conclusions about genes. It is only through the addition of DNA sequencing that genetic assays can approach the directness of cytogenetic assays. In conventional cytogenetics, metaphase analysis is used to detect chromosomal anomalies, especially unstable chromosome and chromatid aberrations. A key factor in the design of cytogenetic assays is obtaining appropriate cell populations for treatment and analysis (Preston et al., 1981; Ishidate et al., 1988; Kirkland et al., 1990; Galloway et al., 1994). Cells with a stable, well-defined karyotype, short generation time, low chromosome number, and large chromosomes are ideal for cytogenetic analysis. For this reason, Chinese hamster cells have been used widely in cytogenetic testing. Other cells are also suitable, and human cells, especially peripheral lymphocytes, have been used extensively. Cells should be treated during a sensitive period of the cell cycle (typically S), and aberrations should be analyzed at the first mitotic division after treatment so that the sensitivity of the assay is not reduced by unstable aberrations being lost during cell division. Examples of chromosome aberrations are shown in Fig. 9-4. Cytogenetic assays require careful attention to growth conditions, controls, doses, treatment conditions, and time intervals between treatment and the sampling of cells for analysis (Kirkland et al., 1990). Data collection is a critical part of cytogenetic analysis. It is essential that sufficient cells be analyzed because a negative result in a small sample is inconclusive. Results should be recorded for specific classes of aberrations, not just an overall index of aberrations per cell. The need for detailed data is all the more important because of nonuniformity in the classification of aberrations and

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Figure 9-4. Chromosome aberrations induced by X-rays in Chinese hamster ovary (CHO) cells. A. A chromatid deletion (). B. A chromatid exchange called a triradial (). C. A small interstitial deletion () that resulted from chromosome breakage. D. A metaphase with more than one aberration: a centric ring plus an acentric fragment () and a dicentric chromosome plus an acentric fragment (→).

disagreement on whether small achromatic (i.e., unstained) gaps in chromosomes are true chromosomal aberrations. Gaps should be quantified but not pooled with other aberrations. In interpreting results on the induction of chromosome aberrations in cell cultures, one must be alert to the possibility of artifacts associated with extreme assay conditions because aberrations induced under such circumstances may not be a reflection of a chemical-specific genotoxicity (Scott et al., 1991; Galloway, 2000). Questionable positive results have been found at highly cytotoxic doses (Galloway, 2000), high osmolality, and pH extremes (Scott et al., 1991). The possibility that metabolic activation systems may be genotoxic also warrants scrutiny (Scott et al., 1991). Although excessively high doses may lead to artifactual positive responses, the failure to test to a sufficiently high dose also undermines the utility of a test. Therefore, testing should be extended to a dose at which there is some cytotoxicity, such as a reduction in the mitotic index (the proportion of cells in division), or to an arbitrary limit of about 10 mM if the chemical is nontoxic (Kirkland et al., 1990).

In vivo assays for chromosome aberrations involve treating intact animals and later collecting cells for cytogenetic analysis (Preston et al., 1981; Kirkland et al., 1990; Tice et al., 1994). The main advantage of in vivo assays is that they include mammalian metabolism, DNA repair, and pharmacodynamics. The target is typically a tissue from which large numbers of dividing cells are easily prepared for analysis. Bone marrow from rats, mice, or Chinese hamsters is most commonly used. Peripheral lymphocytes are another suitable target when stimulated to divide with a mitogen such as phytohemagglutinin. Effective testing requires dosages and routes of administration that ensure adequate exposure of the target cells, proper intervals between treatment and collecting cells, and sufficient numbers of animals and cells analyzed (Kirkland et al., 1990). An important development in cytogenetic analysis is fluorescence in situ hybridization (FISH), in which a nucleic acid probe is hybridized to complementary sequences in chromosomal DNA. The probe is labeled with a fluorescent dye so that the chromosomal location to which it binds is visible by fluorescence microscopy. Composite probes have been developed from sequences unique to

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specific human chromosomes, giving a uniform fluorescent label over the entire chromosome. Slides prepared for standard metaphase analysis are suitable for FISH after they have undergone a simple denaturation procedure. The use of whole-chromosome probes is commonly called “chromosome painting” (Speicher and Carter, 2005). Chromosome painting facilitates cytogenetic analysis, because aberrations are easily detected by the number of fluorescent regions in a painted metaphase. For example, if chromosome 4 were painted with a probe while the other chromosomes were counterstained in a different color, one would see only the two homologues of chromosome 4 in the color of the probe in a normal cell. However, if there were a translocation or a dicentric chromosome and fragment involving chromosome 4, one would see three areas of fluorescence—one normal chromosome 4 and the two pieces involved in the chromosome rearrangement. Aberrations are detected only in the painted portion of the genome, but this disadvantage can be offset by painting a few chromosomes simultaneously with probes of different colors (Tucker et al., 1993b). FISH reduces the time and technical skill required to detect chromosome aberrations, and it permits the scoring of stable aberrations, such as translocations and insertions, that are not readily detected in traditional metaphase analysis without special labeling techniques. Using FISH, some chromosomal analysis can even be conducted in interphase cells. Although FISH is not routinely used in genotoxicity testing, it is a valuable research tool for studying clastogens and is having a substantial impact in monitoring human populations for chromosomal damage.

Micronuclei Metaphase analysis is time consuming and requires considerable skill, so simpler cytogenetic assays have been developed, of which micronucleus assays have become especially important. Micronuclei are membrane-bounded structures that contain chromosomal fragments, or sometimes whole chromosomes, that were not incorporated into a daughter nucleus at mitosis. Because micronuclei usually represent acentric chromosomal fragments, they are most commonly used as simple indicators of chromosomal damage. However, the ability to detect micronuclei containing whole chromosomes has led to their use for detecting aneuploidy as well. Micronucleus assays may be conducted in primary cultures of human lymphocytes (Fenech, 2000; Fenech et al., 2003), mammalian cell lines (Kirsch-Volders et al., 2000, 2003), or mammals in vivo (Heddle et al., 1991; Hayashi et al., 2000; Krishna and Hayashi, 2000; Hamada et al., 2001). Micronucleus assays in lymphocytes have been greatly improved by the cytokinesis-block technique in which cell division is inhibited with cytochalasin B, resulting in binucleate and multinucleate cells (Fenech, 2000; Kirsch-Volders et al., 2000; Fenech et al., 2003). In the cytokinesis-block assay in human lymphocytes, nondividing (G0 ) cells are treated with ionizing radiation or a radiomimetic chemical and then stimulated to divide with the mitogen phytohemagglutinin. Alternatively, the lymphocytes may be exposed to the mitogen first, so that the subsequent mutagenic treatment with radiation or chemicals includes the S period of the cell cycle. In either case, cytochalasin B is added for the last part of the culture period, and micronuclei are counted only in binucleate cells so as to ensure that the cells have undergone a single nuclear division that is essential for micronucleus development. The assay thereby avoids confusion owing to differences in cellular proliferation kinetics. Although micronuclei resulting

Figure 9-5. Micronucleus in a human lymphocyte. The cytochalasin B method was used to inhibit cytokinesis that resulted in a binucleate nucleus. The micronucleus resulted from failure of an acentric chromosome fragment or a whole chromosome being included in a daughter nucleus following cell division. (Figure courtesy of James Allen, Jill Barnes, and Barbara Collins.)

from chromosome breakage comprise the principal endpoint in the cytokinesis-block micronucleus assay, the method can also provide evidence of aneuploidy, chromosome rearrangements that form nucleoplasmic bridges, inhibition of cell division, necrosis, apoptosis, and excision-repairable lesions (Fenech, 2000; Fenech et al., 2003). Micronuclei in a binucleate human lymphocyte are shown in Fig. 9-5. The in vivo micronucleus assay is most often performed by counting micronuclei in immature (polychromatic) erythrocytes in the bone marrow of treated mice, but it may also be based on peripheral blood (Heddle et al., 1991; Hayashi et al., 2000; Krishna and Hayashi, 2000; Hamada et al., 2001). Micronuclei remain in the cell when the nucleus is extruded in the maturation of erythroblasts. In vivo micronucleus assays are increasingly used in genotoxicity testing as a substitute for bone marrow metaphase chromosome analysis. Micronucleus assays in mammalian tissues other than bone marrow and blood are useful for mechanistic studies and research but are less often applied to genotoxicity testing (Hayashi et al., 2000). Sister Chromatid Exchanges Sister chromatid exchanges (SCE), in which there has been an apparently reciprocal exchange of segments between the two chromatids of a chromosome, are visible cytologically through differential staining of chromatids. Figure 9-6 shows SCE in human cells. Many mutagens induce SCE in cultured cells and in mammals in vivo (Tucker et al., 1993a). Despite the convenience and responsiveness of SCE assays, data on SCE are less informative than data on chromosome aberrations. There is uncertainty about the underlying mechanisms by which SCEs are formed and how DNA damage or perturbations of DNA synthesis stimulate their formation (Preston, 1991). SCE assays are therefore best regarded as general indicators of mutagen exposure, analogous to DNA damage and repair assays, rather than measures of a mutagenic effect.

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Figure 9-6. Sister chromatid exchanges (SCEs) in human lymphocytes. A. SCE in untreated cell. B. SCE in cell exposed to ethyl carbamate. The treatment results in a very large increase in the number of SCE. (Figure courtesy of James Allen and Barbara Collins.)

Aneuploidy Although assays based on the underlying mechanisms of the induction of aneuploidy are not yet as refined as those for gene mutations and chromosome aberrations, they are being developed (Aardema et al., 1998). Some of the methods are restricted to specific targets, such as the mitotic spindle in an assay for effects on the polymerization of tubulin in vitro (Parry, 1993). Most, however, measure aneuploidy itself and should therefore encompass all relevant cellular targets. Assays include chromosome counting (Aardema et al., 1998), the detection of micronuclei that contain kinetochores (Natarajan, 1993; Aardema et al., 1998; Fenech, 2000), and the observation of abnormal spindles or spindle–chromosome associations in cells in which spindles and chromosomes have been differentially stained (Parry, 1998). FISH-based assays have also been developed for the assessment of aneuploidy in interphase somatic cells (Rupa et al., 1997) and in sperm (Russo, 2000; Wyrobek et al., 2005). A complication in chromosome counting is that a metaphase may lack chromosomes because they were lost during cell preparation for analysis, rather than having been absent from the living cell. To avoid this artifact, cytogeneticists generally use extra chromosomes (i.e., hyperploidy) rather than missing chromosomes (i.e., hypoploidy) as an indicator of aneuploidy in chromosome preparations from mammalian cell cultures (Galloway and Ivett, 1986; Aardema et al., 1998) or mouse bone marrow (Adler, 1993). A possible means of circumventing this difficulty is growing and treating cells on a glass surface and then making chromosome preparations in situ, rather than dropping cells onto slides from a cell suspension. By counting chromosomes in intact cells, one can collect data for both hyperploidy and hypoploidy (Natarajan, 1993). It has been suggested that counting polyploid cells, which is technically straightforward, may be an efficient way to detect aneugens (Aardema et al., 1998), but there remains some disagreement on the point (Parry, 1998). Micronucleus assays can detect aneugens as well as clastogens. Micronuclei that contain whole chromosomes tend to be somewhat

larger than those containing chromosome fragments, but the two categories are not readily distinguished in typically stained preparations (Natarajan, 1993). However, the presence of the spindle attachment region of a chromosome (kinetochore) or of centromeric DNA in a micronucleus can serve as an indicator that it contains a whole chromosome. Aneuploidy may therefore be detected by means of antikinetochore antibodies with a fluorescent label or FISH with a probe for centromere-specific DNA (Lynch and Parry, 1993; Natarajan, 1993; Fenech, 2000). Micronuclei containing kinetochores or centromeric DNA may be detected in cultured cells (Lynch and Parry, 1993; Aardema et al., 1998; Fenech, 2000) and in mouse bone marrow in vivo (Heddle et al., 1991; Adler, 1993). Frequencies of micronuclei ascribable to aneuploidy and to clastogenic effects may therefore be determined concurrently by tabulating micronuclei with and without kinetochores.

Germ Cell Mutagenesis Gene Mutations Germ-cell mutagenesis assays are of special interest as indicators of genetic damage that can enter the gene pool and be transmitted through generations. Mammalian germ-cell assays provide the best basis for assessing risks to human germ cells and therefore hold a central place in genetic toxicology despite their relative complexity and expense. The design of the test must compensate for the fact that mutations occur at low frequency, and even the simplest animal systems face a problem of there being a sufficiently large sample size. One can easily screen millions of bacteria or cultured cells by selection techniques, but screening large numbers of mice poses practical limitations. Therefore, germ-cell assays must offer a straightforward, unequivocal identification of mutants with minimal labor. The mouse specific-locus test detects recessive mutations that produce easily analyzed, visible phenotypes (coat pigmentation and ear size) conferred by seven genes (Russell and Shelby, 1985;

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Ehling, 1991; Russell and Russell, 1992; Favor, 1999). Mutants may be classified as having point mutations or chromosomal alterations on the basis of genetic and molecular analysis (Favor, 1999). The assay has been important in assessing genetic risks of ionizing radiation and has been used to study various chemical mutagens. Other mouse germ-cell assays use different indicators of gene mutations, such as dominant mutations that cause skeletal abnormalities or cataracts (Ehling, 1991) and recessive mutations that cause electrophoretic changes in proteins (Lewis, 1991). Mammalian assays permit the measurement of mutagenesis at different germ-cell stages (Favor, 1999). Late stages of spermatogenesis are often found to be sensitive to mutagenesis, but spermatocytes, spermatids, and spermatozoa are transitory. Mutagenesis in stem-cell spermatogonia and resting oocytes is of special interest in genetic risk assessment because of the persistence of these stages throughout reproductive life. Chemical mutagens show specificity with respect to germ-cell stages. For example, ethylnitrosourea and chlorambucil are both potent mutagens in the mouse specific-locus test, but the former induces primarily point mutations in spermatogonia, whereas the latter mostly induces deletions in spermatids (Russell and Russell, 1992). The ratio of deletions to point mutations is not only a function of the nature of the mutagen but depends on germ-cell stage, as some mutagens induce higher proportions of gross alterations in late stages of spermatogenesis than in spermatogonia (Lewis, 1991; Favor, 1999; Russell, 2004). There is currently no unequivocal evidence of induced gene mutations in human germ cells, but studies in mice leave little doubt about the susceptibility of mammalian germ cells to mutagenesis by radiation and chemicals. New molecular methods, particularly those involving the assessment of changes in tandem repeat loci (reviewed in Yauk, 2004), hold great promise for the development of systems that will permit the efficient detection of germ-cell mutations in humans. The development of such methods in other species is also important, in that it permits in situ monitoring for environmental mutagens (Yauk, 2004) and the quantification of mutagenesis after controlled exposures of laboratory animals using systems parallel to those being developed for human monitoring (Wu et al., 2006).

Chromosomal Alterations Cytogenetic assays in germ cells are not routinely included in mutagenicity testing, but they are an important source of information for assessing risks to future generations posed by the induction of chromosome aberrations. Metaphase analysis of germ cells is feasible in rodent spermatogonia, spermatocytes, or oocytes (Kirkland et al., 1990; Tease, 1992; Russo, 2000). A germ-cell micronucleus assay, in which chromosomal damage induced in meiosis is measured by observation of rodent spermatids, has also been developed (Hayashi et al., 2000; Russo, 2000). Aneuploidy originating in mammalian germ cells may be detected cytologically through chromosome counting for hyperploidy (Allen et al., 1986; Adler, 1993; Aardema et al., 1998) or genetically in the mouse sex-chromosome loss test (Russell and Shelby, 1985; Allen et al., 1986), but these methods are not widely used in toxicological testing. A promising development is the detection of aneuploidy in the sperm of mice or rats by FISH with chromosomespecific probes (Russo, 2000; Wyrobek et al., 2005). The presence of two fluorescent spots indicates the presence of an extra copy of the chromosome identified by the probe; probes for several chromosomes are used simultaneously so that aneuploid sperm are distinguishable from diploid sperm. Besides cytological observation, indirect evidence for chromosome aberrations is obtained in the mouse heritable transloca-

tion assay, which measures reduced fertility in the offspring of treated males (Russell and Shelby, 1985). This presumptive evidence of chromosomal rearrangements can be confirmed through cytogenetic analysis. Data from the mouse heritable translocation test in postmeiotic male germ cells have been used in an attempt to quantify human germ-cell risk for ethylene oxide, a mutagen used as a fumigant, sterilizing agent, and reactant in chemical syntheses (Rhomberg et al., 1990; Preston et al., 1995). Dominant Lethal Mutations The mouse or rat dominant lethal assay (Adler et al., 1994) offers an extensive database on the induction of genetic damage in mammalian germ cells. In the most commonly used version of the assay, males are treated on an acute or subchronic basis with the agent of interest and then mated with virgin females at appropriate intervals. The females are killed and necropsied during pregnancy so that embryonic mortality may be characterized and quantified. Most dominant lethal mutations, manifested as intrauterine deaths, are thought to arise from chromosomal anomalies.

Development of Testing Strategies Concern about adverse effects of mutation on human health, principally carcinogenesis and the induction of transmissible damage in germ cells, has provided the impetus to identify environmental mutagens. Priorities must be set for testing, because it is not feasible to conduct multiple tests of all chemicals to which people are exposed. Such factors as production volumes, intended uses, the extent of human exposure, environmental distribution, and effects that may be anticipated on the basis of chemical structure or previous testing must be considered in order to ensure that compounds with the greatest potential for adverse effects receive the most comprehensive study. The most obvious use of genetic toxicology assays is screening chemicals to detect mutagens, but they are also used to obtain information on mutagenic mechanisms and dose–responses that contribute to an evaluation of hazards. Besides testing pure chemicals, environmental samples are tested because many mutagens exist in complex mixtures (DeMarini 1998; Ohe et al., 2003; White, 2004). The analysis of complex mixtures often requires a combination of mutagenicity assays and refined analytical methods (White, 2004; Hewitt and Marvin, 2005). The first indication that a chemical is a mutagen often lies in chemical structure. Potential electrophilic sites in a molecule serve as an alert to possible mutagenicity and carcinogenicity, because such sites confer reactivity with nucleophilic sites in DNA (Tennant and Ashby, 1991). Attempts to formalize the structural prediction through automated computer programs have not yet led to an ability to predict mutagenicity and carcinogenicity of new chemicals with great accuracy (Richard, 1998; Snyder et al., 2004), but developmental work on such systems continues (Votano et al., 2004). Moreover, structural alerts in combination with critical interpretation are a valuable adjunct to mutagenicity testing (Tennant and Ashby, 1991; Ashby and Paton, 1993). Although informative, structural alerts cannot eliminate the need for biological data, and they must be used with cognizance of other factors that can influence the effects of a chemical. Factors that may reduce the likelihood of mutagenicity or carcinogenicity of a structurally alerting compound are steric hindrance of reactive or potentially reactive substituents, metabolism, toxicity, and substituents that enhance the chemical’s excretion (Ashby, 1994). Moreover, some agents that lack structural alerts may stimulate mutagenesis indirectly by such

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mechanisms as the generation of radicals that cause oxidative DNA damage (Clayson et al., 1994). Assessment of a chemical’s genotoxicity requires data from well-characterized assays. Assays are said to be validated when they have been shown to perform reproducibly and reliably with many compounds from diverse chemical classes in several laboratories. An evaluation of test performance, however, sometimes extends beyond determining whether the assay effectively detects the specific endpoint that it actually measures to whether it is predictive of other endpoints of interest. For example, there is great interest in the ability of mutagenicity tests, which do not measure carcinogenicity per se, to predict whether chemicals are carcinogens. Mutagenicity testing, combined with an evaluation of chemical structure, has been found to identify a large proportion of transspecies, multiple-site carcinogens (Tennant and Ashby, 1991; Gold et al., 1993). In contrast, some carcinogens are not detected as mutagens. Putatively nongenotoxic carcinogens often give responses that are more specific with respect to species, sites, and conditions (Ashby and Paton, 1993; Gold et al., 1993). In predicting carcinogenicity, one should consider both the sensitivity and the specificity of an assay. Sensitivity refers to the proportion of carcinogens that are positive in the assay, whereas specificity is the proportion of noncarcinogens that are negative (Tennant et al., 1987; McGregor et al., 1999). Sensitivity and specificity both contribute to the predictive reliability of an assay. The commonly held view that deficiencies in the sensitivity or specificity of individual assays may be circumvented by using assays in complementary combinations called tiers or batteries has fallen into disfavor because, rather than offsetting each other’s strengths and weaknesses, genetic toxicology assays are often consistent with one another (Tennant et al., 1987; Ashby and Tennant, 1991; Kim and Margolin, 1999). Strategies for testing have evolved over the last few decades, such that data from a few well-chosen assays are now considered sufficient (MacGregor et al., 2000). Rather than trying to assemble extensive batteries of complementary assays, it is prudent to emphasize mechanistic considerations in choosing assays. Such an approach makes a sensitive assay for gene mutations (e.g., the Ames assay) and an assay for clastogenic effects in mammals pivotal in the evaluation of genotoxicity, and this is the basis for our highlighting these assays in Table 9-1. The Ames assay has performed reliably with hundreds of compounds in laboratories throughout the world. Other bacterial assays and mammalian cell assays also provide useful information on gene mutations. Beyond gene mutations, one should evaluate damage at the chromosomal level with a mammalian in vitro or in vivo cytogenetic assay. Cytogenetic assays in rodents are especially useful for this purpose because they combine a well-validated genetic assay with mammalian pharmacodynamics and metabolism. The other assays in Table 9-1 offer an extensive database on chemical mutagenesis (i.e., Drosophila SLRL), a unique genetic endpoint (i.e., aneuploidy; mitotic recombination), applicability to diverse organisms and tissues (i.e., DNA damage assays, such as the comet assay), or special importance in the assessment of genetic risk (i.e., germ cell assays). The more extensive listing of assays in Table 9-2 provides references that can be helpful in interpreting genetic toxicology data that can be found in the scientific literature.

HUMAN POPULATION MONITORING For cancer risk assessment considerations, the human data utilized most frequently, in the absence of epidemiologic data, are those col-

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lected from genotoxicity/mutagenicity assessments in human populations. The studies conducted most frequently are for chromosome aberrations, micronuclei, and SCEs in peripheral lymphocytes. Cytogenetic alterations have also been assessed in a small number of bone marrow samples. Mutations at the HPRT locus have been assessed in peripheral lymphocytes, and glycophorin A variants have been studied in red blood cells. An important component of any population monitoring study is the selection of the study groups, namely those individuals who are potentially exposed and the matched unexposed controls. The size of each study group should be sufficiently large to avoid any confounder having undue influence. Certain characteristics should be matched among exposed and unexposed groups. These include age, sex, smoking status, and general dietary features. Certain characteristics are exclusionary, namely current or recent medication, radiation exposure, and certain illnesses. It is possible to develop a lengthy list of additional possible confounders of response that would make the selection of suitable study groups very difficult indeed. Study groups of 20 or more individuals can be used as a reasonable substitute for exact matching because confounders will be less influential on chromosome alteration or mutation frequency in larger groups, as mentioned above (discussed in Au et al., 1998). In some instances, it might be informative to compare exposed groups with a historical control, as well as to a concurrent control. The magnitude of different known confounders varies considerably among studies, based in part on the size of the study populations. Some general indication of the magnitude of the effects of age and smoking status on the frequencies of chromosome aberrations and SCE is presented to illustrate the importance of accounting for confounders in the design of a population monitoring study. The comparisons presented are for large studies only. For chromosome aberrations, the frequency of aberrations has been reported in one large study to be about 50% higher in smokers (l.5 aberrations per 100 cells in smokers vs. 1.0 per 100 cells in the nonsmokers) (Galloway et al., 1986) and in another no difference between smokers and nonsmokers (Bender et al., 1988). The complete data set has been reviewed by Au et al. (1998). In general, the frequency of SCE is increased by about one SCE per cell in smokers compared with nonsmokers (Bender et al., 1988; Barale et al., 1998). The study by Barale et al. (1998) also reported a dose–response association between SCE frequency and smoking level. The frequency of chromosome aberrations, particularly chromosome-type (reciprocal) exchanges, has been shown to increase with age of subject (Tucker and Moore, 1996). Galloway et al. (1986) reported an increase from 0.8 per 100 cells at about 25 years of age to about 1.5 at 60. Bender et al. (1988) reported an increase with age only for chromosome-type dicentric aberrations, but the increase over a broad age range was small and just statistically significant. Ramsey et al. (1995), using chromosome painting techniques, reported that individuals 50 years and older had frequencies of stable aberrations, dicentrics, and acentric fragments that were 10.6-fold, 3.3-fold, and 2.9-fold, respectively, greater than the frequency in cord bloods. Bender et al. (1998) did not find an increase in SCE frequency with the increasing age of the subject. The differences among the results from these large control studies emphasize the difficulty of adequately accounting for confounders (age and smoking presented here) when only a small control group is used, as is frequently the case. Similar sources of variation have been identified for the monitoring of individuals for HPRT mutations. The data are reviewed in detail by Albertini and Hayes (1997). There is less information on sources of variation of glycophorin A (GPA) variants, although

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quite considerable interindividual variation exists (reviewed in Cole and Skopek, 1994; Kyoizumi et al., 2005). For cytogenetic assays (chromosome aberrations, SCEs, and micronuclei) the alterations are produced as a consequence of errors of DNA replication, as discussed in previous sections. From the nature of the alterations, assessed in traditional cytogenetic assays, in which nontransmissible alterations are analyzed, it can be established that these alterations were produced at the first in vitro S phase. Irrespective of the duration of exposure, the frequency of cytogenetic alterations will be proportional to that fraction of the DNA damage that remains at the time of in vitro DNA replication. All the DNA damage induced by potent clastogens that results in chromosome alterations is repaired within a relatively short time after exposure for G0 human lymphocytes. Thus, for chronic exposures the lymphocyte cytogenetic assay as typically conducted is insensitive. It is now possible to analyze reciprocal translocations using FISH methods (reviewed in Tucker et al., 1997; Kleinerman et al., 2006), and because this aberration type is transmissible from cell generation to generation, its frequency can be representative of an accumulation over time of exposure. The importance of this is that stable chromosome aberrations observed in peripheral lymphocytes exposed in vivo, but assessed following in vitro culture, are produced in vivo in hematopoietic stem cells or other precursor cells of the peripheral lymphocytes pool. To date, population cytogenetic monitoring studies involving the analysis of reciprocal translocations in chemically exposed individuals or radiation-exposed individuals have been conducted quite rarely (Lucas et al., 1992; Smith et al., 1998; Kleinerman et al., 2006). The overall sensitivity of the FISH analysis of reciprocal translocations for assessing effects of chronic, low level of exposure to chemical clastogens has not been established. However, a cautionary note is provided by the study of Director et al. (1998), who showed that there was no increase in reciprocal translocations assessed by FISH following exposure to cyclophosphamide (0, 32, 64, or 96 ppm) or urethane (0, 5000, 10,000, or 15,000 ppm) for up to 12 weeks. In contrast, recent data on ethylene oxide (Preston et al., unpublished) have shown that exposure of male mice to ethylene oxide at concentrations of 0, 25, 50, 100, 200 ppm for 6, 12, 24, or 48 weeks resulted in a time and concentration-dependent increase in reciprocal translocations assessed by FISH. Another factor that certainly affects the utility of population monitoring data with reciprocal translocations using FISH is that the frequency of reciprocal translocations increases significantly with increasing age (Ramsey et al., 1995), but to a lesser extent for nontransmissible aberrations (Bender et al., 1988). Ramsey et al. (1995) provided data on the influence of other confounders on the frequency of reciprocal translocations in human groups. These confounders include smoking, consumption of diet drinks and/or diet sweeteners, exposure to asbestos or coal products, and having a previous major illness. This reemphasizes the point that the selection of study groups and accounting for confounders is essential for human population cytogenetic monitoring studies to be of utility. Thus, very few of the published studies of cytogenetic population monitoring for individuals have analyzed the appropriate endpoint for detecting the genetic effects of long-term exposure to chemicals. It is quite surprising that positive responses have been reported for increases in unstable, chromatid aberrations because these are nontransmissible, and as noted above are induced at the first in vitro S phase. This anomaly is especially concerning when very low levels of exposure are reported (reviewed for ethylene oxide in Preston, 1999).

The HPRT mutation assay can assess the frequency of induced mutations in stem cells or other precursor cells, because a proportion of the mutations are induced as nonlethal events. The transmissible proportion will be greater for agents that do not induce large deletions; this will include the majority of nonradiomimetic chemicals. Induction of mutations in lymphocyte precursor cells will lead to clonal expansion of mutations in the peripheral pool. However, assessment of the T-cell antigen receptor status of the mutant clones permits a correction for clonal expansion. The population of cells derived from any particular stem cell has a unique antigen receptor status (Albertini and Hayes, 1997). The GPA assay can similarly be used for the assessment of chronic exposures or for estimating exposures at some long time after exposure (Albertini and Hayes, 1997). The predictive value of the assay for adverse health outcome appears to be limited, but it can provide an estimate of exposure. The potential for cytogenetic endpoints being predictive of relative cancer risk has been addressed in recent reports from the European Study Group on Cytogenetic Biomarkers and Health (Hagmar et al., 1998a,b; Bonassi et al., 2004; Norppa et al., 2006). The groups selected for cytogenetic studies consisted of individuals with reported occupational exposure and unexposed controls. The association between cancer and the frequency of unstable chromosome aberrations in the study groups was not based on exposure status, but rather on the relative frequency of chromosome aberrations, namely, low (1–33 percentiles), medium (34–66 percentiles), and high (67–100 percentiles). In general, the higher the relative frequency of unstable aberrations, the greater the risk of cancer death for all tumors combined. The authors make it clear that there is insufficient information on exposure for it to be used as a predictor of cancer development. In fact, the data indicate that individuals with higher frequencies of chromosome aberrations for whatever reason (genetic or environmental) are as a group at greater risk of dying from cancer. This is very different from concluding that exposures to mutagens that result in a higher frequency of chromosome aberrations in peripheral lymphocytes leads to an increased risk of cancer, especially for specific tumor types. The relevance of exposure to mutagenic chemicals in these studies by Hagmar et al. (1998a,b) is uncertain because there was no association between increased SCE frequencies and increased cancer mortality. This latter concern was addressed by the same group (Bonassi et al., 2000) in a more recent study. The study again showed that there was a significantly increased risk for subjects with a high level of chromosome aberrations compared to those with a low level in both Nordic and Italian cohorts. Of particular relevance to risk assessment was the observation that the relationships were not affected by the inclusion of occupational exposure level or smoking. The risk for high versus low levels of chromosome aberrations was similar in individuals heavily exposed to carcinogens and in those who had never, to their knowledge, been exposed to any specific environmental carcinogen. These data highlight the need to use caution when considering the relevance of chromosome aberration data in cancer risk assessment.

NEW APPROACHES FOR GENETIC TOXICOLOGY In the past 15 or so years, the field of genetic toxicology has moved into the molecular era. The potential for advances in our understanding of basic cellular processes and how they can be perturbed is enormous. The ability to manipulate and characterize DNA, RNA, and proteins has been at the root of this advance in knowledge. However, the development of sophisticated molecular biology does not

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in itself imply a corresponding advance in the utility of genetic toxicology and its application to risk assessment. Knowing the types of studies to conduct and knowing how to interpret the data remain as fundamental as always. Measuring finer and finer detail can perhaps complicate the utility of the various mutagenicity assays. There is a need for genetic toxicology to avoid the temptation to use more and more sophisticated techniques to address the same questions and in the end make the same mistakes as have been made previously. How successful we are in designing informative studies based on the most recent molecular techniques perhaps cannot be judged at this time. However, the following examples of recent approaches to obtaining data for enhancing our ability to use noncancer (genotoxicity) data in a mechanistically based cancer (and genetic) risk assessment process provide some encouragement. Several recent developments (e.g., the use of transgenic animals, the comet assay for assessing DNA damages) have already been described in the appropriate assay sections above because they are currently in general use.

Advances in Cytogenetics Until quite recently, the analysis of chromosome alterations relied on conventional chromosome staining with DNA stains such as Giemsa or on the process of chromosome banding. Both approaches require considerable expenditure of time and a rather high level of expertise. However, chromosome banding does allow for the assessment of transmissible aberrations such as reciprocal translocations and inversions with a fairly high degree of accuracy. Knowing the induction frequency of such aberrations is very important, given that they are generally not lethal to the cell and constitute by far the major class observed in inherited genetic defects and a significant fraction of the alterations observed in tumors. In addition, because stable aberrations are transmissible from parent to daughter cell, they represent accumulated effects of chronic exposures. The more readily analyzed but cell lethal, nontransmissible aberrations such as dicentrics and deletions reflect only recent exposures and then only when analyzed at the first division after exposure. A more detailed discussion of these factors can be found in Preston (1998). The relative ease with which specific chromosomes, specific genes, and chromosome alterations can be detected has been radically enhanced by the development of FISH (Trask et al., 1993; Speicher and Carter, 2005). In principle, the technique relies on amplification of DNA from particular genomic regions such as whole chromosomes or gene regions and the hybridization of these amplified DNAs to metaphase chromosome preparations or interphase nuclei. Regions of hybridization can be determined by the use of fluorescent antibodies that detect modified DNA bases incorporated during amplification or by incorporating fluorescent bases themselves during amplification. The fluorescently labeled, hybridized regions are detected by fluorescence microscopy, and the signal can be increased in strength by computer-enhanced processes. The level of sophistication has increased so much that all 24 different human chromosomes (22 autosomes, X and Y) can be individually detected (Macville et al., 1997), as can all mouse chromosomes (Liyanage et al., 1996). Alterations in tumors can also be detected on a whole-genome basis (Coleman et al., 1997; Veldman et al., 1997). A recent example highlights the ability to construct breakpoint profiles of specific tumor types (Trost et al., 2006). In this example, a detailed analysis by spectral karyotyping of specific breakpoints in a set of primary myelodysplastic syndrome and acute myeloid leukemia samples revealed recurrent involvement of specific chromosome bands that contained oncogenes or tumor suppressor genes.

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The aim will be to attempt to reveal the possible prognostic significance of the subgroups linked to these specific markers. There is an extensive literature on the use of FISH for karyotyping tumors and in gene mapping but less on its utility for genetic toxicology studies, especially the assessment of stable chromosome aberrations at long periods after exposure or after long-term exposures. Three particular studies do, however, serve to exemplify the use of FISH in genetic toxicology. Lucas et al. (1992) demonstrated that stable chromosomal aberrations could be detected in individuals decades after exposure to atomic bombs in Japan. How these frequencies relate to frequencies at the time of exposure is not known with any certainty, given the fact that induced frequencies were not measured because appropriate techniques were not available at that time. Studies by Tucker et al. (1997, 2005) provided some assessment of the utility of FISH for the analysis of radiation-induced, stable chromosome alterations at various times after exposure. The frequency of reciprocal translocations induced by gamma rays in rat peripheral lymphocytes decreased with time after exposure, reaching a plateau at four days that was 55–65% of the induced frequency and with a dose dependency (Tucker et al., 1997). Similar results were obtained for human samples (Tucker et al., 2005). These results suggest that reciprocal translocations fall into two classes, stable and unstable (cell-lethal). It is quite possible that these “unstable” translocations are lost because of the presence of other cell-lethal damage in the same cell. Additional work is required to clarify this conclusion and to extend the studies to the effects of chemicals. FISH methods have also allowed for an accurate and sensitive assessment of chromosomal alterations present in tumors. The particular advance that makes this assessment feasible is known as comparative genomic hybridization (CGH) (Kallioniemi et al., 1992). CGH results in the ability to identify the role of chromosomal structural and numerical alterations in tumor development. The genomic instability present in all tumor types appears to have a specific genetic basis, as shown elegantly for colon cancer by Vogelstein and colleagues (Cahill et al., 1998). For CGH, tumor and control DNAs are differentially labeled with fluorescence probes and cohybridized to normal metaphase chromosome preparations. The ratio of the fluorescence intensities of hybridized tumor and control DNA indicates regions of normal genomic content as well as those regions that are over- or underrepresented in tumors. The CGH method is being adapted for automated screening approaches using biochips (Solinas-Tolado et al., 1997; Hosoya et al., 2006). Assessing genetic alterations such as specific gene deletions in single metastatic tumor cells is feasible using a slightly different but complementary approach (Pack et al., 1997, 2005). The types of FISH approaches described here undoubtedly indicate the direction in which cytogenetic analysis will proceed. The types of data collected will affect our understanding of how tumors develop. Data on the dose–response characteristics for a specific chromosomal alteration as a proximate marker of cancer can enhance the cancer risk assessment process by describing effects of low exposures that are below those for which tumor incidence can be reliably assessed. Cytogenetic data of the types described above can also improve extrapolation from data generated with laboratory animals to humans.

Molecular Analysis of Mutations and Gene Expression With the advent of molecular biology techniques, the exact basis of a mutation at the level of the DNA sequence can be established.

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In many cases, the genetic basis of human disease can be determined even though human genes have long DNA sequences and a complex genomic arrangement. Molecular biology techniques have also enabled a distinction to be made between background mutations and those induced by specific agents. The latter observations are addressed by analyzing the mutational spectra in target genes in laboratory animals and in humans (DeMarini, 2000; Hemminki and Thilly, 2004). For reasons of inherent sensitivity of available methods, the genes analyzed for mutations are ones for which mutated forms can be selected. The confounding factor of many normal cells, which far outnumber a few mutant cells in an exposed cellular population, can be removed by mutant selection approaches. Methods to overcome the drawback of only being able to study selectable genes are currently being developed, and particular ones such as ligation-mediated polymerase chain reaction (PCR) are close to the required sensitivity level (Albertini and Hayes, 1998; Makrigiorgos, 2004; Yeh et al., 2006). A giant step forward in the ability to detect and characterize mutations at both the DNA and RNA level has been provided by the development of chip technology (Southern, 1996) and array-based assay systems (Woldicka et al., 1997). With hybridization of test DNAs to oligonucleotide arrays, specific genetic alterations or their cellular consequences can be determined rapidly and automatically (Houlston and Peto, 2004; Vissers et al., 2005). Cost remains a limiting factor, but the potential for assessing specific cellular changes following chemical exposure is enormous. Until quite recently, alterations in gene expression following specific exposures or for specific genotypes were analyzed gene by gene. Such an approach makes it difficult to assess changes in gene expression that occur in a concerted fashion. Recent advances using cDNA microarray technologies have allowed the measurement of changes in expression of hundreds or even thousands of genes at one time (Harrington et al., 2000; Elvidge, 2006). The level of expression at the mRNA level is measured by the amount of hybridization of isolated cDNAs to oligonucleotide fragments from known genes or expressed sequence tags (ESTs) on a specifically laid out grid. Although this technique holds great promise for establishing a cell’s response to exposure to chemical or physical agents in the context of normal cellular patterns of gene expression, it remains to be established how to analyze the vast amounts of data that can and are being obtained and what magnitude of change in gene expression constitutes an adverse effect as far as cellular phenotype is concerned. Extrapolating the responses to organs and whole animals represents a challenge still to be addressed. There are parallel efforts in the area of proteomics and metabolomics whereby changes in a broad range of cellular proteins can be assessed in response to endogenous or exogenous factors, potentially leading to the development of biomarkers of effect (Aebersold et al., 2005; Robertson, 2005; Griffin, 2006; McGregor and Souza, 2006). The biggest hurdle currently is the relative paucity of sequence data available for the world of proteins and their multiple posttranslational modifications. Certainly progress is rapid, and so methodologies akin to gene expression microarrays are likely to be close at hand. The move in the field of genetic toxicology is away from the “yes/no” approach to hazard identification and much more toward a mechanistic understanding of how a chemical or physical agent can produce adverse cellular and tissue responses. The move is clearly toward analysis at the whole genome level and away from single

gene responses. The challenges are apparent and the solutions are being identified.

CONCLUSIONS The field of genetic toxicology has had an overall life of about 70 years and has undergone several rebirths during this period. Genetic toxicology began as a basic research field with demonstrations that ionizing radiations and chemicals could induce mutations and chromosome alterations in plant, insect, and mammalian cells. The development of a broad range of short-term assays for genotoxicity served to identify many mutagens and address the relationship between mutagens and cancer-causing agents, or carcinogens. The inevitable failure of the assays to be completely predictive resulted in the identification of nongenotoxic carcinogens. In the 1980s, genetic toxicology began to move more toward gaining a better understanding of the mutagenic mechanisms underlying carcinogenicity and heritable effects. With this improved understanding, genetic toxicology studies began to turn away from hazard identification alone and move toward quantitative risk assessment. Major advances in our knowledge of mechanisms of cancer formation have been fueled by truly amazing progress in molecular biology. Genetic toxicology has begun to take advantage of the knowledge that cancer is a genetic disease with multiple steps, many of which require a mutation. The identification of chromosome alterations involved in tumor formation has been facilitated greatly by the use of FISH. The ability to distinguish between background and induced mutations can in some cases be achieved by mutation analysis at the level of DNA sequence. Key cellular processes related to mutagenesis have been identified, including multiple pathways of DNA repair, cell cycle controls, and the role of checkpoints in ensuring that the cell cycle does not proceed until the DNA and specific cellular structures are checked for fidelity. These observations have enhanced our knowledge of the importance of genotype in susceptibility to cancer. Recent developments in genetic toxicology have greatly improved our understanding of basic cellular processes and alterations that can affect the integrity of the genetic material and its functions. The ability to detect and analyze mutations in mammalian germ cells continues to improve and can contribute to a better appreciation for the long-term consequences of mutagenesis in human populations. Improvements in the qualitative assessment of mutation in somatic cells and germ cells have been paralleled by advances in the ability to assess genetic alterations quantitatively, especially in ways that enhance the cancer and genetic risk assessment process (Preston, 2005).

ACKNOWLEDGMENTS We would like to recognize the outstanding secretarial support provided by Carolyn Fowler in the preparation of this chapter. The authors also thank Drs. David DeMarini, James Allen, and Les Recio for their valuable comments as part of the review of this chapter. This document has been reviewed in accordance with the U.S. Environmental Protection Agency policy and approved for publication but does not necessarily reflect EPA policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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NRC (National Research Council): Science and Judgment in Risk Assessment. Washington, DC: National Academy Press, 1994. Ohe T, White PA, DeMarini DM: Mutagenic characteristics of river waters flowing through large metropolitan areas in North America. Mutat Res 534:101–112, 2003. Okada N, Masumura K, Nohmi T, Yajima N: Efficient detection of deletions induced by a single treatment of mitomycin C in transgenic mouse gpt delta using the Spi(-) selection. Environ Mol Mutagen 34:106–111, 1999. Osgood CJ, Cyr K: Induction by nitriles of sex chromosome aneuploidy: Tests of mechanism. Mutat Res 403:149–157, 1998. Pack S, Vortmeyer AO, Pak E, et al.: Detection of gene deletion in single metastatic tumor cells in lymph node tissue by fluorescent in-situ hybridization. Lancet 350:264–265, 1997. Pack SD, Weil RJ, Vortmeyer AO, et al.: Individual adult human neurons display aneuploidy: Detection by fluorescence in situ hybridization and single neuron PCR. Cell Cycle 4:1758–1760, 2005. Painter RB: A replication model for sister-chromatid exchange. Mutat Res 70:337–341, 1980. Parry JM: An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential chemical aneugens. Mutat Res 287:23–28, 1993. Parry JM: Detecting and predicting the activity of rodent carcinogens. Mutagenesis 9:3–5, 1994. Parry JM: Detecting chemical aneugens: A commentary to ‘Aneuploidy: A report of an ECETOC task force.’ Mutat Res 410:117–120, 1998. Phillips DH, Farmer PB, Beland FA, et al.: Methods of DNA adduct determination and their application to testing compounds for genotoxicity. Environ Mol Mutagen 35:222–233, 2000. Potter CJ, Turenchalk GS, Xu T: Drosophila in cancer research. Trends Genet 16:33–39, 2000. Prakash S, Johnson RE, Prakash L: Eukaryotic translesion synthesis DNA polymerases: Specificity of structure and function. Annu Rev Biochem 74:317–353, 2005. Preston RJ: Mechanisms of induction of chromosomal alterations and sister chromatid exchanges, in Li AP, Heflich RF (eds.): Genetic Toxicology: A Treatise. Boca Raton, FL: CRC Press, 1991, pp. 41–66. Preston RJ: A consideration of the mechanisms of induction of mutations in mammalian cells by low doses and dose rates of ionizing radiation. Adv Radiat Biol 16:125–135, 1992. Preston RJ: Aneuploidy in germ cells: Disruption of chromosome mover components. Environ Mol Mutagen 28:176–181, 1996. Preston RJ: Chromosomal changes, in McGregor DB, Rice JM, Venitt S (eds.): The Use of Short- and Medium-Term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation. IARC Sci Pub 146. Lyon, France: IARC, 1999, pp. 395–408. Preston RJ: Cytogenetic effects of ethylene oxide, with an emphasis on population monitoring. Crit Rev Toxicol 29:263–282, 1999. Preston RJ: Mechanistic data and cancer risk assessment: The need for quantitative molecular endpoints. Environ Mol Mutagen 45:214–221, 2005. Preston RJ, Au W, Bender MA, et al.: Mammalian in vivo and in vitro cytogenetic assays: A report of the U.S. EPA’s Gene-Tox Program. Mutat Res 87:143–188, 1981. Preston RJ, Fennell TR, Leber AP, et al.: Reconsideration of the genetic risk assessment for ethylene oxide exposures. Environ Mol Mutagen 26:189–202, 1995. Preston RJ, Williams GM: DNA-reactive carcinogens: Mode of action and human cancer hazard. Crit Rev Toxicol 35:673–683, 2005. Prival MJ, Cebula TA: Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium. Genetics 132:303–310, 1992. Quillardet P, Hofnung M: The SOS chromotest: A review. Mutat Res 297:235–279, 1993. Rabbitts TH: Chromosomal translocations in human cancer. Nature 372: 143–149, 1994.

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Ramsey MJ, Moore DH, Briner JF, et al.: The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. Mutat Res 338:95–106, 1995. Rattray AJ, Strathern JN: Error-prone DNA polymerases: When making a mistake is the only way to get ahead. Annu Rev Genet 37:31–66, 2003. Reardon JT, Sancar A: Nucleotide excision repair. Prog Nuclei Acid Res Mol Biol 79:183–235, 2005. Rhomberg L, Dellarco VL, Siegel-Scott C, et al.: Quantitative estimation of the genetic risk associated with the induction of heritable translocations at low-dose exposure: Ethylene oxide as an example. Environ Mol Mutagen 16:104–125, 1990. Richard AM: Structure-based methods for predicting mutagenicity and carcinogenicity: Are we there yet? Mutat Res 400:493–507, 1998. Ripley LS: Mechanisms of gene mutations, in Li AP, Heflich RH (eds.): Genetic Toxicology: A Treatise. Boca Raton, FL: CRC Press, 1991, pp. 13–40. Robertson DG: Metabonomics in toxicology: A review. Toxicol Sci 85:809– 822, 2005. Royds JA, Iacopetta B: p53 and disease: When the guardian angel fails. Cell Death Differ 13:1017–1026, 2006. Rupa DS, Schuler M, Eastmond DA: Detection of hyperdiploidy and breakage affecting the 1cen-1q12 region of cultured interphase human lymphocytes treated with various genotoxic agents. Environ Mol Mutagen 29:161–167, 1997. Russell LB: Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse. Genetica 122: 25–36, 2004. Russell LB, Russell WL: Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: A review. Mutat Res 296:107–127, 1992. Russell LB, Selby PB, Von Halle E, et al.: The mouse specific-locus test with agents other than radiations: Interpretation of data and recommendations for future work. Mutat Res 86:329–354, 1981. Russell LB, Shelby MD: Tests for heritable genetic damage and for evidence of gonadal exposure in mammals. Mutat Res 154:69–84, 1985. Russell WL: X-ray-induced mutations in mice. Cold Spring Harb Symp Quant Biol 16:327–336, 1951. Russo A: In vivo cytogenetics: Mammalian germ cells. Mutat Res 455:167189, 2000. Sankaranarayanan K, Chakraborty R, Boerwinkle EA: Ionizing radiation and genetic risks. VI. Chronic multifactorial diseases: A review of epidemiological and genetical aspects of coronary heart disease, essential hypertension and diabetes mellitus. Mutat Res 436:21–57, 1999. Sankaranarayanan K: Ionizing radiation and genetic risks: IX. Estimates of the frequencies of mendelian diseases and spontaneous mutation rates in human populations: A 1998 perspective. Mutat Res 411:129–178, 1998. Sawada M, Kamataki T: Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays. Mutat Res 411:19–43, 1998. Sax K: Induction by X-rays of chromosome aberrations in Tradescantia microspores. Genetics 23:494–516, 1938. Sax K: The time factor in X-ray production of chromosome aberrations. Proc Natl Acad Sci U S A 25:225–233, 1939. Sax K, Luippold H: The effects of fractional x-ray dosage on the frequency of chromosome aberrations. Heredity 6:127–131, 1952. Scott D, Galloway SM, Marshall RR, et al.: Genotoxicity under extreme culture conditions. Mutat Res 257:147–204, 1991. Seeberg E, Eide L, Bjoras M: The base excision repair pathway. Trends Biochem Sci 20:391–397, 1995. Seed J, Carney EW, Corley RA, et al.: Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol 35:674–672, 2005. Sengstag C: The role of mitotic recombination in carcinogenesis. Crit Rev Toxicol 24:323–353, 1994.

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Shelby MD: Human germ cell mutagens. Environ Mol Mutagen 23(Suppl 24):30–34, 1994. Shinohara A, Ogawa T: Homologous recombination and the roles of doublestrand breaks. Trends Biochem Sci 20:387–391, 1995. Singh NP: Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res 455:111–127, 2000. Smith MT, Zhang L, Wang Y, et al.: Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene. Cancer Res 58:2176–2181, 1998. Snyder RD, Pearl GS, Mandakas G, et al.: Assessment of the sensitivity of the computational programs DEREK, TOPKAT, and MCASE in the prediction of the genotoxicity of pharmaceutical molecules. Environ Mol Mutagen 43:143–158, 2004. Sobels FH: The parallelogram: An indirect approach for the assessment of genetic risks from chemical mutagens, in Bora KC, Douglas GR, Nestman ER (eds.): Progress in Mutation Research, vol. 3. 1982, pp. 323–327. Solinas-Toldo S, Lampel S, Stilgenbauer S, et al.: Matrix-based comparative genomic hybridization: Biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20:399–407, 1997. Sonoda E, Hochegger H, Saberi A, et al.: Differential usage of nonhomologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) Jun 23:[Epub ahead of print], 2006. Sotomayor RE, Sega GA: Unscheduled DNA synthesis assay in mammalian spermatogenic cells: An update. Environ Mol Mutagen 36:255–265, 2000. Southern EM: DNA chips: Analyzing sequence by hybridization to oligonucleotides on a large scale. Trends Genet 12:110–115, 1996. Speicher MR, Carter NP: The new cytogenetics: Blurring the boundaries with molecular biology. Nat Rev Genet 6:782–792, 2005. Stoler DL, Chen N, Basik M, et al.: The onset and extent of genomic instability in sporadic colorectal tumor progression. Proc Natl Acad Sci U S A 96:15121–15126, 1999. Styles JA, Penman MG: The mouse spot test: Evaluation of its performance in identifying chemical mutagens and carcinogens. Mutat Res 154:183– 204, 1985. Sung JS, Demple B: Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA. FEBS J 273:1620–1629, 2006. Swiger RR: Just how does the cII selection system work in MutaTM Mouse? Environ Mol Mutagen 37:290–296, 2001. Swiger RR, Cosentino L, Shima N, et al.: The cII locus in the Muta Mouse system. Environ Mol Mutagen 34:201–207, 1999. Tano K, Shiota S, Collier J, et al.: Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for 06 -alkylguanine. Proc Natl Acad Sci U S A 87:686–690, 1990. Tease C: Radiation- and chemically-induced chromosome aberrations in mouse oocytes: A comparison with effects in males. Mutat Res 296:135–142, 1992. Tennant RW, Ashby J: Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program. Mutat Res 257:209–227, 1991. Tennant RW, Margolin BH, Shelby MD, et al.: Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science 236:933–941, 1987. Thacker J: Radiation-induced mutation in mammalian cells at low doses and dose rates. Adv Radiat Biol 16:77–124, 1992. Tice RR, Agurell E, Anderson D, et al.: Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221, 2000. Tice RR, Hayashi M, MacGregor JT, et al.: Report from the working group on the in vivo mammalian bone marrow chromosomal aberration test. Mutat Res 312:305–312, 1994. Trask BJ, Allen S, Massa H, et al.: Studies of metaphase and interphase chromosomes using fluorescence in situ hybridizaiton. Cold Spring Harb Symp Quant Biol 58:767–775, 1993.

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CHAPTER 10

DEVELOPMENTAL TOXICOLOGY John M. Rogers and Robert J. Kavlock MATERNAL FACTORS AFFECTING DEVELOPMENT

HISTORY

Genetics Disease Nutrition Stress Placental Toxicity Maternal Toxicity

SCOPE OF PROBLEM: THE HUMAN EXPERIENCE Thalidomide Diethylstilbestrol Ethanol Tobacco Smoke Cocaine Retinoids Valproic Acid Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Antagonists

DEVELOPMENTAL TOXICITY OF ENDOCRINE-DISRUPTING CHEMICALS Laboratory Animal Evidence Human Evidence Impact on Screening and Testing Programs

PRINCIPLES OF DEVELOPMENTAL TOXICOLOGY

MODERN SAFETY ASSESSMENT

Critical Periods of Susceptibility and Endpoints of Toxicity Dose–Response Patterns and the Threshold Concept

Regulatory Guidelines for In Vivo Testing Multigeneration Tests Children’s Health and the Food Quality Protection Act Alternative Testing Strategies Epidemiology Concordance of Data Elements of Risk Assessment Contemporary Approaches The Benchmark-Dose Approach Biologically Based Dose–Response Modeling

MECHANISMS AND PATHOGENESIS OF DEVELOPMENTAL TOXICITY Advances in the Molecular Basis of Dysmorphogenesis PHARMACOKINETICS AND METABOLISM IN PREGNANCY RELATIONSHIPS BETWEEN MATERNAL AND DEVELOPMENTAL TOXICITY

PATHWAYS TO THE FUTURE

HISTORY

Hippocrates and Aristotle thought that abnormal development could originate in physical causes such as uterine trauma or pressure, but Aristotle also shared a widespread belief that maternal impressions and emotions could influence the development of the child. He advised pregnant women to gaze at beautiful statuary to increase their child’s beauty. Though this theory may sound fanciful, it is present in diverse cultures throughout recorded history. Indeed, we now know that maternal stress can be deleterious to the developing conceptus (Chernoff et al., 1989). Another belief, the hybrid theory, held that interbreeding between humans and animals was a cause of congenital malformations (Ballantyne, 1904). Again, such hybrid creatures abound in mythology, including centaurs, minotaurs, and satyrs. Into the seventeenth century, cohabitation of humans with demons and witches was blamed for the production of birth defects. Birth defects were also viewed by some to represent God’s retribution on the parents of the malformed infant and on society. In 1649, the French surgeon Ambrois Par´e expounded the theory of Aristotle and Hippocrates by writing that birth defects could result from narrowness of the uterus, faulty posture of the pregnant woman, or physical trauma, such as a fall. Amputations were thought to result from amniotic bands, adhesions, or twisting of

Developmental toxicology encompasses the study of pharmacokinetics, mechanisms, pathogenesis, and outcome following exposure to agents or conditions potentially leading to abnormal development. Manifestations of developmental toxicity include structural malformations, growth retardation, functional impairment, and/or death of the organism. Developmental toxicology so defined is a relatively new science, but teratology, or the study of structural birth defects, as a descriptive science precedes written language. A marble sculpture from southern Turkey, dating to 6500 b.c., depicts conjoined twins (Warkany, 1983), and Egyptian wall paintings of human conditions such as cleft palate and achondroplasia date to as early as 5000 years ago. It is believed that mythological figures such as the Cyclops and Sirens took their origin in the birth of malformed infants (Thompson, 1930; Warkany, 1977). The Babylonians, Greeks, and Romans believed that abnormal infants were reflections of celestial events and as such were considered to be portents of the future. Indeed, the Latin word monstrum, from monstrare (to show) or monere (to warn), is derived from this perceived ability of malformed infants to foretell the future. In turn, derivation of the word teratology is from the Greek word for monster, teras. 415

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the umbilical cord. This conjecture has proven to be accurate. With the blossoming of the biological sciences in the sixteenth and seventeenth centuries, theories of the causation of birth defects with bases in scientific fact began to emerge. In 1651, William Harvey put forth the theory of developmental arrest, which stated that malformations resulted from incomplete development of an organ or structure. An example given by Harvey was harelip in humans, a congenital malformation that represents a normal early developmental stage. Much later, the theory of developmental arrest was solidified by the experiments of Stockard (1921) using eggs of the minnow, Fundulus heteroclitus. By manipulating the chemical constituents and temperature of growth media, he produced malformations in the embryos, the nature of which depended on the stage of the insult. He concluded that developmental arrest explained all malformations except those of hereditary origin (Barrow, 1971). With the advent of the germplasm theory elucidated by Weissmann in the 1880s and the rediscovery of Mendel’s laws in 1900, genetics as the basis for some birth defects was accepted. In 1894, Bateson published his treatise on the study of variations in animals as a tool for understanding evolution, inferring that inheritance of such variations could be a basis for speciation (Bateson, 1894). His study contains detailed descriptions and illustrations of such human birth defects as polydactyly and syndactyly, supernumerary cervical and thoracic ribs, duplicated appendages, and horseshoe (fused) kidneys. Bateson coined the term homeosis to denote morphologic alterations in which one structure has taken on the likeness of another. Studies of such alterations in mutants of the fruit fly Drosophila melanogaster and, more recently, the mouse have served as the basis for much of the recent knowledge of the genetic control of development. Homeobox genes are found throughout the animal and plant kingdoms and direct embryonic pattern formation (Graham et al., 1989; Deschamps and van Nes, 2005). Acceptance of a genetic basis of birth defects was furthered with studies of human inborn errors of metabolism in the first decade of the twentieth century. Modern experimental teratology began in the early nineteenth century with the work of Etienne Geoffrey Saint-Hilaire. SaintHilaire produced malformed chick embryos by subjecting eggs to various environmental conditions including physical trauma (jarring, inversion, pricking) and toxic exposures. In the latter part of the nineteenth century, Camille Dareste experimented extensively with chick embryos, producing a wide variety of malformations by administering noxious stimuli, physical trauma, or heat shock at various times after fertilization. He found that timing was more important than the nature of the insult in determining the type of malformation produced. Among the malformations described and beautifully illustrated by Dareste (1877, 1891) were the neural tube defects anencephaly and spina bifida, cyclopia, heart defects, situs inversus, and conjoined twins. Many of the great embryologists of the nineteenth and twentieth centuries, including Loeb, Morgan, Driesch, Wilson, Spemann, and Hertwig performed teratological manipulations using various physical and chemical probes to deduce principles of normal development. In the early twentieth century, a variety of environmental conditions (temperature, microbial toxins, drugs) were found to perturb development in avian, reptilian, fish, and amphibian species. In contrast, mammalian embryos were thought to be resistant to induction of malformations, protected from adverse environmental conditions by the maternal system. The first reports of induced birth defects in mammalian species were published in the 1930s and were the result of experimental maternal nutrient deficiencies. Hale (1935)

produced malformations including anophthalmia and cleft palate in offspring of sows fed a diet deficient in vitamin A. Beginning in 1940, Josef Warkany and co-workers began a series of experiments in which they demonstrated that maternal dietary deficiencies and other environmental factors could affect intrauterine development in rats (Warkany and Nelson, 1940; Warkany, 1945; Warkany and Schraffenberger, 1944; Wilson et al., 1953). These experiments were followed by many other studies in which chemical and physical agents, e.g., nitrogen mustard, trypan blue, hormones, antimetabolites, alkylating agents, hypoxia, and x-rays, to name a few, were clearly shown to cause malformations in mammals (Warkany, 1965). The first recognized human epidemic of malformations induced by an environmental agent was reported by Gregg (1941), who linked an epidemic of rubella virus infection in Australia to an elevation in the incidence of eye, heart, and ear defects as well as to mental retardation. Heart and eye defects predominated with maternal infection in the first or second months of pregnancy, whereas hearing and speech defects and mental retardation were most commonly associated with infection in the third month. Later, the risk of congenital anomalies associated with rubella infection in the first 4 weeks of pregnancy was estimated to be 61%; in weeks 5–8, 26%; and in weeks 9–12, 8% (Sever, 1967). It has been estimated that in the United States alone approximately 20,000 children were impaired as a consequence of prenatal rubella infections (Cooper and Krugman, 1966). While maternal rubella is now rare in developing countries due to vaccination programs, there are still rubella epidemics in developing countries (De Santis et al., 2006). Although embryos of mammals, including humans, were found to be susceptible to common external influences such as nutritional deficiencies and intrauterine infections, the impact of these findings was not great at the time (Wilson, 1973). That changed, however, in 1961, when the association between thalidomide ingestion by pregnant women and the birth of severely malformed infants was established (see section “Scope of the Problem,” below).

SCOPE OF PROBLEM: THE HUMAN EXPERIENCE Successful pregnancy outcome in the general population occurs at a surprisingly low frequency. Estimates of adverse outcomes include postimplantation pregnancy loss, 31%; major birth defects, 2–3% at birth and increasing to 6–7% at 1 year as more manifestations are diagnosed; minor birth defects, 14%; low birth weight, 7%; infant mortality (prior to 1 year of age), 1.4%; and abnormal neurological function, 16–17% (Schardein, 2000). Thus, less than half of all human conceptions result in the birth of a completely normal, healthy infant. Reasons for the adverse outcomes are largely unknown. Brent and Beckman (1990) attributed 15–25% of human birth defects to genetic causes, 4% to maternal conditions, 3% to maternal infections, 1–2% to deformations (e.g., mechanical problems such as umbilical cord limb amputations), methoxyacetic acid > 2-methoxyethanol) (Smialowicz et al., 1991a,b; Kim and

CHAPTER 12

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Smialowicz, 1997), suggesting a role for metabolism in the observed alterations in immunocompetence. Although there was no effect following 10-day oral exposures to 2-methoxyethanol (50– 200 mg/kg/d) (Smialowicz et al., 1991a), subchronic exposure for 21 days to 2000–6000 ppm (males) or 1600–4800 ppm (females) did produce an enhanced NK cell response (Exon et al., 1991) in addition to suppression of the PFC response and a decrease in IFN-γ production. In that study, it was also determined that 2-methoxyethanol produced greater immunotoxic effects than 2butoxyethanol. 2-Butoxyethanol was observed to enhance NK cell activity, but only at the low doses.

Nitrosamines The nitrosamine family comprises the nitrosamines, nitrosamides, and C-nitroso compounds. Exposure to nitrosamines, especially N -nitrosodimethylamine (dimethylnitrosamine, the most prevalent nitrosamine) comes primarily through industrial and dietary means, and minimally through environmental exposure. N-nitrosodimethylamine is used commonly as an industrial solvent in the production of dimethylhydrazine. It is currently used as an antioxidant, as an additive for lubricants and gasolines, and as a softener of copolymers. The toxicity and immunotoxicity of N -nitrosodimethylamine have been extensively reviewed (Myers and Schook, 1996). Single or repeated exposure to N nitrosodimethylamine inhibits T-dependent humoral immune responses (IgM and IgG), but not T-independent responses. Other symmetrical nitrosamines, such as diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine, demonstrated similar effects on humoral immunity but were not as potent as N nitrosodimethylamine (Kaminski et al., 1989b). In fact, as the length of the aliphatic chain increased, the dose required to suppress the anti-sRBC PFC response by 50% (ED50 ) also increased. In contrast, nonsymmetrical nitrosamines suppressed humoral immunity at comparable concentrations. Overall, the rank order of ED50 values paralleled their LD50 values. T-cell-mediated lymphoproliferative responses (mitogens or MLR) and DTH response are also suppressed following N -nitrosodimethylamine exposure. In vivo exposure to N -nitrosodimethylamine followed by challenge with several pathogens did not produce a pattern of effects that was consistent (decreased resistance to Streptococcus zooepidemicus and influenza, no effects on resistance to herpes simplex types 1 or 2 or Trichinella spiralis, and increased resistance to L . monocytogenes). In contrast, antitumor activity in N nitrosodimethylamine-exposed animals was consistently enhanced. N -Nitrosodimethylamine-exposed animals also have altered development of hematopoietic cells (increased macrophage precursors). Together these data suggest the macrophage (or its developmental precursors) as a primary target. Mechanistic studies have demonstrated that N -nitrosodimethylamine alterations in CMI are associated with enhanced macrophage activity, increased myelopoietic activity, and alterations in TNF-α transcriptional activity. It has been postulated that N -nitrosodimethylamine may cause the enhanced production of GM-CSF, which can have autocrine (enhanced tumoricidal and bactericidal activity) and paracrine (induced secretion of T-cell-suppressing cytokines by macrophages) activities. Mechanistic studies have also indicated a critical role for metabolism in the immune suppression by N -nitrosodimethylamine (Johnson et al., 1987b; Kim et al., 1988; Haggerty and Holsapple, 1990). It is known that N -nitrosodimethylamine is metabolized by the liver cytochrome P-450 system to a strong alkylating agent, and studies have shown that there is a relationship between

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N -nitrosodimethylamine-induced immune suppression, and the anticipated hepatotoxicity. Interestingly, a molecular dissection of N -nitrosodimethylamine-induced hepatotoxicity by mRNA differential display demonstrated an increase in transcripts for the complement protein C3 and serum amyloid A (Bhattacharjee et al., 1998). Previous work by Kaminski and Holsapple (1987) demonstrated the potential immune suppression associated with an increase in serum amyloid A.

Mycotoxins The immunotoxicity of mycotoxins, structurally diverse secondary metabolites of fungi that grow on feed, has been reviewed (IPCS, 1996; Bondy and Pestka, 2000). This class of chemicals comprises such toxins as aflatoxin, ochratoxin, and the tricothecenes, notably T-2 toxin and deoxynivalenol (vomitoxin). As a class, these toxins can produce cellular depletion in lymphoid organs, alterations in T- and B-lymphocyte function, suppression of antibody responses, suppression of NK cell activity, decreased DTH responses, and an apparent increase in susceptibility to infectious disease. T-2 toxin has also been implicated as a developmental immunotoxicant, targeting fetal lymphocyte progenitors leading to the thymic atrophy often observed with these mycotoxins (Holladay et al., 1993). For ochratoxin, at least, the dose, the route of administration, and the species appear to be critical factors in results obtained in immunotoxicity studies. Past studies with aflatoxin B1 suggest that CMI and phagocytic cell functions are affected as evidenced by decreased proliferative responses to PHA and suppression of DTH responses (Raisuddin et al., 1993). In addition, in vitro experiments demonstrated that aflatoxin B1 required metabolic bioactivation in order to produce suppression of antibody responses and mitogeninduced lymphoproliferation (Yang et al., 1986). Studies in laboratory animals have also shown increased risk to secondary infection after aflatoxin B1 treatment. The effects of aflatoxins on the human immune system have not been characterized but are of concern in light of the fact that in many parts of the world, such as in West Africa, exposure to aflatoxins is widespread as recent studies in Benin and Togo found that 99% of children possessed measurable aflatoxin–albumin adducts in blood (Gong et al., 2003). For the extensively studied tricothecenes, the mechanism of immune impairment is related in part to inhibition of protein synthesis. Interestingly, trichothecenes at high doses induce leukocyte apoptosis concomitantly with immune suppression (Pestka et al., 1994). Conversely, at low doses trichothecenes promote expression of a diverse array of cytokines including IL-1, IL-2, IL-5, and IL-6. In addition, tricothecenes activate mitogen-activated protein kinases in vivo and in vitro via a mechanism known as the ribotoxic stress response (Moon and Pestka, 2002; Chung et al., 2003; Zhou et al., 2003). Prolonged consumption of deoxynivalenol by mice was shown to induce elevation of IgA and IgA immune complex formation, and kidney mesangial IgA deposition (Pestka, 2003). It has been postulated that the enhancement in IgA production induced by deoxynivalenol may be associated with the increase in cytokine production described above. The tricothecenes are currently considered among the most potent small-molecule inhibitors of protein synthesis in eukaryotic cells. Adverse health effects have been associated with damp indoor environments following building envelope breech resulting from heavy rains and/or flooding, as occurred during Hurricanes Katrina and Rita in the Gulf Coast of the United States. The adverse health effects have been attributed, at least in part, to the presence of molds, most notably Stachybotrys chartarum, also known as black mold.

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S. chartarum produces the macrocyclic trichothecene toxin, satratoxin G, which like many of the trichothecenes is a potent inhibitor of protein synthesis. In a recent study, satratoxin G exposure of mice, 100 μg/kg for 5 consecutive days by intranasal instillation, induced apoptosis of olfactory sensory neurons, and neutrophilic rhinitis (Islam et al., 2006). Elevated mRNA levels for pro-inflammatory cytokines TNF-α, IL-6, and IL-1, and the chemokine, MIP-2, were detected in nasal airways and the adjacent olfactory bulb of the brain. By Day 7, marked atrophy of the olfactory nerve and glomerular layer of the olfactory bulb was detected. These finding suggest that neurotoxicity and inflammation within the nose may be potential adverse health effects associated with Stachybotrys exposure in indoor air.

Natural and Synthetic Hormones It is well established that a sexual dimorphism exists in the immune system. Females have higher levels of circulating immunoglobulins, a greater antibody response, and a higher incidence of autoimmune disease than do males. Males appear to be more susceptible to the development of sepsis and the mortality associated with soft tissue trauma and hemorrhagic shock. Specific natural sex hormones in this dichotomy have been implicated. Immune effects of androgens and estrogens appear to be very tightly controlled within the physiological range of concentrations, and profound changes in immune activity can result for very slight changes in concentrations of hormones. Estrogens Diethylstilbestrol is a synthetic nonsteroidal compound possessing estrogenic activity. Diethylstilbestrol was used in men to treat prostatic cancer and in women to prevent threatened abortions, as an estrogen replacement, and as a contraceptive drug. Extensive functional and host resistance studies on diethylstilbestrol (mg/kg/d range) have indicated that exposure to this chemical results in alterations in CMI and/or macrophage function and are believed to be mediated by the presence of the estrogen receptor on immune cells (Kalland, 1980; Luster et al., 1980, 1984; Holsapple et al., 1983). Targeted sites of action include the thymus (thymic depletion and alteration in T-cell maturation process), T cells (decreased MLR, DTH, and lymphoproliferative responses), and macrophage (enhanced phagocytic, antitumor, and suppressor function). Pre- and neonatal exposures (mg/kg/d dose range) have also demonstrated immunotoxic effects related to T-cell dysfunction. DTH and inflammatory responses associated with diethylstilbestrol exposure in adult mice have been shown to be reversible upon cessation of exposure (Luster et al., 1980; Holsapple et al., 1983). However, effects from in utero and neonatal exposures appear to have more lasting, possibly permanent effects on immune responses (Kalland et al., 1979; Luster et al., 1979; Ways et al., 1980). Exposure to 17β-estradiol in male rats (63 days of age) intraperitoneally for 15 days (1–50 μg/kg/d) did not alter spleen weight, spleen cellularity, or the humoral immune response to sRBC (Ladics et al., 1998b). As observed with other estrogenic chemicals, thymic weight was decreased following exposure. Serum androgens and luteinizing hormone and male accessory organ weights were depressed, while serum estradiol and prolactin were increased. Dietary exposure (2.5–50 ppm) of male and female rats for 90 days resulted in decreased spleen weights and alterations in hematologic elements suggestive of bone marrow effects. Body weights were also affected. No histological alterations were noted. Decreases in splenic T- and B-cell populations were observed at the higher concentrations. These data suggest the possibility that exposure to

17β-estradiol may have resulted in altered normal immune cell trafficking and distribution, the mechanism of which is not clear. This hypothesis is supported by recent data indicating that the observed anti-inflammatory effects of estrogens may be related to a combination of alterations in homing and the activation of inflammatory cells and their production of TNF-α and IFN-γ (Salem et al., 2000). Recent findings suggest that 17β-estradiol can drive the expansion of the CD4+ CD25+ regulatory T-cell compartment as evidenced by an increase in Foxp3 expression levels in these cells (Polanczyk et al., 2004). Foxp3 is a transcriptional repressor required for the development and function of T-regulatory cells and in combination with surface expression of CD4 and CD25, is currently the most definitive marker of T-regulatory cells. Bisphenol A, a monomer in polycarbonate plastics and a constituent of epoxy and polystyrene resins possessing weak binding affinity for the estrogen receptor, has been recently evaluated by a number of laboratories for its potential to affect various aspects of immune function. The majority of studies to date demonstrate that leukocytes cultured in the presence of very high concentrations (>1 μM) of bisphenol A exhibit a number of alterations, primarily in innate immune function responses, including suppression of lipopolysaccharide-induced nitric oxide production and TNFα secretion by macrophages (Kim and Jeong, 2003). The effects on nitric oxide production where shown to be correlated with a decrease in NF-κB DNA-binding activity, a transcription factor critically involved in the regulation of inducible nitric oxide synthase and TNF-α. In this study, suppression by bisphenol A of lipopolysaccharide-induced nitric oxide production was blocked by the estrogen receptor antagonist, ICI 182,780. Bisphenol A (10– 50 μM) has also been reported to enhance IL-4 production in a model of a secondary immune response (Lee et al., 2003). In vivo treatment of mice with bisphenol A (2.5 mg/kg) for 7 days produced a decrease in ex vivo concanavalin A-induced proliferation and IFNγ secretion, but had no effect on the number of CD4+ , CD8+ , and CD19+ cells in the spleen (Sawai et al., 2003). Presently, the putative effects of bisphenol A on immune function are poorly defined and based on the current literature it is unclear whether the majority of the immunomodulatory effects reported are mediated through an estrogen receptor-dependent mechanism. While it appears that estrogens can affect the maturation and function of the thymus and its components, it has recently been observed that estrogen receptor knockout mice have significantly smaller thymi than do their wild-type littermates, apparently due to the lack of the estrogen receptor-alpha (ERα) (Staples et al., 1999). In addition, it has been suggested that the effects of estrogens on the thymus appear to be mediated not only through ERαs but also through an ERα-independent pathway yet to be elucidated.

Androgens Oxymetholone is a synthetic androgen structurally related to testosterone and used in the past in the treatment of pituitary dwarfism and as an adjunctive therapy in osteoporosis. Its current use is limited to treatment of certain anemias. Oxymetholone was administered orally to male mice daily for 14 consecutive days (50– 300 mg/kg/d) (Karrow et al., 2000). In male mice, oxymetholone exposure resulted in a minimal decrease in CMI (MLR and CTL response); but did not alter the ability of the animals to resist infection in host resistance assays. In contrast, anabolic androgenic steroids have been shown to significantly inhibit the sRBC PFC response and to increase the production of pro-inflammatory cytokines from human PBLs.

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No comprehensive studies evaluating the effects of testosterone on immune parameters have been conducted. However, it is clear that testosterone is capable of contributing to the suppression of immune function; in particular, CMI responses and macrophage activity. There are numerous reports in the clinical literature that males are more susceptible than females to infection following soft tissue trauma and hemorrhagic shock (reviewed in Catania and Chaudry, 1999). Treatment of males with chemicals that block testosterone (e.g., flutamide) can prevent the trauma- and hemorrhage-induced depression of immunity. Similarly, treatment of females with dihydrotestosterone prior to trauma–hemorrhage results in depression of CMI, similar to that of males. Furthermore, gonadectomized mice of either sex have elevated immune responses to endotoxin, which can be attenuated in either sex by the administration of testosterone. The mechanisms in these cases, including influences of the neuroendocrine system, are not clear. Other investigators have reported that, like estrogenic agents, testosterone and other androgens are capable of influencing host defense by altering lymphocyte trafficking in the body and altering the ability of the macrophage to participate in immune responses.

Glucocorticoids The immunosuppressive actions of corticosteroids have been known for years. Following binding to a cytosolic receptor, these chemicals produce profound lymphoid cell depletion in rodent models. In non-human primates and humans, lymphopenia associated with decreased monocytes and eosinophils and increased PMNs are seen. Corticosteroids induce apoptosis in rodents, and T cells are particularly sensitive. In addition, these chemicals inhibit macrophage accessory cell function, the production of IL-1 from macrophages, and the subsequent synthesis of IL-2 by T cells. In general, corticosteroids suppress the generation of CTL responses, MLR, NK cell activity, and lymphoproliferation. Whereas it is clear that these drugs inhibit T-cell function, their effects on B cells are not completely clear. Corticosteroids inhibit humoral responses, but this appears to be due to effects on T cells, as antigen-specific antibody production by B cells to T-independent antigens does not appear to be affected by corticosteroid treatment. In spite of the wide therapeutic use of glucocorticoids, the mechanism of action by which glucocorticoids mediate their antiinflammatory/immunosuppressive activity is not well understood. Several mechanisms have been proposed all of which involve activation of the glucocorticoid receptor. Binding of glucocorticoids to the cytosolic glucocorticoid receptor induces the receptor to function as a ligand-activated transcription factor that undergoes homodimerization and DNA binding to glucocorticoid response elements (GRE) in the regulatory regions of glucocorticoid-responsive genes. Depending on the gene, GRE can either positively or negatively regulate transcription. For example, glucocorticoids induce annexin 1 (lipocortin 1), a calcium and phospholipid-binding protein, which acts to inhibit PLA2 (Goulding and Guyre, 1992; Taylor et al., 1997). Inhibition of PLA2 results in a decrease in arachidonate formation, the precursor in the biosynthesis of inflammatory prostaglandin and leukotriene. Similarly, glucocorticoids induce transcription of IκB, which is the endogenous inhibitor of the transcription factor, NF-κB (Auphan et al., 1995; Scheinman et al., 1995). As transcription of many key inflammatory cytokines is regulated positively by NF-κB, induction of IκB results in retention of NF-κB in the cytosol and thus suppression of inflammatory cytokine production. Ligand-activated glucocorticoid receptors have also been found to physically interact with other transcription factors including AP-1 (Schule et al.,

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1990) and NF-κBg (Ray and Prefontaine, 1994), to inhibit DNA binding and/or their transcriptional activity. Presently, it is believed that all the above mechanisms contribute to the anti-inflammatory and immunosuppressive properties of glucocorticoids.

Therapeutics Historically speaking, very few drugs used today as immunosuppressive drugs were actually developed for that purpose. In fact, if one looks closely enough, most therapeutic agents possess some degree of immunomodulatory activity at some dose (Descotes, 1986). The recent explosion of knowledge regarding the function and regulation of the immune system (at the cellular, biochemical, and molecular levels) has provided investigators with a relatively new avenue for specific drug development. The following discussion focuses on those drugs used primarily for modulating the immune system: the immunosuppressants (corticosteroids are described in the section “Natural and Synthetic Hormones”), AIDS therapeutics, “biologics” (i.e., monoclonal antibodies, recombinant cytokines, and interferons), and anti-inflammatory drugs. Immunosuppressive Drugs Several immunosuppressive drugs are efficacious simply due to their ability to inhibit cellular proliferation, since proliferation is required for lymphocyte clonal expansion and, subsequently, differentiation. Other drugs inhibit specific intracellular proteins that are critical in the activation of the immune response. Originally developed as an antineoplastic agent, cyclophosphamide (Cytoxan, CYP) is the prototypical member of a class of drugs known as alkylating agents. Upon entering the cell, the inactive drug is metabolically cleaved into phosphoramide mustard, a powerful DNA alkylating agent that leads to blockade of cell replication, and acrolein, a compound known to primarily bind to sulfhydral groups. Clinically, CYP has found use in reducing symptoms of autoimmune disease and in the pretreatment of bone marrow transplant recipients. Experimentally, this drug is often used as a positive immunosuppressive control in immunotoxicology studies because it can suppress both humoral immunity and CMI responses. There appears to be preferential inhibition of B-cell responses, possibly due to decreased production and surface expression of immunoglobulins. CMI activities that are suppressed include the DTH response, CTL, graft-versus-host disease, and the MLR. Adminsitration of low doses of CYP prior to antigenic stimulation can produce immune enhancement of cell-mediated and humoral immune responses, which has been attributed, in part, to an inhibition of suppressor T-cell activity (Limpens et al., 1990; Limpens and Scheper, 1991). The immune enhancing properties of CYP where demonstrated to be mediated by acrolein but not by phosphoramide mustard (Kawabata and White, 1988). Azathioprine, one of the antimetabolite drugs, is a purine analog that is more potent than the prototype, 6-mercaptopurine, as an inhibitor of cell replication. Immune suppression likely occurs because of the ability of the drug to inhibit purine biosynthesis. It has found widespread use in the inhibition of allograft rejection, although it is relatively ineffective in attenuating acute rejection reactions. It can also act as an anti-inflammatory drug and can reduce the number of PMNs and monocytes. Clinical use of the drug is limited by bone marrow suppression and leukopenia. Azathioprine inhibits humoral immunity, but secondary responses (IgG) appear more sensitive than primary responses (IgM). Several CMI activities are also reduced by azathioprine treatment, including DTH response,

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MLR, and graft-versus-host disease. Although T-cell functions are the primary targets for this drug, inhibition of NK function and macrophage activities has also been reported. Leflunomide, an isoxazole derivative, is another drug that suppresses cellular proliferation, which has been used in the treatment of rheumatic disease and transplantation (Xiao et al., 1994). Leflunomide inhibits de novo pathways of pyrimidine synthesis, thereby blocking progression from G1 to S of the cell cycle. Thus, direct inhibition of B-cell proliferation may account for the drug’s ability to inhibit both T-cell-dependent and T-cell-independent specific antibody production. Leflunomide can also directly inhibit T-cell proliferation induced by mitogens, antibody directed against CD3, or IL-2. Cyclosporin A (Sandimmune, CsA) is a cyclic undecapeptide isolated from fungal organisms found in the soil. Important to its use as an immunosuppressant is the relative lack of secondary toxicity (e.g., myelotoxicity) at therapeutic concentrations (Calne et al., 1981). However, hepatotoxicity and nephrotoxicity are limiting side effects. CsA acts preferentially on T cells by inhibiting the biochemical signaling pathway emanating from the TCR (reviewed in Ho et al., 1996). The result is inhibition of IL-2 gene transcription and subsequent inhibition of T-cell proliferation and clonal expansion of effector T cells. More specifically, CsA interacts with the intracellular molecule cyclophilin, an intracellular protein with peptidyl proline isomerase activity (although this enzymatic activity has nothing to do with the immunosuppressive effect of CsA). The CsA–cyclophilin complex inhibits the serine/threonine phosphatase activity of a third molecule, calcineurin. The function of calcineurin is to dephosphorylate the cytoplasmic form of the transcription factor, nuclear factor of activated T cells (NFAT), therefore facilitating the transport of NFAT into the nucleus, where it can couple with nuclear components and induce the transcription of the IL-2 gene. Inhibition of calcineurin phosphatase activity by the CsA–cyclophillin complex prevents nuclear translocation of NFAT and the resulting IL-2 gene transcription. FK506 is a cyclic macrolide which is structurally distinct from CsA, but which possesses a nearly identical mechanism of action (reviewed in Ho et al., 1996). Like CsA, FK506 binds intracellularly to proteins with peptidyl proline isomerase activity, the most abundant of which is FK506-binding protein-12 (FKBP12). The FK506– FKBP12 complex also binds to and inhibits calcineurin activity, thereby inhibiting IL-2 gene transcription. Clinically, FK506 inhibits T-cell proliferation, lacks myelotoxicity (although, like CsA, it does cause nephrotoxicity), and induces transplantation tolerance. In addition, the minimum effective dose appears to be approximately one-tenth than that of CsA. Rapamycin (RAP) is also a cyclic macrolide, which is structurally related to FK506. However, the mechanism by which it produces inhibition of proliferation is strikingly distinct. Unlike CsA and FK506, RAP does not inhibit TCR-dependent signaling events and IL-2 gene transcription. Rather, this compound inhibits IL-2stimulated T-cell proliferation by blocking cell-cycle progression from late G1 into S phase (Morice et al., 1993; Terada et al., 1993). Like FK506, RAP binds to the intracellular protein FKBP12. But this RAP–FKBP12 complex does not bind calcineurin. Rather, the RAP–FKBP12 complex binds to the target of rapamycin (TOR) (Sabers et al., 1995), inhibiting its function. Inhibition of TOR results in reduced cell growth, suppression of cell cycle progression and proliferation (reviewed in Fingar and Blenis, 2004). Unlike both CsA and FK506, RAP does not appear to be nephrotoxic. Due to its mechanisms of action, a significant advantage of RAP over CsA and

FK506 is that it is an effective immune suppressant even after T cells have been activated, due to the fact that it blocks signaling through the IL-2 receptor. Conversely, for CsA and FK506 to be effective, T cells must encounter the drug prior to activation, as once IL-2 transcription begins, neither therapeutic provides effective suppression of the already activated T cells and IL-2 production. AIDS Therapeutics Traditionally, antiviral therapies have not been extremely successful in their attempt to rid the host of viral infection. This may be due to the fact that these organisms target the DNA of the host. Thus, eradication of the infection means killing infected cells, which for HIV are primarily CD4+ T cells. Numerous strategies have been developed to combat HIV, including targeting viral reverse transcriptase, viral protease, stimulating immune responses, and targeting the virus-T-cell interaction proteins. The multidrug therapy used currently is referred to as highly active antiretroviral therapy (HAART). However, eradication of this virus, and subsequently AIDS, remains a challenge because the very nature of the infection has significant immunosuppressive consequences. In addition, some of the current therapies also exhibit immunosuppressive actions. One such antiviral drug is zidovudine. Zidovudine (3 -azido-3 -deoxythymidine) is a pyrimidine analog that inhibits viral reverse transcriptase. It was the first drug shown to have any clinical efficacy in the treatment of HIV-1 infection. Unfortunately, its use is limited by myelotoxicity (macrocytic anemia and granulocytopenia) (Luster et al., 1989). Early studies confirmed that the primary action of zidovudine is on innate immunity, although changes in both humoral immunity and CMI have also been observed (reviewed in Feola et al., 2006). In addition, it was shown that oral administration of high doses of zidovudine caused thymic involution and decreased responsiveness of T cells to the HIV protein, gp120 (McKallip et al., 1995). Clinically, zidovudine increases the number of circulating CD4+ cells and can transiently stimulate cell-mediated immune responses (lymphoproliferation, NK cell activity, and IFN-γ production). A final consideration for the immunotoxicity associated with AIDS therapeutics like zidovudine is that they are rarely administered alone and thus, drug interactions likely contribute to various immune effects. Biologics Biologics refers to those therapies that are derived in some manner from living organisms and include monoclonal antibodies, recombinant proteins, and adoptive cell therapies. By its very nature, the immune system is often both the intended therapeutic target and unintended toxicological target of various biologics. Overall, manifestations of toxicity may include exaggerated pharmacology, effects due to biochemical cross-talk, and disruptions in immune regulation by cytokine networks. Monoclonal antibodies can bind normal as well as targeted tissues, and any foreign protein may elicit the production of neutralizing antibodies against the therapeutic protein (i.e., the therapeutic protein may be immunogenic). While certainly many biologics are being utilized safely, the immunotoxicological aspects of an example of a monoclonal antibody (anti-CD3) and a recombinant protein (IFN-α) will be discussed. Monoclonal antibodies have been designed in general to suppress immune function, and include antibodies directed against certain molecules that are critical for inducing or sustaining an immune response (CD3, IL-2 receptor, CD52, or TNF-α), or directed to stimulate certain molecules critical for downregulating activated T cells and in turn the immune response (CTLA-4). Monoclonal antibodies directed against CD3 (OKT3), part of the TCR complex, have been

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used for acute transplant rejection. As all T cells express CD3 as part of the TCR complex, anti-CD3 blockage potently suppresses immune function. Acutely, an adverse effect of OKT3 is “cytokine release syndrome,” in which soon after initial administration, flulike symptoms, pulmonary edema and hematological disorders have been reported (reviewed in Sgro, 1995). Although rare, anaphylactic reactions to the antibody can occur, as it is a murine monoclonal antibody. The majority of recombinant proteins have been used as immunostimulants, including IFN-α, IFN-γ , GM-CSF, erythropoietin, IL-2, and IL-12. IFN-α, which is used as an antiviral drug, is used to treat Hepatitis C and other chronic viral illnesses. The mechanism of the antiviral action of IFN-α involves, in part, direct suppression of viral replication, enhancement of expression of MHC class I on virally infected cells, thus increasing the likelihood of recognition by virus-specific T cells, and activation of NK cells. Administration of IFN-α has been associated with autoimmune diseases, including autoimmune hypothyroidism and lupus (Vial and Descotes, 1995) and hematologic disorders stemming from bone marrow suppression. Anti-inflammatory Agents Anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs), which suppress the production of pro-inflammtory soluble factors, such as prostaglandins and thromboxanes. This mechanism of action has also been demonstrated for other anti-inflammatory drugs, such as aspirin, acetaminophen, and cyclooxygenase (COX) inhibitors, such as rofecoxib or celecoxib. This section also includes a brief discussion of anti-inflammatory biologics, such as anti-TNF-α monoclonal antibodies (adalimumab or infliximab), or recombinant TNF-α receptors (entanercept). Aspirin irreversibly modifies COX enzymes 1 and 2, preventing the formation of prostaglandins and thromboxanes. Aspirin is especially effective as an antiplatelet because platelets possess little biosynthesizing capacity and therefore, aspirin will inhibit COX for the life of the platelet (8–11 days). Acetaminophen and other COX inhibitors are reversible inhibitors of COX enzymes. The COX-2 enzyme, in particular, is induced in response to inflammatory cytokines and mediators and therefore, represents an attractive target to combat inflammatory diseases. Although COX-2 inhibitors are currently available (rofecoxib or celecoxib), their use has been limited due to increased risk of cardiovascular effects in some patients (reviewed in Grosser et al., 2006). Etanercept, adalimumab, and infliximab are a fairly recently developed class of anti-inflammatory drugs that are also biologics. All three drugs target TNF-α, a critical pro-inflammatory cytokine (see “Inflammation”). Etanercept is a recombinant TNF-α receptor, which binds circulating TNF-α levels and prevents TNF-α binding to the endogenous TNF-α receptor. Adalimumab and infliximab, however, are monoclonal antibodies directed against TNF-α, which neutralizes circulating TNF-α and prevents interaction of TNF-α with its receptor. All three drugs suppress TNF-α signaling to prevent (1) induction of other pro-inflammatory cytokines such as IL-1 and IL-6; (2) leukocyte migration to inflammation sites; (3) neutrophil activation; (4) fibroblast activation; and (5) induction of other acute phase proteins.

Drugs of Abuse Drug abuse is a social issue with far-reaching effects on the abuser as well as on friends and family. Whereas drug paraphernalia has

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been directly associated with the spread of HIV, in recent years the actual abuse of some drugs has been linked to the progression, and possibly the onset, of AIDS, suggesting that drugs of abuse may exhibit immunosuppressive actions. Indeed, many studies do suggest that drugs of abuse alter immune competence. Several classes of drugs will be discussed, including cannabinoids, opioids, cocaine, methamphetamine, and ethanol. Reports regarding the immune system effects of many of these drugs, particularly opioids, are often contradictory, so it should be noted that the mechanisms by which drugs of abuse suppresses immune function might depend on the development of tolerance or addiction to the drugs, or the immune, withdrawal, and pain status of the individual as endogenous molecules (i.e., endorphins or endocannabinoids) play critical roles in all of these physiological processes.

Cannabinoids Much attention has been focused on the immunomodulatory effects of the cannabinoids, which can be defined as plant derived (i.e., from the marijuana plant), synthetic, or endogenous. Therapeutically, the primary psychoactive congener of marijuana, 9 -tetrahydrocannabinol is approved for use as an antiemetic in patients undergoing cancer chemotherapy and as an appetite stimulant for cachexia associated with advanced AIDS disease. The mechanism by which 9 -tetrahydrocannabinol produces the high in the CNS is mediated through a G protein-coupled cannabinoid receptor, CB1 (Varvel et al., 2005). Peripheral tissues also express CB1, in addition to a second cannabinoid receptor, CB2. Although both receptors are expressed on immune system cells and are coupled to suppression of adenylate cyclase activity (Schatz et al., 1997), it is not entirely clear the extent to which the receptors and/or suppression of adenylate cyclase activity contributes to immune system effects by cannabinoids. Early studies showed that exposure to 9 -tetrahydrocannabinol decreases host resistance to bacterial and viral pathogens (reviewed in Kaminski, 1994). For example, mice treated with 9 tetrahydrocannabinol exhibited higher mortalities to the opportunistic brain infection Acanthamoeba (Cabral and Marciano-Cabral, 2004). In addition, 9 -tetrahydrocannabinol treatment of mice prior to primary infection with Legionella pneumophila pneumophilia increased mortality following secondary infection (Newton et al., 1994). Recent evidence demonstrates that the mechanism of decreased host resistance to L. pneumophila might involve suppression of dendritic cell function (Lu et al., 2006). Cannabinoids alter both humoral and cell-mediated immune responses as evidenced by suppression of the T-cell-dependent PFC response both in vivo and in vitro (Schatz et al., 1993) and direct suppression of T-cell function (Condie et al., 1996). With respect to the mechanism of T-cell suppression, many plant-derived compounds suppress IL-2 at the transcriptional level which is due, in part, to suppression of transcription factor activation (AP-1, NFAT, and NF-κB) and ERK MAPK activity (Condie et al., 1996; Herring et al., 1998; Faubert and Kaminski, 2000). Although both cannabinoid receptors are expressed on T cells (Galiegue et al., 1995), many of the direct T-cell effects of cannabinoids have been demonstrated to occur independently of either cannabinoid receptor (Kaplan et al., 2003). Interestingly, although endogenous cannabinoids, such as anandamide and 2-arachidonoyl-glycerol, also suppress IL-2, their mechanism has been determined to involve a COX2 metabolite that activates the peroxisome proliferator-activated receptor-γ (Rockwell and Kaminski, 2004; Rockwell et al., 2006).

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Cannabinoid compounds have also been demonstrated to suppress innate immunity, particularly suppression of macrophage function. 9 -Tetrahydrocannabinol exposure impaired lysosomal or cytochrome c processing in macrophages (McCoy et al., 1995; Matveyeva et al., 2000). Interestingly, suppression of macrophage function by 9 -tetrahydrocannabinol seems to be mediated via the CB2 receptor (Buckley et al., 2000; Chuchawankul et al., 2004). Together these studies demonstrate that cannabinoid compounds alter immune function, and the mechanisms involve both cannabinoid receptor-dependent and receptor-independent actions.

Opioids Similar to cannabinoids, opioids refer to plant-derived, synthetic, or endogenous (endorphins) compounds that bind opioid receptors. Although technically “opioid” refers to drugs derived from the poppy plant, and “opiate” refers to agonists and antagonists with morphine-like activity (including plant-derived and synthetic compounds), they are often used interchangeably. It is well established that opioids suppress immune responses (reviewed in Dinda et al., 2005). It is not established, however, whether this action is a direct effect of the drug on immune cells or an indirect effect, and whether the effects are mediated through an opioid receptor (Wei et al., 2003; Sharp, 2006). There are at least three opioid receptors, μ, κ (and subtypes), and δ, which are all G protein coupled. Although many reports have identified opioid receptors on immune cells, controversy still exists as to which receptors and/or subtypes are present on the various immune cell populations and to what extent these receptors mediate immune responses to opioids. Nevertheless, a few reports will be discussed that describe some of the effects and mechanisms of opioid-induced immune modulation, and the reader is referred to several recent reviews for further information (Dinda et al., 2005; Page, 2005; Sharp, 2006). Early studies evaluating the immune competence of heroin addicts revealed a decrease in total T cells and E-rosette capability, which was reversed with the general opioid receptor antagonist, naloxone, suggesting a role for an opioid receptor in mediating immune suppression (McDonough et al., 1980). Later studies demonstrated that although morphine suppressed several immune parameters, there was no dose–response, suggesting the effects were not receptor mediated, but were the result of increased circulating corticosteroids (which were significantly elevated in those animals; LeVier et al., 1994). This conclusion is supported by the findings of other investigators as well (Pruett et al., 1992b). Several investigators have reported decreased host resistance to viral and bacterial infections in opioid-treated animals or heroin addicts. In one recent study, morphine treatment of mice infected with S. pneumoniae demonstrated increased bacterial burden in the lungs and increased mortality. The mechanism by which the immune response was compromised involved, in part, suppression of NF-κB gene transcription, which likely contributes to decreased expression of inflammatory mediators, such as chemokines, reducing recruitment of neutrophils to the infection site (Wang et al., 2005). There is also evidence that opioid use increases susceptibility to HIV infection. Although morphine and/or heroin use is associated with risk of HIV infection through shared needles, opioid use may contribute to progression of AIDS through immune suppression. Specifically, there is evidence that morphine treatment increases CCR5 expression, which is a primary receptor for HIV entry into macrophages (Guo et al., 2002). In addition, chronic morphine treatment shifts the T-cell balance toward Th2 (Roy et al., 2004). Further evidence for

compromised immunity toward HIV is the observation that morphine inhibited the anti-HIV activity in CD8+ cells in an opioid receptor-dependent manner (Wang et al., 2005). Opioids also modulate innate immunity. Earlier studies demonstrated that chronic treatment of mice with morphine suppressed bone marrow cell stimulation in response to macrophage colonystimulating factor (Roy et al., 1991). Consistent with the observations that morphine and/or heroin use contributes to the progression of AIDS, Kupffer cells infected with HIV maintained in vitro in the presence of morphine resulted in a higher number of viral particles relative to untreated HIV-infected cells (Schweitzer et al., 1991). More recent studies demonstrate either suppression (Sacerdote, 2003) or enhancement (Peng et al., 2000) of cytokine production from macrophages. The differences might be due to agonist used, in vitro versus in vivo administration, and dosing regimen (i.e., whether tolerance was induced or not). Overall, it is clear that opioids suppress immune function and that the mechanism by which this occurs is complex and likely involves the CNS, the autonomic nervous system, the hypothalamic–pituitary– adrenal axis, and one or more opioid receptors (Alonzo and Bayer, 2002).

Cocaine Cocaine is a potent local anesthetic and CNS stimulant. This drug and its derivatives have been shown to alter several measures of immunocompetence, including humoral and cell-mediated immune responses and host resistance (Watson et al., 1983; Ou et al., 1989; Starec et al., 1991). Jeong et al. (1996) evaluated the effect of acute in vivo cocaine exposure on the generation of the anti-sRBC PFC, and determined that immune suppression was due to a cytochrome P-450 metabolite of cocaine. Further studies demonstrated that sex, strain and age differences can be detected in cocaine-induced immunodulation as assessed by the anti-SRBC PFC response (Matulka et al., 1996). Similar to other immunotoxic agents, the mechanism by which cocaine alters immune function involves a disruption of the Th1/Th2 balance and the stress response (Stanulis et al., 1997a,b). Cocaine also induces the secretion of TGF-β, which has been linked to the observation that cocaine exposure enhances replication of the HIV-1 virus in human peripheral blood mononuclear cells (Chao et al., 1991; Peterson et al., 1991). More recently, and in agreement with the above studies, cocaine was demonstrated to cause an increased HIV viral burden in a human PBL–SCID animal model, which was mediated through σ 1 receptors, which are informally referred to as psychoactive drug receptors (Roth et al., 2005). Although the function and role of σ 1 receptors still remain to be elucidated, additional studies also suggest that cocaine effects are mediated through these receptors (Maurice and Romieu, 2004).

Methamphetamine Methamphetamine use has been growing over the past several years. Methamphetamine is a stimulant that is similar to amphetamine, although highly addictive. Only recently has the immunotoxicity of methamphetamine been explored (In et al., 2005). Following oral administration to mice, methamphetamine suppressed the anti-sRBC PFC response, IgG production and mitogenic stimulation of T-cell proliferation. Even more striking was the suppression of GM-CSF-stimulated bone marrow colony growth by methamphetamine. These results indicate suppression of both CMI and humoral immunity in vivo following methamphetamine administration.

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Ethanol Ethanol exposure has been studied both in alcoholic patients and in animal models of binge drinking. In humans, alcoholism is associated with an increased incidence of, and mortality from, pulmonary infection and mortality from it (reviewed in Happel and Nelson, 2005). There is also an increased incidence of bacterial infection and spontaneous bacteremia in alcoholics with cirrhosis of the liver (reviewed in Leevy and Elbeshbeshy, 2005). A consistent finding in abusers of ethanol is the significant change in the mononuclear cells of the peripheral blood. In animal models, this is observed as depletion of T- and B cells in the spleen and the T cells in the thymus, particularly CD4+ /CD8+ cells. The latter effect may be related in part to increased levels of corticosteroids (Han et al., 1993). More recently, in a binge-drinking model, ethanol suppresses innate immunity through an inhibition of TLR3 signaling in peritoneal macrophages. The authors also demonstrated suppression of pro-inflammatory cytokines (Pruett et al., 2004a). In addition to suppression of TLR3, ethanol suppresses signaling through other TLRs, contributing to pielotropic effects of ethanol on innate immunity (Pruett et al., 2004b).

Medical Devices and Silicon-Based Materials As reviewed in Rodgers et al. (1997), many of these devices may have intimate and prolonged contact with the body and possible immunologic consequences of this contact could be envisioned to include immune suppression, immune stimulation, inflammation, and sensitization. Concern over the influence of medical devices on the immune system reached a peak during the scientific, medical, social, and legal debate in the late 1990s about the potential impact of silicon breast implants on human health. As such, this section will emphasize studies that have focused on silicon-based materials. Silicon-based materials have known uses in consumer products such as cosmetics, toiletries, food stuffs, household products, and paints as well as in the medical field (e.g., as lubricants in tubing and syringes and as components in numerous implantable devices). Significant interest was focused on the biocompatibility of certain silicon-based materials (silicones) and the potential for these products to produce immunotoxic effects, because of persistent, unsubstantiated speculation that breast implants made with silicone materials were able to provoke connective tissue disease. A committee formed by the Institute of Medicine concluded in 2000 that “a review of the toxicology studies of silicones and other substances known to be in breast implant does not provide a basis for health concerns” (IOM, 2000). Two studies were conducted which extensively evaluated immune status following exposure to dimethylpolysiloxanes used in medical practice (Bradley et al., 1994a,b). In the first study, mice were implanted for 10 days with dimethylpolysiloxane fluid, gel, and elastomer as well as polyurethane as a control. There were no observable alterations in innate or acquired immune function. In fact, the materials tested afforded modest protection to an approximate LD50 challenge with L. monocytogenes. Implantation of the same materials for 180 days resulted in a modest suppression of NK cell activity that did not correlate with altered susceptibility to challenge with B16F10 melanoma. No other alterations in host resistance have been observed. Studies have also been conducted on two low-molecular weight cyclic siloxanes: octamethylcyclotetrasiloxane and decamethylcyclotetrasiloxane. One-month inhalation exposures of rats to high concentrations of octamethylcyclotetrasiloxane (up to 540 ppm) and

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decamethylcyclotetrasiloxane (up to a maximum 160 ppm) did not result in alterations in humoral immunity (Burns-Naas et al., 1998; Klykken et al., 1999). Inhalation exposure of human volunteers to octamethylcyclotetrasiloxane at 10 ppm for 1 hour revealed no effects on several immune parameters (Looney et al., 1998). Finally, under highly specific experimental conditions that do not mimic human exposure, a few silicon-based materials have been observed to act as immunologic adjuvants (reviewed in Potter and Rose, 1996). Under typical exposure conditions, neither octamethylcyclotetrasiloxane, a combination of octamethylcyclotetrasiloxane and decamethylcyclotetrasiloxane, nor dimethylpolysiloxane acts as an immunologic adjuvant (Bradley et al., 1994a,b; Klykken and White, 1996; Klykken et al., 1999). Overall, the studies to date have been largely negative and no link between exposure to silicones and human disease has been established.

Inhaled Substances Pulmonary defenses against inhaled gases and particulates are dependent on both physical and immunologic mechanisms. Immune mechanisms primarily involve the complex interactions between PMNs and alveolar macrophages and their abilities to phagocytize foreign material and produce cytokines, which not only act as local inflammatory mediators, but also serve to attract other cells into the airways. Oxidant Gases It is becoming increasingly clear that exposure to oxidant gases—such as ozone (O3 ), sulfur dioxide (SO2 ), nitrogen dioxide (NO2 ), and phosgene—alters pulmonary immunologic responses and may increase the susceptibility of the host to bacterial infections (reviewed by Selgrade and Gilmour, 1994). Infiltration of both PMNs and macrophages has been observed, resulting in the release of cellular enzyme components and free radicals, which contribute to pulmonary inflammation, edema, and vascular changes. Exposure to O3 has been demonstrated to impair the phagocytic function of alveolar macrophages and to inhibit the clearance of bacteria from the lung. This correlated with decreased resistance to S. zooepidemicus and suggests that other extracellular bacteriostatic factors may be impaired following exposure to these oxidant gases. Short-term NO2 exposure decreases killing of several bacterial pathogens and, like O3 , this decreased resistance is probably related to changes in pulmonary macrophage function. A role for the products of arachidonic acid metabolism (specifically, the prostaglandins) has recently been implied and is supported by the facts that decreased macrophage functions are associated with increased PGE2 production and that pretreatment with indomethacin inhibits O3 -induced pulmonary hyperresponsiveness and related inflammatory responses. It is clear that exposure to oxidant gases can also augment pulmonary allergic reactions. This may be a result of increased lung permeability (leading to greater dispersion of the antigen) and to the enhanced influx of antigen-specific IgE-producing cells in the lungs. In studies involving O3 exposure and challenge with L. monocytogenes, decreased resistance to the pathogen correlated not only with changes in macrophage activity, but also with alterations in T-cell-derived cytokine production (which enhances phagocytosis). In support of an effect on T cells, other cell-mediated changes were observed including changes in the T- to B-cell ratio in the lung, decreased DTH response, enhanced allergic responses, and changes in T-cell proliferative responses.

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Particles: Asbestos and Silica It is believed that alterations in both humoral immunity and CMI occur in individuals exposed to asbestos and exhibiting asbestosis. Decreased DTH response and fewer T cells circulating in the periphery as well as decreased T-cell proliferative responses have been reported to be associated with asbestosis (reviewed in Miller and Brown, 1985; Warheit and Hesterberg, 1994). Autoantibodies and increased serum immunoglobulin levels have also been observed. Within the lung, alveolar macrophage activity has been implicated as playing a significant role in asbestosinduced changes in immune competence. Fibers of asbestos that are deposited in the lung are phagocytized by macrophages, resulting in macrophage lysis and release of lysosomal enzymes and subsequent activation of other macrophages. Recently, it has been hypothesized that the development of asbestosis in animal models occurs by the following mechanism: Fibers of asbestos deposited in the alveolar space recruit macrophages to the site of deposition. Some fibers may migrate to the interstitial space where the complement cascade becomes activated, releasing C5a, a potent macrophage activator and chemoattractant for other inflammatory cells. Recruited interstitial and resident alveolar macrophages phagocytize the fibers and release cytokines, which cause the proliferation of cells within the lung and the release of collagen. A sustained inflammatory response could then contribute to the progressive pattern of fibrosis, which is associated with asbestos exposure. The primary adverse consequence of silica exposure, like that to asbestos, is the induction of lung fibrosis (silicosis). However, several immune alterations have been associated with silica exposure in experimental animals, including decreased antibody- and cell-mediated immune parameters (reviewed in IPCS, 1996). Alterations in both T- and B-cell parameters have been reported, although T-cell-dependent responses appear to be more affected than B-celldependent responses. Dose and route of antigen exposure appear to be important factors in determining silica-induced immunomodulation. Silica is toxic to macrophages and PMNs, and exposure is correlated with increased susceptibility to infectious pathogens. The significance of these immunologic alterations for the pathogenesis of silicosis remains to be determined. The association of this disease with the induction of autoantibodies is covered elsewhere in this chapter. Pulmonary Irritants Chemicals such as formaldehyde, silica, and ethylenediamine have been classified as pulmonary irritants and may produce hypersensitivity-like reactions. Macrophages from mice exposed to formaldehyde vapor exhibit increased synthesis of hydroperoxide (Dean et al., 1984). This may contribute to enhanced bactericidal activity and potential damage to local tissues. Although silica is usually thought of for its potential to induce silicosis in the lung (a condition similar to asbestosis), its immunomodulatory effects have also been documented (Levy and Wheelock, 1975). Silica decreased reticuloendothelial system clearance and suppressed both humoral immunity (PFC response) and the cell-mediated response (CTL) against allogeneic fibroblasts. Both local and serum factors were found to play a role in silica-induced alterations in Tcell proliferation. Silica exposure may also inhibit phagocytosis of bacterial antigens (related to reticuloendothelial system clearance) and inhibit tumoricidal activity (Thurmond and Dean, 1988).

Ultraviolet Radiation Ultraviolet radiation (UVR) is an important environmental factor affecting human health with both beneficial effects, such as vitamin

D production, tanning, and adaptation to UV, and adverse effects, such as sunburn, skin cancer, and ocular damage. UVR has also been demonstrated to modulate immune responses in animals and humans, and the effects of UV exposure on the immune system have been reviewed (Garssen and van Loveren, 2001). UV-induced immunomodulation has been shown to have some beneficial effects on some skin diseases, such as psoriasis, and has been demonstrated to impair some allergic and autoimmune diseases in both animals and humans. However, UV-induced immunomodulation can also lead to several adverse health consequences, including a pivotal role during the process of skin carcinogenesis. UV-induced immunomodulation has been demonstrated in experimental animal studies and human studies utilizing vaccine response rates to impair the resistance to a number of infectious agents, including bacteria, parasites, viruses, and fungi (reviewed in Sleijffers et al., 2004). As noted in the section “Approaches to the Assessment of Human Immunotoxicity,” the parallelogram approach has been used to extrapolate animal to human data in an initial quantitative assessment of the risk for deleterious effects of UV radiation (van Loveren et al., 1995). Importantly, the effects of UVR on host resistance are not limited to skin-associated infections, but are also apparent in systemic infections. There have been a number of recent studies to characterize the mechanism of action for UV-induced immunomodulation. The first step is the absorption of UV photons by chromophores, the socalled “photoreceptors”, such as DNA and urocanic acid (Garssen et al., 1997). As a consequence of UV absorption by chromophores, epidermal and dermal cells, including keratinocytes, melanocytes, Langerhans cells, mast cells, dermal fibroblasts, endothelial cells, as well as skin-infiltrating cells (i.e., granulocytes and macrophages) produce and/or release many immunoregulatory mediators, including cytokine, chemokines, and neurohormones (Sleijffers et al., 2004). The mediators include both pro- and anti-inflammatory cytokines, such as TNF-α, IL-1, IL-6, and IL-10, which can modify directly or indirectly the function of APCs. Langerhans cells, the major APC in the skin, change phenotypically and functionally, which ultimately impacts the activity of T cells at the time of antigen presentation, both locally and systemically. One early explanation for UV-induced immunomodulation is that UVR induced a switch from a predominantly Th1 response (favoring DTH responses) to a Th2 response (favoring antibody responses). This hypothesis was supported by findings of altered cytokine secretion patterns indicative of a Th1 to Th2 switch (Araneo et al., 1989; Simon et al., 1990). Indeed, the majority of studies dealing with the effects of UVR indicated that Th1-mediated immune responses are especially sensitive to UV exposure. However, as noted above, UVR has been demonstrated to be associated with a suppression of certain allergic and autoimmune reactions. Indeed, more recent studies have demonstrated that Ig isotypes that are linked to either Th1 or Th2 cells can be suppressed by UVR and that UV exposure not only impairs Th1 responses; but also some Th2 responses (Sleijffers et al., 2004).

XENOBIOTIC-INDUCED HYPERSENSITIVITY AND AUTOIMMUNITY Hypersensitivity Polyisocyanates Polyisocyanates have a widespread use in industry and are responsible for more cases of occupationally related lung disease than any other class of low-molecular weight compounds. These chemicals are used in the production of adhesives, paint hardeners, elastomers, and coatings. Occupational exposure is

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by inhalation and skin contact. Members of the group are known to induce the full spectrum of hypersensitivity responses, types I–IV, as well as nonimmune inflammatory and neuroreflex reactions in the lung (Grammer, 1985; Bernstein and Bernstein, 1994). Sensitized individuals have shown cross-reactivity between compounds in this group. Toluene diisocyanate is among the most widely used and most studied members of this group. Pulmonary sensitization to this compound can occur through either topical or inhalation exposure. It is a highly reactive compound that readily conjugates with endogenous protein. Laminin, a 70,000-kDa protein, has been identified as one protein that toluene diisocyanate conjugates in the airways, presumably forming one of the neoantigens responsible for hypersensitivity. Studies with guinea pigs have confirmed the need for a threshold level of exposure to be reached in order to obtain pulmonary sensitization. This finding supports the human data in which pulmonary sensitization is frequently the result of exposure to a spill, whereas workers exposed to low levels of vapors for long periods of time fail to develop pulmonary sensitization. Unlike the case in many hypersensitivity reactions, where removal of the antigen alleviates the symptoms of disease, symptoms may persist for years after cessation of exposure in many toluene diisocyanate-induced asthma patients. The molecular mechanism, in part, involves the recognition of neoantigens formed via covalent binding of toluene diisocyanate to airway-associated proteins and their recognition as nonself. The neoantigens drive the clonal expansion of Th2 cells, which upon secondary antigenic stimulation induce a strong Th2 cytokine response involving the production of IL-4, IL-5, and IL-13. Production of IL-4 is especially critical to the hypersensitivity response as it is involved in immunoglobulin class switching by B cells to form IgE, one of the principal mediators responsible for mast cell degranulation and the ensuing airway hyperreactivity typically associated with type I hypersensitivity. In murine models employing intranasal or intratracheal sensitization and challenge with toluene diisocyanate, significant induction of Th2 cytokines, IgE, and eosinophilia has been demonstrated (Matheson et al., 2005; Plitnick et al., 2005; Ban et al., 2006).

Acid Anhydrides The acid anhydrides make up another group of compounds for which nonimmune and IgE, cytotoxic, immune complex, and cell-mediated reactions have been reported (Grammer, 1985; Bernstein and Bernstein, 1994). These reactive organic compounds are used in the manufacturing of paints, varnishes, coating materials, adhesives, and casting and sealing materials. Trimellitic acid anhydride is one of the most widely used compounds in this group. Inhaled trimellitic acid anhydride fumes may conjugate with serum albumin or erythrocytes leading, to type I (trimellitic acid anhydride-asthma), type II (pulmonary disease–anemia), or type III (hypersensitivity pneumonitis) hypersensitivity reactions upon subsequent exposure. Topical exposure to trimellitic acid anhydride may lead to type IV hypersensitivity reactions, resulting in contact dermatitis. Also, re-exposure by inhalation may lead to a cellmediated immune response in the lung, which plays a role in the pathology seen in conjunction with type II and III pulmonary disease. Human and animal testing has supported the clinical findings in trimellitic acid anhydride-exposed workers. Levels of serum IgE can be measured in exposed workers and are predictive of the occurrence of type I pulmonary reactions. Serum titers of IgA, IgG, and IgM have been detected in patients with high levels of exposure

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to trimellitic acid anhydride. Similar findings have been reported in studies with rhesus monkeys, in which exposed animals showed IgA, IgG, and IgM titers to trimellitic acid anhydride-haptenized erythrocytes. Inhalation studies with rats have produced a model corresponding to human trimellitic acid anhydride-induced pulmonary pneumonitis. Other anhydrides known to induce immune-mediated pulmonary disease include phthalic anhydride, himic anhydride, and hexahydrophthalic anhydride. The mechanism responsible for respiratory hypersensitivity by trimellitic acid anhydride is believed to be similar to that described above for toluene diisocyanate and involves induction of Th2 cytokines, IL-4, IL-5, and IL-3, formation of allergen-specific IgE, and eosinophilia. In a murine model, intranasal sensitization and challenge with trimellitic acid anhydrideinduced Th2 cytokine expression in nasal airways and allergic rhinitis as well as mucous cell metaplasia in nasal and pulmonary airways that was not detected in mice only sensitized or challenged with trimellitic acid anhydride. Similarly, intranasal sensitization and challenge with contact sensitizers, dinitrochlorobenzene, or oxazolone, did not induce Th2 cytokines, IgE or mucus cell metaplasia in airways (Farraj et al., 2004). More recent studies compared topical versus intranasal sensitization in mice with trimellitic acid anhydride, in light of the fact that dermal exposure is likely to occur in the occupational setting (Farraj et al., 2006). Intranasal challenge with trimellitic acid anhydride in mice that were either topically or via intranasal sensitized with trimellitic acid anhydride produced a marked allergic rhinitis, of similar severity, characterized by an influx of eosinophils and lymphocytes. Both the topical and the intranasal routes of sensitization also produced significant increases in total serum IgE after intranasal challenge with trimellitic acid anhydride. In addition, both the topical and the intranasal routes of sensitization induced significant increases in the mRNA expression of the Th2 cytokines, IL-4, IL-5, and IL-13 (Farraj et al., 2006). These findings are significant as they suggest that dermal exposure represent a potential route of sensitization of the respiratory tract to chemical allergens. Based on the strong Th2 cytokine responses induced by several model respiratory sensitizers, such as toluene diisocyanate and trimellitic acid anhydride in rodent models, cytokine profiling has been proposed as an approach for identifying potential respiratory sensitizers (Dearman et al., 1996).

Metals Metals and metallic substances, including metallic salts, are responsible for producing contact and pulmonary hypersensitivity reactions. Metallic salts have been implicated in numerous immunologic and nonimmunologic pulmonary diseases. Exposure to these compounds may occur via inhalation or due to their solubility in aqueous media (they can be dissociated and transported into the lungs, where damage due to sensitization or nonimmunologic events takes place). Platinum, cobalt, chromium, nickel, and beryllium (and their salts) are the most commonly implicated allergenic metals. Platinum Exposure to plantinum salts, such as hexachloroplatic acid, occurs occupationally in the mining, and metallurgic industries, in chemical industries where platinum is used as a catalyst, and in the production of catalytic converters. Platinum salts are highly allergenic and induce hypersensitivity reactions such as contact dermatitis and respiratory symptoms. Platinum-reactive T cells and increased IgE have been detected in the peripheral blood of platinum-sensitized patients (Raulf-Heimsoth et al., 2000).

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Cobalt Cobalt exposure comes from metal-on-metal replacement prostheses, or occupationally in superalloy production and pigment manufacturing. Cobalt induces allergic contact dermatitis. Cobalt-reactive lymphocytes can be detected in patients with metal prostheses and the reactivity of the lymphocytes positively correlated with serum levels of cobalt (Hallab et al., 2004). Chromium Chromium is another metal in which exposure occurs either from metal-on-metal protheses, or occupationally in the electroplating, leather tanning, and paint, cement, and paper pulp production industries. Chromium eczema is a type IV hypersensitivity reaction and is the most common form of allergic contact dermatitis to chromium. Chromium exists in several oxidation states and chromium eczema is associated most often with exposure to either chromium (III) or chromium (VI). Evidence of IgE-mediated reactions to chromium has been supported by immediate bronchial hyperreactivity after challenge and the identification of antigenspecific IgE antibodies. Nickel Exposure to nickel occurs via body piercings or clothing fasteners, or occupationally in the mining, milling, smelting, and refinishing industries. Nickel is a common contact sensitizer, but pulmonary hypersensitivity reactions are rare. Nickelreactive T cells can be identified in subjects with contact dermatitis (Sinigaglia et al., 1985) and there is recent evidence that regulatory T cells act to suppress nickel-specific effector T cells in healthy individuals but not in nickel-allergic patients (Moed et al., 2005). Beryllium Beryllium exposure occurs most frequently in hightechnology ceramics and dental alloy manufacturing, and in the electronics, nuclear, and aerospace industries. Although its use in the manufacturing of fluorescent bulbs has been discontinued, chronic beryllium disease was originally identified in 1946 in a group of fluorescent lamp-manufacturing workers. Beryllium is capable of producing both contact and tuberculin type IV hypersensitivity reactions. Skin contact has been found to produce lesions of contact hypersensitivity, whereas lesions produced by penetration of splinters of beryllium under the skin are granulomatous in nature. Inhalation of beryllium can result in disease ranging from acute pneumonitis, tracheobronchitis, and chronic beryllium disease to an increase in the risk of lung cancer. Chronic beryllium disease still exists today with 2–16% of exposed workers developing the disease (reviewed in Fontenot and Maier, 2005). Unlike most hypersensitivity reactions in which removal of the sensitizing agent abates the disease, chronic beryllium disease mortality changes little following lower beryllium exposure. The mechanism of beryllium-induced hypersensitivity involves a pulmonary influx of CD4+ T cells. Th2 and CD8+ cells have minimal roles in chronic beryllium disease. Bronchoalveloar lavage fluid from chronic beryllium disease patients contains CD4+ T cells, which in mouse models, precedes the development of granulomas. The frequency of beryllium-specific T cells in the blood of sensitized individuals is lower than that of chronic beryllium disease patients. Although still unclear at this time, these CD4+ T cells are often from an oliogclonal T-cell population in which the predominant TCRβ chain is encoded by the Vβ3 gene. In addition, there is some evidence that people with a certain MHC class II allele (DPB1*0201) are more susceptible to the development of chronic beryllium disease.

Therapeutic Agents Hypersensitivity responses to therapeutic drugs are among the major types of unpredictable drug reactions, accounting for up to 10% of all adverse effects. Drugs that commonly induce hypersensitivity include sulfa drugs, barbiturates, anticonvulsants, insulin, iodine (used in many X-ray contrast dyes), and platinum-containing chemotherapeutics. Penicillin is the most common agent involved in drug allergy and is discussed here as an example. Exposure to penicillin is responsible for 75% of the deaths due to anaphylaxis in the United States. The route of administration, dosage, and length of treatment all appear to play a role in the type and severity of hypersensitivity reaction elicited. Severe reactions are less likely following oral administration as compared to parenteral, and prolonged treatment with high doses increases the risk of acute interstitial nephritis and immune hemolytic anemia. The high incidence of allergic reaction to penicillin is in part due to widespread exposure to the compound. Not only has there been indiscriminant use of the drug, but exposure also occurs through food products including milk from treated animals and the use of penicillin as an antimicrobial in the production of vaccines. Efforts are still being made to reduce unnecessary exposure. The mechanism by which hypersensitivity to penicillin, and likely other drugs, occurs is through the formation of a neo-antigen, which is then recognized by the immune system as nonself. The formation of the primary penicillin neo-antigen occurs during the break down of penicillin, in which the β-lactam ring opens, forming a reactive intermediate that reacts with other proteins. The resultant penicilloylated protein now acts as a hapten to which the immune system mounts a response. As is the case with other haptens, subsequent exposures to penicillin may not absolutely require the formation of penicilloylated proteins to elicit secondary responses. Reactions to penicillin are varied and may include any of the four types of hypersensitivity reactions. The most commonly seen clinical manifestation of type I reactions is urticaria; however, anaphylactic reactions occur in about 10–40 of every 100,000 patients receiving injections. Clinical signs of rhinitis and asthma are much less frequently observed. Blood dyscrasias can occur due to the production of IgG against penicillin metabolites bound to the surface of red blood cells (type II reaction). Penicillin has also been implicated in type III reactions leading to serum-sickness-like symptoms. Owing to the high frequency of type IV reactions when penicillin is applied topically, especially to inflamed or abraded skin, products are no longer available for topical application. Type IV reactions generally result in an eczematous skin reaction, but a rare, lifethreatening form of dermal necrosis may result. In these cases there is severe erythema and a separation of the epidermis at the basal layer. This reaction, which gives the clinical appearance of severe scalding, is thought to be a severe delayed reaction.

Latex Natural rubber latex is derived from the rubber tree Hevea brasiliensis and is used in the manufacture of over 40,000 products including examination and surgical gloves, among other medical products. Allergic reactions to natural rubber latex products have become an important occupational health concern over the past decade with increased use of universal precautions, particularly latex gloves, to combat the spread of bloodborne pathogens. Hypersensitivity to latex usually occurs via a type I or type IV reaction. Dermatologic reactions to latex include irritant dermatitis due to chemical additives or mechanical abrasion and the occlusive conditions caused by wearing gloves; contact dermatitis (which represents approximately 80% of the allergic responses) due to the chemical

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additives used in the glove manufacturing (e.g., thiurams, carbamates, mercapto compounds, and phenylenediamines), and potentially more serious IgE-mediated responses due to residual latex proteins that remained in the finished products. The IgE responses may manifest as urticaria, asthma, or life-threatening anaphylaxis. Several latex proteins have been identified and antibodies to most can be detected in latex-allergic individuals (Ahmed et al., 2004). Food and Genetically Modified Organisms Awareness of hypersensitivity reactions to foods and genetically modified organisms (or crops; GMOs) has increased in the last several years. The most common food allergens are milk, egg, peanuts and other tree nuts, fish, shellfish, soy, and wheat. Peanut allergies are relatively common, can be severe, and there is much interest sociologically to reduce even airborne exposures to peanuts; thus, current information regarding the mechanism of peanut hypersensitivity is provided as an example. Although many food allergies that develop in infancy or childhood might be outgrown, peanut allergy is often life long. Hypersensitivity to peanuts occurs primarily via a type I reaction and the IgE responses may include shortness of breath, asthma, and anaphylaxis. Several peanut proteins have been identified and antibodies to most can be detected in peanut-allergic patients (reviewed in Palmer and Burks, 2006). The stability of peanut proteins and their ability to interact with IgE contribute to the antigenicity of a particular protein over another. In addition, peanut-reactive T cells have been isolated from the blood of peanut-allergic individuals, suggesting the hypersensitivity to peanuts also involves a type IV reaction (de Jong et al., 1996). Exposure to GMOs are becoming more widespread as biotechnological advances in food production are used, for example, to confer insect resistance or provide desired nutrients. Allergenic determinants in GMOs result from the expression of novel proteins that might be recognized as nonself by the immune system. There are several considerations in determining potential hypersensitivity to a GMO. It is critical to establish whether the introduced protein is allergenic and/or whether its amino acid sequence is similar enough to known allergens to be considered potentially allergenic. In addition, the appropriate test must be selected (e.g., radioallergosorbent tests and immunoglobulin levels) in order to avoid false positives or false negatives. Finally, ideally, hypersensitivity to GMOs will be tested on subjects prior to release, but it is also important to survey reactions in the general public following widespread availability (reviewed in Germolec et al., 2003). Enzymes Detergent enzymes have been implicated in occupationally induced hypersensitivity. Subtilin, a proteolytic enzyme derived from Bacillus subtilis, is used in laundry detergents to enhance their cleaning ability. Both individuals working in the environment where the product is made and those using the product may become sensitized. Subsequent exposure may produce signs of rhinitis, conjunctivitis, and asthma. An alveolar hypersensitivity reaction associated with precipitation antibodies and a type III Arthus reaction from skin testing has also been seen. Papain is another enzyme known to induce IgE-mediated disease. It is a high-molecular weight sulfhydryl protease obtained from the fruit of the papaya tree and most commonly used as a meat tenderizer and a clearing agent in the production of beer. However, it is also used in the production of tooth powders, laxatives, and contact lens cleaning solutions. As emphasized in the section “Assessment of Hypersensitivity Responses,” there is a strong genetic component, and the specific

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nature of the genetic susceptibility and of the T-cell responses to detergent enzymes is largely unknown. In a recent study, transgenic mice with the following human haplotypes, HLA-DQ6, HLA-DQ8, HLA-DR2, HLA-DR3, and HLA-DR4, were generated and used to study the immune and inflammatory components involved in the response to subtilisin. Their results demonstrated that only DQ8 mice showed consistent T-cell responses to subtilisin, developed allergic eosinophilic inflammatory reactions in the airways following intranasal instillations of the enzyme, and responded with a significant IgG1 and IgE production. As noted by the authors, these results offer promise for the use of HLA Class II transgenic mice as models to study the allergenic responses to enzymes in humans, and for the potential to develop modified enzymes to maintain efficient detergent qualities without the allergenic properties (Xue et al., 2005). Sarlo and Kirchner (2002), reviewed the latest developments in the control of enzyme-induced occupational asthma and allergy in the detergent industry. Guidelines have been developed for the safe handling of enzymes, and those manufacturing facilities that follow all of the guidelines enjoy very low or no cases of asthma and allergy among workers exposed to enzymes. The key to the success of these guidelines is the prospective surveillance for the development of enzyme-specific IgE antibody before the onset of allergic symptoms. The results to date have shown that workers with IgE to enzymes can still continue to work in the industry symptom free for their entire career. Mechanistically, these observations suggest that exposures needed to induce sensitization are different, and probably lower, than exposures needed to elicit enzyme allergic symptoms (Sarlo and Kirchner, 2002). More recently, Sarlo (2003) provided an overview of how a comprehensive preclinical, clinical, and industrial hygiene program has been used successfully to control allergy and asthma to enzymes in the detergent industry. These guidelines and industrial hygiene programs designed to minimize sensitization to enzymes and the development of disease can be applied to other industries where occupational allergy and asthma to proteins are common.

Formaldehyde Formaldehyde exposure occurs in the cosmetic industry, the dental industry, and the textile industry, where it is used to improve wrinkle resistance, and in the furniture, auto upholstery, and resins industries. The general public may be exposed to low levels of formaldehyde in products as ubiquitous as newspaper dyes and photographic films and paper. This low-molecular weight compound is extremely soluble in water and forms haptens with human proteins easily (Maibach, 1983). Human predictive testing with 1– 10% formalin (formalin is 37% formaldehyde) for induction and 1% formalin for challenge showed sensitization rates of 4.5–7.8% (Marzulli and Maibach, 1987). Occupational exposure to formaldehyde has been associated with the occurrence of asthma, although it has proven difficult to demonstrate antibodies to formaldehyde in the affected individuals. Animal studies have confirmed the sensitization potential of formaldehyde. Frankild et al. (2000), demonstrated that formaldehyde was a strong sensitizer in both the Buehler test and the guinea pig maximization test. More recently, Basketter et al. (2001) compared 10 aldehydes of varying degrees of allergenicity in man using the LLNA. The results confirmed that the interpolation of the LLNA dose–response data to define the effective concentration of the test chemical required to induce a three-fold stimulation of proliferation (EC3) offers the prospect of a quantitative index of the relative potency of a contact allergen. The comparative study showed that

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Table 12-11 Chemical Agents Known to be Associated with Autoimmunity proposed antigenic chemical Drugs Methyl dopa Hydralazine Isoniazid Procainamide Halothane Nondrug chemicals Vinyl chloride Mercury Silica

clinical manifestations

department

references

Hemolytic anemia SLE-like syndrome SLE-like syndrome SLE-like syndrome Autoimmune hepatitis

Rhesus antigens Myeloperoxidase Myeloperoxidase DNA Liver microsomal proteins

Murphy and Kelton (1991) Cambridge et al. (1994) Jiang et al. (1994) Totoritis et al. (1988) Kenna et al. (1987)

Scleroderma-like syndrome Glomerular neuropathy Scleroderma

Abnormal protein synthesized in liver Glomerular basement membrane protein Most likely acts as an adjuvant

Ward et al. (1976) Pelletier et al. (1994) Pernis and Paronetto (1962)

formaldehyde had the lowest EC3 value, an observation consistent with the fact that it is regarded as the strongest allergen in man. Taken together, the evidence is clear that formaldehyde is a contact sensitizer. It has also been demonstrated to irritate the skin, eyes, and respiratory system, and it is considered a typical air pollutant. One of ongoing debates surrounding formaldehyde is whether it should also be considered to be a respiratory sensitizer. A study by Arts et al. (1997) used the LLNA and serum IgE test in Brown Norway rats to compare the profiles of activity of trimellitic anhydride, a dermal and respiratory sensitizer, dinitrochlorobenzene, a dermal sensitizer with no or limited potential to cause respiratory allergy, formaldehyde, described by the authors as a skin irritant and dermal sensitizer with equivocal evidence for respiratory sensitizing potential, and methyl salicylate, a skin irritant devoid of sensitizing properties. Their results indicated that only exposure to trimellitic anhydride resulted in a significant increase in serum IgE concentration, and that formaldehyde, but not methyl salicylate, caused a dose-dependent activation of the draining lymph nodes. These results would suggest that formaldehyde is not a respiratory sensitizer. Other studies have demonstrated that formaldehyde can augment or enhance the respiratory allergic response to other stimuli. For example, Sadakane et al. (2002) showed that formaldehyde exposure (0.5% mist once a week for 4 weeks) in ICR mice enhanced the eosinophilic airway inflammation following the intratracheal instillation with Der f, the dust mite allergen. Fujimaki and colleagues exposed C3H/He mice to formaldehyde at 0-, 80-, 400-, or 2000-ppb formaldehyde for 12 weeks. When mice were immunized

with ovalbumin and then exposed to formaldehyde, the total number of bronchoalveolar lavage cells, macrophages, and eosinophils were significantly increased at the highest concentration (Fujimaki et al., 2004). Exposure to 400-ppb formaldehyde induced significant decreases in anti-ovalbumin IgG1 and IgG3 antibodies; but there was no effect on anti-ovalbumin IgE antibody. In addition, the ovalbumin-induced increase in plasma nerve growth factor was decreased by 80- and 400-ppb formaldehyde. The authors concluded that this was the first experimental evidence that low levels of longterm formaldehyde inhalation can induce differential immunogenic and neurogenic responses in allergic mice.

Autoimmunity There are numerous reports of xenobiotics that have been associated with autoimmunity. However, firm evidence for their involvement is difficult to obtain, and there are very few human autoimmune diseases for which an environmental trigger has been definitely identified (Rose, 2005). These relationships may be causative through direct mechanisms, or they may be indirect, acting as an adjuvant. In the area of xenobiotic-induced autoimmunity, exact mechanisms of action are not always known. Chemical exposure may also serve to exacerbate a pre-existing autoimmune state (Coleman and Sim, 1994; Kilburn and Warshaw, 1994). Table 12-11 lists chemicals known to be associated with autoimmunity, showing the proposed self-antigenic determinant or stating adjuvancy as the mechanism of action. Table 12-12 shows chemicals that have been implicated

Table 12-12 Chemicals Implicated in Autoimmunity manifestation

implicated chemical

references

Scleroderma

Solvents (toluene, xylene) Tryptophan Silicones Phenothiazines Penicillamine Propylthiouracil Quinidine l-Dopa Lithium carbonate Trichloroethylene Silicones

Walder (1983) Silver et al. (1990) Fock et al. (1984) Canoso et al. (1990) Harpey et al. (1971) DeSwarte (1985) Jiang et al. (1994) DeSwarte (1985) Ananth et al. (1989) Kilburn and Washaw (1992) Fock et al. (1984)

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in autoimmune reactions, but in these cases the mechanism of autoimmunity has not been as clearly defined or confirmed. The list includes both drug and nondrug chemicals. The heterogeneity of these structures and biological activities illustrate the breadth of potential for the induction of chemically mediated autoimmune disease. A number of recent papers have reviewed some specific examples of xenobiotics associated with autoimmune disease, including drugs/immunotherapeutics (D’Cruz, 2000; Vial et al., 2002; Pichler, 2003), vaccines (Descotes et al., 2002; Vial and Descotes, 2004), environmental chemicals (D’Cruz, 2000; Hess, 2002), and pesticides (Holsapple, 2002). A brief discussion of selected drug and nondrug chemicals is provided. Therapeutic Agents Methyldopa Methyldopa is a centrally acting sympatholytic drug that has been widely used for the treatment of essential hypertension; but with the advent of newer antihypertensive drugs, the use of methyldopa has declined. Platelets and erythrocytes are targeted by the immune system in individuals treated with this drug. In the case of thrombocytopenia, antibodies are detected against platelets, which are indicative of immune recognition of a self- or altered self-antigen. Hemolytic anemia occurs in at least 1% of individuals treated with methyldopa, and up to 30% of these individuals develop antibodies to erythrocytes as manifest in a positive Coombs test. Interestingly, the antibodies are not directed against the chemical or a chemical membrane conjugate. Hydralazine, Isoniazid, and Procainamide Hydralazine is a direct-acting vasodilator drug used in the treatment of hypertension. Isoniazid is an antimicrobial drug used in the treatment of tuberculosis. Procainamide is a drug that selectively blocks sodium channels in myocardial membranes, making it useful in the treatment of cardiac arrhythmias. All three drugs produce autoimmunity, which is manifested as a sytemic lupus erythematosus-like syndrome. Indeed, procainamide represents one of the best examples for a clear association between exposure to a xenobiotic and the onset or progression of an autoimmune disease. The association between procainamide and the sytemic lupus erythematosus-like condition is based on the finding that the disease remits when the drug is discontinued and recurs when the drug is re-administered. Antibodies to DNA have been detected in individuals showing this syndrome. Studies with hydralazine and isoniazid indicate that the antigenic determinant is myeloperoxidase. Immunoglobulins are produced against myeloperoxidase in individuals treated with these drugs. DNA is the apparent antigenic determinant for procainamide. For these three drugs, there is no evidence indicating that the immune system is recognizing the chemical or a chemical conjugate. In addition, these drugs have also been shown to produce hypersensitivity responses not associated with the sytemic lupus erythematosus syndrome. Halothane Halothane, one of the most widely studied of the drugs inducing autoimmunity, is an inhalation anesthetic that can induce autoimmune hepatitis. The incidence of this iatrogenic disease in humans is about one in 20,000. The pathogenesis of the hepatitis results from the chemical altering a specific liver protein to such a degree that the immune system recognizes the altered protein and antibodies are produced. Studies using rat microsomes show that halothane has to be oxidized by cytochrome P-450 enzymes to trifluoroacetylhalide before it binds to the protein. Investigations in-

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dicate that in affected individuals antibodies to specific microsomal proteins are produced. Vinyl Chloride Vinyl chloride, which is used in the plastics industry as a refrigerant and in the synthesis of organic chemicals, is a known carcinogen and is also associated with a scleroderma-like syndrome. The disease affects multisystemic collagenous tissues, manifesting itself as pulmonary fibrosis, skin sclerosis, and/or fibrosis of the liver and spleen. Ward et al. (1976) reported on 320 exposed workers, showing that 58 (18%) had a scleroderma-like syndrome. The individuals who showed the disease were in a group genetically similar (i.e., HLA-DR5) to patients with classic idiopathic scleroderma patients. Although the exact mechanism whereby this chemical produces autoimmunity is unclear, it is presumed that vinyl chloride acts as an amino acid and is incorporated into protein. Because this would produce a structurally abnormal protein, which would be antigenic, an immune response would be directed against tissues with the modified protein present. Other occupational exposures suspected to induce scleroderma-like reactions include solvents, particularly organic solvents; but the evidence is still limited. Although several epidemiological studies found an increased relative risk when compared to the general population, the association was weak and not reproduced in other studies, and these studies frequently assessed exposure to solvents in general without providing details on specific solvents (Garabrant and Dumas, 2000; Garabrant et al., 2003). Mercury This widely used metal is now known to have several target systems, including the CNS and renal system. Mercury also has two different actions with respect to the immune system. The first action is direct injury, described previously in the section “Immunomodulation by Xenobiotics, Metals.” The second action was highlighted in the section “Assessment of Autoimmune Responses” as an example of a model in which the autoimmune disease is chemically induced. Indeed, mercury produces an autoimmune disease that is manifested as glomerular nephropathy. Antibodies produced to laminin are believed to be responsible for damage to the basement membrane of the kidney. Mice and rats exposed to mercury also show antinuclear antibodies. The role of these antibodies in the autoimmune disease is not clear; however, they represent a known biomarker of autoimmunity. Studies in the Brown Norway rat point to a mercury-induced autoreactive CD4+ cell as being responsible for the polyclonal antibody response. Mercury chloride induces an increase in the expression of MHC class II molecules on B lymphocytes, as well as shifting the T-helper cell population along the Th2 line. It is the Th2 cell that promotes antibody production. The imbalance between Th1 and Th2 cells is believed to be caused by the depletion of cysteine and the reduced form of gluthathione in Th1 cells. These chemical groups are known to be important in the synthesis of and responsiveness to IL-2 in T cells. Thus, Th1 cells that synthesize and respond to IL-2 would be at a greater risk than Th2 cells. Mercury-induced autoimmunity has a strong genetic component. This has been extensively studied in the rat. Some strains of rats, such as the Lewis rat, are completely resistant, while others, such as the Brown Norway, are exquisitely sensitive. Susceptibility appears to be linked to three or four genes, one of which is the MHC. A number of reviews addressing the role of mercury in autoimmunity have been prepared (Pelletier et al., 1994; Bigazzi, 1999).

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Silica Crystalline silica (silicon dioxide) is a primary source of elemental silicon and is used commercially in large quantities as a constituent of building materials, ceramics, concretes, and glasses. Experimental animals as well as humans exposed to silica may have perturbations in the immune system. Depending on the length of exposure, dose, and route of administration of silica, it may kill macrophages or may act as an immunostimulant. Silica has been shown to be associated with an increase in scleroderma in silicaexposed workers (Kilburn and Warshaw, 1994). This effect is believed to be mediated via an adjuvant mechanism. Adjuvancy as a mechanism of causing autoimmunity has been implicated with a number of other chemicals, including paraffin and silicones. Inherent in adjuvancy as a mechanism of producing autoimmunity is that the population affected by these chemicals must already be at risk for the autoimmune disease. This is supported by the data indicating a genetic component to many autoimmune diseases. More recently, Brown and colleagues have developed a model in which apoptosis plays a critical role in silica-induced autoimmune diseases. As described by the authors, inhalation of crystalline silica results in concurrent activation and apoptosis of the alveolar macrophage resulting in an environment of inflammation and apoptosis (Brown et al., 2004). This environment may provide excess antigen that is further ingested by activated macrophages or dendritic cells that are able to migrate to local lymph nodes. Within these local lymph nodes, these APCs, laden with apoptotic material, activate T- and B cells thereby inducing an autoimmune response.

of activity in rats, where the predominant effect was immune enhancement, and in mice, where the predominant effect was immune suppression (Michielsen et al., 1999). Only the former results will be discussed further in this chapter. Exposure to a variety of strains of rats produced increases in the following types of parameters: peripheral blood counts, serum IgM and IgG levels, autoantibodies, spleen and lymph node weights, marginal zones and follicles of spleens, and primary and secondary antibody responses to tetanus toxoid. Interestingly, exposure to hexachlorobenzene caused opposite effects on two induced autoimmune models in Lewis rats, causing an increase in the severity of experimental allergic encephalomyelitis, and a decrease in the severity of adjuvant arthritis (Michielsen et al., 1999). This finding suggests that comparative studies using different genetically autoimmune-prone models may be needed to investigate the role of xenobiotics in the onset and progression of autoimmunity. In terms of a possible mechanism of action, Ezendam et al. (2004) proposed that after exposure to hexachlorobenzene, its deposition can directly induce cell damage or elicit damage by interfering with the integrity of cell membranes due to its lipophilic nature. Ultimately, hexachlorobenzene exposure triggers pro-inflammatory mediators, such as TNF-α, IL-1, IL-6, reactive oxygen species, and chemokines. These pro-inflammatory mediators serve as adjuvant signals that induce a systemic inflammatory response with influxes of neutrophils and macrophages into various nonimmune and immune organs. Subsequently, this leads to polyclonal activation of Tand B cells, eosinophilia, and eventually to visible clinical effects.

Hexachlorobenzene As noted above, a critical review of the state-of-the-science of autoimmunity by pesticides was prepared (Holsapple, 2002). There is little doubt that the pesticide that has been most extensively studied in the context of autoimmunity is hexachlorobenzene. Hexachlorobenzene is a low-molecular weight compound that was used in the past as a fungicide for seed grains. Even though its use as a pesticide was prohibited in most countries in the 1970s, it is still generated as a by product of several industrial processes and trace amounts of hexachlorobenzene are present as contaminants in some chlorine-containing pesticides. Finally, although emissions of hexachlorobenzene have decreased dramatically compared to the 1970s, residues can still be found throughout the environment due to its stability and persistence. One of the drivers for including hexachlorobenzene in this brief presentation of examples of xenobiotics associated with autoimmune disease is based on an accidental poisoning incident that occurred in Turkey in 1955–1959. Approximately 3000–5000 people ingested seed grain contaminated with the fungicide, hexachlorobenzene. Patients developed a disease characterized by hepatic porphyria, called porphyria turcica, which was manifested as bullous skin lesions, mainly in sun-exposed skin that ultimately healed with severe scars. The skin lesions have been attributed to the phototoxicity associated with the elevated levels of porphyrins. In addition to the dermatological changes, other clinical manifestations included neurologic symptoms, hepatomegaly, enlarged thyroid, splenomegaly, hyperpigmentation, hirsutism, enlarged lymph nodes and painful arthritis of the hands. For many of the clinical symptoms exhibited by the victims of the hexachlorobenzene poisoning incident in Turkey, an immune etiology was considered. Indeed, the autoimmunogenic potential of hexachlorobenzene has been characterized in a number of laboratory studies, which have been reviewed (Michielsen et al., 1999; Ezendam et al., 2004). The former review emphasized a striking difference in the profile

NEW FRONTIERS AND CHALLENGES IN IMMUNOTOXICOLOGY As noted throughout this chapter, the immune system has unquestionably been identified as a potential target organ for drugs and chemicals. With the demonstration that (1) chemicals can perturb the immune system of animals; (2) perturbation of immune function is correlated with an increased risk of infectious disease; and (3) perturbations in immune function can occur in the absence of any clinically observable effect, attention has focused, and will continue to focus, on the risk to the human population following exposure to chemicals that can alter immune function in animals. In fact, the characterization of the risk associated with xenobiotic-induced immunotoxicity arguably represents one of the key challenges for this discipline in the immediate future. Risk can be defined as the probability that an adverse event/effect will manifest itself. Risk must also incorporate the hazard, including dose–response relationships, and exposure. Exposure is a function of the amount of chemical involved and the time of its interaction with people and/or the environment. As such, assessment of risk is often an assessment of the probability for exposure. However, most papers in the immunotoxicology literature that are identified as “risk assessment” papers have focused on just one of the above components, most often, hazard identification. Thus, risk assessment in immunotoxicology must still be considered a “New Frontier.” The science of immunotoxicology continues to evolve, and any overview, including this chapter, must consider the discipline as a “snapshot” in time. Just during the period of time since this chapter was last published, immunotoxicology has experienced significant advancement. This, in part, has been driven by the tremendous growth in knowledge within immunology and cell biology coupled with an explosion in methodological and technological capabilities.

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New tests reflecting a variety of potential impacts of immunotoxicity have emerged, and traditional tests have been improved. In spite of these advances, significant challenges remaining to be addressed within the discipline of immunotoxicology and include: (1) how to interpret the significance of minor or moderate immunotoxic effects in animal models in relation to human risk assessment; (2) how to better integrate a consideration of exposure, especially to multiple agents simultaneously, into immunotoxicological risk assessment; (3) how to design better human studies to assess the

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impact on the immune system in the species of greatest interest in the context of risk assessment; (4) how to identify and establish sensitive human biomarkers of immunotoxicity; and (5) how to gain a better understanding of the role of genetics in identifying sensitive subpopulations to immune-altering agents. Many of the challenges identified above are not unique to immunotoxicology, but nevertheless are critical, and will need to be addressed through concerted and systematic efforts to improve human immune testing strategies.

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Immunotoxicology and Immunopharmacology. New York: Raven Press, 1994, pp. 617–642. Bhattacharjee A, Lappi VR, Rutherford MS, Schook LB: Molecular dissection of dimethylnitrosamine (DMN)-induced hepatotoxicity by mRNA differential display. Toxicol Appl Pharmacol 150:186–195, 1998. Biagini RE: Epidemiology studies in immunotoxicity evaluations. Toxicology 129:37–54, 1998. Bigazzi PE: Metals and kidney autoimmunity. Environ Health Perspect 107(suppl 5):753–765, 1999. Bondy GS, Pestka JJ: Immunomodulation by fungal toxins. J Toxicol Environ Health B Crit Rev 3:109–143, 2000. Bradley SG, Munson AE, McCay JA, et al.: Subchronic 10 day immunotoxicity of polydimethylsiloxane (silicone) fluid, gel and elastomer and polyurethane disks in female B6C3F1 mice. Drug Chem Toxicol 17:175–220, 1994a. Bradley SG, White KL, Jr., McCay JA, et al.: Immunotoxicity of 180 day exposure to polydimethylsiloxane (silicone) fluid, gel and elastomer and polyurethane disks in female B6C3F1 mice. Drug Chem Toxicol 17:221–269, 1994b. Brown JM, Pfau JC, Pershouse MA, Holian A: Silica, apoptosis, and autoimmunity. J Immunotoxicol 1:177–188, 2004. Buckley NE, McCoy KL, Mezey E, et al.: Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor. Eur J Pharmacol 396:141–149, 2000. Buehler EV: Delayed contact hypersensitivity in the guinea pig. Arch Dermatol 91:171–177, 1965. Burbach CM, Poland A, Bradfield CA: Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc Natl Acad Sci USA 89:8185–8189, 1992. Burchiel SW, Kerkvliet NL, Gerberick GF, Lawrence DA, Ladics GS: Assessment of immunotoxicity by multiparameter flow cytometry. Fundam Appl Toxicol 38:38–54, 1997. Burchiel SW, Lauer FT, Gurule D, Mounho BJ, Salas VM: Uses and future applications of flow cytometry in immunotoxicity testing. Methods 19:28–35, 1999. Burleson GR, Lebrec H, Yang YG, Ibanes JD, Pennington KN, Birnbaum LS: Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundam Appl Toxicol 29:40–47, 1996. Burns LA, Bradley SG, White KL, Jr., et al.: Immunotoxicity of mononitrotoluenes in female B6C3F1 mice: I. Para-nitrotoluene. Drug Chem Toxicol 17:317–358, 1994a. Burns LA, Bradley SG, White KL, Jr., et al.: Immunotoxicity of nitrobenzene in female B6C3F1 mice. Drug Chem Toxicol 17:271–315, 1994b. Burns LA, Bradley SG, White KL, et al.: Immunotoxicity of 2,4diaminotoluene in female B6C3F1 mice. Drug Chem Toxicol 17:401– 436, 1994c.

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CHAPTER 12

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TOXIC RESPONSES OF THE IMMUNE SYSTEM

Sadakane K, Takano H, Ichinose T, Yanagisawa R, Shibamoto T: Formaldehyde enhances mite allergen-induced eosinophilic inflammation in the murine airway. J Environ Pathol Toxicol Oncol 21:267–276, 2002. Salem ML, Hossain MS, Nomoto K: Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma. Int Arch Allergy Immunol 121:235–245, 2000. Sanders VM, Kohm AP: Sympathetic nervous system interaction with the immune system. Int Rev Neurobiol 52:17–41, 2002. Sanders VM, Tucker AN, White KL, Jr., et al.: Humoral and cell-mediated immune status in mice exposed to trichloroethylene in the drinking water. Toxicol Appl Pharmacol 62:358–368, 1982. Sanders VM, White KL, Jr., Shopp GM, Jr., Munson AE: Humoral and cell-mediated immune status of mice exposed to 1,1,2-trichloroethane. Drug Chem Toxicol 8:357–372, 1985. Sandy P, Ventura A, Jacks T: Mammalian RNAi: Aa practical guide. Biotechniques 39:215–224, 2005. Sarlo K: Control of occupational asthma and allergy in the detergent industry. Ann Allergy Asthma Immunol 90:32–34, 2003. Sarlo K, Kirchner DB: Occupational asthma and allergy in the detergent industry: New developments. Curr Opin Allergy Clin Immunol 2:97–101, 2002. Sawai C, Anderson K, Walser-Kuntz D: Effect of bisphenol A on murine immune function: Modulation of interferon-gamma, IgG2a, and disease symptoms in NZB × NZW F1 mice. Environ Health Perspect 111:1883–1887, 2003. Schatz AR, Koh WS, Kaminski NE: Delta 9-tetrahydrocannabinol selectively inhibits T-cell dependent humoral immune responses through direct inhibition of accessory T-cell function. Immunopharmacology 26:129–137, 1993. Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE: Cannabinoid receptors CB1 and CB2: A characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Pharmacol 142:278–287, 1997. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS, Jr.: Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science 270:283–286, 1995. Schule R, Umesono K, Mangelsdorf DJ, Bolado J, Pike JW, Evans RM: JunFos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene. Cell 61:497–504, 1990. Schwab CL, Fan R, Zheng Q, Myers LP, Hebert P, Pruett SB: Modeling and predicting stress-induced immunosuppression in mice using blood parameters. Toxicol Sci 83:101–113, 2005. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD: Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208, 2003. Schweitzer C, Keller F, Schmitt MP, et al.: Morphine stimulates HIV replication in primary cultures of human Kupffer cells. Res Virol 142:189–195, 1991. Selgrade MK: Use of immunotoxicity data in health risk assessments: uncertainties and research to improve the process. Toxicology 133:59–72, 1999. Selgrade MK, Cooper GS, Germolec DR, Heindel JJ: Linking environmental agents and autoimmune disease: An agenda for future research. Environ Health Perspect 107 Suppl 5:811–813, 1999. Selgrade MK, Cooper KD, Devlin RB, van Loveren H, Biagini RE, Luster MI: Immunotoxicity—bridging the gap between animal research and human health effects. Fundam Appl Toxicol 24:13–21, 1995. Selgrade MK, Gilmour MI: Effects of gaseous air pollutants on immune responses and susceptibility to infectious and allergic diseases, in Dean JH, Luster MI, Munson AE, Kimber I (eds.): Immunotoxicology and Immunopharmacology. New York: Raven Press, 1994, pp. 395–411. Selgrade MK, Lemanske RF, Jr., Gilmour MI, et al.: Induction of asthma and the environment: What we know and need to know. Environ Health Perspect 114:615–619, 2006. Serhan CN, Savill J: Resolution of inflammation: The beginning programs the end. Nat Immunol 6:1191–1197, 2005.

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UNIT 4

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CHAPTER 13

TOXIC RESPONSES OF THE LIVER Hartmut Jaeschke Disruption of the Cytoskeleton Fatty Liver Fibrosis and Cirrhosis Tumors Critical Factors in Toxicant-Induced Liver Injury Uptake and Concentration Bioactivation and Detoxification Regeneration Inflammation Immune Responses Idiosyncratic Liver Injury

INTRODUCTION LIVER PHYSIOLOGY Hepatic Functions Structural Organization Bile Formation LIVER PATHOPHYSIOLOGY Mechanisms and Types of Toxin-induced Liver Injury Cell Death Canalicular Cholestasis Bile Duct Damage Sinusoidal Damage

FUTURE DIRECTIONS

INTRODUCTION

processes involved in the excretory functions of the liver, and mechanisms of cell and organ injury. Each of these aspects can contribute to mechanisms of drug- and chemical-induced hepatotoxicities.

The liver is the main organ where exogenous chemicals are metabolized and eventually excreted. As a consequence, liver cells are exposed to significant concentrations of these chemicals, which can result in liver dysfunction, cell injury, and even organ failure. If an industrial chemical, e.g., carbon tetrachloride, bromobenzene, or vinyl chloride, is identified as a hepatotoxant, the use of the chemical may be restricted, the exposure may be minimized by mandating protective clothing and respirators, and attempts are made to replace it with a safer alternative. In the pharmaceutical industry, adverse effects on the liver are one of the most frequently cited reasons for discontinuing the development of drug candidates. In addition, hepatotoxicity recognized during the postmarketing phase is one of the main causes for withdrawing drugs from the market r ), a new an(Temple and Himmel, 2002). Troglitazone (Rezulin tidiabetic drug, was removed from the market after close to 100 of the 1.9 million patients treated with the drug suffered from liver failure (Chojkier, 2005). Thus, predictable and idiosyncratic hepatotoxicities severely restrict drug discovery efforts and drug development (Lee and Senior, 2005). Furthermore, the increasing popularity of herbal medicines, which are generally plant extracts, enhances the incidence of drug-induced liver injury and liver failure (Stickel et al., 2005). Since these medicines are mixtures of sometimes hundreds of compounds, it remains a difficult task to identify the causative agent and the mechanism of injury (Lee and Senior, 2005). Basic science and clinical aspects of drug- and chemical-induced liver injury was discussed in detail in several monographs and reviews (McCuskey and Earnest, 1997; Zimmerman, 1999; Jaeschke et al., 2002; Kaplowitz and DeLeve, 2002; Boyer et al., 2006b). Given the unprecedented speed of drug discovery and the increasing demand and use of “natural products” as food supplements and medicine, the early identification of hepatotoxins remains a formidable challenge for the future. The liver, with its multiple cell types and numerous functions, can respond in many different ways to acute and chronic insults. To recognize potential liver cell dysfunction and injury, it is necessary to have a general knowledge of basic liver functions, the structural organization of the liver, the

LIVER PHYSIOLOGY Hepatic Functions The liver’s strategic location between intestinal tract and the rest of the body facilitates the performance of its enormous task of maintaining metabolic homeostasis of the body (Table 13-1). Venous blood from the stomach and intestine flows into the portal vein and then through the liver before entering the systemic circulation. Thus the liver is the first organ to encounter ingested nutrients, vitamins, metals, drugs, and environmental toxicants as well as waste products of bacteria that enter portal blood. Efficient scavenging or uptake processes extract these absorbed materials from the blood for catabolism, storage, and/or excretion into bile. All the major functions of the liver can be detrimentally altered by acute or chronic exposure to toxicants (Table 13-1). When toxicants inhibit or otherwise impede hepatic transport and synthetic processes, dysfunction can occur without appreciable cell damage. Loss of function also occurs when toxicants kill an appreciable number of cells and when chronic insult leads to replacement of cell mass by nonfunctional scar tissue. Alcohol abuse is the major cause of liver disease in most western countries (Crawford, 1999); thus ethanol provides a highly relevant example of a toxicant with multiple functional consequences (Lieber, 1994). Early stages of ethanol abuse are characterized by lipid accumulation (fatty liver) due to diminished use of lipids as fuels and impaired ability to synthesize the lipoproteins that transport lipids out of the liver. As alcohol-induced liver disease progresses, appreciable cell death occurs, the functioning mass of the liver is replaced by scar tissue, and hepatic capacity for biotransformation of certain drugs progressively declines. People with hepatic cirrhosis due to chronic alcohol abuse frequently become deficient at detoxifying both the ammonia formed by catabolism of amino acids and the bilirubin derived from breakdown of hemoglobin. Uncontrollable hemorrhage due to 557

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Table 13-1 Major Functions of Liver and Consequences of Impaired Hepatic Functions type of function

examples

consequences of impaired functions

Nutrient homeostasis

Glucose storage and synthesis Cholesterol uptake Products of intestinal bacteria (e.g., endotoxin) Clotting factors Albumin Transport proteins (e.g., very low density lipoproteins) Bilirubin and ammonia Steroid hormones Xenobiotics

Hypoglycemia, confusion Hypercholesterolemia Endotoxemia

Filtration of particulates Protein synthesis

Bioactivation and detoxification

Formation of bile and biliary secretion

Bile acid–dependent uptake of dietary lipids and vitamins Bilirubin and cholesterol Metals (e.g., Cu and Mn) Xenobiotics

inadequate synthesis of clotting factors is a common fatal complication of alcoholic cirrhosis. A consequence of liver injury that merits emphasis is that loss of liver functions can lead to aberrations in other organ systems and to death.

Structural Organization Two concepts exist for organization of the liver into operational units, namely, the lobule and the acinus (McCuskey, 2006b). Classically, the liver was divided into hexagonal lobules oriented around terminal hepatic venules (also known as central veins). At the corners of the lobule are the portal triads (or portal tracts), containing a branch of the portal vein, a hepatic arteriole, and a bile duct (Fig. 13-1). Blood entering the portal tract via the portal vein and hepatic artery is mixed in the penetrating vessels, enters the sinusoids, and percolates along the cords of parenchymal cells (hepatocytes), eventually flows into terminal hepatic venules, and exits the liver via the hepatic vein. The lobule is divided into three regions known as centrolobular, midzonal, and periportal. The acinus is the preferred concept for a functional hepatic unit. The terminal branches of the portal vein and hepatic artery, which extend out from the portal tracts, form the base of the acinus. The acinus has three zones: zone 1 is closest to the entry of blood, zone 3 abuts the terminal hepatic vein, and zone 2 is intermediate. Despite the utility of the acinar concept, lobular terminology is still used to describe regions of pathologic lesions of hepatic parenchyma. Fortunately, the three zones of the acinus roughly coincide with the three regions of the lobule (Fig. 13-1). Acinar zonation is of considerable functional consequence regarding gradients of components both in blood and in hepatocytes (Jungermann and Kietzmann, 2000). Blood entering the acinus consists of oxygen-depleted blood from the portal vein (60–70% of hepatic blood flow) plus oxygenated blood from the hepatic artery (30– 40%). Enroute to the terminal hepatic venule, oxygen rapidly leaves the blood to meet the high metabolic demands of the parenchymal cells. Approximate oxygen concentrations in zone 1 are 9–13%, compared with only 4–5% in zone 3. Therefore, hepatocytes in zone 3 are exposed to substantially lower concentrations of oxygen than

Excess bleeding Hypoalbuminemia, ascites Fatty liver Jaundice, hyperammonemia-related coma Loss of secondary male sex characteristics Diminished drug metabolism Inadequate detoxification Fatty diarrhea, malnutrition, Vitamin E deficiency Jaundice, gallstones, hypercholesterolemia Mn-induced neurotoxicity Delayed drug clearance

hepatocytes in zone 1. In comparison to other tissues, zone 3 is hypoxic. Another well-documented acinar gradient is that of bile salts (Groothuis et al., 1982). Physiologic concentrations of bile salts are efficiently extracted by zone 1 hepatocytes with little bile acids left in the blood that flows past zone 3 hepatocytes. Heterogeneities in protein levels of hepatocytes along the acinus generate gradients of metabolic functions. Hepatocytes in the mitochondria-rich zone 1 are predominant in fatty acid oxidation, gluconeogenesis, and ammonia detoxification to urea. Gradients of enzymes involved in the bioactivation and detoxification of xenobiotics have been observed along the acinus by immunohistochemistry (Jungermann and Katz, 1989). Notable gradients for hepatotoxins are the higher levels of glutathione in zone 1 and the greater amounts of cytochrome P450 proteins in zone 3, particularly the CYP2E1 isozyme inducible by ethanol (Tsutsumi et al., 1989). Hepatic sinusoids are the channels between cords of hepatocytes where blood percolates on its way to the terminal hepatic vein. Sinusoids are larger and more irregular than normal capillaries. The three major types of cells in the sinusoids are endothelial cells, Kupffer cells, and stellate cells (Fig. 13-2) (McCuskey, 2006b). In addition, there are rare pit cells, which are liver-associated lymphocytes with natural killer (NK) activity (Bouwens and Wisse, 1992). Sinusoids are lined by thin, discontinuous endothelial cells with numerous fenestrae (or pores) that allow molecules smaller than 250 kDa to cross the interstitial space (known as the space of Disse) between the endothelium and hepatocytes. Sinusoidal endothelial cells are separated from the hepatocytes by a basement membrane-like matrix, which is not as electron-dense as a regular basement membrane (Friedman, 2000). However, this subendothelial extracellular matrix is important for the normal function of all resident liver cells (Friedman, 2000). The numerous fenestrae and the lack of basement membrane facilitate exchanges of fluids and molecules, such as albumin, between the sinusoid and hepatocytes, but hinder movement of particles larger than chylomicron remnants. Endothelial cells are important in the scavenging of lipoproteins via the apo E receptor and of denatured proteins and advanced glycation endproducts by the scavenger receptor (Smedsrod et al., 1994). Hepatic endothelial cells also secrete cytokines, prostanoids, nitric

CHAPTER 13

TOXIC RESPONSES OF THE LIVER

559

Lobule

Bile Duct Hepatic Artery Terminal Hepatic Vein

PV HA

Portal Vein

els

Penetrating vess

Hepatocytes ZONES

THV

3

2

1

1

2

3

THV

ZONES

HA

PV

PV BD BD

ACINUS Figure 13-1. Schematic of liver operational units, the classic lobule and the acinus. The lobule is centered around the terminal hepatic vein (central vein), where the blood drains out of the lobule. The acinus has as its base the penetrating vessels, where blood supplied by the portal vein and hepatic artery flows down the acinus past the cords of hepatocytes. Zones 1, 2, and 3 of the acinus represent metabolic regions that are increasingly distant from the blood supply.

oxide, and endothelins (Smedsrod et al., 1994) and express intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the cell surface (Jaeschke, 1997). Kupffer cells are the resident macrophages of the liver and constitute approximately 80% of the fixed macrophages in the body (McCuskey, 2006b). Kupffer cells are situated within the lumen of the sinusoid. The primary function of Kupffer cells is to ingest and degrade particulate matter. Also, Kupffer cells are a major source of cytokines and eicosanoids and can act as antigen-presenting cells (Laskin, 1990). Hepatic stellate cells (also known as Ito cells or by the more descriptive terms of fat-storing cells) are located between endothelial cells and hepatocytes (Wake, 1980). Stellate cells are the major sites for vitamin A storage in the body (Wake, 1980). Upon activation, these cells can synthesize and excrete collagen and other extracellular matrix proteins and express smooth muscle actin (Friedman, 2000).

Bile Formation Bile is a yellow fluid containing bile acids, glutathione, phospholipids, cholesterol, bilirubin and other organic anions, proteins, metals, ions, and xenobiotics (Klaassen and Watkins, 1984). Formation of this fluid is a specialized function of the liver. Adequate bile formation is essential for uptake of lipid nutrients from the small

intestine (Table 13-1), for protection of the small intestine from oxidative insults (Aw, 1994), and for excretion of endogenous and xenobiotic compounds. Hepatocytes begin the process of bile formation by transporting bile acids, glutathione, and other osmolytically active compounds including xenobiotics and their metabolites into the canalicular lumen. These molecules are the major driving force for the passive movement of water and electrolytes across the tight junctions and the hepatocyte epithelium. The canalicular lumen is a space formed by specialized regions of the plasma membrane between adjacent hepatocytes (Fig. 13-2). The canaliculi are separated from the intercellular space between hepatocytes by tight junctions, which form a barrier permeable only to water, electrolytes, and to some degree to small organic cations. Under physiological conditions, tight junctions are impermeable to organic anions allowing the high concentrations of bile acids, glutathione, bilirubin-diglucuronide, and other organic anions in bile. The structure of the biliary tract is analogous to the roots and trunk of a tree, where the tips of the roots equate to the canalicular lumens. Canaliculi form channels between hepatocytes that connect to a series of larger and larger channels or ducts within the liver. The large extrahepatic bile ducts merge into the common bile duct. Bile can be stored and concentrated in the gallbladder before its release into the duodenum. However, the gallbladder is not essential to life and is absent in several species, including the horse, whale, and rat.

560

Kupffer Cell

Sinusoidal Lumen Endothelial Cell

RBC

Stellate Cell

Space of Disse

Hepatocytes

Bile Canaliculi Figure 13-2. Schematic of liver sinusoidal cells. Note that the Kupffer cell resides within the sinusoidal lumen. The stellate cell is located in the space of Disse between the thin, fenestrated endothelial cells and the cord of hepatocytes.

With the identification of specific transporters, substantial progress has been made in the understanding of the molecular mechanisms of bile formation (reviewed in Jansen and Groen, 2006; Pauli-Magnus and Meier, 2006). On the basolateral (sinusoidal) side of the hepatocytes, there are sodium-dependent and sodiumindependent uptake systems. Most conjugated bile acids (taurineand glycine-conjugates) and some of the unconjugated bile acids are transported into hepatocytes by NTCP (sodium-dependent taurocholate cotransporting polypeptide) (Fig. 13-3) (Trauner and Boyer, 2003; Hagenbuch and Dawson, 2004; Stieger et al., 2007). Sodiumindependent uptake of conjugated and unconjugated bile acids is performed by members of the organic anion-transporting polypeptides (OATPs) (Hagenbuch and Meier, 2004). OATP1B1 and OATP1B3, which are predominantly expressed in the liver and OATP1A2 are all capable of transporting conjugated and unconjugated bile acids and steroids, bromosulfophthalein and many other organic anions (Hagenbuch and Meier, 2004). Furthermore, the OATPs are transporting numerous drugs and also some hepatotoxins, e.g., phalloidin, microcystin, and amanitin (Pauli-Magnus and Meier, 2006). In addition to the uptake systems, there are ATP-dependent efflux pumps located on the basolateral membrane of hepatocytes. These carriers are members of the multidrug resistance-associated proteins (MRPs; ABCC), which are multispecific transporters for many different anions (Homolya et al., 2003) (Fig. 13-3). All unconjugated bile acids in hepatocytes are conjugated before being transported by the bile salt export pump (BSEP) across the canalicular membrane. Bile acid excretion is a major driving force of bile formation (bile salt-dependent bile flow). Other constituents of bile are transported by members of the multidrug resistance (MDR) P-glycoprotein family such as MDR3 (ABCC2), which transports phospholipids, and the heterodimeric transporters ABCG5/ABCG8, which transport cholesterol and plant sterols into bile (Pauli-Magnus and Meier, 2006). In addition, MRP2 (a member of the multidrug resistance-associated proteins) transports glutathione (GSH), which is the main compound responsible for the bile salt-independent bile flow, as well as sulfated and glucuronidated bile acids, glutathione disulfide and glutathione conjugates, bilirubin diglucuronide, and many other conjugated drugs and chemicals (Gerk and Vore, 2002; Borst et al., 2000). Other transport systems of the canalicular mem-

brane include the breast cancer resistance protein (BCRP; ABCG2), which can contribute to the biliary excretion of bile acids and xenobiotics. Metals are excreted into bile by a series of processes that include (1) uptake across the sinusoidal membrane by facilitated diffusion or receptor-mediated endocytosis; (2) storage in binding proteins or lysosomes; and (3) canalicular secretion via lysosomes, a glutathione-coupled event, or use of specific canalicular membrane transporter, e.g. MRP2 (Ballatori, 2002). Biliary excretion is important in the homeostasis of multiple metals, notably copper, manganese, cadmium, selenium, gold, silver, and arsenic (Klaassen, 1976; Gregus and Klaassen, 1986). Species differences are known for biliary excretion of several toxic metals; for example, dogs excrete arsenic into bile much more slowly than rats. Inability to export Cu into bile is a central problem in Wilson’s disease, a rare autosomal recessive inherited disorder characterized by a defect or the absence of a copper transporting P-type ATPase (ATP7B). This carrier is located in the trans-Golgi network and transports copper into the secretory pathway for binding to ceruplasmin and then exretion into bile (Loudianos and Gitlin, 2000). Because biliary excretion is the only way to eliminate Cu, a defect in ATP7B results in excessive Cu accumulation in hepatocytes, which causes chronic hepatitis and cirrhosis (Loudianos and Gitlin, 2000). Canalicular lumen bile is propelled forward into larger channels by dynamic, ATP-dependent contractions of the pericanalicular cytoskeleton (Watanabe et al., 1991). Bile ducts, once regarded as passive conduits, modify bile by absorption and secretion of solutes (Lira et al., 1992). Bile acids are taken up into biliary epithelial cells (cholangiocytes) by OATP1A2 (sodium-independent uptake) and by the sodium-dependent bile acid transporter (Hagenbuch and Dawson, 2004; Hagenbuch and Meier, 2004). These bile acids are then excreted on the basolateral side mainly by MRP3 and heterodimeric organic solute transporter (OSTα/OSTβ) (Ballatori et al., 2005). The bile acids excreted from cholangiocytes return to the portal circulation via the peribiliary plexus (cholehepatic shunt pathway). Biliary epithelial cells also express a variety of phase I and phase II enzymes, which may contribute to the biotransformation of chemical toxicants present in bile (Lakehal et al., 1999).

CHAPTER 13

MRP1

TOXIC RESPONSES OF THE LIVER

MRP3

MRP5

MRP4

561

MRP6 OATP1A2

MDR3 NTCP

ABCG5/G8 MDR1 BCRP

Bile

OATP1B3

BSEP

OCT1

OATP1B1 FIC1

MRP2

OAT2

OATP2B1 Hepatocyte

Cholehepatic Shunt Pathway Cholangiocyte MRP3 OstαOstβ

OATP1A2 ASBT MRP2

Figure 13-3. Transport proteins in human hepatocytes and cholangiocytes. Efflux transporters (blue symbols): BSEP, bile salt export pump; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; ABCG5/8; BCRP, breast cancer resistance protein; Ostα/Ostβ. Uptake transporters (red symbols): ASBT, apical sodium dependent bile salt transporter; NTCP, sodium taurocholate cotransporting polypeptide; OATP, organic anion-transporting polypeptide; OCT, organic cation transporter; OAT, organic anion transporter. Transporters localized to the sinusoidal membrane extract solutes from the blood. Exporters localized to canalicular membrane move solutes into the lumen of the canaliculus. Exporters of particular relevance to canalicular secretion of toxic chemicals and their metabolites are the canalicular multiple organic anion transporter (MOAT) system and the family of multiple-drug resistant (MDR) P-glycoproteins. (From Pauli-Magnus C, Meier PJ: Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44:778–787, 2006. Reprinted with permission of John Wiley & Sons, Inc.)

Secretion into biliary ducts is usually but not always a prelude to toxicant clearance by excretion in feces or urine. Exceptions occur when compounds such as arsenic are repeatedly delivered into the intestinal lumen via bile, efficiently absorbed from the intestinal lumen, and then redirected to the liver via portal blood, a process known as enterohepatic cycling. A few compounds, such as methyl mercury, are absorbed from the biliary tract; the extensive reabsorption of methyl mercury from the gallbladder is thought to contribute to the long biological half-life and toxicity of this toxicant (Dutczak et al., 1991). Alternatively, secretion into bile of toxicant metabolites can be a critical prelude to the development of injury in extrahepatic tissues. A clinically relevant example of bile as an important delivery route for a proximate toxicant is that of diclofenac, a widely prescribed nonsteroidal anti-inflammatory drug (NSAID) that causes small intestinal ulceration. Experiments with mutant rats lacking a

functional MRP2 exporter (Fig. 13-3) have shown that these mutants secrete little of the presumptive proximate toxicant metabolite into bile and are resistant to the intestinal toxicity of diclofenac (Seitz and Boelsterli, 1998). Toxicant-related impairments of bile formation are more likely to have detrimental consequences in populations with other conditions where biliary secretion is marginal. For example, neonates exhibit delayed development of multiple aspects of bile formation, including synthesis of bile acids and the expression of sinusoidal and canalicular transporters (Arrese et al., 1998). Neonates are more prone to develop jaundice when treated with drugs that compete with bilirubin for biliary clearance. Individuals with genetic deficiency of certain transporters are not only at risk for chronic liver injury and fibrosis but may also be more susceptible to drugs and hepatotoxins (Jansen and Sturm, 2003; Jansen and Groen, 2006). Patients

562

with sepsis frequently develop cholestasis, which is mainly caused by downregulation of multiple canalicular transport systems (Geier et al., 2006). In addition, direct inhibition of BSEP, as was shown for the endothelin receptor antagonist bosentan, can lead to retention of bile acids in the liver and potentially cell injury (Fattinger et al., 2001).

LIVER PATHOPHYSIOLOGY Mechanisms and Types of Toxin-induced Liver Injury The response of the liver to chemical exposure depends on the intensity of the insults, the cell population affected, and the duration of the chemical exposure (acute vs. chronic). Milder stresses may just cause reversible cellular dysfunction, e.g., temporary cholestasis after exposure to estrogens. However, acute poisoning with acetaminophen or carbon tetrachloride triggers parenchymal cell necrosis. Exposure to ethanol induces steatosis, which may enhance the susceptiblity to subsequent inflammatory insults (Table 13-2). Note that the representative hepatotoxins listed in Table 13-2 include pharmaceuticals (valproic acid, cyclosporin A, diclofenac, acetaminophen, tamoxifen), recreational drugs (ethanol, ecstasy), a vitamin (vitamin A), metals (Fe, Cu, Mn), hormones (estrogens, androgens), industrial chemicals (dimethylformamide, methylene dianiline), compounds found in teas (germander) or foods (phalloidin, pyrrolidine alkaloids), and toxins produced by fungi (sporidesmin) and algae (microcystin). Cell Death Based on morphology, liver cells can die by two different modes, oncotic necrosis (“necrosis”) or apoptosis. Necrosis is characterized by cell swelling, leakage of cellular contents, nuclear disintegration (karyolysis), and an influx of inflammatory cells. Because necrosis is generally the result of an exposure to a toxic chemical or other traumatic conditions, e.g., ischemia, large numbers of contiguous hepatocytes and nonparenchymal cells may be affected. Cell contents released during oncotic necrosis includes proteins such a high mobility group box-1 (HMGB1) and other alarmins, which are a subset of the larger class of damage-associated molecular patterns (DAMPs) (Bianchi, 2007). These molecules are recognized by cells of the innate immune system including Kupffer cells through their toll-like receptors and trigger cytokine formation, which orchestrate the inflammatory response after tissue injury (Scaffidi et al., 2002). Thus, an ongoing oncotic necrotic process can be identified by the release of liver-specific enzymes such as alanine (ALT) or aspartate (AST) aminotransferase into the

plasma and by histology, where areas of necrosis with loss of nuclei and inflammatory infiltrates are easily detectable in H&E sections. In contrast, apoptosis is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, formation of apoptotic bodies, and, generally a lack of inflammation. The characteristic morphological features of apoptosis are caused by the activation of caspases, which trigger the activation of enzymes such as caspase-activated DNase (CAD) responsible for internucleosomal DNA fragmentation (Nagata et al., 2003). In addition, caspases can directly cleave cellular and nuclear structural proteins (Fischer et al., 2003). Apoptosis is always a single cell event with the main purpose of removing cells no longer needed during development or eliminating aging cells during regular tissue turnover. Under these conditions, apoptotic bodies are phagocytosed by Kupffer cells or taken up by neighboring hepatocytes. In the absence of cell contents release, the remnants of apoptotic cells disappear without causing an inflammatory response. Because of effective regeneration, apoptotic cell death during normal tissue turnover or even a moderately elevated rate of apoptosis is of limited pathophysiological relevance in the liver. However, if the rate of apoptosis is substantially increased, the apoptotic process cannot be completed. In this case, cells undergo secondary necrosis with breakdown of membrane potential, cell swelling, and cell contents release (Ogasawara et al., 1993). The fundamental difference between oncotic necrosis and secondary necrosis is the fact that during secondary necrosis many apoptotic cells can still be identified based on morphology, many apoptotic characteristics such as activation of various caspases are present, and the process can be completely inhibited by potent pancaspase inhibitors (Jaeschke et al., 2004). Oncotic necrosis does not involve relevant caspase activation and is not inhibitable by caspase inhibitors. In recent years, signaling mechanisms of apoptosis were elucidated in great detail (Fig. 13-4) (reviewed in Jaeschke, 2006a; Malhi et al., 2006; Schulze-Bergkamen et al., 2006). In the extrinsic pathway of apoptosis, ligands (e.g., Fas ligand, TNF-α) bind to their respective death receptor (Fas receptor, TNF receptor type I), which triggers the trimerization of the receptor followed by recruitment of various adapter molecules and procaspases to the cytoplasmic tail of the receptor. The assembly of this death-inducing signaling complex (DISC) leads to the activation of initiator caspases (caspase-8 or -10). In hepatocytes, the active initiator caspase cleaves Bid, a member of the Bcl-2 family of proteins, and the truncated Bid (tBid) translocates together with other Bcl-2 family members such as Bax to the mitochondria. These proteins form pores in the outer membrane of the mitochondria and cause the release of intermembrane proteins such as cytochrome c and the second mitochondria-derived

Table 13-2 Types of Hepatobiliary Injury type of injury or damage

representative toxins

Fatty liver Hepatocyte death Immune-mediated response Canalicular cholestasis Bile duct damage Sinusoidal disorders Fibrosis and cirrhosis Tumors

Amiodarone, CCl4 , ethanol, fialuridine, tamoxifen, valproic acid Acetaminophen, allyl alcohol, Cu, dimethylformamide, ethanol Diclofenac, ethanol, halothane, tienilic acid Chlorpromazine, cyclosporin A, 1,1-dichloroethylene, estrogens, Mn, phalloidin Alpha-naphthylisothiocyanate, amoxicillin, methylene dianiline, sporidesmin Anabolic steroids, cyclophosphamide, microcystin, pyrrolizidine alkaloids CCl4 , ethanol, thioacetamide, vitamin A, vinyl chloride Aflatoxin, androgens, arsenic, thorium dioxide, vinyl chloride

TOXIC RESPONSES OF THE LIVER

FAS-L

C C

C Bax

EndoG

Apaf 1 Casp 9

dATP C CARD Casp 9

EndoG

DD

FLIP

FADD Cas 8

FADD

Bax

Bax

Bid

Casp 8

C

Apaf 1

C

Casp 10

C

C

Smac

Cas 10

Bcl-2

C

Bax

EndoG

FADD

FADD FADD

AIF

C

Smac

EndoG

C

C

DISC

C

C

EndoG

AIF

C

C

R1

FADD

R1

C C EndoG

Smac

C

FAS-L

FAS-L

FADD

DD

R1

DD

R1

DD

R1

DD

FAS-L

563

FADD

CHAPTER 13

C

CARD dATP

Smac

cIAP

Casp 3 Casp 6 Casp 7 CAD

AIF

Substrates

CAD

ICAD Degradation

Figure 13-4. Fas receptor-mediated apoptotic signaling pathways in hepatocytes. AIF, apoptosis-inducing factor; Apaf1, apoptosis protease-activating factor-1; CARD, caspase-activating and -recruiting domain; Casp, caspase; c, cytochrome c; cIAP, cellular inhibitor of apoptosis proteins; DD, death domain; Smac, second mitochondria-derived activator of caspases; DISC, deathinducing signaling complex; EndoG, endonuclease G; FADD, Fas-associated death domain; FAS-L, Fas-ligand; FLIP, FLICE-inhibitory protein. (Adapted from Jaeschke H: Mechanisms of liver cell destruction, in Boyer TD, Wright TL, Manns M (eds): Zakim and Boyer’s Hepatology. 5th edn. Philadelphia: Saunders-Elsevier, 2006a, p 37.)

activator of caspases (Smac). Cytochrome c, together with apoptosis protease activating factor-1 (APAF-1), ATP, and procaspase-9, forms the apoptosome causing the activation of caspase-9, which then processes (and activates) downstream effector caspases, e.g. caspase-3. The effector caspases can propagate the apoptosis signal by activating CAD to initiate nuclear DNA fragmentation and by cleaving numerous cellular proteins critical to cellular function and the structural integrity of the cell and the nucleus (Fischer et al., 2003; Nagata et al., 2003). In addition to downstream substrates, caspase-3 can also process more procaspase-8 and further amplify the apoptotic signal. Although hepatocytes constitutively express Fas and TNF receptors, the death signal generated with ligation of the receptor is clearly insufficient to trigger apoptosis. Inhibitor studies and experiments with gene-deficient mice support the hypothesis that only the amplification of the receptor-derived signal through multiple mitochondrial cycles can successfully induce apoptosis in hepatocytes (Yin et al., 1999; Bajt et al., 2000, 2001). In addition to the direct propagation of the apoptosis signal by mitochondrial cytochrome c release, the simultaneous release of Smac ensures that the cytosolic inhibitors of apoptosis proteins (IAPs) are inactivated and do not interfere with the promotion of apoptosis (Li et al.,

2002). Thus, mitochondria are an indispensable part of the extrinsic (receptor-mediated) apoptotic signal transduction pathway in liver cells. In contrast to the extrinsic pathway, the intrinsic or mitochondrial pathway of apoptosis is initiated independent of the TNF receptor family, caspase-8 activation, and formation of the DISC. Despite the upstream differences, the postmitochondrial effects are largely similar to the extrinsic pathway. The intrinsic pathway is generally triggered by a cytotoxic stress or DNA damage, which activates the tumor suppressor p53 (Sheikh and Fornace, 2000). This protein acts as transcription factor to promote the formation of pro-apoptotic Bcl-2 family members, e.g., Bax. The increased Bax translocation to the mitochondria induces the release of mitochondrial intermembrane proteins including cytochrome c, Smac, endonuclease G, and apoptosis-inducing factor (AIF) (Saelens et al., 2004). An intrinsic mechanism of apoptosis has been discussed for cell death in aging livers (Zhang et al., 2002), prolonged treatment with alcohol (Ishii et al., 2003), or toxicity of benzo(a)pyrene and acetaminophen in hepatoma cells (Boulares et al., 2002; Ko et al., 2004). For other hepatotoxic chemicals, such as carbon tetrachloride (Cai et al., 2005), galactosamine (Gomez-Lechon et al., 2002), and microcystin (Ding

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et al., 2000), evidence for mitochondria-dependent apoptosis has been reported in cultured hepatocytes and relevant apoptotic cell death was observed after in vivo exposure to these chemicals (Shi et al., 1998; Hooser, 2000; Gujral et al., 2003b). The dramatically increased knowledge of the signaling mechanisms of apoptotic cell death in hepatocytes lead to the identification of many biochemical “apoptosis” parameters, most of which turned out to be not as specific for apoptosis as originally thought. Prominent examples of these tests are the DNA ladder on agarose gels and the terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay, which demonstrate internucleosomal DNA fragmentation and DNA strandbreaks, respectively. Originally thought to specifically identify apoptotic cells, both assays are positive for most mechanisms of necrotic cell death (Grasl-Kraupp et al., 1995; Gujral et al., 2002; Jaeschke and Lemasters, 2003). As a result of the misinterpretation of many of these assays, the contribution of apoptosis to the overall pathophysiology processes and toxicological liver injuries is controversially debated (Jaeschke and Lemasters, 2003; Jaeschke et al., 2004; Malhi et al., 2006; Schulze-Bergkamen et al., 2006). However, the controversy can be avoided if the decision to label the process as apoptosis is based primarily on the morphological features of the dying cells. Because the characteristic morphology is caused by the caspase-mediated cleavage of structural proteins within the cell, relevant caspase activation, especially of downstream effector caspases such as caspase-3 or -6, is another hallmark of apoptosis. As a result, pancaspase inhibitors can effectively prevent apoptosis-induced liver injury in vivo and in isolated hepatocytes. Once the process is identified as apoptosis, additional parameters can be used to further characterize the signaling mechanism. In addition, the use of positive controls, e.g., Fas ligand- or TNF-mediated hepatocellular apoptosis, can be helpful in assessing qualitative and quantitative changes of many parameters relative to a proven apoptotic process (Jaeschke et al., 2004). Another critical issue to consider is the model system that is being used. For example, both the antidiabetic drug troglitazone and the analgesic acetaminophen clearly induce apoptosis in hepatoma cell lines (Yamamoto et al., 2001; Boulares et al., 2002). However, there is no evidence for a relevant role of apoptotic cell death in animals or patients for both drugs (Gujral et al., 2002; Jaeschke et al., 2004; Chojkier, 2005). Thus, characterization of cell death after chemical exposure has to be primarily based on morphology and a number of additional biochemical parameters, which need to quantitatively correlate with the number of apoptotic cells. In addition, the relevance of the model system for the human pathophysiology needs to be considered. The mechanisms of oncotic necrosis are more diverse and depend on the chemical insult to the cell (a detailed example of the mechanism of acetaminophen-induced hepatocellular necrosis is discussed later). However, a general trend is emerging. Independent of the initial insult and signaling pathways, mitochondria are almost always involved in the pathophysiology. The opening of the mitochondrial membrane permeability transition pore with collapse of the membrane potential and depletion of cellular ATP is a common final step of the mechanism of necrotic cell death (Kim et al., 2003). The loss of ATP inhibits ion pumps of the plasma membrane resulting in the loss of cellular ion homeostasis, which causes the characteristic swelling of oncotic necrosis.

Canalicular Cholestasis This form of liver dysfunction is defined physiologically as a decrease in the volume of bile formed or an

impaired secretion of specific solutes into bile. Cholestasis is characterized biochemically by elevated serum levels of compounds normally concentrated in bile, particularly bile salts and bilirubin. When biliary excretion of the yellowish bilirubin pigment is impaired, this pigment accumulates in skin and eyes, producing jaundice, and spills into urine, which becomes bright yellow or dark brown. Because drug-induced jaundice reflects a more generalized liver dysfunction, it is considered a more serious warning sign in clinical trials than mild elevations of liver enzymes (Zimmerman, 1999). The histologic features of cholestasis can be very subtle and difficult to detect without ultrastructural studies. Structural changes include dilation of the bile canaliculus and the presence of bile plugs in bile ducts and canaliculi. Toxicant-induced cholestasis can be transient or chronic; when substantial, it is associated with cell swelling, cell death, and inflammation. Cell injury is generally caused by the accumulation of chemicals in the liver, i.e., the cholestasiscausing chemical and, as a consequence, potentially cytotoxic bile acids, bilirubin, and other bile constituents. Many different types of chemicals, including metals, hormones, and drugs, cause cholestasis (Table 13-2) (Zimmerman, 1999). The molecular mechanisms of cholestasis are related to expression and function of transporter systems in the basolateral and canalicular membranes (reviewed by Pauli-Magnus and Meier, 2006) (Fig. 13-3). In principle, an increased hepatic uptake, decreased biliary excretion, and increased biliary reabsorption (cholehepatic shunting) of a drug may contribute to its accumulation in the liver. Although no case of drug toxicity has been reported in response to modifications of basolateral uptake, OATPs can contribute to the liver injury potential of toxins. The hepatotoxicity of phalloidin, microcystin, and amanitin is facilitated by the uptake through OATPs (Pauli-Magnus and Meier, 2006). Furthermore, there is a growing list of drugs including rifampicin, bosentan, and troglitazone, which are known to directly inhibit BSEP (Stieger et al., 2000; Fattinger et al., 2001). However, estrogen and progesterone metabolites inhibit BSEP from the canalicular side after excretion by MRP2 (Stieger et al., 2000). A substantial inhibition of bile salt excretion can lead to accumulation of these compounds in hepatocytes and may directly cause cell injury (Palmeira and Rolo, 2004). In addition, the initial tissue injury can be aggravated by the subsequent inflammatory response (Gujral et al., 2003a). However, the increased bile acid levels can trigger compensatory mechanisms, which limit the injury potential of cholestasis (Zollner et al., 2006). Bile acids are substrates for the nuclear receptor farnesoid X receptor (FXR). FXR activation stimulates the small heterodimeric partner 1 (SHP1), which downregulates NTCP and limits bile acid uptake (Denson et al., 2001). In addition, FXR activation causes the increased expression of BSEP and MDR3, which enhances the transport capacity for bile acids and phospholipids, respectively, at the canalicular membrane (Ananthanarayanan et al., 2001; Huang et al., 2003). Furthermore, the FXR-independent upregulation of the basolateral transporters MRP3 and MRP4 reduces intracellular bile acid and drug concentrations (Schuetz et al., 2001; Wagner et al., 2003; Fickert et al., 2006). Recent findings indicate that agonists of the nuclear xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) can not only induce MRP3 and -4 expression but also induce bile acid hydroxylation by Cyp3a11 and Cyp2b10 resulting in improved export and detoxification of bile acids during cholestasis (Wagner et al., 2005). In cholangiocytes, OSTα/OSTβ is upregulated at the basolateral membrane during cholestasis (Boyer et al., 2006a). This response, which is dependent on FXR, mediates the enhanced return of bile

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acids from bile to the plasma (Boyer et al., 2006a). Thus, the pharmacological modulation of transporter expression may counteract some of the detrimental effects of cholestasis with various etiologies (Zollner et al., 2006). Bile Duct Damage Another name for damage to the intrahepatic bile ducts is cholangiodestructive cholestasis (Cullen and Ruebner, 1991; Zimmerman, 1999). A useful biochemical index of bile duct damage is a sharp elevation in serum activities of enzymes localized to bile ducts, particularly alkaline phosphatase. In addition, serum levels of bile acids and bilirubin are elevated, as observed with canalicular cholestasis. Initial lesions following a single dose of cholangiodestructive chemicals include swollen biliary epithelium, debris of damaged cells within ductal lumens, and inflammatory cell infiltration of portal tracts. Chronic administration of toxins that cause bile duct destruction can lead to biliary proliferation and fibrosis resembling primary biliary cirrhosis (PBC). A number of drugs have been implicated to cause prolonged cholestasis with features of PBC (Zimmerman, 1999). However, only in rare cases will there be permanent damage or even loss of bile ducts, a condition known as vanishing bile duct syndrome. Cases of this persisting problem have been reported in patients receiving antibiotics (Davies et al., 1994), anabolic steroids, contraceptive steroids, or the anticonvulsant carbamazepine (Zimmerman, 1999). Sinusoidal Damage The sinusoid is, in effect, a specialized capillary with numerous fenestrae for high permeability (Braet and Wisse, 2002). The functional integrity of the sinusoid can be compromised by dilation or blockade of its lumen or by progressive destruction of its endothelial cell wall. Dilation of the sinusoid will occur whenever efflux of hepatic blood is impeded. The rare condition of primary dilation, known as peliosis hepatis, has been associated with exposure to anabolic steroids and the drug danazol. Blockade will occur when the fenestrae enlarge to such an extent that red blood cells become caught in them or pass through with entrapment in the interstitial space of Disse. Endothelial cell gaps and injury have been shown after exposure to acetaminophen (Ito et al., 2005), galactosamine/endotoxin (Ito et al., 2006), or an antiFas antibody (Ogasawara et al., 1993). These gaps can be caused by direct injury to endothelial cells by acetaminophen (DeLeve et al., 1997) and the Fas antibody (Bajt et al., 2000) or could be just the result of detachment from the extracellular matrix (Ito et al., 2006). In general, matrix metalloproteinase inhibitors prevent the gap formation (McCuskey, 2006a). A consequence of endothelial cell injury is the loss of barrier function with extensive blood accumulation in the liver resulting in hypovolemic shock. Microcystin produces this effect within hours in rodents (Hooser et al., 1989). Microcystin dramatically deforms hepatocytes by altering cytoskeleton actin filaments, but it does not affect sinusoidal cells (Hooser et al., 1991). Thus, the deformities that microcystin produces on the cytoskeleton of hepatocytes likely produce a secondary change in the structural integrity of the sinusoid owing to the close proximity of hepatocytes and sinusoidal endothelial cells (Fig. 13-2). Progressive destruction of the endothelial wall of the sinusoid will lead to gaps and then ruptures of its barrier integrity, with entrapment of red blood cells. These disruptions of the sinusoid are considered the early structural features of the vascular disorder known as veno-occlusive disease (DeLeve et al., 1999). Well established as a cause of veno-occlusive disease are the pyrrolizidine alkaloids (e.g., monocrotaline, retrorsine, and seneciphylline) found

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in some plants used for herbal teas and in some seeds that contaminate food grains. Numerous episodes of human and animal poisoning by pyrrolizidine alkaloids have been reported around the world, including massive problems affecting thousands of people in Afghanistan in 1976 and 1993 (Huxtable, 1997). Veno-occlusive disease is also a serious complication in about 15% of the patients given high doses of chemotherapy (e.g., cyclophosphamide) as part of bone-marrow transplantation regimens (DeLeve et al., 1999). Selective depletion of glutathione within sinusoidal endothelial cells and activation of matrix metalloproteinases are critical events in the mechanism of endothelial cell injury in the pathophysiology of veno-occlusive disease (Wang et al., 2000; DeLeve et al., 2003b). Endothelial cell gap formation and injury and the resulting microcirculatory disturbances have been well established as the cause of veno-occlusive disease (DeLeve et al., 2003a).

Disruption of the Cytoskeleton Phalloidin and microcystin disrupt the integrity of hepatocyte cytoskeleton by affecting proteins that are vital to its dynamic nature. The detrimental effects of these two potent hepatotoxicants are independent of their biotransformation and are exclusive for hepatocytes, because there is no appreciable uptake of either toxin into other types of cells. Tight binding of phalloidin to actin filaments prevents the disassembly phase of the normally dynamic rearrangement of the actin filament constituent of the cytoskeleton. Phalloidin uptake into hepatocytes leads to striking alterations in the actin-rich web of cytoskeleton adjacent to the canalicular membrane; the actin web becomes accentuated and the canalicular lumen dilates (Phillips et al., 1986). Experiments using time-lapse video microscopy have documented dose-dependent declines in the contraction of canalicular lumens between isolated hepatocyte couplets after incubation with a range of phalloidin concentrations (Watanabe and Phillips, 1986). Microcystin uptake into hepatocytes leads to hyperphosphorylation of cytoskeletal proteins secondary to this toxicant’s covalent binding to the catalytic subunit of serine/threonine protein phosphatases (Runnegar et al., 1995b). Reversible phosphorylations of cytoskeletal structural and motor proteins are critical to the dynamic integrity of the cytoskeleton. Extensive hyperphosphorylation produced by large amounts of microcystin leads to marked deformation of hepatocytes due to a unique collapse of the microtubular actin scaffold into a spiny central aggregate (Hooser et al., 1991). Lower doses of microcystin, insufficient to produce the gross structural deformations, diminish uptake and secretory functions of hepatocytes in association with preferential hyperphosphorylation of the cytoplasmic motor protein dynein (Runnegar et al., 1999). Dynein is a mechanicochemical protein that drives vesicles along microtubules using energy from ATP hydrolysis; central to the hydrolysis of the dynein-bound ATP is a cycle of kinase phosphorylation and phosphatase dephosphorylation. Thus, hyperphosphorylation of dynein freezes this motor pump. Chronic exposure to low levels of microcystin has raised new concerns about the health effects of this water contaminant. Specifically, low levels of microcystin promote liver tumors and kill hepatocytes in the zone 3 region, where microcystin accumulates (Solter et al., 1998). Information about the binding of phalloidin and microcystin to specific target molecules is valuable for two reasons. First, the linkages of specific binding to loss of target protein functions provide compelling evidence that such binding constitutes a defined molecular mechanism of injury. Second, the demonstrations of high-affinity binding to a target molecule without confounding effects on other

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processes or tissues have translated into applications of these toxins as tools for cell biology research. For example, phalloidin complexed with a fluorochrome (e.g., rhodamine phalloidin or Texas Red phalloidin) is used to visualize the actin polymer component of the cytoskeleton in all types of permeabilized cells. The collapse of actin filaments into spiny aggregates after microcystin treatment was visualized by fluorescence microscopy of cells stained with rhodamine phalloidin (Hooser et al., 1991). Low levels of microcystin are being used to discriminate the roles of dynein from other cytoskeletal motor proteins (Runnegar et al., 1999).

Fatty Liver Fatty liver (steatosis) is defined biochemically as an appreciable increase in the hepatic lipid (mainly triglyceride) content, which is 90% of a therapeutic dose of APAP is conjugated with sulfate or glucuronide, the limited formation of a reactive

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metabolite, i.e., N-acetyl-p-benzoquinone imine (NAPQI), poses no risk for liver injury. In fact, long-term studies with acetaminophen in osteoarthritis patients did not reveal any evidence of liver dysfunction or cell injury even in patients consuming the maximal recommended daily dose of APAP for 12 months (Kuffner et al., 2006; Temple et al., 2006). In contrast, after an overdose, the formation of large amounts of NAPQI leads first to depletion of cellular glutathione (GSH) stores and subsequently causes covalent binding of NAPQI to intracellular proteins (Jollow et al., 1973; Mitchell et al., 1973) (Fig. 13-5). The generally higher levels of P450 enzymes combined with the lower GSH content in centrilobular hepatocytes are the main reasons for the predominant centrilobular necrosis observed after APAP poisoning. Consistent with the critical role of protein binding for cell injury are the findings that APAP protein adducts are located only in centrilobular hepatocytes undergoing necrosis (Roberts et al., 1991) and that no APAP hepatotoxicity is observed without protein binding (Nelson, 1990). Because protein binding can be prevented by conjugation of NAPQI with GSH, any manipulation that reduces hepatic GSH levels, e.g., fasting or protein malnutrition, potentially enhances the toxicity of APAP. In contrast, interventions such as the supply of cysteine, the ratelimiting amino acid for GSH synthesis, promote the detoxification of NAPQI and limits cell injury (Mitchell et al., 1973). Based on this fundamental insight into the mechanism of APAP hepatotoxicity, N-acetylcysteine was introduced in the clinic as intervention therapy (Smilkstein et al., 1988). This highly successful approach, which saved the lives of many patients who took an APAP overdose, is still the most effective treatment available (Lee, 2004). A significant factor in APAP hepatotoxicity can be the consumption of alcoholic beverages. In addition to potential malnutrition in alcoholics, ethanol is a potent inducer of CYP2E1, which is the main enzyme responsible for the metabolic activation of APAP in humans (Gonzalez, 2007). Whereas the simultaneous exposure of ethanol and APAP competitively inhibits NAPQI formation and therefore prevents APAP-induced toxicity (Sato and Lieber, 1981), the increased expression of CYP2E1 can enhance APAP toxicity after ethanol metabolism (Gonzalez, 2007). In addition, the presence of higher-chain alcohols, e.g., isopentanol, in alcoholic beverages can induce additional P450 isoenzymes such as CYP3A, which can significantly enhance APAP hepatotoxicity (Sinclair et al., 2000; Guo et al., 2004). Despite the clear experimental evidence that alcohol consumption can increase the susceptibility to APAP (Sato et al., 1981) and the clinical observation of severe APAP hepatotoxicity in alcoholics, it remains controversial whether alcohol can actually induce hepatotoxicity at therapeutic doses of APAP as suggested by some case reports (Zimmerman and Maddrey, 1995). However, extensive review of the literature involving APAP consumption in alcoholics suggests no relevant risk for APAP hepatotoxicity at therapeutic levels in this patient population (Dart et al., 2000). In addition, a randomized, double-blind, placebo-controlled trial with multiple therapeutic doses of APAP showed no evidence of liver dysfunction or cell injury in alcoholics (Kuffner et al., 2001). Thus, alcohol consumption does not increase the risk for liver injury after therapeutic doses of APAP. This finding may apply to the potential interaction with other drugs and dietary chemicals. Nevertheless, consistent with experimental data and clinical experience, inducers of CYPs aggravate liver injury after a hepatotoxic dose of APAP. Although the focus of early mechanistic investigations was on the role of covalent binding in APAP-induced hepatotoxicity, it became apparent during the last decade that protein adduct formation

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GSH ↓

Protein Arylation Ca2+ – ATPase ↓

P450

NAPQI

AAP

Protein Nitration iNOS NO

Bax / Bid

ONOO–

–

O2 [Ca2+]cyt ↑

[Ca2+]↑

Bax / tBid

Mitochondria

MPT

Calpain Activation AIF

ATP ↓ ΔΨm ↓

Endonuclease G

Cyt c / Smac ATP↓ Caspase Activation

Chromatin Condensation DNAFragmentation

DNAStrandbreaks PARP Activation DNA Repair

Proteolysis of Structural Proteins

CME Nucleus

NAD+ Depletion

ATP ↓ ΔΨm ↓

Protein Arylation and Nitration

Figure 13-5. Intracellular signaling pathways of acetaminophen-induced liver cell necrosis. Proposed sequence of events leading to acetaminophen (APAP)-induced hepatotoxicity (see text for details). Abbreviations: AIF, apoptosis-inducing factor; CME, nuclear Ca2+ /Mg2+ dependent endonuclease (DNAS1L3); cyt, cytosolic; cyt c, cytochrome c; Δ Ψm , mitochondrial membrane potential; GSH, reduced glutathione; iNOS, inducible NO synthase; MPT, mitochondrial membrane permeability transition; NAPQI, N-acetyl-p-benzoquinone – imine; NO, nitric oxide; O− 2 , superoxide; ONOO , peroxynitrite; PARP, poly(ADP-ribose) polymerase; Smac, second mitochondria-derived activator of caspases; tBid, truncated form of Bid. (Adapted from Jaeschke H, Bajt ML: Intracellular signaling mechanisms of acetaminophen-induced liver cell death. Toxicol Sci 89:31–41, 2006, with permission from Oxford University Press.)

is an important biomarker for APAP overdose (Davern et al., 2006), but protein binding alone was not sufficient to explain cell injury (Fig. 13-5). Because no APAP-induced cell injury is observed without covalent binding of NAPQI to cellular proteins, in particular mitochondrial proteins (Tirmenstein and Nelson, 1989), it is considered a critical initiating event of the toxicity that requires amplification (Jaeschke et al., 2003). Mitochondrial protein binding causes inhibition of mitochondrial respiration, a selective mitochondrial oxidant stress, mitochondrial peroxynitrite formation, and declining ATP levels in the liver (Jaeschke and Bajt, 2006). The early mitochondrial translocation of Bax and Bid, members of the Bcl-2 family of proteins, trigger the release of mitochondrial intermembrane proteins including endonuclease G and apoptosisinducing factor (AIF) (Jaeschke and Bajt, 2006). These endonucle-

ases, which translocate to the nucleus after APAP exposure, cause the initial nuclear DNA fragmentation (Bajt et al., 2006). Recent findings suggest that activation of c-Jun N-terminal kinase could induce the mitochondrial Bax translocation (Gunawan et al., 2006). However, the continued exposure of GSH-depleted mitochondria to peroxynitrite results in nitration of mitochondrial proteins and mitochondrial DNA modifications (Cover et al., 2005). The continued oxidant stress will eventually trigger the mitochondrial membrane permeability transition (MPT) pore opening with breakdown of the membrane potential, mitochondrial swelling, and rupture of the outer membrane (Kon et al., 2004). These events lead to the loss of mitochondrial ATP synthesis capacity, more extensive nuclear DNA fragmention, and eventually oncotic necrosis (Gujral et al., 2002). In addition to these intracellular signaling mechanisms

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leading to cell death, additional events may expand the area of necrosis. The release of calpains, which are Ca2+ -activated proteases, during necrosis can promote further cell injury in neighboring cells (Limaye et al., 2003). Likewise, the release of DNase-1 enhances nuclear DNA fragmentation in adjacent cells and aggravates the injury after APAP overdose (Napirei et al., 2006). Also, the release of intracellular proteins such as the nuclear protein HMGB-1 from necrotic cells can stimulate macrophages to produce proinflammatory cytokines (Scaffidi et al., 2002). This way, the necrotic cell death during APAP hepatotoxicity can promote an innate immune response with recruitment of neutrophils and other inflammatory leukocytes, which may clear cell debris and prepare for regeneration of the lost tissue (Jaeschke, 2005; Cover et al., 2006) but, under certain conditions, may cause additional injury (see section “Inflammation”). Although many details of the mechanism still remain to be elucidated, the newly gained insight into signaling events in response to APAP overdose suggests two fundamentally new developments: First, necrotic cell death is in most cases not caused by a single catastrophic event but can be the result of a cellular stress, which is initiated by metabolic activation and triggers sophisticated signaling mechanisms culminating in cell death (Fig. 13-5). Second, the multitude of events following the initial stress offers many opportunities for therapeutic interventions at later time points. Because these events are not occurring in all cells to the same degree and at the same time, delayed interventions may not completely prevent cell damage but limit the area of necrosis enough to prevent liver failure. Delayed treatment with GSH to accelerate the recovery of mitochondrial GSH levels effectively scavenged peroxynitrite, reduced the area of necrosis, and promoted regeneration resulting in improved survival after APAP overdose (Bajt et al., 2003). Overexpression of calpastatin, an inhibitor of calpains, attenuated APAP-induced liver injury and enhanced survival (Limaye et al., 2006). A similar effect on the progression of APAP-induced liver injury was also observed in animals deficient in DNase-1 (Napirei et al., 2006). Furthermore, inhibiting the innate immune response exerted beneficial effects against APAP hepatotoxicity (Liu et al., 2004). Together these findings underscore the concept that the later stages of APAP-induced liver injury can be affected at the level of intracellular signaling in hepatocytes, during the propagation of the injury to neighboring cells and the inflammatory response. Ethanol Morbidity and mortality associated with the consumption of alcohol is mainly caused by the toxic effects of ethanol on the liver (Stewart and Day, 2006). This targeted toxicity is due to the fact that >90% of a dose of ethanol is metabolized in the liver. Three principal pathways of ethanol metabolism are known (Fig. 13-6). Alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde with a Km of 1 mM; the electrons are transferred to NAD+ , which leads to the production of NADH. Acetaldehyde is further oxidized to acetate in a NAD-dependent reaction by acetaldehyde dehydrogenase (ALDH). This pathway is mainly regulated by the mitochondrial capacity to utilize NADH and regenerate NAD+ (Stewart and Day, 2006). The formation of excess reducing equivalents and acetate stimulates fatty acid synthesis and is a major factor in the development of alcohol-induced steatosis. Both ADH and ALDH exhibit genetic polymorphisms and ethnic variations, which play a role in the development of alcoholism and liver damage (Agarwal, 2001; Day, 2006). A toxicologically relevant polymorphism involves the mitochondrial ALDH2, where the ALDH2*2 form shows little or no catalytic activity. The increased levels of acetaldehyde present in individuals that carry this polymorphism is thought to cause the

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“flushing” syndrome after ethanol exposure. The inactive form of ALDH is found in 50% of Asians but is absent in Caucasians. This may be the reason for the overall reduced incidence of alcoholism in Asia compared to Europe and North America (Chen et al., 1999). However, heterozygotes of ALDH2*2 were found to develop more severe liver injury in response to lower alcohol consumption, suggesting a higher susceptibility to alcoholic liver disease (Enomoto et al., 1991). These findings underscore the importance of acetaldehyde in the pathophysiology. The second major pathway involves the alcohol-inducible enzyme CYP2E1, which oxidizes ethanol to acetaldehyde (Fig. 13-6). The enzyme is located predominantly in hepatocytes of the centrilobular region and requires oxygen and NADPH. Because the Km of CYP2E1 for ethanol is >10 mM, this reaction is most relevant for high doses of ethanol and, due to the enzyme’s inducibility, for chronic alcoholism (Stewart and Day, 2006). The third pathway involves catalase in peroxisomes. In this reaction, ethanol functions as electron donor for the reduction of hydrogen peroxide to water. Thus, the capacity of this pathway is limited due to the low levels of hydrogen peroxide. It is estimated that 99.9 99.4 99.1 99.1 50 0

Glomerular filtration rate: 125 mL/min = 180 L/24 h.

Catabolism and apical transport of glutathione (GSH) occurs to a much greater extent in the S3 segment, where the brush-border enzyme γ -glutamyl transpeptidase is present in greater amounts. Chemically induced injury to distinct proximal tubular segments therefore may be related in part to their segmental differences in biochemical properties (see section “Site-Selective Injury”).

Loop of Henle The thin descending and ascending limbs and the thick ascending limb of the loop of Henle are critical to the processes involved in urinary concentration (Fig. 14-2). Approximately 25% of the filtered Na+ and K+ and 20% of the filtered water are reabsorbed by the segments of the loop of Henle. The tubular fluid entering the thin descending limb is iso-osmotic to the renal interstitium; water is freely permeable and solutes, such as electrolytes and urea, may enter from the interstitium. In contrast, the thin ascending limb is relatively impermeable to water and urea, and Na+ and Cl– are reabsorbed by passive diffusion. The thick ascending limb is impermeable to water, and active transport of Na+ and Cl– is mediated by the Na+ /K+ -2Cl– cotransport mechanism, with the energy provided by the Na+ , K+ -ATPase. The relatively high rates of Na+ , K+ -ATPase activity and oxygen demand, coupled with the meager oxygen supply in the medullary thick ascending limb, are believed to contribute to the vulnerability of this segment of the nephron to hypoxic injury. The close interdependence between metabolic workload and tubular vulnerability has been demonstrated, revealing that selective damage to the thick ascending limb in the isolated perfused kidney can be blunted by reducing tubular work and oxygen consumption (via inhibition of the Na+ , K+ -ATPase with ouabain) or by increasing oxygen supply (via provision of an oxygen carrier, hemoglobin) (Brezis and Epstein, 1993). Conversely, increasing the tubular workload (via the ionophore amphotericin B) exacerbates hypoxic injury to this segment (Brezis et al., 1984).

Distal Tubule and Collecting Duct The macula densa comprises specialized cells located between the end of the thick ascending limb and the early distal tubule, in close proximity to the afferent arteriole (Fig. 14-2). This anatomic arrangement is ideally suited for a feedback system whereby a stimulus received at the macula densa is transmitted to the arterioles of the same nephron. Under normal physiologic conditions, increased solute delivery or concentration at the macula densa triggers a signal resulting in afferent arteriolar constriction leading to decreases in GFR (and hence decreased solute delivery). Thus, increases in fluid/solute out of the proximal tubule, due to impaired tubular re-

absorption, will activate this feedback system, referred to as tubuloglomerular feedback (TGF) and resulting in decreases in the filtration rate of the same nephron. This regulatory mechanism is viewed as a powerful volume-conserving mechanism, designed to decrease GFR in order to prevent massive losses of fluid/electrolytes due to impaired tubular reabsorption. Humoral mediation of TGF by the renin-angiotensin system has been proposed, and evidence suggests that other substances may be involved. The distal tubular cells contain numerous mitochondria but lack a well-developed brush border and an endocytotic apparatus characteristic of the pars convoluta of the proximal tubule. The early distal tubule reabsorbs most of the remaining intraluminal Na+ , K+ , and Cl– but is relatively impermeable to water. The late distal tubule, cortical collecting tubule, and medullary collecting duct perform the final regulation and fine-tuning of urinary volume and composition. The remaining Na+ is reabsorbed in conjunction with K+ and H+ secretion in the late distal tubule and cortical collecting tubule. The combination of medullary and papillary hypertonicity generated by countercurrent multiplication and the action of antidiuretic hormone (vasopressin, ADH) serve to enhance water permeability of the medullary collecting duct. Agents that interfere with ADH synthesis, secretion, or action therefore may impair concentrating ability. Additionally, because urinary concentrating ability is dependent upon medullary and papillary hypertonicity, agents that increase medullary blood flow may impair concentrating ability by dissipating the medullary osmotic gradient. Table 14-1 illustrates the efficiency of the nephrons in the conservation of electrolytes, substrates, and water and excretion of nitrogenous wastes (urea). The reader may refer Brenner and Rector’s The Kidney (2004) and Diseases of the Kidney and Urinary Tract (2006) for further review of renal physiology.

PATHOPHYSIOLOGIC RESPONSES OF THE KIDNEY Acute Kidney Injury One of the most common manifestations of nephrotoxic damage is acute renal failure—an abrupt decline in kidney function secondary to an injury that leads to a functional or structural change in the kidney (American Society of Nephrology, 2005). While acute renal failure is the commonly used terminology, it has numerous definitions; the American Society of Nephrology Research Report (2005) recently suggested that the term acute kidney injury (AKI) be used to describe the entire spectrum of the disease. AKI is defined as a complex disorder that comprises multiple causative factors and occurs

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Figure 14-4. Mechanisms of reduction of the GFR. A. GFR depends on four factors: (1) adequate blood flow to the glomerulus; (2) adequate glomerular capillary pressure; (3) glomerular permeability; and (4) low intratubular pressure. B. Afferent arteriolar constriction decreases GFR by reducing blood flow, resulting in diminished capillary pressure. C. Obstruction of the tubular lumen by cast formation increases tubular pressure; when tubular pressure exceeds glomerular capillary pressure, filtration decreases or ceases. D. Back-leak occurs when the paracellular space between cells increases and the glomerular filtrate leaks into the extracellular space and bloodstream. [From Molitoris BA, Bacallao R: Pathophysiology of ischemic acute renal failure: Cytoskeletal aspects, in Berl T, Bonventre JV (eds.): Atlas of Diseases of the Kidney. Philadelphia: Current Medicine, 1999, p. 13.5, with permission.]

in a variety of settings with varied clinical manifestations ranging from a minimal elevation in serum creatinine to anuric renal failure. Any decline in GFR is complex and may result from prerenal factors (renal vasoconstriction, intravascular volume depletion, and insufficient cardiac output), postrenal factors (ureteral or bladder obstruction), and intrarenal factors (glomerulonephritis, tubular cell injury, death, and loss resulting in back leak; renal vasculature damage, interstitial nephritis) (Fig. 14-4). Figure 14-5 illustrates the pathways that lead to diminished GFR following chemical exposure. As discussed above, pre- and postrenal factors can lead to decreased GFR. If a chemical causes tubular damage directly, then tubular casts can cause tubular obstruction, increased tubular pressure, and decreased GFR. The tubular damage may result in epithelial cell death/loss, leading to back leak of glomerular filtrate and a decrease in GFR. If a chemical causes intrarenal vascular damage with hemodynamic alterations that lead to vasoconstriction, the resulting medullary hypoxia may cause tubular damage and/or decreases in perfusion pressure, glomerular hydrostatic pressure, and GFR. If a chemical causes intrarenal inflammation, then tubular and vascular damage may follow with decreases in GFR. Finally, a chemical may disrupt glomerular function, resulting in decreased glomerular ultrafiltration and GFR. It has been estimated that prerenal factors are responsible for AKI in 55–60% of patients, intrarenal

factors are responsible for AKI in 35–40% of patients, and postrenal factors are responsible for AKI in 2 weeks), but—by comparison to the in vivo condition—exhibit differentiated functions and similarity to a lesser degree; this is particularly true of immortalized renal cell lines. The reader is referred to several excellent reviews for further details on the utility and limitations of these preparations (Tarloff and Kinter, 1997; Ford, 1997; Hart and Kinter, 2005; Kirkpatrick and Gandolfi, 2005; Ford, 2005). Such approaches may be used to distinguish between an effect on the kidney due to a direct chemical insult and one caused by extrarenal effects such as extrarenally generated metabolites, hemodynamic effects, immunologic effects, and so forth. Care must be taken to ensure that the cell type affected in the in vitro model is the same as that affected in vivo. In addition, concentrations of the nephrotoxicant to be used in the in vitro preparations must be comparable to those observed in vivo, as different mechanisms of toxicity may be operative at concentrations that saturate metabolic pathways or overwhelm detoxification mechanisms. Once a mechanism has been identified in vitro, the postulated mechanism must be tested in vivo. Thus, appropriately designed in vivo and in vitro studies should provide a complete characterization of the biochemical, functional, and morphologic effects of a chemical on the kidney and an understanding of the underlying mechanisms in the target cell population(s).

In Vitro Renal slices Freshly isolated and purified glomeruli Freshly isolated and purified tubular segments Freshly isolated and purified proximal tubular epithelial cells Primary cultures of renal cells Tubular epithelial cells Glomerular cells Fibroblasts Immortalized renal epithelial cell lines LLC-PK1 MDCK NRK-52E OK HK-2

BIOCHEMICAL MECHANISMS/MEDIATORS OF RENAL CELL INJURY Cell Death In many cases, renal cell injury may culminate in cell death. In general, cell death is thought to occur through either oncosis or apoptosis (Levin et al., 1999). The morphologic and biochemical characteristics of oncosis (“necrotic cell death”) and apoptosis are very different. For example, apoptosis is a tightly controlled, organized process that usually affects scattered individual cells. The organelles retain integrity while cell volume decreases. Ultimately, the cell breaks into small fragments that are phagocytosed by adjacent cells or macrophages without producing an inflammatory response. In contrast, oncosis often affects many contiguous cells; the organelles swell, cell volume increases, and the cell ruptures with the release of cellular contents, followed by inflammation. The reader is encouraged to see Brady et al. (2004) and Cummings and Schnellmann (2006) for additional details of apoptosis and oncosis. With many toxicants, lower but injurious concentrations produce cell death through apoptosis (Fig. 14-11). As the concentration of the toxicant increases, oncosis plays a predominant role. However, because apoptosis is an ATP-dependent process, for those toxicants that target the mitochondrion, oncosis may be the predominant pathway with only limited apoptosis occurring. In general, nephrotoxicants produce cell death through apoptosis and oncosis, and it is likely that both forms of cell death contribute to AKI.

Mediators of Toxicity A chemical can initiate cell injury by a variety of mechanisms (Fig. 14-12). In some cases the chemical may initiate toxicity due to its intrinsic reactivity with cellular macromolecules. For example,

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Figure 14-11. The general relationship between oncosis and apoptosis after nephrotoxicant exposure. For many toxicants, low concentrations primarily cause apoptosis and oncosis occurs principally at higher concentrations. When the primary mechanism of action of the nephrotoxicant is ATP depletion, oncosis may be the predominant cause of cell death, with limited apoptosis occurring. [From Schnellmann RG, Kelly KJ: Pathophysiology of nephrotoxic acute renal failure, in Berl T, Bonventre JV (eds.): Atlas of Diseases of the Kidney. Philadelphia: Current Medicine, 1999, p. 15.6, with permission.]

amphotericin B reacts with plasma membrane sterols, increasing membrane permeability; fumonisin B1 inhibits sphinganine (sphingosine) N -acyltransferase; and Hg2+ binds to sulfhydryl groups on cellular proteins. In contrast, some chemicals are not toxic until they are biotransformed to a reactive intermediate. Biologically reactive intermediates, also known as alkylating agents, are electron-deficient compounds (electrophiles) that bind to cellular nucleophiles (electron-rich compounds) such as proteins and lipids. For example, acetaminophen and chloroform are metabolized in the mouse kidney by cytochrome P450 to the reactive intermediates, N -acetyl- p-benzoquinoneimine and phosgene, respectively (see sections “Chloroform” and “Acetaminophen”). The covalent binding of the reactive intermediate to critical cellular macromolecules is thought to interfere with the normal biological activity of the macromolecule and thereby initiate cellular injury. In other instances, extrarenal biotransformation may be required prior to the delivery of the penultimate nephrotoxic species to the proximal tubule, where it is metabolized further to a reactive intermediate. Finally, chemicals may initiate injury indirectly by inducing oxidative stress via increased production of ROS, such as superoxide anion, hydrogen peroxide, and hydroxyl radicals. ROS can react with a variety of cellular constituents to induce toxicity. For example, ROS are capable of inducing lipid peroxidation, which may result in altered membrane fluidity, enzyme activity, and membrane permeability and transport characteristics; inactivating cellular enzymes by directly oxidizing critical protein sulfhydryl or amino groups; depolymerizing polysaccharides; and inducing DNA strand breaks and chromosome breakage. Each of these events could lead to cell injury and/or death. Oxidative stress has been proposed to contribute, at least in part, to the nephrotoxicity associated with ischemia/reperfusion injury, gentamicin, cyclosporine, cisplatin, and haloalkene cysteine conjugates (Chen et al., 1990; Groves et al., 1991; Ueda et al., 2001). While nitric oxide is an important second messenger in a number of physiologic pathways, recent studies suggest that in the presence of oxidative stress, nitric oxide can be converted into reactive nitrogen species that contribute to cellular injury and death. For

Figure 14-12. Covalent and noncovalent binding versus oxidative stress mechanisms of cell injury. Nephrotoxicants are generally thought to produce cell injury and death through one of two mechanisms, either alone or in combination. In some cases the toxicant may have a high affinity for a specific macromolecule or class of macromolecules that results in altered activity (increase or decrease) of these molecules and cell injury. Alternatively, the parent nephrotoxicant may not be toxic until it is biotransformed into a reactive intermediate that binds covalently to macromolecules and, in turn, alters their activity, resulting in cell injury. Finally, the toxicant may increase ROS in the cells directly, after being biotransformed into a reactive intermediate or through redox cycling. The resulting increase in ROS results in oxidative damage and cell injury. [From Schnellmann RG, Kelly KJ: Pathophysiology of nephrotoxic acute renal failure, in Berl T, Bonventre JV (eds.): Atlas of Diseases of the Kidney. Philadelphia: Current Medicine, 1999, p. 15.7, with permission.]

example, in the presence of superoxide anion, nitric oxide can be transformed into peroxynitrite (ONOO– ), a strong oxidant and nitrating species (Pryor and Squadrito, 1995). Proteins, lipids, and DNA are all targets of peroxynitrite. The primary evidence for a role of peroxynitrite in renal ischemia/reperfusion injury is the formation of nitrotyrosine-protein adducts and the attenuation of renal dysfunction through the inhibition of the inducible form of nitric oxide synthase (Ueda et al., 2001).

Cellular/Subcellular and Molecular Targets A number of cellular targets have been identified to play a role in cell death. It is generally thought that an intracellular interaction (e.g., an alkylating agent or ROS with a macromolecule) initiates a sequence of events that leads to cell death. In the case of oncosis, a “point of no return” is reached in which the cell will die regardless of any intervention. The idea of a single sequence of events is probably simplistic for most toxicants, given the extensive number of targets available for alkylating species and ROS. Rather, multiple pathways, with both distinct and common sequences of events, may lead to cell death.

Cell Volume and Ion Homeostasis Cell volume and ion homeostasis are tightly regulated and are critical for the reabsorptive properties of the tubular epithelial cells.

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Toxicants generally disrupt cell volume and ion homeostasis by interacting with the plasma membrane and increasing ion permeability or by inhibiting energy production. The loss of ATP, for example, results in the inhibition of membrane transporters that maintain the internal ion balance and drive transmembrane ion movement. Following ATP depletion, Na+ , K+ -ATPase activity decreases, resulting in K+ efflux, Na+ and Cl– influx, cell swelling, and ultimately cell membrane rupture. Miller and Schnellmann (1993, 1995) have proposed that ATP depletion in rabbit renal proximal tubule segments initially results in K+ efflux and Na+ influx followed by a lag period before Cl– influx occurs. Cl– influx occurs during the late stages of cell injury produced by a diverse group of toxicants and appears to be due to the volume-sensitive, outwardly rectifying (VSOR) Cl– channel (Okada et al., 2004). Cl– influx may be a trigger for cell swelling, because decreasing Cl– influx decreased cell swelling and cell death, and inhibition of cell swelling decreased cell lysis but not Cl– influx. Meng and Reeves (2000) have reported similar findings using hydrogen peroxide as the toxicant and LLCPK1 cells. In contrast, the cell shrinkage that occurs during apoptosis is mediated by K+ and Cl− efflux through respective channels and inhibition of these channels is cytoprotective (Okada et al., 2004).

Cytoskeleton and Cell Polarity Toxicants may cause early changes in membrane integrity such as loss of the brush border, blebbing of the plasma membrane, or alterations in membrane polarity. These changes can result from toxicantinduced alterations in cytoskeleton components and cytoskeletalmembrane interactions, or they may be associated with perturbations in energy metabolism or calcium and phospholipid homeostasis. Marked changes in the polarity of tubular epithelium occur following an ischemic insult. Under controlled conditions, the tubular epithelial cell is polarized with respect to certain transporters and enzymes. During in vivo ischemia and in vitro ATP depletion there is a dissociation of Na+ , K+ -ATPase from the actin cytoskeleton and redistribution from the basolateral membrane to the apical domain in renal proximal tubule cells (Molitoris, 1997). The redistribution of this enzyme has been postulated to explain decreased Na+ and water reabsorption during ischemic injury.

Mitochondria Many cellular processes depend on mitochondrial ATP and thus become compromised simultaneously with inhibition of respiration. Conversely, mitochondrial dysfunction may be a consequence of some other cellular process altered by the toxicant. Numerous nephrotoxicants cause mitochondrial dysfunction (Schnellmann and Griner, 1994). For example, following an in vivo exposure, HgCl2 altered isolated renal cortical mitochondrial function and mitochondrial morphology prior to the appearance of tubular necrosis (Weinberg et al., 1982a). Furthermore, HgCl2 produced similar changes in various respiratory parameters when added to isolated rat renal cortical mitochondria (Weinberg et al., 1982b). Different toxicants also produce different types of mitochondrial dysfunction. For example, pentachlorobutadienyl-l-cysteine initially uncouples oxidative phosphorylation in renal proximal tubular cells by dissipating the proton gradient, whereas TFEC does not uncouple oxidative phosphorylation but rather inhibits state 3 respiration by inhibiting sites I and II of the electron transport chain (Schnellmann et al., 1987, 1989; Wallin et al., 1987; Hayden and Stevens, 1990).

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Whether toxicants target mitochondria directly or indirectly, it is clear that mitochondria play a critical role in determining whether cells die by apoptosis or oncosis. The mitochondrial permeability transition (MPT) is characterized by the opening of a highconductance pore that allows solutes of 3 years) results in an often irreversible form of nephrotoxicity known as analgesic nephropathy (Palmer and Heinrich, 2004; Tarloff, 2005; De Broe, 2005). The incidence of analgesic nephropathy varies widely in the western world, ranging from less than 2–5% of all end-stage renal disease patients in countries where analgesic consumption is low (e.g., U.S. Canada), and up to 20% of all end-stage renal disease patients in countries with the highest analgesic consumption (e.g., Australia, Sweden). Impaired urinary concentration and acidification are the earliest clinical manifestations. The primary lesion in this nephropathy is papillary necrosis with chronic interstitial nephritis. Initial changes are to the medullary interstitial cells and are followed by degenerative changes to the medullary loops of Henle and medullary capillaries. Well-defined clinical signs have been associated with analgesic nephropathy and are helpful in the diagnosis thereof. De Broe (2005) and colleagues have developed an effective computed tomography (CT) protocol that does not use contrast media to diagnose analgesic nephropathy. While analgesic nephropathy is associated with a number of well-defined effects, the mechanism by which NSAIDs produce analgesic nephropathy is not known, but may result from chronic medullary/papillary ischemia secondary to renal vasoconstriction. Other studies have suggested that a reactive intermediate is formed in the cells that, in turn, initiates an oxidative stress, or binds covalently to critical cellular macromolecules. The third albeit rare type of nephrotoxicity associated with NSAIDs is an interstitial nephritis with a mean time of NSAID exposure to development of approximately 5 months (Tarloff, 2005; Palmer and Heinrich, 2004). This nephrotoxicity is characterized by a diffuse interstitial edema with mild to moderate infiltration of inflammatory cells. Patients normally present with elevated serum creatinine, proteinuria, and nephritic syndrome. If NSAIDs are discontinued, renal function improves in 1–3 months.

Aminoglycosides The aminoglycoside antibiotics are so named because they consist of two or more amino sugars joined in a glycosidic linkage to a central hexose nucleus. Whereas they are drugs of choice for many gram-negative infections, their use is primarily limited by their nephrotoxicity. The incidence of renal dysfunction following aminoglycoside administration ranges from 5 to 25%, but seldom leads to a fatal outcome (Servais et al., 2005; Palmer and Heinrich, 2004).

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Renal dysfunction by aminoglycosides is characterized by a nonoliguric renal failure with reduced GFR and an increase in serum creatinine and BUN. Polyuria is an early event following aminoglycoside administration and may be due to inhibition of chloride transport in the thick ascending limb (Kidwell et al., 1994). Within 24 hours, increases in urinary brush-border enzymes, glucosuria, aminoaciduria, and proteinuria are observed. Histologically, lysosomal alterations are noted initially, followed by damage to the brush border, endoplasmic reticulum, mitochondria, and cytoplasm, ultimately leading to tubular cell necrosis. Interestingly, proliferation of renal proximal tubule cells can be observed early after the onset of nephrotoxicity. Aminoglycosides are highly polar cations; they are almost exclusively filtered by the glomerulus and excreted unchanged. Filtered aminoglycosides undergo proximal tubular reabsorption by binding to anionic phospholipids in the brush border, followed by endocytosis and sequestration in lysosomes of the S1 and S2 segments of proximal tubules (Fig. 14-15). Basolateral membrane binding and uptake also may occur, but this is a minor contribution to the total proximal tubular uptake of aminoglycosides. The earliest lesion observed following clinically relevant doses of aminoglycosides is an increase in the size and number of lysosomes. These lysosomes contain myeloid bodies, which are electron-dense lamellar structures containing undergraded phospholipids. The renal phospholipidosis produced by the aminoglycosides is thought to occur through their inhibition of lysosomal hydrolases, such as sphingomyelinase and phospholipases. Whereas phospholipidosis plays an important role in aminoglycoside nephrotoxicity, the steps between the phospholipid accumulation in the lysosomes and tubular cell death are less clear. One hypothesis suggests that the lysosomes become progressively distended until they rupture, releasing lysosomal enzymes and high concentrations of aminoglycosides into the cytoplasm (Fig. 14-15). The released lysosomal contents can interact with various membranes and organelles and trigger cell death. Another mechanism of aminoglycoside nephrotoxicity includes a decrease in K f and GFR (see above).

Amphotericin B Amphotericin B is a very effective antifungal agent whose clinical utility is limited by its nephrotoxicity (Bernardo and Branch, 1997; Palmer and Heinrich, 2004). Renal dysfunction associated with amphotericin B treatment is dependent on cumulative dose and is due to both hemodynamic and tubular effects. With respect to hemodynamics, Amphotericin B administration is associated with decreases in RBF and GFR secondary to renal arteriolar vasoconstriction or activation of TGF. Amphotericin B nephrotoxicity is characterized by ADH-resistant polyuria, renal tubular acidosis, hypokalemia, and either acute or chronic renal failure. Amphotericin B nephrotoxicity is unusual in that it impairs the functional integrity of the glomerulus and of the proximal and distal portions of the nephron. Some of the renal tubular cell effects of amphotericin B are due to the ability of this polyene to bind to cholesterol in the plasma membrane and form aqueous pores. In the presence of amphotericin B, cells of the turtle and rat distal tubule do not produce a normal net outward flux of protons due to an increase in proton permeability (Steinmetz and Husted, 1982; Gil and Malnic, 1989). This results in impaired proton excretion and renal tubular acidosis. The hypokalemia observed with amphotericin B may be due to an increase in luminal potassium ion permeability in the late distal tubule and the cortical collecting duct and the loss of potassium ions in the urine.

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Figure 14-15. Renal handling of aminoglycosides: (1) glomerular filtration, (2) binding to the brush-border membranes of the proximal tubule, (3) pinocytosis, and (4) storage in the lysosomes. [From De Broe ME: Renal injury due to environmental toxins, drugs, and contrast agents, in Berl T, Bonventre JV (eds.): Atlas of Diseases of the Kidney. Philadelphia: Current Medicine, 1999, p. 11.4, with permission.]

Cyclosporine Cyclosporine is an important immunosuppressive agent and is widely used to prevent graft rejection in organ transplantation (Charney et al., 2005; Palmer and Heinrich, 2004). Cyclosporine is a fungal cyclic polypeptide and acts by selectively inhibiting T-cell activation. Nephrotoxicity is a critical side effect of cyclosporine, with nearly all patients who receive the drug exhibiting some form of nephrotoxicity. Clinically, cyclosporine-induced nephrotoxicity may manifest as (1) acute reversible renal dysfunction, (2) acute vasculopathy, and (3) chronic nephropathy with interstitial fibrosis (Mason and Moore, 1997; Dieperink et al., 1998). Acute renal dysfunction is characterized by dose-related decreases in RBF and GFR and increases in BUN and serum creatinine. These effects are lessened by reducing the dosage or by cessation of therapy. The decrease in RBF and GFR is related to marked vasoconstriction induced by cyclosporine; and it is probably produced by a number of factors, including an imbalance in vasoconstrictor and vasodilatory prostaglandin production. In particular, increased production of the vasoconstrictor thromboxane A2 appears to play a role in cyclosporine-induced ARF. Endothelin may contribute to constriction of the afferent arteriole because endothelin receptor antagonists inhibit cyclosporine-induced vasoconstriction (Lanese and Conger, 1993). Whereas cyclosporine can produce proximal tubular epithelial changes (many small equally sized vacuoles in the cytosol), it is still not clear whether a direct effect of cyclosporine on tubular cells plays a significant role in the nephrotoxicity. Acute vasculopathy or thrombotic microangiopathy is a rather unusual nephrotoxic lesion that affects arterioles and glomerular capillaries, without an inflammatory component, following cyclosporine treatment. The lesion consists of fibrin-platelet thrombi and fragmented red blood cells occluding the vessels (Charney et al., 2005). The pathogenesis of this lesion is poorly understood. Whereas the characteristics of this lesion differ from the vascular changes of acute rejection, a variety of factors may contribute to this lesion in the clinical transplant setting. Long-term treatment with cyclosporine can result in chronic nephropathy with interstitial fibrosis and tubular atrophy. Modest elevations in serum creatinine and decreases in GFR occur along with hypertension, proteinuria, and tubular dysfunction. Histologic changes are profound; they are characterized by arteriolopathy,

global and segmental glomerular sclerosis, striped interstitial fibrosis, and tubular atrophy. These lesions may not be reversible if cyclosporine therapy is discontinued and may result in end-stage renal disease. Whereas the mechanism of chronic cyclosporine nephropathy is not known, vasoconstriction probably plays a contributing role. Studies by Wang and Salahudeen (1994, 1995) indicated that rats treated with cyclosporine and an antioxidant lazaroid for 30 days exhibited increased GFR and RBF and less tubulointerstitial fibrosis and lipid peroxidation than rats treated with cyclosporine alone, suggesting that oxidative stress plays a role in cyclosporine nephrotoxity in rats. The marked interstitial cell proliferation and increased procollagen secretion that occurs following cyclosporine administration may contribute to the interstitial fibrosis (Racusen and Solez, 1993). Tacrolimus (FK-506) is a newer immunosuppressive agent that also exhibits nephrotoxicity. At this time, the degree and incidence of nephrotoxicity and morphologic changes associated with tacrolimus exposure are similar to that exhibited with cyclosporine, suggesting similar modes of toxic action.

Cisplatin Cisplatin is a valuable drug in the treatment of solid tumors, with nephrotoxicity limiting its clinical use. The kidney is not only responsible for the majority of cisplatin excreted but is also the primary site of accumulation. The effects of cisplatin on the kidney are several, including acute and chronic renal failure, renal magnesium wasting, and polyuria and patients treated with cisplatin regimens permanently lose 10–30% of their renal function (Bonegio and Lieberthal, 2005). Early effects of cisplatin are decreases in RBF and GFR produced by vasoconstriction and is followed by tubular injury with enzymuria. The early polyuria, has been suggested, to result from the inhibition of vasopressin release (Clifton et al., 1982). Although the primary cellular target associated with ARF is the proximal tubule S3 segment in the rat, in humans the S1 and S2 segments, distal tubule, and collecting ducts can also be affected. The chronic renal failure observed with cisplatin is due to prolonged exposure and is characterized by focal necrosis in numerous segments of the nephron without a significant effect on the glomerulus. Considerable

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effort has been expended in the development of measures to prevent cisplatin nephrotoxicity. These efforts include the use of extensive hydration and mannitol diuresis and the development of less nephrotoxic platinum compounds such as carboplatin. The mechanism by which cisplatin produces cellular injury is not known but may involve metabolites of cisplatin. For example, in a mouse model of cisplatin-induced nephrotoxicity the inhibition of γ -glutamyl transpeptidase or cysteine S-conjugate β-lyase blocked toxicity, suggesting that cisplatin-glutathione conjugates may be important in targeting cisplatin to the kidney and its resulting nephrotoxicity (Townsend and Hanigan, 2002). Interestingly, the trans isomer of cisplatin is not nephrotoxic even though similar concentrations of platinum are observed in the kidney after dosing. Thus, it is not the platinum atom per se that is responsible for the toxicity but rather the geometry of the complex or a metabolite. The antineoplastic and perhaps the nephrotoxic effects of cisplatin may be due to its intracellular hydrolysis to the reactive mono-chloro-mono-aquodiammine-platinum or diaquo-diammine-platinum species and the ability of these metabolites to alkylate purine and pyrimidine bases. In vitro studies using primary cultures of mouse proximal tubular cells revealed that the type of cell death produced by cisplatin is dependent on the concentration (Lieberthal et al., 1996). At cisplatin concentrations less than 100 μM, the primary form of cell death is apoptosis. As the concentration increases above 100 μM, a greater percentage of the cells die by oncosis. Using rabbit renal proximal tubule cells, Courjault et al. (1993) showed that while DNA synthesis, protein synthesis, glucose transport, Na+ , K+ -ATPase activity,

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and cell viability were all inhibited by cisplatin, DNA synthesis was the most sensitive. These results suggest that cisplatin may produce nephrotoxicity through its ability to inhibit DNA synthesis as well as transport functions. Finally, primarily through the use of antioxidants, in vivo and in vitro studies support a role for oxidative stress in cisplatin-induced nephrotoxicity (Bonegio and Lieberthal, 2005). The lack of complete return of renal function following cisplatin treatment in vivo may result from the interference of cisplatin with the normal proliferative response that occurs after injury. Radiocontrast Agents Iodinated contrast media are used for the imaging of tissues, with two major classes of compounds currently in use. The ionic compounds, diatrizoate derivatives, are (1) ionized at physiologic pH, (2) not significantly bound to protein, (3) restricted to the extracellular space, (4) almost entirely eliminated by the kidney, and (5) freely filtered by the glomerulus and neither secreted nor reabsorbed. These agents have a very high osmolality (>1200 mOsm/L) and are potentially nephrotoxic, particularly in patients with existing renal impairment, diabetes, or heart failure or who are receiving other nephrotoxic drugs. The newer contrast agents (e.g., iotrol, iopamidol) are nonionic owing to the addition of an organic side chain, their low osmolality, and their lower nephrotoxicity. The nephrotoxicity of these agents is due to both hemodynamic alterations (vasoconstriction) and proximal tubular injury (Kaperonis et al., 2005). The vasoconstriction is prolonged and is probably produced by more than one mediator while ROS are thought to play a role in the proximal tubular injury.

REFERENCES American Society of Nephrology: Renal research report. J Am Soc Nephrol 16:1886–1903, 2005. Arany I, Megyesi JK, Kaneto H, et al.: Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells. Am J Physiol Renal Physiol 87:F543, 2004. Arrington D, Van Vleet T, Schnellmann RG: Calpain 10: A mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am J Physiol 291:C1159–C1171, 2006. Bach PH: The renal medulla and distal nephron toxicity, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology, Vol 7. Oxford, England: Elsevier, 1997, pp. 279–298. Ballatori N: Mechanisms of metal transport across liver cell plasma membrane. Drug Metab Rev 23:83–132, 1991. Basnakian AG, Ueda N, Hong X, Galitovsky VE, Yin X, Shah SV: Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation. Am J Physiol 288:F308, 2005. Bernardo JF, Branch RA: Amphotericin B, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology, Vol 7. Oxford, England: Elsevier, 1997, pp. 475–494. Bonegio R, Lieberthal W: Cisplatin-induced nephrotoxicity, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca Raton, FL: CRC Press, 2005, pp. 779–815. Bonventre JV, Zuk A: Ischemic acute renal failure: An inflammatory disease? Kidney Int 66:480–485, 2004. Brady HR, Clarkson W, Lieberthal W: Acute renal failure, in Brenner BM (ed.): Brenner and Rector’s The Kidney, 7th ed., Philadelphia: WB Saunders, 2004, pp. 1215–1292. Brenner BM (ed.): Brenner and Rector’s The Kidney, 7th ed., Philadelphia: WB Saunders, 2004.

Brenner BM, Bohrer MP, Baylis C, Deen WM: Determinants of glomerular permselectivity: Insights derived from observations in vivo. Kidney Int 12:229–237, 1977. Brenner BM, Meyer TH, Hotstetter TH: Dietary protein intake and the progressive nature of kidney disease: The role of hemodynamically mediated glomerular injury in the pathogenesis of glomerular sclerosis in agina, renal ablation and intrinsic renal disease. N Engl J Med 307:652– 659, 1982. Brezis M, Epstein FH: Pathophysiology of acute renal failure, in Hook JB, Goldstein RS (eds.): Toxicology of the Kidney, 2nd ed. New York: Raven Press, 1993, pp. 129–152. Brezis M, Rosen S, Silva P: Transport activity modifies thick ascending limb damage in isolated perfused kidney. Kidney Int 25:65–72, 1984. Bruschi SA, West K, Crabb JW, et al.: Mitochondrial HSP60 (P1 protein) and a HSP-70 like protein (mortalin) are major targets for modification during S-(1,1,2,2-tetrafluorethyl)-l-cysteine induced nephrotoxicity. J Biol Chem 268:23157–23161, 1993. Bucci TJ, Howard PC, Tolleson WH, et al.: Renal effects of fumonisin mycotoxins in animals. Toxicol Pathol 26:160–164, 1998. Burne MJ, Daniels F, El Ghandour A, et al.: Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure. J Clin Invest 108:1065–1073, 2001. Burne-Taney MJ, Ascon DB, Daniels F, Racusen L, Baldwin W, Rabb H: B cell deficiency confers protection from renal ischemia reperfusion injury. J Immunol 171:3210–3215, 2003. Charney D, Solez K, Racusen LC: Nephrotoxicity of cyclosporine and other immunosuppressive and immunotherapeutic agents, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 687–777.

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Chen Q, Jones TW, Brown PC, Stevens JL: The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells. J Biol Chem 265:21603– 21611, 1990. Chen Q, Yu K, Stevens JL: Regulation of the cellular stress response by reactive electrophiles: The role of covalent binding and cellular thiols in transcriptional activation of the 70-kDa heat shock protein gene by nephrotoxic cysteine conjugates. J Biol Chem 267:24322–24327, 1992. Clifton GG, Pearce C, O’Neill WM Jr, Wallin JD: Early polyuria in the rat following single-dose cis-dichlorodiammineplatinum (II): Effects on plasma vasopressin concentration and posterior pituitatary function. J Lab Clin Med 100:659–670, 1982. Conner EA, Fowler BA: Mechanisms of metal-induced nephrotoxicity, in Hook JB, Goldstein RS (eds.): Toxicology of the Kidney, 2nd ed. New York: Raven Press, pp. 437–457, 1993. Counts RS, Nowak G, Wyatt RD, Schnellmann RG: Nephrotoxicants inhibition of renal proximal tubule cell regeneration. Am J Physiol 269:F274– F281, 1995. Courjault F, Leroy D, Coquery I, Toutain H: Platinum complex-induced dysfunction of cultured renal proximal tubule cells. Arch Toxicol 67:338– 346, 1993. Cummings BS, Kinsey GR, Bolchoz LJ, Schnellmann RG: Identification of caspase-independent apoptosis in epithelial and cancer cells. J Pharmacol Exp Ther 310:126, 2004a. Cummings BS, McHowat J, Schnellmann RG: Phospholipase A2 s in cell injury and death. J Pharmacol Exp Ther 294(3):793–799, 2000. Cummings BS, McHowat J, Schnellmann RG: Role of an endoplasmic reticulum Ca2+ -independent phospholipase A2 in oxidant-induced renal cell death. Am J Physiol 283:F492, 2002. Cummings BS, McHowat J, Schnellmann RG: Role of an endoplasmic reticulum Ca2+ -independent phospholipase A2 in cisplatin-induced renal cell apoptosis. J Pharmacol Exp Ther 308:921, 2004b. Cummings BS, Schnellmann RG: Pathophysiology of nephrotoxic cell injury, in Schrier RW (ed.): Diseases of the Kidney and Urinary Tract, 8th ed. Philadelphia: Lippincott, 2006, pp. 962–985. Daemen MARC, van ‘tVeer C, Denecker G, et al.: Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104:541–549, 1999. De Broe ME: Renal injury due to environmental toxins, drugs, and contrast agents, in Berl T, Bonventre JV (eds.): Atlas of Diseases of the Kidney. Philadelphia: Current Medicine, 1999, pp. 11.2–11.14. De Broe ME: Anagesic nephropathy, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., CRC Press: Boca Raton, FL, 2005, pp. 619–634. Dekant W: Chemical-induced nephrotoxicity mediated by glutathione Sconjugate formation, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 995–1020. Dieperink H, Perico N, Nielsen FT, Remuzzi G: Cyclosporine/tacrolimus (FK-506), in DeBroe ME, Porter GA, Bennett AM, Verpooten GA (eds.): Clinical Nephrotoxicants, Renal Injury from Drugs and Chemicals. The Netherlands: Kluwer, 1998, pp. 275–300. Schrier RW (ed.): Diseases of the Kidney and Urinary Tract, 8th ed., Philadelphia: Lippincott, 2006. Dong J, Ramachandiran S, Tikoo K, Jia Z, Lauu SS, Monks TJ: EGFRindependent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 are required for ROS-induced renal cell death. Am J Physiol Renal Physiol 287:F1049–1058, 2004. Duffield JS, Park KW, Hsiao LL, et al.: Restoration of tubular epithelial cells during repair of the postischemic kidney occurs undependently of bone marrow-derived stem cells. J Clin Invest 115:1743–1755, 2005. Dunn RB, Anderson S, Brenner B: The hemodynamic basis of progressive renal disease. Semin Nephrol 6:122–138, 1986. Edelstein CL, Yaqoob MM, Schrier RW: The role of calcium-dependent enzymes nitric oxide synthase and calpain in hypoxia-induced proximal tubule injury. Ren Fail, 501–511, 1996. Eknoyan G, Bulger RE, Dobyan DC: Mercuric chloride-induced acute renal failure in the rat: I. Correlation of functional and morphologic changes and their modification by clonidine. Lab Invest 46:613–620, 1982.

Elfarra AA: Halogenated hydrocarbons, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology, Vol 7. Oxford, England: Elsevier, 1997, pp. 601–616. Emeigh Hart SG, Wyand DS, Khairallah EA, Cohen SD: A role for the glutathione conjugate and renal cytochrome P450 in acetaminophen (APAP) induced nephrotoxicity in the CD-1 mouse. Toxicologist 11:57, 1990. Emeigh Hart SGE, Beierschmitt WP, Wyand DS, et al.: Acetaminophen nephrotoxicity in CD-1 Mice. I. Evidence of a role for in situ activation in selective covalent binding and toxicity. Toxicol Appl Pharmacol 126:267–275, 1994. Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A: Cystatin C as a maker of GFR-history, indications and future research. Clin Biochem 38:1–8, 2005. Ford SM: In vitro toxicity systems, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7. Oxford, England: Elsevier, 1997, pp. 121–142. Ford SM: In vitro techniques in screening and mechanistic studies: Cell culture, cell-free systems, and molecular and cell biology, in Tarloff JB,Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 191–213. Friedewald JJ, Rabb H: Inflammatory cells in ischemic acute renal failure. Kidney Int 66:486–491, 2004. Fukino H, Hirai M, Hsueh YM, Yamane Y: Effect of zinc pretreatment on mercuric chloride–induced lipid peroxidation in the rat kidney. Toxicol Appl Pharmacol 73:395–401, 1984. Gil FZ, Malnic G: Effect of amphotericin B on renal tubular acidification in the rat. Pflugers Arch 413:280–286, 1989. Goering PL, Fisher BR, Chaudhary PP, Dick CA: Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity. Toxicol Appl Pharmacol 113:184–191, 1992. Goering PL, Fisher BR, Noren BT, et al: Mercury induces regional and cellspecific stress protein expression in rat kidney. Toxicol Sci 53:447–457, 2000. Goldstein RS: Biochemical heterogeneity and site-specific tubular injury, in Hook JB, Goldstein RS (eds.): Toxicology of the Kidney, 2nd ed. New York: Raven Press, 1993, pp. 201–248. Goligorsky MS, Lieberthal W, Racusen L, Simon EE: Integrin receptors in renal tubular epithelium: New insights into pathophysiology of acute renal failure. Am J Physiol 264:F1–F8, 1993. Gopee NV, He Q, Sharma RP: Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-kappaB and tumor necrosis factor alpha in LLC-PK1 cells. Chem Biol Interact 146:131–145, 2003. Groves CE, Hayden PJ, Lock EA, Schnellmann RG: Differential cellular effects in the toxicity of haloalkene and haloalkane cysteine conjugates to rabbit renal proximal tubules. J Biochem Toxicol 8:49–56, 1993. Groves CE, Lock EA, Schnellmann RG: The role of lipid peroxidation in renal proximal tubule cell death induced by haloalkene cysteine conjugates. J Toxicol Appl Pharmacol 107:54–62, 1991. Hammerman MR, Miller SB: Therapeutic use of growth factors in renal failure. J Am Soc Nephrol 5:1–11, 1994. Harriman JF, Liu XL, Aleo MD, Machaca K, Schnellmann RG: Endoplasmic reticulum Ca2+ signaling and calpains mediate renal cell death. Cell Death Differ 9:734–741, 2002. Hart SE, Kinter LB: Assessing renal effects of toxicants in vivo, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 81–147. Hayden PJ, Stevens JL: Cysteine conjugate toxicity, metabolism and binding to macro-molecules in isolated rat kidney mitochondria. Mol Pharmacol 37:468–476, 1990. Hayden PJ, Welsh CJ, Yang Y, et al.: Formation of mitochondrial phospholipid adducts by nephrotoxic cysteine conjugate metabolites. Chem Res Toxicol 5:231–237, 1992. Hayden PJ, Yang Y, Ward AJ, et al.: Formation of diflourothionoacetylprotein adducts by S-(1,1,2,2-tetrafluoroethyl)-l-cysteine metabolites: Nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine. Biochemistry 30:5935–5943, 1991.

CHAPTER 14

TOXIC RESPONSES OF THE KIDNEY

Holmes E, Cloarec O, Nicholson JK: Probing latent biomarker signatures and in vivo pathway activity in experimental disease states via statistical total correlation spectroscopy (STOCSY) of biofluids: Application to HgCl2 toxicity. J Proteome Res 5:1313–1320, 2006. Ikeda M, Prachasilchai W, Burner-Taney MJ, Rabb H, Yokota-Ikeda N: Ischemic acute tubular necrosis models and drug discovery: A focus on cellular inflammation. Drug Discov Today 11:364–370, 2006. Jarnberg P: Renal toxicity of anesthetic agents, in DeBroe ME, Porter GA, Bennett AM, Verpooten GA (eds.): Clinical Nephrotoxicants, Renal Injury from Drugs and Chemicals. The Netherlands: Kluwer, 1998, pp. 413–418. Kanwar YS, Liu ZZ, Kashihara N, Wallner EI: Current status of the structural and functional basis of glomerular filtration and proteinuria. Semin Nephrol 11:390–413, 1991. Kaperonis N, Krause M, Bakris GL: The pathogenesis and prevention of radiocontrast medium induced renal dysfunction, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca Raton, FL: CRC Press, 2005, pp. 817–860. Kaushal GP, Singh AB, Shah SV: Identification of gene family cf caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol 274:F587–F595, 1998. Kelly KJ: Heat shock (stress response) proteins and renal ischemia/ reperfusion injury. Contrib Nephrol 148:86–106, 2005. Kelly KJ, Williams WW, Colvin RB, Bonventre JV: Antibody to intercellular adhesion molecule 1 protects the kidney against ischemic injury. Proc Natl Acad Sci U S A 91:812–816, 1994. Kido T, Nordberg G: Cadmium-induced renal effects in the general environment, in DeBroe ME, Porter GA, Bennett AM, Verpooten GA (eds.): Clinical Nephrotoxicants, Renal Injury from Drugs and Chemicals. The Netherlands: Kluwer, 1998, pp. 345–362. Kidwell DT, KcKeown JW, Grider JS, et al.: Acute effects of gentamicin on thick ascending limb function in the rat. Eur J Pharmaco Environ Toxicol Pharmacol Section 270:97–103, 1994. Kinsey GR, McHowat J, Beckett C, Schnellmann RG: Identification of calcium-independent phospholipase A2γ in mitochondria and its role in mitochondrial oxidative stress. Am J Physiol 292:F853–F860, 2007. Kirkpatrick DS, Gandolfi AJ: In vitro techniques in screening and mechanistic studies: Organ perfusion, slices, and nephron components, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 149–189. Klaassen CD, Liu J, Choudhuri S: Metallothionein: An intracellular protein to protect cadmium toxicity. Ann Rev Pharmacol Toxicol 39:267–294, 1999. Komatsuda A, Wakui H, Satoh K, et al.: Altered localization of 73-kilodalton heat shock protein in rat kidneys with gentamicin–induced acute tubular injury. Lab Invest 68:687–695, 1993. Lanese DM, Conger JD: Effects of endothelin receptor antagonist on cyclosporine-induced vasoconstriction in isolated rat renal arterioles. J Clin Invest 91:2144–2149, 1993. Lauwerys RR, Bernard AM, Roels HA, Buchet JP: Cadmium: Exposure markers as predictors of nephrotoxic effects. Clin Chem 40:1391–1394, 1994. Lehman-McKeeman LD: α2u -globulin nephropathy, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology, Vol 7. Oxford, England: Elsevier, 1997, pp. 677–692. Leiberthal W, Triaca V, Levine J: Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: Apoptosis vs. necrosis. Am J Physiol 270:F700–F708, 1996. Lemasters JJ, Qian T, Bradham CA, et al.: Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr 31:305–319, 1999. Leonard I, Zanen J, Nonclercq D, et al.: Modification of immunoreactive EGF and EGF receptor after acute tubular necrosis induced by tobramycin or cisplatin. Ren Fail 16(5):583–608, 1994. Levin S, Bucci TJ, Cohen SM, et al.: The nomenclature of cell death: Recommendations of an ad hoc committee of the society of toxicologic pathologists. Tox Pathol 27:484–490, 1999.

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Lin F, Moran A, Igarashi P: Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney. J Clin Invest 115:1756–1764, 2005. Lindon JC, Holmes E, Nicholson JK: Metabonomics techniques and applications to pharmaceutical research and development. Pharm Res 23:1075–1088, 2006. Liu X, Van Vleet T, Schnellmann RG: The role of calpain in oncotic cell death. Ann Rev Pharmacol Toxicol 44:349–370, 2004. Liu X, Schnellmann RG: Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death. J Pharmacol Exp Ther 304:63–70, 2003. Lock EA, Reed CJ: Renal xenobiotic metabolism, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology, Vol 7. Oxford, England: Elsevier, 1997, pp. 77–98. Lund BO, Miller DM, Woods JS: Studies in Hg(II)-induced H2 O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria. Biochem Pharmacol 45:2017–2024, 1993. Maddox DA, Brenner BM: Glomerular ultrafiltration, in Brenner BM, Rector FC (eds.): The Kidney, 4th ed. Philadelphia: WB Saunders, 1991, pp. 205–244. Mason J, Moore LC: Cyclosporine, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7 Oxford, England: Elsevier, 1997, pp. 567–582. Melnick R: An alternative hypothesis on the role of chemically induced protein droplet (α2u -globulin) nephropathy in renal carcinogenesis. Reg Toxicol Pharmacol 16:111–125, 1992. Meng X, Reeves WB: Effects of chloride channel inhibitors on H2 O2 induced renal epithelial cell injury. Am J Physiol Renal Physiol 278:F83–F90, 2000. Miller GW, Schnellmann RG: Cytoprotection by inhibition of chloride channels: The mechanism of action of glycine and strychnine. Life Sci 53:1211–1215, 1993. Miller GW, Schnellmann RG: Inhibitors of renal chloride transport do not block toxicant-induced chloride influx in the proximal tubule. Toxicol Lett 76:179–184, 1995. Molitoris BA: Alterations in surface membrane composition and fluidity: Role in cell injury, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7. Oxford, England: Elsevier, 1997, pp. 317–328. Nowak G: Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatininduced apoptosis in renal cells. J Biol Chem 277:43377–43388, 2002. Nowak G: Protein kinase C mediates repair of mitochondrial and transport functions after toxicant-induced injury in renal cells. J Pharmacol Exp Ther 306:157–165, 2003. Nowak G, Bakajsova D, Clifton GL: Protein kinase C-{epsilon} modulates mitochondrial function and active Na+ transport after oxidant injury in renal cells. Am J Physiol Renal Physiol 286:F307, 2004. O’Brien E, Dietrich DR: Mycotoxins affecting the kidney, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 895–936. Okada Y, Maeno E, Shimizu T, Manabe K, Mori S, Nabekura T: Dual roles of plasmalemmal chloride channels in induction of cell death. Pflugers Arch 448:287–295, 2004. Palmer BF, Heinrich WL: Toxic nephropathy, in Brenner BM (ed.): Brenner and Rector’s The Kidney, 7th ed., Philadelphia: WB Saunders, 2004, pp. 1625–1658. Pryor WA, Squadrito GL: The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide Am J Physiol 268:L699–L722, 1995. Rabb H, Daniels F, O’Donnell M, et al.: Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice. Am J Physiol 279:F525–F531, 2000. Rabb H, Mendiola CC, Dietz J, et al.: Role of CD11a and CD11b in ischemic acute renal failure in rats. Am J Physiol 267:F1052–F1058, 1994.

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Racusen LC, Solez K: Nephrotoxicity of cyclosporine and other immunotherapeutic agents, in Hook JB, Goldstein RS (eds.): Toxicology of the Kidney, 2nd ed. New York: Raven Press, 1993, pp. 319– 360. Ramachandiran S, et al.: Mitogen-activated protein kinases contribute to reactive oxygen species-induced cell death in renal proximal tubule epithelial cells. Chem Res Toxicol 15:1635, 2002. Rankin GO, Valentovic MA: Role of xenobiotic metabolism, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca Raton, FL: CRC Press, 2005, pp. 217–243. Safirstein R, Deray G: Anticancer, cisplatin/carboplatin, in DeBroe ME, Porter GA, Bennett AM, Verpooten GA (eds.): Clinical Nephrotoxicants, Renal Injury from Drugs and Chemicals. The Netherlands: Kluwer, 1998, pp. 261–272. Sapirstein A, Spech RA, Witzgall R, et al.: Cytosolic phospholipase A2 (PLA2 ) but not secretory PLA2 , potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells. J Biol Chem 271(35):21505–21513, 1996. Servais H, Mingeot-Leclercq M-P, Tulkens PM: Antibiotic-induced nephrotoxicity, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca Raton, FL: CRC Press: 2005 pp. 635–685. Schnellmann RG: Analgesic nephropathy in rodents. J Toxicol Environ Health, Part B 1:81–90, 1998. Schnellmann RG, Cross TJ, Lock EA: Pentachlorabutadienyl-l-cysteine uncouples oxidative phosphorylation by dissipating the proton gradient. Toxicol Appl Pharmacol 100:498–505, 1989. Schnellmann RG, Cummings BS: Pathophysiology of nephrotoxic cell injury, in Schrier RW (ed.): Diseases of the Kidney and Urinary Tract, 8th ed. Philadelphia: Lippincott, 2006, pp. XXX-YYY. Schnellmann RG, Griner RD: Mitochondrial mechanisms of tubular injury, in Goldstein RS (ed.): Mechanisms of Injury in Renal Disease and Toxicity. Boca Raton, FL: CRC, 1994, pp. 247–265. Schnellmann RG, Lock EA, Mandel LJ: A mechanism of S-(1,2,3,4,4pentachloro-1,3-butadienyl)-l-cysteine toxicity to rabbit renal proximal tubules. Toxicol Appl Pharmacol 90:513–521, 1987. Schnellmann RG, Williams SW: Proteases in renal cell death: Calpains mediate cell death produced by diverse toxicants. Ren Fail 20(5):679–686, 1998. Sheikh-Hamad D, Cacini W, Buckley AR, et al.: Cellular and molecular studies on cisplatin-induced apoptotic cell death in rat kidney. Arch Toxicol 78:147–155, 2004. Smith JH, Maita K, Sleight SD, Hook JB: Effect of sex hormone status on chloroform nephrotoxicity and renal mixed function oxidases in mice. Toxicology 30:305–316, 1984. Smith MW, Ambudkar IS, Phelps PC, et al.: HgCl2 -induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells. Biochem Biophys Acta 931:130–142, 1987. Steinmetz PR, Husted RF: Amphotericin B toxicity for epithelial cells, in Porter GA (ed.): Nephrotoxic Mechanisms of Drugs and Environmental Toxins. New York, London: Plenum, 1982, pp. 95–98. Stevens LA, Levey AS: Measurement of Kidney Function. Med Clinics of North America 89:457–473, 2005. Takaoka M, Itoh M, Hayashi S, Kuro T, Matsumura Y: Proteasome participates in the pathogenesis of ischemic acute renal failure in rats. Eur J Pharmacol 384:43–46, 1999. Tarloff JB: Analgesics and nonsteroidal anti-inflammatory drugs, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7. Oxford, England: Elsevier, 1997, pp. 583–600. Tarloff JB: Analgesics and nonsteroidal anti-inflammatory drugs, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed., Boca Raton, FL: CRC Press, 2005, pp. 861–894.

Tarloff JB, Kinter LB: In vivo methodologies used to assess renal function, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7. Oxford, England: Elsevier, 1997, pp. 99–120. Townsend DM, Hanigan MH: Inhibition of γ tglutamyl transpeptidase or cysteine S-conjugate β-lyase activity blocks the nephrotoxicity of cisplain in mice. J Pharmacol Exptl Ther 300:142–148, 2002. Ueda N, Mayeux PR, Baglia R, Shah SV: Oxidant mechanisms in acute renal failure, in Molitoris BA, Finn W (eds.): Acute Renal Failure: A Companion to Brenner’s and Rector’s The Kidney. St. Louis: WB Saunders, 2001, pp. F853–F860. Ueda N, Walker PD, Hsu SM, Shah SV: Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis. Proc Natl Acad Sci USA 92:7202–7206, 1995. van de Water B, de Graauw M, Le Devedec S, Alderliesten M: Cellular stress responses and molecular mechanisms of nephrotoxicity. Toxicol Lett 162:83–93, 2006. van de Water B, Tijdens IB, Verbrugge A, et al.: Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatininduced apoptosis of renal epithelial cells. J Biol Chem 275:25805– 25813, 2005. Wallin A, Jones TW, Vercesi AE, et al.: Toxicity of S-pentachorobutadienyll-cysteine studied with isolated rat renal cortical mitochondria. Arch Biochem Biophys 258:365–372, 1987. Wang C, Salahudeen AK: Cyclosporine nephrotoxicity: Attenuation by an antioxidant-inhibitor of lipid peroxidation in vitro and in vivo. Transplantation 58:940–946, 1994. Wang C, Salahudeen AK: Lipid peroxidation accompanies cyclosporine nephrotoxicity: Effects of vitamin E. Kidney Int 47:927–934, 1995. Wang Y, Bollard ME, Nicholson JK, Holmes E: Exploration of the direct metabolic effects of mercury II chloride on the kidney on SpragueDawley rats using high-resolution magic angle spinning 1H NMR spectroscopy of intact tissue and patter recognition. J Pharm Biomed Anal 40:375–381, 2006. Waters SL, Sarang SS, Wang KKW, Schnellmann RG: Calpains mediate calcium and chloride influx during the late phase of cell injury. J Pharmacol Exp Ther 283:1177–1184, 1997a. Waters SL, Wong JK, Schnellmann RG: Depletion of endoplasmic reticulum calcium stores protects against hypoxia- and mitochondrial inhibitor– induced cellular injury and death. Biochem Biophys Res Commun 240:57–60, 1997b. Weinberg JM, Harding PG, Humes HD: Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury: I. Direct effects of in vitro mercuric chloride on renal cortical mitochondrial function. J Biol Chem 257:60–67, 1982b. Weinberg JM, Harding PG, Humes HD: Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury: II. Functional alterations of renal cortical mitochondria isolated after mercuric chloride treatment. J Biol Chem 257:68–74, 1982a. Woo KR, Shu WP, Kong L, Liu BC: Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells. J Urol 155:1779–1783, 1996. Zalups RK: Renal toxicity of mercury, in Sipes IG, McQueen CA, Gandolfi AJ (eds.): Comprehensive Toxicology. Vol 7. Oxford, England: Elsevier, 1997, pp. 633–652. Zalups RK, Diamond GL: Nephrotoxicology of metal, in Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca Raton, FL: CRC Press, 2005, pp. 937–994. Zalups RK, Lash LH: Advances in understanding the renal transport and toxicity of mercury. J Toxicol Environ Health 42:1–44, 1994.

CHAPTER 15

TOXIC RESPONSES OF THE RESPIRATORY SYSTEM Hanspeter R. Witschi, Kent E. Pinkerton, Laura S. Van Winkle, and Jerold A. Last Emphysema Fibrosis Asthma Lung Cancer The Developing Lung

LUNG STRUCTURE AND FUNCTION Nasal Passages Conducting Airways Gas Exchange Region Gas Exchange Ventilation Perfusion Diffusion Distribution of Metabolic Competence in the Respiratory Tract

AGENTS KNOWN TO PRODUCE LUNG INJURY IN HUMANS Airborne Agents That Produce Lung Injury in Humans Asbestos Silica Naphthalene Blood-borne Agents That Cause Pulmonary Toxicity in Humans Bleomycin Cyclophosphamide and 1,3 Bis (2-Chloroethyl)-1-Nitrosourea (BCNU)

GENERAL PRINCIPLES IN THE PATHOGENESIS OF LUNG DAMAGE CAUSED BY CHEMICALS Toxic Inhalants, Gases, and Dosimetry Particle Deposition Particle Size Nanotoxicology Deposition Mechanisms Particle Clearance Nasal Clearance Tracheobronchial Clearance Pulmonary Clearance

EVALUATION OF TOXIC LUNG DAMAGE

CHRONIC RESPONSES OF THE LUNG TO INJURY

Studies Being Done in Humans Studies Being Done in Animals Inhalation Exposure Systems Pulmonary Function Tests in Experimental Animals Morphologic Techniques Pulmonary Lavage In Vitro Approaches Isolated Perfused Lung Microdissection Organotypic Cell Culture Systems Isolated Lung Cell Populations

Lung injury caused by chemicals was first recognized as an occupational disease. In 1713, the Italian physician Bernardino Ramazzini provided detailed and harrowing accounts of the sufferings of miners. Two of his quotations remain noteworthy. With regard to miners of metals, he stated, “the lungs and brains of that class of workers are badly affected, the lungs especially, since they take in with the air mineral spirits and are the first to be keenly aware of injury.” Ramazzini was also aware of the important concept of exposure: “They (workers who shovel, melt, cast and refine mined material) are liable of the same diseases, though in less acute form, because they perform their tasks in open air (Ramazzini, 1964).” Thus, exposure to chemicals by inhalation can have two effects: on the lung tissues and on distant organs that are reached after chemicals enter the body by means of inhalation. Indeed, “inhalation toxicology” refers to the route of exposure, whereas “respiratory tract toxicology” refers to target organ toxicity, in this case abnormal changes in the respiratory tract produced by airborne (and occasionally blood-borne) agents.

We now know of numerous lung diseases prompted by occupational exposures, many crippling and some fatal. Examples include black lung in coal miners, silicosis and silicotuberculosis in sandblasters and tunnel miners, and asbestosis in shipyard workers and asbestos miners. Occupational exposures to asbestos or metals such as nickel, beryllium, and cadmium can also cause lung cancer. In the twentieth century, it has become obvious that disease caused by airborne agents may not be limited to certain trades. The ubiquitous presence of airborne chemicals is a matter of concern, since “air pollution” adversely affects human health and may be an important contributor to morbidity and mortality. To better understand environmental lung disease, we need more precise knowledge about the doses of toxic inhalants delivered to specific sites in the respiratory tract and an understanding of the extent to which repeated and often intermittent low-level exposures eventually may initiate and propagate chronic lung disease. Inhalation and respiratory tract toxicology cover a field in which

ACUTE RESPONSES OF THE LUNG TO INJURY Mechanisms of Respiratory Tract Injury Oxidative Burden Mediators of Lung Toxicity Airway Reactivity Pulmonary Edema

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Figure 15-1. Predicted fractional deposition of inhaled particles in the nasopharyngeal, tracheobronchial, and alveolar region of the human respiratory tract during nose breathing. Based on data from the International Commission on Radiological Protection (1994). (Drawing courtesy of J. Harkema.) [From Fig. 8 in Oberdorster et al., 2005a. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect Vol. 113, No 7. 823–839, 2005.]

epidemiologists, physiologists, toxicologists, and cell and molecular biologists must closely work together. Epidemiologists now use a variety of pulmonary function tests to assess decrements in lung function in people. Many of these tests have been adapted for animal studies. Bronchoalveolar lavage is widely utilized in experimental animals and human subjects to examine airways contents after exposure. When similar data can be obtained in both experimental animals and humans, these direct comparisons assist in extrapolation and modeling. Progress has been made in understanding some mechanisms that underlie the response of the lung to toxic agents. In response to toxic insult, pulmonary cells are known to release a variety of potent chemical mediators that may critically affect lung function. Biochemical data from the study of cells taken from exposed animals and in vitro exposure of cells are also useful in assessing the toxic potential of many agents. This chapter will discuss how pulmonary toxicologists profit from these methods to study the biochemical, structural, and functional changes produced by the inhalation of gases and particles in both humans and experimental animals.

LUNG STRUCTURE AND FUNCTION Nasal Passages Figure 15-1 shows a schematic overview of the different regions of the respiratory tract. Air enters the respiratory tract through the nasal and oral regions. Many species, particularly small laboratory rodents, are obligate nose breathers in which air passes almost exclusively through the nasal passages. Other species, including humans, monkeys, and dogs can inhale air through both the nose and the mouth (oronasal breathers). Air is warmed and humidified while passing through the nose. The nasal passages function as a filter for particles, which may be collected by diffusion or impaction on the nasal mucosa. Highly water soluble gases are absorbed efficiently in the nasal passages, which reach from the nostril to the pharynx. The nasal turbinates thus form a first defensive barrier against many toxic inhalants. The nasal passages are lined by distinctive epithelia: stratifiedsquamous epithelium in the vestibule, nonciliated cuboidal/

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columnar epithelium in the anterior chamber, and ciliated pseudostratified respiratory epithelium. Located in the superior part is the olfactory epithelium, which contains sensory cells. Nerve endings in the nasal passages are associated mostly with the fifth cranial (trigeminal) nerve. Nasal epithelia are competent to metabolize foreign compounds and P-450 isozymes have been localized in the nose of several species by immunohistochemical procedures. The nasal cavity is thus a ready target site for metabolite-induced lesions.

Conducting Airways The proximal airways (trachea and bronchi) of humans have a pseudostratified epithelium containing ciliated cells and two types of nonciliated cells: mucous and serous cells. Mucous cells (and glandular structures) produce respiratory tract mucus, a family of high-molecular-weight glycoproteins with a sugar content of 80% or more. They coat the epithelium with a viscoelastic sticky protective layer that traps pollutants and cell debris. Serous cells produce a fluid in which mucus may be dissolved, or upon which a mucus layer may be floated. The action of the respiratory tract cilia continuously drives the mucus layer toward the pharynx, where it is removed from the respiratory system by swallowing or expectoration. The mucus layer is also thought to have antioxidant, acid-neutralizing, and free radical scavenging functions that protect the epithelial cells (Cross et al., 1998). Conducting airways have a characteristic branched bifurcating structure, with successive airway generations containing approximately twice the number of bronchi progressively decreasing in internal diameter. Thus, the conducting airways contain a continuously increasing total surface area from the trachea to the distal airways. Bifurcations are flow dividers and airway branch points serve as sites of impaction for particles. Successively narrower diameters also favor the collection of gases and particles on airway walls. Eventually a transition zone is reached where cartilaginous airways (bronchi) give way to noncartilaginous airways (bronchioles), which in turn give way to gas exchange regions, respiratory bronchioles, and alveoli. In the bronchiolar epithelium, mucus-producing cells and glands give way to Clara cells.

Gas Exchange Region Human lungs are divided into five lobes: the superior and inferior left lobes and the superior, middle, and inferior right lobes. In small laboratory animals such as rats, mice, and hamsters, the left lung consists of a single lobe, whereas the right lung is divided into four lobes: cranial, middle, caudal, and ancillary. In the guinea pig and rabbit, the left lung is divided into two lobes. Dogs have two left and four right lobes. The lung can be further subdivided at the periphery of the bronchial tree into distinct anatomic bronchopulmonary segments, then into lobules, and finally into acini. An acinus includes a terminal bronchiole and all its respiratory bronchioles, alveolar ducts, and alveolar sacs. An acinus may be made up of 2–8 ventilatory units. A ventilatory unit is defined as an anatomical region that includes all alveolar ducts and alveoli distal to each bronchiolaralveolar duct junction (Mercer and Crapo, 1991). The ventilatory unit is important because it represents the smallest common denominator when the distribution of inhaled gases to the gas-exchanging surface of the lung is modeled (Fig. 15-2). Gas exchange occurs in the alveoli, which comprise approximately 80–90% of the total parenchymal lung volume; adult human lungs contain an estimated 300 million alveoli. The ratio of total

Figure 15-2. Centriacinar region (ventilatory unit) of the lung. An airway (AW) and an arteriole [blood vessel (BV)] are in close proximity to the terminal bronchiole (TB) opening into alveolar ducts (AD) at the bronchiole–alveolar duct junction (BADJ). A number of the alveolar septal tips (arrows) close to the BADJ are thickened after a brief (4 hour) exposure to asbestos fibers, indicating localization of fiber deposition. Other inhalants, such as ozone, produce lesions in the same locations. (Photograph courtesy of Dr. Kent E. Pinkerton, University of California, Davis.)

capillary surface to total alveolar surface is slightly less than 1. Capillaries, blood plasma, and formed blood elements are separated from the air space by a thin layer of tissue formed by epithelial, interstitial, and endothelial components (Fig. 15-3). Type I and type II alveolar cells represent approximately 25% of all the cells in the alveolar septum (Fig. 15-3). Type I cells cover a large surface area (approximately 90% of the alveolar surface). They have an attenuated cytoplasm and appear to be poor in organelles but probably are as metabolically competent as are the more compact type II cells. Preferential damage to type I cells by various agents may be explained by the fact that they constitute a large percentage of the total target (surface of the epithelium). Type II cells are cuboidal and show abundant perinuclear cytoplasm. They produce surfactant and, in the case of damage to the type I epithelium, may undergo mitotic division and replace damaged cells (Witschi, 1997). The shape of type I and type II cells is independent of alveolar size and is remarkably similar in different species. A typical rat alveolus (14,000 μm2 surface area) contains an average of two type I cells and three type II cells, whereas a human alveolus with a surface area of 200,000–300,000 μm2 contains an average of 32 type I cells and 51 type II cells (Pinkerton et al., 1991).The mesenchymal

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Figure 15-3. Micrograph of four alveoli (A) separated by the alveolar septum. The thin air-to-blood tissue barrier of the alveolar septal wall is composed of squamous alveolar type I cells and occasional alveolar type II cells (II), a small interstitial space, and the attenuated cytoplasm of the endothelial cells that form the wall of the capillaries (C). (Photograph courtesy of Dr. Kent E. Pinkerton, University of California, Davis.)

interstitial cell population consists of fibroblasts and myofibroblasts that produce collagen and elastin as well as other cell matrix components and various effector molecules. Pericytes, monocytes, and lymphocytes also reside in the interstitium and so do macrophages before they enter the alveoli. Endothelial cells have a thin cytoplasm and cover about one-fourth of the area covered by type I cells.

Gas Exchange The principal function of the lung is gas exchange, which consists of ventilation, perfusion, and diffusion. The lung is superbly equipped to handle its main task: bringing essential oxygen to the organs and tissues of the body and eliminating its most abundant waste product, CO2 (Weibel, 1983).

Ventilation During inhalation, fresh air is moved into the lung through the upper respiratory tract and conducting airways and into the terminal respiratory units when the thoracic cage enlarges and the diaphragm moves downward; the lung passively follows this expansion. After diffusion of oxygen into the blood and that of CO2 from the blood into the alveolar spaces, the air (now enriched in CO2 ) is expelled by exhalation. Relaxation of the chest wall and diaphragm diminishes the internal volume of the thoracic cage, the elastic fibers of the lung parenchyma contract, and air is expelled from the alveolar zone through the airways. Any interference with the elastic properties of the lung, for example, the decrease in elastic fibers that occurs in emphysema, adversely affects ventilation, as do the decrease in the diameters of, or blockage of, the conducting airways, as in asthma. The total volume of air in an inflated human lung, approximately 5700 cm3 , represents the total lung capacity (TLC). After a maximum expiration, the lung retains approximately 1200 cm3 of air, the residual volume (RV). The air volume moved into and out of the lung with a maximum inspiratory and expiratory movement, which is called the vital capacity (VC), is thus approximately 4500 cm3 . Under resting conditions, only a fraction of the VC, the tidal volume (TV), is moved into and out of the lung. In resting humans, the TV measures approximately 500 cm3 with each breath (Fig. 15-4). The respiratory frequency, or the number of breaths per minute, is approximately 12–20. If an augmented metabolic demand of the body requires the delivery of increased amounts of oxygen, for example, during heavy and prolonged exercise, both the TV and the respiratory rate can be greatly increased. The amount of air moved into and out of the human lung may increase to up to 60 L/min. Increased ventilation in a polluted atmosphere increases the deposition of inhaled toxic material. For this reason, it is often stated that people, particularly children, should not exercise during episodes of heavy air pollution. The TLC, as well as the ratio of RV to VC, changes when the lung is diseased. In emphysema, the alveoli overextend and more air is trapped. While the TLC may stay the same or even increase, the volume of air that is actually moved during breathing is diminished.

RESPONSES OF THE RESPIRATORY SYSTEM TO TOXIC AGENTS Paper

Liters

6

Total lung capacity

Spirometer Vital capacity

4 Tidal volume

Pen

2 Functional residual Residual capacity volume 0 Figure 15-4. Lung volumes. Note that the functional residual capacity and RV cannot be measured with spirometer but require special procedures (e.g., nitrogen or helium outwash).

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This results in decreased VC with a concomitant increase in RV. If part of the lung collapses or becomes filled with edema fluid, TLC and VC are reduced. Pulmonary function tests give quantitative information on such changes. Perfusion The lung receives the entire output from the right ventricle, approximately 70–80 cm3 of blood per heartbeat, and thus may be exposed to substantial amounts of toxic agents carried in the blood. A chemical placed onto or deposited under the skin (subcutaneous injection) or introduced directly into a peripheral vein (intravenous injection) travels through the venous system to the right ventricle and then comes into contact with the pulmonary capillary bed before distribution to other organs or tissues in the body. Diffusion Gas exchange takes place across the entire alveolar surface. Contact to an airborne toxic chemical thus occurs over a surface of approximately 140 m2 . This surface area is second only to the small intestine (approximately 250 m2 ) and considerably larger than the skin (approximately 1.75 m2 ), two other organs that are in direct contact with the outside world. A variety of abnormal processes may severely compromise the unhindered diffusion of oxygen to the erythrocytes. Acute events may include collection of liquid or of inflammatory cells in the alveolar space. Chronic toxicity can impair diffusion due to abnormal increase in formation and deposition of extracellular substances such as collagen in the interstitium or through interstitial accumulation of edema fluid.

Distribution of Metabolic Competence in the Respiratory Tract Often overlooked as an organ involved in metabolism of chemicals, in favor of the liver, the lung has substantial capabilities for both metabolic activation as well as detoxification. Total lung P-450 activity is roughly one-tenth to one-third of that in the liver. However, when specific activity in a few cell types is considered, the difference is only twofold for many enzymes and in the case of nasal mucosa higher enzyme activity is reported per cell. Metabolic competence in the lung and nasal tissues is concentrated in a few cell types and these have a defined, and sometimes limited, distribution in the respiratory tract that can vary substantially by species (Table 15-1). The balance of activation and detoxification is a critically important determinant of lung protection as well as of lung injury (Buckpitt and Cruikshank, 1997). Protection from oxidation is another important ongoing function of detoxifying enzymes in light of the high tissue oxygen concentration that occurs in the respiratory tract. Other factors that can influence the role of Phase-I and Phase-II systems in lung toxicity include: age, sex, diet, local inflammation, and the history of prior exposure (Plopper et al., 2001a). Interestingly, many xenobiotic metabolizing enzymes have different patterns of induction (less) in the respiratory tract than in the liver, leading to the concept that regulation of these systems may be different depending on where they are located (Buckpitt and Cruikshank, 1997). The major Phase-I enzyme system, the cytochrome P-450 monooxygenases, are concentrated into a few lung cells: nonciliated bronchiolar (Clara) cells, Type 2 cells, macrophages and endothelial cells, predominantly. Of these cell types, the Clara cell has the most P-450 followed by the Type 2 cell. The amount of total lung P-450 contributed by Clara cells is species dependent, with humans having less P-450 in their lungs from Clara cells than rats or mice. Further, the isoforms of P-450 present and their loca-

613

tion also vary by species. Nasal epithelia also metabolize foreign compounds via the cytochrome P-450 monooxygenase system. Cytochrome P-450s are expressed at some of the highest levels for an extrahepatic tissue in the nasal mucosa and this pattern of expression varies by nasal region and cell type (Ding and Kaminsky, 2003). The olfactory mucosa is considered to be a “metabolic hot spot.” Most species have P-450 in nasal tissue and some of these P-450s are predominantly, possibly even solely, expressed in the olfactory mucosa (e.g., CYP2G1, CYP2A3, and CYP2A13) (Ling et al., 2004). Metabolism by the olfactory epithelium may play a role in providing, or preventing, access of inhalants directly to the brain; for example, inhaled xylene may be converted to metabolites that move to the brain by axonal transport. The presence of the following cytochrome P-450 isozymes in the respiratory tract of at least one species has been reported: CYP1A1, CYP1B1, CYP2A3, CYP2A10/2A11, CYP2B1/4, CYP2B6, CYP2B7, CYP2E1, CYP2F1/2/4, CYP2S1, CYP2J2, CYP2G1, CYP3A, and CYP4B1 (for a review of CYP expression in human lung see Hukkanen et al., 2002). Other enzymes found in lung tissue include epoxide hydrolases, flavin monooxygenases, prostaglandin synthases, glucuronsyl transferases, sulfotransferases, and glutathione S-transferases (alpha, mu, and pi). The only constant feature of the expression of these enzymes is lack of uniformity in their expression by cell type and airway level throughout the lung and their tendency to concentrate in epithelia. Both microsomal and cytosolic epoxide hydrolases are found in the lung and nasal tissues and the activity of microsomal epoxide hydrolase can be greater in the distal airways of the lung than even in the liver (Bond et al., 1988). Flavin monooxygenase activity (FMO1 and FMO2) is found in rodent and human lung and nasal tissue. The isoforms present in the lung (FMO2) are different from that found in the liver (FMO1). FM01 is the predominant isoform in the nasal mucosa (Shehin-Johnson et al., 1995). There are polymorphisms in the expression of FMO2 in humans. The gene for FMO2 in human lung contains a premature stop codon encoding production of an inactive protein, but some ethnic groups have at least one copy of an allele that expresses the full length protein (Whetstine et al., 2000). Prostaglandin synthases (aka cyclooxygenases) oxidize substrates at a much lower rate than the cytochrome P-450 monooxygenases but may have a role in human pulmonary metabolism due to the relatively lower P-450 activity in human lung tissue compared to rodents (Smith et al., 1991). Despite the fact that activity of glucuronosyl transferase has been reported in both rodent and human pulmonary tissues, these proteins have received little attention. An olfactory-specific glucoronosyl transferase has been reported and is suggested to have a role in termination of odorant signals (Lazard et al., 1991). There is little information on sulfotransferases in respiratory tract tissues although some studies have demonstrated immunochemical localization of these enzymes to the conducting airway epithelium (He et al., 2005) and activity has been demonstrated in human bronchoscopy samples (Gibby and Cohen, 1984). Sulphotransferases have been localized to the sustentacular cells of the olfactory epithelium and some isoforms may be specific to the olfactory epithelium (Miyawaki et al., 1996; Tamura et al., 1998). Glutathione S-transferases (and glutathione) play a major role in the modulation of both acute and chronic chemical toxicity in the lung (West et al., 2003). A major determinant of the potential for detoxification may also be the cellular localization of, and ability to synthesize, glutathione in the lung (Plopper et al., 2001b, West et al., 2000). Pulmonary glutathione S-transferase activity is 5–15% that of the liver in rodents and about 30% of that in human liver (Buckpitt and Cruikshank, 1997). The distribution of

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Table 15-1 Distribution of Xenobiotic Metabolizing Enzymes in the Respiratory Tract proximal airways trachea, bronchial (epithelium)

enzyme

nasal tissue

Cytochrome P450s

+++ Olfactory mucosa: sustentacular cells and Bowmans glands +/− Trasitional epithelium Respiratory epithelium + Other glands/ducts

++ Secretory cell types (2J2 only is ++ in ciliated cells)

Miceosomal Epoxide Hydrolases

+++

Flavin Monooxygenases

+

Prostaglandin synthase (Cyclooxygenase1,-2)

distal airways (bronchiolar epithelium)

alveoli

other

+++ Clara cells and secretory cell types

++ Type II cells

++ Macrophages Endothelium (3A5, 1A1)

++ Clara cells, ciliated cells

+++ Clara cells, ciliated cells

++

+

++ Clara cells + Ciliated cells

+++ Clara cells + Ciliated cells

++ Type II cells + Type I cells

+

++ Basal and ciliated cells (cox-1 and cox-2)

++ Basal cells, columnar epithelial cells (Cox-1 > Cox2)

++ Clara cells

+/− Type II cells, alveolar septa

+++ Macrophages (Cox-1 < Cox2), mast cells (Cox2) eosinophils and neutrophils (Cox2), vascular smooth muscle (Cox1), endothelium (Cox1)

Glutathione S-Transferases

+

++ Cell types vary with isoform & include ciliated and Clara cells

+++ Cell types vary with isoform & include ciliated and Clara cells

+

?

Glucuronsyl Transferases

+

?

++ Clara cells, Ciliated cells

+

Sulfotransferases

++ Olfactory epithelium, sustentacular cells

+

+/−

Olfactory epithelium

Olfactory epithelium +

Macrophages ++ Blood vessels

Capillary endothelial cells

Type II cells

? Type II cells ?

Code for labels: +++, most isoforms expressed highly; +/−, some expression in some studies but not in others, generally low; ++, some isoforms expressed highly; ?, unknown/not found; +, low level of expression.

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the isoforms of glutathione S-transferase varies by lung region with the alpha, mu, and pi isoforms (the most abundant) and the alpha and pi classes predominate in the airway epithelia of human lung. In nasal tissue, glutathione S-transferases are found in the olfactory mucosa. The mu isoform demonstrates a zonal pattern of expression increased in the lateral olfactory turbinates of the mouse (WhitbyLogan et al., 2004). The glutathione transferases have recently been the focus of acute interest because of widespread polymorphisms in their expression in humans and the potential for correlation of this with lung cancer, particularly in smokers. A key point to keep in mind is that these enzyme systems work in concert with one another (i.e., a decrease in one enzyme may result in a concomitant increase in another) and it is the balance of all of them, and their location, that determines toxicity.

GENERAL PRINCIPLES IN THE PATHOGENESIS OF LUNG DAMAGE CAUSED BY CHEMICALS Toxic Inhalants, Gases, and Dosimetry The sites of deposition of gases in the respiratory tract define the pattern of toxicity of those gases. Water solubility is the critical factor in determining how deeply a given gas penetrates into the lung. Highly soluble gases such as SO2 do not penetrate farther than the nose unless doses are very high, and are therefore relatively nontoxic to animals, especially obligatory nose breathers, such as the rat. Relatively insoluble gases such as ozone and NO2 penetrate deeply into the lung and reach the smallest airways and the alveoli (centriacinar region), where they can elicit toxic responses. Mathematical models of gas entry and deposition in the lung that are based solely on the aqueous solubility of a gas predict sites of lung lesions fairly accurately. These models may be useful for extrapolating findings made in laboratory animals to humans (Kimball and Miller, 1999; Medinsky et al., 1999). Very insoluble gases such as CO and H2 S efficiently pass through the respiratory tract and are taken up by the pulmonary blood supply to be distributed throughout the body.

615

a given size. Inhaled ambient particles and aerosols are most frequently polydisperse in size. The size distribution of many aerosols approximates a log-normal distribution that may be described by the median or geometric mean and the geometric standard deviation. A plot of the frequency of occurrence of a given size against the log of the size produces a bell-shaped probability curve. Particle data frequently are analyzed by plotting the cumulative percentage of particles smaller than a stated size on log-probability paper. This results in a straight line that may be fitted by eye or mathematically. In actual practice, it is not unusual to have some deviation from a straight line at the largest or smallest particle sizes measured. The geometric mean is the 50% size as the mean bisects the curve. The geometric standard deviation (σ g ) is calculated as σg = 84.1% size/50% size The σ g of the particle size distribution is a measure of the polydispersity of the aerosol. In the laboratory, values for σ g of 1.8–3.0 are encountered frequently. In the field, value for σ g may range up to 4.5. An aerosol with a σ g below 1.2 may be considered monodispersed. Particles that are nonspherical in shape are frequently characterized in terms of equivalent spheres on the basis of equal mass, volume, or aerodynamic drag. The MMAD takes into account both the density of the particle and aerodynamic drag. It represents the diameter of a unit density sphere with the same terminal settling velocity as the particle, regardless of its size, shape, and density. Aerodynamic diameter is the proper measurement for particles that are deposited by impaction and sedimentation. For very small particles, which are deposited primarily by diffusion, the critical factor is particle size, not density. It must be kept in mind that the size of a particle may change before its deposition in the respiratory tract. Materials that are hygroscopic, such as sodium chloride, sulfuric acid, and glycerol, take on water and grow in size in the warm, saturated atmosphere of the upper and lower respiratory tract.

Nanotoxicology Particle Deposition Particle size is usually the critical factor that determines the region of the respiratory tract in which a particle or an aerosol will be deposited. Deposition of particles on the surface of the lung and airways is brought about by a combination of lung anatomy and the patterns of airflow in the respiratory system (Raabe, 1999; Miller, 1999).

Particle Size The median diameter that is determined may reflect the number of particles, as in the count median diameter (CMD), or reflect mass, as in the mass median aerodynamic diameter (MMAD). If more particles actually reach the deep lung, the higher is the probability of a toxic effect. Particle surface area is of special importance when toxic materials are adsorbed on particles and thus are carried into the lung. Large particles (larger than 5 μm MMAD) are usually trapped in the upper respiratory tract (nasopharyngeal region and large conducting airways), whereas smaller particles (0.2–5 μm MMAD) can be transported to the smaller airways and the alveoli (Fig. 15-5). Patterns of breathing can change the site of deposition of a particle of

There is intense current interest in the lung toxicity of nanoparticles, particles with diameters of 70%) inhibition occurs and the inhibited enzyme undergoes aging. Thus, in the case of NTE and OPIDN, inhibition alone is insufficient to precipitate toxicity. It appears that the biochemical lesion is not simply a blockade of the active site. Instead, axonopathy is triggered by specific chemical modification of the NTE protein (Fig. 16-6). Neuropathic (aging) inhibitors of NTE include compounds from the phosphate, phosphonate, and phosphoramidate classes of OP compounds (Richardson, 1992; Kropp et al., 2004) (Fig. 16-7). Certain NTE inhibitors, including members of the phosphinate, carbamate, and sulfonylfluoride classes, do not age and do not cause OPIDN (Fig. 16-7). However, pretreatment with a nonaging NTE inhibitor prevents OPIDN from occurring after a challenge dose of a neuropathic (aging) NTE inhibitor. It has been proposed that these nonaging compounds protect against OPIDN by blocking the

CHAPTER 16

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645

Figure 16-6. NTE and OPIDN.

Figure 16-5. AChE and cholinergic toxicity. (A) Inhibition by an ageable organophosphate produces cholinergic toxicity, treatable with atropine and 2-PAM. Aging does not alter the type of toxicity, but it obviates 2-PAM therapy. (B) Inhibition by a nonageable phosphinate produces cholinergic toxicity, treatable with atropine and 2-PAM. R, R : substituted or unsubstituted alkyl or aryl groups; X: primary leaving group displaced by the AChE active site serine.

active site of NTE, and preventing inhibition and aging by a subsequent dose of a neuropathic (aging) inhibitor (Richardson, 2005) (Fig. 16-6). In contrast, when protective NTE inhibitors are administered following exposure to a near-threshold subclinical dose of a neuropathic OP compound, OPIDN is fully expressed (Pope et al., 1993). Because the initial treatment involves a compound that can produce OPIDN on its own and the disease is likely to be incipient rather than absent, this effect should be called potentiation; however, some authors refer to the phenomenon as promotion (Lotti, 2002). Although the potentiating agents inhibit NTE, this enzyme is not thought to be the target of potentiation. The level of NTE inhibition produced by the potentiator is not related to the level of potentiation observed, and these potentiators appear to exacerbate axonopathies from other causes as well, such as trauma and 2,5-hexanedione exposure. These results have been interpreted to indicate that potentiation enhances progression of the axonopathic process, inhibits repair, or both (Lotti, 2002; Randall et al., 1997). Axonal degeneration does not commence immediately after acute exposure to a neuropathic OP compound, but is delayed for at least 8 days between the acute high-dose exposure and clinical signs of axonopathy. Some effective regeneration of axons occurs in the

(A) Inhibition by an ageable organophosphate; rapid aging yields a negatively charged phosphoryl conjugate resulting in OPIDN. (B) Inhibition by a nonageable phosphinate does not produce OPIDN but provides protection against neuropathic (ageable) NTE inhibitors. R, R : substituted or unsubstituted alkyl or aryl groups. X: primary leaving group displaced by the NTE active site serine.

PNS, for example, excitatory inputs to skeletal muscle from lower motor neurons in the spinal cord. In contrast, axonal degeneration is progressive and persistent in long tracts of the spinal cord, for example, inhibitory pathways from upper motor neurons in the motor cortex to lower motor neurons in the spinal cord anterior horn. Accordingly, the clinical picture of OPIDN changes from flaccid to spastic paralysis during a course of months to years (Lotti and Moretto, 2005; Richardson, 2005). Fortunately, studies of the initiation steps of OPIDN and structure-activity relationships of neuropathic OP compounds have led to highly accurate prediction of the neuropathic potential of these chemicals. Consequently, human cases of OPIDN are now rare and usually arise from intentional ingestion of massive doses of OP insecticides in suicide attempts. Nevertheless, the fact remains that OPIDN is a debilitating and incurable condition. Moreover, the mechanism linking aged NTE to axonopathy is unknown. Accordingly, research continues in order to enhance mechanistic understanding that could be applied to improving biosensors and biomarkers of exposure, high-throughput testing of new compounds, and prophylaxis and treatment for OPIDN (Makhaeva et al., 2003; Malygin et al., 2003; Richardson, 2005). The foregoing discussion has been limited to organic compounds of pentacovalent phosphorus, which are by far the most common and best studied of the OP compounds. However, organic

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Figure 16-7. NTE inhibitors. (A). Neuropathic (ageable). (B) Nonneuropathic (nonageable). R, R : substituted or unsubstituted alkyl or aryl groups; X: primary leaving group displaced by the NTE active site serine.

compounds of trivalent phosphorus, such as triphenylphosphine (TPPn) and triphenylphosphite (TPPi), have relatively widespread use, particularly as antioxidants, chemical intermediates, and polymer enhancers. Both TPPn and TPPi produce axonal degeneration in the CNS and PNS, but the spatial-temporal distributions of lesions are different from that of classical OPIDN produced by organic pentacovalent phosphorus compounds and the pathogenic mechanisms are unknown (Abou-Donia, 1992). In particular, it appears that the mechanism of initiation of axonopathy by TTPn is independent of NTE inhibition and aging (Davis et al., 1999), and this relationship is unclear for TPPi (Padilla et al., 1987). Pyridinethione This compound is a chelating agent that is usually encountered as the zinc complex. Two molecules of pyridinethione are complexed with zinc to form bis[1-hydroxy-2(1H)-pyridinethionato] zinc, commonly known as zinc pyridinethione or zinc pyrithione (ZPT) (Bond et al., 2002; Lewis et al., 2005). ZPT is a biocide that has antibacterial and antifungal properties. It is the active ingredient in shampoos and other preparations for the treatment of seborrheic dermatitis and dandruff. ZPT is also used as an antifouling agent for ship paints, drywall, and tarps, and as an antibacterial agent for incorporation into cleaning sponges. Thus, the intended uses of ZPT can lead to human exposures through direct dermal contact and potential exposure to biota through leaching into marine and freshwater environments (Grunnet and Dahllof, 2005; Pierard-Franchimont et al., 2002). Because the compound is directly applied to the human scalp in antidandruff shampoos, the finding that ZPT produced limb weakness and peripheral neuropathy in rodents after oral administration raised concern about potential neurotoxicity in humans (Sahenk and Mendell, 1979). Rats, rabbits, and guinea pigs all develop a distal axonopathy when exposed to ZPT in the diet. Fortunately, however, dermal absorption of ZPT is minimal, and there have been no reports of neurological findings in humans attributable to occupational or consumer ZPT exposures (Sahenk and Mendell, 2000). Although the zinc ion appears to be an important component of the therapeutic action of ZPT, only the pyridinethione moiety is absorbed following ingestion, with the majority of zinc eliminated in the feces. In addition, oral sodium pyridinethione is also neurotoxic, indicating that the pyridinethione moiety is responsible for the

neurotoxicity. Pyridinethione chelates zinc, copper, and other metal ions and, once oxidized to the disulfide, may lead to the formation of protein-pyridinethione mixed disulfides. However, which of these properties, if any, is responsible for the molecular mechanism of its neurotoxicity remains unknown (Sahenk and Mendell, 2000). Although these molecular issues remain to be resolved, pyridinethione appears to interfere with the fast axonal transport systems. While the fast anterograde system is less affected, pyridinethione impairs the turnaround of rapidly transported vesicles and slows their retrograde transport (Sahenk and Mendell, 1980). This aberration of the fast axonal transport systems is the most likely physiologic basis of the accumulation of tubulovesicular structures in the distal axon. As these materials accumulate in one region of the axon, they distend the axonal diameter, resulting in axonal swellings filled with membranous profiles. As in many other distal axonopathies, the axon degenerates in its more distal regions beyond the accumulated structures. The earliest signs are diminished grip strength and electrophysiologic changes of the axon terminal, with normal conduction along the proximal axon in the early stages of exposure (Ross and Lawhorn, 1990). Microtubule-Associated Neurotoxicity A number of plant alkaloids alter the assembly and depolymerization of microtubules in nerve axons, causing neurotoxicity. The oldest known of these are colchicine and the vinca alkaloids, which bind to tubulin and cause depolymerization of microtubules. Colchicine is an alkaloid pharmaceutical used in the treatment of gout, familial Mediterranean fever, and other disorders. A common side effect of treatment in patients with abnormal renal function is a peripheral axonal neuropathy. While this neuropathy is generally mild, it is often accompanied by a disabling myopathy that can lead to the inability to walk (Riggs et al., 1986). A number of vinca alkaloids, including vincristine and vinblastine, both chemotherapeutic agents, produce a peripheral axonopathy very similar to that induced by colchicine. Vincristine is commonly used to treat leukemias and lymphomas, and also has greater potential for adverse toxic effects than vinblastine. The agent binds to tubulin subunits and prevents the polymerization into microtubules (Prakash and Timasheff, 1992). Nearly all evidence of vincristine-induced neuropathy has been observed in humans. Most

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treated patients develop neurotoxicity to some extent, beginning with parasthesias of the fingers. General weakness and clumsiness is common, but this improves quickly with removal of treatment. Parasthesias may persist, however, and some distal sensory loss may be permanent (Schaumburg, 2000). More recently paclitaxel (Taxol), another plant alkaloid, has become a popular chemotherapeutic agent used to treat a variety of neoplasms. However, side effects include a predominantly sensory neuropathy, beginning in the hands and feet (Sahenk et al., 1994). Like colchicine and the vinca alkaloids, paclitaxel binds to tubulin; however, instead of leading to depolymerization, it promotes the formation of microtubules. Once formed, these microtubules remain stabilized by paclitaxel even in conditions that normally lead to dissociation of tubulin subunits, including cold temperatures or the presence of calcium (Schiff and Horowitz, 1981). When paclitaxel is injected directly into the sciatic nerve of rats, microtubules aggregate along the axon, causing axonal degeneration, demyelination, and impairment of regeneration (Lipton et al., 1989; Mielke et al., 2006). The pathologies of the axon induced by these drugs are different. While colchicine leads to atrophy of the axon and a decrease in the number of microtubules, paclitaxel causes the aggregation to form a matrix that may inhibit fast axonal transport, which has been demonstrated with both colchicine and paclitaxel. A change in the number of microtubules has been observed in some reports and absent from others (Roytta et al., 1984; Nakata and Yorifuji, 1999). While the mechanisms may differ slightly, both exposures result in a peripheral neuropathy which must be taken into account in medical treatments.

Hexachlorophene Hexachlorophene, or methylene 2,2 -methylenebis(3,4,6-trichlorophenol), resulted in human neurotoxicity when newborn infants, particularly premature infants, were bathed with the compound to avoid staphylococcal skin infections (Mullick, 1973). Following skin absorption of this hydrophobic compound, hexachlorophene enters the NS and results in intramyelinic edema, splitting the intraperiod line of myelin in both the CNS and the PNS. The intramyelinic edema leads to the formation of vacuoles, creating a “spongiosis” of the brain (Purves et al., 1991). Experimental studies with erythrocyte membranes show that hexachlorophene binds tightly to cell membranes, resulting in the loss of ion gradients across the membrane (Flores and Buhler, 1974). This loss of the ability to exclude ions from between the layers of myelin leads to water and ion entry, which separates the myelin layers as “edema.” Another, perhaps related, effect is the uncoupling of mitochondrial oxidative phosphorylation by hexachlorophene (Cammer and Moore, 1972), because this process is dependent on a proton gradient. Intramyelinic edema is reversible in the early stages, but with increasing exposure, hexachlorophene causes segmental demyelination. Swelling of the brain causes increased intracranial pressure, which may be fatal. With high-dose exposure, axonal degeneration is seen, along with degeneration of photoreceptors in the retina. It has been postulated that the pressure from severe intramyelinic edema may also injure the axon, leading to axonal degeneration; endoneurial pressure measurements support this idea (Myers et al., 1982). The toxicity of hexachlorophene expresses itself functionally in diffuse terms that reflect the diffuse process of myelin injury. Humans exposed acutely to hexachlorophene may have generalized weakness, confusion, and seizures. Progression may occur to include coma and death.

Myelinopathies

Tellurium Although human exposures have not been reported, neurotoxicity of tellurium has been demonstrated in animals. Young rats exposed to tellurium in their diet develop a severe peripheral neuropathy. Within the first 2 days of dietary exposure, the synthesis of myelin lipids in Schwann cells displays striking changes (Harry et al., 1989). These include decreased synthesis of cholesterol and cerebrosides (lipids richly represented in myelin), and downregulated myelin protein mRNA (Morell et al., 1994). However, the synthesis of phosphatidylcholine, a more ubiquitous membrane lipid, is unaffected. The synthesis of free fatty acids and cholesterol esters increases to some degree, and there is a marked elevation of squalene, a precursor of cholesterol. These biochemical findings demonstrate a variety of lipid abnormalities, and the simultaneous increase in squalene and decrease in cholesterol suggest that tellurium or one of its derivatives may interfere with the normal conversion of squalene to cholesterol. Squalene epoxidase, a microsomal monooxygenase that utilizes NAPDH cytochrome P450 reductase, has been strongly implicated as the target of tellurium, because its inhibition by tellurium as well as certain other oranotellurium compounds shows a correlation between the potency of enzyme inhibition and demyelination in vivo (Goodrum, 1998). In conjunction with these biochemical changes, lipids accumulate in Schwann cells within intracytoplasmic vacuoles; shortly afterwards, these Schwann cells lose their ability to maintain myelin. Axons and the myelin of the CNS are impervious to the effects of tellurium. However, individual Schwann cells in the PNS disassemble their concentric layers of myelin membranes, depriving the adjacent intact axon of its electrically insulated status. Not all Schwann cells are equally affected by the process; rather, those Schwann cells that encompass the greatest distances appear to be the most affected.

Myelin provides electrical insulation of neuronal processes, and its absence leads to a slowing of and/or aberrant conduction of impulses between adjacent processes, so-called ephaptic transmission. Toxicants exist that result in the separation of the myelin lamellae, termed intramyelinic edema, and in the selective loss of myelin, termed demyelination. Intramyelinic edema may be caused by alterations in the transcript levels of myelin basic protein-mRNA (Veronesi et al., 1991) and early in its evolution is reversible. However, the initial stages may progress to demyelination, with loss of myelin from the axon. Demyelination may also result from direct toxicity to the myelinating cell. Remyelination in the CNS occurs to only a limited extent after demyelination. However, Schwann cells in the PNS are capable of remyelinating the axon after a demyelinating injury. Interestingly, remyelination after segmental demyelination in peripheral nerve involves multiple Schwann cells and results, therefore, in internodal lengths (the distances between adjacent nodes of Ranvier) that are much shorter than normal and a permanent record of the demyelinating event. The compounds in Table 16-3 all lead to a myelinopathy. Some of these compounds have created problems in humans, and many have been used as tools to explore the process of myelination of the NS and the process of remyelination following toxic disruption of myelin. In general, the functional consequences of demyelination depend on the extent of the demyelination and whether it is localized within the CNS or the PNS or is more diffuse in its distribution. Those toxic myelinopathies in which the disruption of myelin is diffuse generate a global neurological deficit, whereas those that are limited to the PNS produce the symptoms of peripheral neuropathy.

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Table 16-3 Compounds Associated with Injury of Myelin (Myelinopathies) neurotoxicant

neurologic findings

basis of neurotoxicity

Acetylethyltetramethyl tetralin (AETT)

Not reported in humans; hyperexcitability, tremors (rats)

Intramyelinic edema; pigment accumulation in neurons

Amiodarone

Peripheral neuropathy

Cuprizone

Ethidium bromide

Not reported in humans; encephalopathy (experimental animals) Peripheral neuropathy, predominantly sensory Insufficient data (humans)

Hexachlorophene

Irritability, confusion, seizures

Lysolecithin

Effects only on direct injection into PNS or CNS (experimental animals) Peripheral neuropathy

Axonal degeneration and demyelination; lipid-laden lysosomes in Schwann cells Status spongiosis of white matter, intramyelinic edema (early stages); gliosis (late) Axonal degeneration, swellings in distal axons Intramyelinic edema, status spongiosis of white matter Brain swelling, intramyelinic edema in CNS and PNS, late axonal degeneration Selective demyelination

Disulfiram

Perhexilene

Tellurium

Hydrocephalus, hind-limb paralysis (experimental animals)

Triethyltin

Headache, photophobia, vomiting, paraplegia (irreversible)

These cells are associated with the largest-diameter axons, encompass the longest intervals of myelination, and provide the thickest layers of myelin. Thus, it appears that the most vulnerable cells are those with the largest volume of myelin to support (Bouldin et al., 1988). As the process of remyelination begins, several cells cooperate to reproduce the myelin layers that were previously formed by a single Schwann cell. Perhaps this diminished demand placed upon an individual cell is the reason that remyelination occurs even in the presence of continued exposure to tellurium (Bouldin et al., 1988). The expression of the neurological impairment is also short in duration, reflecting the transient cellular and biochemical events. The animals initially develop severe weakness in the hind limbs but then recover their strength after 2 weeks on the tellurium-laden diet.

Lead Lead exposure in animals results in a peripheral neuropathy with prominent segmental demyelination, a process that bears a strong resemblance to tellurium toxicity (Dyck et al., 1977). However, the neurotoxicity of lead is much more variable in humans than in rats, and there are also a variety of manifestations of lead toxicity in other organ systems. The neurotoxicity of lead has been appreciated for centuries. In current times, adults are exposed to lead in occupational settings through lead smelting processes and soldering and in domestic

Demyelinating neuropathy, membrane-bound inclusions in Schwann cells Demyelinating neuropathy, lipofuscinosis (experimental animals) Brain swelling (acute) with intramyelinic edema, spongiosis of white matter

reference Graham and Lantos, 1997; Spencer and Schaumburg, 2000 Graham and Lantos, 1997; Spencer and Schaumburg, 2000 Graham and Lantos, 1997; Spencer and Schaumburg, 2000 Graham and Lantos, 1997 Spencer and Schaumburg, 2000 Graham and Lantos, 1997; Spencer and Schaumburg, 2000 Graham and Lantos, 1997

Graham and Lantos, 1997; Spencer and Schaumburg, 2000 Graham and Lantos, 1997

Chang and Dyer, 1995; Graham and Lantos, 1997; Spencer and Schaumburg, 2000

settings through lead pipes or through the consumption of “moonshine” contaminated with lead. In addition, some areas contain higher levels of environmental lead, resulting in higher blood levels in the inhabitants. Children, especially those below 5 years of age, have higher blood levels of lead than adults in the same environment, due to the mouthing of objects and the consumption of substances other than food. In addition, children absorb lead more readily, and the very young do not have the protection of the blood–brain barrier. The most common acute exposure in children, however, has been through the consumption of paint chips containing lead pigments (Perlstein and Attala, 1966), a finding that has led to public efforts to prevent the use of lead paints in homes with children. In young children, acute massive exposures to lead result in severe cerebral edema, perhaps from damage to endothelial cells. Children seem to be more susceptible to this lead encephalopathy than adults (Johnston and Goldstein, 1998); however, adults may also develop an acute encephalopathy in the setting of massive lead exposure. Chronic lead intoxication in adults results in peripheral neuropathy, often accompanied by manifestations outside the NS, such as gastritis, colicky abdominal pain, anemia, and the prominent deposition of lead in particular anatomic sites, creating lead lines in the gums and in the epiphyses of long bones in children. The effects of lead on the peripheral nerve of humans (lead neuropathy) are not entirely understood. Electrophysiologic studies have demonstrated

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Table 16-4 Compounds Associated with Neurotransmitter-Associated Toxicity neurotoxicant

neurologic findings

basis of neurotoxicity

reference

Amphetamine and methamphetamine

Tremor, restlessness (acute); cerebral infarction and hemorrhage; neuropsychiatric disturbances

Spencer and Schaumburg, 2000; Hardman et al., 1996

Atropine

Restlessness, irritability, hallucinations Increased risk of stroke and cerebral atrophy (chronic users); increased risk of sudden cardiac death; movement and psychiatric abnormalities, especially during withdrawal Decreased head circumference (fetal exposure) Headache, memory loss, hemiparesis, disorientation, seizures

Bilateral infarcts of globus pallidus, abnormalities in dopaminergic, serotonergic, cholinergic systems Acts at adrenergic receptors Block cholinergic receptors (anticholinergic) Infarcts and hemorrhages; alteration in striatal dopamine neurotransmission

Cocaine

Domoic acid

Kainate

Insufficient data in humans; seizures in animals (selective lesioning compound in neuroscience)

β-N -Methylamino-lalanine (BMAA)

Weakness, movement disorder (monkeys)

Muscarine (mushrooms)

Nausea, vomiting, headache

Nicotine

Nausea, vomiting, convulsions

β-N -Oxalylamino-lalanine (BOAA)

Seizures

a slowing of nerve conduction. Whereas this observation is consistent with the segmental demyelination that develops in experimental animals, pathologic studies in humans with lead neuropathy typically have demonstrated an axonopathy. Another finding in humans is the predominant involvement of motor axons, creating one of the few clinical situations in which patients present with predominantly motor symptoms. The basis for the effect on the brain (lead encephalopathy) is also unclear, although an effect on the membrane structure of myelin and myelin membrane fluidity has been shown (Dabrowska-Bouta et al., 1999). The etiologies associated with the pathogenesis of peripheral neuropathy are, like that of central neuropathy, rather speculative. One hypothesis postulates that the effects of lead on the blood–nerve–barrier are similar to those on

Structural malformations in newborns Neuronal loss, hippocampus and amygdala, layers 5 and 6 of neocortex Kainate-like pattern of excitotoxicity Degeneration of neurons in hippocampus, olfactory cortex, amygdala, thalamus Binds AMPA/kainate receptors Degenerative changes in motor neurons (monkeys) Excitotoxic probably via NMDA receptors Binds muscarinic receptors (cholinergic) Binds nicotinic receptors (cholinergic) low-dose stimulation; high-dose blocking Excitotoxic probably via AMPA class of glutamate receptors

Spencer and Schaumburg, 2000; Hardman et al., 1996 Spencer and Schaumburg, 2000; Hardman et al., 1996

Graham and Lantos, 1997; Spencer and Schaumburg, 2000

Graham and Lantos, 1997

Graham and Lantos, 1997; Spencer and Schaumburg, 2000

Hardman et al., 1996 Spencer and Schaumburg, 2000; Hardman et al., 1996

Graham and Lantos, 1997; Spencer and Schaumburg, 2000

the blood–brain barrier, providing a unitary mechanism for lead’s effects on both the peripheral and central nervous systems. Another theory on the mechanisms associated with lead-induced effects on the peripheral nervous system suggests a toxic effect on Schwann cells, and the ensuing demyelination of nerves (Powell et al., 1982). Although the manifestations of acute and chronic exposures to lead have been long established, the effects of low level exposures on infants and children have also become known. Initial reports noted a relationship between mild elevations of blood lead in children and school performance; more recently, correlations between elevated lead levels in decidual teeth and performance on tests of verbal abilities, attention, and behavior (nonadaptive) have been demonstrated (Needleman and Gatsonis, 1990; Needleman, 1994).

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Although there is a clear association between lead level and intellectual performance, there has been some discussion as to whether lead is causal. Children with higher blood levels tend to share certain other environmental factors, such as socioeconomic status and parental educational level. However, in spite of these complex social factors, it appears that lead exposure has an adverse effect on the intellectual abilities of children (Needleman, 1994), although a threshold for these effects has not yet been determined.

Astrocytes Rather than being the passive glue described by Virchow and other pathologists, astrocytes are now known to perform and regulate a wide range of physiological functions in the central nervous system. Perturbations in the function of these important cells are frequently reflected in abnormal neuronal physiology, even in the absence of altered nerve cell morphology. Indeed, the astrocyte appears to be a primary means of defense in the CNS following exposure to neurotoxicants, as a spatial buffering system for osmotically active ions, and as a depot for the sequestration and metabolic processing of endogenous molecules and xenobiotics. While in its relative infancy, investigations into the role of astrocytes in normal/abnormal function of the nervous system will be crucial to a better understanding of neurotoxicity and its pathological sequelae. Ammonia Hepatic encephalopathy (HE) or congenital and acquired hyperammonemia lead to excessive brain ammonia (ammonium, NH+ 4 ) accumulation. The condition results from liver failure. The effects of ammonia on the CNS vary with its concentration. At high CNS concentrations ammonia produces seizures, resulting from its depolarizing action on cell membranes, whereas, at lower concentrations, ammonia produces stupor and coma, consistent with its hyperpolarizing effects. Ammonia intoxication is associated with astrocytic swelling and morphological changes, yielding the so-called Alzheimer type II astrocytes, which precede any other morphological change (Mossakowski et al., 1970). The exclusive site for brain detoxification of glutamate to glutamine occurs within the astrocytes. This process requires ATP-dependent amidation of glutamate to glutamine, and it is mediated by the astrocyte-specific enzyme, glutamine synthetase (GS) and catalyzed by ammonia. Increased intracellular ammonia concentrations have also been implicated in the inhibition of neuronal glutamate precursor synthesis, resulting in diminished glutamatergic neurotransmission, changes in neurotransmitter uptake (glutamate), and changes in receptormediated metabolic responses of astrocytes to neuronal signals (Albrecht, 1996). Nitrochemicals The therapeutic potential of organic nitrates has been recognized for more than a century and began with the use of nitroglycerine for the management of acute anginal episodes. The resulting peripheral vasodilation and reduction in blood pressure, while useful in treating cardiovascular disease, has recently been shown to be only one of the pharmacologic properties of this class of chemicals. The mitochondrion features prominently as a target for nitrochemicals; however, the causal relationship between mitochondrial dysfunction and initiation of the neurotoxic state remains to be established for many of the chemicals. The dinitrobenzenes are important synthetic intermediates in the industrial production of dyes, plastics, and explosives. The neurotoxic compound, 1,3-dinitrobenzene (DNB), produces gliovascu-

lar lesions that specifically target astrocytes in the periaqueductal gray matter of the brainstem and deep cerebellar roof nuclei (Philbert et al., 1987). Though the molecular basis for the remarkable sensitivity of this cell population is unclear, it has been proposed that bioactivation of DNB by NADPH-dependent cytochrome c reductase (Hu et al., 1997; Romero et al., 1991) and subsequent induction of oxidative stress underlies its toxicity (Romero et al., 1995; Ray et al., 1992, 1994; Hu et al., 1999). Brainstem nuclei with high glucose requirements, such as the vestibular and deep cerebellar roof nuclei are affected more severely than forebrain and mesencephalic structures that have similar or higher requirements for glucose and oxygen (Calingasan et al., 1994; Bagley et al., 1989; Mastrogiacomo et al., 1993). The molecular basis of the susceptibility of brainstem astrocytes is unknown but growing evidence suggests that differences in mitochondrial respiratory capacity, cellular antioxidant levels, and the expression of proteins that regulate the mitochondrial permeability transition pore all contribute to the observed regional and cellular differences in susceptibility. In vitro studies indicate that DNB is a potent inducer of the mitochondrial permeability transition pore (mtPTP) (for reviews of the mtPTP see Lemasters et al., 1998; Crompton, 1999) in cultured C6 glioma cells (Tjalkens et al., 2000). Mitochondrial inner membrane permeabilization in an in vitro model of DNB exposure is dependent on generation of reactive oxygen species, in agreement with the reported capacity of DNB to deplete reduced pyridine nucleotides and glutathione due to redox-cycling (Romero et al., 1995). Metronidazole, a 5-nitroimidazole [1-(2-hydroxyethyl)-2methyl-5-nitroimidazole], is an antimicrobial, antiprotozoal agent that is commonly used for the treatment of a wide variety of infections. Prolonged treatment with metronidazole is associated with a peripheral neuropathy characterized by paraesthesias and dysaesthesias. In addition, headaches, glossitis, urticaria, pruritis, and other somatosensory disorders are also seen. Long-term administration of mitronidazole produces an irreversible sensorimotor deficit in the lower extremities of humans (Kapoor et al., 1999). Metronidazole is readily reduced to the highly reactive and toxic hydroxylamine intermediate and binds to cellular macromolecules including proteins and DNA (Coxon and Pallis, 1976). Use of higher intravenous doses of metronidazole for extended periods results in the expression of epileptiform seizures, hallucination, and attendant encephalopathy. The distribution of lesions is similar to preceding descriptions for DNB with the exception that both neurons and glia appear to be equally susceptible to the effects of metronidazole (Schentag, 1982). The mechanism of toxicity is well linked to the fact that metronidazole and its reduced metabolites bear close structural resemblance to the antineuritic nutrient, thiamine. Thiamine triphosphate (Vitamin B1) is an essential coenzyme in the mitochondrial metabolism of α-ketoglutarate and pyruvate, and also modulates the activity of sodium channels. Given the similarity in the lesions produced by metronidazole and pyrithiamine, a common antimetabolite mode of action has been proposed as the primary mechanism of neurotoxicity (Evans et al., 1975; Watanabe and Kanabe, 1978; Kapoor et al., 1999).

Methionine Sulfoximine Methionine sulfoximine (MSO) is an irreversible inhibitor of the astrocyte-specific enzyme, glutamine synthetase (GS) (Albrecht and Norenberg, 1990). Ingestion of large amounts of MSO leads to neuronal cell loss in the hippocampal fascia dentata and pyramidal cell layer, in the short association fibers and lower layers of the cerebral cortex, and in cerebellar

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Purkinje cells. MSO also leads to large increases of glycogen levels (Folbergrova, 1973), primarily within astrocytic cell bodies (Phelps, 1975), as well as swollen and damaged astrocytic mitochondria (Hevor, 1994). Although it is generally accepted that MSO inhibits GS, it remains unclear whether this inhibition represents the primary mechanism of MSO neurotoxicity. The relationship between inhibition of GS by MSO and seizure generation is also not well understood but is believed to be associated with inhibition of glutamate and GABA in seizure generation, since glutamine provides the precursor for these neurotransmitters. Rothstein and Tabakoff (1986) have demonstrated that the calcium-dependent, potassiumstimulated release of glutamate and aspartate is inhibited in striatal tissue after intracerebroventricular injection of MSO, and that their release correlates over time with the inhibition of GS. Fluroacetate and Fluorocitrate The Krebs cycle inhibitor fluorocitrate (FC) and its precursor fluoroacetate (FA) are preferentially taken up by glia. FA occurs naturally in a number of plants, and has been used as a rodenticide (Compound 1080). It is prevalent in the South African plant Dichapetalum cymosum, commonly referred to as the Gifblaar plant. Exposure to FA may also occur via exposure to the anticancer drug 5-fluorouracil (Okeda et al., 1990). Ingestion of large amounts of FA results in ionic convulsions. Animals consuming FA commonly seize within minutes, and those surviving these episodes frequently die later on due to respiratory arrest or heart failure. The actions of FC and FA have been attributed to both the disruption of carbon flux through the Krebs cycle and to impairment of ATP production (Swanson and Graham, 1994). FA can be metabolized to fluoroacetyl CoA, followed by condensation with oxaloacetate to form FC by citrate synthase. A second hypothesis implies that FA toxicity is associated with the inhibition of a bi-directional citrate carrier in mitochondrial membranes, which leads to elevated intramitochondrial citrate and could affect citrate-dependent ATP synthesis (Kirsten et al., 1978). Finally, it has been suggested that elevated citrate, secondary to inhibition of aconitase, is associated with the cytotoxicity of these compounds. FA selectively lowers the level of glutamine and inhibits glutamine formation in the brain, not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase (Hassel et al., 1994). After the inhibition of aconitase, citrate accumulates, whereas the levels of isocitrate and α-ketoglutarate decrease. The reversible enzyme glutamate dehydrogenase begins to work in the opposite direction feeding more α-ketoglutarate into the TCA cycle (Martin and Waniewski, 1996).

Neurotransmission-Associated Neurotoxicity A wide variety of naturally occurring toxins, as well as synthetic chemicals, alters specific mechanisms of intercellular communication. Some chemicals that have neurotransmitter-associated toxicity are listed in Table 16-4. Whereas neurotransmitter-associated actions may be well understood for some chemicals, the specificity of the mechanisms should not be assumed. For example, organophosphorus (OP) and carbamate pesticides produce their insecticidal actions by inhibiting acetylcholinesterase, the catalytic enzyme that ends the postsynaptic action of acetylcholine. The resultant cholinergic overstimulation produces signs of acute toxicity ranging from flu-like symptoms to gastrointestinal distress, ataxia, twitching, convulsions, coma, and death. These effects are not as well correlated with acetyl-

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cholinesterase inhibition as might be expected for all such pesticides, leading to suggestions of additional mechanisms of actions that have since been verified in animal and in vitro studies. These include direct actions on pre- and postsynaptic cholinergic receptors and altered reuptake of choline; such actions serve to modulate the downstream impact of cholinergic overstimulation (reviewed in Pope, 1999). Thus, multiple neurotransmitter targets may be more common than was once expected. Nicotine Widely available in tobacco products and in certain pesticides, nicotine has diverse pharmacologic actions and may be the source of considerable toxicity. These toxic effects range from acute poisoning to more chronic effects. Nicotine exerts its effects by binding to a subset of cholinergic receptors, the nicotinic receptors. These receptors are located in ganglia, at the neuromuscular junction, and also within the CNS, where the psychoactive and addictive properties most likely reside. Smoking and “pharmacologic” doses of nicotine accelerate heart rate, elevate blood pressure, and constrict blood vessels within the skin. Because the majority of these effects may be prevented by the administration of α- and β-adrenergic blockade, these consequences may be viewed as the result of stimulation of the ganglionic sympathetic nervous system (Benowitz, 1986). At the same time, nicotine leads to a sensation of “relaxation” and is associated with alterations of electroencephalographic (EEG) recordings in humans. These effects are probably related to the binding of nicotine with nicotinic receptors within the CNS, and the EEG changes may be blocked with mecamylamine, a nicotinic antagonist. Acute overdose of nicotine has occurred in children who accidentally ingest tobacco products, in tobacco workers exposed to wet tobacco leaves (Gehlbach et al., 1974), and in workers exposed to nicotine-containing pesticides. In each of these settings, the rapid rise in circulating levels of nicotine leads to excessive stimulation of nicotinic receptors, a process that is followed rapidly by ganglionic paralysis. Initial nausea, rapid heart rate, and perspiration are followed shortly by marked slowing of heart rate with a fall in blood pressure. Somnolence and confusion may occur, followed by coma; if death results, it is often the result of paralysis of the muscles of respiration. Such acute poisoning with nicotine fortunately is uncommon. Exposure to lower levels for longer duration, in contrast, is very common, and the health effects of this exposure are of considerable epidemiologic concern. In humans, however, it has been difficult to separate the effects of nicotine from those of other components of cigarette smoke. The complications of smoking include cardiovascular disease, cancers (especially malignancies of the lung and upper airway), chronic pulmonary disease, and attention deficit disorders in children of women who smoke during pregnancy. Nicotine may be a factor in some of these problems. For example, an increased propensity for platelets to aggregate is seen in smokers, and this platelet abnormality correlates with the level of nicotine. Nicotine also places an increased burden on the heart through its acceleration of heart rate and blood pressure, suggesting that nicotine may play a role in the onset of myocardial ischemia (Benowitz, 1986). In addition, nicotine also inhibits apoptosis and may play a direct role in tumor promotion and tobacco-related cancers (Wright et al., 1993). Cocaine and Amphetamines While nicotine is a legal and readily available addictive compound, cocaine and amphetamines are

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illegal, although still widely used. The number of adults using these drugs in the United States was approximately 9 million in 1972. That number grew to near 33 million in 1982, and in the 2001 National Household Survey on Drug Abuse it was reported that just over 10% of those surveyed had ever used cocaine, while approximately 2.6% claimed to have used it in the past 12 months (Fishburne et al., 1983; U.S. Department of Health and Human Services, 2001). Cocaine use is abundant in urban settings. It is estimated that from 10–45% of pregnant women take cocaine (Volpe, 1992), and metabolites can be detected in up to 6% of newborns in suburban hospitals (Schutzman et al., 1991). Cocaine blocks the reuptake of dopamine, norepinephrine, and serotonin at the nerve terminal in the CNS, and also causes release of dopamine from storage vesicles. The primary event responsible for the addictive properties and euphoric feeling when intoxicated is a block on the dopamine reuptake transporter (DAT) (Giros et al., 1996). This leads to enhanced dopaminergic transmission, and can result in a variety of symptoms in the user. Many individuals report a euphoric feeling and increased self-confidence, in addition to racing thoughts and a feeling of pressure. In other users, a period of paranoid psychosis ensues. The mechanism of altered neurotransmission has been linked to the dopamine D1 receptor, as mice lacking this receptor fail to exhibit many of the same characteristic behaviors as wild-type mice (Xu et al., 2000). Cocaine abuse also puts individuals at risk for cerebrovascular defects. Habitual users exhibit a greater degree of cerebral atrophy, compared by CT scan, and are more at risk of stroke and intracranial hemorrhage (Berliner, 2000). Cerebrovascular resistance assessed by Doppler sonography has also been found to be higher in cocaine abusers than controls (Herning et al., 1999). In chronic cocaine users, neurodegenerative disorders have been observed, similar to those observed with amphetamine use. Amphetamines also affect catecholamine neurotransmission in the CNS, but also have the potential to damage monoaminergic cells directly. Amphetamines, including methylenedioxymethamphetamine (MDMA, or “ecstasy”), have become popular with young adults in recent decades due to the belief that it is a “safe” drug, and its ability to increase energy and sensation in adults. However, they also exert serious side effects. Similar to cocaine, the most pronounced effect of amphetamines is on the dopaminergic neurons, but they can also damage 5-HT axons and axon terminals (McCann and Ricaurte, 2004). The result is a distal axotomy of DA and 5-HT neurons. The exact mechanism of amphetamine neurotoxicity is still unknown, but several clues have emerged recently. It seems that oxidative stress plays a key role in toxicity. Following amphetaminetriggered dopamine release from the neuron, the dopamine is oxidized to produce free radicals (Lotharius and O’Malley, 2001). Chronic use can affect superoxide dismutase and catalase balance in rodents (Frey et al., 2006), and amphetamine neurotoxicity is attenuated by antioxidants (DeVito and Wagner, 1989). It also induces hyperthermia when given at ambient temperatures, and it has been shown that increasing environmental temperature increases the associated neurotoxicity (Miller and O’Callaghan, 2003). Because drug use and HIV infection have been linked, the effect of cocaine and amphetamine use on toxicity induced by HIV-1 proteins Tat and gp120 has also been investigated recently. HIV associated dementia (HAD) is a neurological disorder afflicting many AIDS patients. Exposure to cocaine and amphetamines in AIDS patients results in a synergistic neurotoxicity, which is attenuated by

β-estradiol (Turchan et al., 2001). Oxidative stress has been implicated in this mechanism as well. When a normally nontoxic dose of cocaine is administered, Tat-induced oxidative stress is enhanced (Aksenov et al., 2006). Excitatory Amino Acids Glutamate and certain other acidic amino acids are excitatory neurotransmitters. The discovery that these excitatory amino acids can be neurotoxic at concentrations that can be achieved in the brain has generated a great amount of interest in these “excitotoxins.” In vitro systems have established that the toxicity of glutamate can be blocked by certain glutamate antagonists (Rothman and Olney, 1986), and the concept has emerged that the toxicity of excitatory amino acids may be related to such divergent conditions as hypoxia, epilepsy, and neurodegenerative diseases (Meldrum, 1987; Choi, 1988; Lipton and Rosenberg, 1994; Beal, 1992, 1995, 1998). Glutamate is the main excitatory neurotransmitter of the brain and its effects are mediated by several subtypes of receptors (Fig. 16-8) called excitatory amino acid receptors (EAARs) (Schoepfer et al., 1994; Hollmann and Heinemann, 1994; Lipton and Rosenberg, 1994). The two major subtypes of glutamate receptors are those that are ligand-gated directly to ion channels (ionotropic) and those that are coupled with G proteins (metabotropic). Ionotropic receptors may be further subdivided by their specificity for binding kainate, quisqualate, α-amino3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and N -methyl-d-aspartate (NMDA). The entry of glutamate into the CNS is regulated at the blood–brain barrier and, following an injection of a large dose of glutamate in infant rodents, glutamate exerts its effects in the area of the brain in which the blood–brain barrier is least developed, the circumventricular organ. Within this site of limited access, glutamate injures neurons, apparently by

Figure 16-8. Excitatory synapse. Synaptic vesicles are tranported to the axonal terminus, and released across the synaptic cleft to bind to the postsynaptic receptors. Glutamate, as an excitatory neurotransmitter, binds to its receptor and opens a calcium channel, leading to the excitation of the postsynaptic cell.

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opening glutamate-dependent ion channels, ultimately leading to neuronal swelling and neuronal cell death (Olney, 1978; Coyle, 1987). The toxicity affects the dendrites and neuronal cell bodies but seems to spare axons. The only known related human condition is the “Chinese restaurant syndrome,” in which consumption of large amounts of monosodium glutamate as a seasoning may lead to a burning sensation in the face, neck, and chest in sensitive individuals. The cyclic glutamate analog kainate was initially isolated in Japan from seaweed as the active component of a herbal treatment of ascariasis. Kainate is extremely potent as an excitotoxin, being 100-fold more toxic than glutamate and is selective at a molecular level for the kainate receptor (Coyle, 1987). Like glutamate, kainate selectively injures dendrites and neurons and shows no substantial effect on glia or axons. As a result, this compound has found use in neurobiology as a tool. Injected into a region of the brain, kainate can destroy the neurons of that area without disrupting the fibers that pass through the same region. Neurobiologists, with the help of this neurotoxic tool, are able to study the role of neurons in a particular area independent of the axonal injuries that occur when similar lesioning experiments are performed by mechanical cutting. Development of permanent neurological deficits in individuals accidentally exposed to high doses of an EAAR agonist has underscored the potential importance of excitatory amino acids in disease (Perl et al., 1990; Teitelbaum et al., 1990). A total of 107 individuals in the Maritime Provinces of Canada were exposed to domoic acid, an analog of glutamate, and suffered an acute illness that most commonly presented as gastrointestinal disturbance, severe headache, and short-term memory loss. A subset of the more severely afflicted patients was subsequently shown to have chronic memory deficits and motor neuropathy. Neuropathologic investigation of patients who died within 4 months of intoxication showed neurodegeneration that was most prominent in the hippocampus and amygdala but also affected regions of the thalamus and cerebral cortex. Other foci of unusual neurodegenerative diseases also have been evaluated for being caused by dietary exposure to EAARs. Perhaps the best known of these is the complex neurodegenerative disease in the indigenous population of Guam and surrounding islands that shares features of amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. Early investigations of this Guamanian neurodegenerative complex suggested that the disorder may be related to an environmental factor, perhaps consumption of seeds of Cycas circinalis (Kurland, 1963). Subsequently, α-amino-methylaminopropionic acid (or B-N -methylamino-l-alanine, BMAA) was isolated from the cycad and was shown to be neurotoxic in model systems. The toxicity of BMAA is similar to that of glutamate in vitro and can be blocked by certain EAAR antagonists (Nunn et al., 1987). Studies in vivo, however, have not demonstrated a relationship between BMAA and the Guamanian neurodegenerative complex (Spencer et al., 1987; Hugon et al., 1988; Seawright et al., 1990; Duncan, 1992). Therefore, it remains unresolved what role cycad consumption and environmental factors play in this cluster of atypical neurodegenerative disease.

Models of Neurodegenerative Disease MPTP Because of an error on the part of a so-called designer chemist, people who injected themselves with a meperidine derivative that was intended to serve as a substitute for heroin

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also received a contaminant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Fig. 16-9) (Langston et al., 1983). Over hours to days, dozens of these patients developed the signs and symptoms of irreversible Parkinson’s disease (PD), some becoming immobile with rigidity (Langston and Irwin, 1986). Autopsy studies demonstrated marked degeneration of dopaminergic neurons in the substantia nigra, with degeneration continuing many years after exposure (Langston et al., 1999). It was initially surprising to find not only that MPTP is neurotoxic, but also that it is a substrate for the B isozyme of monoamine oxidase (MAO-B) (Gerlach et al., 1991). MPTP, an uncharged species at physiologic pH, crosses the blood–brain barrier (BBB) and diffuses into cells, including astrocytes. The MAO-B of astrocytes catalyzes a two-electron oxidation to yield MPDP+ , the corresponding dihydropyridinium ion. A further two-electron oxidation yields the pyridinium ion, MPP+ (Fig. 16-9). MPP+ enters DAergic neurons of the substantia nigra via the dopamine (DA) uptake system, resulting in injury or death of the neuron. Noradrenergic neurons of the locus ceruleus are also vulnerable to repeated exposures of MPTP (Langston and Irwin, 1986), although they are less affected by single exposures than the DAergic neurons are. Once inside neurons, MPP+ acts as a general mitochondrial toxin, blocking respiration at complex I (Di Monte and Langston, 2000). MPP+ may also lead to the production of reactive oxygen species (ROS) and the release of DA from vesicles due to the higher pH environment of the cytosol, where the neurotransmitter undergoes autoxidation (Lotharius and O’Malley, 2000). Consistent with the role of MAO-B in the bioactivation of MPTP to MPP+ , inhibitors of this enzyme, such as l-(-)-deprenyl (selegiline) protect against MPTP neurotoxicity. However, the protection afforded by deprenyl does not appear to arise from its inhibition of MAO-B alone, but also upon other properties, including its ability to act as an antioxidant and free-radical scavenger (Ebadi et al., 2002; Magyar and Szende, 2004; Mandel et al., 2003; Muralikrishnan et al., 2003). Thus, mice deficient in Cu,Znsuperoxide dismutase or glutathione peroxidase show increased vulnerability to MPTP neurotoxicity (Zhang et al., 2000), while overexpression of Mn-superoxide dismutase attenuates the toxicity (Klivenyi et al., 1998). It should be noted that the general toxicity of the proximate neurotoxicant, MPP+ , is considerable when it is administered to animals, although systemic exposure to MPP+ does not result in neurotoxicity, because it does not cross the BBB. Moreover, compared to primates, rats are relatively resistant to DAergic neurotoxicity from systemic administration of the parent compound, MPTP, even when the compound is injected directly in the cerebral circulation via the carotid artery. This resistance appears to be conferred, at least in part, by a high level of biotransformation of MPTP by rat endothelial cells to MPP+ and other polar metabolites, such as MPTP-N -oxide, that are retained in endothelial cells and do not readily traverse the BBB (Mushiroda et al., 2001; Riachi et al., 1990; Scriba and Borchardt, 1989). Although not identical, MPTP neurotoxicity and PD are strikingly similar. The symptomatology of each reflects a disruption of the nigrostriatal pathway. Thus, masked facies, difficulties in initiating and terminating movements, resting “pill-rolling” tremors, rigidity (including characteristic “cogwheel rigidity”), and bradykinesias are all features of both conditions. Pathologically, there is an unusually selective degeneration of neurons in the substantia nigra and depletion of striatal DA in both diseases (Di Monte and Langston, 2000). However, positron emission tomography (PET)

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Figure 16-9. MPTP toxicity. MPP+ , either formed elsewhere in the body following exposure to MPTP or injected directly into the blood, is unable to cross the blood–brain barrier. In contrast, MPTP gains access and is oxidized in situ to MPDP+ and MPP+ . The same transport system that carries dopamine into the dopaminergic neurons also transports the cytotoxic MPP+ .

scanning studies employing the DAergic probe, [18 F]-fluorodopa, show that while patients with idiopathic PD demonstrate greater loss of DAergic function in the putamen than the caudate nucleus, the loss from these two nuclei was the same in patients who had taken MPTP (Snow et al., 2000). The discovery of the relationship between MPTP intoxication and Parkinsonism provided researchers with a new model for studying the pathogenic mechanism of PD and prompted investigations to reveal environmental and occupational exposures that might be associated with the disease (Dauer and Przedborski, 2003). Thus, epidemiological studies have implicated exposures to herbicides, other pesticides, or metals as risk factors for PD (Ferraz et al., 1988; Gorell et al., 1997, 1998, 1999; Liou et al., 1997). Accordingly, the scope of neurotoxicants employed in experimental studies of PD has been enlarged beyond 6-hydroxydopamine (6-OHDA) and MPTP to include agricultural chemicals such as maneb, paraquat, and rotenone (Bove et al., 2005; Uversky, 2004). Epidemiological studies have also found apparent protective effects of cigarette smoking or coffee consumption on the development of PD (Lai et al., 2002; Logroscino, 2005), and experimental studies indicate that nicotine and caffeine are protective in animal models of PD (Quik, 2004; Quik et al., 2006; Ross and Petrovitch, 2001). It is interesting that PET studies of smokers show a marked reduction in brain MAO-B activity, similar to that produced by administration of the MAO-B inhibitor, L-deprenyl (Fowler et al., 1996). Other MAO-B inhibitors are being developed as anti-PD drugs (Mandel et al., 2005), and MAO-B inhibitors have been isolated from tobacco smoke (Khalil et al., 2006). However, it appears that MAO-B inhibition is not essential for the neuropro-

tective activity of these agents; instead, their effectiveness stems from their overall ability to preserve mitochondrial integrity and function. Although several families with early-onset PD demonstrate autosomal dominant inheritance and candidate genes have been identified (Agundez et al., 1995; Kurth et al., 1993; Polymeropoulos et al., 1997), environmental exposures play a more significant role than genetics in the vast majority of PD patients, particularly those with late-onset disease (Kuopio et al., 1999; Tanner et al., 1999). Nevertheless, the delineation of specific genes involved in familial forms of PD (e.g., those encoding α-synuclein, parkin, ubiquitin C-terminal hydrolase L1, DJ-1, PTEN-induced putative kinase 1, and leucine-rich repeat kinase 2) has provided a rational basis for research concerning gene-environment interactions in the etiology of sporadic PD (Benmoyal-Segal and Soreq, 2006; Gosal et al., 2006). Several hypotheses on the loss of DAergic neurons in PD suggest that mitochondrial damage is a primary cause of DAergic neuronal death (Mandel et al., 2005; Abou-Sleiman et al., 2006). These include the following: (1) mitochondria of DAergic neurons are selectively vulnerable to environmental contaminants that cause mitochondrial dysfunction (Przedborski et al., 2004; AmiryMoghaddam et al., 2005), (2) DAergic neurons produce an endogenous mitochondrial toxin (Naoi et al., 2002), and (3) mitochondria harbor defects in enzymes, such as complex I, that lead to impaired energy metabolism (Greene et al., 2005; Gu et al., 1998). The centrality of mitochondria in these hypotheses arises primarily from findings that mitochondrial poisons, such as MPP+ and rotenone can induce a Parkinson-like syndrome in humans, non-human

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primates, and rodents. These neurotoxicants are all capable of inhibiting mitochondrial complex I and appear to model the pathology of PD. Moreover, neuropathological studies reveal a ∼30% decrease in complex I function in deceased PD patients, as compared with age-matched controls (Adam-Vizi, 2005). Mitochondrial complex I inhibitors, such as MPP+ and rotenone, damage nigral neurons by mechanisms involving oxidation. Oxidative damage also plays a significant role in DAergic neuronal cell death induced by intracranial injection of 6-OHDA, another experimental model of PD (Cannon et al., 2006). Both enzymatic and auto-oxidation of 6-OHDA generate ROS, including H2 O2 , superoxide ions, and hydroxyl radicals (reactive oxygen species, or ROS). These ROS and the direct inhibition of complex I lead to lipid peroxidation, protein denaturation, and a decrease in reduced glutathione: all hallmark features of post mortem PD (Jenner, 2003). It is remarkable that the poisoning of drug addicts by MPTP has led to major advances in our understanding of PD, which is the second most prevalent neurodegenerative disorder in the western world (Alzheimer’s disease being first) (Landrigan et al., 2005). Although much remains to be done, knowing that most cases of PD arise from environmental exposures that promote mitochondrial dysfunction and oxidative damage provides us with promising avenues for prevention and treatment of this debilitating disease.

Manganese Manganese (Mn) is an essential metal in both humans and animals. Although Mn is present in almost all diets, animals maintain stable tissue Mn levels by tightly regulating absorption and excretory processes. As an essential trace metal that is found in all tissues, Mn is required for normal metabolism of amino acids, proteins, lipids, and carbohydrates. Mn acts as a cofactor for a variety of enzymes, such as manganese metalloenzymes and Mn-dependent enzyme families. Mn metalloenzymes include arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and Mn superoxide dismutase (Mn-SOD). Mn-dependent enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Therefore, Mn is necessary for the function of many organ systems. In rare Mn deficiencies, clinical manifestations can be seizures, impaired growth, skeletal abnormalities, and impaired reproductive function (Critchfield et al., 1993; Wedler, 1993). At the other end of the spectrum, it is well established that excessive exposure to Mn causes neurotoxicity (McMillan, 1999; Aschner et al., 2005). The most common commercial sources of Mn include the fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT), pesticides such as Maneb, steel factories, welding and mining plants. Occupational exposure to toxic levels of Mn in industrial workers results in psychological and neurological disturbances, including delusions, hallucinations, depression, disturbed equilibrium, compulsive or violent behavior, weakness and apathy, followed by extrapyramidal motor system defects such as tremors, muscle rigidity, ataxia, bradykinesia, and dystonia. Although Mn toxicity has been under investigation for many years, the underlying primary molecular mechanisms of its neurotoxicity remain to be elucidated. Very few potential biomarkers have been established with no early detection markers available. The epidemiological associations and similarities in symptoms between Mn neurotoxicity and dopamine (DA) neuropathology suggest that exposure and accumulation of this metal may be an environmental factor that contributes to idiopathic Parkinson’s disease (IPD). Mn toxicity causes a loss of DA neurons in the substantia

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nigra, and as in Parkinson’s disease (PD), oxidative stress appears to play a significant role in the disorder (Oestreicher et al., 1994; Kienzl et al., 1995; Montgomery, 1995). The brain areas most susceptible to Mn injury are also highly sensitive to oxidative stress. Many metabolically active cell types, particularly tonically active motor neurons in the substantia nigra (SN), require high levels of ATP for optimal function and survival. Mn accumulates in the SN, globus pallidus (GP) and striatum and interferes with ATP synthesis, analogous to effects seen with mitochondrial inhibitors or ischemia. Appraisal of the literature strongly suggests that in addition to targeting similar brain areas, dopaminergic (DAergic) neurodegeneration associated with PD and Mn exposure share multiple common mechanisms, namely mitochondrial dysfunction, aberrant signal transduction, oxidative stress, protein aggregation, and the activation of cell death pathways. The accumulation of Mn in the striatum causes damage to the SN, reduction in tyrosine hydroxylase (TH) activity, and loss of DA neurons (Parenti et al., 1988; Tomas-Camardiel et al., 2002). Intracellular Mn is sequestered by mitochondria through the Ca2+ uniporter (Gavin et al., 1999). Intrastriatal injections of Mn result in excitotoxic brain injury similar to that caused by mitochondrial poisons, such as aminooxyacetic acid and 1-methyl-4-phenyl-pyridinium (Brouillet et al., 1993b). The specificity for Mn accumulation in GP and striatum likely correlates with Mn transporter distribution and the metabolic activity of these basal ganglia nuclei.

Guamanian Cycad-Induced Parkinsonism/ALS syndrome An unusual prevalence of “hereditary paralysis” among the native Guam Chamorros was first reported in the early 1900s. This led to the formation of the National Institute of Neurological and Communicative Disorders and Stroke Research Center on Guam in 1939 (Rodgers-Johnson et al. 1986). In the mid-1950s the incidence of amyotrophic lateral sclerosis (ALS) and parkinsonism–dementia complex (PDC) was up to 100 times higher in Guam than anywhere else in the world (Hirano et al. 1961a). While Guam ALS is clinically indistinguishable from ALS that occurs elsewhere in the world, Guam PDC is a distinct neurodegenerative disorder where parkinsonism and dementia may occur simultaneously in affected Chamorros. Hirano and colleagues first described the clinical features and pathology in 1961 (Hirano et al., 1961a,b). The main clinical features included mental deterioration, parkinsonism, and evidence of motor neuron involvement. The duration of clinical symptoms was about 4 years with the average age of diagnosis being approximately 52 years and a higher incidence in men. The main macroscopic neuropathological features are the presence of cortical atrophy and depigmentation of the substantia nigra. Microscopic evaluations revealed widespread ganglion cell degeneration and neurofibrillary tangles throughout the central nervous system. The lack of uniformity in the clinical presentation of these diseases has made it difficult to determine a possible causative agent. Through the years, the focus has shifted from a genetic to an environmental causative agent. Due to the formation of the case registrar, established in 1958, entire pedigrees have been developed using Chamorros from the same village as controls. Although several investigators have reported a high degree of familial occurrence, no definitive inheritance pattern has been established (Plato et al., 2002). Support for an environmental hypothesis includes a decrease in the incidence of the diseases and an increase in the age of onset with the westernization of Guam (Plato et al., 2003).

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Cycads are primitive plants that contain several toxins, including cycasin and L-β-methylaminoalanine (BMAA) (Schneider et al., 2002). Cycasin, which is metabolized to methylazoxymethanol (MAM), is known to be carcinogenic and cause hepatotoxicity (Sieber et al., 1980). BMAA is a nonprotein amino acid that functions as a glutamate excitotoxin (Rakonczay et al., 1991). In 1987, Spencer and colleagues reported that BMAA, when fed to primates in high concentrations (>100 mg/kg), produced a syndrome that closely resembled the neurological disorder observed in Guam. Traditionally, native Chamorros prepare food from cycads by washing the seeds several times, then grinding them into flour (Kisby et al., 1992). However, it appears that the washing process is sufficient to remove the cycad toxins (Duncan et al., 1990). In 2002, Cox and Sacks suggested “the Chamorro population of Guam ingested large quantities of cycad toxins indirectly by eating flying foxes.” This study demonstrated that the sharp decrease in the flying fox population was followed by a sharp decrease in the incidence of ALS. The flying fox, Pteropus mariannus, is a fruit bat with a wing span of 3 feet and is known to eat three times its weight in fruit, cycad seeds, or beetles which are known to bioaccumulate cycad toxins for protective purposes (Schneider et al., 2002). The consumption of the flying foxes by the Chamorros is not only included in social, but ceremonial settings (Banack and Cox, 2003). Traditionally, the men consume the animal in its entirety, while the women only consume the breast meat. Accumulation of the neurotoxin, BMAA, in the flying fox, Pteropus mariannus, may result in concentrations that are sufficient to cause behavioral and neuropathological changes in primates similar to those observed in the Guam ALS-PDC. Furthermore, BMAA-induced neurodegeneration may occur by an excitotoxic mechanism involving the mitochondria permeability transition pore (mtPTP). It has been shown that BMAA increases the intracellular calcium concentration through the formation of a β-carbamate intermediate (Brownson et al., 2002). This intermediate can act as a glutamate agonist and induce glutamate excitotoxicity. Studies have also demonstrated that an increase in intracellular calcium (Dubinsky and Levi, 1998) and glutamate excitotoxicity are able to induce mtPTP (Schinder et al., 1996).

Developmentally Neurotoxic Chemicals Replication, migration, differentiation, myelination, and synapse formation are the basic processes that underlie development of the NS. There are a variety of insults known to disrupt NS development, the outcomes of which may be very different depending on the time of exposure, including exposures to certain metals, solvents, antimetabolites, persistent organic pollutants, pesticides, pharmaceuticals, and ionizing radiation. Multiple mechanisms of action may be present, producing a wide array of effects in the offspring. The impact on the developing NS may be very different, and often cannot be predicted, from effects observed in adults. Ethanol exposure during pregnancy can result in abnormalities in the fetus, including abnormal neuronal migration and facial development, and diffuse abnormalities in the development of neuronal processes, especially the dendritic spines (Stoltenburg-Didinger and Spohre, 1983). While the exposure may be of little consequence to the mother, it can be devastating to the fetus. There is an effect on NMDA glutamate receptors and excessive activation of GABA receptors, with induction of apoptosis throughout the brain (Ikonomidou et al., 2000). The clinical result of fetal alcohol exposure is

often mental retardation, with malformations of the brain and delayed myelination of white matter (Riikonen et al., 1999). Although there remains a great deal of uncertainty concerning the molecular basis of this developmental aberration, it occurs in a variety of experimental animals, and it appears that acetaldehyde, a product of ethanol catabolism, can produce migration defects in developing animals similar to those that occur in the fetal alcohol syndrome (O’Shea and Kaufman, 1979). Some developmental neurotoxicants have been revealed by human studies or tragic poisoning occurrences. The methyl mercury contamination of fish in Minamata Bay, Japan, led to the birth of many children with developmental disabilities, including cerebral palsy,mental retardation, and seizures. Since then, it was shown that children exposed to methyl mercury in utero show widespread neuronal loss, disruption of cellular migration, profound mental retardation, and paralysis (Costa et al., 2004; Reuhl and Chang, 1979). Studies on primates exposed in utero also have demonstrated abnormal social development (Burbacher et al., 1990). The earlier the exposure, the more generalized the damage that is observed. As with methyl mercury, ethanol and lead are known to produce frank neuropathology in highly exposed populations. However, in recent years the concept has emerged that extremely low levels of exposure to these substances in “asymptomatic” children may have an effect on their behavioral and cognitive development. The association between lead exposure and brain dysfunction has received experimental support in animal models and has prompted screening for lead in children (Benjamin and Platt, 1999). There is no proven safe lower limit for lead, and recent studies have attributed lower IQ scores to blood lead levels less than 5–10 μg/dl (Canfield et al., 2003; Winneke et al., 1994). Similarly, the debate regarding “safe” level of drinking during pregnancy is ongoing, with recent reports of no threshold for subtle cognitive effects (Sampson et al., 2000). There is considerable evidence that chronic exposure to nicotine has effects on the developing fetus (reviewed in Slikker et al., 2005). Along with decreased birth weights, attention deficit disorders are more common in children whose mothers smoke cigarettes during pregnancy, and nicotine has been shown to lead to analogous neurobehavioral abnormalities in animals exposed prenatally to nicotine (Lichensteiger et al., 1988). Nicotinic receptors are expressed early in the development of the NS, beginning in the developing brainstem and later expressed in the diencephalon. The role of these nicotinic receptors during development is unclear; however, it appears that prenatal exposure to nicotine alters the development of nicotinic receptors in the CNS (van de Kamp and Collins, 1994)— changes that may be related to subsequent attention and cognitive disorders in animals and children. Cocaine use during pregnancy is a major concern, especially in urban areas, where use can lead to a variety of acute and chronic adverse events in offspring. Cocaine is able to cross the placental barrier and the fetal blood–brain barrier, and also causes reduced blood flow in the uterus. In severe events at large doses taken by the mother, the fetus may develop hypoxia, leading to a higher rate of birth defects (Woods et al., 1987). Maternal cocaine use is associated with low birth weight and behavioral defects, including a decreased awareness of the surroundings and altered response to stress and pain sensitivity (Chasnoff et al., 1985; Huber et al., 2001). Several epidemiological studies have reported deficits in neurodevelopment and psychological performance in children exposed to polychlorinated hydrocarbons (PCBs) and/or dioxins

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(Seegal, 1996). Research in animals has shown that these persistent pollutants produce endocrine disruptions, cognitive deficits, and changes in activity levels in exposed offspring; however, the specific outcomes depend on the congener or mixture tested as well as the timing of exposure. Changes in estrogen or thyroid hormone, neurotransmitter function, and second messenger systems have been proposed as cellular bases for PCB toxicity (Seegal, 1996; Tilson and Kodavanti, 1998). Recent studies with another persistent class of hydrocarbons, polybrominated diphenyl ethers (PBDEs), have shown similarities in altering thyroid hormone metabolism and cholinergic function, and it has thus been proposed that this chemical class would also be developmentally neurotoxic (Branchi et al., 2003). Thyroid hormone is critical to NS development, and animal studies have suggested that the developing brain may be vulnerable to environmental thyrotoxicants of all sorts (Porterfield, 1994). Finally, it has been shown that even reversible changes in neurotransmission, such as those produced by nicotine or cholinesterase inhibitors, may alter specific growth processes and produce long-lasting deficits (Slotkin, 2004).

CHEMICALS THAT INDUCE DEPRESSION OF NERVOUS SYSTEM FUNCTION Generalized depression of central nervous system function is produced by a variety of volatile solvents. These solvents include several chemical classes—aliphatic and aromatic hydrocarbons, halogenated hydrocarbons, ketones, esters, alcohols, and ethers—that are small, lipophilic molecules. They are widely found in industry, medicine, and commercial products. Human exposures range from chronic low-level concentrations encountered in environmental or occupational setting to high-level concentrations intentionally generated through solvent abuse.

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There are several theories as to the mechanism of this generalized depression, but none is fully explanatory. Solvent potency correlates well with the olive oil:water or octanol:water partition coefficients, leading to the once-popular Meyer-Overton hypothesis that CNS depressants exert their actions through nonspecific disruption of the lipid portions of cell membranes (e.g., Janoff et al., 1981). Anesthesia could occur as a consequence of membrane expansion or perturbations of mitochondrial calcium transport. More recent research has implicated interactions with ligand-gated ion channels as well as voltage-gated calcium channels. Specific receptors regulating these channels include gamma-aminobutyric acid type A (GABAA ), N-methyl-D-aspartate (NMDA), and glycine receptors. These actions relate the effects of solvents to those of pharmaceutical agents such as barbiturates and benzodiazepines. While these targets have been demonstrated mostly for ethanol (Davies, 2003), recent in vitro studies have extended this generality to other volatile solvents (e.g., Cruz et al., 2000). The CNS maintains balance via interplay between inhibitory and excitatory influences. With general depressants, initial suppression of inhibitory systems at low doses produces excitation, such as intoxication observed with ethanol. Thus, acutely, solvents produce a continuum of effects from excitation to sedation, motor impairment, coma, and ultimately death by depression of respiratory centers. A syndrome known as solvent-induced chronic toxic encephalopathy has been described for some populations with longterm and/or high-level exposure. Somewhat vague presenting symptoms include irritability, fatigue, impaired memory or concentration, leading to the need for widely-accepted diagnostic criteria (van der Hoek et al., 2000). The absence of corroborating animal studies have prevented studies of molecular changes which may underlie these long-term effects, and indeed, have raised doubt as to the existence of such a syndrome (Ridgway et al., 2003). Specific solvents also produce other neurotoxicological actions, such as peripheral neuropathy, which are described elsewhere in this text.

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Albrecht J, Norenberg MD: L-methionine-DL-sulfoximine induces massive efflux of glutamine from cortical astrocytes in primary culture. Eur J Pharmacol 182:587–599, 1990. Altman J, Sudarshan K: Postnatal development of locomotion in the laboratory rat. Anim Behav 23:896–920, 1975. Amarnath V, Anthony DC, Valentine WM, et al.: The molecular mechanism of the carbon disulfide mediated crosslinking of proteins. Chem Res Toxicol 4:148–150, 1991. Amiry-Moghaddam M, Lindland H, Zelenin S, et al.: Brain mitochondria contain aquaporin water channels: Evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane. FASEB J 19:1459–1467, 2005. Anderson JP, Carroll Z, Smulowitz M, et al.: A possible mechanism of action of the neurotoxic agent iminodipropionitrile (IDPN): A selective aggregation of the medium and heavy neurofilament polypeptides (NF-M and NF-H). Brain Res 547:353–357, 1991. Anthony DC, Boekelheide K, Graham DG: The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione: I. Accelerated clinical neuropathy is accompanied by more proximal axonal swellings. Toxicol Appl Pharmacol 71:362–371, 1983. Aschner M, Erikson KM, Dorman DC: Manganese dosimetry: Species differences and implications for neurotoxicity. Crit Rev Toxicol 35:1–32, 2005. Atchison WD, Hare MF: Mechanisms of methylmercury-induced neurotoxicity. FASEB J 8:622–629, 1994.

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Turchan J, Anderson C, Hauser KF, et al.: Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine. BMC Neurosci 2:3–13, 2001. U.S. Department of Health and Human Services, Substance Abuse and Mental Health Services Administration, Office of Applied Studies: National Household Study on Drug Abuse, 2001. Washington, DC. U.S. Environmental Protection Agency Water Quality Criterion for the Protection of Human Health: Methylmercury, Office of Science and Technology, Office of Water, Environmental Protection Agency Washington, DC 20460, EPA-823-R-01-001, 2001. Uversky VN: Neurotoxicant-induced animal models of Parkinson’s disease: Understanding the role of rotenone, maneb and paraquat in neurodegeneration. Cell Tissue Res 318:225–241, 2004. Valentine WM, Amarnath V, Amarnath K, et al.: Carbon disulfide-mediated protein cross-linking by N ,N -diethyldithiocarbamate. Chem Res Toxicol 8:96–102, 1995. Valentine WM, Graham DG, Anthony DC: Covalent cross-linking of erythrocyte spectrin in vivo. Toxicol Appl Pharmacol 121:71–77, 1993. Valentine WM, Amarnath V, Graham DG, et al.: Covalent cross-linking of proteins by carbon disulfide. Chem Res Toxicol 5:254–262, 1992. Valentine WM, Amarnath V, Graham DG, et al.: CS2 mediated cross-linking of erythrocyte spectrin and neurofilament protein: Dose response and temporal relationship to the formation of axonal swellings. Toxicol Appl Pharmacol 142:95–105, 1997. Van de Kamp JL, Collins AC: Prenatal nicotine alters nicotinic receptor development in the mouse brain. Pharmacol Biochem Behav 47:889–900, 1994. Van der Hoek JA, Verberk MM, Hageman G: Criteria for solvent-induced chronic toxic encephalopathy: A systematic review. Int Arch Occup Environ Health 73:362–368, 2000. Veronesi B, Jones K, Gupta S, et al.: Myelin basic protein-messenger RNA (MBP-mRNA) expression during triethyltin-induced myelin edema. Neurotoxicology 12:265–276, 1991. Volpe JJ: Effect of cocaine use on the fetus. N Engl J Med 327:399–407, 1992. Wang L, Ho CL, Sun D, et al.: Rapid movement of axonal neurofilaments interrupted by prolonged pauses. Nat Cell Biol 2:137–141, 2000. Watanabe I, Kanabe S: Early edematous lesion of pyrithiamine-induced acute thiamine deficient encephalopathy in the mouse. J Neuropathol Exp Neurol 37:401–413, 1978. Wedler FC: Biological significance of manganese in mammalian systems. Prog Med Chem 30:89–133, 1993. Winneke G, Altmann L, Kramer U, et al.: Neurobehavioral and neurophysiological observations in six-year-old children with low lead levels in East and West Germany. Neurotoxicology 15:705–713, 1994. Wood SL, Beyer BK, Cappon GD: Species comparison of postnatal CNS development: Functional measures. Birth Defects Res (Part B) 68:391– 407, 2003. Woods JR, Plessinger MA, Clark KE: Effect of cocaine on uterine blood flow and fetal oxygenation. JAMA 257:957–961, 1987. Wright SC, Zhong J, Zheng H, et al.: Nicotine inhibition of apoptosis suggests a role in tumor production. FASEB J 7:1045–1051, 1993. Xu M, Guo Y, Vorhees CV, et al.: Behavioral responses to cocaine and amphetamine administration in mice lacking the dopamine D1 receptor. Brain Res 852:198–207, 2000. Yamamura Y: n-Hexane polyneuropathy. Folia Psychiatr Neurol 23:45–57, 1969. Zhang J, Graham DG, Montine TS, et al.: Enhanced N-methyl-4-tetrahydropyridine toxicity in mice deficient in CuZn-superoxide dismutase or glutathione peroxidase. J Neuropathol Exp Neurol 59:53–61, 2000.

CHAPTER 17

TOXIC RESPONSES OF THE OCULAR AND VISUAL SYSTEM Donald A. Fox and William K. Boyes Retinotoxicity of Systemically Administered Therapeutic Drugs Cancer Chemotherapeutics Chloroquine and Hydroxychloroquine Digoxin and Digitoxin Indomethacin Sildenafil Citrate Tamoxifen Retinotoxicity of Known Neurotoxicants Inorganic Lead Methanol Organic Solvents: n-Hexane, Perchloroethylene, Styrene, Toluene, Trichloroethylene, Xylene and Mixtures Organophosphates

INTRODUCTION TO OCULAR AND VISUAL SYSTEM TOXICOLOGY EXPOSURE TO THE EYE AND VISUAL SYSTEM Ocular Pharmacodynamics and Pharmacokinetics Ocular Drug Metabolism Central Visual System Pharmacokinetics TESTING VISUAL FUNCTION Evaluation of Ocular Irritancy and Toxicity Ophthalmologic Evaluations Electrophysiologic Techniques Behavioral and Psychophysical Techniques TARGET SITES AND MECHANISMS OF ACTION: CORNEA Acids Bases or Alkalies Organic Solvents Surfactants

TARGET SITES AND MECHANISMS OF ACTION: OPTIC NERVE AND TRACT Acrylamide Carbon Disulfide Cuban Epidemic of Optic Neuropathy Ethambutol

TARGET SITES AND MECHANISMS OF ACTION: LENS Light and Phototoxicity Corticosteroids Naphthalene Phenothiazines

TARGET SITES AND MECHANISMS OF ACTION: THE CENTRAL VISUAL SYSTEM Lead Methylmercury

TARGET SITES AND MECHANISMS OF ACTION: RETINA

ACKNOWLEDGEMENTS

INTRODUCTION TO OCULAR AND VISUAL SYSTEM TOXICOLOGY

fact that these alterations often occur in the absence of any clinical signs of toxicity (Baker et al., 1984; Anger and Johnson, 1985). This suggests that sensory systems, and in particular the retina and central visual system, may be especially vulnerable to toxic insult. In fact, alterations in the structure and/or function of the eye or central visual system are among the criteria utilized for setting permissible occupational or environmental exposure levels for many different chemicals in the United States (http://www.cdc.gov/niosh/npg/, http://www.epa.gov/iris/index.html). In addition, numerous new drugs used for the treatment of ocular diseases or ocular complications of systemic diseases recently entered the marketplace (Novack, 2003). Moreover, subtle alterations in visual processing of information (e.g., visual perceptual, visual motor) can have profound immediate, long-term, and—in some cases—delayed effects on the mental, social, and physical health and performance of an individual. Finally, ocular and visual system impairments can lead to increased occupational injuries, loss of productive work time, costs for providing medical and social services, lost

Environmental and occupational exposure to toxic chemicals, gases, and vapors as well as side effects resulting from systemic and ocular therapeutic drugs frequently result in structural and functional alterations in the eye and central visual system (Grant, 1986; Anger and Johnson, 1985; Grant and Schuman, 1993; Otto and Fox, 1993; Jaanus et al., 1995; Fox, 1998; Santaella and Fraunfelder, 2007). Almost half of all neurotoxic chemicals affect some aspect of sensory function (Crofton and Sheets, 1989). The most frequently reported sensory system alterations occur in the visual system (Anger and Johnson, 1985; Crofton and Sheets, 1989; Fox, 1998; Grant and Schuman, 1993). Grant (1986) lists approximately 2800 substances that are reportedly toxic to the eye. In many cases, alterations in visual function are the initial symptoms following chemical exposure (Hanninen et al., 1978; Damstra, 1978; Baker et al., 1984; Mergler et al., 1987; Iregren et al., 2002a). Even more relevant is the 665

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Table 17-1 Ocular and Central Visual System Sites of Action of Selected Xenobiotics Following Systemic Exposure xenobiotic Acrylamide Amiodarone Carbon disulfide Chloroquine Chlorpromazine Corticosteroids Digoxin and digitoxin Ethambutol Hexachlorophene Indomethacin Isotretinoin Lead Methanol Methyl mercury, mercury n-Hexane Naphthalene Organic solvents Organophosphates Styrene Tamoxifen

cornea lens + + + +

+ + ++ +

+ + +

+ + + + + +

+ + +

outer retina: rpe

+

outer retina: rods and cones

inner retina: bcs, acs, ipcs

rgcs, optic nerve or tract

lgn, visual cortex

–

–

++

+ + +

–

++ + ++ + + + ++ +

++ + + + ++ ++ + + + + + + +

+ – –

+ ++ – + +

+

+ + + + ++ + +

+

key: RPE = retinal pigment epithelium; BC = bipolar cell; AC = amacrine cell; IPC = interplexiform cell; RGC = retinal ganglion cell; LGN = lateral geniculate nucleus. “+” indicates that this site of action was cited in one or more case reports, review articles, clinical or animal studies. “–” indicates that this site of action showed no adverse effect as cited in one or more case reports, review articles, clinical or animal studies.

productivity, and a distinct decrease in the overall quality of life. The overall goal of this chapter is to review the structural and functional alterations in the mammalian eye and central visual system commonly produced by environmental and workplace chemicals, gases, and vapors and by therapeutic drugs. Except where noted, all these compounds are referred to as chemicals and drugs. The adverse effects of these agents on the different compartments of the eye [i.e., cornea, lens, retina, and retinal pigment epithelium (RPE)], central visual pathway [i.e., optic nerve and optic tract], and the central processing areas [i.e., lateral geniculate nucleus (LGN), visual cortex] are addressed (Table 17-1). To further understand the disposition and effects of these chemicals and drugs on the eye and central visual system, the pharmacodynamics and pharmacokinetics of these compartments are briefly reviewed (Table 17-2). Furthermore, the ophthalmologic evaluation of the eye and the testing of visual function are discussed, as the results from these clinical, behavioral, and electrophysiologic studies form the basis of our diagnosis and understanding of adverse visual system effects in patients and animals. Many of the chemicals discussed below initially appear to have a single site and, by inference, mechanism of action, whereas others have several sites and corresponding mechanisms of action. However, a more in-depth examination reveals that, depending upon dose (concentration), many of these chemicals have multiple sites of action. A few examples illustrate the point. First, as described below in more detail, carbon disulfide produces optic nerve and optic tract degeneration and also adversely affects the neurons and vasculature of the retina, resulting in photoreceptor and retinal ganglion cell (RGC) structural and functional alterations (Hotta et al., 1971; Raitta et al., 1974; Palacz et al., 1980; Seppalainen et al., 1980; Raitta et al., 1981; De Rouck et al.,

1986; Eskin et al., 1988; Merigan et al., 1988; Fox, 1998). Second, gestational and postnatal exposure to inorganic lead clearly affects rod photoreceptors in developing and adult mammals, resulting in rod-mediated (or scotopic) vision deficits; however, structural and functional deficits at the level of the RGCs, visual cortex, and oculomotor system are also observed (Fox and Sillman, 1979; Costa and Fox, 1983; Fox, 1984; Glickman et al., 1984; Lilienthal et al., 1988, 1994; Reuhl et al., 1989; Ruan et al., 1994; Fox et al., 1997; Rice, 1998; Rice and Hayward, 1999; He et al., 2000; and see reviews by Otto and Fox, 1993; Fox, 1998; Rothenberg et al., 2002). Although both gestational and postnatal lead exposure produce scotopic electroretinographic (ERG) deficits, the amplitude changes are in opposite directions and their underlying mechanisms are distinctly different. Finally, some environmental and occupational neurotoxicants (e.g., acrylamide, lead) have been utilized for in vivo and in vitro animal models to examine the pathogenesis of selected retinal, neuronal, or axonal diseases; the basic functions of the retinocortical pathways; and/or the molecular mechanisms of apoptosis (Fox and Sillman, 1979; Vidyasagar, 1981; Lynch et al., 1992; He et al., 2000). The conceptual approach, format, and overall organization of this chapter on ocular and visual system toxicology for the 7th edition of Casarett and Doull’s Toxicology: The Basic Science of Poisons were designed in anticipation that the main audience would be graduate and medical school students, ophthalmologists and occupational physicians, basic and applied science researchers interested in ocular and visual system toxicology, and those interested in having a basic reference source. To write this chapter, information was synthesized and condensed from several excellent resources on different aspects of ocular, retinal, and visual system anatomy, biochemistry, cell and molecular biology, histology, pharmacology,

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Table 17-2 Distribution of Ocular Xenobiotic-Biotransforming Enzymes

Phase I reactions

Phase II reactions

enzymes

tears

Acetylcholinesterase (AChE) Alcohol dehydrogenase Aldehyde dehydrogenase Aldehyde reductase Aldose reductase Carboxylesterase Catalase Cu/Zn superoxide dismutase CYP1A1 or CYP1A2 CYP1B1 CYP2B1 or CYP2B2 CYP2C11 CYP3A1 CYP4A1 or CYP4B2 CYP27A1 MAO-A or MAO-B Glutathione peroxidase Glutathione reductase Glutathione-S-transferase Sulfotransferases UDP-glucuronosyl transferases N -Acetyltransferase

+

+ – + +

cornea

lens

+ + + + + + + + + +

+ + –

iris/ciliary body

+ + + + +

+ + +

+ + +

+

+

+

– + + + + –/+ – + + + +

+ + + + + +

retina

choriod

+

+

+ + + + + + + + + +

+ + +

+ + + + + + + + + +

+ + +

+ +

+

key: “+” and “–” indicate that the enzyme was present (localized by immunohistochemistry, immunogold electron microscopy, Western blot or gene expression) or absent, respectively, in human, monkey or rodent tissues.

physiology, and toxicology (Hogan et al., 1971; Merigan and Weiss, 1980; Fox et al., 1982; Sears, 1984; Dayhaw-Barker et al., 1986; Grant, 1986; Dowling, 1987; Fraunfelder and Meyer, 1989; Ogden and Schachat, 1989; Berman, 1991; Boyes, 1992; Chiou, 1992; Hart, 1992; Hockwin et al., 1992; Bartlett and Jaanus, 1995; Herr and Boyes, 1995; Potts, 1996; Fox, 1998; Rodieck, 1998; Ballantyne, 1999; Kaufman and Alm, 2002; Schuman and Grant, 2003). The interested reader should consult these sources for more detail than is provided below. We gratefully acknowledge the use of the information in these sources as well as those cited in the text below.

EXPOSURE TO THE EYE AND VISUAL SYSTEM Ocular Pharmacodynamics and Pharmacokinetics Toxic chemicals and systemic drugs can affect all parts of the eye (Fig. 17-1; Table 17-1). Several factors determine whether a chemical can reach a particular ocular site of action, including the physiochemical properties of the chemical, concentration and duration of exposure, route of exposure, and the movement of the chemical into and across the different ocular compartments and barriers. The cornea and external adnexa of the eye, including the conjunctiva (the delicate membranes covering the inner surface of the eyelids and the exposed surface of the sclera) and eyelids are often exposed directly to chemicals (i.e., acids, bases, solvents), gases and particles, and drugs. The first site of action is the tear film—a threelayered structure with both hydrophobic and hydrophilic properties.

The outermost tear film layer is a thin (0.1 μm) hydrophobic layer that is secreted by the meibomian (sebaceous) glands. This superficial lipid layer protects the underlying thicker (7 μm) aqueous layer that is produced by the lacrimal glands. The third layer, which has both hydrophobic and hydrophilic properties, is the very thin (0.02 to 0.05 μm) mucoid layer. It is secreted by the goblet cells of the conjunctiva and acts as an interface between the hydrophilic layer of the tears and the hydrophobic layer of the corneal epithelial cells. Thus, the aqueous layer is the largest portion of the tear film, and therefore water-soluble chemical compounds more readily mix with the tears and gain access to the cornea. However, a large proportion of the compounds that are splashed into the eyes is washed away by the tears and thus not absorbed. The cornea, an avascular tissue, is considered the external barrier to the internal ocular structures. Once a chemical interacts with the tear film and subsequently contacts the cornea and conjunctiva, the majority of what is absorbed locally enters the anterior segment by passing across the cornea. In contrast, a greater systemic absorption and higher blood concentration occurs through contact with the vascularized conjunctiva (Sears, 1984; Pepose and Ubels, 1992; Fig. 17-2). The human cornea, which is approximately 500 μm thick, has several distinct layers, or barriers, through which a chemical must pass in order to reach the anterior chamber (see discussion on the cornea, below). The first is the corneal epithelium. It is a stratified squamous, nonkeratinized, and multicellular hydrophobic layer. These cells have a relatively low ionic conductance through apical cell membranes, and due to the tight junctions (desmosomes), they have a high resistance paracellular pathway. The primary barrier to chemical penetration of the cornea is the set of tight junctions

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Figure 17-1. Diagrammatic horizontal cross section of the eye, with medium-power enlargement of details for the cornea, iris and ciliary body, lens, and retina. The morphologic features, their role in ocular pharmacodynamics, pharmacokinetics, drug metabolism, and the adverse effects of drugs and chemical agents on these sites are discussed in the text.

at the superficial layer of the corneal epithelial cells. Thus, the permeability of the corneal epithelium as a whole is low and only lipid soluble chemicals readily pass through this layer. Bowmann’s membrane separates the epithelium from the stroma. The corneal stroma makes up 90% of the corneal thickness and is composed of water, collagen, and glycosaminoglycans. It contains approximately 200 lamellae, each about 1.5 to 2.0 μm thick. Due to the composition and structure of the stroma, hydrophilic chemicals eas-

ily dissolve in this thick layer, which can also act as a reservoir for these chemicals. The inner edge of the corneal stroma is bounded by a thin, limiting basement membrane, called Descemet’s membrane, which is secreted by the corneal endothelium. The innermost layer of the cornea, the corneal endothelium, is composed of a single layer of large diameter hexagonal cells connected by terminal bars and surrounded by lipid membranes. The endothelial cells have a relatively low ionic conductance through apical cell surface and

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Figure 17-2. Ocular absorption and distribution of drugs and chemicals following the topical route of exposure. The details for movement of drugs and chemicals between compartments of the eye and subsequently to the optic nerve, brain, and other organs are discussed in the text. The conceptual idea for this figure was obtained from Lapalus and Garaffo (1992).

a high-resistance paracellular pathway. Although, the permeability of the corneal endothelial cells to ionized chemicals is relatively low, it is still 100 to 200 times more permeable than the corneal epithelium. The Na+ ,K+ -pump is located on the basolateral membrane while the energy-dependent Na+ ,HCO− 3 -transporter is located on the apical membrane (Sears, 1984; Pepose and Ubels, 1992). There are two separate vascular systems in the eye: (1) the uveal blood vessels, which include the vascular beds of the iris, ciliary body, and choroid, and (2) the retinal vessels (Hogan, 1971; Alm, 1992). In humans, the ocular vessels are derived from the ophthalmic artery, which is a branch of the internal carotid artery. The ophthalmic artery branches into (1) the central retinal artery, which enters the eye and then further branches into four major vessels serving each of the retinal quadrants; (2) two posterior ciliary arteries; and (3) several anterior arteries. In the anterior segment of the eye, there is a blood–aqueous barrier that has relatively tight junctions between the endothelial cells of the iris capillaries and nonpigmented cells of the ciliary epithelium (Hogan, 1971; Alm, 1992). The major function of the ciliary epithelium is the production of aqueous humor from the plasma filtrate present in the stroma of the ciliary processes. In humans and several widely used experimental animals (e.g., monkeys, pigs, dogs, rats, mice), the retina has a dual circulatory supply: choroidal and retinal. The retinal blood vessels are distributed within the inner or proximal portion of the retina, which consists of the outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). The endothelial cells of capillaries of the retinal vessels have tight junctions similar to those that form the blood–brain barrier in the cerebral capillaries. These capillaries form the blood-retinal barrier

and under normal physiologic conditions, they are largely impermeable to chemicals such as glucose and amino acids (Alm, 1992). However, at the level of the optic disk, the blood-retinal barrier lacks these tight-junction types of capillaries and thus hydrophilic molecules can enter the optic nerve head by diffusion from the extravascular space (Alm, 1992) and cause selective damage at this site of action. The outer or distal retina, which consists of the retinal pigment epithelium (RPE), rod, and cone photoreceptor outer segments (ROS, COS) and inner segments (RIS, CIS), and the photoreceptor outer nuclear layer (ONL), are avascular. These areas of the retina are supplied by the choriocapillaris: a dense, one-layered network of fenestrated vessels formed by the short posterior ciliary arteries and located next to the RPE. Consistent with their known structure, these capillaries have loose endothelial junctions and abundant fenestrae; they are highly permeable to large proteins. Thus, the extravascular space contains a high concentration of albumin and γ -globulin (Sears, 1992). Following systemic exposure to drugs and chemicals by the oral, inhalation, dermal, or parenteral route, these compounds are distributed to all parts of the eye by the blood in the uveal blood vessels and retinal vessels (Fig. 17-3). Most of these drugs and chemicals can rapidly equilibrate with the extravascular space of the choroid where they are separated from the retina and vitreous body by the RPE and endothelial cells of the retinal capillaries, respectively. Hydrophilic molecules with molecular weights less than 200 to 300 Da can cross the ciliary epithelium and iris capillaries and enter the aqueous humor (Sears, 1992). Thus, the corneal endothelium—the cells responsible for maintaining normal hydration and transparency of the corneal stroma—could be exposed to chemical compounds by the aqueous humor and limbal capillaries.

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Similarly, the anterior surface of the lens can also be exposed as a result of its contact with the aqueous humor. The most likely retinal target sites following systemic drug and chemical exposure appear to be the RPE and photoreceptors in the distal retina because the endothelial cells of the choriocapillaris are permeable to proteins smaller than 50 to 70 kDa. However, the cells of the RPE are joined on their basolateral surface by tight junctions—zonula occludens— that limit the passive penetration of large molecules into the neural retina. The presence of intraocular melanin plays a special role in ocular toxicology. First, it is found in several different locations in the eye: pigmented cells of the iris, ciliary body, RPE, and uveal tract. Second, it has a high binding affinity for polycyclic aromatic hydrocarbons, electrophiles, calcium, and toxic heavy metals such as aluminum, iron, lead, and mercury (Meier-Ruge, 1972; Potts and Au, 1976; Dr¨ager, 1985; Ulshafer et al., 1990; Eichenbaum and Zheng, 2000). Although this initially may play a protective role, it also results in the excessive accumulation, long-term storage, and slow release of numerous drugs and chemicals from melanin. For example, atropine binds more avidly to pigmented irides and thus its duration of action is prolonged (Bartlett and Jaanus, 1995). In addition, the accumulation of chloroquine in the RPE produces an 80-fold higher concentration of chloroquine in the retina relative to liver (Meier-Rouge, 1972). Similarly, lead accumulates in the human retina such that its concentration is 5 to 750 times that in other ocular tissues (Eichenbaum and Zheng, 2000).

Ocular Drug Metabolism Metabolism of xenobiotics occurs in all compartments of the eye by well-known Phase I and II xenobiotic-biotransforming enzymes. Drug metabolizing enzymes such as acetylcholinesterase, carboxylesterase (also known as pseudocholinesterase: see chap. 6 entitled “Biotransformation of Xenobiotics”), alcohol and aldehyde dehydrogenase, aldehyde and aldose reductase, catalase, monoamine oxidase A and/or B, and Cu2+ /Zn2+ superoxide dismutase as well as several types of proteases are present in the tears, iris-ciliary body, choroid, and retina of many different species (Shanthaverrappa and Bourne, 1964; Waltmann and Sears, 1964; Bausher, 1976; Anderson, 1980; Puro, 1985; Atalla et al., 1998; Berman, 1991; Crouch et al., 1991; Watkins et al., 1991; Gondhowiardjo and van Haeringen, 1993; Downes and Holmes, 1995; Gaudet et al., 1995; Behndig et al., 1998; King et al., 1999; Table 17-2). Although there are new data on the number and activity of cytochrome P450 (CYP) isoforms (Phase I metabolism) and Phase II conjugating enzymes in ocular tissues since our first edition of this chapter, there is still less known about xenobiotic metabolism in ocular tissues than in other tissues (Shichi et al., 1975; Shichi and Nebert, 1982; Sears, 1992; Zhao and Shichi, 1995; Shichi, 1996; Srivastava et al., 1996; Schwartzman, 1997; Singh and Shichi, 1998; Mastyugin et al., 1999; Nakamura et al., 2005; Doshi et al., 2006; Lee et al., 2006). CYP1A1 and CYP1A2—previously known as aryl hydrocarbon hydroxylase activity—are found in all bovine and mouse ocular tissues except the lens and can be induced by 3-methylcholanthrene and βnaphthoflavone (Shichi et al., 1975; Shichi et al., 1982; Zhao and Shichi, 1995). Moreover, a CYP4 family member—CYP4A1 in the mouse and CYP4B1 in the rabbit—is present in corneal epithelium and can be induced by phenobarbital and the peroxisome proliferator clofibrate (Zhao et al., 1996; Mastyugin et al., 1999). This corneal epithelial CYP monooxygenase metabolizes arachidonic acid to two of its major metabolites: 12(R)-HETE [12(R)-hydroxy-5,8,10,14-

eicosatrienoic acid] and 12(R)-HETrE [12(R)-hydroxy-5,8,14eicosatrienoic acid] (Schwartzman, 1987; Asakura et al., 1994; Mastyugin et al., 1999). In the corneal epithelium, 12(R)-HETE is a potent inhibitor of Na+ ,K+ -ATPase, whereas 12(R)-HETrE is a potent angiogenic and chemotactic factor (Schwartzman, 1997). The Phase II conjugating enzymes found in bovine, rabbit, and rat ocular tissues include UDP glucuronosyltransferase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and N acetyltransferase (Awasthi et al., 1980; Shichi and Nebert, 1982; Penn et al., 1987; Watkins et al., 1991: Srivastava et al., 1996; Singh and Shichi, 1998; Nakamura et al., 2005; see Table 17-2). Whereas the activity of these enzymes varies between species and ocular tissues, the whole lens appears to have low biotransformational activity. Metabolically, the lens is a heterogeneous tissue, with glutathione S-transferase activity found in the lens epithelium and not in the lens cortex or nucleus (Srivastava et al., 1996). Overall, these findings suggest that ocular tissues that contact the external environment have a blood supply possessing both CYPs and Phase II conjugating enzymes, especially those enzymes related to glutathione conjugation. The presence and need for a competent glutathione conjugation system is clearly understandable in ocular tissues that directly interact with UV radiation, light, and xenobiotics and that have high rates of metabolism and high lipid content. Further work is needed to determine the presence and activity of other CYP family members in ocular tissue, the various factors (i.e., age, gender, tissue-specific, xenobiotics, etc.) that regulate their expression, and their endogenous and exogenous substrates.

Central Visual System Pharmacokinetics The penetration of potentially toxic compounds into visual areas of the central nervous system (CNS) is governed, like other parts of the CNS, by the blood–brain barrier (Fig. 17-3). The blood–brain barrier is formed through a combination of tight junctions in brain capillary endothelial cells and foot processes of astrocytic glial cells that surround the brain capillaries. Together these structures serve to limit the penetration of blood-borne compounds into the brain and in some cases actively exclude compounds from brain tissue. The concept of an absolute barrier is not correct, however, because the blood–brain barrier is differentially permeable to compounds depending on their size, charge, and lipophilicity. Compounds that are large, highly charged, or otherwise not very lipid soluble tend to be excluded from the brain, whereas smaller, uncharged, and lipidsoluble compounds more readily penetrate into the brain tissue. In addition to entering the CNS through this nonspecific semipermeable diffusion barrier, some specific nutrients including, ions, amino acids, and glucose enter the CNS through selective transport mechanisms. In some cases, toxic compounds may be actively transported into the brain by mimicking the natural substrates of active transport systems. A few areas of the brain lack a blood–brain barrier; consequently, blood-borne compounds readily penetrate into the brain tissue in these regions. Interestingly, one such area is the optic nerve near the lamina cribrosa (Alm, 1992), which could cause this part of the central visual system to be vulnerable to exposures that do not affect much of the remainder of the brain.

TESTING VISUAL FUNCTION Testing for potential toxic effects of compounds on the eye and visual system can be divided into tests of ocular toxicity and tests of visual function. Alternatively, such tests could be grouped according

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Figure 17-3. Distribution of drugs and chemicals in the anterior and posterior segments of the eye, optic nerve, brain, and other organs following the systemic route of exposure. The details for movement of drugs and chemicals between compartments of the eye are discussed in the text. The conceptual idea for this part of the figure was obtained from Lapalus and Garaffo (1992). The solid and dotted double lines represent the different blood–tissue barriers present in the anterior segment of the eye, retina, optic nerve, and brain. The solid double lines represent tight endothelial junctions, whereas the dotted double lines represent loose endothelial junctions.

to the professional training of the individual conducting the evaluation. Such a categorization might include tests of contact irritancy or toxicity akin to dermatologic procedures, ophthalmologic evaluations, neurophysiologic studies of the function of the visual system, and behavioral or psychophysical evaluations of visual thresholds and aspects of perception.

Evaluation of Ocular Irritancy and Toxicity Standard procedures for evaluating ocular irritation have been based on a method originally published by Draize et al. over a half a century ago (Draize et al., 1944). Over this time, the Draize test with some additions and revisions formed the basis of safety evaluations in data submitted to several government regulatory bodies including the European Economic Community and several federal agencies within the United States. Traditionally, albino rabbits were the subjects evaluated in the Draize test, although the Environmental Protection Agency (EPA) protocol allows different test species to be used if sufficient justification is provided. The standard procedure involves instillation of 0.1 mL of a liquid or 100 mg of a solid into the conjunctival sac of one eye and then gently holding the eye closed for 1 second. The untreated eye serves as a control. Both eyes are evaluated at 1, 24, 48, and 72 hours, respectively, after treatment. If there is evidence of damage in the treated eye at 72 hours, the examination time may be extended. The cornea, iris, and conjunc-

tiva are evaluated and scored according to a weighted scale. The cornea is scored for both the degree of opacity and area of involvement, with each measure having a potential range from 0 (none) to 4 (most severe). The iris receives a single score (0 to 2) for irritation, including degree of swelling, congestion, and degree of reaction to light. The conjunctiva is scored for the redness (0 to 3), chemosis (swelling: 0 to 4), and discharge (0 to 3). The individual scores are then multiplied by a weighting factor: 5 for the cornea, 2 for the iris, and 5 for the conjunctiva. The results are summed for a maximum total score of 110. Photographic examples of lesions receiving each score are provided in Datson and Freeberg (1991). In this scale, the cornea accounts for 80 (73%) of the total possible points, in accordance with the severity associated with corneal injury. The Draize test, although a standard for decades, has been criticized on several grounds, including high interlaboratory variability, the subjective nature of the scoring, poor predictive value for human irritants, and most significantly, for causing undue pain and distress to the tested animals. These criticisms have spawned a concerted effort to develop alternative methods or strategies to evaluate compounds for their potential to cause ocular irritation. These alternatives include modifications of the traditional Draize test to reduce the number of test animals required, reduce the volume of the compound administered, and increase objectivity of scoring (e.g., Kennah et al., 1989; Bruner et al., 1992; Lambert et al., 1993). In addition, several alternative test procedures have been proposed, including the use of

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skin irritancy tests as substitutes for ocular irritancy and the use of in vitro assays (Datson and Freeberg, 1991; Chamberlain et al., 1997; Kruszewski et al., 1997). Additional research efforts are developing quantitative structure activity relationships to better predict ocular irritancy (Barratt, 1995; Sugai et al., 1990; Kulkarni and Hopfinger, 1999). To date, there is no general consensus as to which of the alternative test strategies, alone or in combination, provides a suitable alternative to the Draize procedure. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), a committee representing 14 agencies of the U.S. Federal government, has established criteria for validating methods to substitute for whole animal testing of ocular corrosiveness or irritancy (NIEHS, 1997). ICCVAM was established in 1997 by the Director of the National Institute of Environmental Health Sciences (NIEHS) to implement NIEHS directives in Public Law 103-43 (http://iccvam.niehs.nih.gov/ home.htm). In March of 2005, ICCVAM released a summary evaluation of the current status of validation of in vitro test methods for identifying ocular corrosives and irritants (http://iccvam. niehs.nih.gov/methods/ocudocs/EPreport/ocureport.htm). The report considered methods that utilize isolated rabbit, chicken, or bovine eyes and one that utilized the hen egg chorioallantoic membrane (ICCVAM, 2005). Among the most important concerns in developing alternative test approaches is the predictive ability of the assay as compared to the existing results of the Draize test. The Draize results are used as a standard because information available from human ocular exposures almost invariably comes from accidental exposure episodes in which the dose levels, durations and conditions of exposure are unknown. However, the Draize test cannot be considered a “gold standard” due to the variability of results within and between studies, subjectivity of scoring outcomes, and inter-species differences in the ability to predict human ocular toxic potency from testing rabbit eyes. On the positive side, however, a large number of substances have been assessed using the Draize test and consequently, there is an ample list of chemicals against which alternative test procedures can be assessed. A list of positive and negative control substances against which alternative tests can be assessed was reviewed by ICCVAM. Factors considered included the presence of important classes of substances, quality and reliability of the data, ready availability of the chemicals in a reasonably pure form, and the range and types of lesions produced. None of the alternative tests reviewed reproduced injurious responses that produced an inflammatory response, nor did they reflect the time course of lesion development and recovery, as does the Draize test. On the other hand, most of the alternative tests were reasonably consistent with the Draize test at identifying irritant or corrosive substances. The alternative tests were less consistent with regard to classification of surfactants, alcohols, organic solvents, or solids.

Ophthalmologic Evaluations There are many ophthalmologic procedures for evaluating the health of the eye. These should be conducted by a trained ophthalmologist or optometrist experienced in evaluating the species of interest. Procedures available range from fairly routine clinical screening evaluations to sophisticated techniques for very targeted purposes, the latter of which are beyond the scope of this chapter. A clinical evaluation of the eye addresses the adnexa and both the anterior and posterior structures in the eye. Examination of the adnexa includes evaluating the eyelids, lacrimal apparatus, and palpebral (covering the eyelid) and bulbar (covering the eye) conjunctiva. The anterior

structures or anterior segment include the cornea, iris, lens, and anterior chamber. The posterior structures, referred to as the ocular fundus, include the retina, retinal vasculature, choroid, optic nerve, and sclera. The adnexa and surface of the cornea can be examined initially with the naked eye and a hand-held light. Closer examination requires a slit-lamp biomicroscope, using a mydriatic drug (causes pupil dilation) if the lens is to be observed. The width of the reflection of a thin beam of light projected from the slit lamp is an indication of the thickness of the cornea and may be used to evaluate corneal edema. Lesions of the cornea can be better visualized with the use of fluorescein dye, which is retained where there is an ulceration of the corneal epithelium. Examination of the fundus requires use of a mydriatic drug. Fundoscopic examination is conducted using a direct or an indirect ophthalmoscope, as described (Gelatt, 1981; Harroff, 1991; Hockwin et al., 1992). Several recently developed techniques are described in Peiffer et al. (2000). An ophthalmologic examination of the eye may also involve, prior to introducing mydriatics, an examination of the pupillary light reflex. The direct pupillary reflex involves shining a bright light into the eye and observing the reflexive pupil constriction in the same eye. The consensual pupillary reflex is observed in the eye not stimulated. Both the direct and consensual pupillary light reflexes are dependent on function of a reflex arc involving cells in the retina, which travel through the optic nerve, optic chiasm, and optic tract to project to neurons in the pretectal area. Pretectal neurons travel to both ipsilateral (for the direct reflex) and contralateral (for the consensual reflex) parasympathetic neurons of the midbrain accessory oculomotor (Edinger–Westphal) nucleus. Preganglion neurons from the Edinger–Westphal nucleus project through the oculomotor nerve to the ciliary ganglion. Postganglionic neurons from the ciliary ganglion then innervate the smooth muscle fibers of the iridal pupillary sphincter. The absence of a pupillary reflex is indicative of damage somewhere in the reflex pathway, and differential impairment of the direct or consensual reflexes can indicate the location of the lesion. The presence of a pupillary light reflex, however, is not synonymous with normal visual function. Pupillary reflexes can be maintained even with substantial retinal damage. In addition, lesions in visual areas outside of the reflex pathway, such as in the visual cortex, may also leave the reflex function intact.

Electrophysiologic Techniques Many electrophysiologic or neurophysiologic procedures are available for testing visual function in a toxicologic context. In a simple sense, most of these procedures involve stimulating the eyes with visual stimuli and electrically recording potentials generated by visually responsive neurons. Different techniques and stimuli are used to selectively study the function of specific retinal or visual cortical neurons. In the study of the effects of potential toxic substances on visual function, the most commonly used electrophysiologic procedures are the flash-evoked ERG, visual-evoked potentials (VEPs), and, less often, the electrooculogram (EOG). ERGs are typically elicited with a brief flash of light and recorded from an electrode placed in contact with the cornea. A typical ERG waveform (see Fox and Farber, 1988; Rosolen et al., 2005) includes an initial negative-going waveform, called the a-wave, that reflects the activation of photoreceptors, and a following positive b-wave that reflects the activity of retinal bipolar cells and associated membrane potential changes in M¨uller cells—a type of retinal glial cell that buffers extracellular potassium ions and glutamate (Dowling, 1987; Rodieck, 1998). In addition, a series of oscillatory

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potentials can be observed overriding the b-wave, of which the neural generators are somewhat uncertain, but they presumably reflect various stages of intraretinal signal processing. A standard set of ERG procedures has been recommended and updated for screening assessments of human clinical patients (Marmor et al., 1989; Marmor and Zrenner, 1995). These procedures include the recording of (1) a response reflective of only rod photoreceptor function in the dark-adapted eye, (2) the maximal response in the dark-adapted eye, (3) a response developed by cone photoreceptors, (4) oscillatory potentials, and (5) the response to rapidly flickered light. These recommendations were used to create a protocol for screening the retinal function of dogs in toxicologic studies (Jones et al., 1994). For testing retinal function beyond a screening level evaluation, ERG amplitude and latency versus log stimulus intensity functions are very useful (e.g., Fox and Farber, 1988; Rosolen et al., 2005). Although flash-evoked ERGs do not reflect the function of the RGC layer, ERGs elicited with pattern-reversal stimuli (PERGs) do reflect the activation of RGCs. To date, PERGs have not been used widely in toxicological evaluations. VEPs are elicited with stimuli similar to those used to evoke ERGs; however, VEPs are recorded from electrodes overlying visual (striate) cortex. Consequently, VEPs reflect the activity of the retinogeniculostriate pathway and the activity of cells in the visual cortex. Flash-elicited VEPs have been used in a number of studies of potentially neurotoxic compounds in laboratory animals (Fox et al., 1977, 1982; Dyer et al., 1982; Rebert, 1983; Dyer, 1985; Boyes, 1992; Mattsson et al., 1992; Herr and Boyes, 1995). Pattern-elicited VEPs (PEPs) are more widely used in human clinical evaluations because of their diagnostic value. However, they are infrequently used in laboratory animals because albino rats do not produce usable PEPs (Boyes and Dyer, 1983). Recording PEPs and conducting psychophysical studies with pigmented Long-Evans hooded rats, Fox (1984) found that the PEP spatial frequency functions yielded almost the same visual acuity values (1.4 cycles per degree) as the psychophysically determined spatial resolution limit values (1.8 cycles per degree). These values are in good agreement with those obtained by others using single-cell electrophysiological and behavioral techniques (Powers and Green, 1978; Birch and Jacobs, 1979; Dean, 1981). Moreover, PEPs and flash-elicited VEPs have exhibited differential sensitivity to some neurotoxic agents (Boyes and Dyer, 1984; Fox, 1984). The U.S. Environmental Protection Agency has published guidelines for conducting visual evoked potential testing in a toxicological context, along with analogous sensory evoked potential procedures for evaluating auditory and somatosensory function (EPA, 1998a). The EOG is generated by a potential difference between the front and back of the eye, which originates primarily within the RPE (Berson, 1992). Metabolic activity in the RPE generates a lightinsensitive potential (the standing potential) and a light-sensitive potential; the difference in amplitude between these two potentials is easily measured as the EOG. The magnitude of the EOG is a function of the level of illumination and health status of the RPE. Electrodes placed on the skin on a line lateral or vertical to the eye measure potential changes correlated with eye movements as the relative position of the ocular dipole changes. Thus, the EOG finds applications in assessing both RPE status and measuring eye movements. The EOG is also used in monitoring eye movements during the recording of other brain potentials, so that eye movement artifacts are not misinterpreted as brain generated electrical activity (Berson, 1992). In addition, EOG measurements have been used to detect eye movement deficits caused by exposures to toxic

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substances, such as those altering the function of the cerebellum (reviewed by Geller et al., 1995). As noted in the introduction to this chapter, there is a clear need for testing visual function as a component of the toxicological evaluation of commercial chemicals. The magnitude of the potential threat posed by visual system toxicity is not known, because sensory function has not been evaluated in a systematic fashion. Currently, the EPA screening batteries for neurotoxicological evaluation of laboratory animals include only minimal evaluations of visual function. Standard screening procedures such as those included in EPA’s neurotoxicity screening battery (EPA, 1998b) include a functional observational battery (FOB)—an automated measurement of motor activity and neuropathology. Of the screening procedures, only the FOB evaluates visual function and the extent of these measures is very limited. The entire assessment of visual function includes observing the animal’s response to an approaching object such as a pencil and observing the pupil’s response to a light. These procedures are limited in not exploring responsiveness over a range of stimulus features such as luminance, color, spatial frequency, and temporal frequency. In addition, they do not evaluate rod or cone sensory thresholds, nor do they isolate potential motor or integrative contributions to task performance. Despite these shortcomings, the FOB has been successful at detecting visual deficits produced by exposure to 3,3 -iminodipropionitrile (Moser and Boyes, 1993) or carbon disulfide (Moser et al., 1998), which illustrates the importance of conducting at least this level of visual function screening in routine testing programs. Some studies include ophthalmologic and/or ocular pathologic evaluations. Comprehensive visual toxicity studies should include ophthalmologic and pathologic evaluation of ocular tissues and assessments of visual function. Another potentially limiting problem in product safety testing is the routine use of albino animals, whose capacity for visual function is limited at best. The toxicity of many polycyclic aromatic compounds is mediated through interactions with melanin (Potts, 1964, 1996; Meier-Ruge, 1972; see discussion of the retina, below), which is absent in the eyes of albino strains. Furthermore, lightrelated ocular lesions, including cataracts and retinal degeneration, are often observed in control albino rats and mice used in 2-year product-safety evaluations. It is well known that normal photoreceptor physiology and susceptibility to chemical damage in rats and mice are mediated by light (LaVail, 1976; Williams et al., 1985; Williams, 1986; Penn and Williams, 1986; LaVail et al., 1987; Penn et al., 1987, 1989; Rapp et al., 1990; Backstrom et al., 1993). For example, one study showed that the proportion of Fisher 344 rats in the control group with photoreceptor lesions ranged from less than 10% of rats housed on the bottom row of the cage racks to over 55% of rats housed on the top row, where the luminance was greater (Rao, 1991). Even under reduced light levels, the incidence of these effects was as high as 15%. If albino animals are used as the test subjects, it is important to control the overall level of illumination in the animal colony and also to periodically rotate the animals among the rows of the cage racks. Even under these conditions, it is extremely difficult to interpret pathologic changes in albino rats and mice exposed to test compounds against such high rates of background retinal lesions. Sensory dysfunction can confound other measures of neurotoxicity. Many behavioral and observational evaluations of neurotoxicity involve presentation of sensory stimuli to human or animal subjects followed by the observation or measurement of a behavioral or motor response. In many cases, the inferences drawn from such measures are stated in terms of the cognitive abilities of the test

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subject, such as whether learning or memory have been compromised as a function of exposure to the test compound. If the subject was unable to clearly and precisely perceive the test stimuli, which are often complex patterns or contain color, task performance may be affected independently of any effect on cognition. Controlling for visual deficits may alter the interpretation of performance or cognitive tasks (Anger et al., 1994; Hudnell et al., 1996; Walkowiak et al., 1998; Cestnick and Coltheart, 1999).

Behavioral and Psychophysical Techniques Behavioral and psychophysical testing procedures typically vary the parameters of the visual stimulus and then determine whether the subject can discriminate or perceive the stimulus (Woodhouse and Barlow, 1982; Maurissen, 1995). Many facets of visual function in humans and laboratory animals have been studied using these procedures. Often, the goal of these procedures is to resolve the spatial or temporal limits of visual discrimination; however, most visual scenes and targets in our daily life involve discrimination of objects with low to middle spatial and temporal frequencies (Woodhouse and Barlow, 1982). Contrast sensitivity functions are used to assess these parameters. In addition, as discussed below, other visual parameters also have been investigated. Contrast sensitivity refers to the ability to resolve small differences in luminance contrast, such as the difference between subtle shades of gray. Contrast sensitivity should be measured for a series of visual patterns that differ in pattern size. Typically, such patterns are a series of sine-wave gratings (striped patterns where the luminance changes across the pattern in a sinusoidal profile) where the spatial frequency of the sinusoidal pattern (i.e., the width of the bars in the pattern) varies in octave steps. The contrast of the patterns (i.e., the difference between the brightest and darkest parts of the pattern that is adjusted for mean luminance) also varies. The resulting data, when plotted on log/log coordinates, forms a contrast sensitivity function that is representative of the ability to detect visual patterns over the range of visible pattern sizes. Contrast sensitivity functions are dependent primarily on the neural as opposed to the optical properties of the visual system. The contrast sensitivity functions generally form an inverted U-shaped profile with highest sensitivity to contrast at intermediate spatial frequencies. Sensitivity to high spatial frequencies is equivalent to a measure of visual acuity, whereas sensitivity to mid-range spatial frequencies is important for facial recognition (Ginsburg, 2003). The peak of the function as well as the limits of resolution on both the spatial frequency and contrast axes vary across species. For example, at relatively mesopic (rod- and cone-mediated) luminance levels, the peak of the spatial contrast sensitivity function for the albino rat, hooded rat, cat, and human are 0.1, 0.3, 0.3, and 2 cycles per degree, respectively (Birch and Jacobs, 1979; Fox, 1984). Some of the visual parameters that have been investigated include: (1) the absolute luminance threshold, which is the threshold value for detecting an illuminated target by a dark-adapted subject in a dark-adapted environment; (2) visual acuity, which is the spatial resolution of the visual system [approximately 50 cycles per degree in humans (Woodhouse and Barlow, 1982) and 1.1 to 1.8 cycles per degree in albino and hooded rats (Fox, 1984; Birch and Jacobs, 1979; Dean, 1981)]; (3) color and spectral discriminations (Porkony et al., 1979); (4) critical flicker fusion frequency, which is the threshold value for detecting a flickering light at different luminance intensities; and (5) the peak of the spatial and temporal contrast sensitivity functions at different luminance levels (Wood-

house and Barlow, 1982). Most of these tests are dependent upon the quality of the ocular optics and the ability to obtain a sharply focused visual image on the retina. Thresholds for detecting luminance, contrast, flicker, and color are primarily dependent on retinal and central mechanisms of neural function, although optical impairments (e.g., cataracts) interfere with these functions. The assessment of visual acuity and contrast sensitivity has been recommended for field studies of humans potentially exposed to neurotoxic substances (ATSDR, 1992). Color vision deficits are either inherited or acquired. Hereditary red-green color deficits occur in about 8% of males (X-linked), while only about 0.5% of females show similar congenital deficits (Porkony et al., 1979). Inherited color deficiencies take two common forms: protan, a red-green confusion caused by abnormality or absence of the long-wavelength (red) sensitive cones; and deutan, concomitant confusion of red-green and blue-yellow caused by abnormality or absence of the middle-wavelength (green) sensitive cones. Congenital loss of short-wavelength cones, resulting in a blue-yellow confusion (tritanopia, or type III), is extremely rare. Most acquired color vision deficits, such as those caused by drug and chemical exposure, begin with a reduced ability to perform blue-yellow discriminations (Porkony et al., 1979; Jaanus et al., 1995). With increased or prolonged low-level exposure, the color confusion can progress to the red-green axis as well. Because of the rarity of inherited tritanopia, it is generally assumed that blueyellow deficits, when observed, are acquired deficits. K¨ollner’s rule of thumb states that disorders of the outer retina produce blue-yellow deficits, whereas disorders of the inner retina and optic nerve produce red-green perceptual deficits (Porkony et al., 1979). Bilateral lesions in the area V4 of visual cortex can also lead to color blindness (prosopagnosia). Several reviews discuss and/or list the effects of drugs and chemicals on color vision (Lyle, 1974; Porkony, 1979; Grant, 1986; Mergler, 1990; Grant and Schuman, 1993; Jaanus et al., 1995; Lessel, 1998). Recently, the assessment of color vision by rapid screening procedures has been used to evaluate occupationally and environmentally exposed populations (Mergler, 1990; Geller and Hudnell, 1997; Iregren et al., 2002a; Iregren et al., 2002b: see discussion below of the retina and optic nerve/tract for specific references and details). Color vision may be evaluated using several different testing procedures. Commonly used procedures in human toxicologic evaluations include the Ishihara color plates and chip arrangement tests such as the Farnsworth–Munson 100 Hue (FM-100) test and the simplified 15-chip tests using either the saturated hues of the Farnsworth D-15 or the desaturated hues of the Lanthony Desaturated Panel D-15. The Ishihara plates involve a series of colored spots arranged in patterns that take advantage of perceived difference in shades resulting from congenital protan or deutan anomalies. Normal observers perceive different sets of embedded numbers than do those with color vision deficits. The Farnsworth–Munson procedure involves arrangement of 85 chips in order of progressively changing color. The relative chromatic value of successive chips induces those with color perception deficits to abnormally arrange the chips. The pattern is indicative of the nature of the color perception anomaly. The FM-100 is considered more diagnostically reliable but takes considerably longer to administer than the similar but more efficient Farnsworth and Lanthony tests. The desaturated hues of the Lanthony D-15 are designed to better identify subtle acquired color vision deficits. For this reason, and because it requires only a few minutes to administer, the Lanthony D-15 was recommended as the procedure for color vision screening of potentially

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occupationally and environmentally exposed populations (ATSDR, 1992). A critical review of this procedure, its test–retest reliability and its use in toxicologic applications should be consulted prior to its use (see Geller and Hudnell, 1997; Good et al., 2005).

TARGET SITES AND MECHANISMS OF ACTION: CORNEA The cornea provides the anterior covering of the eye and as such must provide three essential functions. First, it must provide a clear refractive surface. The air-to-fluid/tissue interface at the cornea is the principal refractive surface of the eye, providing approximately 48 diopters of refraction. The curvature of the cornea must be correct for the visual image to be focused at the retina. Second, the cornea provides tensile strength to maintain the appropriate shape of the globe. Third, the cornea protects the eye from external factors, including potentially toxic chemicals. The anatomy is reviewed in the discussion of ocular pharmacodynamics and pharmacokinetics above. The cornea is transparent to wavelengths of light ranging between 310 nm (UV) to 2500 nm (IR) in wavelengths. Exposure to UV light below this range can damage the cornea. It is most sensitive to wavelengths of approximately 270 nm. Excessive UV exposure leads to photokeratitis and corneal pathology, the classic example being welder’s arc burns. The cornea can be damaged by topical or systemic exposure to drugs and chemicals. Reports of such adverse reactions have been catalogued and reviewed by Diamante and Fraunfelder (1998). One summary analysis, of approximately 600 agricultural and industrial chemicals (raw materials, intermediates, formulation components, and sales products), evaluated using the Draize procedure, reported that over half of the materials tested caused no (18–31%) or minimal (42–51%) irritation. Depending on the chemical category, 9–17% of compounds were graded as slightly irritant, whereas 1–6% were graded as strong or extreme irritants (Kobel and Gfeller, 1985). Direct chemical exposure to the eye requires emergency medical attention. Acid and alkali chemicals that come into contact with the cornea can be extremely destructive. Products at pH extremes ≤2.5 or ≥11.5 are considered as extreme ocular irritants (Potts, 1996). They can cause severe ocular damage and permanent loss of vision. Damage that extends to the corneal endothelium is associated with poor repair and recovery. The most important therapy is immediate and adequate irrigation with large amounts of water or saline, whichever is most readily available. The extent of damage to the eye and the ability to achieve a full recovery are dependent upon the nature of the chemical, the concentration and duration of exposure, and the speed and magnitude of the initial irrigation.

Acids Strong acids with a pH ≤2.5 can be highly injurious. Among the most significant acidic chemicals in terms of the tendency to cause clinical ocular damage are hydrofluoric acid, sulfurous acid, sulfuric acid, chromic acid, hydrochloric acid and nitric acid and acetic acid (McCulley, 1998). Injuries may be mild if contact is with weak acids or with dilute solutions of strong acids. Compounds with a pH between 2.5 and 7, produce pain or stinging; but with only brief contact, they will cause no lasting damage (Grant and Schuman, 1993). Following mild burns, the corneal epithelium may become turbid as the corneal stroma swells. Mild burns are typically followed by rapid regeneration of the corneal epithelium and full recovery.

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In more severe burns, the epithelium of the cornea and conjunctiva become opaque and necrotic and may disintegrate over the course of a few days. In severe burns, there may be no sensation of pain because the corneal nerve endings are destroyed (Grant, 1986; Potts, 1996). Acid chemical burns of the cornea occur through hydrogen ion–induced denaturing and coagulation of proteins. As epithelial cell proteins coagulate, glycosaminoglycans precipitate and stromal collagen fibers shrink. These events cause the cornea to become cloudy. The protein coagulation and shrinkage of the collagen is protective in that it forms a barrier and reduces further penetration of the acid. The collagen shrinkage, however, contracts the eye and can lead to a dangerous acute increase in intraocular pressure. The pH of the acid is not the only determinant of the severity of injury; however, as equimolar solutions of several chemicals adjusted to the same pH of 2 produce a wide range of outcomes. Both the hydrogen ion and anionic portions of the acid molecules contribute to protein coagulation and precipitation. The tissue proteins also tend to act as buffers (Grant, 1986; Potts, 1996).

Bases or Alkalies Compounds with a basic pH are potentially even more damaging to the eye than are strong acids. Among the compounds of clinical significance in terms of frequency and severity of injuries are ammonia or ammonium hydroxide, sodium hydroxide (lye), potassium hydroxide (caustic potash), calcium hydroxide (lime), and magnesium hydroxide (McCulley, 1998). One of the reasons that caustic agents are so dangerous is their ability to rapidly penetrate the ocular tissues. This is particularly true for ammonia, which has been measured in the aqueous humor just seconds after application to the cornea. The toxicity of these substances is a function of their pH, being more toxic with increasing pH values. As with acid burns, the concentration of the solution and the duration of contact with the eye are important determinants of the eventual clinical outcome. Rapid and extensive irrigation after exposure and removal of particles, if present, is the immediate therapy of choice (Grant, 1986; Potts, 1996). A feature of caustic burns that differentiates them from acid burns is that two phases of injury may be observed. There is an acute phase from exposure up to 1 week. Depending on the extent of injury, direct damage from exposure is observed in the cornea, adnexia, and possibly in the iris, ciliary body, and lens. The presence of strong hydroxide ions causes rapid necrosis of the corneal epithelium and, if sufficient amounts are present, penetration through and/or destruction of the successive corneal layers. Strong alkali substances attack membrane lipids, causing necrosis and enhancing penetration of the substance to deeper tissue layers. The cations also react with the carboxyl groups of glycosaminoglycans and collagen, the latter reaction leading to hydration of the collagen matrix and corneal swelling. The cornea may appear clouded or become opaque immediately after exposure as a result of stromal edema and changes to, or precipitation of, proteoglycans. The denaturing of the collagen and loss of protective covering of the glycosoaminoglycans is thought to make the collagen fibrils more susceptible to subsequent enzymatic degradation. Intraocular pressure may increase as a result of initial hydration of the collagen fibrils and later through the blockage of aqueous humor outflow. Conversely, if the alkali burn extends to involve the ciliary body, the intraocular pressure may decrease due to reduced formation of aqueous humor. The acute phase of damage is typically followed by initiation of corneal repair. The

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repair process may involve corneal neovascularization along with regeneration of the corneal epithelium. Approximately, 2 to 3 weeks after alkali burns, however, damaging ulceration of the corneal stroma often occurs. The formation of these lesions is related to the inflammatory infiltration of polymorphonuclear leukocytes and fibroblasts and the release of degratory proteolytic enzymes. Clinically, anti-inflammatory therapy limits ulcerative damage. Stromal ulceration usually stops when the corneal epithelium is restored (Grant, 1986; Potts, 1996).

Organic Solvents When organic solvents are splashed into the eye, the result is typically a painful immediate reaction. As in the case of acids and bases, exposure of the eye to solvents should be treated rapidly with abundant water irrigation. Highly lipophilic solvents can damage the corneal epithelium and produce swelling of the corneal stroma. Most organic solvents do not have a strongly acid or basic pH and therefore cause little in the way of chemical burns to the cornea. In most cases, the corneal epithelium will be repaired over the course of a few days and there will be no residual damage. Exposure to solvent vapors may produce small transparent vacuoles in the corneal epithelium, which may be asymptomatic or associated with moderate irritation and tearing (Grant, 1986; Potts, 1996).

Surfactants These compounds have water-soluble (hydrophilic) properties at one end of the molecule and lipophilic properties at the other end that help to dissolve fatty substances in water and also serve to reduce water surface tension. The widespread use of these chemicals in soaps, shampoos, detergents, cosmetics, and similar consumer products leads to abundant opportunities for exposure to ocular tissues. Many of these agents may be irritating or injurious to the eye. The hydrophilic portion of these compounds may be anionic, cationic, or neutral. In general, the cationic substances tend to be stronger irritants and more injurious than the other types, and anionic compounds more so than neutral ones (Grant, 1986; Potts, 1996). Because these compounds are by design soluble in both aqueous and lipid media, they readily penetrate the sandwiched aqueous and lipid barriers of the cornea (see discussion of ocular pharmacodynamics and pharmacokinetics, above). This property has implications in drug delivery; for example, low concentrations of the preservative benzalkonium chloride to ophthalmic solutions enhance ocular penetration of topically applied medications (Jaanus et al., 1995).

TARGET SITES AND MECHANISMS OF ACTION: LENS The lens of the eye plays a critical role in focusing the visual image on the retina. While the cornea is the primary refractive surface for bending incoming light rays, the lens is capable of being reshaped to adjust the focal point to adapt for the distance of visual objects. The lens is a biconvex transparent body, encased in an elastic capsule, and located between the pupil and the vitreous humor (Fig. 17-1). The mature lens has a dense inner nuclear region surrounded by the lens cortex. The high transparency of the lens to visible wavelengths of light is a function of its chemical composition, approximately twothirds water and one-third protein, and the special organizational structure of the lenticular proteins. The water-soluble crystallins are a set of proteins particular to the lens that, through their close inter-

molecular structure, give the lens both transparency and the proper refractive index. The lens fibers are laid down during development, as the epithelial cells grow and elongate along meridian pathways between the anterior and posterior poles of the lens. As the epithelial cells continue to grow, the nuclei recede and, in the central portions of the lens, disappear, such that the inner lens substance is composed of nonnucleated cells that form long proteinaceous fibers. The lens fibers are arranged within the lens in an onion-like fashion of concentric rings that have a prismatic arrangement in cross section. The regular geometric organization of the lens fibers is essential for the refractive index and transparency of the lens. At birth, the lens has no blood supply and no innervation. Nutrients are provided from the aqueous and vitreous fluids, and are transported into the lens substance through a system of intercellular gap-type junctions. The lens is a metabolically active tissue that maintains careful electrolyte and ionic balance. The lens continues to grow throughout life, with new cells added to the epithelial margin of the lens as the older cells condense into a central nuclear region. The dramatic growth of the lens is illustrated by increasing its weight, from approximately 150 mg at 20 years of age to approximately 250 mg at 80 years of age (Patterson and Delamere, 1992). Cataracts are decreases in the optical transparency of the lens that ultimately can lead to functional visual disturbances. They are the leading cause of blindness worldwide, affecting an estimated 30 to 45 million people. In the United States, approximately 400,000 people develop cataracts each year. This accounts for about 35% of existing visual impairments (Patterson and Delamere, 1992). Cataracts can occur at any age; they can also be congenital (Rogers and Chernoff, 1988). However, they are much more frequent with advancing age. Senile cataracts develop most frequently in the cortical or nuclear regions of the lens and less frequently in the posterior subcapsular region. Senile cataracts in the cortical region of the lens are associated with disruptions of water and electrolyte homeostasis, whereas nuclear cataracts are characterized by an increase in the water-insoluble fraction of lens proteins (Patterson and Delamere, 1992). Recent studies indicate that both genetic and environmental factors contribute to age-related and environmentally mediated cataracts and that these involve several different mechanisms of action (Hammond et al., 2000; Ottonello et al., 2000; Spector, 2000). Risk factors for the development of cataracts include aging, diabetes, low antioxidant levels, and exposure to a variety of environmental factors. Environmental factors include exposure to UV radiation and visible light, trauma, smoking, and exposure to a large variety of topical and systemic drugs and chemicals (e.g., Grant, 1986; Leske et al., 1991; Taylor and Nowell, 1997; Spector, 2000). Several different mechanisms of action have been hypothesized to account for the development of cataracts. These include the disruption of lens energy metabolism, hydration and/or electrolyte balance, the occurrence of oxidative stress due to the generation of free radicals and reactive oxygen species, and the occurrence of oxidative stress due a decrease in antioxidant defense mechanisms such as glutathione, superoxide dismutase, catalase, ascorbic acid, or vitamin E (Giblin, 2000; Ottonello et al., 2000; Spector, 2000). The generation of reactive oxygen species leads to oxidation of lens membrane proteins and lipids. A critical pathway in the development of highmolecular-weight aggregates involves the oxidation of protein thiol groups, particularly in methionine or cysteine amino acids, that leads to the formation of polypeptide links through disulfide bonds, and in turn, high-molecular-weight protein aggregates (Patterson and Delamere, 1992; Ottonello et al., 2000; Spector, 2000). These large

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aggregations of proteins can attain a size sufficient to scatter light, thus reducing lens transparency. Oxidation of membrane lipids and proteins may also impair membrane transport and permeability.

Light and Phototoxicity The most important oxidizing agents are visible light and UV radiation, particularly UV-A (320 to 400 nm) and UV-B (290 to 320 nm), and other forms of electromagnetic radiation. Light- and UV-induced photooxidation leads to generation of reactive oxygen species, and oxidative damage that can accumulate over time. Higher-energy UV-C (100 to 290 nm) is even more damaging. At sea level, the atmosphere filters out virtually all UV-C and all but a small fraction of UV-B derived from solar radiance (AMA Report, 1989). The cornea absorbs about 45% of light with wavelengths below 280 nm, but only about 12% between 320 and 400 nm. The lens absorbs much of the light between 300 and 400 nm and transmits 400 nm and above to the retina (Patterson and Delamere, 1992). Absorption of light energy in the lens triggers a variety of photoreactions, including the generation of fluorophores and pigments that lead to the yellow-brown coloration of the lens. Sufficient exposure to infrared radiation, as occurs to glassblowers, or microwave radiation will also produce cataracts through direct heating of the ocular tissues. Drugs and other chemicals can serve as mediators of photoinduced toxicity in the cornea, lens or retina (Dayhaw-Barker et al., 1986; Roberts, 2001; Glickman, 2002; Roberts, 2002). This occurs when the chemical structure allows absorption of light energy in the UV or visible spectrum and the subsequent generation of activated intermediates, free radicals and reactive oxygen species. Chemical structures likely to participate in such phototoxic mechanisms include those with tricyclic, heterocyclic or porphyrin ring structures because, with light, they produce stable triplet reactive molecules leading to free radicals and reactive oxygen species. The propensity of chemicals to cause phototoxic reactions can be predicted using photo-physical and in vitro procedures (Roberts, 2001; Glickman, 2002; Roberts, 2002).

Corticosteroids Systemic treatment with corticosteroids causes cataracts (Urban and Cotlier, 1986). Observable opacities begin in the posterior subcapsular region of the lens and progress into the cortical region as the size of the lesion increases. Development of cataracts in individuals varies as a function of total dose of the drug, age, and the nature of the individual’s underlying disease. It was estimated that 22% of patients receiving corticosteroid immunosuppressive therapy for renal transplants experienced cataracts as a side effect of therapy (Veenstra et al., 1999). The use of inhaled corticosteroids—commonly prescribed asthma therapy—was once thought to be without this risk, but subsequent epidemiologic evidence documented a significant association between inhaled steroidal therapy and development of nuclear and posterior subcapsular cataracts (Cumming et al., 1997; Cumming and Mitchell, 1999). There are two proposed mechanisms through which corticosteroids might cause cataracts. One proposal involves disruption of the lens epithelium electrolyte balance through inhibition of Na+ ,K+ -ATPase. The regular hexagonal array structure of normal lens epithelial cells is disrupted and appears reticulated, while gaps appear between the lateral epithelial cell borders in lenses of humans with steroidinduced cataracts (Karim et al., 1989). Another theory is that cor-

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ticosteroid molecules react with lens crystallin proteins through Schiff base reactions between the carbonyl group of the steroid and protein amino groups, with subsequent rearrangement into stable products (Urban and Cotlier, 1986). The resulting covalent corticosteroid–crystallin adducts would be high-molecularweight light-scattering complexes. Whatever mechanism is responsible, these results illustrate the importance of routine ophthalmologic screening of patients receiving chronic corticosteroid therapy.

Naphthalene Accidental exposure to naphthalene results in cortical cataracts and retinal degeneration (Grant, 1986; Potts, 1996). Naphthalene itself is not cataractogenic; instead, the metabolite 1,2-dihydro-1,2dihydroxynaphthalene (naphthalene dihydrodiol) is the cataractinducing agent (van Heyningen and Pirie, 1967). Subsequent studies using biochemical and pharmacologic techniques, in vitro assays, and transgenic mice showed that aldose reductase in the rat lens is a major protein associated with naphthalene dihydrodiol dehydrogenase activity and that lens aldose reductase is the enzyme responsible for the formation of naphthalene dihydrodiol (Sato, 1993; Lee and Chung, 1998; Sato et al., 1999). In addition, in vivo and in vitro studies have shown that aldose reductase inhibitors prevent naphthalene-induced cataracts (Lou et al., 1993; Sato et al., 1999). Finally, there is a difference in naphthalene-induced cataract formation between albino and pigmented rats, with the latter showing a faster onset and more uniform cataract (Murano et al., 1993).

Phenothiazines It has been known since the 1950s that schizophrenics receiving phenothiazine drugs as anti-psychotic medication develop pigmented deposits in their eyes and skin (Grant, 1986; Potts, 1996). The pigmentation begins as tiny deposits on the anterior surface of the lens and progresses, with increasing dose, to involve the cornea as well. The phenothiazines combine with melanin to form a photosensitive product that reacts with sunlight, causing formation of the deposits. The amount of pigmentation is related to the dose of the drug, with the annual yearly dose being the most predictive dose metric (Thaler et al., 1985). More recent epidemiologic evidence demonstrates a dose-related increase in the risk of cataracts from use of phenothiazine-like drugs, including both antipsychotic drugs such as chlorpromazine and nonantipsychotic phenothiazines (Isaac et al., 1991).

TARGET SITES AND MECHANISMS OF ACTION: RETINA The adult mammalian retina is a highly differentiated tissue containing nine distinct layers plus the RPE, ten major types of neurons, and three cells with glial functions (Fig. 17-1). The nine layers of the neural retina, which originate from the cells of the inner layer of the embryonic optic cup, are the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), rod and cone photoreceptor inner segment layer (RIS; CIS), and the rod and cone photoreceptor outer segment layer (ROS; COS). The RPE, which originates from the cells of the outer layer of the embryonic optic cup, is a single layer of cuboidal

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epithelial cells that lies on Bruch’s membrane adjacent to the vascular choroid. Between the RPE and photoreceptor outer segments lies the subretinal space, which is similar to the brain ventricles. The ten major types of neurons are the rod (R) and cone (C) photoreceptors, (depolarizing) ON-rod and ON-cone bipolar cells (BC), (hyperpolarizing) OFF-cone bipolar cells, horizontal cells (HC), numerous subtypes of amacrine cells (AC), an interplexiform cell (IPC), and ON-RGCs and OFF-RGCs. The three cells with glial functions are the M¨uller cells (MC), fibrous astrocytes, and microglia. The somas of the MCs are in the INL. The end feet of the MCs in the proximal or inner retina along with a basal lamina form the internal limiting membrane (ILM) of the retina, which is similar to the pial surface of the brain. In the distal retina, the MC end feet join with the photoreceptors and zonula adherens to form the external limiting membrane (ELM), which is located between the ONL and RIS/CIS. The interested reader is referred to the excellent references in the Introduction as well as to numerous outstanding websites devoted exclusively to the retina (e.g., http://webvision.umh.es/webvision/ intro.html; http://cvs.anu.edu.au/; http://retina.anatomy.upenn.edu/ ∼lance/retina/retina.html) for basic information on the anatomic, biochemical, cell and molecular biological, and physiologic aspects of retinal structure and function. The mammalian retina is highly vulnerable to toxicant-induced structural and/or functional damage due to (1) the presence of a highly fenestrated choriocapillaris that supplies the distal or outer retina as well as a portion of the inner retina; (2) the very high rate of oxidative mitochondrial metabolism, especially that in the photoreceptors (Linsenmeier, 1986; Ahmed et al., 1993; Medrano and Fox, 1994, 1995; Braun et al., 1995; Winkler, 1995; Shulman and Fox, 1996); (3) high daily turnover of rod and cone outer segments (LaVail, 1976; Rodieck, 1998); (4) high susceptibility of the rod and cones to degeneration due to inherited retinal dystrophies as well as associated syndromes and metabolic disorders (Ogden and Schachat, 1989; Hart, 1992; von Soest et al., 1999); (5) presence of specialized ribbon synapses and synaptic contact sites (Dowling, 1987; Ogden and Schachat, 1989; Cohen, 1992; Rodieck, 1998); (6) presence of numerous neurotransmitter and neuromodulatory systems, including extensive glutamatergic, GABAergic and glycinergic systems (Rauen et al., 1996; Brandst¨atter et al., 1998; Rodieck, 1998; Kalloniatis and Tomisich, 1999; Winkler et al., 1999); (7) presence of numerous and highly specialized gap junctions used in the information signaling process (Cohen, 1992; Cook and Becker, 1995; Rodieck, 1998); (8) presence of melanin in the choroid and RPE and also in the iris (Meier-Rouge, 1972; Potts, 1996); (9) a very high choroidal blood flow rate, as high as ten times that of the gray matter of the brain (Alm, 1992; Cohen, 1992); and (10) the additive or synergistic toxic action of certain chemicals with light (Dayhaw-Barker et al., 1986; Backstrom et al., 1993; Roberts, 2001; Glickman, 2002; Roberts, 2002). The retina is also an excellent model system for studying the effects of chemicals on the developing and mature CNS. Its structure– function relations are well established. The histogenic steps of development of the neurons and glial components are well characterized. The development of the CNS and most retinal cells occurs early during gestation in humans (Hendrickson, 1992; Hendrickson and Drucker, 1992) and continues for an additional 7 to 14 days postnatally in the rat (Dobbing and Sands, 1979; Raedler and Sievers, 1975). Therefore, toxicological effects in the rodent retina have relevance for chemical exposure during the early gestation period in humans as well as during early postnatal development. The retina contains a wide diversity of synaptic transmitters and second messengers whose developmental patterns are well described.

Moreover, the rodent retina is easily accessible, it has most of the same anatomical and functional features found in the developing and mature human retina, and the rat rod pathway is similar to that in other mammals (Dowling, 1987; Finlay and Sengelbaub, 1989; Berman, 1991; Chun et al., 1993). Finally, rat rods have similar dimensions, photochemistry, and photocurrents as human and monkey rods (Baylor et al., 1984; Schnapf et al., 1988). These general and specific features underscore the relevance and applicability of using the rodent retina to investigate the effects of chemicals on this target site as well as a model to investigate the neurotoxic effects of chemicals during development. Each of the retinal layers can undergo specific as well as general toxic effects. These alterations and deficits include, but are not limited to visual field deficits, scotopic vision deficits such as night blindness and increases in the threshold for dark adaptation, cone-mediated (photopic) deficits such as decreased color perception, decreased visual acuity, macular and general retina edema, retinal hemorrhages and vasoconstriction, and pigmentary changes. The list of chemicals and drugs that cause retinal alterations is extensive, as evidenced by an examination of Grant’s Toxicology of the Eye (Grant, 1986) and Dr. Potts’ chapter entitled “Toxic Responses of the Eye” in an earlier edition of this volume (1996). In addition, a review by Jaanus et al. (1995) discusses the adverse retinal effects of therapeutic systemic drugs. Another review by Wolfensberger (1998) concentrates on toxic effects on the retinal pigment epithelium. The main aim of this section is to discuss in detail several chemicals and drugs (1) that are currently used as pharmacological agents or environmentally relevant neurotoxicants; (2) whose behavioral, physiologic, and/or pathologic effects on retina are known; and (3) whose retinal site(s) and/or mechanism of action are well characterized. In addition, the effects of organic solvents and organophosphates on retinal function and vision are discussed, as these are emerging areas of concern. The chemical- and drug-induced alterations in retinal structure and function are grouped into two major categories. The first category focuses on retinotoxicity of systemically administered therapeutic drugs. Four major drugs are discussed in detail: chloroquine/hydroxychloroquine, digoxin/digitoxin, indomethacin, and tamoxifen. The second category focuses on well-known neurotoxicants that produce retinotoxicity: inorganic lead, methanol, selected organic solvents, and organophosphates. See chapters 16 and 23 entitled “Toxic Responses of the Nervous System” and “Toxic Effects of Metals” for information on the effects of lead on the brain and other target organs and chapter 24 entitled “Toxic Effects of Solvents and Vapors,” for additional information on methanol and the organic solvents discussed below.

Retinotoxicity of Systemically Administered Therapeutic Drugs Cancer Chemotherapeutics Ocular toxicity is a common side effect of cancer chemotherapy (Imperia et al., 1989; Schmid et al., 2006). Symptoms include blurred vision, diplopia, decreased color vision, decreased visual acuity, optic/retrobular neuritis, transient cortical blindness, and demyelination of the optic nerves (Imperia et al., 1989; Schmid et al., 2006). The retina due to its high metabolic activity and choroidal circulation (vide infra) appears to be particularly vulnerable to numerous cytotoxic agents such as the alkylating agents cisplatin, carboplatin, and carmustine; the antimetabolites cytosine arabinoside, 5-fluorouracil and methotrexate; and the mitotic inhibitors such as docetaxel. The ocular toxicity of different drugs is dependent upon the dose, duration of dosage, and route of

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administration. However, if not detected at an early stage of toxicity, the ocular complications are often irreversible even after chemotherapy is discontinued (Imperia et al., 1989; Schmid et al., 2006). One strategy to avoid such retinal complications is to conduct prospective ophthalmological exams as well as scotopic and photopic ERG testing prior to the onset and during chemotherapy. The ocular side effects of tamoxifen, an estrogen antagonist used in oncology, are discussed below.

Chloroquine and Hydroxychloroquine Two of the most extensively studied retinotoxic drugs are chloroquine (Aralen) and hydroxychloroquine (Plaquenil). The first case of chloroquine-induced retinopathy was reported more than 40 years ago (Jaanus et al., 1995; Potts, 1996). These 4-aminoquinoline derivatives are used as antimalarial and anti-inflammatory drugs. The low-dose therapy used for malaria is essentially free from toxic side effects; however, the chronic, high-dose therapy used for rheumatoid arthritis, and discoid and systemic lupus erythematosus (initially 400 to 600 mg/day for 4 to 12 weeks and then 200 to 400 mg/day; Ellsworth et al., 1999) can cause irreversible loss of retinal function. Chloroquine, its major metabolite desethylchloroquine, and hydroxychloroquine have high affinity for melanin, which results in very high concentrations of these drugs accumulating in the choroid and RPE, ciliary body, and iris during and following drug administration (Rosenthal et al., 1978; Potts, 1996). Prolonged exposure of the retina to these drugs, especially chloroquine, may lead to an irreversible retinopathy. In fact, small amounts of chloroquine and its metabolites were excreted in the urine years after cessation of drug treatment (Bernstein, 1967). Approximately, 20–30% of patients who received high doses of chloroquine exhibited some type of retinal abnormality, while 5–10% showed severe changes in retinal function (Burns, 1966; Potts, 1996; Shearer and Dubois, 1967; Sassaman et al., 1970; Krill et al., 1971). Hydroxychloroquine is now the drug of choice for treatment of rheumatic diseases because it has fewer side effects and less ocular toxicity. Doses less than 400 mg per day appear to produce little or no retinopathy even after prolonged therapy (Johnson and Vine, 1987). The clinical findings accompanying chloroquine retinopathy can be divided into early and late stages. The early changes include (1) the pathognomonic “bull’s-eye retina” visualized as a dark, central pigmented area involving the macula, surrounded by a pale ring of depigmentation, which, in turn, is surrounded by another ring of pigmentation; (2) a diminished EOG; (3) possible granular pigmentation in the peripheral retina; and (4) visual complaints such as blurred vision and problems discerning letters or words. Late-stage findings, which can occur during or even following cessation of drug exposure, include (1) a progressive scotoma, (2) constriction of the peripheral fields commencing in the upper temporal quadrant, (3) narrowing of the retinal artery, (4) color and night blindness, (5) absence of a typical retinal pigment pattern, and (6) very abnormal EOGs and ERGs. These late-stage symptoms are irreversible. Interestingly, dark adaptation is relatively normal even during the late stages of chloroquine retinopathy, which helps distinguish the peripheral retinal changes from those observed in patients with retinitis pigmentosa (Bernstein, 1967). In humans and monkeys, long-term chloroquine administration results in sequential degeneration of the RGCs, photoreceptors, and RPE and the eventual migration of RPE pigment into the ONL and OPL. In addition, in the RPE there is a thickening of the RPE layer, an increase in the mucopolysaccharide and sulfhydryl group content, and a decrease in activity of several enzymes (Potts, 1996; Ramsey

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and Fine, 1972; Rosenthal et al., 1978). Although the molecular mechanism of action is unknown, it has been suggested that the primary biochemical mechanism is inhibition of protein synthesis (Bernstein, 1967).

Digoxin and Digitoxin The cardiac glycosides digoxin (Lanoxin) and digitoxin (Crystodigin) are digitalis derivatives used in the treatment of congestive heart disease and in certain cardiac arrhythmias. As part of the extract of the plant foxglove, digitalis was recommended for heart failure (dropsy) over 200 years ago. Digitalisinduced visual system abnormalities such as decreased vision, flickering scotomas, and altered color vision were documented during that time (Withering, 1785). Approximately, 20–60% of patients with cardiac glycoside serum levels in the therapeutic range and 50–80% of the patients with cardiac glycoside serum levels in the toxic range complain of visual system disturbances within 2 weeks after the onset of therapy (Robertson et al., 1966a; Aronson and Ford, 1980; Rietbrock and Alken, 1980; Haustein et al., 1982; Piltz et al., 1993; Duncker et al., 1994). Digoxin produces more toxicity than digitoxin due to its greater volume of distribution and plasma protein binding (Haustein and Schmidt, 1988). The most frequent visual complaints are color vision impairments and hazy or snowy vision, although complaints of flickering light, colored spots surrounded by bright halos, blurred vision, and glare sensitivity also are reported. The color vision disturbances have been confirmed with the Farnsworth-Munsell 100 Hue Test (Aronson and Ford, 1980; Rietbrock and Alken, 1980; Haustein et al., 1982; Haustein and Schmidt, 1988; Duncker and Krastel, 1990). Clinical examinations show that these patients have decreased visual acuity and central scotomas but no funduscopic changes. ERG analysis revealed reduced rod and cone amplitudes, increased rod and cone implicit times, and elevated rod and cone thresholds (Robertson et al., 1966a; Robertson et al., 1966b; Alken and Belz, 1984; Duncker and Krastel, 1990; Madreperla et al., 1994). Taken together, these ophthalmologic, behavioral, and electrophysiologic findings demonstrate that the photoreceptors are a primary target site of the cardiac glycosides digoxin and digitoxin. The above results suggest that cone photoreceptors are more susceptible to the effects of cardiac glycosides than rod photoreceptors. To directly test this hypothesis, Madreperla et al. (1994) conducted electrophysiologic (suction electrode) experiments with isolated tiger salamander (Ambystoma tigrinum) rods and cones exposed to different physiologically and toxicologically relevant concentrations of digoxin in the bathing solution. Following a single light flash of saturating intensity, rods and cones exhibited concentration-dependent decreases in the peak light (current) response. The cones, however, were about 50 times more sensitive to digoxin and were impaired to a greater degree at the same digoxin concentration than the rods. Neither the rods nor the cones recovered to their dark-adapted baseline following the short-duration saturating light flash. The rods, however, appeared to recover faster and more completely than the cones. Moreover, following a return to control Ringer’s solution, the photoreceptors still did not recover to their dark-adapted baseline response level. This latter finding correlates with the slow recovery of the ERG seen in patients following termination of digoxin exposure (Robertson et al., 1966b; Duncker and Krastel, 1990; Madreperla et al., 1994) and is most likely due to the high affinity and slow off-rate of digoxin binding to the cardiac glycoside site located on the extracellular side of the catalytic α-subunit of the Na+ ,K+ -ATPase enzyme (Sweadner, 1989).

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Digitalis glycosides, like ouabain, are potent inhibitors of retinal Na+ ,K+ -ATPase (Winkler and Riley, 1977; Fox et al., 1991b; Ottlecz et al., 1993; Shulman and Fox, 1996). Digoxin binding studies show that the retina has the highest number of Na+ ,K+ ATPase sites of any ocular tissue, even higher than those of brain (Lissner et al., 1971; Lufkin et al., 1967). There are three different isoforms of the α subunit of Na+ ,K+ -ATPase (i.e., α1, α2, and α3), and they differ significantly in their sensitivity to cardiac glycoside inhibition (Sweadner, 1989). In the rat retina, the α1-low and α3-high ouabain affinity isoforms of the enzyme account for ≥97% of the Na+ ,K+ -ATPase mRNA. The α3 isoform is localized to rat photoreceptors, horizontal cells, and bipolar cells. Photoreceptors predominantly express the α3 mRNA (approximately 85%), a small amount of α1 mRNA (approximately15%), and almost no detectable α2 mRNA. Electron microscopic immunocytochemistry studies reveal that the α3 isoform is localized exclusively to the plasma membrane of the rat photoreceptor inner segments (McGrail and Sweadner, 1989; Schneider and Kraig, 1990; Schneider et al., 1990). The α3 isozyme accounts for most of the rod Na+ ,K+ -ATPase activity (Shulman and Fox, 1996). The rat rod photoreceptor Na+ ,K+ -ATPase–specific activity is approximately threefold higher than whole retinal (Fox et al., 1991b; Shulman and Fox, 1996) or whole brain values (Marks and Seeds, 1978). This is also reflected in the two- to threefold greater ouabain-sensitive oxygen consumption in the dark-adapted outer retina relative to the whole or inner retina, respectively (Medrano and Fox, 1995; Shulman and Fox, 1996). Indomethacin Indomethacin is a nonsteroidal anti-inflammatory drug with analgesic and antipyretic properties that is frequently used for the management of arthritis, gout, and musculoskeletal discomfort. It inhibits prostaglandin synthesis by inhibiting cyclooxygenase. The first cases of indomethacin-induced retinopathy were reported approximately 30 years ago (Jaanus et al., 1995; Potts, 1996). Chronic administration of 50 to 200 mg per day of indomethacin for 1 to 2 years has been reported to produce corneal opacities, discrete pigment scattering of the RPE perifoveally, paramacular depigmentation, decreases in visual acuity, altered visual fields, increases in the threshold for dark adaptation, blue-yellow color deficits, and decreases in ERG and EOG amplitudes (Burns, 1966; Burns, 1968; Henkes et al., 1972; Koliopoulos and Palimeris, 1972; Palimeris et al., 1972). Decreases in the ERG a- and b-wave amplitudes, with larger changes observed under scotopic dark-adapted than light-adapted conditions, have been reported. Upon cessation of drug treatment, the ERG waveforms and color vision changes return to near normal, although the pigmentary changes are irreversible (Burns, 1968; Henkes et al., 1972; Palimeris et al., 1972). The mechanism of retinotoxicity is unknown; however, it appears likely that the RPE is a primary target site. Sildenafil Citrate Sildenafil citrate (Viagra) is a cGMP-specific phosphodiesterase (PDE) type 5 inhibitor that is utilized in the treatment of erectile dysfunction (Corbin et al., 2002). Sildenafil is also a weak cGMP PDE type 6 inhibitor, which is present in rod and cone photorecepotrs (Corbin et al., 2002; Zhang et al., 2005). Transient visual symptoms such as a blue tinge to vision, increased brightness of lights and blurry vision as well as alterations in scotopic and photopic ERGs have been reported following sildenafil usage (Laties and Zrenner, 2002; Jagle et al., 2004). More recently, sildenafil has been associated with the occurrence of nonarteritic anterior

ischemic optic neuropathy (NAION) in at-risk patients (i.e., those with small cup-to-disc ratios and/or arteriosclerotic risk profiles) within minutes to hours after the ingestion of the drug (Fraunfelder et al., 2006). However, available data suggest that the risk of occurrence of NAION in patients taking sildenafil is not significantly different from the general population (Laties and Zrenner, 2002; Fraunfelder et al., 2006; Gorkin et al., 2006). Tamoxifen Tamoxifen (Nolvadex, Tamoplex), a triphenylethylene derivative, is a nonsteroidal antiestrogenic drug that competes with estrogen for its receptor sites. It is a highly effective antitumor agent used for the treatment of metastatic breast carcinoma in postmenopausal women. Tamoxifen-induced retinopathy following chronic high-dose therapy (180 to 240 mg per day for approximately 2 years) was first reported 20 years ago (Kaiser-Kupfer et al., 1981). At this dose, there is widespread axonal degeneration in the macular and perimacular area, as evidenced by the presence of different sized yellow–white refractile opacities in the IPL and NFL observed during fundus examination. Macular edema may or may not be present. Clinical symptoms include a permanent decrease in visual acuity and abnormal visual fields, as the axonal degeneration is irreversible (reviewed by Jaanus et al., 1995; Potts, 1996; Ah-Song and Sasco, 1997). Several prospective studies, with sample sizes ranging from 63 to 303 women with breast cancer, have shown that chronic low-dose tamoxifen (20 mg per day) can result in a small but significant increase in the incidence (≤10%) of keratopathy (Pavlividis et al., 1992; Gorin et al., 1998; Lazzaroni et al., 1998; Noureddin et al., 1999). In addition, these studies showed that retinopathy is much less frequently observed than with high-dose therapy and, except for a few reports of altered color vision and decreased visual acuity, there were no significant alterations in visual function. Following cessation of low-dose tamoxifen therapy, most of the keratopathy and retinal alterations except the corneal opacities and retinopathy were reversible (Pavlividis et al., 1992; Gorin et al., 1998; Noureddin et al., 1999).

Retinotoxicity of Known Neurotoxicants Inorganic Lead Inorganic lead is probably the oldest known and most studied environmental toxicant. For almost 100 years, it has been known that overt lead poisoning [mean blood lead (BPb) ≥80 μg/dL] in humans produces visual system pathology and overt visual symptoms (Grant, 1986; Otto and Fox, 1993; Fox, 1998). Clinical manifestations include amblyopia, blindness, optic neuritis or atrophy, peripheral and central scotomas, paralysis of eye muscles, and decreased visual function. Moderate to high level lead exposure produces scotopic and temporal visual system deficits in occupationally exposed factory workers and developmentally lead-exposed monkeys and rats (Bushnell et al., 1977; Guguchkova, 1972; Cavelleri et al., 1982; Betta et al., 1983; Signorino et al., 1983; Campara et al., 1984; Jeyaratnam et al., 1986; Fox and Farber, 1988; Lilienthal et al., 1988; Fox et al., 1991a; Fox and Katz, 1992; Otto and Fox, 1993; Lilienthal et al., 1994; Fox, 1998; Rice, 1998). Early work in monkeys exposed to moderate to high levels of lead during and following gestation reveal that this lead exposure regimen produces irreversible retinal deficits (Lilienthal et al., 1988; Lilienthal et al., 1994; Kohler et al., 1997). A recent study in 7–10 year old children reveals that low-level gestational lead exposure produces long-lasting scotopic supernormal ERG deficits (Rothenberg et al., 2002). However, relatively little effort has been made to understand the impact of lead-induced alterations on retinal and central visual

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information processing on learning and memory in children. These types of visual deficits can adversely affect learning and memory as well as experimental procedures used to assess these cognitive parameters (Anger et al., 1994; Hudnell et al., 1996; Walkowiak et al., 1998; Cestnick and Coltheart, 1999). Studies in Occupationally Exposed Lead Workers Clinical and electrophysiological studies in lead-exposed factory workers have assessed both the site of action and extent of injury. Several cases of retrobulbar optic neuritis and optic nerve atrophy have been observed following chronic moderate-level or acute high-level lead exposure (Sherer, 1935; Baghdassarian, 1968; Baloh et al., 1979; Karai et al., 1979). Most of these cases presented with fundus lesions, peripheral or paracentral scotomas while the most severe cases also had a central scotoma. Generally, the scotomas were not observed until approximately 5 years of continuous lead exposure. Interestingly, the earliest observable scotomas were not detected under standard photopic viewing conditions but became evident only under scotopic or mesopic (rod- and cone-mediated) viewing conditions. These ophthalmologic findings correlate directly with the ERG data observed in similarly exposed lead workers. No alterations in the critical flicker fusion threshold (i.e., temporal resolution) were observed when the test was conducted under photopic conditions or when using red lights. However, consistent decreases in temporal resolution were observed when the test was conducted under scotopic conditions or when green lights were used (Cavelleri et al., 1982; Betta et al., 1983; Signorino et al., 1983; Campara et al., 1984; Jeyaratnam et al., 1986). Moreover, in occupationally lead-exposed workers with or without visual acuity deficits or no observable alterations following ophthalmologic examination, the sensitivity and amplitude of the a-wave and/or bwave of the dark-adapted ERG were decreased (Guguchkova, 1972; Scholl and Zrenner, 2000). In other lead-exposed workers, one funduscopic study noted the presence of a grayish lead pigmentary deposit in the area peripheral to the optic disk margins (Sonkin, 1963). In addition to the retinal deficits, oculomotor deficits occur in chronically lead-exposed workers who have no observable ophthalmologic abnormalities. Results from three independent studies, including a follow-up, show that the mean accuracy of saccadic eye movements is lower in lead-exposed workers and the number of overshoots is increased (Baloh et al., 1979; Spivey et al., 1980; Specchio et al., 1981; Glickman et al., 1984). In addition, these studies also revealed that the saccade maximum velocity was decreased. Moreover, one study also observed abnormal smooth pursuit eye movements in lead-exposed workers (Specchio et al., 1981). Although the site and mechanism of action underlying these alterations are unknown, they most likely result from CNS-mediated deficits. In summary, these results suggest that occupational lead exposure produces concentration- and time-dependent alterations in the retina such that higher levels of lead directly and adversely affect both the retina and optic nerve, whereas lower levels of lead appear to primarily affect the rod photoreceptors and their pathway. Interestingly, these latter clinical findings showing preferential lead-induced rod-selective deficits in sensitivity and temporal resolution are observed in both in vivo and in vitro animal studies (see below). Furthermore, these retinal and oculomotor alterations were, in most cases, correlated with the blood lead levels and occurred in the absence of observable ophthalmologic changes, CNS symptoms, and abnormal performance test scores. Thus, these measures of temporal visual function may be among the most sensi-

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tive for the early detection of the neurotoxic effects of inorganic lead. In Vivo and in Vitro Animal Studies Lead exposure to adult animals and postnatally developing animals produces retinal damage and functional deficits. The degree and extent of these alterations depends upon the dose, age, and duration of lead exposure. Highlevel lead exposure to adult rabbits for 60 to 300 days (Hass et al., 1964; Brown, 1974; Hughes and Coogan, 1974) and to newborn rats for 60 days (Santos-Anderson et al., 1984) resulted in focal necrosis of the rod inner and outer segments, necrosis in the inner nuclear layer and M¨uller cells, and lysosomal inclusions in the RPE. In addition, high-level lead exposure to mice and rats from birth to weaning resulted in hypomyelination of the optic nerve and a reduction in its diameter; but, interestingly, there were no changes in the sciatic nerve (Tennekoon et al., 1979; Toews et al., 1980). Newborn monkeys exposed to high levels of lead for 6 years had no changes in optic nerve diameter or myelination, although visual cortex neuronal volume and branching were decreased (Reuhl et al., 1989). Rhesus monkeys exposed prenatally and postnatally to moderate or high levels of lead for 9 years, followed by almost 2 years of no lead exposure, had decreased tyrosine hydroxylase immunoreactivity in the large dopaminergic amacrine cells and a complete loss of tyrosine hydroxylase immunoreactivity in small subset of amacrine cells (Kohler et al., 1997). These results suggest that long-term lead exposure produces a decrease in tyrosine hydroxylase synthesis, a finding consistent with other studies (Lasley and Lane, 1988; Jadhav and Ramesh, 1997), and/or a loss of a subset of tyrosine hydroxylase–positive amacrine cells, a finding consistent with recent in vitro work (Scortegagna and Hanbauer, 1997). In contrast to these studies, 6 weeks of moderate-level lead exposure to adult rats (Fox et al., 1997) and 3 weeks of low- or moderate-level lead exposure to neonatal rats from birth to weaning produced rod- and bipolar cell–selective apoptotic cell death (Fox and Chu, 1988; Fox et al., 1997). Moreover, recent results reveal that brief (15-min) exposure of isolated adult rat retinas to nanomolar to micromolar Pb2+ , concentrations regarded as pathophysiologically relevant (Cavalleri et al., 1984; Al-Modhefer et al., 1991), resulted in rod-selective apoptosis (He et al., 2000). By extension, these results suggest that the triggering event (initiating phase) and the execution phase of rod and bipolar cell death share common underlying biochemical mechanisms. Results from several studies suggest that an elevated level of rod photoreceptor Ca2+ and/or Pb2+ plays a key role in the process of apoptotic rod cell death in humans and animals during inherited retinal degenerations, retinal diseases and injuries, chemical exposure, and lead exposure. These include patients with retinitis pigmentosa and cancer-associated retinopathy (Thirkill et al., 1987; van Soest et al., 1999), mice with retinal degeneration (rd) (Chang et al., 1993; Fox et al., 1999), rats injected with antirecoverin monoclonal antibodies (Adamus et al., 1998), rats with hypoxic-ischemic injury (Crosson et al., 1990), rats with light-induced damage (Edward et al., 1991), and lead-exposed rats (Fox and Chu, 1988; Fox et al., 1997, 1999). In addition, moderate-level Pb2+ exposure produces apoptotic neuronal cell death in primary cultured cells (Oberto et al., 1996; Scortegagna et al., 1997). In vivo and in vitro data suggest that Pb2+ produces a dose (concentration)- dependent inhibition of rod cGMP phosphodiesterase (PDE), a resultant elevation of rod cGMP (Fox and Farber, 1988; Fox et al., 1991a; Srivastava et al., 1995a; Srivastava et al., 1995b; Fox et al., 1997), which gates the nonselective cation channel of the rod photoreceptor outer segments (Yau and

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Baylor, 1989), and an elevation of the rod Ca2+ concentration (Fox and Katz, 1992; Medrano and Fox, 1994; He et al., 2000). Detailed kinetic analysis revealed that picomolar Pb2+ competitively and directly inhibits rod cGMP PDE relative to millimolar concentrations of Mg2+ (Srivastava et al., 1995a, 1995b). In addition, nanomolar Pb2+ can elevate the rod Ca2+ (and Pb2+ ) concentration via its competitive inhibition of retinal Na+ ,K+ -ATPase relative to MgATP (Fox et al., 1991b). Once inside the rod, both Ca2+ and Pb2+ enter the mitochondria via the ruthenium red-sensitive Ca2+ uniporter and induce mitochondrial depolarization, swelling, and cytochrome c release (He et al., 2000). The effects of Ca2+ and Pb2+ were additive and blocked completely by the mitochondrial permeability transition pore inhibitor cyclosporin A. Following cytochrome c release, caspase-9 and caspase-3 are sequentially activated. There was no evidence of caspase-8, oxidative stress or lipid peroxidation in this model. These results demonstrate that rod mitochondria are the target site for Ca2+ and Pb2+ . This is consistent with numerous studies from different tissues demonstrating that lead is preferentially associated with mitochondria and particularly with the inner membrane and matrix fractions (Barltrop et al., 1971; Bull, 1980; Pounds, 1984). Taken together, the results suggest that Ca2+ and Pb2+ bind to the internal divalent metal binding site of the mitochondrial permeability transition pore (Szabo et al., 1992) and subsequently open it, which initiates the cytochrome c–caspase cascade of apoptosis in rods (He et al., 2000). In vitro extracellular and intracellular electrophysiologic recordings in isolated whole retinas or photoreceptors reveal that nanomolar to micromolar Pb2+ selectively depress the amplitude and absolute sensitivity of the rod but not cone photoreceptor potential (Fox and Sillman, 1979; Sillman et al., 1982; Tessier-Lavigne et al., 1985; Frumkes and Eysteinsson, 1988). These electrophysiologic results are similar to the ERG alterations observed in occupationally lead-exposed workers (Cavelleri et al., 1982; Betta et al., 1983; Signorino et al., 1983; Campara et al., 1984; Jeyaratnam et al., 1986) and in adult rats exposed to low and moderate levels of lead only during development (Fox and Farber, 1988; Fox and Rubinstein, 1989; Fox et al., 1991a; Fox and Katz, 1992). In addition, these postnatally lead-exposed rats exhibit rod-mediated increases in dark and light adaptation time, decreases in critical flicker fusion frequency (i.e., temporal resolution), decreases in relative sensitivity, and increases in a- and b-wave latencies (Fox and Farber, 1988; Fox and Rubinstein, 1989; Fox et al., 1991a; Fox and Katz, 1992) and decreases in the temporal response properties of both sustained (X-type) and transient (Y-type) RGCs, such as decreased optimal temporal frequency and temporal resolution (Ruan et al., 1994). By extension, these results suggest that there is a common underlying biochemical mechanism responsible for these rodmediated deficits. In vivo and in vitro data suggest that lead-induced inhibition of cGMP PDE and resultant elevation of rod Ca2+ underlies the ERG deficits (Fox and Katz, 1992; Medrano and Fox, 1994; Fox et al., 1997; He et al., 2000). Finally, rod-mediated alterations in dark adaptation and b-wave amplitude are also observed in adult rats and monkeys with prenatal and lifetime moderate- and highlevel lead exposure (Hennekes et al., 1987; Lilienthal et al., 1988; Lilienthal et al., 1994). In the gestationally and postnatally leadexposed monkeys and children, the amplitude of the scotopic bwave was increased (Lilienthal et al., 1988; Lilienthal et al., 1994; Rothenberg et al., 2002): an effect hypothesized to result from the loss of dopaminergic amacrine cells or their processes (Kohler et al., 1997). If rods and blue-sensitive cones in humans exhibit the same sensitivity to a lead-induced inhibition of cGMP-PDE as

they do to the drug-induced inhibition of cGMP-PDE (Zrenner and Gouras, 1979; Zrenner et al., 1982), Fox and Farber (1998) predicted that blue-cone color vision deficits as well as scotopic deficits may be found in adults and children exposed to lead. S- (or blue-) cone deficits have been observed in an occupationally lead-exposed worker (Scholl and Zrenner, 2000).

Methanol Methanol is a low-molecular-weight (32), colorless, and volatile liquid that is widely used as an industrial solvent; a chemical intermediate; a fuel source for picnic stoves, racing cars, and soldering torches; an antifreeze agent; and an octane booster for gasoline. The basic toxicology and references can be found in two thorough reviews (Tephly and McMartin, 1984; Eells, 1992). Briefly, methanol is readily and rapidly absorbed from all routes of exposure (dermal, inhalation, and oral), easily crosses all membranes, and thus is uniformly distributed to organs and tissues in direct relation to their water content. Following different routes of exposures, the highest concentrations of methanol are found in the blood, aqueous and vitreous humors, and bile as well as the brain, kidneys, lungs, and spleen. In the liver, methanol is oxidized sequentially to formaldehyde by alcohol dehydrogenase in human and nonhuman primates or by catalase in rodents and then to formic acid. It is excreted as formic acid in the urine or oxidized further to carbon dioxide and then excreted by the lungs. Formic acid is the toxic metabolite that mediates the metabolic acidosis as well as the retinal and optic nerve toxicity observed in humans, monkeys, and rats with a decreased capacity for folate metabolism (Tephly and McMartin, 1984; Murray et al., 1991; Eells, 1992; Lee et al., 1994a; Lee et al., 1994b; Garner et al., 1995a; Garner et al., 1995b; Eells et al., 1996; Seme et al., 1999). Human and nonhuman primates are highly sensitive to methanol-induced neurotoxicity due to their limited capacity to oxidize formic acid. The toxicity occurs in several stages. It first occurs as a mild CNS depression, followed by an asymptomatic 12- to 24-hour latent period, then by a syndrome consisting of formic acidemia, uncompensated metabolic acidosis, ocular and visual toxicity, coma, and possibly death (Tephly and McMartin, 1984; Eells, 1992). The treatment of methanol poisoning involves both combating acidosis and preventing methanol oxidation, but it is not discussed further here. Experimental rats were made as sensitive to acute methanol exposure as primates by using two different, but related, procedures that effectively reduce the levels of hepatic tetrahydrofolate. One study acutely inhibited methionine synthase and reduced the level of hepatic tetrahydrofolate (Murray et al., 1991; Eells et al., 1996; Seme et al., 1999), while the other fed rats a folate-deficient diet for 18 weeks (Lee et al., 1994a; Lee et al., 1994b). Administration of methanol to rats with a decreased capacity for folate metabolism resulted in toxic blood formate concentrations of 8 to 16 mM (Murray et al., 1991; Lee et al., 1994a; Lee et al., 1994b; Garner et al., 1995a; Garner et al., 1995b; Eells et al., 1996; Seme et al., 1999). Permanent visual damage occurred in humans and monkeys when the blood folate levels exceeded 7 mM (Tephly and McMartin, 1984; Eells, 1992). Acute methanol poisoning in humans, monkeys, and experimental rats resulted in profound and permanent structural alterations in the retina and optic nerve and visual impairments ranging from blurred vision to decreased visual acuity and light sensitivity to blindness. Ophthalmologic studies of exposed humans and monkeys reveal varying degrees of edema of the papillomacular bundle and optic nerve head (Benton and Calhoun, 1952; Potts, 1955;

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Baumbach et al., 1977; Hayreh et al., 1980). Histopathologic and ultrastructural investigations in methanol-exposed monkeys and folate-modified rats showed retinal edema, swollen and degenerated photoreceptors, degenerated RGCs, swollen retinal pigment epithelial cells, axonal (optic nerve) swelling, and mitochondrial swelling and disintegration in each of these cells but especially in the photoreceptors and optic nerve (Baumbach et al., 1977; Hayreh et al., 1980; Murray et al., 1991; Seme et al., 1999). Considering the differences in species, methanol exposures, time course of analysis, and procedures utilized, the overall data for the acute effects of methanol on the ERG are remarkably consistent. Following methanol exposure, the ERG b-wave amplitude in humans, monkeys, and folatemodified rats starts to decrease significantly when the blood formate concentration exceeded 7 mM (Potts, 1955; Ruedeman, 1961; Ingemansson, 1983; Murray et al., 1991; Lee et al., 1994b). These ERG b-wave alterations, as well as flicker-evoked ERG alterations (Seme et al., 1999), occur at lower formate concentrations than those associated with structural changes in the retina and optic nerve, as discussed above. Decreases in the a-wave amplitude are delayed, relative to the b-wave and occur when blood formate concentrations further increase (Ruedeman, 1961; Ingemansson, 1983; Murray et al., 1991; Eells et al., 1996). In addition, it has been shown that intraretinal metabolism of methanol is necessary for the formate-mediated alterations in the ERG (Garner et al., 1995a), although intravenous infusion of formate in monkeys does induce optic nerve edema (Martin-Amat et al., 1978). Finally, in the folatemodified rats, it appears that photoreceptors that respond to a 15-Hz flicker/510-nm wavelength mesopic-photopic stimulus [i.e., rods and middle wavelength–sensitive (M) cones] are more sensitive to methanol than the ultraviolet-sensitive (UV) cones (Seme et al., 1999). The retinal sources of the ERG a-wave and b-wave were previously discussed. Thus, the data from the ERG b-wave methanol studies suggest that the initial effect of formate is directly on the ONtype rod bipolar cells, M¨uller glial cells, and/or synaptic transmission between the photoreceptors and bipolar cells. A well-designed series of pharmacologic, ERG, and potassium-induced M¨uller cell depolarization studies using several controls and folate-modified rats revealed a direct toxic effect of formate on M¨uller glial cell function (Garner et al., 1995a; Garner et al., 1995b). These studies also provided evidence that formate does not directly affect depolarizing rod bipolar cells or synaptic transmission between the photoreceptors and bipolar cells. Formate also appears to directly and adversely affect the rod and cone photoreceptors as evidenced by the markedly decreased ERG a-wave and flicker response data (Ruedeman, 1961; Ingemansson, 1983; Murray et al., 1991; Eells et al., 1996; Seme et al., 1999). Although there are no direct data on the underlying molecular mechanism responsible for the toxic effects of formate on M¨uller glial cells and photoreceptors, several findings suggest that the mechanism involves a disruption in oxidative energy metabolism. First, the whole retinal ATP concentration is decreased in folatedeficient rats 48 hour following methanol exposure, the time point when the b-wave was lost (Garner et al., 1995b). Second, both formate (10 to 200 mM) and formaldehyde (0.5 to 5 mM) inhibited oxygen consumption in isolated ox retina, and formaldehdye was considerably more potent (Kini and Cooper, 1962). Third, similar concentrations of formaldehyde inhibited oxidative phosphorylation of isolated ox retinal mitochondria, with greater effects observed using FAD-linked than NADH-linked substrates (Kini and Cooper, 1962). Unfortunately, the effects of formate were not exam-

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ined. Fourth, and consistent with the above results, formate inhibits succinate-cytochrome c reductase and cytochrome oxidase activity (Ki = 5 to 30 mM), but not NADH-cytochrome c reductase activity in isolated beef heart mitochondria and/or submitochondrial particles (Nicholls, 1976). Fifth, ultrastructural studies reveal swollen mitochondria in rat photoreceptor inner segment and optic nerve 48 to 72 hour after nitrous oxide/methanol exposure (Murray et al., 1991; Seme et al., 1999). To date, there are no such studies conducted on the M¨uller glial cells. Taken together, these results suggest formate is a mitochondrial poison that inhibits oxidative phosphorylation of photoreceptors, M¨uller glial cells, and optic nerve. The evidence for this hypothesis and establishment of subsequent steps resulting in retinal and optic nerve cell injury and death remain to be elucidated.

Organic Solvents: n-Hexane, Perchloroethylene, Styrene, Toluene, Trichloroethylene, Xylene and Mixtures The neurotoxicity of organic solvents is well established. However, there is a paucity of mechanistic studies on the adverse effects of organic solvents on the retina and visual system despite findings of structural alterations in rods and cones as well as functional alterations such as color vision deficits, decreased contrast sensitivity, and altered visual-motor performance (Raitta et al., 1978; Odkvist et al., 1983; Baker and Fine, 1986; Larsby et al., 1986; Mergler, 1990; ArlienS¨oborg, 1992; Backstrom and Collins, 1992; Backstrom et al., 1993; Broadwell et al., 1995; Fox, 1998; Iregren et al., 2002a; Paramei et al., 2004; Benignus et al., 2005; chapter 24 entitled “Toxic Effects of Solvents and Vapors”). Dose-response color vision loss (acquired dyschromatopsia) and decreases in the contrast sensitivity function occur in workers exposed to organic solvents such as alcohols, n-hexane, toluene, trichlorethylene, xylene, and mixtures of these and others. Adverse effects usually occur only at concentrations above the occupational exposure limits (Raitta et al., 1978; Baird et al., 1994; Mergler et al., 1987, 1988, 1991; Nakatsuka et al., 1992). A large percentage of workers in microelectronic plants, print shops, and paint manufacturing facilities, who were exposed to concentrations of solvents that exceeded the threshold limit values, had acquired dyschromatopsia as assessed by the Lanthony D-15 desaturated color arrangement panel (Mergler et al., 1987, 1988, 1991). These workers had no observable clinical abnormalities as assessed by biomicroscopy, funduscopy, and peripheral visual field tests. The color vision losses were mainly blue-yellow losses, although more severe red-green losses were reported. As a rule, acquired blue-yellow losses generally result from lens opacification or outer retinal alterations, whereas red-green losses are associated with inner retinal, retrobular, or central visual pathway alterations (Porkony et al., 1979). Moreover, these occupationally exposed workers also exhibited lower contrast sensitivity at intermediate spatial frequencies, which likely reflects alterations in neural function (Mergler et al., 1991). The data from the Mergler et al. (1987, 1988, 1991) studies appear to show gender differences in these adverse visual effects. A study of female workers, where the Lanthony D-15 desaturated test was used to assess color vision, showed a trend toward increased prevalence of color vision impairment following exposure to low to moderate concentrations of toluene (Zavalic et al., 1996). Similar blue–yellow deficits as well as macular changes were observed in workers exposed to n-hexane for 5 to 21 years (Raitta et al., 1978). These findings correlate with the rod and cone degeneration observed in rats exposed to 2,5-hexanedione (Backstrom

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and Collins, 1992; Backstrom et al., 1993). Clearly more detailed, well-designed, and well-executed studies are needed to determine (1) which solvent(s) cause alterations in color vision, (2) are spatial and temporal contrast sensitivity affected, (3) the dose (concentration)-response relations between exposure and effects, (4) possible gender differences, and (5) whether these deficits are reversible. Perchloroethylene Deficits in visual function, such as contrast sensitivity, have been observed in residents of neighborhoods containing dry cleaners using perchloroethylene (Altmann et al., 1995), and in residents of apartment buildings with co-located dry cleaners (Schreiber et al., 2002). For both of these residential studies, the atmospheric concentrations of perchloroethylene were well below those typical of occupational settings. Laboratory experiments with human subjects exposed to perchloroethylene for four hours for four days revealed increased peak latency delays in the N75, P100, and N150 of the VEP as well as decreases in contrast sensitivity at low and intermediate spatial frequencies (Altmann et al., 1990). Interestingly, there were no changes in the brainstem auditory evoked potential during the exposure period. Moreover, gestational exposure to perchloroethylene produced clinical red-green color loss and decreased visual acuity in the children of occupationally exposed mothers (Till et al., 2001). The above results reveal that perchloroethylene is toxic to both the developing and adult visual system. Styrene Six independent studies report that workers exposed to mean atmospheric concentrations of styrene ranging from 20 to 70 ppm exhibit concentration-dependent alterations in color vision (Gobba et al., 1991; Fallas et al., 1992; Chia et al., 1994; Eguchi et al., 1995; Campagna et al., 1995: Iregren et al., 2005). A combined data analysis from two of the above studies (Gobba et al., 1991; Campagna et al., 1995) suggests that the threshold for color visual impairments is ≤4 ppm styrene (Campagna et al., 1996). This is well below the threshold limit value–time weighted average (TLV-TWA) value for any country: range 20 to 50 ppm. The findings of similar blue-yellow color vision deficits by five different groups of investigators in different countries argue convincingly for the reproducibility and validity of these styrene-induced color vision deficits. The reversibility of these impairments has not been thoroughly studied, although in one study no recovery was found after a 1-month period of no exposure (Gobba et al., 1991). The findings reveal the potential sensitivity of the visual system and especially the photoreceptors to toxicant exposure. The results overall demonstrate that the Lanthony D-15 desaturated test can be a sensitive and reliable test for detecting color vision abnormalities in solvent-exposed workers. In summary, the evidence indicates that organic solvents can produce color vision deficits in occupationally exposed workers. In addition to these articles, two meta-analyses of the solvent literature have been presented. Paramei et al. (2004) focused on color confusion index (CCI) measures, and evaluated studies of CCI scores from workers exposed to toluene, styrene, or mixed solvents from a total of 15 studies. To combine the data for their meta-analysis a z-score transformation was conducted on the CCI values. While 13 of the original 15 studies reported CCI values indicative of poorer performance in the exposed group than in controls, the meta-analysis showed large variations among the effect sizes across studies. This obscured any statistically significant association between the level of exposure and CCI values for toluene

and mixtures, however, a borderline significant difference was found for styrene. Benignus et al. (2005) only focused on styrene exposure from six independent studies. They conducted a meta-analysis on behavioral reaction time data as well as CCI values. Z-score transformations were not conducted under the rationale that z-score transformations confound the magnitude of an outcome with its variability. Instead, Benignus et al. (2005) used proportional changes with respect to the control group as a measure of the effect magnitude and utilized the data variability to estimate confidence limits around the estimates of association between cumulative exposure and CCI. Using this approach, the meta-analysis revealed statistically significant relationships in the digitized individual subject data between cumulative styrene exposure and increased CCI scores, as well as between cumulative styrene exposure and prolonged choice reaction times. In summary, the above studies indicate that occupational styrene exposure causes long-lasting color vision deficits in humans. The severity of such impairments needs to be considered, especially in relation to public health and regulatory issues. An example is to compare the loss of visual function from occupational styrene exposure to that associated with normal aging. Thus, it was estimated that loss of visual function from 8 years of occupational exposure to 20 ppm styrene would be equivalent to 1.7 additional years of aging (Benignus et al., 2005). Analyses such as this may lead to better understanding of the severity of subtle changes in visual function. The mechanism of action underlying these color vision changes is unknown. One rodent study suggests that a styrene-induced loss of tyrosine hydroxylase immunoreactive amacrine cells and subsequent decrease in retinal dopamine may contribute to this deficit (Vettori et al., 2000). At present, it is not clear how this mechanism would work.

Organophosphates The neurotoxicity of organophosphates is well established (see chapter 16 entitled “Toxic Responses of the Nervous System”); however, the link between organophosphate exposure and retinotoxicity is presently unresolved. Clinical studies conducted in Japan, report on ocular toxicity from laboratory animals exposed to organophosphates, and reports to the EPA by pesticide manufacturers suggest that various organophosphates produce retinotoxicity and chronic ocular damage (Ishikawa, 1973; Dementi, 1994). However, many of the early clinical reports were poorly designed and remain unconfirmed. The evidence for organophosphateinduced retinal toxicity is strongest for fenthion (dimethyl 3-methyl4-methylthiophenyl phosphorothionate) (Imai et al., 1983; Misra et al., 1985; Boyes et al., 1994; Tandon et al., 1994). Two recent epidemiologic studies of licensed pesticide applicators and their spouses in two states, North Carolina and Iowa, did not find a statistically increased risk of retinal degeneration from use of organophosphate insecticides as a class, but risks were increased for some individual members of the chemical class (Kamel et al., 2000; Kirrane et al., 2005). Interestingly, both studies identified an increased risk of retinal degeneration in individuals exposed to fungicides. One report cites a high incidence of myopia in Japenese children exposed to organophosphates (Ko et al., 1988) and one experimental study found that visual control of ocular growth was impaired in the eyes of chicks exposed to the organophosphate insecticide chlorpyrifos (Geller et al., 1998). Until further detailed, well-designed, and replicated clinical and basic science studies are conducted; an adequate discussion of the sites and mechanisms of action of organophosphates must be delayed. In the interim, the references noted above

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will provide the interested reader with a synopsis of the current status in this area.

TARGET SITES AND MECHANISMS OF ACTION: OPTIC NERVE AND TRACT The optic nerve consists primarily of RGC axons carrying visual information from the retina to several distinct anatomic destinations in the CNS. Both myelinated and nonmyelinated axons are present and grouped into bundles of axons that maintain a topographic distribution with respect to the site of origin in the retina. At the optic chiasm, the fibers split, so that, in humans and other primates, those fibers originating from the temporal retina continue in the optic tract toward the ipsilateral side of the brain, while those fibers originating in the nasal half of the retina, cross the midline and project to the contralateral side of the brain. In species with sideward-facing eyes such as the rat, a larger proportion of the optic nerve fibers (up to 90%) cross the midline. Fibers from the optic nerve terminate in the dorsal LGN, the superior colliculus, and pretectal areas. Information passing through the LGN to visual cortex gives rise to conscious visual perception. Information traveling to the superior colliculus is used to generate eye movements. Pathways leading to the pretectal areas subserve the pupil response. The LGN of primates contains six histologic layers that are alternately innervated by cells from the contralateral and ipsilateral eyes. The cells projecting to and from the ventral two layers of the LGN have large cell bodies, and consequently, this pathway is referred to as the magnocellular system. Retinal ganglion cells projecting to the magnocellular layers of the LGN are referred to as either M-type or Pα cells. Magnocellular neurons are sensitive to fast moving stimuli and to low levels of luminance contrast, but are insensitive to differences in color. The cells from the magnocellular pathway are involved in motion perception. On the dorsal side of the LGN, the cells are smaller and form the parvocellular pathway. Retinal ganglion cells projecting to the parvocellular layers of the LGN are referred to as P-type or Pβ cells. Parvocellular neurons are sensitive to color and to fine detailed patterns, have slower conduction velocities, and are involved in perception of color and form (Horton, 1992; Rodieck, 1998). Disorders of the optic nerve may be termed optic neuritis, optic neuropathy, or optic nerve atrophy, referring to inflammation, damage, or degeneration, respectively, of the optic nerve. Retrobulbar optic neuritis refers to inflammation of the portion of the optic nerve posterior to the globe. Among the symptoms of optic nerve disease are reduced visual acuity, contrast sensitivity, and color vision. Toxic effects observed in the optic nerve may originate from damage to the optic nerve fibers themselves or to the RGC somas that provide axons to the optic nerve. A number of nutritional disorders can adversely affect the optic nerve. Deficiency of thiamine, vitamin B12 , or zinc results in degenerative changes in optic nerve fibers. Nutritional and toxic factors can interact to produce optic nerve damage. A condition referred to as alcohol–tobacco amblyopia or simply as toxic amblyopia is observed in habitually heavy users of these substances and is associated nutritional deficiency. Dietary supplementation with vitamin B12 is therapeutically helpful, even when patients continue to consume large amounts of alcohol and tobacco (Grant, 1986; Anderson and Quigley, 1992; Potts, 1996).

Acrylamide Acrylamide monomer is used in a variety of industrial and laboratory applications, where it serves as the basis for the production of

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polyacrylamide gels and other polyacrylamide products. Exposure to acrylamide produces a distal axonopathy in large-diameter axons of the peripheral nerves and spinal cord that is well documented in humans and laboratory animals (Spencer and Schaumburg, 1974a, 1974b). Visual effects of acrylamide exposure occur at dose levels sufficient to cause substantial peripheral neuropathy, but the selective nature of the visual deficits and associated neuropathology is very instructive. Whereas the large-diameter and long axons are most vulnerable to acrylamide in the peripheral nerve and spinal cord, this is not the case in the optic tract. The middle diameter axons of the Pβ -type RGCs that project to the parvocellular layers of the LGN of New- and Old-World primates degenerate after prolonged treatment with acrylamide (Eskin and Merigan, 1986; Lynch et al., 1989). The larger-diameter Pα -type RGCs that project to the magnocellular layers of the LGN are apparently spared. Visual function testing in these primates, without a functional parvocellular system, revealed selective perceptual deficits in detecting visual stimuli with high spatial-frequency components (i.e., fine visual patterns) and low temporal-frequency components (i.e., slowly modulating sine waves) (Merigan and Eskin, 1986). However, the monkeys’ perception of larger visual patterns, modulated at higher temporal rates, was not impaired. These toxicologic experiments helped elucidate the functional differentiation of primate parvocellular and magnocellular visual systems. Why the axons of the optic nerve and tract show a different size-based pattern of vulnerability than do axons of the peripheral nerve and spinal cord is not currently understood.

Carbon Disulfide Carbon disulfide (CS2 ) is used in industry to manufacture viscose rayon, carbon tetrachloride, and cellophane. The neurotoxicity of CS2 is well known and involves damage to the peripheral and central nervous systems as well as profound effects on vision (Beauchamp et al., 1983). The peripheral neuropathy results from a distal axonal degeneration of the large-caliber and long axons of the peripheral nerves and spinal cord, probably through the reactions with the sulfhydryl groups of axonal neurofilament proteins, yielding covalent cross linkages that lead to filamentous tangles and axonal swellings (Graham and Valantine, 2000). The mechanism of action through which inhalation of high concentrations of CS2 vapors leads to psychotic mania is not currently established but may result from alterations in catecholamine synthesis or neuronal degeneration in several brain areas (Beauchamp et al., 1983). Workers exposed to CS2 experience loss of visual function accompanied by observable lesions in the retinal vasculature. Among the changes in visual function reported in viscose rayon workers are central scotoma, depressed visual sensitivity in the peripheral visual field, optic atrophy, pupillary disturbances, blurred vision, and disorders of color perception. A workplace study of 123 Belgian viscose rayon workers found a statistical association between a weighted cumulative CS2 exposure score, deficits in color vision measured using the Farnsworth-Munsell 100-HUE test, and observations of excess microaneurysms observed ophthalmoscopically and in fundus photographs (Vanhoorne et al., 1996). This association was not observed in the 42 workers who were never exposed to levels above the TLV value of 31 mg/m3 . The coexistence of retinal microaneurysms with functional loss has led to the presumption that the visual deficits were a secondary consequence of vascular disease and perhaps of retinal hemorrhages. This association was addressed in carbon disulfide–exposed macaque monkeys used in visual psychophysical,

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fluorescein angiography, and fundus photography studies as well as postmortem neuropathologic evaluations (Merigan et al., 1988; Eskin et al., 1988). They observed markedly decreased contrast sensitivity functions, decreased visual acuity, and degeneration of the RGCs, all of which occurred in the absence of retinal microaneurysms or hemorrhages. There was little evidence of effects on the other retinal neurons. These findings indicate that the retinal and optic nerve pathology produced by CS2 are likely a direct neuropathologic action and not the indirect result of vasculopathy. Interestingly, and importantly, after cessation of exposure, the visual acuity measures recovered temporarily in two of the CS2 -treated monkeys; however, the contrast sensitivity measures did not recover. This demonstrates the independence of these two measures and the utility and importance of independent evaluations of contrast sensitivity and visual acuity.

Cuban Epidemic of Optic Neuropathy During 1992 and 1993, an epidemic occurred in Cuba in which over 50,000 people suffered from optic neuropathy, sensory and autonomic peripheral neuropathy, high-frequency neural hearing loss, and myelopathy. This is the largest epidemic of neurologic disease in the twentieth century (Roman, 1998). The affected individuals were characterized as having bilateral low visual acuity, impaired color perception, impaired visual contrast sensitivity, central scotoma, optic disk pallor, and, in particular, loss of nerve fibers from the papillomacular bundle (Sadun et al., 1994a; Hedges et al., 1997). Individuals with neurologic findings demonstrated stockingglove sensory deficits, leg cramps, sensory ataxia, altered reflexes, and complaints of memory loss (Mojon et al., 1997). Various authors noticed similarities between the Cuban cases and nutritional or alcohol–tobacco amblyopia, Leber’s hereditary optic neuropathy, and Strachan’s disease (Hedges et al., 1994; Hirano et al; 1994; Espinosa et al., 1994; Sadun et al., 1994b; Mojon et al., 1997). The optic neuropathy resembled methanol poisoning (Roman, 1998; Sadun, 1998). The outbreak of the epidemic was linked to nutritional deficiencies (Hedges et al., 1997; Mojon et al., 1997; Roman, 1998). In most cases, aggressive supplementation of the diet with B vitamins and folic acid led to a significant clinical improvement (Mojon et al., 1997). Nutritional deficiencies were a primary contributor to the epidemic; however, it was not clear whether they were solely responsible or whether dietary insufficiency served to make individuals more susceptible to other factors. Genetic susceptibility factors and viral exposures have been considered (Johns et al., 1994; Johns and Sadun, 1994; Newman et al., 1994; Mas et al., 1997; Hedges et al., 1997). One likely contributing factor was co-exposure to low levels of neurotoxic compounds that would otherwise have been tolerated (Sadun, 1998). In addition to low food intake, risk factors for the development of optic neuropathy included use of tobacco, in particular the frequent smoking of cigars, and high cassava consumption (Roman, 1998). The mitochondrial toxicant cyanide may be a contaminant of both cassava and tobacco products. Moderate to severe folic acid deficiency was observed in more than half of the cases (Roman, 1998). Samples of local home-brewed rum showed approximately 1 percent contamination with methanol, a level that would not produce optic nerve toxicity in normal healthy individuals (Sadun et al., 1994). However, onequarter of the Cuban patients showed elevated serum formate concentrations, probably a result of folic acid deficiency. The maximum serum formate concentrations observed (approximately 4 mM) were similar to levels that produce retinal and optic nerve toxicity in a

rodent model of methanol toxicity (Eells et al., 1996). Sadun (1998) postulated that mitochondrial impairment, created by the combination of low nutritional status and toxic exposures, was responsible for the neurologic impairments. The nutritional deficiency would lead to ATP depletion. Exposure to either cyanide or formic acid, the toxic metabolite of methanol, causes inhibition of cytochrome oxidase, which further depletes ATP levels (Nicholls, 1976; also see retinal section for additional references and details). Because axoplasmic transport of new mitochondria from nerve cell bodies to distal axonal segments is energy-dependent (Vale et al., 1992), the lowered ATP levels would be expected to impair mitochondrial transport and start a cycle of further ATP depletion and reduced mitochondrial transport to the nerve terminal regions. Sadun proposed that the nerve fibers most sensitive to this type of damage would be the long peripheral nerve axons, which have high transport demands, and the small caliber fibers of the optic nerve, in particular at the papillomacular bundle, which have physical constrictions to transporting mitochondria. Exposure to toxicants could not be documented in most of the people identified late in the epidemic, suggesting nutritional deficit as the principal cause. However, coexposure to low levels of mitochondrial toxicants or other factors may have pushed individuals over a threshold for causing nerve damage.

Ethambutol The dextro isomer of ethambutol is widely used as an antimycobacterial drug for the treatment of tuberculosis. It is well known that ethambutol produces dose-related alterations in the visual system, such as blue-yellow and red-green dyschromatopsias, decreased contrast sensitivity, reduced visual acuity, and visual field loss. The earliest visual symptoms appear to be a decrease in contrast sensitivity and color vision, although impaired red-green color vision is the most frequently observed and reported complaint. However, the loss of contrast sensitivity may explain why some patients with normal visual acuity and color perception still complain of visual disturbance. These visual system alterations can occur with a few weeks of doses equal to or greater than 20 mg/kg body weight; however, they usually become manifest after several months of treatment (Koliopoulos and Palimeris, 1972; Polak et al., 1985; Salmon et al., 1987; Jaanus et al., 1995). The symptoms are primarily associated with one of two forms of retrobulbar optic neuritis (i.e., optic neuropathy). The most common form, seen in almost all cases, involves the central optic nerve fibers and typically results in a central or paracentral scotoma in the visual field and is associated with impaired red-green color vision and decreased visual acuity, whereas the second form involves the peripheral optic nerve fibers and typically results in a peripheral scotoma and visual field loss (Jaanus et al., 1995; Lessell, 1998). In experimental animals, ethambutol causes RGC and optic nerve degeneration, discoloration of the tapetum lucidum (in dogs), retinal detachment (in cats), and possibly amacrine and bipolar cell alterations (van Dijk and Spekreijse, 1983; Grant and Schuman, 1993; Sjoerdsma et al., 1999). Although the mechanism responsible for producing the RGC and optic nerve degeneration is unknown, recent in vivo studies in rats and in vitro rat RGC cell culture experiments suggest that ethambutol causes RGC death secondary to glutamate-induced excitotoxicity (Heng et al., 1999). Pharmacologic studies, using the in vivo and in vitro models, show that although ethambutol is not a direct NMDA-receptor agonist, it makes RGCs more sensitive to endogenous levels of glutamate. Using the

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fluorescent Ca2+ dyes calcium green 1-AM and rhod-2, Heng et al. (1999) showed that following application of ethambutol in the presence, but not absence, of glutamate to isolated RGCs, there was a decrease in cytosolic Ca2+ and a subsequent increase in mitochondrial Ca2+ . Interestingly, the increase in mitochondrial Ca2+ resulted in an increase in the mitochondrial membrane potential as measured by the mitochondrial membrane potential sensitive dye JC-1. The authors (Heng et al., 1999) postulate that this latter phenomenon occurs as a result of an ethambutol-mediated chelation of Zn2+ from the mitochondrial ATPase inhibitor protein IF1 (Rouslin et al., 1993) that subsequently results in the inhibition of mitochondrial ATP synthesis and elevation of mitochondrial membrane potential. These intriguing ideas have merit; however, many additional experiments will be needed to prove this hypothesis. In addition, the authors suggest that some glutamate antagonists may be useful in decreasing the side effects of ethambutol—a practical suggestion that appears worthy of clinical investigation.

TARGET SITES AND MECHANISMS OF ACTION: THE CENTRAL VISUAL SYSTEM Many areas of the cerebral cortex are involved in the perception of visual information. The primary visual cortex—called V1, Brodmann’s area 17, or striate cortex—receives the primary projections of visual information from the LGN and also from the superior colliculus. Neurons from the LGN project to visual cortex maintaining a topographic representation of the receptive field origin in the retina. The receptive fields in the left and right sides of area 17 reflect the contralateral visual world and representations of the upper and lower regions of the visual field are separated below and above, respectively, the calcarine fissure. Cells in the posterior aspects of the calcarine fissure have receptive fields located in the central part of the retina. Cortical cells progressively deeper in the calcarine fissure have retinal receptive fields that are located more and more peripherally in the retina. The central part of the fovea has tightly packed photoreceptors for resolution of fine detailed images, and the cortical representation of the central fovea is proportionately larger than the peripheral retina in order to accommodate a proportionately larger need for neural image processing. The magnocelluar and parvocellular pathways project differently to the histologically defined layers of primary striate visual cortex and then to extrastriate visual areas. The receptive fields of neurons in visual cortex are more complex than the circular center-surround arrangement found in the retina and LGN. Cortical cells respond better to lines of a particular orientation than to simple spots. The receptive fields of cortical cells are thought to represent computational summaries of a number of simpler input signals. As the visual information proceeds from area V1 to extrastriate visual cortical areas, the representation of the visual world reflected in the receptive fields of individual neurons becomes progressively more complex (Horton, 1992).

Lead In addition to the well-documented retinal effects of lead (see above), lead exposure during adulthood or perinatal development produces structural, biochemical, and functional deficits in the visual cortex of humans, nonhuman primates, and rats (Fox et al., 1977; Winneke, 1979; Costa and Fox, 1983; Sborgia et al., 1983; Fox, 1984; Otto et al., 1985; Lilienthal et al., 1988; Reuhl et al., 1989; Murata et al., 1993; Otto and Fox, 1993; Altmann et al., 1994,

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1998; Winneke et al., 1994). Quantitative morphometric studies in monkeys exposed to either high levels of lead from birth or infancy to 6 years of age revealed a decrease in visual cortex (areas V1 and V2), cell volume density, and a decrease in the number of initial arborizations among pyramidal neurons (Reuhl et al., 1989). The former results may be due to an absolute decrease in total cell numbers, possibly resulting from lead-induced apoptosis as observed in the retina (Fox et al., 1997; He et al., 2000). This may also account for the decreased density of cholinergic muscarinic receptors found in the visual cortex of adult rats following moderate level developmental lead exposure (Costa and Fox, 1983). The morphometric results on neuronal branching are reminiscent of earlier findings in the neocortex of rats following high level developmental lead exposure (Petit and LeBoutillier, 1979), and recent findings in the somatosensory cortex of rats following low or moderate level developmental lead exposure (Wilson et al., 2000). These alterations could partially contribute to the decreases in contrast sensitivity observed in lead-exposed rats and monkeys (Fox, 1984; Rice, 1998), the alterations in the amplitude and latency measures of the flash and pattern-reversal evoked potentials in lead-exposed children, workers, monkeys, and rats (Fox et al., 1977; Winneke, 1979; Sborgia et al., 1983; Otto et al., 1985; Lilienthal et al., 1988; Murata et al., 1993; Altmann et al., 1994, 1998; Winneke et al., 1994), and the alterations in tasks assessing visual function in lead-exposed children (Winneke et al., 1983; Hansen et al., 1989; Mu˜noz et al., 1993).

Methylmercury Methyl mercury became notorious in two episodes of mass poisoning (see chapter. 16 entitled “Toxic Responses of the Nervous System”). In the 1950s, industrial discharges of mercury into Minamata Bay in Japan became biomethylated to form methyl mercury, which then accumulated in the food chain and reached toxic concentrations in the fish and shellfish consumed in the surrounding communities. Hundreds of people were poisoned, showing a combination of sensory, motor, and cognitive deficits. A more widespread episode of methyl mercury poisoning affected thousands of Iraqi citizens who mistakenly ground wheat grain into flour that had been treated with methyl mercury as a fungicide and that was intended for planting and not for direct human consumption. Visual deficits are a prominent feature of methyl mercury intoxication in adult humans, along with several other neurologic manifestations such as difficulties with sensation, gait, memory, and cognition. Methyl mercury poisoned individuals experienced a striking and progressive constriction of the visual field (peripheral scotoma) as patients became progressively less able to see objects in the visual periphery (Iwata, 1977). The narrowing of the visual field gives impression of looking through a long tunnel, hence the term tunnel vision. Visual field constrictions also have been observed in methyl mercury–poisoned monkeys (Merigan, 1979). On autopsy of some of the Minamata patients, focal neurologic degeneration was observed in several brain regions including motor cortex, cerebellum, and calcarine fissure of visual cortex (Takeuchi and Eto, 1977). The histopathologic feature was a destruction of the cortical neural and glial cells, with sparing of the subcortical white matter, optic radiations, and LGN. Monkeys and dogs that were treated experimentally with methyl mercury showed greater damage in the calcarine fissure, associated with higher regional concentrations of proteinbound mercury, than in other brain regions (Yoshino et al., 1966; Berlin et al., 1975). In the Minamata patients, there was a regional distribution of damage observed within striate cortex, such that the

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most extensive damage occurred deep in the calcarine fissure and was progressively less in the more posterior portions. Thus, the damage was most severe in the regions of primary visual cortex that subserved the peripheral visual field, with relative sparing of the cortical areas representing the central vision. This regional distribution of damage corresponded with the progressive loss of peripheral vision while central vision was relatively preserved. Methyl mercury–poisoned individuals also experienced poor night vision (i.e., scotopic vision deficits), also attributable to peripheral visual field losses. Similar changes were observed in adult monkeys exposed to methyl mercury (Berlin et al., 1975). Mercury also accumulates in the retina of animals exposed to methylmercury (DuVal et al., 1987). Acute exposure of isolated retinas to mercury or methylmercury produces rod-selective electrophysiologic and morphologic alterations (Fox and Sillman, 1979; Braekevelt, 1982), whereas subacute dosing with methylmercury alters the photopic ERG prior to the scotopic ERG (Goto et al., 2001). The neurologic damage in adult cases of Minamata disease was focally localized in the calcarine cortex and other areas but was more globally distributed throughout the brain in those developmentally exposed. The levels of methyl mercury exposure experienced by people in Minamata Bay were undoubtedly high. Studies of visual function in nonhuman primates exposed to methyl mercury during perinatal development demonstrate a decrease in visual contrast sensitivity, visual acuity, and temporal flicker resolution at dose levels lower

than those associated with constriction of the visual fields (Rice and Gilbert, 1982; Rice and Gilbert, 1990; Rice, 1996). Monkeys exposed to methyl mercury from birth onward or in utero plus postnatally exhibited spatial vision deficits under both high and low luminance conditions, although the deficits were greater under scotopic illumination (Rice and Gilbert, 1982; Rice, 1990). The effects on temporal vision were different. That is, monkeys exposed from birth displayed superior low-luminance temporal vision, whereas high-luminance temporal vision was not impaired. In contrast, monkeys exposed to methyl mercury in utero plus postnatally exhibited deficits in low-frequency high-luminance temporal vision, while low-luminance temporal vision was superior to that of control monkeys (Rice, 1990). These data indicate that the spatial and temporal vision deficits produced by developmental exposure to methyl mercury are different from those produced during adulthood. The underlying mechanisms have yet to be determined.

ACKNOWLEDGEMENTS We thank Dr. Andrew M. Geller and Jean-Claude Mwanza for review and editorial comments. Partial support for writing this chapter came from a NIEHS RO1 Grant ES012482 to Donald A. Fox. This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Mention of trade names and commercial products does not constitute endorsement or recommendation for use.

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Veenstra DL, Best JH, Hornberger J, et al.: Incidence and long-term cost of steroid-related side effects after renal transplantation. Am J Kidney Dis 33:829–839, 1999. Vettori MV, Corradi D, Coccini T, et al.: Styrene-induced changes in amacrine retinal cells: an experimental study in the rat. Neurotoxicology 21:607–614, 2000. Vidyasagar TR: Optic nerve components may not be equally susceptible to damage by acrylamide. Brain Res 224:452–455, 1981. Walkowiak J, Altmann L, Kramer U, et al.: Cognitive and sensorimotor functions in 6-year-old children in relation to lead and mercury levels: Adjustment for intelligence and contrast sensitivity in computerized testing. Neurotoxicol Teratol 20:511–521, 1998. Waltman S, Sears ML: Catechol-O-methyltransferase and monoamine oxidase activity in the ocular tissues of albino rabbits. Invest Ophthalmol 3:601–605, 1964. Watkins JB III, Wirthwein DP, Sanders RA: Comparative study of Phase II biotransformation in rabbit ocular tissues. Drug Metab Dispos 19:708– 713, 1991. Williams RA, Howard AG, Williams TP: Retinal damage in pigmented and albino rats exposed to low levels of cyclic light following a singlemydriatic treatment. Curr Eye Res 4:97–102, 1985. Wilson MA, Johnston MV, Goldstein GW, Blue ME: Neonatal lead exposure impairs development of rodent barrel field cortex. Proc Natl Acad Sci USA 97:5540–5545, 2000. Winkler BS, Kapousta-Bruneau N, Arnold MJ, Green DG: Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina. Vis Neurosci 16:345–353, 1999. Winkler BS, Riley MV: Na+,K+ and HCO3 ATPase activity in retina: Dependence on calcium and sodium. Invest Ophthalmol Vis Sci 16:1151–1154, 1977. Winkler BS: A quantitative assessment of glucose metabolism in the rat retina, in Christen Y, Doly M, Droy-Lefaix MT (eds):Vision and Adaptation, Les Seminaires Ophtamologiques d’IPSEN. Vol 6. Paris: Elsevier, 1995, pp. 78–96. Winneke G: Modification of visual evoked potentials in rats after long-term blood lead elevation. Act Nerv Sup (Praha) 4:282–284, 1979. Winneke G, Beginn U, Ewert T, et al.: Study to determine the subclinical effects of lead on the nervous system in children with known prenatal exposure in Nordenham. Schr-Reine Verein Wabolu 59:215–230, 1984. Winneke G, Kramer U, Brockhaus A, et al.: Neuropsychological studies in children with elevated tooth-lead concentrations: II. Extended study. Int Arch Occup Environ Health 51:231–252, 1983. Winneke G, Altmann L, Kr¨amer U, Turfeld M, Behler R, Gutsmuths FJ, Mangold M: Neurobehavioral and neurophysiological observations in six year old children with low lead levels in East and West Germany. Neurotoxicology 15:705–713, 1994. Withering W: An Account of the Foxglove and Some of Its Medicinal Uses: With Practical Remarks on Dropsy and Other Diseases. London: Broomsleigh Press, 1785. Wolfensberger TJ: Toxicology of the retinal pigment epithelium. In Marmor MF, Wolfensberger TJ (eds): The Retinal Pigment Epithelium Function and Disease. New York, NY. Oxford University Press, 1998, pp. 621– 647. Woodhouse JM, Barlow HB: Spatial and temporal resolution and analysis, in Barlow HB, Mollon JD (eds): The Senses. Cambridge: Cambridge University Press, 1982, pp. 133–164. Yau KW, Baylor DA: Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu Rev Neurosci 12:289–327, 1989. Yoshino Y, Mozai T, Nako K: Distribution of mercury in the brain and its subcellular units in experimental organic mercury poisonings. J Neurochem 13:397–406, 1966. Zavalic M, Turk R, Bogadi-Sare A, Skender L: Colour vision impairment in workers exposed to low concentrations of toluene. Arh Hig Rada Toksikol 47:167–175, 1996. Zhao C, Schwartzman ML, Shichi H: Immunocytochemical study of cytochrome P450 4A induction in mouse eye. Exp Eye Res 63:747–751, 1996.

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CHAPTER 18

TOXIC RESPONSES OF THE HEART AND VASCULAR SYSTEM Y. James Kang

Biomarkers for Cardiac Toxicity Validation of Biomarkers Availability of Biomarkers Biomarker Applications and Limitations

INTRODUCTION OVERVIEW OF THE HEART Overview of Cardiac Structural and Physiological Features Review of Cardiac Structure Electrophysiology Contractility Electrotonic Cell-to-Cell Coupling Electrocardiograph Neurohormonal Regulation Overview of Cardiac Energy Metabolism and Biochemistry ATP and the Heart Phosphocreatine and the Heart Metabolic Pathways Calcium and Calcineurin AMP-Activated Protein Kinase Mitogen-Activated Protein Kinases Protein Kinase C Transcription Factors

CARDIAC TOXIC CHEMICALS Alcohol and Alcoholic Cardiomyopathy Pharmaceutical Chemicals Antiarrhythmic Agents Inotropic Drugs Central Nervous System Acting Drugs Local Anesthetics Anthracyclines and Other Antineoplastic Agents Antimicrobial and Antiviral Agents Anti-Inflammatory Agents Antihistamines Immunosuppressants Miscellaneous Drugs Natural Products Catecholamines Steroids and Related Hormones Cytokines Animal and Plant Toxins Environmental Pollutants and Industrial Chemicals Particulate Matters Solvents Alcohols and Aldehydes Halogenated Alkanes Metals and Metalloids

CARDIAC TOXIC RESPONSES Basic Concepts and Definitions Cardiac Arrhythmia Cardiac Hypertrophy Heart Failure Acute Cardiac Toxicity Chronic Cardiac Toxicity Myocardial Degeneration and Regeneration Myocardial Degenerative Responses Toxic Effect on Myocardial Regeneration Myocardial Cell Death and Signaling Pathways Apoptosis and Necrosis Mitochondrial Control of Cell Death Death Receptors and Signaling Pathways Cardiac Hypertrophy and Heart Failure Adaptive and Maladaptive Responses Hypertrophic Signaling Pathways Transition from Cardiac Hypertrophy to Heart Failure QT prolongation and Sudden Cardiac Death Definition of QT Prolongation Molecular Basis of QT Prolongation Torsade De Pointes and Sudden Cardiac Death Parameters Affecting QT Prolongation and Torsadogenesis

OVERVIEW OF VASCULAR SYSTEM Vascular Physiology and Structural Features Arterial System and Physiological Function Capillaries and Microcirculation Venous System and Physiological Function Lymphatic System and Physiological Function Regulatory Mechanisms of the Vascular System Neurohormonal Regulation Local Metabolic Regulation VASCULAR SYSTEM TOXIC RESPONSES Mechanisms of Vascular Toxicity Responses of Vascular Endothelial Cells to Toxic Insults Responses of Smooth Muscle Cells to Toxic Insults Oxidative Stress and Vascular Injury Inflammatory Lesions

699 Copyright © 2008 by The McGraw-Hill Companies, Inc. Click here for terms of use.

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Toxic Responses of Blood Vessels Hypertension and Hypotension Atherosclerosis Hemorrhage Edema VASCULAR SYSTEM TOXIC CHEMICALS Pharmaceutical Chemicals Sympathomimetic Amines Nicotine Cocaine Psychotropic Agents Antineoplastic Agents Analgesics and Nonsteroidal Anti-Inflammatory Agents Oral Contraceptives

INTRODUCTION Cardiovascular toxicology is concerned with the adverse effects of extrinsic and intrinsic stresses on the heart and vascular system. Extrinsic stress involves exposure to therapeutic drugs, natural products, and environmental toxicants. Intrinsic stress refers to exposure to toxic metabolites derived from nontoxic compounds such as those found in food additives and supplements. The intrinsic exposures also include secondary neurohormonal disturbance such as overproduction of inflammatory cytokines derived from pressure overload of the heart and counter-regulatory responses to hypertension. These toxic exposures result in alterations in biochemical pathways, defects in cellular structure and function, and pathogenesis of the affected cardiovascular system. The manifestations of toxicologic response of the heart include cardiac arrhythmia, hypertrophy, and overt heart failure. The responses of the vascular system include changes in blood pressure and lesions in blood vessels in the form of atherosclerosis, hemorrhage, and edema. This chapter is divided into two parts: the heart and the vascular system. For a better understanding of the toxic manifestations of the cardiovascular system, an overview of the physiology and biochemistry of the heart and the vascular system is presented in relation to toxicologic concerns. The toxicologic responses of the heart and the vascular system and the mechanisms of these responses are the major focus of this chapter. This chapter also presents a brief discussion of chemicals that affect the heart and the vascular system.

OVERVIEW OF THE HEART Overview of Cardiac Structural and Physiological Features Cardiac muscle, along with nerve, skeletal muscle, and smooth muscle, is one of the excitable tissues of the body. It shares many bioelectrical properties with other excitable tissues, but also has unique features associated with cardiac structural and physiological specificities. Figure 18-1 illustrates the basic anatomy of the heart. With regard to cardiac toxicology, this section will only review some features of cardiac physiology and structures. There are many textbooks of cardiac anatomy and physiology that provide extensive knowledge basis of cardiac physiology and structural properties, which will not be repeated in this section.

Natural Products Bacterial Endotoxins Homocysteine Hydrazinobenzoic Acid T-2 Toxin Vitamin D β-Amyloid Environmental Pollutants and Industrial Chemicals Carbon Monoxide Carbon Disulfide 1,3-Butadiene Metals and Metalloids Aromatic Hydrocarbons Particulate Air Pollution

Review of Cardiac Structure The primary contractile unit within the heart is the cardiac muscle cell, or cardiac myocyte. Cardiac myocytes are composed of several major structural features and organelles, as illustrated in Fig. 18-2. A primary component is the contractile elements known as the myofibril. Each myofibril consists of a number of smaller filaments (the thick and thin myofilaments). The thick filaments are special assemblies of the protein myosin, whereas the thin filaments are made up primarily of the protein actin. Cardiac myosin is a hexamer composed of one pair of myosin heavy chains (MHCs) and two pairs of myosin light chains (MLC). Two isoforms of MHC, α and β, are expressed in cardiac muscle; the expression of these is under developmental control and may be altered by a variety of physiologic, pathologic, and pharmacologic stimuli (Martin et al., 1996; Metzger et al., 1999). In addition, the predominant isoform expressed in normal adult cardiac tissue also depends on the species examined. Similarly, two isoforms of actin are expressed in the heart (cardiac and skeletal α-actin), and, as with MHC, actin isoform expression is influenced by developmental, physiologic, pathologic, and pharmacologic stimuli, and the primary isoform of actin found in normal adult cardiac muscle also depends on the species examined. Under electron microscopy, these essential structural components of myocardial contractile proteins display alternating dark bands (A bands, predominantly composed of myosin) and light bands (I bands, predominantly composed of actin). Visible in the middle of the I band is a dense vertical Z line. The area between two Z lines is called a sarcomere, the fundamental unit of muscle contraction. Although cardiac and skeletal muscle share many similarities, a major difference lies in the organization of cardiac myocytes into a functional syncytium where cardiac myocytes are joined end-to-end by dense structures known as intercalated disks. Within these, there are tight gap junctions that facilitate action potential propagation and intercellular communication. About 50% of each cardiac myocyte is composed of myofibrils. The rest of the intracellular space contains the remaining major components of the cell: mitochondria (33%), one or more nuclei (5%), the sarcoplasmic reticulum (SR) (2%), lysosomes (very low), glycogen granules, a Golgi network, and cytosol (12%) (Opie, 1996). Cardiac myocyte is the largest cell in the heart and contributes to the majority of cardiac mass. However, cardiac myocytes make

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Figure 18-1. Diagram illustrating the basic anatomy of the heart.

up only about one-quarter of all the cells in the heart. Cardiac fibroblasts, vascular cells, Purkinje cells, and other connective tissue cells make up the majority of cell number in the heart. Cardiac fibroblasts make up approximately 90% of these “nonmuscle” cells. Cardiac myocytes are generally considered to be terminally differentiated, although this view has been challenged recently (Anversa et al., 2006). These cells may be multinucleated, but they may not divide after birth unless under certain circumstances in some species such as mice (Anversa et al., 2006). The heart undergoes a significant increase in size and mass throughout growth of the organism, but the increase in heart size and mass is produced by enlargement (or hypertrophy) of preexisting cardiac myocytes (Li et al., 1996). With regard to this developmental period, cardiac hypertrophy is considered to be a normal physiological process. Under pathological conditions, hypertrophy of remaining cardiac myocytes is a hallmark of cardiac remodeling following myocardial injuries, such as myocardial infarction. Cardiac fibroblasts may continue to proliferate after birth, particularly in response to injury. Cardiac fibroblasts also contribute to cardiac remodeling following myocardial infarction and are believed to promote fibrosis and scarring of injured cardiac tissue. Thus, from a toxicologic perspective, the heart is vulnerable to injury because of limited proliferative capacity of cardiac myocytes, and promotion of cardiac fibroblast proliferation and remodeling following injury. Electrophysiology The electrophysiology of the heart is concerned with bioelectricity and its related cardiac physiological function. Bioelectricity is the result of charge generated from the movement of positively and negatively charged ions in tissues. In cardiac myocytes, three major positively charged ions make a significant contribution to the bioelectricity of the heart; calcium (Ca2+ ), sodium (Na+ ), and potassium (K+ ). Each of the ions has specific channels and transporters (pumps) on the membrane of cardiac myocytes. Through the movement of these ions across the cell membrane, an action potential is generated and propagated from one cell to another, so that electric conductance is produced in the heart.

Figure 18-2. Structural organization of cardiac muscle tissue.

Action Potential Cardiac myocytes produce an action potential when activated. In the resting state, the resting potential of a myocyte is about −60 to −90 mV relative to the extracellular fluid potential. A sudden depolarization changes the membrane potential from negative inside to positive inside, followed by a repolarization

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Figure 18-3. Characteristic cardiac action potential recorded from sinoatrial node and Purkinje fibers as indicated. (From Berne RM, Levy MN (eds.), Physiology pp. 454–457, 1983. With permission from Elsevier.)

to reset the resting potential. The process of an action potential from depolarization to the completion of repolarization is divided into five phases in cardiac Purkinje fibers as shown in Fig. 18-3. Phase 0 represents a rapid depolarization due to the inward current of Na+ . Phase 1 is associated with an immediate rapid repolarization, during which the Na+ inward current is inactivated and a transient K+ outward current is activated, followed by an action potential plateau or phase 2, which is dominated by slowly decreasing inward Ca2+ current and a slow activation of an outward K+ current. Phase 3 reflects a fast K+ outward current and inactivation of the plateau Ca2+ inward current, and phase 4 is the diastolic interval for the resetting of resting potential. Automaticity A group of specialized cells in the heart are capable of repetitively spontaneous self-excitation, which generate and distribute each impulse through the heart in a highly coordinated manner to control the normal heart beat. These cells include the sinus node P cells and Purkinje fibers in the ventricles. Other cells such as atrial-specialized fibers under normal conditions do not have automaticity, but can become automatic under abnormal conditions. The sinus node P cells or pacemaker cells have only three distinct phases of action potential (Fig. 18-3): phase 0, rapid depolarization; phase 3, plateau and repolarization; and phase 4, slow depolarization or often referred to as pacemaker potential. It is the pacemaker potential that brings the membrane potential to a level near the threshold for activation of the inward Ca2+ current, which triggers the phase 0 rapid depolarization and makes the pacemaker cells of automaticity. In pacemaker cells, phase 0 is mediated almost entirely by increased conductance of Ca2+ ions. Contractility Cardiac myocytes like other muscle cells have a unique functional feature, contractility. Myocyte contraction occurs when an action potential causes the release of Ca2+ from the SR

as well as the entry of extracellular Ca2+ into the cell. This action potential-triggered Ca2+ increase in the plasma and myocyte contraction is called excitation–contraction coupling (Fig. 18-4). The increase in Ca2+ concentrations in the cell allows Ca2+ to bind to troponin and tropomysin leading to some conformational change in the contractile unit of the cardiac myocyte, thin filament. This conformational change permits interaction between the actin and myosin filaments through the crossbridges (myosin heads). ATP is hydrolyzed by ATPase present in the crossbridges to release energy for the movement of the crossbridges in a ratchet-like fashion. This action increases the overlap of the actin and myosin filaments, resulting in shortening of the sarcomeres and contraction of the myocardium. Electrotonic Cell-to-Cell Coupling Myocardium as a whole has to synchronize the contraction and relaxation of individual myocytes in order to perform its pump function. This is achieved by a special structural feature of cell-to-cell interaction, electrotonic cell-to-cell coupling via the gap junction. Through the gap junction, major ionic fluxes between adjacent cardiomyocytes are spread, thus allowing electrical synchronization of contraction. Each single gap junction is composed of 12 connexin 43 (Cx43) units, assembled in two hexametric connexons (hemichannels) that are contributed, one each, by the two participating cells. The connexins interact with other proteins within the cell so that connexons are not only important for cell-to-cell coupling, but also involved in cell signaling and volume regulation. An important feature of the connexon-controlled electrotonic cell-to-cell coupling is the electrotonic current flow that attenuates the differences in action potential duration of individual cardiac myocytes. Electrocardiograph The electrophysiological features of cardiac myocytes and the electrotonic cell-to-cell coupling give rise to

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Figure 18-4. Overview of excitation–contraction coupling in cardiac myocytes. Upon rapid depolarization (rapid influx of Na+ through fast channels; phase 0 of the action potential), L-type Ca2+ channels are opened allowing a slower but sustained influx of Ca2+ down the electrochemical gradient (phase 2 of the action potential). During the process of Ca2+ -induced Ca2+ release (CICR), slight elevation in intracellular-free Ca2+ stimulates Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR). The SR provides the majority of Ca2+ required for contraction. The mitochondria provide energy for contraction in the form of ATP. Repolarization of the cell occurs largely by activation of K+ channels and efflux of K+ (phase 3 of the action potential). For relaxation, the SR Ca2+ ATPase (SERCA) actively pumps Ca2+ back into the SR, although some Ca2+ may be removed by the Na+ /Ca2+ exchanger or by sarcolemmal Ca2+ pumps.

charge at any given locus in the heart with a magnitude and a direction. Therefore, at any given moment in the cardiac cycle, there is a complex pattern of electrical charges across the membranes of myriad cells in the heart. The sum of all the individual cells that exist at any given time within the heart is the resultant cardiac vector. The changes in the resultant cardiac vector throughout the cardiac cycle can be recorded as a vector cardiograph. Lead systems are used to record certain projections of the resultant cardiac vector. The potential difference between two recoding electrodes represents the projection of the vector on the line between the two leads. Components of vectors projected on such lines eliminate their directions and have a sum of magnitude, being scalar quantities. Thus, a recording of the changes with time in the potential differences between two points on the surface of the skin is the so-called scalar electrocardiograph. In general, the pattern of the scalar electrocardiograph consists of P, QRS, and T waves, as shown in Fig. 18-5. The PR interval is a measure of the time from the onset of atrial activation to the onset of ventricular activation. The QRS complex represents the conduction pathways through the ventricles. The ST segment is the interval during which the entire ventricular myocardium is depolarized, and lies on the isoelectric line under normal conditions. The QT interval is sometimes referred to as the period of “electrical systole” of the ventricles, and reflects the action potential duration. The QT interval prolongation is recognized as a major life-threatening factor of drug cardiac toxicity, which is brought about by a reduction of outward currents and/or enhanced inward currents during phase 2 and 3 of the action potential. Neurohormonal Regulation Although the heartbeat is governed by the automaticity of the sinus node P cells, neurohormonal regu-

R

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QRS Q-T Figure 18-5. A typical electrocardiogram (ECG) with the illustration of important deflections and intervals.

lation of cardiac electrophysiology and contraction controls cardiac function under normal and abnormal conditions. Toxicants often exert their effects on the cardiac system through interference with the neurohormonal regulation, thus this regulatory system is of significant relevance to cardiac toxicology. There are many neurohormonal systems that have significant impact on the heart. A detailed description of the regulatory system will be provided in the following sections in association with specific discussion of cardiac functional regulation, compensatory and maladaptive responses to toxic exposures.

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Overview of Cardiac Energy Metabolism and Biochemistry ATP and the Heart It is easy to understand that the need of energy for the heart is high. The chemical energy in the form of ATP is absolutely needed to support the systolic and diastolic work of the heart (Ventura-Clapier et al., 2004). In the heart, the primary ATP-utilizing reactions are catalyzed by actomyosin ATPase in the myofibril, the Ca2+ -ATPase in the SR, and the Na+ ,K+ -ATPase in the sarcolemma (Ingwall and Weiss, 2004). ATP is also needed for molecular synthesis and degradation in the heart as in other organ systems. ATP synthesis by oxidative phosphorylation in the mitochondria is usually sufficient to support the normal needs of the heart, even when the work output of the heart increases three- to fivefold (Ingwall and Weiss, 2004). In addition, the glycolytic pathway and the tricarboxylic acid cycle also make small contributions to ATP synthesis. The concentration of ATP does not define the energetic state of the heart. The amount of ATP made and used at any given time is many times greater than the size of the measurable ATP pool (Ingwall and Weiss, 2004). Thus, cardiac myocytes contain high concentrations of mitochondria, which ensure that ATP remains constant through oxidation of a variety of carbon-based fuels for ATP synthesis under different conditions. Phosphocreatine and the Heart A unique feature in energy metabolism of the heart is the use of energy reserve systems, such as phosphocreatine (PCr), to maintain a high phosphorylation potential to drive ATPase reactions under highly demanding conditions (Ingwall et al., 1985). PCr exists in the heart at twice the ATP concentration (Bittl and Ingwall, 1985). The enzyme creatine kinase (CK) transfers the phosphoryl group between ATP and PCr at a rate about 10 times faster than the rate of ATP synthesis by oxidative phosphorylation. The reaction catalyzed by CK is: PCr + ADP + H+ ↔ creatine + ATP. Under the conditions when ATP demand exceeds ATP supply, the use of PCr is a major pathway to maintain a constant supply of ATP. The CK reaction is also important to maintain low ADP and Pi concentrations, thereby retaining high phosphorylation potential (Saupe et al., 2000). Creatine is not made in the heart but accumulates against a large concentration gradient facilitated by a creatine transporter. In the normal heart, about two-thirds of the total creatine pool is phosphorylated through the CK reaction to form PCr (Wallimann et al., 1998; Neubauer et al., 1999). Metabolic Pathways The continuous synthesis of ATP via mitochondrial oxidative phosphorylation is mandatory for the work of the heart (Huss and Kelly, 2005). Under normal conditions, the oxidation of fatty acid (FA) is the major pathway, providing about 65% of the total energy demand. In contrast, the oxidation of glucose provides about 30% of the total energy demand (Shipp et al., 1961; Wisneski et al., 1987). In hypertrophic and failing hearts, there is a metabolic shift from FA- to glucose-dependent energy supply. Thus, decreased FA oxidation and increased glucose utilization in association with depressed FA deposition and increased glucose uptake are observed in hypertrophic and failing hearts (van Bilsen et al., 2004). This shift enhances the glycolytic pathway, and thus increases the anaerobic metabolism. However, it remains debated whether this metabolic shift to the so-called “fetal phenotype” is adaptive or maladaptive. It is important to note that the “shift” is only partial, and even when the proportion of ATP synthesized

from glucose increases many fold, aerobic metabolism still remains dominant (van Bilsen et al., 2004). With regard to the concern of cardiac toxicology, the metabolic shift is often observed with mitochondrial dysfunction. In response to toxic exposure, mitochondrial damage leads to impaired oxidative phsphorylation and a metabolic shift from aerobic to anaerobic and a reliance on glucose utilization. Calcium and Calcineurin The role of calcium in cardiac toxic responses has been extensively investigated. However, our understanding of the role of calcium in cardiac toxicity remains superficial. When carefully examining the current literature, one can find that there are very few mechanistic studies that specifically probe the role of calcium in cardiac toxicity, although numerous studies have implicated intracellular Ca2+ as a signal for cardiac responses to environmental toxic insults (Shier et al., 1992; Toraason et al., 1997; Buck et al., 1999). In response to myocardial stress by environmental toxic exposures, calcium concentrations are increased in the myocardial cells (Sleight, 1996). This is consistent with the speculation that Ca2+ coordinates physiological responses to stresses. There are many other speculations that are derived from the studies examining the role of calcium in toxicologic responses in other systems. The unique action of calcium in cardiac toxicity, however, has to be studied specifically. The role of calcium in mediating myocardial hypertrophic signals has been extensively studied (Stemmer and Klee, 1994). A sustained increase in intracellular Ca2+ concentrations activates calcineurin. Calcineurin is a ubiquitously expressed serine/threonine phosphatase that exists as a heterodimer, comprises a 59-kDa calmodulin (CaM)-binding catalytic A subunit and a 19-kDa Ca2+ binding regulatory B subunit (Molkentin et al., 1998). Activation of calcineurin is mediated by binding of Ca2+ to the regulatory subunit and CaM to the catalytic subunit (Fig. 18-6). Of toxicologic relevance is that calcineurin is activated by a sustained increase in Ca2+ concentration and is insensitive to transient Ca2+ fluxes, such as in response to cardiomyocyte contraction (Stemmer and Klee, 1994). Numerous studies have demonstrated important roles for Ras, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways in myocardial responses to hypertrophic stimuli (Jalili et al., 1999). All of these signal transduction pathways are associated with increase in intracellular Ca2+ concentrations (Ho et al., 1998). The coordinating role of calcium in cardiac hypertrophic response has been demonstrated (Stemmer and Klee, 1994) as follows. Hypertrophic stimuli, such as angiotensin II and phenylephrine, cause an elevation of intracellular Ca2+ that results in activation of calcineurin. A series of reactions occur through activated calcineurin, including dephosphorylation of nuclear factor of activated T-cell (NFAT) and its translocation to nucleus, where it interacts with GATA4 (Fig. 18-6). Calcineurin also acts through an NFAT-independent mechanism to regulate hypertrophic gene expression. AMP-Activated Protein Kinase Activation of AMP-activated protein kinase (AMPK) often occurs when the myocardial metabolic phenotype shifts to the fetal form. Activation of AMPK occurs with changes in high-energy phosphate metabolism in hypertrophic and failing hearts. The increase in AMP/ATP ratio occurs when the PCr/ATP ratio decreases due to a decrease in PCr, with or without a concomitant decrease in ATP. The decrease in PCr/ATP ratio is an index of decreased energy reserve and correlates with the severity of heart failure and is of prognostic value (Neubauer et al., 1997).

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MAPKKK

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c-jun ATF2 MEF2C APK2/3

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NFAT

Hsp25/27 Figure 18-7. Schematic representation of the hierarchy constituents of the MAPK signaling pathways. MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase.

Figure 18-6. Calcineurin signal transduction pathways in regulation of transcription factors involved in hypertrophic growth of cardiac myocytes. Sustained increases in intracellular Ca2+ concentrations, along with calmodulin (CaM), activate calcineurin, which in turn causes dephosphorylation of nuclear factor of activated T cells (NFAT), enabling NFAT to translocate to the nucleus where it interacts with GATA4 to regulate gene expression. Phosphorylation of NFAT is stimulated by glycogen synthase kinase 3β (GSK3β), whose activity is inhibited by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Calcineurin also regulates CaM activation of CaM-dependent kinase (CaMK), which activates GATA transcription factor. CaMK also phosphorylates histone deacetylases (HDAC), leading to HDAC translocation from nucleus to cytoplasm. Otherwise, HDAC in the nucleus inhibits myocyte-enhancer factor 2 (MEF2) transcription activity. Cyclosporine A (CsA) and modulatory calcineurin-interacting protein (MCIP) inhibit calcineurin activation.

Activation of AMPK leads to translocation of the insulin-dependent glucose transporter (GLUT4) from intracellular stores to the sarcolemma (Russell et al., 1999). Mice overexpressing an active form of AMPK suffer from pathological cardiac glucogen accumulation (Arad et al., 2003). Furthermore, the AMPK-dependent phosphorylation of the enzyme 6-phosphofructo-2-kinase stimulates glycolysis (Marsin et al., 2000). These pathways indicate the importance of AMPK activation for the cardiac metabolic shift to the energy supply reliance on glucose metabolism. Mitogen-Activated Protein Kinases MAPKs play a major role in cardiac response to toxic insults. A generalized diagram for MAPK signaling is presented in Fig. 18-7. The MAPKs consist of a series of successively acting kinases and three major branches are involved in the classic MAKP signaling pathway. These branches are divided based on their terminal effector kinases: the extracellular signal-regulated protein kniases (ERK), the c-jun NH2-terminal kinases (JNK), and p38 MAPKs (Sugden and Clerk, 1998). Each branch of the MAPKs has a hierarchy control system beginning at the MAPK kinase kinase (MAPKKK), as shown in Fig. 18-7. Among the MAPKs, p38 MAPKs have been extensively studied in myocardial apoptosis. This subfamily consists of p38α, p38β, p38γ, and p38δ (Sugden and Clerk, 1998). Several studies have identified p38 MAPKs as an important group of signaling molecules that mediate environmental stress responses in various cell types (Tibbles and Woodgett, 1999). In noncardiac cells, p38 MAPKs have been implicated in gene expression, morphological changes, and cell death in response to endotoxin, cytokines, physical stress, and chemical

insults (Tan et al., 1996; Wang and Ron, 1996). In cardiac cells, p38 MAPKs are associated with the onset of apoptosis in ischemia– reperfusion (Yin et al., 1997). In particular, transfection experiments using primary cultures of neonatal rat cardiomyocytes have shown that p38α is critically involved in myocyte apoptosis (Wang et al., 1998). Adriamycin is a cardiac toxicant and two observations indicate that the p38 MAPK is involved in Adriamycin-induced myocyte apoptosis (Kang et al., 2000a). First, a time–course analysis revealed that p38 MAPK activation preceded the onset of apoptosis. As early as 30 minutes after Adriamycin administration, myocyte apoptosis occurs, detected by a sensitive method of fluorescein isothiocyanate (FITC) conjugation of Annexin V (Annexin V-FITC). However, p38 MAPK activation detected by the FITC-conjugated anti-phosphop38 antibody and confocal microscopy is observed 20 minutes after Adriamycin treatment (Kang et al., 2000a). Second, SB203580, a specific inhibitor of p38 MAPKs, inhibits Adriamycin-induced myocyte apoptosis (Kang et al., 2000a). Because SB203580 acts as a specific inhibitor of p38α and p38β, but not p38γ and p38δ, the involvement of the former specific isoforms of the p38 MAPK in the Adriamycin-induced myocyte apoptosis are implicated. p38α is involved in apoptosis of neonatal rat cardiomyocytes in primary cultures and p38β mediates hypertrophy of these cells (Wang et al., 1998). Protein Kinase C PKC is among the most extensively studied signaling molecules in the heart. Many cardiac toxicologic studies have examined the role of PKC in mediating toxic signals. Several excellent reviews on PKC in myocardial signaling pathways leading to cardiac hypertrophy and heart failure are available (Puceat and Vassort, 1996). PKC is a ubiquitously expressed serine/threonine kinase, which is activated predominantly by Gq /G11 -coupled receptors. The PKC family consists of 11 isoforms, which are divided into three subgroups: conventional PKCs (cPKCs) including α, β (I and II), and γ, novel PKCs (nPKCs) including ε, δ, η, ζ, and θ, and atypical PKCs (aPKCs) including ι, λ, and μ (Newton, 1995). The cPKCs are activated by Ca2+ and diacylglycerol (DAG), as well as phorbol esters. The nPKCs do not bind Ca2+ , but respond to DAG and phorbol ester stimulation. The aPKCs do not respond to either Ca2+ , DAG, or phorbol esters. PKC has been demonstrated to participate in the regulation of transcription, the maintenance of cell growth and membrane structure, and modulation of immune responses. Disturbances in PKC signaling pathways

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lead to cardiac hypertrophy and heart failure, which is of toxicologic significance.

interacts with GATA4 (Fig. 18-6). NFAT3 can also activate some hypertrophic responsive genes through mechanisms independent of GATA4.

Transcription Factors Transcription factors activate or deactivate myocardial gene expression, which affects the function and phenotype of the heart. Many transcription factors have been studied in myocardial tissue. Several of them are of toxicologic significance as described below:

GATA GATA factors are a family of nuclear transcriptional regulatory proteins that are related structurally within a central DNAbinding domain, but are restricted in expression to distinct sets of cell types (Yamamoto et al., 1990). Currently, six different family members have been characterized in vertebrate species. They are GATA1, 2, 3, 4, 5, and 6. Each protein contains two similar repeats of a highly conserved zinc finger of the form CXNCX6 LWRRX7 CNAC. The c-terminal repeat constitutes a minimal DNA-binding domain sufficient for sequence-specific recognition of a “GATA” cis-element, usually (A/T)GATA(A/G) or a related DNA sequence, present in promoters and/or enhancers of target genes (Evans et al., 1988). It has been shown that GATA-1/2/3 regulate various aspects of hematopoiesis (Orkin, 1992), whereas GATA 4/5/6 regulates cardiogenesis (Yamamoto et al., 1990). The significance of GATA4 in regulation of hypertrophic response in myocardial cells has been demonstrated (Evens, 1997). Cardiac hypertrophy induced by angiotensin II is mediated by an angiotensin II type1α receptor (AT1α R). A GATA motif exists in the AT1α R promoter. Mutations introduced to the consensus-binding site for GATA factor abolished the pressure overload response (Evens, 1997). Moreover, interactions between AP-1 and GATA-4, and between NFAT3 and GATA-4 are essential in myocardial hypertrophic responses.

Activator Protein-1 AP-1 is a transcription factor composed of Jun and Fos gene family members (McMahon and Monroe, 1992). The AP-1-binding site is the TRE (12-O-tetradecanoyl phorbol 13-acetate response element), and the binding of AP-1 to the TRE initiates transcription of the target genes (Diamond et al., 1990). Elevated levels of c-Jun are seen in cardiomyocytes with ischemia– reperfusion (Brand et al., 1992). In volume-overload hypertrophy, AP-1 plays an important role in the regulation of Fas and FasL activities (Wollert et al., 2000). Over-stretching of myocardium induces Fas expression (Cheng et al., 1995). Subsequently, Fas-dependent signaling pathways can lead to myocardial cell apoptosis. However, there are other studies that indicate activation of AP-1 is not associated with the induction of apoptosis (Lenczowski et al., 1997). AP-1 has been implicated in transcriptional regulation of several genes associated with a hypertrophic response (Paradis et al., 1996). Myocyte-Enhancer Factor-2 MEF-2 is a transcription factor that binds to A-/T-rich DNA sequences within the promoter regions of a number of cardiac genes, including muscle CK gene, β-MHC, MLC1/3, MLC2v, skeletal α-actin, SR Ca2+ -ATPase, cardiac troponin T, C, and I, desmin, and dystrophin (Black and Olsen, 1998). MEF2 is critically involved in the regulation of inducible gene expression during myocardial hypertrophy. The activation of MEF2 involves phosphorylation of the transcription factor by p38 MAPK or ERK5-MAPK. The ERK5-MEF2 pathway has been observed in the generation of cardiac hypertrophy. An important function of MEF2 is the convergence in the binary downstream pathway of Ca2+ signaling. Increased intracellular Ca2+ binds to and activates Ca2+ binding proteins including CaM, which regulates calcineurin and Ca2+ /CaM-dependent protein kinase (CaMKs). Activation of either calcineurin or CaMKs induces cardiac hypertrophy. CaMKs stimulate MEF2 through phosphorylation of the transcriptional suppressor, histone deacetylases (HDACs). CaMK is considered as HDAC kinase whose activity is enhanced by calcineurin. Thus, MEF2 converges the stimulating signaling of both CaMKs and calcineurin leading to activation of hypertrophic gene expression (Fig. 18-6). Nuclear Factor of Activated T Cells 3 NFAT3 is a member of a multigene family that contains four members, NFATc, NFATp, NFAT3, and NFAT4 (Rao et al., 1997). These transcription factors bind to the consensus DNA sequence GGAAAAT as monomers or dimers through a Rel homology domain (Rooney et al., 1994). Unlike the other three members that are restricted in their expression to T cells and skeletal muscle, NFAT3 is expressed in a variety of tissues including the heart. NFAT3 plays a major role in cardiac hypertrophy (Pu et al., 2003). Hypertrophic stimuli, such as angiotensin II and phenylephrine, cause an increase in intracellular Ca2+ levels in myocardial cells. This elevation in turn results in activation of calcineurin. NFAT3 is localized within the cytoplasm and is dephosphorylated by the activated calcineurin. This dephosphorylation enables NFAT3 to translocate to the nucleus where it

CARDIAC TOXIC RESPONSES Basic Concepts and Definitions The ultimate functional effect of cardiac toxic manifestations is decreased cardiac output and peripheral tissue hypoperfusion, resulting from alterations in biochemical pathways, energy metabolism, cellular structural and function, electrophysiology, and contractility of the heart. These morphological and functional alterations induced by toxic exposure are referred to as toxicologic cardiomyopathy. The critical cellular event leading to toxicologic cardiomyopathy is myocardial cell death and extracellular matrix (ECM) remodeling. The recognition of the role of apoptosis in the development of heart failure during the last decade has significantly enhanced our knowledge of myocardial cell death (James, 1994; Haunstetter and Izumo, 1998; Sabbah and Sharov, 1998). Manipulation of genes responsible for cardiac function began in the mid-1990s (Robbins, 2004). The most important conclusion of these studies is that a sustained expression of any single mutated functional gene, either in the form of gain-of-function or loss-offunction, can lead to a significant phenotype, often in the form of cardiac hypertrophy and heart failure (Robbins, 2004; Olson, 2004). However, it is difficult to apply this knowledge to patients: first, acquired cardiac disease such as heart failure is the result of interaction between environmental factors and genetic susceptibility, indicating the role of polymorphisms. Second, extrinsic and intrinsic stresses produce lesions that cannot be explained by a single gene or a single pathway, suggesting complexity between deleterious factors and the heart. Cardiac toxicity is the critical link between environmental factors and myocardial pathogenesis. For a better understanding of cardiac toxicology, a triangle model of cardiac toxicity is presented in Fig. 18-8. In this model, complexity of the interaction between environmental stresses and the heart, and the balance between myocardial protection and

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Drugs or Xenobiotics Ca2+ Fetal gene expression

Heart Hypertrophy

Apoptosis

Cell death

Survival (gp130)

Dilation ANP, ET-1, TNF Apoptosis

Dilation

Heart Failure

Figure 18-8. Triangle analytical model of cardiac responses to drugs and xenobiotics. Drugs or xenobiotics can directly cause both heart failure and heart hypertrophy. Under severe acute toxic insults, myocardial cell death becomes the predominant response leading to cardiac dilation and heart failure. In most cases, myocardial survival mechanisms can be activated so that myocardial apoptosis is inhibited. The survived cardiomyocytes often become hypertrophy through activation of calcium-mediated fetal gene expression and other hypertrophic program. If toxic insult continues, the counter-regulatory mechanisms against heart hypertrophy such as activation of cytokine-medicated pathways eventually lead to myocardial cell death through apoptosis or necrosis, dilated cardiomyopathy, and heart failure.

deleterious dose and time effects are considered. First, it is important to recognize that chemicals can lead to heart failure without heart hypertrophy. Second, a chemical can lead to activation of both protective and destructive responses in the myocardium. Third, longterm toxicologic responses often result in maladaptive hypertrophy, which primes the heart for malignant arrhythmia, leading to sudden death or transition to heart failure. In the study of cardiac toxicology, the manifestations of cardiac toxicity in human patients and animal models are critical parameters serving as indices of cardiac toxicity. These manifestations are expressed in the forms of cardiac arrhythmia, hypertrophy, and heart failure. These abnormal changes reflect myocardial functional alterations resulting from both acute and chronic cardiac toxicity. Although some changes, such as cardiac hypertrophy, was viewed as a compensatory response to hemodynamic changes in the past, more recent studies suggest that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses (van Empel et al., 2004; Berenji et al., 2005; Dorn and Force, 2005). Cardiac hypertrophy is a risk factor for sudden cardiac death and has a high potential to progress to overt heart failure. Therefore, a distinction between compensatory and maladaptive responses is critical for treatment of patients with toxicologic cardiomyopathy. Cardiac Arrhythmia Cardiac rhythms under physiological conditions are set by pacemaker cells that are normally capable of developing spontaneous depolarization and responsible for generating the cardiac rhythm, the so-called automatic rhythm. A cardiac rhythm that deviates from the normal automatic rhythm is called cardiac arrhythmia, often manifested in the form of tachycardia (fast heart rate). There are several classes of tachycardia, including sinus tachycardia, atrial tachycardia, ventricular tachycardia, and torsade de pointes (TdP) (a life-threatening ventricular tachycardia). In addition, subclasses such as atrial fibrillation, atrial flutter, and accelerated idioventricular rhythm provide further description of the manifestations of arrhythmia. Mechanisms for different classes

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of arrhythmia will be discussed in the section of QT-prolongation and sudden cardiac death. According to the cause of the tachycardia, it is divided into abnormal automatic arrhythmia and triggered arrhythmia, which will be discussed in other sections. Cardiac Hypertrophy There are two basic forms of cardiac hypertrophy: concentric hypertrophy, which is often observed during pressure overload and is characterized by new contractile-protein units assembled in parallel resulting in a relative increase in the width of individual cardiac myocytes (De Simone, 2003). By contrast, eccentric hypertrophy is characterized by the assembly of new contractile-protein units in series resulting in a relatively greater increase in the length than in the width of individual myocytes, occurring in human patients and animal models with dilated cardiomyopathy (Kass et al., 2004). Toxicologic cardiomyopathy is often manifested in the form of eccentric hypertrophy. The development of cardiac hypertrophy can be divided into three stages: Developing hypertrophy, during which period the cardiac workload exceeds cardiac output; compensatory hypertrophy, in which the workload/mass ratio is normalized and normal cardiac output is maintained; decompensatory hypertrophy, in which ventricular dilation develops and cardiac output progressively declines, and overt heart failure occurs (Richey and Brown, 1998). Heart Failure A traditional definition of heart failure is the inability of the heart to maintain cardiac output sufficient to meet the metabolic and oxygen demands of peripheral tissues. This definition has been modified recently to include changes in systolic and diastolic function that reflect specific alterations in ventricular function and abnormalities in a variety of subcellular processes (Piano et al., 1998). Therefore, a detailed analysis to distinguish right ventricular from left ventricular failure can provide a better understanding of the nature of the heart failure and predicting the prognosis. Acute Cardiac Toxicity Acute cardiac toxicity is referred to as cardiac response to a single exposure to a high dose of cardiac toxic chemicals. It is often manifested by cardiac arrhythmia. However, myocardial apoptosis is also involved in acute cardiac toxicity. It is not difficult to define acute cardiac toxicity; however, it sometimes is technically difficult to measure acute cardiac toxicity. In particular, the impact of acute cardiac toxicity on the ultimate outcome of cardiac function is not often easily recognized. For instance, a single high dose of arsenic can lead to cardiac arrhythmia and sudden cardiac death, which is easy to measure (Goldsmith and From, 1980). However, that a single oral dose of monensin (20 mg/kg) leads to a diminished cardiac function progressing to heart failure in calves requires a long-term observation; often a few months for clinical signs of heart failure (van Vleet, et al., 1983; Litwak et al., 2005), which is difficult to measure. As shown in Fig. 18-8, toxic exposure can directly lead to heart failure, which is different from an often-observed hypertrophic response, which may or may not progress to heart failure. Chronic Cardiac Toxicity Chronic cardiac toxicity is the cardiac response to long-term exposure to chemicals, which is often manifested by cardiac hypertrophy and the transition to heart failure. About 25% of human patients with cardiomyopathy are categorized as having idiopathic cardiomyopathy. At least a portion of these patients with idiopathic cardiomyopathy are due to chemical exposure. Environmental exposure to particulate matter (PMs) in the air

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can lead to cardiomyopathy, which has only been recognized recently (Dockery, 2001; Gordon and Reinman, 2002). Recognition of chronic cardiac toxicity in the pathogenesis of cardiomyopathy is of clinical relevance, and this knowledge can be used to prevent and treat patients with toxicologic cardiomyopathy.

Myocardial Degeneration and Regeneration Myocardial degeneration is the ultimate response of the heart to toxic exposure, which can be measured by both morphological and functional degenerative phenotypes. However, myocardial degeneration should not be considered an irreversible toxic response. In the past, the heart has been considered incapable of regenerating, so that cardiac injury in the form of cell loss or scar tissue formation was considered permanent damage to the heart. However, evidence now indicates myocardial regeneration and recovery from cardiomyopathy. Cardiac toxic responses or damage are now divided into reversible and irreversible. Myocardial Degenerative Responses Myocardial cell death, fibrosis (scar tissue formation), and contractile dysfunction are considered as degenerative responses, which can result in cardiac arrhythmia, hypertrophy, and heart failure. If acute cardiac toxicity does not affect the capacity of myocardial regeneration, the degenerative phenotype is reversible. Both acute and chronic toxic stresses can lead to irreversible degeneration, depending on whether or not the cardiac repair mechanisms are overwhelmed. Cell death is the most common phenotype of myocardial degeneration. Both apoptosis and necrosis occur in the process of myocardial cell death, which will be discussed in the next section. Myocardial cell death is accompanied by hypertrophy of the remaining cardiac myocytes so that in the hypertrophic heart, the total number of cardiac myocytes is reduced but the size or volume of individual cells is increased. During myocardial remodeling after cell death, not only is there an increase in the size of cardiac myocytes, but also cardiac fibrosis occurs. Myocardial fibrosis results from excess accumulation of ECM, which is mainly composed of collagens. The net accumulation of ECM connective tissue results from enhanced synthesis or diminished break down of the matrix, or both. Collagen, predominately type I and III, are the major fibrous proteins in ECM and their synthesis may increase in response to toxic insults. The degradation of ECM is dependent on the activity of matrix metalloproteinases (MMPs). According to their substrate specificity, MMPs fall into five categories: collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-7, MMP-10, and MMP-11), membrane-type MMPs (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, and MMP-25), and metalloelastase (MMP-12). These MMPs are organ specific so that not all are present in the heart. The activities of these enzymes are altered during the processes of fibrogenesis and fibrinolysis. Under toxic stress condition, the imbalance between fibrogenesis and fibrinolysis leads to enhanced fibrogenesis and excess collagen accumulation— fibrosis. Toxic Effect on Myocardial Regeneration The mainstay of cardiac medicine and therapy has centered on the concept that the heart is a terminally differentiated organ and that cardiac myocytes are incapable of proliferating. Thus, cell death would lead to a permanent loss of the total number of cardiac myocytes. However, this view has been challenged recently due to the identification of cardiac

progenitor cells (Anversa et al., 2006). These cells are characterized and proposed to be responsible for cardiac repair because these cells can make myocytes and vascular structures. These cells possess the fundamental properties of stem cells, therefore, they are also called cardiac stem cells. They are self-renewing, clonogenic, and multipotent, as demonstrated by reconstitution of infarcted heart by intramyocardial injection of cardiac progenitor cells or the local activation of these cells by growth factors. It is important to note that toxicologic studies of the cardiac progenitor cells have not been done and it is important to determine the potential of cardiac stem cells to help recover from toxic insults. The effect of chemicals on the cardiac progenitor cells is unknown. One speculation is that when severe damage to cardiac progenitor cells occurs, the potential for recovery from severe cardiac injury would be limited. The removal of scar tissue or fibrosis in the myocardium in the past has been considered impossible. Although there are no studies that have shown scar tissue is removable, there are observations in animal models of hypertensive heart disease that myocardial fibrosis is recoverable (Weber, 2005). Myocardial vascularization is required for myocardial regeneration. Many toxic insults affect the capacity of angiogenesis in the myocardium, so that cardiac ischemia occurs. The combination of cardiac ischemia and the direct toxic insults to cardiomyocytes constitute synergistic damage to the heart. During regeneration, coronary arterioles and capillary structures are formed to bridge the dead tissue (scar tissue) and supply nutrients for the survival of the regenerated cardiomyocytes. There is an orderly organization of myocytes within the myocardium and a well-defined relationship between the myocytes and the capillary network. This proportion is altered under cardiac toxic conditions; either toxicologic hypertrophy or diminished capillary formation can lead to hypoperfusion of myocytes in the myocardium. Unfortunately, our understanding of the toxic effects on myocardial angiogenesis is limited. Reversible and irreversible toxic response. Cardiomyopathy was viewed not to be recoverable in the past, but there is cumulative evidence that demonstrates reversibility of cardiomyopathy. The issue related to whether or not toxicologic cardiac lesions are reversible has not been explored. However, it can be speculated that there would be reversible and irreversible manifestations of the cardiac response to toxic insults.

Myocardial Cell Death and Signaling Pathways Apoptosis and Necrosis Toxic insults trigger a series of reactions in cardiac cells leading to measurable changes. Mild injuries can be repaired. However, severe injuries will lead to cell death in the modes of apoptosis and necrosis. If the cell survives the insults, structural and functional adaptations will take place. Apoptosis was found to be involved in cardiomyopathy in 1994 (Gottlieb et al., 1994). The loss of cardiac myocytes is a fundamental part of myocardial injury, which initiates or aggravates cardiomyopathy. An important mode of myocardial cell loss is apoptosis, which has been demonstrated in heart failure patients (Olivetti et al., 1997). Myocardial apoptosis has been shown to play an important role in cardiac toxic effects induced by Adriamycin (Kang et al., 2000a; Wang et al., 2001a), an important anticancer drug whose clinical application is limited by its major toxic effect, cardiotoxicity. Exposure of primary cultures of cardiomyocytes to cadmium also induces apoptosis (El-Sherif et al., 2000). Many in vivo studies have shown that only a very small percentage of myocardial cell populations undergo apoptosis under

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pathological conditions. For example, less than 0.5% of cells appeared apoptotic in myocardial tissue under the stress of dietary copper deficiency in mice (Kang et al., 2000b). At first glance, this number seems to be too insignificant to account for myocardial pathogenesis. In a carefully designed time–course study (Kajstura et al., 1996), it was estimated that cardiomyocyte apoptosis is completed in less than 20 hours in rats. Myocytes that undergo apoptosis are lost and may not be replaced under toxicologic conditions. Although the possibility of myocardial regeneration has been identified (Anversa et al., 2006), xenobiotics often cause degenerative effect through apoptosis as well as inhibitory effect on regeneration. Adriamycin-induced cardiomyopathy is a good example for the pathogenesis resulting from both degeneration and inhibition of regeneration. If apoptosis occurs at a constant rate of about 0.5% myocytes a day (Kang et al., 2000b), the potential contribution of apoptosis to the overall loss of myocytes over a long period of time is significant under Adrimaycin toxic exposure. Necrosis is a term that had been widely used to describe myocardial cell death in the past. Myocardial infarction, in particular, had been considered as a consequence of necrosis (Eliot et al., 1977). It is now recognized that apoptosis contributes significantly to myocardial infarction (Yaoita et al., 2000). However, the importance of necrosis in myocardial pathogenesis cannot be underestimated. The contribution of necrosis to cardiomyopathy induced by environmental toxicants and pollutants is particularly important. A critical issue is how to distinguish apoptosis from necrosis. Apoptosis and necrosis were originally described as two distinct forms of cell death that can be clearly distinguished (Wyllie, 1994). However, these two modes of cell death can occur simultaneously in tissues and cultured cells. The intensity and duration of insults may determine the outcome. Triggering events can be common for both types of cell death. A downstream controller, however, may direct cells toward a programmed execution of apoptosis. If the apoptotic program is aborted before this control point and the initiating stimulus is severe, cell death may occur by necrosis (Leist et al., 1997). To distinguish apoptosis from necrosis, specific oligonucleotide probes have been developed to recognize different aspects of DNA damage (Didenko et al., 1998), and have been successfully applied, in combination with confocal microscopy, to identify apoptotic and necrotic cell death in the heart with different pathogenic challenges. Single-Strand DNA Breaks A monoclonal mouse anti-ssDNA antibody has been developed that is specifically reactive with ssDNA, but does not recognize dsDNA. An immunohistochemical assay for detection of ssDNA using this antibody in combination with a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling(TUNEL) assay can distinguish repairable ssDNA breaks from apoptotic DNA damage in the heart. Apoptotic DNA damage produces end products that are fragments of double-strand DNA cleavage with 3 overhang (Didenko et al., 1998), which can be specifically identified by Tag polymerasegenerated probe (Didenko et al., 1998). The specificity of this molecular probe to identify apoptosis has been confirmed by other methods such as dual labeling of TdT and caspase-3 (Frustaci et al., 2000). In addition, this apoptotic specific probe in combination with fluorescence labeling of different cellular components allows quantitative detection of apoptotic cells, with the possibility of identifying the origin of the apoptotic cells, such as myocytes (stained with α-sarcomeric actin), endothelial cells (stained with factor VIII),

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and fibroblasts (stained with vimentin) in the heart (Anversa, 2000). Necrotic DNA damage is characterized by double-strand DNA cleavage with blunt ends. That is because during necrosis, the release of lysosomal proteases degrades histones, resulting in loss of DNA protection and exposure to endonucleases and exonucleases. Endonucleases produce double-strand DNA cleavage with 3 overhangs, but exonucleases remove terminal nucleotides, leading to a bunt end of the damaged DNA. A probe generated by pfu polymerase can specifically recognize these blunt-end DNAs (Anversa, 2000). Its specific reaction with necrotic DNA has been confirmed by other methods such as the permeability of myosin antibody into necrotic cells (Guerra et al., 1999), and the disruption of the sarcolemma by vinculin staining, which can clearly define the continuity of the sarcolemmal surface (Yamashita et al., 2001). Proportion of apoptotic and necrotic cell death in the heart can be estimated by the combination of the above procedures. First, a conventional TUNEL procedure can be used to identify the total TUNEL-positive cells. Second, the procedure to define doublestrand DNA breaks with blunt ends can be used to quantify the proportion of necrotic cells in the total TUNEL-positive population. Finally, the combination of the procedure to identify double-strand DNA breaks with 3 overhangs, and the specific antibody to identify total ssDNA breaks can distinguish the proportion of apoptotic cells from those with ssDNA breaks only. Distinguishing apoptotic myocytes from non-myocytes in the myocardium is another problem to overcome. An in situ TUNEL assay in combination with a dual immunohistochemical detection of α-sarcomeric actin has been used to distinguish apoptotic myocytes from non-myocytes (Kang et al., 2000a). Apoptotic myocytes are dually stained by TUNEL and α-sarcomeric actin, and apoptotic non-myocytes are stained only by TUNEL. Another procedure is immuno-gold TUNEL and electron microscopic examination of the apoptotic cells (Kang et al., 2000a). The gold standard for identification of apoptotic cells is morphological examination by electron microscopy. The immuno-gold TUNEL and electron microscopic procedure defines cell type and morphological characteristics of apoptotic cells.

Mitochondrial Control of Cell Death The role of mitochondria in myocardial response to toxicants as well as therapeutic drugs has long been a focus of investigation. Mitochondrial control of cell death is an important topic of apoptotic research during the last decade. Factors affecting mitochondrial control of cell death is presented in Fig. 18-9. These factors have the same target effect: modification of mitochondrial permeability transition (MPT). Mitochondrial permeability transition occurs under toxic insults (Kroemer et al., 1997). This MPT behaves like a membrane pore that allows diffusion of solutes 90 g/d of alcohol for more than 5 years (Lazarevic et al., 2000). For the symptomatic ACM, some limited data have shown that more than 10 years of excessive alcohol consumption in alcoholics produces congestive heart failure (Mathews et al., 1981). The pathogenesis of heart failure begins after an index event such as alcohol-induced cardiac muscle injury that produces an initial decline in pumping capacity of the heart. Following this initial decline, a variety of compensatory mechanisms are activated, including the adrenergic nervous system, the renin–angiotensin

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system, and the cytokine system. Some of these compensatory changes have been detected in alcoholic patients (Adams and Hirst, 1986). However, with time, the sustained activation of these systems can lead to secondary end-organ damage within the ventricle by activating and accelerating the left ventricle remodeling and subsequent cardiac decompensation, resulting in the transition from asymptomatic to symptomatic heart failure (Mathews et al., 1981). It was proposed that the metabolite acetaldehyde is responsible for some of the cardiac injury associated with ethanol consumption. The metabolic enzyme responsible for the conversion of ethanol to acetaldehyde is alcohol dehydrogenase, which is absent in cardiac myocytes. Studies have indicated that the impaired liver function of alcoholics may be sufficient to generate quantities of acetaldehyde that can reach the heart. The direct effects of acetaldehyde on the myocardium include inhibition of protein synthesis, inhibition of Ca2+ sequestration by the SR, alterations in mitochondrial respiration, and disturbances in the association of actin and myosin. The exact mechanism of ACM is unresolved. It has been suggested that a combination of multiple factors is involved, including malnutrition, cigarette smoking, systemic hypertension, and beverage additives, in addition to a long-term consumption of alcohol in the ACM patients (Ahmed and Regan, 1992). The generation of reactive oxidative metabolites from the biotransformation of ethanol has been suggested to be a major contributing factor for ACM, because these metabolites lead to lipid peroxidation of cardiac myocytes or oxidation of cytosolic and membraneous protein thiols (Ribiere et al., 1992; Kannan et al., 2004). Most experimental approaches involve alcohol-containing liquid diet feeding to rodent models of ACM for several weeks to several months. However, a key factor for the development of ACM in humans is the duration of excessive consumption of alcohol. The simulation of daily excessive amount of alcohol consumption in rodents without disturbances in the food intake to produce nutritional deficiency is a constant challenge. One of the difficulties in using rodent models is that the short life span of the animals does not allow a sufficient long period of alcohol exposure to produce some of the critical pathological changes such as myocardial fibrosis observed in humans. A recent study using a mouse model in which alcohol-induced heart hypertrophy and fibrosis were all produced, may have been a breakthrough. In this mouse model, a zinc-regulatory protein, metallothionein (MT), is genetically deleted and when zinc was exogenously added to the alcohol-containing liquid diet, alcohol-induced cardiac fibrosis, but not heart hypertrophy, in the MT knockout (MTKO) mice was prevented (Wang et al., 2005). Zinc deficiency is an important feature in alcoholic patients and animal models (McClain and Su, 1983; Bogden et al., 1984). Zinc homeostasis within the cell is dependant on MT, which under physiological conditions binds 7 atoms of zinc (Kagi, 1991). The role of MT in the regulation of zinc homeostasis has been revealed only recently (Maret, 2000; Kang, 2006). The most important feature of MT is that under oxidative stress conditions, it releases zinc (Maret, 2000; Kang, 2006). In the MT-KO mice, alcohol-induced myocardial pathological changes are either accelerated or severely altered relative to those in WT mice. These pathological changes, in particular the myocardial fibrosis, resemble the pathology observed in patients with ACM. There are several interesting clues that have been provided from the MT-KO mouse studies. First, the link between the deficiency in endogenously stored zinc due to the lack of MT and the alcohol-induced myocardial fibrosis is suggested by the fact that supplementation with zinc inhibits alcoholic myocardial fibrosis.

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Second, the dissociation between alcohol-induced heart hypertrophy and myocardial fibrosis is suggested by the fact that supplementation with zinc only inhibits fibrosis but heart hypertrophy. Third, possible involvement of oxidative stress in the fibrogenesis is suggested by the fact that MT functions as an antioxidant (Kang, 1999) and zinc release from MT is an essential response of MT to oxidative stress (Maret, 2000; Kang, 2006), suggesting that oxidative stress is involved in the fibrogenesis. Further studies following the same direction will provide more comprehensive insights into the pathogenesis of ACM.

Pharmaceutical Chemicals Cardiac toxicity of pharmaceutical chemicals is a major problem in drug development and their clinical application. The pharmaceutical chemicals that cause cardiac toxic responses can be simply classified as drugs that are used to treat cardiac disease, and others that are used to treat noncardiac disease. In the category of drugs used to treat cardiac disease, cardiac toxicity is often produced by overexpression of the principal pharmaceutical effects. Although overdosing of these drugs can be a major factor for untoward effects, cardiac toxicity is often inevitable for this group of drugs. Drugs such as digitalis, quinidine, and procainamide often cause acute cardiac toxicity in the form of arrhythmia, which is reversible upon cessation of their use. Other cardiac drugs that may cause cardiac toxicity by mechanisms different from that of the therapeutic action. For instance, catecholamines may cause cardiac toxicity through oxidative stress, rather than by their pharmaceutical action on the sympathetic nervous system. The other category is noncardiac drugs that produce cardiac toxicity. For instance, anthracyclines, such as Adriamycin, are effective anticancer drugs, but their ability to produce severe cardiac toxicity limits their use in cancer patients. Vioxx is a selective COX-2 inhibitor used as an anti-inflammatory drug, but it causes QT prologation and increases the risk for sudden cardiac death. Antiarrhythmic Agents Antiarrhythmic drugs have historically been classified based upon a primary mechanism of action: Na+ channel blockers (class I), β-adrenergic blockers (class II), drugs that prolong action potential duration, especially K+ channel blockers (class III), and Ca2+ channel blockers (class IV). However, this classification is artificial because most of the drugs have multiple mechanisms of action. Class I antiarrhythmic agents are primarily Na+ channel blockers, including disopyramide, encainide, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, propafenone, quinidine, and tocainide. Blockade of cardiac Na+ channels results in reduction of conduction velocity, prolonged QRS duration, decreased automaticity, and inhibition of triggered activity from delayed afterdepolarizations or early afterdepolarizations (Roden, 1996). The primary concern of Na+ channel blocker toxicity is that proarrhythmic effects are seen at a much higher incidence in those patients with a previous history of myocardial infarction or with acute myocardial ischemia (Nattel, 1998). The proarrhythmic effects of these drugs would also be more prevalent in patients with other cardiac complications. Class II antiarrhythmic drugs are β-adrenergic receptorblocking drugs, including acebutolol, esmolol, propranolol, and sotalol. The catecholamines increase contractility, heart rate, and conduction through activation of β-adrenergic receptors in the heart. These effects can be explained by increased adenylyl cyclase

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activity, increased cyclic AMP, activation of protein kinase A, and phosphorylation and activation of L-type Ca2+ channels, thereby increasing intracellular Ca2+ , and particularly the amplitude of the Ca2+ transient. Therefore, antagonists of β-adrenergic receptors in the heart lead to effects that are opposite that of catecholamines, and are useful for the treatment of supraventricular tachycardia. The main adverse cardiovascular effect of β-adrenergic receptor antagonists is hypotension. These drugs may also exacerbate AV conduction deficits (e.g., heart block) and promote arrhythmias during bradycardia. Class III antiarrhythmic drugs are primarily K+ channel blockers. These drugs include amiodarone, bretylium, dofetilide, ibutilide, quinidine, and sotalol. Blockade of K+ channels increases action potential duration and increases refractoriness. Prolonged action potential duration contributes to the development of early afterdepolarizations and promotion of tachycardia, especially polymorphic ventricular tachycardia (torsades de pointes). The most noticeable adverse effect of these drugs is QT prolongation and torsadogenesis. Most of the drugs in this class also affect other ion channels and/or receptors. Amiodarone and quinidine also block Na+ channels, whereas sotalol inhibits β-adrenergic receptors in the heart. Amiodarone prolongs action potential duration and effective refractory period of Purkinje fibers and ventricular myocytes, and the most common adverse cardiovascular effect of amiodarone is bradycardia. Amiodarone may also have cardiotoxic effects by stimulating excessive Ca2+ uptake, especially in the presence of procaine (Gotzsche and Pedersen, 1994). Class IV antiarrhythmic drugs are Ca2+ channel blockers and include diltiazem and verapamil. The dihydropyridine Ca2+ channel blockers are not used to treat arrhythmias because they have a greater selectivity for vascular cells; however, these drugs may also alter cardiac ion homeostasis when plasma concentrations of the drugs are elevated. The dihydropyridines interact with Ca2+ channels in the inactivated state of the channel, and because vascular smooth muscle resting potentials are lower than cardiac cells, the time spent in the inactivated state is relatively longer in vascular smooth muscle, thus providing some preference of dihydropyridines for the vasculature (Galan et al., 1998). Bepridil, verapamil, and diltiazem exert negative inotropic and chronotropic effects. These drugs also exert a negative chronotropic effect, thus they may produce bradycardia. In contrast, the dihydropyridine Ca2+ channel blockers typically induce a reflex tachycardia subsequent to peripheral vascular dilation and baroreceptor reflex leading to increased sympathetic outflow from the medulla.

Inotropic Drugs Drugs involved in this category include the cardiac glycosides, Ca2+ sensitizing agents, catecholamines, and other sympathomimetic drugs. As with the antiarrhythmic drugs, inotropic drugs may exert cardiotoxic effects through extensions of their pharmacologic action. Cardiac glycosides (digoxin and digitoxin) are inotropic drugs used for the treatment of congestive heart failure. Ouabain is a cardiac glycoside commonly used in the laboratory for electrophysiological experiments in cardiac myocytes. The mechanism of inotropic action of cardiac glycosides involves inhibition of Na+ , K+ -ATPase, elevation of intracellular Na+ , activation of Na+ /Ca2+ exchange, and increased availability of intracellular Ca2+ for contraction. Consequently, cardiotoxicity may result from Ca2+ overload, potentially including reduction in resting membrane potential (less negative), delayed afterdepolarizations, and premature ven-

tricular contraction or ectopic beats. Cardiac glycosides also exhibit parasympatho-mimetic activity through vagal stimulation and facilitation of muscarinic transmission; however, at higher doses, sympathomimetic effects may occur as sympathetic outflow is enhanced. The principal adverse cardiac effects of cardiac glycosides include slowed AV conduction with potential block, ectopic beats, and bradycardia. During overdose, when the resting membrane potential is significantly altered and ectopic beats are prevalent, ventricular tachycardia may develop and can progress to ventricular fibrillation. A wide variety of drug interactions with digoxin have been reported, including both pharmacokinetic interactions (drugs that alter serum concentrations of digoxin) and pharmacodynamic interactions (drugs that alter the cardiac effects of digoxin). Ca2+ -sensitizing drugs including adibendan, levosimendan, and pimobendan are useful as inotropic drugs for the treatment of heart failure. In contrast to the main mechanism by which many other inotropic drugs act through elevating intracellular-free Ca2+ ([Ca2+ ]i ) during the Ca2+ transient (i.e., increase the amplitude of the Ca2+ transient), these Ca2+ -sensitizing drugs increase Ca2+ sensitivity of cardiac myocytes, thereby avoiding Ca2+ overload (Lee and Allen, 1997). Although cardiotoxicity resulting from Ca2+ overload would not be expected following administration of these new drugs, some experimental data suggest that they may still exert proarrhythmic effects (Lee and Allen, 1997). The possibility that such Ca2+ -sensitizing drugs interfere with diastolic function (relaxation) requires further investigation but may contribute to the ventricular arrhythmias associated with these drugs. Other Ca2+ -sensitizing drugs include the xanthine oxidase inhibitors allopurinol and oxypurinol, which have been shown to increase contractile force but decrease Ca2+ transient amplitude (Perez et al., 1998). Catecholamines and sympathomimetics Catecholamines represent a chemical class of neurotransmitters synthesized in the adrenal medulla (epinephrine and norepinephrine) and in the sympathetic nervous system (norepinephrine). These neurotransmitters exert a wide variety of cardiovascular effects. Because of their ability to activate α- and β-adrenergic receptors, especially in the cardiovascular system, a number of synthetic catecholamines have been developed for the treatment of cardiovascular disorders and other conditions such as asthma and nasal congestion. Inotropic and chronotropic catecholamines used to treat bradycardia, cardiac decompensation following surgery, or to increase blood pressure (e.g., hypotensive shock) include epinephrine, isoproterenol, and dobutamine, and these drugs typically display nonselective activation of adrenergic receptors. More selective β2 -adrenergic receptor agonists used for bronchodilatory effects in asthma include albuterol, bitolterol, fenoterol, formoterol, metaproterenol, pirbuterol, procaterol, salmeterol, and terbutaline. High oral doses of albuterol or terbutaline or inhalation doses (i.e., enhanced delivery to the stomach instead of the lungs with subsequent systemic absorption) of these drugs may lead to nonselective activation of β1 -adrenergic receptors in the heart with subsequent tachycardia. Sympathomimetic drugs that are more selective for αadrenergic receptors include the nasal decongestants ephedrine, phenylephrine, phenylpropanolamine, and pseudoephedrine. As with the asthma drugs, at high doses these nasal decongestants can produce tachycardia, and a number of deaths have been reported. Of particular interest is the high concentration of ephedra alkaloids that may be present in some herbal remedies or “neutraceuticals,” especially in products containing ma huang (Gurley et al., 1998). Tachycardia may occur from the consumption of large amounts of

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ephedra alkaloids, which may predispose the myocardium to ventricular arrhythmias. High circulating concentrations of epinephrine (adrenaline) and norepinephrine (noradrenaline) and high doses of synthetic catecholamines, such as isoproterenol, may cause cardiac myocyte death. Many of the catecholamines and related drugs have been shown to induce cardiac myocyte hypertrophic growth in vitro. Catecholamine-induced cardiotoxicity involves pronounced pharmacologic effects, including increased heart rate, enhanced myocardial oxygen demand, and an overall increase in systolic arterial blood pressure. Other possible mechanisms for the cardiotoxicity of high concentrations of catecholamines include coronary insufficiency resulting from coronary vasospasm, decreased levels of high-energy phosphate stores caused by mitochondrial dysfunction, increased sarcolemmal permeability leading to electrolyte alterations, altered lipid metabolism resulting in the accumulation of FAs, and intracellular Ca2+ overload (Dhalla et al., 1992).

Central Nervous System Acting Drugs Some of central nervous system (CNS)-acting drugs have considerable effects on the cardiovascular system, including tricyclic antidepressants (TCAs), general anesthetics, some of the opioids, and antipsychotic drugs. TCAs including amitriptyline, desipramine, doxepin, imipramine, and protriptyline have significant cardiotoxic effects, particularly in cases of overdose. The effects of TCAs on the heart include ST segment elevation, QT prolongation, supraventricular and ventricular arrhythmias (including torsades de pointes), and sudden cardiac death. In addition, as a result of peripheral αadrenergic blockade, TCAs cause postural hypotension—the most prevalent cardiovascular effect. Although many of these adverse effects are related to the quinidine-like actions, anticholinergic effects, and adrenergic actions of these drugs, the tricyclics also have direct actions on cardiac myocytes and Purkinje fibers, including depression of inward Na+ and Ca2+ and outward K+ currents (Pacher et al., 1998). Furthermore, the risk of TCAinduced cardiotoxicity is significantly enhanced in children and by concomitant administration of other drugs that alter ion movement or homeostasis in the heart (e.g., the Na+ channel-blocking class I antiarrhythmic agents), or use in patients with cardiovascular disease. Antipsychotic drugs include the phenothiazines (acetophenazine, chlorpromazine, fluphenazine, mesoridazine, perphenazine, thioridazine, and trifluoperazine), chlorprothixene, thiothixene, and other heterocyclic compounds (clozapine, haloperidol, loxapine, molindone, pimozide, and risperidone). As with TCAs, the most prominent adverse cardiovascular effect of antipsychotic drugs is orthostatic hypotension. However, the phenothiazines (e.g., chlorpromazine and thioridazine) may exert direct effects on the myocardium, including negative inotropic actions and quinidine-like effects (Baldessarini, 1996). Some ECG changes induced by these drugs include prolongation of the QT and PR intervals, blunting of T waves, and depression of the ST segment. Through anticholinergic actions, clozapine can produce substantial elevations in heart rate (tachycardia). General anesthetics as exemplified by enflurane, desflurane, halothane, isoflurane, methoxyflurane, and sevoflurane have adverse cardiac effects, including reduced cardiac output by 20–50%, depression of contractility, and production of arrhythmias (generally benign in healthy myocardium but more serious in cardiac dis-

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ease). These anesthetics may sensitize the heart to the arrhythmogenic effects of endogenous epinephrine or to β-receptor agonists. Halothane has been found to block the L-type Ca2+ channel by interacting with dihydropyridine-binding sites, to disrupt Ca2+ homeostasis associated with the SR, and to modify the responsiveness of the contractile proteins to activation by Ca2+ (Bosnjak, 1991). Propofol is an intravenously administered general anesthetic that also decreases cardiac output and blood pressure. In addition, propofol causes a negative inotropic effect by its direct action on cardiac myocytes. Propofol has been shown to antagonize β-adrenergic receptors, inhibit L-type Ca2+ current, and reduce Ca2+ transients (Zhou et al., 1997, 1999; Guenoun et al., 2000). Local Anesthetics In general, local anesthetics have few undesirable cardiac effects. However, when high systemic concentrations of cocaine and procainamide are attained, these chemicals may have prominent adverse effects on the heart. Cocaine acts as a local anesthetic agent by blocking conduction in nerve fibers through reversibly inhibiting Na+ channels and stopping the transient rise in Na+ conductance. In the heart, cocaine decreases the rate of depolarization and the amplitude of the action potential, slows conduction speed, and increases the effective refractory period. The other major pharmacologic action of cocaine is its ability to inhibit the reuptake of norepinephrine and dopamine into sympathetic nerve terminals (sympathomimetic effect). Cocaine also, indirectly through its actions on catecholamine reuptake, stimulates β- and α-adrenergic receptors, leading to increased cyclic AMP and inositol triphosphate levels. These second messengers will, in turn, provoke a rise in cytosolic Ca2+ , which causes sustained action potential generation and extrasystoles. The net effect of these pharmacologic actions is to elicit and maintain ventricular fibrillation. In addition, cocaine causes cardiac myocyte death and myocardial infarction, but the mechanism of action remains to be elucidated. Other local anesthetic drugs include benzocaine, bupivacaine, etidocaine, lidocaine, mepivacaine, pramoxine, prilocaine, procaine, procainamide, proparacaine, ro-pivacaine, and tetracaine. Lidocaine and procainamide are also used as antiarrhythmic drugs. Extremely high doses of these drugs cause decreases in electrical excitability, conduction rate, and force of contraction likely through inhibition of cardiac Na+ channels (Catterall and Mackie, 1996). Anthracyclines and Other Antineoplastic Agents Cardiotoxicity is recognized as a serious side effect of chemotherapy for malignant cancers, especially with well-known antitumor agents such as doxorubicin, daunorubicin, 5-fluorouracil, and cyclophosphamide (Havlin, 1992). Anthracyclines (doxorubicin or Adriamycin and daunorubicin) are widely used antineoplastic drugs for the treatment of breast cancer, leukemias, and a variety of other solid tumors. Unfortunately, the clinical usefulness of these drugs is limited because of acute and chronic cardiotoxic effects. The acute effects mimic anaphylactictype responses, such as tachycardia and various arrhythmias. These effects are usually manageable and most likely are due to the potent release of histamine from mast cells sometimes observed in acute dosing. In addition, large acute doses can also cause left ventricular failure. The greatest limiting factor of the anthracyclines is associated with long-term exposure, which usually results in the development of cardiomyopathies and, in severe cases, congestive heart failure (Havlin, 1992).

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Two new anthracyclines were introduced to the U.S. market in 1999, and a lipid formulation of doxorubicin (liposomal doxorubicin) is under development. Valrubicin is a semisynthetic derivative of doxorubicin approved for treatment of carcinoma in situ of the bladder. It is administered locally for bladder cancer and therefore induces only mild systemic toxicities; however, systemic absorption from the bladder may occur, but valrubicin seems to exhibit a lower propensity for cardiotoxicity than doxorubicin (Hussar, 2000). Epirubicin is a semisynthetic derivative of daunorubicin approved for treatment of breast cancer. Like doxorubicin, epirubicin is given systemically and may induce cardiotoxicity. However, epirubicin is more lipophilic than doxorubicin and is biotransformed by the conjugative pathways in the liver, resulting in a shorter half-life and a lower incidence of cardiotoxicity than with doxorubicin (Hussar, 2000). Several major hypotheses have been suggested to account for the onset of anthracycline-induced cardiomyopathy: (1) oxidative stress from redox cycling or mitochondrial Ca2+ cycling, (2) defects in mitochondrial integrity and subsequent deterioration of myocardial energetics, (3) alterations in both SR Ca2+ currents and mitochondrial Ca2+ homeostasis, and (4) altered cardiac myocyte gene expression and induction of apoptosis. The cause-andeffect relationships of the proposed mechanisms of cardiotoxicity have not been determined, and no single theory adequately explains the exact mechanism for anthracycline-induced cardiomyopathy. The free radical hypothesis has received the most attention in the understanding of anthrocycline-induced cardiotoxicity. The formation of ROS by doxorubicin (Fig. 18-16) has been attributed to redox cycling of the drug (Powis, 1989). Doxorubicin can undergo futile redox cycling that results in the production of oxygen free radicals; these ROS may then oxidize proteins, lipids, and nucleic acids and potentially cause DNA strand scission. The quinone-like structure of doxorubicin permits this molecule to accept an electron and form a semiquinone radical. Oxidation of the semiquinone back to the parent quinone by molecular oxygen results in the formation of superoxide radical ions that are believed to initiate oxidative stress. The enzymatic reduction that is believed to be responsible for the generation of superoxide by doxorubicin has been proposed to occur between complexes I and III of the mitochondrial respiratory chain. Doxorubicin has high affinity for cardiolipin, a phospholipid found on the inner mitochondrial membrane, where NADH dehydrogenase converts the drug to a semiquinone radical (Marcillat et al., 1989). In the presence of oxygen, this radical is responsible for the generation of ROS, which then may peroxidize unsaturated membrane lipids and initiate myocardial cell injury. Several alternate hypotheses to explain the cardiotoxicity of doxorubicin have been proposed and tested. For example, several studies have tested the hypothesis that doxorubicin induces a cycling of mitochondrial Ca2+ that is associated with the production of ROS and dissipation in the mitochondrial membrane potential, which in turn may result in depletion of cellular ATP (Chacon and Acosta, 1991; Solem et al., 1994). It is important to note that the observed changes in ROS accumulation, disruption of Ca2+ homeostasis, and mitochondrial damage are not isolated, but rather these changes occur sequentially or simultaneously. It is extremely difficult to dissect the sequences of these changes, leading to several alternate hypotheses, which may in fact occur sequentially. One of the ultimate consequences of these changes is myocardial cell death. Many studies have demonstrated that anthracycline-induced cardiotoxicity includes induction of apoptosis (Kang et al., 2000a; Wang et al.,

Figure 18-16. Production of superoxide anions by oxidation-reduction cycling of doxorubicin at the level of the mitochondria. NADH dehydrogenase (NAD-DH), which is located within complex I, has been proposed as the enzyme that catalyzes the one-electron reduction of doxorubicin (1) to a semiquinone radical (2). The semiquinone then may be reoxidized back to the parent compound by means of the reduction of molecular oxygen (O2 ) to the superoxide anion (O− 2 ).

2001a; Sawyer et al., 1999; Andrieu-Abadie et al., 1999; Arola et al., 2000). 5-Fluorouracil Clinical evidence of 5-fluorouracil cardiotoxicity ranges from mild precordial pain and ECG abnormalities (ST segment elevation, high peaked T waves, T-wave inversions, and sinus tachycardia) to severe hypotension, atrial fibrillation, and abnormalities of ventricular wall motion. The mechanism of cardiotoxicity of fluorouracil is unknown, but it may relate to impurities present in commercial products of the drug, one of which is metabolized to fluoroacetate, a compound that might participate in fluorouracilinduced cardiotoxicity. Cyclophosphamide High doses of cyclophosphamide given to cancer or transplant patients may lead to severe hemorrhagic cardiac necrosis. The mechanism of the cardiotoxicity of this drug is not clear, but there is suggestive evidence that the toxic metabolite of cyclophosphamide, 4-hydroperoxycyclophosphamide, may alter the ion homeostasis in cardiac myocytes, resulting in increased Na+ and Ca2+ content and reduced K+ levels (Levine et al., 1993). Antimicrobial and Antiviral Agents Cardiotoxicity associated with the clinical use of antimicrobial and antiviral drugs is often observed in overdosage and in patients with preexisting cardiovascular dysfunction.

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Aminoglycosides include amikacin, gentamicin, kanamycin, netilmicin, streptomycin, and tobramycin. Gentamicin is a representative aminoglycoside and has an inhibitory action on slow inward Ca2+ channels in heart muscle. Aminoglycosides inhibit the uptake or binding of Ca2+ at sarcolemmal sites, thus reducing the concentration of membrane-bound Ca2+ available for movement into the myoplasm during depolarization of the sarcolemma. The principle mechanism of cardiodepression by gentamicin is the dislocation of Ca2+ from slow-channel-binding sites on the external surface of the sarcolemma, which results in a blockade of the channels (Hino et al., 1982). Macrolides include azithromycin, clarithromycin, dirithromycin, and erythromycin. Erythromycin is associated with QT prolongation and cardiac dysrhythmias characterized by polymorphic ventricular tachycardia (torsades de pointes). These effects occur primarily in patients with underlying cardiac disease. Fluoroquinolones are a group of rapid growing antibacterial chemicals in terms of numbers of new drugs released into the market in the United States. Fluoroquinolone antibacterial drugs include ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin (levo-rotatory isomer of ofloxacin), lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, sparfloxacin, and trovafloxacin (Pickerill et al., 2000). Grepafloxacin, moxifloxacin, and sparfloxacin are associated with QT prolongation in perhaps a higher incidence than macrolides. In fact, grepafloxacin was voluntarily removed from the U.S. market because of the relatively high incidence of QT prolongation and risk of torsades de pointes. Tetracycline and chloramphenicol have been reported to depress myocardial contractility by direct cardiac myocyte interaction or an indirect effect that lowers Ca2+ concentrations in the plasma or extracellular spaces. Tetracylcines are Ca2+ chelating agents, which explain the action of tetracyclines on myocardial contractility. Antifungal agents, such as amphotericin B, may depress myocardial contractility by blocking activation of slow Ca2+ channels and inhibiting the influx of Na+ . Ventricular tachycardia and cardiac arrest have been reported in patients treated with amphotericin B. Flucytosine is another antifungal drug that has been associated with cardiotoxicity. In fungal cells, flucytosine is converted to 5-fluorouracil, which then exerts antifungal effects. However, flucytosine may be converted to 5-fluorouracil by gastrointestinal microflora in humans, which then may be absorbed systemically and induce cardiotoxicity as discussed above. Cardiac arrest has been reported in individuals receiving flucytosine. Antiviral drugs that are potentially cardiotoxic include the nucleoside analog reverse transcriptase inhibitors used for the treatment of human immunodeficiency virus (HIV) infections. Clinical studies of direct cardiotoxicity of these drugs in HIV patients are complicated by cardiomyopathy related to disease progression. The direct evidence for cardiotoxicity of zidovudine (AZT) has been obtained from a study using transgenic mice expressing replicationincompetent HIV (Lewis et al., 2000). AZT-induced cardiotoxicity in this model is related to alteration in Ca2+ homeostasis and/or mitochondrial toxicity. The mitochondrial toxicity of AZT has also been shown in skeletal muscle biopsy samples from AIDS patients (Dalakas, et al., 1990). Although cardiotoxicity of individual antiviral drugs is rare in clinical setting, the combination of several antiviral drugs for highly active anti-retroviral therapy (HAART) has generated a major concern of cardiotoxicity (Bozkurt, 2004). HAART has dramatically improved the life expectancy of patients with HIV. The majority of the studies examining the incidence of cardiac effects demon-

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strated an increase in cardiac adverse event rate with HAART in the HIV-infected population. Overall, the cardiotoxicity risk appears to be greater in the HIV-infected population than in the general population, and the increased cardiac risk is associated with HAART, particularly with protease inhibitor use. However, there is general consensus that the benefits of HAART far outweigh toxicity-related risks of the treatment with HAART. Anti-Inflammatory Agents Nonsteroidal anti-inflammatory drugs (NSAIDs) include aspirin, Motrin, and Naprosyn, which are classified as nonselective NSAIDs because they are inhibitors for both COX-1 and COX-2. Inhibition of COX-1 is associated with gastrointestinal toxicity because COX-1 exerts a protective effect on the lining of the stomach. A newer class of NSAIDs has been developed; including rofecoxib (Vioxx), celecoxib (Celebrex), and valdecoxib (Bextra), witch are selective inhibitors of COX-2. In September 2004, Vioxx was voluntarily withdrawn from the market based upon the data from a clinical trial that showed after 18 months of use Vioxx increased the relative risk for cardiovascular events, such as heart attack and stroke (Arellano, 2005). In April 2005, Bextra was removed from the market based on the potential increased risk for serious cardiovascular adverse events and increased risk of serious skin reactions (e.g., toxic epidermal necrolysis, Stevens– Johnson syndrome, erythema multiforme) (Talhari et al., 2005). Emerging information indicates the risk of cardiovascular events may be increased in patients receiving Celebrex (Solomon et al., 2005). The cardiovascular events induced by COX-2 inhibitors are presumably related to thrombotic events. Studies have also indicated the link of Vioxx to long QT syndrome and the increased risk for TdP and sudden cardiac death (Arellano 2005; Fitzgerald 2004). Antihistamines The most severe adverse effect of the secondgeneration histamine H1 receptor antagonists (antihistamines) is their association with life-threatening ventricular arrhythmias and sudden cardiac death (Simons, 1994). Terfenadine and astemizole cause altered repolarization, notched inverted T waves, prominent TU waves, prolonged QT interval, first- and second-degree AV block, ventricular tachycardia or fibrillation, and torsades de pointes. These antihistamines produce cardiac arrhythmias by blocking the delayed rectifier K+ channel and prolonging action potential duration in cardiac myocytes. The prolonged action potential duration promotes early afterdepolarizations and predisposes the myocardium to ventricular arrhythmias. However, terfenadine also inhibits L-type Ca2+ channels in rat ventricular myocytes at concentrations near or below that required to inhibit delayed rectifier K+ current (Liu et al., 1997). Therefore, both inhibition of Ca2+ and inhibition of K+ current likely contribute to the cardiotoxic actions of terfenadine. As a result of cardiotoxicity, both astemizole and terfenadine have been removed from the United States market. However, the understanding of astemizole- and terfenadine-induced cardiotoxicity continues to be an important consideration in drug development, and other drugs have demonstrated similar clinical limitations (e.g., cisapride and fluoroquinolone antibacterial agents). Immunosuppressants Rapamycin and tacrolimus may produce adverse cardiovascular effects, including hypertension, hypokalemia, and hypomagnesemia. Rapamycin and tacrolimus (FK506) interact with a protein that associates with ryanodine receptors (RyRs), and the protein carries the tacrolimus- or FK506binding protein (FKBP). When rapamycin or tacrolimus binds to

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FKBP in cardiac myocytes, RyR becomes destabilized, resulting in Ca2+ leak from the SR (Marks, 1997). Tacrolimus has been shown to be associated with hypertrophic cardiomyopathy in pediatric patients, a condition that was reversed by discontinuation of tacrolimus and administration of cyclosporin A; some of these patients developed severe heart failure (Atkison et al., 1995). Miscellaneous Drugs Several drugs that are not included in the categories discussed above have significant cardiotoxic concerns and are briefly discussed below, including cisapride, methylxanthines, and sildenafil. Cisapride is a chemical that has been used as a prokinetic drug for gastrointestinal hypomotility. However, cisapride has been removed from the U.S. market because of risk of potentially lifethreatening arrhythmias (torsades de pointes) associated with its use. Like astemizole and terfenadine, cisapride inhibits delayed rectifier K+ current, prolongs action potential duration, prolongs the QT interval, and predisposes the heart to ventricular arrhythmias. Methylxanthines (including caffeine, theobromine, and theophylline), can be found in significant quantities in coffee, tea, chocolate, soft-drinks, and other foods. Theophylline has been used for many decades for the treatment of asthma, although the mechanism of action has not been fully understood. Overdose of theophylline or rapid intravenous administration of therapeutic doses of aminophylline (theophylline complexed with ethylenediamine to increase water solubility) may produce life-threatening ventricular arrhythmias; these effects may in part be explained by direct actions of theophylline on cardiac myocyte SR or by inhibition of phosphodiesterase and elevation of cyclic AMP. The cardiac effects of methylxanthines observed in vivo (including increases in cardiac output and heart rate) may also be explained by elevated catecholamines, as theophylline has been shown to increase plasma epinephrine concentrations (Vestal et al., 1983). High concentrations of caffeine stimulate massive release of Ca2+ from the SR, an effect that is often utilized experimentally to determine SR function. Although it rarely occurs, caffeine-associated ventricular arrhythmias have been reported. Sildenafil is a relatively specific inhibitor of phosphodiesterase 5, which is responsible for the degradation of cyclic GMP (a vasodilatory second messenger). Interestingly, sildenafil was originally developed as a potential drug for treating angina; however, it was not very effective for this purpose and was subsequently developed for treatment of erectile dysfunction, where it produces vasodilation and filling of the corpus cavernosum. The primary concern regarding adverse effects of sildenafil is nonspecific inhibition of PDE3 in the heart and vasculature (Hussar, 1999). In vitro studies have revealed that sildenafil increases cyclic AMP in cardiac tissue without significant effects on cyclic GMP (Stief et al., 2000); however, whether these effects are associated with cardiotoxicity is not known.

Natural Products Natural products include naturally occurring catecholamines, hormones, and cytokines, as well as animal and plant toxins. Many of these products have been shown to cause cardiac toxic responses. However, it is difficult to define whether or not the cardiac toxicity results directly from the action of these products in vivo, although these products cause deleterious effects on cultured cardiomyocytes. The exposure levels of these chemicals tested in vitro in general are much higher than the concentration reached in cardiac tissue under

in vivo exposure conditions. Therefore, extrapolation of in vitro data related to cardiac toxicity of natural products to in vivo conditions is challenging. However, there are some products that have clearly demonstrated cardiac toxic effects, and mechanisms of action of these products have been determined. Catecholamines The naturally occurring sympathomimetic amines, such as epinephrine and norepinepherine, are potent and can cause deleterious effects to the heart. The synthetic catecholamine, isoproterenol, is able to cause massive necrotic changes in the myocardium and is often used as a prototype compound for the study of catecholamine cardiotoxicity, which has been discussed in the therapeutic drugs that cause cardiotoxicity. Steroids and Related Hormones Estrogens, progestins, androgens, and adrenocortical steroids are major steroid hormones produced by mammals including humans. Myocardial tissue contains steroid receptors; therefore the heart serves as a target organ for steroid effects. It also has been shown that cardiac tissue can synthesize steroid hormones, although the capacity for synthesis may be much lower than more classic steroid synthesizing tissues. There are two major mechanisms of action of the hormones: the first is to alter gene expression and the second is to change signaling transduction pathways. Estrogens are synthesized in ovaries, testes, and adrenal glands, and estrogen is an active metabolite of testosterone. Endogenous estrogens include 17β-estradiol (E2 ), estrone, and estriol. Synthetic estrogens include diethylstilbestrol (nonsteroidal), equilin, esterified versions of E2 , ethinyl estradiol, mestranol, and quinestrol. In addition, many other synthetic chemicals have been shown to exert estrogenic activity, including the pesticides DDT and methoxychlor, the plasticizer bisphenol A, other industrial chemicals including polychlorinated biphenyls, and some compounds found in soybeans and tofu (e.g., phytoestrogens). Estrogens (frequently in combination with progestins) have been used for over 40 years as oral contraceptive drugs. The older versions of estrogenic oral contraceptives that contained high amounts of estrogens were associated with increased risk of coronary thrombosis and myocardial infarction; however, lower doses of estrogens have been found by numerous investigators to impart protective effects on the cardiovascular system, including antiapoptotic effects, and beneficial effects on lipid metabolism such as decreased low-density lipoproteins (LDL cholesterol) and increased high-density lipoproteins (HDL cholesterol). Estrogens alter cardiac fibroblast proliferation, but they can either increase or decrease proliferation of these cells. Progestins are also synthesized in the ovaries, testes, and adrenal glands. Naturally occurring and synthetic progestins include desogestrel, hydroxyprogesterone, medroxyprogesterone, norethindrone, norethynodrel, norgestimate, norgestrel, and progesterone. As part of hormone replacement therapy, progestins serve an opposing role to estrogens. Unfortunately, estrogen treatment opposed with progestins may negate the cardiovascular benefits of estrogens on lipid metabolism (Kalin and Zumoff, 1990). Very little is known about the direct effects of progestins on the heart. Although progestins could exert deleterious effects on the heart, more studies are required to investigate mechanisms. Androgens cause adverse cardiovascular effects (Rockhold, 1993; Melchert and Welder, 1995). The principal androgens are testosterone and its active metabolite dihydrotestosterone.

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TOXIC RESPONSES OF THE HEART AND VASCULAR SYSTEM

Testosterone is synthesized in the testes, ovaries, and adrenal glands and dihydrotestosterone mediates most androgen actions. Synthetic anabolic-androgenic steroids include the alkylated and orally available drugs danazol, fluoxymesterone, methandrostenolone, methenolone, methyltestosterone, oxandrolone, oxymetholone, and stanozolol. The nonalkylated drugs with poor oral bioavailability include androstenedione and dehydroepiandrosterone (both sold in various “nutraceutical” formulations), boldenone (veterinary product), nandrolone (19-nortestosterone), and testosterone. Nearly all of these chemicals have received significant illicit use, particularly in extremely high doses with attempts to improve physical appearance or performance. Anabolic-androgenic steroids have been associated with alterations in lipid metabolism, including increased LDL cholesterol and decreased HDL cholesterol; therefore these chemicals may predispose individuals to atherosclerosis (Melchert and Welder, 1995). Evidence indicating the direct cardiac toxic effect of anabolic-androgenic steroids includes alteration of Ca2+ fluxes in cardiac myocytes induced by testosterone (Koenig et al., 1989), hypertrophic growth of neonatal rat cardiac myocytes stimulated by testosterone and dihydrotestosterone (Marsh et al., 1998), and mitochondrial abnormalities and myofibrillar lesions induced by methandrostenolone given intramuscularly to rats (Behrendt and Boffin, 1977). In humans, high-dose anabolic-androgenic steroid use has been associated with cardiac hypertrophy and myocardial infarction. However, the mechanisms responsible for the cardiotoxic effects of anabolic-androgenic steroids remain poorly understood. Glucocorticoids and mineralocorticoids are primarily synthesized in the adrenal glands. Naturally occurring glucocorticoids include corticosterone, cortisone, and hydrocortisone (cortisol), and the mineralocorticoid is aldosterone. A large number of synthetic glucocorticoids are used for treatment of various autoimmune and inflammatory diseases. These drugs include alclometasone, amcinonide, beclomethasone, betamethasone, clobetasol, desonide, desoximetasone, dexamethasone, diflorasone, fludrocortisone, flunisolide, fluocinolone, fluocinonide, fluorometholone, flurandrenolide, halcinonide, medrysone, methylprednisolone, mometasone, paramethasone, prednisolone, prednisone, and triamcinolone. Most of these agents are primarily used topically, intranasally, or inhalationally. The primary glucocorticoids used systemically include cortisone, hydrocortisone, dexamethasone, methylprednisolone, prednisolone, and prednisone. The mineralocorticoid aldosterone is not used clinically; however, the aldosterone receptor antagonist spironolactone has been used for years to treat hypertension and is now thought to decrease morbidity and mortality associated with congestive heart failure. Both aldosterone and glucocorticoids appear to stimulate cardiac fibrosis by regulating cardiac collagen expression independently of hemodynamic alterations (Young et al., 1994; Robert et al., 1995). Furthermore, aldosterone and glucocorticoids induce hypertrophic growth and alter expression of Na+ , K+ -ATPase, Na+ /H+ antiporter, and chloride/bicarbonate exchanger of cardiac myocytes in vitro. Clinically relevant cardiac hypertrophy has been observed in premature infants undergoing dexamethasone treatment. The mechanisms responsible for the direct effects of these chemicals remain poorly understood. Thyroid hormones include thyroxine (T4 ) and triiodothyronine (T3 ). These hormones exert profound effects on the cardiovascular system. Hypothyroid states are associated with decreased heart rate, contractility, and cardiac output; whereas hyperthyroid states are associated with increased heart rate, contractility, cardiac output,

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ejection fraction, and heart mass. Patients with underlying cardiovascular disease may display arrhythmias under the treatment of thyroid hormones. Thyroid hormones also alter expression of cardiac SR Ca2+ handling proteins including increased expression of SR Ca2+ ATPase (SERCA) and decreased expression of phospholamban, an inhibitory protein of SERCA (Kaasik et al., 1997).

Cytokines More than 100 different cytokines have been found, and the cardiovascular effects of these substances can be classified as proinflammatory, anti-inflammatory, or cardioprotective (Pulkki, 1997). Members of the proinflammatory class include TNF-α; interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), Fas ligand, and chemokines (e.g., CC chemokines such as MCP-1, macrophage chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; and RANTES, regulated on activation normally T-cell-expressed and secreted). Members of the anti-inflammatory class typically downregulate expression of proinflammatory cytokines and include interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), and transforming growth factor-beta (TGF-β). The cardioprotective cytokines include cardiotrophin-1 (CT-1) and leukemia inhibitor factor, which have been shown to inhibit cardiac myocyte apoptosis from a number of different stimuli. Many of these cytokines are elevated during cardiovascular diseases such as I/R injury, myocardial infarction, and congestive heart failure. IL-1β is known to exert negative inotropic actions and induce apoptosis of cardiac myocytes. The effects of IL-1β on cardiac myocytes are likely mediated through induction of nitric oxide synthase (NOS) and/or increased production of nitric oxide (NO) (Arstall et al., 1999). Superoxide anion and peroxynitrite formation was associated with reduced left ventricular ejection fraction in dogs treated with microspheres containing IL-1β, suggesting that involvement of peroxynitrite in IL-1β cardiotoxicity (Cheng et al., 1999b). TNF-α induces apoptotic cell death in myocardium (Krown et al., 1996). The mechanisms responsible for TNF-α-induced apoptosis of cardiac myocytes are not entirely clear. TNF-α also exerts negative inotropic effects on cardiac myocytes at least potentially through increased production of sphingosine (Sugishita et al., 1999). However, it has been shown TNF-α is essential for the cardiac protective response to stresses such as ischemic cardiac injury (Kurrelmeyer et al., 2000). IL-6 has been shown to induce negative inotropic effects on cardiac myocytes, possibly through induction of NOS expression and increased NO production (Sugishita et al., 1999). IL-2 may decrease the mechanical performance and metabolic efficiency of the heart, and these myocardial effects may be related to changes in NO synthesis and Na+ /H+ exchange. Interferon may result in cardiac arrhythmias, dilated cardiomyopathy, and signs of myocardial ischemia. Interferon-γ acts synergistically with IL-1β to increase NO formation in the heart, and induce Bax expression and apoptosis in cardiac myocyte cultures (Arstall et al., 1999).

Animal and Plant Toxins Animal toxins in the venom of snakes, spiders, scorpions, and marine organisms have profound effects on the cardiovascular system. There are also a number of plants— such as foxglove, oleander, and monkshood—that contain toxic constituents and have adverse effects on the cardiovascular system.

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Environmental Pollutants and Industrial Chemicals There are many chemicals classified in this category that cause cardiac toxicity. Metals and metalloids can be found both in environmental pollutants and industrial chemicals. Some heavy metals, such as cadmium, block calcium channels that affect cardiac rhythm leading to arrhythmia, others such as arsenic have high affinity for sulfhydryl groups, and interfere with sulfhydryl-containing proteins, such as receptors, regulatory proteins, and transporters. During the last decade, epidemiological and experimental studies have identified an association of air pollution of PMs (particulate matters) and cardiac toxicity, however, mechanistic insights into cardiac toxicity induced by PM remain elusitive. In this section, a brief discussion of selected industrial agents with their prominent cardiotoxic effects and proposed mechanisms of cardiotoxicity is presented. For a more comprehensive review of industrial chemicals and their cardiotoxic potential, the reader is referred to Zakhari (1992). Particulate Matters There are several obstacles in the systemic study of cardiac toxicity caused by particulate air pollution. One of the major challenges is the complexity of the particulate components of air pollution. Current consensus in the field is to divide the airborne particulates into classes according to aerodynamic diameters. There are three major classes: coarse (PM10 , 2.5–10 μm), fine (PM2.5 , forehead > abdomen (Scheuplein and Blank, 1971). Scrotal skin reportedly has the highest permeability for some topical chemicals (Fisher, 1989). Under ordinary conditions, absorption through the epidermal appendages is generally neglected, despite the ability of chemicals to bypass the stratum corneum by this route, because the combined appendageal surface area is such a small fraction of the total available for uptake. However, because loading of the stratum corneum is slow, penetration through the appendages can constitute an appreciable fraction of the total for short exposures. In some cases, the effects of appendages can even be dominant. For instance, benzo(a)pyrene

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penetrates the skin of haired mice several-fold faster than that of hairless strains (Kao and Hall, 1987). As indicated below, in some cases these properties have been exploited for therapeutic uses. Transdermal Drug Delivery The ability of the stratum corneum to serve as a reservoir for exogenously applied chemicals is well illustrated by the recent development of methods for the delivery of pharmaceuticals. Application of drugs to the skin can produce systemic effects, a phenomenon observed unintentionally before the ability of the skin to serve as a delivery system was appreciated. For example, topical exposure of young girls to estrogens has led to reports of pseudoprecocious puberty, whereas in young or adult males, such exposure has produced gynecomastia (Amin et al., 1998). Specially designed patches are currently in use to deliver estradiol, testosterone, nitroglycerin, scopolamine, clonidine, fentanyl, and nicotine for therapeutic purposes, and others are under development. The advantages of this approach over oral dosing include providing a steady infusion for extended periods (typically 1–7 days), thereby avoiding large variations in plasma concentration, preventing exposure to the acidic pH of the stomach, and avoiding biotransformation in the gastrointestinal tract or from first-pass removal by the liver. The contrast in plasma concentration kinetics between different methods of delivery is particularly evident for agents that are rapidly metabolized, such as nitroglycerin, which has a half-life of a few minutes. A variety of chemicals, chosen carefully to minimize irritation or allergenicity, have been incorporated into pharmaceutical preparations to enhance absorption and penetration. In addition, encapsulating a drug in small vesicles of phospholipid or nonionic surfactant can be effective at least in part by targeting hair follicles (Choi and Maibach, 2005). Measurements of Penetration For many purposes, including risk assessment and pharmaceutical design, the most useful subject for experimentation is human skin. Volunteers are dosed, plasma and/or urine concentrations are quantified at suitable intervals, and amounts excreted from the body are estimated. Previous measurements of penetration often used 14 C-labeled agents. This approach is not preferred, but use of isotopic labels now is readily feasible when coupled to ultrasensitive detection by accelerator mass spectrometry (Buchholz et al., 1999). For in vitro work, excised split-thickness skin can be employed in special diffusion chambers, though care is needed to preserve the viability of the living layer of epidermis. The chemical is removed for quantification from the underside by a fluid into which it partitions, thereby permitting continued penetration. Commonly employed is a simpler set up, using cadaver skin with the lower dermis removed. This lacks biotransformation capability but retains the barrier function of the stratum corneum. The phamacokinetic approach with intact subjects is most commonly employed with experimental animals. Without verification using human skin, such measurements are subject to large uncertainties due to species differences in density of epidermal appendages, stratum corneum properties (e.g., thickness, lipid composition), and biotransformation rates. Because penetration through rodent skin is usually faster than through human skin, the former can provide an overestimate for the behavior of the latter. To simplify determination of penetration kinetics, skin flaps may be employed and the capillary blood flow monitored to measure penetration. For this purpose, pig skin has particular utility (Riviere and Brooks, 2005). A promising variation minimizing species differences is to use skin grafts on experimental animals for these measurements. Human skin persists well

on athymic mice and retains its normal barrier properties (Krueger and Pershing, 1993). More recently, penetration of chemicals can be tracked through the skin using confocal microscopy (AlvarezRoman et al., 2004). In any case, accurate testing of percutaneous absorption of poorly soluble agents from environmental substrates requires attention to details of particle size, component complexes, application rate and skin contact.

Biotransformation The ability of the skin to metabolize chemicals that diffuse through it contributes to its barrier function. This influences the potential biological activity of xenobiotics and topically applied drugs, leading to their degradation or their activation as skin sensitizers or carcinogens (Hotchkiss, 1998). To this end, the epidermis and pilosebaceous units are the most relevant and, indeed, are the major sources of such activity in the skin. On a body-weight basis, phase I metabolism in this organ usually is only a small fraction (∼2%) of that in the liver, but its importance should not be underestimated. For example, when the epidermis of the neonatal rat is treated with benzo(a)pyrene or Aroclor 1254, the arylhydrocarbon hydroxylase (P450) activity in the skin can exceed 20% of that in the whole body (Mukhtar and Bickers, 1981). As illustrated in this example, cytochrome P4501A1 is inducible in the epidermis by chemicals that are inducers in other tissues—TCDD (tetrachlorodibenzo- pdioxin), polycyclic aromatic hydrocarbons, PCBs (polychlorinated biphenyls), and crude coal tar, which is used in dermatological therapy. Thus, exposure to such inducers could influence skin biotransformation and even sensitize epidermal cells to other chemicals that are not good inducers themselves, a phenomenon observable in cell culture (Walsh et al., 1995). Biotransformation of a variety of compounds in the skin has been detected, including arachidonic acid derivatives, steroids, retinoids, and 2-amino-anthracene, suggesting that multiple P450 activities are expressed. With the advent of DNA microarray and real-time polymerase chain reaction technologies, it is now evident that over a dozen distinct isozymes are expressed at widely varying levels. A recent survey of those in the CYP1-4 families indicated that half were expressed at substantially higher levels in differentiating keratinocytes (similar to spinous cells) than in basallike cells (Du et al., 2006). In addition to influencing our response to the natural environment, these activities are also important in influencing our response to pharmaceuticals used in clinical dermatology inasmuch as a large fraction of the latter are P450 inducers, inhibitors, or substrates (Ahmad and Mukhtar, 2004). Species differences are apparent in the amounts of P450 activities detectable. For example, measured ethoxycoumarin-O-deethylase activity is 20-fold higher in mouse than human (or rat) skin. Differences of such magnitude help rationalize the observation that the rate of penetration of ethoxycoumarin is sufficient to saturate its metabolism in some species (e.g., the human) but not in others (e.g., the mouse or guinea pig) (Storm et al., 1990). To the extent that phase I (and II) metabolism influences sensitization to exogenous chemicals (Merk et al., 2004), they may also help rationalize species differences in allergic response. Enzymes participating in phase II metabolism are expressed in skin. For example, multiple forms of epoxide hydrolase and UDP-glucuronosyl transferase have been detected in human and rodent skin. In general, this activity occurs at a much lower rate than observed in the liver, but exceptions are evident, as in the case of quinone reductase (Khan et al., 1987). Human and rodent skin

CHAPTER 19

TOXIC RESPONSES OF THE SKIN

exhibit qualitatively similar phase II reactions, but rodent skin often has a higher level of activity. An additional consideration is that different species express different relative amounts of the various isozymes, which could alter resulting target specificities or degree of responsiveness. Glutathione transferase, for instance, catalyzes the reaction of glutathione with exogenous nucleophiles or provides intracellular transport of bound compounds in the absence of a reaction. It also facilitates the reaction of glutathione with endogenous products of arachidonate lipoxygenation (leukotrienes) to yield mediators of anaphylaxis and chemotaxis, which are elements of the inflammatory response in the skin. Of the first three major transferase forms characterized in the liver, the major form in the skin of humans and rodents is the P isozyme. Human skin also expresses the A isozyme, whereas rat and mouse skin express the M isozyme and, in much smaller amounts, the A isozyme (Raza et al., 1992). A variety of other metabolic enzyme activities have also been detected in human epidermal cells, including sulfatases, β-glucuronidase, N -acetyl transferases, esterases, and reductases (Hotchkiss, 1998). The intercellular region of the stratum corneum has catabolic activities (e.g., proteases, lipases, glycosidases, phosphatases) supplied by the lamellar bodies along with their characteristic lipid (Elias, 1992). The report that cholesterol sulfotransferase is regulated by ligands of LXR and PPAR illustrates the potential for exogenous agents, including pharmaceuticals, to influence such activities and thereby the barrier function of the skin (Jiang et al., 2005), where penetration can be influenced by cell differentiation and biotransformation. For example, methyl salicylate readily diffuses through the epidermis and is detected in the dermis, but only as de-esterified salicylate, illustrating first-pass metabolism (Cross et al., 1997). The influence of hydroxysteroid dehydrogenases and microsomal reductase activities during percutaneous absorption is evident in studies on mouse skin in organ culture. In one study (Kao and Hall, 1987), 8 hours after topical application of testosterone, 59% of the permeated steroid was collected unchanged and the rest was transformed into metabolites. In parallel, estrone was converted substantially to estradiol (67%), while only 23% was collected as the parent compound. By contrast, estradiol was metabolized to a much lower extent (21%).

CONTACT DERMATITIS In the occupational arena, where records are compiled on large workforces, contact dermatitis is by far the largest category (∼90%) of compensated skin disease. Using eczema of the hand as a sentinel condition, since 80% of the total reported dermatitis occurs at that location (10% on the face), reveals a prevalence of 7–10% among workers. Attributed to better diagnosis, more accurate identification of offending chemicals, and more effective prevention and worker education, the fraction of afflicted workers recovering without impairment has improved nearly to 80% with proper management (Belsito, 2005). However, whereas certain conditions carry a favorable prognosis, others (e.g., chronic cumulative irritant contact dermatitis or contact allergy to nickel, chromate, formaldehyde, or rubber) frequently result in chronic disease in which changing jobs is of limited or no benefit (Emmett, 2003; Belsito, 2005). Overall, contact dermatitis falls into the two major categories of irritant and allergic forms. Both involve inflammatory processes and can have indistinguishable clinical characteristics of erythema (redness), induration (thickening and firmness), scaling (flaking), and vesiculation (blistering) in areas of direct contact with the chemical. Biopsies from affected sites reveal a mixed-cell inflammatory infil-

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trate of lymphocytes and eosinophils and spongiosis (intercellular edema), but are insufficient to distinguish the two conditions from each other or from certain other common syndromes.

Irritant Dermatitis Accounting for ∼80% of contact dermatitis cases, this condition arises from the direct action of chemicals on the skin. A chemical in this category is anticipated to give an adverse reaction to anyone if the concentration is high enough and the exposure time long enough. Certain chemicals at sufficient concentration produce an acute irritation, sometimes called a second-degree chemical burn, which can even result in scarring in serious cases. These include strong acids and alkalies and powerful oxidizing and reducing agents that substantially disrupt the cornified layer, produce cytotoxicity directly, and stimulate release of proinflammatory cytokines. More common is chronic cumulative irritation from repeated exposures to mild irritants such as soaps, detergents, solvents, and cutting oils. An example of eczema from cutting oil is shown in Fig. 19-2A. The chronic friction and production of small-scale trauma can wear away the lipid barrier of the stratum corneum, leading gradually to further damage (loss of cohesion, desquamation) that facilitates penetration of exogenous chemicals, and may be detectable as increased transepidermal water loss. In some cases, epidermal thickening occurs without much inflammation. In any case, increased penetrance can facilitate exposure to chemicals that elicit a subsequent allergic reaction. For example, because wet cement is alkaline and often contains chromates (commonly allergenic), chronic exposure can produce a composite response. The skin at some anatomic sites is more sensitive than at other sites. Eyelids have a thin epidermis and are quite sensitive, for example, and the back is more sensitive than the forearm or the scalp of individuals with male pattern baldness (Zhai et al., 2004). Individuals vary greatly in sensitivity to irritant dermatitis. Figure 19-2B shows an irritant reaction on the inside of the elbow on an atopic person. Comprising up to 20% of school children (Laughter et al., 2000), atopic individuals are the most sensitive to irritants and exhibit a propensity to produce specific IgE antibodies to allergens. They typically suffer from hay fever but do not seem more prone to allergic contact dermatitis (Belsito, 2005). These individuals usually have a poorer prognosis than nonatopics and have a higher frequency of persistent dermatitis. The best preventive measure for atopics and others is to avoid exposure to contact irritants, but in practice this strategy is difficult to implement. Lipid-rich moisturizers and barrier creams containing dimethicone or perfluoropolyethers may be useful in protecting skin from offending agents (Saary et al., 2005). Information on the irritancy of chemicals toward human skin may be obtained as part of differential diagnosis by patch testing for allergic response. The skin of laboratory animals (mice, rats, rabbits, guinea pigs) can be used for testing, but it is thinner and more sensitive than human skin to irritants. For development of new pharmaceuticals, cosmetics, and other consumer products, a great need exists for an in vitro system to determine the potential for irritant responses. Use of human epidermal cell cultures has been increasing as reconstructed epidermal and skin models come closer to the native differentiated state. For example, a recent study compared 50 chemicals for which data on 30 are available from patch testing (Tornier et al., 2006). The tests provided useful comparative data and, judging by viability (mitochondrial function), histology, and release of inflammatory mediators (IL-1α) suggested a parallel with natural skin. Such models offer advantages in convenience

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Figure 19-2. Examples of occupational skin toxicity. (See the colored insert.) The panels, available at the NIOSH website (http://www.cdc.gov/niosh/ocderm1.html), are a small selection from the 140-slide NIOSH program “Occupational Dermatoses—A Program for Physicians” prepared by Drs. E. Shmunes, M.M. Key, J.B. Lucas, and J.S. Taylor. (A, eczema from cutting oil; B, atopic irritant dermatitis; C, burn from ethylene oxide; D, burn from alkali exposure; E, sensitization to dichromate; F, beryllium granulomas; G, phototoxicity from lime juice; H, acne from cutting oil; I, leukoderma from rubber antioxidants; J, hyperpigmentation from mercaptobenzothiazole.)

and cost, appear applicable to photoxicity as well, and are more uniform in response than skin in the human population. Though advanced, the state of maturation in such culture models is not complete, as seen by histology and barrier function, resulting in their greater sensitivity than the skin (Netzlaff et al., 2005). In addition, extrapolation of the models to cumulative insult dermatitis presents a challenge.

Chemical Burns A chemical that is extremely corrosive can produce immediate coagulative necrosis resulting in considerable tissue damage with ulceration and sloughing. Sometimes referred to as a third-degree chemical burn, the damage does not have a primary inflammatory component and thus may not be classified as an irritant reaction. Examples of burns from ethylene oxide and alkali are shown in Fig. 19-2C and 2D. If the chemical is not quickly and completely removed, damage to the skin may continue and, with increased access to the circulation, systemic injury can occur. Table 19-2 lists some important corrosive compounds giving chemical burns in the occupational arena. Certain chemical warfare agents first used in

combat nearly a century ago and intermittently since, such as bis(2-chloroethyl)sulfide (sulfur mustard) or 2-chlorovinyl dichloroarsine (Lewisite), are potent vesicants upon skin contact and produce considerable damage when inhaled. Exposure today is rare, but the threat of use remains (McManus and Huebner, 2005).

Allergic Contact Dermatitis Allergic contact dermatitis is a delayed (T-cell mediated) hypersensitive reaction. To induce sensitization through the skin, chemical haptens generally penetrate the lipid barrier and, to be detected by the immune system, become attached to carrier proteins. The complete antigens are then processed by Langerhans cells (resident macrophages) and displayed on their surfaces with major histocompatibility complex II molecules. The Langerhans cells present the processed peptides to T helper type 1 cells in regional lymph nodes, thereby stimulating interleukin release and proliferation of the sensitive T helper cells. Over a 1–3 week period, memory T cells are thus generated and enter the circulation. Upon subsequent exposure to a specific antigen previously encountered, allergen presentation by the Langerhans cells results in a much greater response due to

CHAPTER 19

TOXIC RESPONSES OF THE SKIN

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Table 19-2 Selected Chemicals Causing Skin Burns chemical

comment

Ammonia

Potent skin corrosive Contact with compressed gas can cause frostbite Severe chemical burns Extremely exothermic reaction—dissolving in water can cause heat burns Liquid and concentrated vapors cause cell death and ulceration Solutions and vapors may burn Compressed gas can cause frostbite Severe burning with scar formation Severe, painful, slowly healing burns from high concentration Lower concentration causes delayed cutaneous injury Systemic absorption can lead to electrolyte abnormalities and death Calcium-containing topical medications and quaternary ammonium compounds are used to limit damage High concentration causes severe burns and blistering Liquid exposure produces blistering, deep burns Moist skin facilitates the formation of nitric acid causing severe yellow-colored burns White phosphorus continues to burn on skin in the presence of air Extremely corrosive even in low concentrations Systemic absorption through burn sites may result in cardiac arrhythmias, renal disease, and death High concentration causes deep burns, readily denatures keratin Severe burns with contact Skin contact rarely may result in respiratory sensitization

Calcium oxide (CaO) Chlorine Ethylene oxide Hydrogen chloride (HCl) Hydrogen fluoride (HF)

Hydrogen peroxide Methyl bromide Nitrogen oxides Phosphorus Phenol

Sodium hydroxide Toluene diisocyanate

homing by the memory cells to the skin, their clonal proliferation and their release of cytokines chemotactic for inflammatory cells and stimulatory for their further production. Because this process takes time, the characteristic dermal infiltration and spongiosis result after a delay (latent period) of 0.5–2 days (Mark and Slavin, 2006). Thousands of chemicals have been reported to give rise to allergic contact dermatitis, many across a variety of occupations and consumer products. Table 19-3 lists some common contact allergens, several of which are shown in Fig 19-3. Because most compounds in the chemical universe are only weakly active or infrequently encountered, much effort has focused on finding the major allergens in the population by systematic patch testing of dermatology patients.

Although not measuring sensitivity in the population at large, the results are quite useful. The panel of compounds tested can vary with geographic location to accommodate local usage, or it can be directed to specific anatomic sites such as the foot (Holden and Gawkrodger, 2005). Panels also are adapted to emerging trends as new products appear and others decline in use. Table 19-4 lists the chemicals (22) giving positive reactions in at least 3% of the subjects in a recent test from the North American Contact Dermatitis Group (Pratt et al., 2004). Other testing groups show similar results but often include some different chemicals or use them at different concentrations, which can affect the measured frequencies (Wetter et al., 2005). A number of these chemicals (nickel, dichromate, p-phenylenediamine, formaldehyde) have shown high prevalences

Figure 19-3. Structural formulas of some potent contact sensitizers.

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Table 19-3 Common Contact Allergens source Topical medications/ hygiene products

Plants and trees

Antiseptics

Rubber products

Leather Paper products

Glues and bonding agents

Metals

common allergens Antibiotics Bacitracin Neomycin Polymyxin Aminoglycosides Sulfonamides Preservatives Benzalkonium chloride Formaldehyde Formaldehyde releasers Quaternium-15 Imidazolidinyl urea Diazolidinyl urea DMDM Hydantoin Methylchloroisothiazolone Abietic acid Balsam of Peru Rosin (colophony) Chloramine Chlorhexidine Chloroxylenol Dichlorophene Dodecylaminoethyl glycine HCl Diphenylguanidine Hydroquinone Mercaptobenzothiazole p-Phenylenediamine Formaldehyde Glutaraldehyde Abietic acid Formaldehyde Nigrosine Bisphenol A Epichlorohydrin Formaldehyde Acrylic monomers Cyanoacrylates Chromium Cobalt

of reactivity for several decades, whereas others (e.g., gold salts), once thought innocuous, have recently become recognized as reactive (Cohen, 2004). An example of contact allergy to dichromate in cement is shown in Fig. 19-2E. Reduction in use of the most prevalent allergenic chemicals and their replacement by less allergenic substitutes is now advocated. Caution in using less characterized agents as replacements must be exercised, however, because their allergenicity may not become evident until they reach large populations of users, as has happened in several prominent cases (Uter et al., 2005). For example, methylchloroisothiazolinone/methylisothiazolinone, used in cosmetics, was replaced with the biocide methyldibromo glutaronitrile, which did not cause allergic contact dermatitis in initial screens. Upon widespread use, however, the latter also was shown to be a potent contact allergen (Kynemund Pedersen et al., 2004).

Therapeutics Benzocaine Idoxuridine α-Tocopherol (vitamin E) Corticosteroids Others Cinnamic aldehyde Ethylenediamine Lanolin p-Phenylenediamine Propylene glycol Benzophenones Fragrances Thioglycolates Pentadecylcatechols Sesquiterpene lactone Tuliposide A Glutaraldehyde Hexachlorophene Thimerosal (Merthiolate) Mercurials Triphenylmethane dyes Resorcinol monobenzoate Benzothiazolesulfenamides Dithiocarbamates Thiurams Potassium dichromate Rosin (Colophony) Triphenyl phosphate Dyes Epoxy resins p-(t-Butyl)formaldehyde resin Toluene sulfonamide resins Urea formaldehyde resins Mercury Nickel

Unlike contact irritants, where the response is generally proportional to the applied dose and time, contact allergens can elicit reactions at very small doses. Nevertheless, careful analysis from human and animal testing (Boukhman and Maibach, 2001; Arts et al., 2006) shows that a higher dose confers a greater likelihood of sensitization and that doses below a threshold for sensitization can have a cumulative effect. In addition, the dose required to elicit a reaction is lower after sensitization with a higher dose. Moreover, the dose dependence for sensitization displays nonlinearity, suggesting that the response of individual dendritic cells is sublinear, probably sigmoidal. Thus, more stimulation can produce a more than proportionally larger response, although at high doses saturation and sometimes even inhibition of the response become evident. This result emphasizes the importance of minimizing individual exposures. The findings also reveal a wide variation in human

CHAPTER 19

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Table 19-4 Prevalence of Positive Reactions in Patch Test Patients allergen Nickel sulfate Neomycin Balsam of Peru Fragrance mix Thimerosal Sodium gold thiosulfate Quaternium-15 Formaldehyde Bacitracin Cobalt chloride Methyldibromoglutaronitrile/ phenoxy ethanol Carba mix p-Phenylenediamine Thiuram mix Potassium dichromate Benzalkonium chloride Propylene glycol 2-Bromo-2-nitropropane Diazolidinyl urea Imidazolidinyl urea Tixocortol-21-pivalate Disperse blue 106

patients with positive patch tests (%) 16.7 11.6 11.6 10.4 10.2 10.2 9.3 8.4 7.9 7.4 5.8 4.9 4.8 4.5 4.3 4.3 4.2 3.3 3.2 3.0 3.0 3.0

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Table 19-5 Common Cross-Reacting Chemicals chemical

cross reactor

Abietic acid Balsam of Peru Bisphenol A

Pine resin (colophony) Pine resin, cinnamates, benzoates Diethylstilbestrol, hydroquinone monobenzyl ether Benzyl salicylate Chloroxylenol Imidazolidinyl urea, formaldehyde Aminophylline, piperazine Arylsulfonamide resin, chloroallyl-hexaminium chloride Resorcinol Parabens, hydroquinone monobenzyl ether p-Aminosalicylic acid, sulfonamide Parabens, p-aminobenzoic acid Hydroquinone monobenzyl ether Resorcinol, cresols, hydroquinone Tetraethylthiuram mono- and disulfide

Canaga oil Chlorocresol Diazolidinyl urea Ethylenediamine di-HCl Formaldehyde

Hydroquinone Methyl hydroxybenzoate p-Aminobenzoic acid Phenylenediamine Propyl hydroxybenzoate Phenol Tetramethylthiuram disulfide

source: Data from Pratt et al. (2004).

response to sensitization, which appears to have at least in part a genetic basis. Diagnosis and Testing When a patient exhibits allergic contact dermatitis, finding the responsible chemical is important to avoid continued exposure. For this purpose, patch testing is commonly employed by procedures refined over many years of practice since it was first employed a century ago (Mark and Slavin, 2006). On the washed backs of patients, who are not currently exhibiting contact dermatitis or using corticosteroids or other immunosuppressives, are placed patches each containing a small amount of a potential allergen. Conveniently, many of the materials are commercially available at standardized concentrations too low to produce irritant reactions. Certain chemicals normally are not tested because they induce too strong a response (urushiol from poison ivy) or might produce sensitization (beryllium). After 2–3 days, during which time a maximal reaction usually develops, the patches are removed and sites of exposure are scored for degree of response. Relevance to the pa-

Figure 19-4. Structural formulas of selected para-amino compounds that show cross-reactions in allergic contact sensitization.

tient’s actual environment must be considered so that exposure in daily life can be minimized to appropriate chemicals. Interpretation of the results and environmental modification should take into account the phenomenon of cross-sensitivity, where reactivity to a compound may be evident if it shares functional groups that have provoked sensitization in another compound. Figure 19-4 illustrates the principle with three amine compounds, and Table 19-5 lists some common cross-reacting chemicals. Animal testing to predict allergenicity has an extended history. A chemical is applied to intact or abraded skin or through intradermal injection with or without adjuvant to enhance sensitization. The reaction of the skin to subsequent challenge with the chemical is then observed and graded. This approach has successfully identified some strong sensitizers relevant to human exposures, but detection of weak sensitizers on a large scale is hampered by the usual difficulties in animal testing, including small animal numbers and limited experiment time to reduce expense. In addition, extrapolation of sensitivity measurements from laboratory animals to humans presents large uncertainties. Nevertheless, the local lymph node assay performed in mice has gained attention as a way to measure the pool of sensitized T cells by their proliferation in draining lymph nodes, illustrated by a comparison of potencies of Disperse Blue 106 and 2,4-dinitrochlorobenzene (Betts et al., 2005). Because sensitizers differ in potency by at least four orders of magnitude, a quantitative assay has a distinct advantage. Other steps in the sensitization process (e.g., percutaneous absorption) would be expected to influence the potency, but incorporation of structure–activity data may improve the validity of predictions for certain chemicals (Uter et al., 2005). To this end, a structure–activity study to streamline testing of new chemical products has identified

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15 classes of reactive functional groups, as well as several subject to activation by metabolism, as especially suspect of bestowing allergenicity (Gerner et al., 2004). That water solubility, octanol/water partitioning, and molecular weight (15% of the cortex), which secretes minute quantities of adrenal sex hormones. The adrenal cortical cells contain large cytoplasmic lipid droplets which consist of cholesterol and other steroid hormone precursors. The lipid droplets are in close proximity to the smooth endoplasmic reticulum and large mitochondria which contain the specific hydroxylase and dehydrogenase enzyme systems required to synthesize the different steroid hormones. Unlike polypeptidehormone-secreting cells, there are no secretory granules in the cytoplasm because there is direct secretion without significant storage of preformed steroid hormones.

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Steroid-hormone-producing cells of the adrenal cortex synthesize a major parent steroid with one to four additional carbon atoms added to the basic 17-carbon steroid nucleus. Because steroid hormones are not stored in any significant amount, a continued rate of synthesis is required to maintain a normal secretory rate. Once in the circulation, cortisol or corticosterone are bound reversibly to plasma proteins (such as transcortin, albumin). Under normal conditions 10% of the glucocorticoids are in a free unbound state. Adrenal steroids are synthesized from cholesterol by specific enzyme-catalyzed reactions and involve a complex shuttling of steroid intermediates between mitochondria and endoplasmic reticulum. The specificity of mitochondrial hydroxylation reactions in terms of precursor acted upon and the position of the substrate which is hydroxylated is confined to a specific cytochrome P-450. The common biosynthetic pathway from cholesterol is the formation of pregnenolone, the basic precursor for the three major classes of adrenal steroids. Pregnenolone is formed after two hydroxylation reactions at the carbon 20 and 22 positions of cholesterol and a subsequent cleavage between these two carbon atoms. In the zona fasciculata, pregnenolone is first converted to progesterone by two microsomal enzymes. Three subsequent hydroxylation reactions occur involving, in order, carbon atoms at the 17, 21, and 11 positions. The resulting steroid is cortisol, which is the major glucocorticoid in teleosts, hamsters, dogs, nonhuman primates, and humans. Corticosterone is the major glucocorticoid produced in amphibians, reptiles, birds, rats, mice, and rabbits. It is produced in a manner similar to the production of cortisol, except that progesterone does not undergo 17α-hydroxylation and proceeds directly to 21-hydroxylation and 11β-hydroxylation. In the zona glomerulosa, pregnenolone is converted to aldosterone by a series of enzyme-catalyzed reactions similar to those involved in cortisol formation; however, the cells of this zone lack the 17α-hydroxylase and thus cannot produce 17α-hydroxyprogesterone which is required to produce cortisol. Therefore, the initial hydroxylation product is corticosterone. Some of the corticosterone is acted on by 18-hydroxylase to form 18-hydroxycorticosterone, which in turn interacts with 18-hydroxysteroid dehydrogenase to form aldosterone. Since 18-hydroxysteroid dehydrogenase is found only in the zona glomerulosa, it is not surprising that only this zone has the capacity to produce aldosterone. In addition to the aforementioned steroid hormones, cells in the zona reticularis also produce small amounts of sex steroids including progesterone, estrogens, and androgens. The mineralocorticoids (e.g., aldosterone) are the major steroids secreted from the zona glomerulosa under the control of the rennin-angiotensin II system. The mineralocorticoids have their effects on ion transport by epithelial cells, particularly renal cells, resulting in conservation of sodium (chloride and water) and loss of potassium. In the distal convoluted tubule of the mammalian nephron, a cation exchange exists which promotes the resorption of sodium from the glomerular filtrate and the secretion of potassium into the lumen. Under conditions of decreased blood flow or volume, the enzyme renin is released into the circulation at an increased rate by cells of the juxtaglomerular apparatus of the kidney. Renin release has also been associated with potassium loading or sodium depletion. Renin in the peripheral circulation acts to cleave a plasma globulin precursor (angiotensinogen produced by the liver) to angiotensin-I. An angiotensin-converting enzyme (ACE) subsequently hydrolyzes angiotensin-I to angiotensin-II, which acts as a trophic hormone to stimulate the synthesis and secretion of aldosterone. Under normal

conditions negative feedback control to inhibit further renin release is exerted by the elevated levels of angiotensin (principally angiotensin-II) as well as the expanded extracellular fluid volume resulting from the increased electrolyte (sodium and chloride) and water reabsorption by the kidney. The principal control for the production of glucocorticoids by the zona fasciculata and zona reticularis is exerted by adrenocorticotropin (ACTH), a polypeptide hormone produced by corticotrophs in the adenohypophysis of the pituitary gland. ACTH release is largely controlled by the hypothalamus through the secretion of corticotropin-releasing hormone (CRH). An increase in ACTH production results in an increase in circulating levels of glucocorticoids and under certain conditions also can result in weak stimulation of aldosterone secretion. Negative feedback control normally occurs when the elevated blood levels of cortisol act either on the hypothalamus, anterior pituitary, or both to cause a suppression of ACTH secretion (Rothuizen et al., 1991). Fetal Adrenal Cortex A specialized fetal adrenal cortex exists in primates during late gestation (Mesiano and Jaffe, 1997). The cortex is composed of large polyhedral cells that produce abundant cortisol and estrogen precursors. The hormones secreted by the cortex are important for normal development of the fetus, and the steroid precursor dihydroepiandrosterone is converted to estrogen by the placenta. The cells of the fetal cortex are produced in the outer cortex and migrate medially, where they undergo hypertrophy and eventually apoptosis. After birth, there is a rapid regression, apoptosis, and lysis of the fetal cortex with dilatation of cortical capillaries and replacement by the typical three cortical zones. It is important not to misinterpret this as a lesion in neonatal primates since it represents physiological replacement of the fetal cortex with the definitive postnatal adrenal cortex. X-Zone of Adrenal Gland The X-zone in the mouse adrenal cortex is a similar unique physiologic phenomenon as the fetal cortex in primates. In contrast to the fetal cortex of primates, the X-zone develops postnatally in the inner cortex of mice and is fully formed at weaning. Its function is unknown, but it may be similar to the fetal zone in primates. After weaning, the X-zone degenerates at variable rates, depending on the sex of the mouse. In male mice, the X-zone undergoes degeneration at puberty with accumulation of intracellular fat globules. In unbred females, the zone undergoes slow regression and degeneration during the first pregnancy. As with the fetal zone in primates, it is important not to misinterpret the degeneration associated with regression of the X-zone in mice as a lesion.

Mechanisms of Adrenal Cortical Toxicity The reason the adrenal cortex is predisposed to the toxic effects of xenobiotic chemicals appears to be related to at least two factors. First, adrenal cortical cells of most animal species contain large stores of lipids used primarily as substrate for steroidogenesis. Many adrenal cortical toxic compounds are lipophilic and therefore can accumulate in these lipid-rich cells. Second, adrenal cortical cells have enzymes capable of metabolizing xenobiotic chemicals, including enzymes of the cytochrome P450 family. Many of these enzymes function in the biosynthesis of endogenous steroids and are localized in membranes of the endoplasmic reticulum or mitochondria. A number of toxic xenobiotic chemicals serve as

CHAPTER 21

TOXIC RESPONSES OF THE ENDOCRINE SYSTEM

815

Lysosome Endocytosis

LDL

SER

Acetyl CoA Steroidogenesis

HDL

nCEH Mitochondrion ACAT

Cholestrol

Steroid Hormone

nCEH

Storage Pathway Lipid Droplet CE

Nucleus

Figure 21-5. Cholesterol metabolism and steroid biosynthesis in adrenocortical and ovarian interstitial cells. Cholesterol is the substrate for steroid biosynthesis. Conversion of cholesterol to pregnenolone occurs in the mitochondria and oxidative reactions catalyzed by P450 enzymes occur in the smooth endoplasmic reticulum and mitochondria. Sources of cholesterol include lipoprotein uptake from serum (LDL and HDL), de novo synthesis from acetate via the acetyl coenzyme A pathway, and hydrolysis of cholesteryl ester (CE) by neutral CE hydrolase (nCEH). The storage pool in the form of lipid droplets is derived principally from the conversion of free cholesterol to CE catalyzed by acyl coenzyme A:cholesterol acyltransferase (ACAT). Direct uptake of CE from serum to the storage pool is minimal in the rat. (From Latendresse et al., 1993).

pseudosubstrates for these enzymes and can be metabolized to reactive toxic compounds. These reactive compounds result in direct toxic effects by covalent interactions with cellular macromolecules or through oxygen activation with the generation of free radicals (Colby, 1988; Hinson and Raven, 2006). Impaired Steroidogenesis Impaired steroidogenesis is an important mechanism of toxicity in the adrenal cortex. It can occur by inhibition of cholesterol biosynthesis or metabolism and by disruption of cytochrome P-450 enzymes. Both these mechanisms will lead to the accumulation of increased cytoplasmic lipid in the form of discrete droplets. Toxin Activation by CYP-450 Enzymes Toxins may be activated by many of the cytochrome P-450 enzymes in the cortical cells. Activation of toxins can result in the generation of reactive oxygen metabolites, membrane damage, and produce phospholipidosis in the cells. Exogenous Steroids Exogenous steroids can disrupt normal function and structure of the adrenal cortex. Exogenous agonists will induce negative feedback inhibition of ACTH secretion by the pituitary and will result in atrophy of the zona fasciculata and reticularis. Some steroids, such as the sex steroids, can induce proliferative lesions in the adrenal cortex. Exogenous steroid antagonists will block steroid hormone action, lead to increased ACTH secretion, and diffuse hyperplasia of the cortex. There is considerable species variation in the response of the adrenal cortex to exogenous chemicals. This is due to both inherent differences in the sensitivity to certain drugs and differences in the metabolic pathways of steroidogenesis. An interesting example is o, p  -DDD (Mitotane) which was originally developed to treat metastatic adrenal cortical cancer in humans; however, humans are relatively insensitive to the effects of o, p  -DDD, and the drug was not useful in the treatment of adrenal cancer. In contrast, dogs are more sensitive to the effects of o, p  -DDD, and it has been used ef-

fectively to treat pituitary-dependent hyperadrenocorticism due to autonomous secretion of ACTH by pituitary (corticotroph) tumors in a dose-dependent manner. o, p  -DDD is a selective toxin for the zona fasciculata and reticularis, thereby sparing the important functions of the zona glomerulosa (Vilar and Tullner, 1959). The zonae reticularis and fasciculata appear to be the principal targets of xenobiotic chemicals in the adrenal cortex. Classes of chemicals known to be toxic for the adrenal cortex include short chain (3 or 4 carbon) aliphatic compounds, lipidosis-inducers, and amphiphilic compounds (Yarrington et al., 1981, 1983, 1985). A variety of other compounds also may affect the medulla. The most potent aliphatic compounds are of 3-carbon length with electronegative groups at both ends. These compounds frequently produce necrosis, particularly in the zonae fasciculata and reticularis. Examples include acrylonitrile, 3-aminopropionitrile, 3-bromopropionitrile, 1-butanethiol, and 1,4-butanedithiol (Szabo et al., 1980). By comparison, lipidosis inducers can cause the accumulations, often coalescing, of neutral fats which may be of sufficient quantity to cause a reduction or loss of organellar function and eventual cell destruction. Lipidosis-Producing Compounds Cholesterol is the precursor substrate required to synthesize steroid hormones. Steroidogenic cells obtain cholesterol exogenously from serum lipoproteins and endogenously from de novo synthesis via the acetyl coenzyme A pathway (Fig. 21-5). The adrenal cortical cells and OI cells in the rat preferentially utilize serum high-density lipoproteins (HDLs) for their primary source of cholesterol and resort to de novo synthesis if HDL does not meet the demand of steroidogenesis. This is in contrast to Leydig cells of the testis, which preferentially utilizes de novo synthesis of cholesterol and uses an exogenous source only when intracellular synthesis does not meet the demand and the cholesterol pool has been depleted (Payne et al., 1985). Examples of the compounds causing lipidosis include aminoglutethimide, amphenone, and anilines. Tricresyl phosphate (TCP) and other triaryl phosphates cause a defect in cholesterol metabolism by blocking both the uptake from

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Lysosome Endocytosis

LDL

SER

Acetyl CoA Steroidogenesis

HDL

nCEH Mitochondrion Cholestrol ACAT

Steroid Hormone

nCEH

CE

Storage Pathway Lipid Droplet CE

Nucleus

CE

Figure 21-6. Pathogenesis of cholesteryl lipidosis in adrenocortical cells and ovarian interstitial cells. The defect in cholesterol metabolism occurs in the uptake from serum and storage pathways. An inhibition of neutral cholesteryl ester hydrolase (nCEH) by a xenobiotic chemical results in the accumulation of CE in the form of lipid droplets in the cytoplasm. Acyl coenzyme A:cholesterol acyltransferase (ACAT) (catalyzes the formation of CE from cholesterol) activity remains near normal levels. (From Latendresse et al., 1993).

serum and storage pathways. An inhibition of cytosolic neutral cholesteryl ester hydroxylase (nCEH) by triaryl phosphate (97% inhibition compared to controls) results in the progressive accumulation of cholesteryl ester in the form of lipid droplets in the cytoplasm of adrenal cortical and ovarian interstitial (OI) cells (Fig. 21-6) but not in testicular Leydig cells of rats (Latendresse et al., 1993). Acyl coenzyme A: cholesterol acyl transferase (ACAT), an enzyme that esterifies cholesterol to make cholesteryl ester, was depressed only 27% (compared to controls) resulting in elevated intracellular storage of cholesterol in the form of lipid droplets (Fig. 21-7) (Latendresse et al., 1994a,b, 1995). Disruption of Membrane Organellar Membrane Turnover Biologically active cationic amphiphilic compounds produce a generalized phospholipidosis that involves primarily the zonae reticularis and fasciculata and produce microscopic phospholipid-rich inclusions. These compounds affect the functional integrity of lyso-

somes, which appear ultrastructurally to be enlarged and filled with membranous lamellae or myelin figures. Examples of compounds known to induce phospholipidosis include chloroquine, triparanol, and chlorphentermine. Selective Destruction of Mitochondria and Smooth Endoplasmic Reticulum In addition, there is a miscellaneous group of chemicals that affect hydroxylation and other functions of mitochondrial and microsomal fractions (e.g., smooth endoplasmic reticulum) in the adrenal cortex. Examples of these compounds include o, p  -DDD and α-(1,4-dioxido-3-methylquinoxalin-2-yl)N -methylnitrone (DMNM). Other compounds in this miscellaneous category cause their effects by means of cytochrome P-450 metabolism and the production of toxic metabolites. A classic example is the activation of carbon tetrachloride, resulting in lipid peroxidation and covalent binding to cellular macromolecules of the adrenal cortex.

Figure 21-7. The effects of tricresyl phosphate (TCP) and butylated triphenyl phosphate (BTH) on the concentration of cholesteryl ester (CE) and cholesterol in adrenal gland and ovary of rats. Mean ± SEM (n = 8–9) mg/g wet weight of adrenal gland and ovary. ∗∗∗ Different (p ≤ 0.01, p ≤ 0.05, respectively) from control. (From Latendresse et al., 1993).

CHAPTER 21

TOXIC RESPONSES OF THE ENDOCRINE SYSTEM

Studies with o, p  -DDD have shown a dose-dependent mitochondrial degeneration with vacuolization in the zonae fasciculata and reticularis (cortisol- and sex hormone-producing zone) with sparing of the aldosterone-producing zona glomerulosa (multiformis) (Hart et al., 1971). The canine adrenal cortex is unusually sensitive to the cytotoxic effects of o, p  -DDD, which has led to the development of an effective drug (Lysodren or Mitotane) for the medical management of pituitary-dependent hypercortisolism (Cushing’s disease). Cessation of weekly maintenance treatment with o, p  -DDD after regression of clinical signs often results in a recrudescence of functional disturbances of cortisol-excess due to chronic ACTH over-stimulation of the adrenal cortex leading to hypertrophy and hyperplasia of the cortisol-producing zones. The canine adrenal cortex is considerably more sensitive to the effects of o, p  -DDD than the adrenal cortices of humans.

Selective Inhibition of Angiotensin Converting Enzyme (ACE) Drugs such as Captopril, used in the medical management of hypertension, selectively inhibit ACE and result in atrophy of the outer zona glomerulosa (multiformis) in the adrenal cortex. Inhibition of ACE decreases the synthesis of angiotensin II from angiotensin I leading to trophic atrophy of the zona glomerulosa. The decreased production of aldosterone results in decreased renal tubular reabsorption of sodium and chloride, increased serum potassium due to decreased tubular excretion, and decreased tubular reabsorption of water resulting in lower blood volume and pressure.

Selective Inhibition of ACYL-CoenzymeA: Cholesterol ACYL Transferase (ACAT) Compounds that inhibit ACAT often result in degeneration of the zonae fasciculata and reticularis in the adrenal cortex. There is a distinct species sensitivity with dogs > guinea pigs > rabbits > monkeys > rats. It is uncertain whether the mechanism of toxicity is due to a direct cytotoxic effect of the compound or the result of the selective ACAT inhibition (Dominick et al., 1993a,b; Reindel et al.; 1994; Junquero et al., 2001; Robertson et al., 2001). Many of the chemicals that cause morphological changes in the adrenal glands also affect cortical function. Chemically induced changes in adrenal function result either from blockage of the action of adrenocorticoids at peripheral sites or by inhibition of synthesis and/or secretion of hormone. In the first mechanism, many antisteroidal compounds (antagonists) act by competing with or binding to steroid hormone-receptor sites; thereby, either reducing the number of available receptor sites or by altering their binding affinity. Cortexolone (11α-deoxycortisol) an antiglucocorticoid and spironolactone, an antimineralocorticoid, are two examples of peripherally acting adrenal cortical hormone antagonists. Xenobiotic chemicals affecting adrenal function often do so by altering steroidogenesis and result in histologic and ultrastructural changes in adrenal cortical cells. For example, chemicals causing increased lipid droplets often inhibit the utilization of steroid precursors, including the conversion of cholesterol to pregnenolone. Chemicals that affect the fine structure of mitochondria and smooth endoplasmic reticulum often impair the activity of 11α-, 17α-, and 21α-hydroxylases, respectively, and are associated with lesions primarily in the zonae reticularis and fasciculata. Atrophy of the zona glomerulosa may reflect specific inhibition of aldosterone synthesis or secretion, either directly (e.g., inhibition of 18α- hydroxylation) or indirectly (e.g., suppression of the rennin-angiotensin system II) by chemicals such as spironolactone and captopril.

817

It is well documented that synthetic and naturally occurring corticosteroids are potent teratogens in laboratory animals. The principal induced defect is cleft lip or palate; however, there is a paucity of information on the direct effect of xenobiotic chemicals on the development of the adrenal cortex. For example, adrenal aplasia occurred in 7.6 of 9.8% of white Danish rabbits when thalidomide was given to their dams.

Pathologic Alterations and Proliferative Lesions in Cortical Cells Macroscopic lesions of chemically affected adrenal glands are characterized either by enlargement or reduction in size that often is bilateral. Cortical hypertrophy due to impaired steroidogenesis or hyperplasia due to long-term stimulation often is present when the adrenal cortex is increased in size. Small adrenal glands often are indicative of degenerative changes or trophic atrophy of the adrenal cortex. Midsagittal longitudinal sections of adrenal glands under the above conditions will reveal either a disproportionately wider cortex relative to the medulla or vice versa, resulting in an abnormal cortical:medullary ratio. Nodular lesions that distort and enlarge one or both adrenal glands suggest that a neoplasm is present. A single well-demarcated nodular lesion suggests a cortical adenoma whereas widespread incorporation of the entire adrenal gland by a proliferative mass is suggestive of cortical carcinoma, especially if there is evidence of local invasion into periadrenal connective tissues or into adjacent blood vessels and the kidney. Non-neoplastic lesions of the adrenal cortex induced by xenobiotic chemicals are characterized by changes ranging from acute progressive degeneration to reparative processes such as multifocal hyperplasia. Early degenerative lesions characterized by enlarged cortical cells filled with cytoplasmic vacuoles (often lipid) may result in a diffuse hypertrophy of the cortex. A lesion of this type has been observed in rats treated with an antibacterial compound α-(1,4-dioxido-3-methylquinoxalin-2-yl)-N -methylnitrone (DMNM). This type of vacuolar degeneration is a reflection of impaired steroidogenesis resulting in an accumulation of steroid precursors. More destructive lesions such as hemorrhage and/or necrosis are associated with an inflammatory response in the cortex. If the zona glomerulosa remains functional there will be no life-threatening electrolyte disturbances and no signs of hypoadrenocorticism (Addison’s disease). While many chemical agents that affect the adrenal cortex initially involve the zona reticularis and inner zona fasciculata, certain chemicals such as DMNM can cause a progressive degeneration of the entire adrenal cortex. Occasionally, a chemical’s effect is limited to a specific zone of the adrenal cortex and may be species specific (Yarrington and Johnston, 1994). Ultrastructural alterations of adrenal cortical cells associated with chemical injury are quite diverse in nature. The zonae reticularis and fasciculata typically are most severely affected, although eventually the lesions involve the zona glomerulosa. These lesions may be classified as follows: endothelial damage (e.g., acrylonitrile), mitochondrial damage (e.g., DMNM, o, p  -DDD, amphenone), endoplasmic reticulum disruption (e.g., triparanol), lipid aggregation (e.g., aniline), lysosomal phospholipid aggregation (e.g., chlorophentermine), and secondary effects due to embolization by medullary cells (e.g., acrylonitrile). Mitochondrial damage with vacuolization and accompanying changes in the endoplasmic reticulum and autophagocytic responses appear to be among the most common ultrastructural changes observed following chemical injury in the adrenal cortex. Because mitochondria and smooth endoplasmic

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reticulum form an intimate subcellular organellar network in cortical cells with important hydroxylases and dehydrogenase enzymes, it is not surprising that many chemical agents altering the ultrastructural morphology of cortical cells inhibit steroidogenesis. Chemically induced proliferative lesions of the adrenal cortex are less frequently reported and include hyperplasia, adenoma, and carcinoma. Unlike the diffuse cortical hyperplasia/hypertrophy associated with excess ACTH stimulation, chemically induced hyperplasia usually is nodular in type, often multiple in distribution, and composed of increased numbers of normal or vacuolated cortical cells. A variety of different chemicals are associated with an increased incidence of adrenal cortical neoplasia. Most of the reported tumors tend to be benign (adenomas) although an occasional tumor may be malignant (carcinomas). The zonae reticularis and fasciculata are more prone to develop tumors following chemical injury whereas the zona glomerulosa is spared unless invaded by an expanding tumor in the adjacent zones of the cortex. The tumorigenic agents of the adrenal cortex have a diverse chemical nature and use. Spontaneous proliferative lesions may be found in all zones of the adrenal cortex but in adult rats are found most frequently in the zona fasciculata. Spontaneous nodular hyperplasia of the adrenal cortex is common in the rabbit, golden hamster, rat, mouse, dog, cat, horse, and baboon. Naturally occurring adrenal cortical tumors are found infrequently in domestic animals, except adult dogs and castrated male goats. However, cortical adenomas and (to a lesser extent) cortical carcinomas have been reported in moderately high incidence in certain strains of laboratory hamsters (e.g., BIO 4.24 and BIO 45.5 strains) and rat (e.g., Osborne–Mendel, WAG/Rij, BUF, and BN/Bi strains). The incidence often increases markedly in rats over 18 months of age. Adrenal cortical neoplasms in mice are uncommon but the incidence may be increased by gonadectomy.

Assessment of Function of the Adrenal Cortex It may be useful to measure the function of the adrenal cortex. This can be accomplished by measuring glucocorticoid hormone concentrations in the blood or urine (expressed as a ratio to creatinine). It is important to remember the diurnal variations in secretion. Provocative testing is a useful tool to evaluate the functional capacity of the zonae fasciculata and reticularis by measuring the increase in secretion of glucocorticoids in response to exogenous ACTH. Light and electron microscopy and histomorphometry (cortico:medullary ratio and width of different cortical zones) are useful to characterize lesions that may disrupt the function of the cortical cells.

ADRENAL MEDULLA Normal Structure and Function The medulla constitutes approximately 10% of the volume of the adrenal gland. In the normal rodent adrenal gland and in most other laboratory animal species the medulla is sharply demarcated from the surrounding cortex. The bulk of the medulla is composed of chromaffin cells, which are the sites of synthesis and storage of the catecholamine hormones (Tischler et al., 1988a). In the rat and mouse, norepinephrine and epinephrine are stored in separate cell types that can be distinguished ultrastructurally after fixation in glutaraldehyde and postfixation in osmium tetroxide. The norepinephrine-containing core of the secretory granules appears electron-dense and is surrounded by a wide submembranous space

whereas epinephrine-containing granules are less dense, have a finely granular core, and a narrow space beneath the limiting membrane. Granules of varying densities may be found in the same cell types in the adrenal medulla of immature rats (Tischler and DeLellis, 1988a). Human adrenal medullary cells may contain both norepinephrine and epinephrine within a single chromaffin cell. The adrenal medulla contains variable numbers of ganglion cells in addition to chromaffin cells (Reznik et al., 1980). A third cell type has been described in the medulla and designated the small granule-containing (SGC) cell or small intensely fluorescent (SIF) cell. These cells morphologically appear intermediate between chromaffin cells and ganglion cells, and may function as inter-neurons. The adrenal medullary cells also contain serotonin and histamine, but it has not been determined if these products are synthesized in situ or taken up from the circulation. A number of neuropeptides also are present in rat chromaffin cells including enkephalins, neurotensin, and neuropeptide Y. In the catecholamine biosynthetic pathway, tyrosine is acted on by tyrosine hydroxylase to produce dopa, which is converted to dopamine by dopa decarboxylase. Dopamine in turn, is acted on by dopamine beta-hydroxylase to form norepinephrine, which is converted to epinephrine by phenylethanolamine-N -methyltransferase (PNMT). Tyrosine hydroxylase and PNMT are the principal rate limiting steps in catecholamine synthesis. The conversion of tyrosine into dopa and dopamine occurs within the cytosol of chromaffin cells. Dopamine then enters the chromaffin granule where it is converted to norepinephrine. Norepinephrine leaves the granule and is converted into epinephrine in the cytosol and epinephrine re-enters and is stored in the chromaffin granule. In contrast to the synthesis of catecholamines, which occurs in the cytosol, neuropeptides and chromogranin-A proteins are synthesized in the granular endoplasmic reticulum and are packaged into granules in the Golgi apparatus. Innervation plays an important role in regulating the functions of chromaffin cells. During adult life, stresses such as insulininduced hypoglycemia or reserpine-induced depletion of catecholamines produces a reflex increase in splanchnic nerve discharge, resulting both in catecholamine secretion and transsynaptic induction of catecholamine biosynthetic enzymes, including tyrosine hydroxylase. These effects become apparent during the first week of life, following an increase in the number of nerve terminals in the adrenal medulla. Other environmental influences including growth factors, extracellular matrix, and a variety of hormonal signals that generate cyclic AMP, may also regulate the function of chromaffin cells.

Mechanisms of Adrenal Medullary Toxicity Proliferative lesions of the medulla, particularly in the rat, have been reported to develop as a result of a variety of different mechanisms (Rosol et al., 2001; Tischler et al., 1985, 1988b, 1991). Warren et al. (1966) studied over 700 pairs of rats with parabiosis and found that more than 50% of male irradiated rats developed adrenal medullary tumors. A relationship exists between adenohypophyseal (anterior pituitary) hormones and the development of adrenal medullary proliferative lesions (Manger et al., 1982). For example, the long-term administration of growth hormone is associated with an increased incidence of pheochromocytomas as well as the development of tumors at other sites. Prolactin-secreting pituitary tumors, which occur commonly in many rat strains, also play a role in the development of proliferative medullary lesions. In addition,

LABELLED CHROMAFFIN CELL NUCLEI (% ± S.E.)

CHAPTER 21

TOXIC RESPONSES OF THE ENDOCRINE SYSTEM

Week 4

Week 8

20

*(6)

*

(6)

*(6)

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819

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(7)

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(6)

0

0 5,000 10,000 20,000

0

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Week 12

Week 26 20

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10 (6)

* (6)

* (6)

* (6)

10 (11)

(12)

* (10)

*

(9)

0

0 0

5,000 10,000 20,000

0

5,000 10,000 20,000

Vitamin D3 (IU/kg/day) Figure 21-8. Effects of vitamin D3 on percent of chromaffin cells labeled with BrdU during weeks 4, 8, 12, and 26 of dietary supplementation. Asterisks indicate statistically significant increases over corn oil controls. Numbers in parenthesis indicate numbers of rats scored for each point. Premature deaths or euthanasia caused loss of three animals from the 20,000 IU/kg/day group, two from the 10,000 IU/kg/day group and one control. Histological examination of these animals’ adrenal glands showed no detectable abnormalities. One extra animal at the start of the experiment was assigned to the control group at week 1. At least 2500 chromaffin cells were scored for each rat. (From Tischler et al., 1999).

several neuroleptic compounds that increase prolactin secretion by inhibiting dopamine production have been associated with an increased incidence of proliferative lesions of medullary cells in chronic toxicity studies in rats. Both nicotine and reserpine have been implicated in the development of adrenal medullary proliferative lesions in rats. Both chemicals act by a shared mechanism since nicotine directly stimulates nicotinic acetylcholine receptors whereas reserpine causes a reflex increase in the activity of cholinergic nerve endings in the adrenal (Tischler et al., 1995). A short dosing regimen of reserpine administration in vivo stimulates proliferation of chromaffin cells in the adult rat and the mechanism may involve a reflex increase in neurogenic stimulation via the splanchnic nerve. Several other drugs have been reported to increase the incidence of adrenal medullary proliferative lesions. These include zomepirac sodium (a nonsteroidal inflammatory drug) (Mosher and Kircher, 1988), isoretinoin (a retinoid), and nafarelin (LHRH analog), atenolol (β-adrenergic blocker), terazosin (α-adrenergic blocker), ribavirin (antiviral), and pamidronate (bisphosphonate) (Davies and Monro, 1995). Lynch et al. (1996) have reported that nutritional factors have an important modulating effect on the spontaneous incidence of adrenal medullary proliferative lesions in rats. Several sugars and sugar alcohols have produced adrenal medullary tumors at high dosages (concentrations of 10–20% in the diet), including xylitol, sorbitol, lacitol, and lactose (Baer, 1988). Although the exact mechanism involved is not completely understood, an important role for calcium has been suggested. High doses of slowly absorbed sugars and starches increase the intestinal absorption and urinary excretion of calcium. Hypercalcemia is known to increase catecholamine synthesis in response to stress, and low-calcium diets will reduce the incidence of adrenal medullary tumors in xylitol-treated rats. Other

compounds that may act by a similar mechanism of altered calcium homeostasis include the retinoids (which produce hypercalcemia) and conditions such as progressive nephrocalcinosis in aging male rats treated with nonsteroidal anti-inflammatory agents. Roe and Bar (1985) have suggested that environmental and dietary factors may be more important than genetic factors as determinants of the incidence of adrenal medullary proliferative lesions in rats. The incidence of adrenal medullary lesions can be reduced by lowering the carbohydrate content of the diet. Several of the chemicals that increase the incidence of adrenal medullary lesions, including sugar alcohols, increase absorption of calcium from the gut. Calcium ions as well as cyclic nucleotides and prostaglandins may act as mediators capable of stimulating both hormonal secretion and cellular proliferation. Tischler et al. (1996, 1999) presented data that vitamin D is the most potent in vivo stimulus yet identified for chromaffin cell proliferation in the adrenal medulla. Vitamin D3 (5000, 10,000, or 20,000 IU/kg/day in corn oil) resulted in a four- to fivefold increase in bromodeoxyuridine (BrdU) labeling at week 4 that diminished to a twofold increase by week 26 (Fig. 21-8). An initial preponderance of epinephrine-labeled (PNMT-positive) cells subsequently gave way to norepinephrine-labeled cells. By week 26, 89% of rats receiving the two highest doses of vitamin D3 had focal medullary proliferative lesions (BrdU-labeled focal hyperplasia, “hot spots”) and pheochromocytomas in contrast to absence of proliferative lesions in controls. This increase in medullary cell proliferation was associated with a significant increase in circulating levels of both calcium and phosphorus after vitamin D administration (Fig. 21-9). The nuclei of hyperplastic chromaffin cells were labeled by BrdU but were phenylethanolamine-N -methyl transferase-negative indicating that they most likely were norepinephrine-producing cells

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Figure 21-10. Photomicrographs of adrenal medullary sections stained for BrdU (dark nuclei) and PNMT (dark cytoplasm) to compare BrdU labeling of epinephrine (E) and norepinephrine (NE) cells. Vitamin D3 (20,000 IU/kg/day) caused a dramatic increase in BrdU labeling of predominantly E-cells at week 4. The response was greatly reduced at week 26 and there was no longer an E-cell predominance. A representative hyperplasic nodule in the vitamin D3 -stimulated adrenal medulla at week 26 (top left) is densely labeled with BrdU and is PNMT-negative (bar = 100 μm). (From Tischler et al., 1999).

Pathologic Alterations and Proliferative Lesions of Adrenal Medullary Cells

Figure 21-9. Effects of vitamin D3 on serum calcium and phosphorus levels at weeks 4, 8, 12, and 26. Sustained perturbation of both Ca++ and PO4 concentrations persisted throughout the time course. (From Tischler et al., 1999).

of the rat medulla (Fig. 21-10). The proliferative lesions usually were multicentric, bilateral, peripheral in location in the medulla, nearly all were PNMT negative, and appeared to represent morphologic continuum rather than separate entities. Earlier studies reported by the same research group had demonstrated that the vitamin D3 (20,000, 40,000 IU/kg/day) increase in chromaffin cell proliferation was observed as early as 1 week and had declined by 4 weeks. These findings support the hypothesis that altered calcium homeostasis is involved in the pathogenesis of pheochromocytomas in rodents, most likely through effects on increasing chromaffin cell proliferation (Fig. 21-11). Vitamin D3 , calcitrol (active metabolite of D3 ), lactose, and xylitol all failed to stimulate directly the proliferation of rat chromaffin cells in vitro. In summary, three dietary factors have been suggested to lead to an increased incidence of adrenal medullary proliferative lesions in chronic toxicity studies in rats (Roe and Bar, 1985). These are (1) excessive intake of food associated with feeding ad libitum; (2) excessive intake of calcium and phosphorus, since commercial diets contain 2–3 times more calcium and phosphorus than needed by young rats; and (3) excessive intake of other food components (e.g., vitamin D and poorly absorbable carbohydrates) which increase calcium absorption.

Adrenal Medullary Hyperplasia and Neoplasia The adrenal medulla undergoes a series of proliferative changes ranging from diffuse hyperplasia to benign and malignant neoplasia. The latter neoplasms have the capacity to invade locally and to metastasize to distant sites. Diffuse hyperplasia is characterized by symmetrical expansion of the medulla with maintenance of the usual sharp demarcation between the cortex and the medulla. The medullary cell cords often are widened, but the ratio of norepinephrine to epinephrine cells is similar to that of normal glands. Focal hyperplastic lesions are often juxtacortical but may occur within any area of the medulla. The small nodules of hyperplasia in general are not associated with compression of the adjacent medulla; however, the larger foci may be associated with limited medullary compression. The foci of adrenal medullary hyperplasia are typically composed of small cells with round to ovoid nuclei and scanty cytoplasm. At the ultrastructural level, the cells composing these focal areas of hyperplasia contain small numbers of dense core secretory granules resembling the granules of SIF or SGC cells (Tischler and DeLellis, 1988b). Larger benign adrenal medullary proliferative lesions are designated as pheochromocytomas. These lesions may be composed of relatively small cells similar to those found in smaller hyperplastic foci or larger chromaffin cells or a mixture of small and large cells. According to some authors, the lack of a positive chromaffin reaction in these focal proliferative lesions precludes the diagnosis of pheochromocytoma; however, the chromaffin reaction is quite insensitive and catecholamines (particularly norepinephrine) can be demonstrated in these proliferative lesions by biochemical extraction studies and by the formaldehyde- or glyoxylic-acidinduced fluorescence methods. Even in some of the larger medullary lesions, the chromaffin reaction is equivocal but catecholamines can be demonstrated both biochemically and histochemically.

CHAPTER 21

TOXIC RESPONSES OF THE ENDOCRINE SYSTEM

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Figure 21-11. Pathogenesis of adrenal medullary proliferative lesions associated with ingestion of polyols resulting in elevation of the blood calcium concentration. (Modified from Lynch et al., 1996).

Malignant pheochromocytomas invade the adrenal capsule and often grow in the periadrenal connective tissues with or without distant metastases. Proliferative lesions occur with high frequency in many strains of laboratory rats. The incidence of these lesions varies with strain, age, sex, diet, and exposure to drugs, and a variety of environmental agents (Nyska et al., 1999; Roe et al., 1995). Studies from the NTP historical database of two-year-old F344 rats have reported that the incidence of pheochromocytomas was 17.0% and 3.1% for males and females, respectively. Malignant pheochromocytomas were detected in 1% of males and 0.5% of females. In addition to F344 rats, other strains with a high incidence of pheochromocytomas include Wistar, NEDH (New England Deaconess Hospital), Long– Evans, and Sprague–Dawley. Pheochromocytomas are considerably less common in Osborne–Mendel, Charles River, Holtzman, and WAG/Rij rats. Most studies have revealed a higher incidence in males than in females (Figs. 21-12 and 21-13). Crossbreeding of animals with high and low frequencies of adrenal medullary proliferative lesions results in F1 animals with an intermediate tumor frequency. Pheochromocytomas are less common in mice than in most strains of rats.

There is a conspicuous relationship between increasing age and the frequency, size, and bilateral occurrence of adrenal medullary proliferative lesions in the rat. In the Long–Evans strain, medullary nodules have been found in less than 1% of animals under 12 months of age. The frequency increases to almost 20% in 2-year-old animals and to 40% in animals between 2 and 3 years of age. The mean tumor size increases progressively with age as does the frequency of bilateral and multicentric occurrence. A variety of techniques may be used for the demonstration of catecholamines in tissue sections. The chromaffin reaction is the oxidation of catecholamines by potassium dichromate solutions and results in the formation of a brown-to-yellow pigment in medullary tissue. The chromaffin reaction as traditionally performed possesses a low level of sensitivity and should not be used for the definitive demonstration of the presence of catecholamines in tissues. Similarly, both the argentaffin and argyrophil reactions, which have been used extensively in the past for the demonstration of chromaffin cells, also possess low sensitivity and specificity. Fluorescence techniques using formaldehyde or glyoxylic acid represent the methods of choice for the demonstration of catecholamines at the cellular level. These aldehydesform highly fluorescent derivatives

Figure 21-12. Rat strains with a high incidence of pheochromocytomas. (Modified from Lynch et al., 1996).

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Figure 21-13. Rat strains with a low incidence of pheochromocytomas. (Modified from Lynch et al., 1996).

with catecholamines, which can be visualized by ultraviolet microscopy. Immunohistochemistry provides an alternative approach for the localization of catecholamines in chromaffin cells and other cell types. Antibodies are available that permit epinephrine- and norepinephrine-containing cells to be distinguished in routinely fixed and embedded tissue samples. Several of the important enzymes involved in the biosynthesis of catecholamine hormones also may be demonstrated by immunohistochemical procedures. Antibodies to chromogranin-A can be used for the demonstration of this unique protein in chromaffin cells (Puchacz et al., 1996; Tischer et al., 1990, 1994). Pheochromocytomas in rats and human beings are both composed of chromaffin cells with variable numbers of hormonecontaining secretory granules (Fig. 21-14). The incidence is high in many strains of rats by comparison to human patients where pheochromocytomas are uncommon except in patients with inherited clinical syndromes of multiple endocrine neoplasia (MEN). These tumors in rats usually do not secrete excess amounts of catecholamines whereas human pheochromocytomas episodically secrete increased amounts of catecholamines leading to hypertension

and other clinical disturbances. There appears to be a striking species difference in the response of medullary chromaffin cells to mitogenic stimuli with rats being very sensitive compared to humans (Fig. 21-14). Tischler and Riseberg (1993) reported that adult rat chromaffin cells had a marked (10–40%) increase in bromodeoxyuridine (BrdU) labeled nuclei in vitro following the addition of the following mitogens: nerve growth factor (NGF), fibroblast growth factor (FGF), forskolin, and phorbol myristate (PMA) whereas human chromaffin cells had a minimal (1000 >1000 5 >1000 3 0.002

source: Kelce et al. (1995).

etc.) is weak at best, and inconclusive (Golden et al., 1998; Safe, 2005). Nevertheless, the possibility that these chemicals, as well as several other endocrine disruptors, may contribute to hormonally mediated cancers, reproductive effects, and developmental abnormalities, should not be discounted, and additional research in this area is warranted. DDT and Public Health: Risk-Benefit Considerations The Stockholm Convention on Persistent Organic Pollutants, ratified in 2004 by 50 states, outlawed the use of 12 industrial chemicals (the “Dirty Dozen”), including DDT. Yet, an exemption clause allows malaria-endemic nations to continue utilizing DDT for indoor residual wall spraying. The United Nations Environment Program estimates that about 25 countries would use DDT under this exemption from its ban. This situation is keeping the debate on the risks and benefits of DDT usage very much alive. On one hand, the environmental and human health effects of DDT are evident; on the other, one has to come to grips with the burden of mortality from malaria worldwide. Indeed, each year up to three million deaths due to malaria occur throughout the world, with 90% occurring in Africa (Breman et al., 2004). Most such mortality occurs in the first five years of life (Rogan and Chen, 2005). Thus, use of DDT might reduce mortality from malaria and overall infant mortality, if spraying is carried out according to planned schedule, which is not always the case, and if malaria-transmitting mosquitoes do not become resistant to DDT, a problem that in the past has forced

switching to other insecticides. The successful results obtained in South Africa in recent years (see earlier section “Economics and Public Health”), would support a continuous use of DDT. Indoor residual spraying might not cause the ecological effects that caused the ban of DDT, but could expose humans to amounts of DDT that may cause adverse health effects. In this regard, reproductive outcomes are of most concern. In particular, preterm births and early weaning (decreased duration of lactation), which can lead to increased infant mortality, have been associated with DDT exposure (Chen and Rogan, 2003; Rogan and Chen, 2005), though findings are controversial (Roberts et al., 2004). DDT remains, however, a public health intervention that is cheap, long lasting, and effective. Its judicious use should be combined with that of insecticide-treated bed nets, to prevent mosquito biting, and with a better availability of therapeutic interventions in affected populations.

Other Old and New Insecticides Rotenoids The roots of the East Asian Derris plants, particularly Derris Elliptica, and those of Lonchocarpus utilis and Lonchocarpus urucu in South America contain at least six rotenoid esters, among which the most abundant is rotenone. Rotenone is used as an agricultural insecticide/acaricide, particularly in organic farming (Isman, 2006). It is rather persistent in food crops after treatment, as indicated by half-life of 4 days in olives (Cabras et al., 2002). Rotenone is very toxic to fish; root extracts were used to paralyze fish for capture and consumption, and rotenone is still used in fishery management. Toxicity of rotenone in target and nontarget species is due to its ability to inhibit, at nanomolar concentrations, the mitochondrial respiratory chain, by blocking electron transport at NADH-ubiquinone reductase, the energy conserving enzyme complex commonly known as Complex I. Insect and fish mitochondria are particularly sensitive to Complex I inhibition (Degli Esposti, 1998). Purified rotenone has a high acute toxicity in rodents and dogs, and is less toxic to rabbits and birds (Ujvary, 2001). Poisoning symptoms include initial increased respiratory and cardiac rates, clonic and tonic spasms, and muscular depression, followed by respiratory depression. Acute intoxication in humans is rare, and the lethal dose in adults has been estimated at >140 mg/kg. However, a case report describes a fatal case in a 3.5-year-old girl who ingested an estimated 40 mg/kg of rotenone. Of note is that the label

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on the insecticide, which was manufactured in France and recommended for external use on animals, had stated “Natural Product – Non Toxic” (De Wilde et al., 1986). In recent years, rotenone has received much attention because of its potential role in the etiology of Parkinson’s disease. An earlier study by Heikkila et al. (1985) showed that stereotaxic administration to rats of rotenone damaged the dopaminergic nigrostriatal pathway, same as what observed with MPP+ (1-methyl-4-phenyl pyridinium, the active metabolite of 1-methyl-4-phenyl- 1,2,3,6tetrahydropyridine, MPTP), a known parkinsonism-causing chemical, which is also a Complex I inhibitor (Degli Esposti, 1998). More recent studies have shown that administration of rotenone to rats (2–3 mg/kg/d for 1–5 weeks) caused selective nigrostriatal degeneration, though inhibition of Complex I was observed uniformly in brain (Betarbet et al., 2000; Sherer et al., 2003). The finding that rotenone also produced protein inclusions, similar to Lewy bodies, that stained positively for ubiquitin and alpha-synuclein, suggests that the rotenone model for Parkinson’s disease would be even better than the MPTP model (Betarbet et al., 2002). However, the severe systemic toxicity of rotenone, the high variability across and within strains, and reports on nonspecific CNS effects (LaPointe et al., 2004), have also pointed out the limitations of the rotenone model (Li et al., 2005). There is no evidence of Parkinson’s diseaselike clinical signs or neurodegenerative pathology in chronic dietary studies (Hollingworth, 2001). Thus, although rotenone may represent an useful experimental model, its primary role, if any, in the etiology of Parkinson’s disease in the general population is still unproven (Hollingworth, 2001; Li et al., 2005).

Nicotine The tobacco plant (Nicotiana tabacum, Nicotiana rustica) was introduced in Europe in 1559 from the Americas where it had long been cultivated primarily for smoking. Tobacco extracts have been used to repel and kill insects since 1690, and tobacco smoke was also used for fumigation (Ujvary, 2001). Nicotine is an alkaloid extracted from the leaves of tobacco plants, and is used as a free base or as the sulfate salt. The most notorious commercial preparation, Black Leaf 40, has been discontinued. Very little nicotine is used currently in the United States, but nicotine is still used as a minor insecticide in some Asian countries. It is a systemic insecticide effective toward a wide range of insects, including aphids, thrips, and whiteflies (Ujvary, 1999). As the primary component of tobacco used for smoking or chewing, nicotine’s pharmacology and toxicology have been thoroughly investigated (Benowitz, 1996; Taylor, 1996). Nicotine exerts its pharmacological and toxic effects in mammals and insects by activating nicotinic acetylcholine receptors (nAChRs). In vertebrates, nAChRs are expressed at neuromuscular junctions, in the PNS and in the CNS; in insects, nAChRs are confined to the nervous system (Eldefrawi and Eldefrawi, 1997). Interaction of nicotine with nAChRs produces initial stimulation followed by protracted depolarization, which results in receptor paralysis. The overall effect is the summation of stimulatory and inhibitory effects of nicotine at all sites expressing nAChRs. At high doses, parasympathetic stimulation and ganglionic and neuromuscular blockade predominate (Matyunas and Rodgers, 2001). Nicotine has a high acute toxicity in vertebrates, with LD50 s usually below 50 mg/kg (Ujvary, 2001). Signs and symptoms of poisoning include nausea, vomiting, muscle weakness, respiratory effects, headache, lethargy, and tachycardia. Most cases of poisoning with nicotine occur after exposure to tobacco products, or gum or patches.

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Workers who cultivate, harvest, or handle tobacco may experience green tobacco sickness, caused by dermal absorption of nicotine. Neonicotinoids By various chemical modifications of nicotine and other nAChRs agonists, new classes of insecticides have been developed that contain a nitromethylene, nitroimine or cyanoimine group, and are referred to as neonicotinoids. One of the first compounds synthesized was nithiazine, a nitromethylenyl heterocyclic compound highly toxic toward insects but with low mammalian toxicity. Nithiazine was not developed commercially because of its photo-instability. Further structure-activity studies led to the development of imidacloprid, nitenpyram, acetamiprid, and other neonicotinoid compounds (Fig. 22-14; Matsuda et al., 2001). The insecticidal activity of neonicotinoids is attributed to activation of nAChRs. They are used primarily for crop protection as systemic insecticides, but are also effective against fleas in cats and dogs (Schenker et al., 2003). The mammalian toxicity of neonicotinoids is similar to that of nicotine, and correlates with agonist action and binding affinity at the nAChRs. Acute oral toxicity (LD50) in rats ranges from 180 to >2000 mg/kg (Fig. 22-14), while dermal toxicity is much lower (2000–5000 mg/kg), likely because of the low lipophilicity (Tomizawa and Casida, 2005). Signs and symptoms of toxicity are attributable to stimulation of nAChRs. Some neonicotinoids (imidocloprid, thiacloprid) are particularly toxic to birds, others (thiacloprid) to fish. Most neonicotinoids are not mutagenic or carcinogenic, nor teratogenic. Neonicotinoids undergo limited biotransformation in mammals, involving mostly cytochrome P450-mediated oxidative reactions (Sheets, 2001; Tomizawa and Casida, 2005). Neonicotinoids account for 10–15% of the total insecticide market, and their use is increasing faster than other insecticides (Matsuda et al., 2001; Tomizawa and Casida, 2005). The main reason for their success lies in their selectivity profile, which is largely attributable to their specificity toward insect vs. mammalian nAChRs. The nAChR consists of diverse subtypes assembled in combination from ten α and four β, γ, δ, ε subunits. The most abundant subtypes in the vertebrate nervous system are α4β2 and α7, which are insensitive and sensitive, respectively, to α-bungarotoxin. In insects, neonicotinoids have been shown to bind to at least three pharmacologically distinct nAChRs (Sheets, 2001; Matsuda et al., 2001). Table 22-16 shows the in vitro effects of some neonicotinoids toward insect nAChRs and mammalian α4β2 nAChRs, and compares them to nicotine. Structural features of neonicotinoids that contribute to their selective actions at insect nAChRs have been described (Nakayama and Sukekawa, 1998; Matsuda et al., 2001; Tomizawa and Casida, 2005). Formamidines Formamidines, such as chlordimeform [(N -(4chloro-o-tolyl)-N , N -dimethylformamidine] or amitraz [N  -2,4(dimethyl-phenyl)-N -N ((2,4-dimethylphenyl) imino) methyl-N methanimidamide] are used in agriculture and in veterinary medicine as insecticides/acaricides (Hollingworth, 1976). Their structures are closely related to the neurotransmitter norepinephrine (Fig. 22-15). In invertebrates, these compounds exert their toxicity by activating an octopamine-dependent adenylate cyclase (Nathanson, 1985). In mammals, symptoms of formamidines poisoning are sympathomimetic in nature (Beeman and Matsumura, 1973). The similarity between insect octopamine receptors and mammalian α2 -adrenergic receptors had suggested the latter as a possible target for formamidines. In vivo and in vitro studies have indeed shown that formamidines act as rather selective

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Figure 22-14. Structures of nicotine and of neonicotinoid insecticides with indication of their acute oral toxicity in rat and their octanol/water partition (P). Data are derived from Tomizawa and Casida (2005).

agonists at α2 –adrenergic receptors (Hsu and Lu, 1984; Hsu and Kakuk, 1984; Costa et al., 1988; Altobelli et al., 2001). Chlordimeform’s metabolism plays a most relevant role in its toxicity. The N -demethylated metabolite (desmethylchlordimeform) is more acutely toxic than chlordimeform, and displays a >400-fold higher potency toward α2 –adrenoceptors (Ghali and Hollingsworth, 1985; Costa and Murphy, 1987). Two other metabolites of chlordimeform, 4-chloro-toluidine and N -formyl-4-chloro-o-toluidine, are believed to be responsible for the observed haemangioendotheliomas in mice observed in carcinogenicity studies (IPCS, 1998). Chlordimeform was classified as a probable human carcinogen (Group 2A) by IARC

in 1990. Given the increasing evidence of an association between exposure to chlordimeform and 4-chloro-o-toluidine and bladder cancer (Popp et al., 1992), chlordimeform was withdrawn form the market in 1992. Amitraz, in contrast, remains on the market and is still used worldwide for the control of ectoparasites in farm animals and crops. In recent years several cases of acute amitraz poisoning have been reported, particularly in Turkey, and most involved children (Yaramis et al., 2000; Caksen et al., 2003; Elinav et al., 2005; Proudfoot, 2003). Signs and symptoms of poisoning mimicked those of α2 -adrenergic receptor agonists such as clonidine, and included nausea, hypotension, hyperglycemia, bradycardia, and

CHAPTER 22

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Table 22-16 Specificity of Neonicotinoids for Insect and Vertebrate nAChRs IC50 (nM)

vertebrate insecticide

insect

α4β2

selectivity ratio

Imidacloprid Acetamiprid Thiacloprid Nitenpyram (-)Nicotine

4.6 8.3 2.7 14.0 4000

2,600 700 860 49,000 7

565 84 319 3,500 0.002

source: Tomizawa and Casida, 2005.

Figure 22-15. Structures of the formamidine insecticides/acaricides amitraz and chlordimeform. Structures of the mammalian neurotransmitter norepinephrine and of the insect neurotransmitter octopamine are also shown.

miosis. No deaths occurred. Though α2 -adrenoceptor antagonists such as yohimbine have proven useful as antidotes in animals (Andrade and Sakate, 2003), their usefulness in managing amitraz poisoning in humans has not been evaluated. Avermectins The avermectins are macrocyclic lactones, first isolated in 1975 from the fermentation broth of the actinomycete Streptomyces avermitilis, which originated from a Japanese soil

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sample (Campbell, 1989; Fisher and Mrozik, 1992). This fungus synthesizes eight individual avermectins, of which avermectin B1a displays the highest antiparasitic activity. Currently, abamectin (a mixture of 80% avermectin B1a and 20% avermectin B1b ) is used as an insecticide, whereas the semisynthetic derivatives of avermectin B1a , emamectin benzoate, and ivermectin, are used as insecticides, and for parasite control in human and veterinary medicine, respectively (Stevens and Breckenridge, 2001). Abamectin is used primarily to control mites, whereas emamectin benzoate is effective at controlling lepidopterian species in various crops. Ivermectin is used as an antihelmintic and antiparasitic drug in veterinary medicine, and in humans it has proven to be an effective treatment for infection of intestinal threadworms, onchocerciasis (river blindness), and lymphatic filariasis (Stevens and Breckenridge, 2001). In insects and nematodes, avermectins exert their toxic effects by binding to, and activating, glutamate-dependent chloride channels (Arena et al., 1995). Avermectins have a high acute toxicity, with oral LD50 s in rats of 11 (abamectin) to 80 (emamectin) mg/kg. Toxicity is higher in neonate animals, possibly because of a deficient blood-brain barrier (Stevens and Breckenridge, 2001). Signs and symptoms of intoxication include hyperexcitability, tremors, and incoordination, followed by ataxia and coma-like sedation. These effects are due to the ability of avermectins to activate GABAA receptor-gated chloride channels in the vertebrate CNS (Pong et al., 1982; Fisher and Mrozik, 1992). Activity at GABAA receptors also mediates the anticonvulsant effects of avermectins, but because the same target seems to mediate both pharmacological and toxic effects, the potential of avermectins as anticonvulsants is limited (Dawson et al., 2000). Avermectins are also strong inhibitors of P-glycoprotein, a plasma membrane protein whose main function is the ATPase-dependent transport of foreign substances from the cell (Didier and Loor, 1996). As such, avermectins are being investigated for their potential ability to inhibit multidrug resistance of tumor cells (Korystov et al., 2004). In this respect, the complete sequencing of the S. avermitilis genome would allow the definition of the precise biosynthetic pathways and regulatory mechanisms for avermectins, which in turn may lead to engineering of this fungus to produce pharmacological compounds of interest (Yoon et al., 2004). Given the wide use of avermectins, and particularly that of ivermectin in Africa, there is little evidence of adverse health effects in humans. The major effect following administration of active doses of ivermectin (0.1–0.2 mg/kg) is a severe inflammatory response (the Mazzotti reaction), characterized by pruritis, erithema, vesicle and papulae formation, and attributable to the killing of microfiliae which dislodge from their site of infestation and are transported in the blood and body fluids (Ackerman et al., 1990).

Phenylpyrazoles A relatively new class of insecticides is that of phenylpyrazole derivatives, of which fipronil, commercialized in the mid-1990s, was the first one brought to market. Fipronil is a broadspectrum insecticide with moderate mammalian toxicity (LD50 in rat: oral, 97 mg/kg; dermal, >2000 mg/kg), and a high selectivity for target species. Fipronil acts as a blocker of the GABAA -gated chloride channel, but binds to a site different from the picrotoxin binding site used by organochlorine insecticides. It also has a much higher specificity for insect receptors over mammalian receptors (Hainzl et al., 1998). There is no evidence that fipronil is an eye or skin irritant, or has any mutagenic, carcinogenic, or teratogenic effects. A number of human poisonings with fipronil have been reported that resulted from accidental or intentional ingestion. Less than 20% of

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the patients developed seizures, and all recovered (Mohamed et al., 2004). Bacillus Thuringiensis The past decade has seen increasing research and development in the area of biopesticides, that is pesticides derived from natural materials such as plants, bacteria, and fungi. As of 2001, there were 195 registered biopesticide active ingredients in the United States, and 780 products. Biopesticides fall into three major classes: (1) Microbial pesticides, which consist of a microorganism (e.g., a bacterium, fungus or protozoan) as the active ingredient. The most widely used microbial pesticides are subspecies and strains of B. thuringiensis (Bt) which act as insecticides. Other microbial pesticides can control different kinds of pests; for example there are fungi that can control certain weeds, and others that can kill specific insects. (2) Plant-incorporated-protectants, which are pesticidal substances that plants produce from genetic material that has been added to the plant. For example, a plant can be genetically manipulated to produce the Bt pesticidal protein. (3) Biochemical pesticides, that are naturally occurring substances that control pests by nontoxic mechanisms. Examples are sex pheromones, that interfere with mating of insects, or various scented plant extracts that attract insect pests to traps (Sudakin, 2003). Biopesticides represent a little more than 1% of the world pesticide market, but Bt products represent 80% of all biopesticides sold (Whalon and Wingend, 2003). Bt is a soil microorganism that produces proteins that are selectively toxic to certain insects. Its name comes from the German region of Thuringia, where this strain was found in 1915. Bt-based microbial insecticides were commercialized in France in 1938 and in the United States in 1961. Over 150 insecticidal crystal proteins (Cry) have been identified in Bt and in the closely related Bacillus cereus (Schnepf et al., 1998). After ingestion of Bt by an insect, the crystal proteins are solubilized, and proteolytically processed to active toxins (δ-endotoxin) in the insect’s midgut. Here they bind to specific receptors in the epithelial cells and insert into the cellular membrane. Next, aggregation of inserted crystal protein occurs, resulting in the formation of pores, which lead to changes in K+ fluxes across the epithelial cells, and to changes in pH. Ultimately, cells of the midgut epithelium are destroyed by the high pH and by osmotic lysis. Insects eventually die as a result of gut paralysis and feeding inhibition, and subsequent starvation and septicemia (Gringorten, 2001). Bt targets primarily leaf-feeding lepidoptera, breaks down rapidly in UV light, and exhibits low mammalian toxicity. The basis for the selective toxicity of Bt is attributed to the fact that crystalline Bt endotoxins require activation by alkalis and/or digestion, conditions absent in the mammalian stomach (Ujvary, 2001). A summary of the toxicology studies in mammals of Btbased insecticides is provided by McClintock et al. (1995). Adverse health effects in humans are infrequent and include allergic reactions and infections (Ujvary, 2001). Bt genes are also expressed in a variety of crop plants, most notably cotton and corn. Thus, the plant, instead of the Bt bacterium, produces the substance that affects the insect upon feeding. Resistance can develop to Bt toxins that involves alterations in the processing of Cry toxin in the insect’s gut or in its binding to receptors (Whalon and Wingerd, 2003).

INSECT REPELLENTS Insect-transmitted diseases remain a major source of illness and death worldwide, as mosquitoes alone transmit disease to more than 700 million persons annually (Fradin and Day, 2002). Though insect-borne diseases represent a greater health problem in tropical

and subtropical climates, no part of the world is immune to their risks. For example, in 1999, the West Nile virus, transmitted by mosquitoes, was detected for the first time in the western hemisphere. In the New York City area, 62 persons infected with the West Nile virus were hospitalized and seven died (Nash et al., 2001). Other arthropod-borne viral diseases (e.g., equine encephalitis) and tick-borne diseases (e.g., Lyme disease) are also of concern; additionally, other insect bites can be associated with variable adverse health effects, from mild irritation and discomfort to possible allergic reactions. Insect repellents are thus widely used to provide protection toward insect bites. The best known and most widely used insect repellent is DEET. Botanical insect repellents based on citronella or oil of eucalyptus, and a biopesticide structurally similar to the aminoacid alanine are also commercialized in Europe and the United States (Fradin and Day, 2002). A new compound, picaridin, has been recently approved for use as an insect repellent; it is effective against biting flies, mosquitoes, ticks and fleas, and has a very favorable toxicological profile (USEPA, 2005).

DEET DEET (N , N -diethyl-m-toluamide or N , N -diethyl-3-methylbenzamide) was first developed by the USDA in 1946 for use in the military, and was registered as an insect repellent for the general public in 1957. The USEPA estimates that 30% of the U.S. population uses DEET every year. More than 200 formulations exist with varying concentrations of DEET (commonly 4.75–40%) which are applied directly to the skin or on clothing. DEET is very effective at repelling insects, flies, fleas, and ticks, and protection time increases with increasing concentrations (Fradin and Day, 2002). Percutaneous absorption of DEET varies from 7.9 to 59%, depending on the species tested and the conditions of the study (Osimitz and Murphy, 1997). DEET undergoes oxidative biotransformation catalyzed by various cytochromes P450, and is excreted mostly in the urine (Sudakin and Trevathan, 2003). DEET has low acute toxicity, with LD50 values in the rat of 1892 mg/kg (oral) and >5,000 mg/kg (dermal) (Schoenig and Osimitz, 2001). From 1961 to 2002, eight deaths were reported related to DEET: three resulted from deliberate ingestion, while two were reported following dermal exposure (Tenenbein, 1987; Bell et al., 2002). The remaining three cases were children, age 17 months to 6 years (Zadikoff, 1979). Subchronic toxicity studies in various species did not reveal major toxic effects, with the exception of renal lesions in male rats; these were considered to be reflective of α2u -globulin induced nephropathy, a condition unique to male rats and not occurring in humans (Schoenig and Osimitz, 2001). No significant effects of DEET were seen in mutagenicity, reproductive toxicity, and carcinogenicity studies. Acute and chronic neurotoxicity studies also provided negative results (Schoenig et al., 1993). Yet, several case reports over the past 40 years have indicated neurological effects of DEET, and most of these were in children (Petrucci and Sardini, 2000; Osimitz and Murphy, 1997; Hampers et al., 1999; Sudakin and Trevathan, 2003; Briassoulis et al., 2001; MMWR, 1989; Gryboski et al., 1961). The most common symptoms reported were seizures. Given that seizure disorders occur in 3–5% of children, and almost 30% of children in the United States are utilizing DEET, an association just by chance is certainly possible. Possible mechanism(s) responsible for neurotoxic effects of DEET are unknown, though it has been suggested that DEET’s structure is similar to that of nikethamide, a convulsant (Briassoulis et al., 2001). It has also been suggested that DEET may disrupt the permeability of the blood–brain barrier,

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Table 22-17 Some Mechanisms of Action of Herbicides mechanism

chemical classes (example)

Inhibition of photosynthesis

Triazines (atrazine), Substitued ureas (diuron), Uracils (bromacil) Dinitrophenols Phenoxy acids (2,4-D), Benzoic Acids (dicamba), Pyridine acids (picloram) Dinitroanilines Aryloxyphenoxyproprionates (diclofop)

Inhibition of respiration Auxin growth regulators

Inhibition of protein synthesis Inhibition of lipid synthesis Inhibition of specific enzymes • Glutamine synthetase • Enolpyruvylshikimate -3phosphate synthetase • Acetalase synthase Cell membrane disruptors

Glufosinate Glyphosate Sulfonylureas Bipyridyl derivatives (Paraquat)

but results are inconclusive (Abdel-Rahman et al., 2002). Overall, given its long-standing and widespread use, DEET appears to be relatively safe when used as recommended (Osimitz and Murphy, 1997; Koren et al., 2003). A risk assessment by the Canadian Pest Management Regulatory agency has recommended, however, that toddlers and children, up to 12 years old, should only be exposed to products with up to 10% DEET (Sudakin and Trevathan, 2003). For all other individuals, products with up to 30% DEET can be used, as they appear safe and effective (Fradin and Day, 2002).

HERBICIDES Herbicides are chemicals that are capable of either killing or severely injuring plants. They represent a very broad array of chemical classes and act at a large number of sites of metabolic functions and energy transfer in plant cells (Duke, 1990). Some of the various mechanisms by which herbicides exert their biological effects are shown in Table 22-17, together with examples for each class. Another method of classification pertains to how and when herbicides are applied. Thus, preplanting herbicides are applied to the soil before a crop is seeded; preemergent herbicides are applied to the soil before the time of appearance of unwanted vegetation; and postemergent herbicides are applied to the soil or foliage after the germination of the crop and/or weeds (Ecobichon, 2001a). Herbicides are also divided according to the manner they are applied to plants. Contact herbicides are those that affect the plant that was treated, while translocated herbicides are applied to the soil or to above-ground parts of the plant, and are absorbed and circulated to distant tissues. Nonselective herbicides will kill all vegetation, while selective compounds are those used to kill weeds without harming the crops. In the past decade, the development of herbicide-resistant crops through transgenic technology has allowed the use of nonselective compounds as selective herbicides (Duke, 2005). A final classification, of relevance to adverse health effects in nontarget species, relies, on the other hand, on chemical structures, as indicated below. For the past several decades, herbicides have represented the most rapidly growing sector of the agrochemical market, and these compounds now represent almost half of the pesticides used in the

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United States, and more than one-third of those utilized worldwide (Table 22-3). This can be ascribed in part to movement to monocultural practices, where the risk of weed infestation has increased, and to mechanization of agricultural practices because of increased labor costs. In addition to agriculture and home and garden uses, herbicides are also widely utilized in forestry management and to clear roadsides, utilities’ rights of way, and industrial areas. In terms of general toxicity, herbicides, as a class, display relatively low acute toxicity, compared for example to most insecticides. There are exceptions, however, such as paraquat. A number of herbicides can cause dermal irritation and contact dermatitis, particularly in individuals prone to allergic reactions. Other compounds have generated much debate for their suspected carcinogenicity or neurotoxicity. The principal classes of herbicides associated with reported adverse health effects in humans are discussed below.

Chlorophenoxy Compounds Chlorophenoxy herbicides are characterized by an aliphatic carboxylic acid moiety attached to a chlorine-or-methyl-substituted aromatic ring. The most commonly used compound of this class is 2,4-dichlorophenoxyacetic acid (2,4-D), while others are 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 4-chloro-2methylphenoxyacetic acid (MCPA) (Fig. 22-16). Chlorophenoxy herbicides are chemical analogues of auxin, a plant growth hormone, and produce uncontrolled and lethal growth in target plants. Because the auxin hormone is critical to the growth of many broad-leaved plants, but is not used by grasses, chlorophenoxy compounds can suppress the growth of weeds (e.g., dandelions) without affecting the grass. Once absorbed, they selectively eliminate broad-leaved plants, due to their larger leaf area and greater absorption. 2,4-D is one of the most widely used herbicides throughout the world, and is primarily used in agriculture to control weeds in corn and grain, in forestry, and in lawn care practices. 2,4,5-T has been largely withdrawn from use because of concerns that arose from contamination of some formulations with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), which can derive from the reaction of two molecules of 2,4,5-trichlorophenol (Fig. 22-17). A 50:50 mixture of the n-butyl esters of 2,4,-D and 2,3,5-T, known as Agent Orange (from the color of the barrels which contained it), was extensively used as a defoliant during the Vietnam War, and was found to be contaminated with TCDD to a maximum of 47 μg/g. Exposure of military personnel and Vietnamese population to Agent Orange has raised concerns on possible long-term health effects, particularly carcinogenicity and reproductive toxicity (IOM, 1996), which are ascribed to the presence of TCDD. 2,4-D is a compound of low to moderate acute toxicity, with oral LD50 s in rodents of 300–2000 mg/kg. The dog is more sensitive, possibly because of its lower ability to eliminate organic acids via the kidney. Upon oral exposure, 2,4-D is rapidly absorbed, and its salts and esters rapidly dissociate or hydrolyze in vivo, so that toxicity depends primarily on the acid form. It binds extensively to serum albumin, but does not accumulate in tissue, and is excreted almost exclusively through the urine. Ingestion of 2,4-D has caused several cases of acute poisoning in humans, usually at doses above 300 mg/kg, though lower doses have been reported to elicit symptoms. Vomiting, burning of the mouth, abdominal pain, hypotension, myotonia and CNS involvement including coma, are among the clinical signs observed (Bradberry et al., 2000; 2004a). Management of 2,4-D poisoning appears to be aided by urine alkalinization, through intravenous administration of bicarbonate (Proudfoot et al., 2004;

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Figure 22-17. Formation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during the synthesis of 2,4,5-T because of reaction between two molecules of 2,4,5-trichlorophenol.

Table 22-18 Effect of Urine Alkalinization on Renal Clearance and Plasma Half-Life of 2,4-D urine pH

renal clearance (mL/min)

half-life (h)

5.10–6.5 6.55–7.5 7.55–8.8

0.28 1.14 9.60

219 42 4.7

source: Park et al., (1977).

Figure 22-16. Structures of three chlorophenoxy acid herbicides.

Bradberry et al., 2004a). Ionization of an acid, such as 2,4-D, is increased in an alkaline environment. Though, at pH 5.0, practically all 2,4-D would already be ionized, further alkalinization of urine, to pH 7.5 or above, would reduce the non-ionized fraction from approximately 0.53 to 0.0017%. As a result, the fraction prone to reabsorption would be >300-times lower (Bradberry et al., 2004a). This explains why alkalinization of the urine diminishes reabsorption and increases 2,4-D elimination. An example of the effect of urine alkalinization on 2,4-D renal clearance and plasma half-life is given in Table 22-18. Dermal exposure is by far the major route of unintentional exposure to 2,4-D in humans. Dermal absorption studies in rats, mice and rabbit, indicate an absorption of 12–36%; the absorption in humans, however, is lower (2–10%), and is usually less than 6% (Ross et al., 2005). Acute poisoning by 2,4-D via the dermal route is thus uncommon; no reports of systemic toxicity following dermal exposure have been reported for over 20 years, and no fatalities have ever occurred (Bradberry et al., 2004a). The precise mechanisms of toxicity of chlorophenoxy herbicides have not been completely elucidated, but experimental studies indicate the possible involvement of three actions: (1) Cell membrane damage; (2) Interference with metabolic pathways involving acetyl-coenzyme A; (3) Uncoupling of oxidative phosphorylation (Bradberry et al., 2000). The toxicity of chlorophenoxy herbicides has been summarized in several reviews (Sterling and Arundel, 1986; Munro et al., 1992; Garebrant and Philbert, 2002). 2,4-D and its salts and esters are not teratogenic in mice, rats, or rabbits,

unless the ability of the dam to excrete the chemical is exceeded. There is also no convincing evidence that 2,4-D is associated with human reproductive toxicity (Garabrant and Philbert, 2002). Subchronic and chronic toxicity studies have not provided evidence of immunotoxicity, and there is very limited evidence that 2,4-D may affect the nervous system (Mattsson et al., 1997). There are, however, several case reports suggesting an association between exposure to 2,4-D and neurologic effects like peripheral neuropathy, demyelination and ganglion degeneration in the CNS, reduced nerve conduction velocity, mytonia and behavioral alterations (Garabrant and Philbert, 2002). Numerous in vitro and in vivo studies with 2,4-D indicate that it has very little genotoxic potential (Munro et al., 1992). Long-term bioassays in rats, mice and dogs provided no evidence to suggest that 2,4-D is a carcinogen in any of these species. An earlier study in rat reported an increase in the incidence of brain astrocytomas in male animals, only at the highest dose tested (45 mg/kg/d) (Serota et al., 1986). However, a review of this study concluded that the observed tumors were not treatment-related (Munro et al., 1992), and more recent studies did not replicate the original finding (Charles et al., 1996). Nevertheless, the chlorophenoxy herbicides have attracted much attention because of the association between exposure and non-Hodgkin’s lymphoma or soft-tissue sarcoma, found in a small number of epidemiological studies (Hoar et al., 1986; Hardell et al., 1994). In a recent review that follows several previous ones discussing this topic (Woods et al., 1987; Munro et al., 1992; USEPA, 1994), Garabrant and Philbert (2002) evaluated all cohort and case-control studies available to date, and concluded that the evidence from epidemiological studies was not adequate

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Figure 22-18. Structures of the bipyridyl herbicides paraquat and diquat, marketed as the dichloride and dibromide salts, respectively.

to conclude that exposure to 2,4-D is associated with soft-tissue sarcoma, non-Hodgkin’s lymphoma or Hodgkin’s disease. 2,4-D is classified as a Group D agent (not classifiable as to human carcinogenicity) (USEPA, 1997).

Bipyridil Compounds This class of herbicides comprises paraquat and diquat (Fig. 22-18). Of these, paraquat (1,1 -dimethyl-4,4 -bipyridilium dichloride) is of most toxicological concern, and will be discussed in more detail. First described in 1882, paraquat’s redox properties were discovered in 1933, when the compound was called methyl viologen. Paraquat, introduced as a herbicide in 1962, is formulated as an aqueous solution or as a granular formulation. Paraquat is a fastacting, nonselective contact herbicide, used to control broad-leaved weeds and grasses in plantations and fruit orchards, and for general weed control (Lock and Wilks, 2001a). Paraquat has one of the highest acute toxicity among herbicides; its oral LD50 in rat is approximately 100 mg/kg, while guinea pigs, rabbits, and monkeys are more sensitive. Paraquat is more toxic when given by the i.p. route (LD50 in rats = 10–20 mg/kg), suggesting that it is poorly absorbed from the gastrointestinal tract. Paraquat is also poorly absorbed through the skin. Upon absorption, independent of the route of exposure, paraquat accumulates in the lung and the kidney, and these two organs are the most susceptible to paraquat-induced injury. Paraquat is very poorly metabolized, and is excreted almost unchanged in the urine. Paraquat has minimal to no genotoxic activity, is not carcinogenic in rodents, has no effect on fertility, is not teratogenic, and only produces fetotoxicity at maternally toxic doses (Lock and Wilks, 2001a). Thus, the major toxicological concerns for paraquat are related to its acute systemic effects, particularly in the lung, and secondarily, the kidney. Rose et al. (1974) first described an energy-dependent accumulation of paraquat in lung tissue, particularly, but not exclusively, in type II alveolar epithelial cells. The loss of paraquat from lung tissue following in vivo administration is slow. Thus, the basis for the selective

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toxicity of paraquat to the lung resides in its ability to concentrate in alveolar type II and I cells and Clara cells. The mechanism(s) by which paraquat is toxic to living cells have been extensively investigated (Autor, 1974; Bus and Gibson, 1984; Smith, 1987). Paraquat can be reduced to form a free radical, which in the presence of oxygen, rapidly reoxidizes to the cation, with a concomitant production of superoxide anion (O2− ). Thus, once paraquat enters a cell, it undergoes alternate reduction followed by reoxidation, a process known as redox cycling (Adam et al., 1990). Superoxide dismutases (SOD) are a family of metalloenzymes that can dismutate superoxide anions to hydrogen peroxide and oxygen. The finding that transgenic mice lacking copper/zinc SOD show marked increase in sensitivity to paraquat (Ho et al., 1998), supports a role for superoxide anions in paraquat’s cellular toxicity. Nevertheless, superoxide anion itself is unlikely to be the ultimate toxic species (Lock and Wilks, 2001a). Three hypotheses have been proposed to account for the ensuing cytotoxicity, which are not mutually exclusive. The generation of superoxide anion and subsequently of hydroxy radicals, would initiate lipid peroxidation, ultimately leading to cell death (Bus and Gibson, 1984). Intracellular redox cycling of paraquat would also result in the oxidation of NADPH, leading to its cellular depletion, which is augmented by the detoxification of hydrogen peroxide formed in the glutathione peroxidase/reductase enzyme system to regenerate GSH (Fig. 22-19). A third hypothesis is that paraquat toxicity is due to mitochondrial damage; however, paraquat does not affect complex I in isolated brain mitochondria (Richardson et al., 2005). Upon acute exposure to lethal doses of paraquat, mortality may occur 2–5 days after dosing, though death can also occur after longer periods (Clark et al., 1966). Damage to alveolar epithelial cells is seen within 24 hours after exposure. Damage progresses in the following 2–4 days with large areas of the alveolar epithelium completely lost. This is followed by alveolar edema, extensive infiltration of inflammatory cells into the alveolar interstitium, and finally death due to severe anoxia (Smith and Heath, 1976). Survivors of this first phase, called the destructive phase, show extensive proliferation of fibroblasts in the lung. The second phase, called the proliferative phase, is characterized by attempts by the alveolar epithelium to regenerate and restore normal architecture, and presents itself as an intensive fibrosis (Smith and Heath, 1976). Some individuals who survive the first phase may still die from the progressive loss of lung function several weeks after exposure. Attempts to develop treatments for paraquat poisoning have focused on prevention of absorption from the gastrointestinal tract, removal from the bloodstream, prevention of its accumulation in the lung, use of free radical scavengers, and prevention of lung fibrosis (Lock and Wilks, 2001a). Though some approaches have shown promises in vitro or in isolated lung tissue preparations, only the first one, removal of the ingested material by emesis or purgation of the gastrointestinal tract, has been shown to be effective in vivo in animals. Since its introduction as a herbicide, there have been thousands of episodes of acute poisoning with paraquat in humans, a large percentage of which results in death (Malone et al., 1971; Casey and Vale, 1994; Wesseling et al., 2001). Most cases involved ingestion of a 20% paraquat concentrate solution for suicidal purposes, or as a result of accidental poisoning due to decanting in unlabeled drink bottles or containers. To avoid the latter, in the 1980s, the manufacturers added a blue pigment, a stenching compound, and an emetic substance to the formulation, to make severe unintentional poisoning due to oral intake virtually impossible (Sabapathy, 1995). As

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Figure 22-19. Mechanism of toxicity of paraquat. (1) Redox cycling of paraquat utilizing NADPH; (2) formation of hydroxy radicals leading to lipid peroxidation (3); (4) detoxication of H2 O2 via glutathione reductase/peroxidase couple, utilizing NADPH. Modified from Smith (1987), with permission from Palgrave Macmillan.

said, absorption of paraquat across the human skin is very low (but is increased by damage to skin, and paraquat is a skin irritant), and inhalation exposure is even lower. Few cases of paraquat poisoning have been reported following dermal exposure. Signs and symptoms of paraquat poisoning in humans reflect those previously described. A dose of 20–30 mg/kg can cause mild poisoning, while 30–50 mg/kg can cause delayed development of pulmonary fibrosis, which can be lethal. Higher doses usually cause death within a few days due to pulmonary edema, and renal and hepatic failure (Smith and Heath, 1976). No single therapeutic intervention, among those outlined earlier, has proven efficacious in case of severe acute paraquat poisoning (Bismuth et al., 1982). In rare instances, heart/lung transplant has been used to treat severely paraquat-poisoned patients. Chronic exposure of experimental animals to paraquat affects the same target organs of acute toxicity, i.e., the lung and the kidney, and no-effect levels have been established. Under normal use conditions, exposure to paraquat is very low and can be monitored by measuring paraquat levels in urine, as the compound is excreted unchanged. In the late 1970s concern was raised about possible exposure of marijuana smokers to paraquat by inhalation. Paraquat was indeed used to destroy marijuana fields, and residues were still

present in the final products (Landrigan et al., 1983). However, no clinical cases were identified. Chronic paraquat exposure has also been suggested as a possible etiological factor in the development of Parkinson’s disease, and the first suggestion came from a study in the Canadian province of Quebec (Barbeau et al., 1986). The hypothesis arose by the structural similarity of paraquat to MPP+ (1-methyl-4-phenylpyridinium ion), the toxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,5-tetrahydropyridine). MPP+ itself was initially developed as a possible herbicide, but was never commercialized. It has been argued that paraquat, being positively charged, cannot easily pass the blood–brain barrier. Yet, animal studies have shown that paraquat can cause CNS effects, most notably a neurodegeneration of dopaminergic neurons (McCormack et al., 2002). Paraquat may be transported into the brain by a neutral amino acid transporter, such as the system L carrier (LAT-1) (Shimizu et al., 2001). The ability of paraquat to cause oxidative damage through a free radical mechanism may explain the selective vulnerability of dopaminergic neurons, which are per se more susceptible to oxidative damage (McCormack et al., 2005). Paraquat neurotoxicity is, however, distinct from that of MPTP or rotenone (Richardson et al., 2005). Whereas these animal studies are of interest, there is still no solid evidence that paraquat may be associated with Parkinson’s disease in humans (Li et al., 2005). Furthermore, a 5–10 years follow-up of individuals who survived paraquat poisoning did not provide any evidence of parkinsonism (Zilker et al., 1988). Despite the chemical similarity to paraquat (Fig. 22-18), the herbicide diquat presents a different toxicological profile. Acute toxicity is somewhat lower, with oral LD50 in rats of approximately 200 mg/kg (Lock and Wilks, 2001b). Diquat is not a skin sensitizer, has minimal or no genotoxic activity, is not carcinogenic in rodents, has no effect on fertility, and is not teratogenic. In contrast to paraquat, it does not accumulate in the lung, and no lung toxicity is seen upon acute or chronic exposure. Upon chronic exposure, target organs for toxicity are the gastrointestinal tract, the kidney, and particularly the eye. Diquat indeed causes a dose- and time-dependent appearance of cataracts in both rats and dogs (Lock and Wilks, 2001b). Like paraquat, diquat can be reduced to form a free radical and then reoxidized in the presence of oxygen, with the concomitant production of superoxide anion. This process of redox cycling occurs in the eye and is believed to be the likely mechanism of cataract formation (Lock and Wilks, 2001b). A limited number of cases of human poisoning with diquat have occurred. Clinical symptoms include nausea, vomiting, diarrhea, ulceration of mouth and esophagus, decline of renal functions and neurologic effects, but no pulmonary fibrosis. As for paraquat, therapy for intoxication is directed at preventing absorption and enhancing elimination (Vanholder et al., 1981; Lock and Wilks, 2001b).

Chloroacetanilides Representative compounds of this class of herbicides are alachlor, acetochlor, and metolachlor (Fig. 22-20), which are used to control herbal grasses and broad-leaved weeds in a number of crops (corn, soybeans, peanuts). Chloroacetanilides display moderate to low acute toxicity, with oral LD50 in rat ranging from about 600 mg/kg (propachlor) to 2800 mg/kg (metolachlor). Dermal LD50 values are higher, indicating poor absorption of these compounds across the skin (Heydens et al., 2001). Subchronic and chronic toxicity studies, carried out in multiple species, have identified the liver and kidney as principal target organs, and no-adverse-effect

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ity studies with alachlor, acetochlor, and butachlor, while glandular stomach tumors were found with alachlor and butachlor. These findings led to the classification of alachlor, acetochlor, and butachlor as probable human carcinogens (Group B2). The discovery of alachlor in well water led to cancellation of its registration in some countries, and to its restriction in others. A series of mechanistic studies in the past decade has provided evidence that tumors observed in rats may be species-specific, show a threshold, and are not due to genotoxic mechanisms, and hence may not be relevant to humans (Ashby et al., 1996; Heydens et al., 1999, 2001). Alachlor and other chloroacetanilides are extensively metabolized in rats; more than 30 metabolites have been identified in this species, whereas in primates alachlor is metabolized to a limited number of glutathione and glucuronide metabolites. Furthermore, in rats, biliary excretion and enterohepatic recirculation of metabolites is observed, whereas in primates excretion is primarily via the kidneys. Thus, in rats, alachlor and butachlor are initially metabolized in the liver via the cytochrome P450 pathway and by glutathione conjugation, and the metabolites undergo enterohepatic circulation and further metabolism in liver and nasal tissue, to form the putative carcinogenic metabolite diethyl quinoneimine (DEIQ) (Feng et al., 1990). In the case of acetochlor, the carcinogenic metabolite is EMIQ, an acetochlor quinoneimine compound analogous to DEIQ. Quinoneimines are electrophilic, deplete glutathione and exert cellular toxicity, which is followed by regenerative cell proliferation. Whole-body autoradiography studies have shown that alachlor-derived radioactivity specifically localizes in the nasal mucosa of rats, but not mice or monkeys. Furthermore, the ability of rat nasal tissue to form DEIQ is much higher than human nasal tissue (Wilson et al., 1995). With regard to stomach and thyroid tumors, it is suggested that these result from tissue-specific toxicity, leading to compensatory cell proliferation in the fundic mucosa, and to alterations of thyroid stimulating hormone homeostasis. Both are believed to be threshold-sensitive phenomena. Epidemiological investigations in workers involved in the manufacturing of alachlor have not demonstrated any evidence of increased mortality or cancer incidence (Leet et al., 1996).

Triazines Figure 22-20. Structures of chloroacetanilide herbicides.

levels have been established. Alachlor was found to produce an ocular lesion, termed “progressive uveal degeneration syndrome,” in rats (Heydens, 1998). However, an investigation of similar eye abnormalities in alachlor production workers considered to have the highest alachlor exposure, provided no evidence of ocular disease (Ireland et al., 1994). None of the chloroacetanilides appears to be teratogenic or cause reproductive or developmental toxicity (Heydens et al., 2001). Chloroacetanilides have been extensively tested for genotoxicity in vitro and in vivo, and these studies indicate, on the basis of a weight-of-evidence approach, that these compounds are not genotoxic (Ashby et al., 1996; Heydens et al., 1999, 2001). Yet these compounds have been shown to induce tumors at various sites in rats. Tumors in mice (lung adenomas), found with alachlor and acetochlor, were considered not to be treatment-related (Ashby et al., 1996; Heydens et al., 1999). In rats, nasal epithelial (olfactory) tumors and thyroid follicular tumors were observed in carcinogenic-

The family of triazine herbicides comprises several compounds (atrazine, simazine, propazine; Fig. 22-21), that are extensively used for the preemergent control of broad-leaved weeds (Stevens et al., 2001). Triazines have low acute oral and dermal toxicity, and chronic toxicity studies indicate primarily decreased body weight gain. In dogs, cardiotoxicity is seen with atrazine, and was used to derive the NOEL for this compound (0.5 mg/kg/d) (Gammon et al., 2005). There is no evidence that triazines are teratogenic, nor developmental or reproductive toxicants. Evidence from in vitro and in vivo studies indicate that atrazine is not genotoxic (Brusick, 1994), although a more recent study has suggested a possible clastogenic effect (Taets et al., 1998). Oncogenicity studies found that triazines cause an increased incidence of mammary carcinomas in female Sprague–Dawley rats (Stevens et al., 2001; Gammon et al., 2005). Such tumors were not observed in male Sprague–Dawley rats, or in Fischer 344 rats or CD-1 mice of either sex (IARC, 1999; Stevens et al., 2001). It is believed that such mammary tumors arise from an endocrine effect that might be expected to show a threshold. In contrast to most rodent species, female Sprague–Dawley rats have a high degree of spontaneous mammary tumors, due to their reproductive aging; they display prolonged or persistent estrus associated with high estrogen levels (Eldridge et al., 1996). This is supported by the

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Figure 22-22. Structures of the phosphomethyl amino acids glyphosate and glufosinate. Note that though having a P=O moiety like organophosphates, these compounds are not acetylcholinesterase inhibitors.

et al., 2003). Both atrazine and simazine are classified as Group 3 carcinogens (not classifiable as to its carcinogenicity to humans) by IARC. Though exposure to atrazine through residues in food commodities is very low, contamination of ground water and drinking water is common (Villanueva et al., 2005). Several European countries have banned the use of atrazine in recent years, mainly because it was often detected at levels exceeding the 0.1 mg/L standard for drinking water. Recent publications have reported a possible feminization of frogs, measured in laboratory and field studies, by atrazine levels around 0.1 mg/L (Hayes et al., 2002). However, other investigators failed to reproduce these findings (Carr et al., 2003). The USEPA has concluded that the conflicting results do not allow any firm conclusion in this regard, and that further studies are needed (USEPA, 2003). Nevertheless, the known hormonal effects of triazines call for careful evaluation of endocrine disrupting effects of these herbicides.

Phosphonomethyl Amino Acids

Figure 22-21. Structures of triazine herbicides.

finding that ovariectomy eliminates mammary tumors in Sprague– Dawley rats that arise both spontaneously and as a result of atrazine administration (Stevens et al., 1999). The possible mechanisms for the effect of atrazine involve an action on pituitary luteinizing hormone, regulated by hypothalamic gonadothropin-releasing hormone, in turn controlled by hypothalamic norepinephrine (Cooper et al., 2000). Some regulatory agencies have thus concluded that mammary tumors in female Sprague–Dawley rats are formed via a secondary, hormone-mediated mechanism, of little or no toxicological relevance to humans. Epidemiological studies of triazine herbicides and cancer have provided inconclusive results (Sathiakumar and Delzell, 1997). More recent studies have suggested an increased risk of prostate cancer associated with triazine herbicides (Mills and Yang, 2003), but this has not been substantiated by others (Alavanja

The two compounds of this class are glyphosate (N-phosphonomethyl glycine) and glufosinate (N-phosphonomethyl homoalanine). Both are broad-spectrum nonselective systemic herbicides used for postemergent control of annual and perennial plants, and are marketed primarily as the isopropylamine salt (glyphosate) or ammonium salt (glufosinate). Though both compounds contain a P=O moiety (Fig. 22-22), they are not organophosphates, but rather organophosphonates, and do not inhibit AChE (Farmer, 2001). Glyphosate Glyphosate exerts its herbicidal action by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, responsible for the synthesis of an intermediate in the biosynthesis of various amino acids. Although important in plant growth, this metabolic pathway is not present in mammals. The toxicity profile of technical grade glyphosate is unremarkable (Williams et al., 2000; Farmer, 2001). Oral and dermal LD50 are >5000 mg/kg, and chronic toxicity studies show only nonspecific effects, such as failure to gain weight. It has no teratogenic, developmental or reproductive effects. Genotoxicity and carcinogenicity studies in animals were negative; on the basis of all available evidence glyphosate has been classified as a Group E compound (evidence of noncarcinogenicity in humans) by the USEPA (Farmer, 2001).

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Glyphosate is one of the most widely used herbicides in the United States and worldwide (Table 22-4), and the development of transgenic crops that can tolerate glyphosate treatment have expanded its utilization. Given its widespread use, including the home and garden market, accidental or intentional exposure to glyphosate is inevitable. For example, in the period 2001–2003, there were over 13,000 reports to the American Association of Poisons Control Centers’ Toxic Exposure Surveillance System relating to glyphosate exposure (Bradberry et al., 2004b). There was a major adverse outcome in 18 patients, and five died. Several other cases of glyphosate ingestions have been published, with a 10–15% mortality rate (Sawada et al., 1988; Talbot et al., 1991; Lee et al., 2000; Stella and Ryan, 2004; Bradberry et al., 2004b). Given the low acute toxicity of glyphosate itself, the attention has been focused on its formulation, which contains surfactants to aid its penetration. The most widely used glyphosate product is Roundup® which is formulated as a concentrate containing water, 41% glyphosate (as isopropylamine salt) and 15% polyoxethyleneamine (POEA). Animal studies suggest that the acute toxicity of this glyphosate formulation is due to the surfactant POEA, which has an oral LD in rat of 1200 mg/kg (Bradberry et al., 2004b). Mild intoxication results mainly in transient gastrointestinal symptoms, while moderate or severe poisoning presents with gastrointestinal bleeding, hypotension, pulmonary dysfunction and renal damage (Talbot et al., 1991).

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an infestation has already begun. Still others are used as systemic fungicides, that are absorbed and distributed throughout the plant. With few exceptions, fungicides have low acute toxicity in mammals. However, several produce positive results in genotoxicity tests and some have carcinogenic potentials. The effects are often associated with the mechanisms by which these compounds act on their targets, the fungi. A 1987 evaluation by the National Research Council concluded that fungicides, though accounting for only 7% of all pesticide sales, and less than 10% of all pounds of pesticides applied, accounted for about 60% of estimated dietary oncogenic risk (NRC, 1987). Some fungicides have been associated with severe epidemics of poisoning, and have thus been banned. Methylmercury was associated with poisoning in Iraq, when treated grains were consumed (Bakir et al., 1973). Hexachlorobenzene (HCB), used in the 1940–1950s to treat seed grains, was associated with an epidemic of poisoning in Turkey from 1955 to 1959 (Cam and Nigogosyan, 1963). HCB has a high cumulative toxicity and caused a syndrome called black sore, characterized by blistering and epidermolysis of the skin, pigmentation and scarring. HCB also causes porphyria as well as hepatomegaly and immunosuppression (Ecobichon, 2001a). The main classes of fungicides currently in use are discussed below; additional discussions of fungicide use and toxicity can be found in Hayes (1982) and in Edwards et al. (1991).

Captan and Folpet Glufosinate Glufosinate is a nonselective contact herbicide that acts by irreversibly inhibiting glutamine synthetase (Ebert et al., 1990). Plants die as a consequence of the increased levels of ammonia. Mammals have other metabolizing systems that can cope with the effects on glutamine synthetase activity to a certain limit. In brain, however, inhibition of >10% of this enzyme activity is considered an adverse effect (EFSA, 2005). Glufosinate has relatively low acute toxicity, and chronic toxicity studies provided a NOAEL of 4.5 mg/kg/d, based on decreased glutamine synthetase activity. There is no evidence of genotoxicity or carcinogenicity, or direct effects on reproductive performance and fertility. Developmental toxic effects were found in rabbits (premature deliveries, abortions, dead fetuses). The most commonly used form of glufosinate is as ammonium salt, which is formulated with an anionic surfactant. Several cases of acute human poisoning from glufosinate ammonium-containing products have been reported, particularly in Japan, due to suicidal intent or accidental misuse. Symptoms include gastrointestinal effects, impaired respiration, neurological disturbance, and cardiovascular effects (Koyama et al., 1994; Watanabe and Sano, 1998; Ujvary, 2001). Though glufosinate does not inhibit cholinesterase, a reduction of red blood cell and plasma cholinesterase was found in poisoned patients. As is the case for glyphosate, a role for the surfactant in the acute toxicity has been proposed, particularly with regard to the cardiovascular effects (Ujvary, 2001).

FUNGICIDES Fungal diseases are virtually impossible to control without chemical application. Fungicidal chemicals are derived from a variety of structures, from simple inorganic compounds, such as copper sulfate, to complex organic compounds. The majority of fungicides are surface or plant protectants, and are applied prior to potential infection by fungal spores, either to plants or to postharvest crops. Other fungicides can be used therapeutically, to cure plants when

Captan and folpet are broad-spectrum protectant fungicides; together with captafol, which was taken off the market in 1988, they are called chloroalkylthio fungicides, due to the presence of side chains containing chlorine, carbon, and sulfur (Fig. 22-23). Captan was first registered in the United States in 1949, and folpet followed a few years later. As for most fungicides, captan and folpet have low acute oral and dermal toxicity (LD50 = ∼5g/kg). They are potent eye irritants, but only mild skin irritants. Dermal absorption is low. Both are extensively and rapidly metabolized in mammals, through hydrolysis and thiol interactions, with thiophosgene being a common metabolite (Gordon, 2001). Captan and folpet, as well as thiophosgene, are mutagenic in in vitro tests; however, in vivo mutagenicity tests are mostly negative, possibly because of the rapid degradation of these compounds. Both fungicides induce the development of duodenal tumors in mice, and on this basis, they are classified by the USEPA as probable human carcinogens. The mode of action for these tumors is thought to be dependent on irritation and cell loss in the intestinal villi, followed by a compensatory increase in proliferation in the crypt compartment (Gordon, 2001). Captan and folpet share a common mechanism of toxicity with regard to the development of duodenal tumors in mice, as well as other toxicity end points, and are considered for cumulative risk assessment under the FQPA (Bernard and Gordon, 2000). Tumors observed in rats (renal adenomas and uterine sarcomas) are suggested not to be treatment-related (Gordon, 2001), but are considered in the carcinogenicity classification of these fungicides. Because of their structural similarity to the potent teratogen thalidomide (Fig. 22-23), chloroalkylthio fungicides have been extensively tested in reproductive/developmental studies in multiple species, but no evidence of teratogenicity has been found (McLaughlin et al., 1969).

Dithiocarbamates Dithiocarbamates are a group of fungicides that have been widely used since the 1940s to control about 400 fungal pathogens in a

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Figure 22-23. Structures of the phthalimide fungicides captan and folpet. The structure of thalidomide is also shown, though phthalimides have been shown not to be teratogenic, despite structural similarities.

variety of crops. The nomenclature of many of these compounds arises from the metal cations with which they are associated; thus, there are for example Maneb (Mn), Ziram and Zineb (Zn), and Mancozeb (Mn and Zn) (Fig. 22-24). Thiram is an example of dithiocarbamate without a metal moiety (Fig. 22-24). The dithiocarbamates have low acute toxicity by the oral, dermal, and respiratory route (Hurt et al., 2001). However, chronic exposure is associated with adverse effects that may be due to the dithiocarbamate acid or the metal moiety. These compounds are metabolized to a common metabolite, ethylenethiourea (ETU), which is responsible for the effects of dithiocarbamates on the thyroid. ETU produced thyroid tumors in rats and mice, that result from the inhibition of the synthesis of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). This leads to elevated serum levels of thyroid stimulating hormone (TSH), via feedback stimulation of the hypothalamus and the pituitary, and subsequent hypertrophy and hyperplasia of thyroid follicular cells, that progresses to adenomas and carcinomas (Chhabra et al., 1992). Similarly, dithiocarbamates alter thyroid hormone levels, and cause thyroid hypertrophy. The hormonal mechanism of thyroid tumors would imply a threshold model for hazard assessment. ETU also causes liver tumors in mice, by yet unknown mechanisms, although levels of ETU resulting from fungicide metabolism at maximum tolerated doses are believed to be insufficient to produce hepatic tumors (Hurt et al., 2001). Neither dithiocarbamates nor ETU are genotoxic in in vitro and in vivo tests. Developmental toxicity and teratogenicity is observed with dithiocarbamates and ETU at maternally toxic doses. These effects are ascribed to an effect of ETU on the thyroid. A key concern with chemicals affecting thyroid functions, is their potential developmental neurotoxicity, given the essential role of thyroid hormones in brain development (Chan and Kilby, 2000), and this deserves further investigation. There is also some evidence that dithiocarbamates may cause neurotoxicity by mechanisms not involving ETU.

Figure 22-24. Structures of three dithiocarbamate fungicides.

High doses of several of these compounds cause hind limb paralysis, which is possibly related to the release of the carbon disulfide moiety (Johnson et al., 1998). Chronic exposure to Maneb has been associated with parkinsonism, which is likely ascribed to exposure to the manganese moiety, rather than the dithiocarbamate (Ferraz et al., 1988; Meco et al., 1994). Maneb has also been shown to produce nigrostriatal degeneration when given in combination with paraquat (Thiruchelvam et al., 2000), and to potentiate the neurotoxicity of MPTP (McGrew et al., 2000). It has been shown that Maneb affects dopaminergic neurons by inhibiting mitochondrial functions (Zhang et al., 2003). The structure of dithiocarbamate fungicides resembles that of disulfiram, a compound used therapeutically to produce intolerance to alcohol, by virtue of its ability to inhibit aldehyde dehydrogenase. Interactions of dithiocarbamates with alcohol, leading to elevation in acetaldehyde levels, have been reported (Edwards et al., 1991).

Chlorothalonil Chlorothalonil (Bravo® ) is a halogenated benzonitrile fungicide (Fig. 22-25), widely used to treat vegetable, ornamental, and orchard diseases (Table 22-4). Whereas oral and dermal toxicities are low (LD50 s = 5–10 g/kg), it is highly toxic by the intraperitoneal and inhalation routes. It also causes severe irreversible eye lesions in the rabbit, because of its irritant properties. Dermal absorption is low, but following oral administration, chlorothalonil is rapidly

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soluble copper salts was discovered as early as 1807, and by 1890 copper sulfate found extensive use, particularly in the formulation known as Bordeaux mixture (copper sulfate and calcium hydroxide). Copper sulfate has overall low toxicity and remains one of the most widely used fungicides (Table 22-4). Among organotin compounds, triphenyltin acetate is used as a fungicide, while tributyltin is utilized as an antifouling agent. Triphenyltin has moderate to high acute toxicity, but may cause reproductive toxicity and endocrine disruption (Golub and Doherty, 2004). Organic mercury compounds, such as methylmercury, were used extensively as fungicides in the past for the prevention of seed-borne diseases in grains and cereals. Given their high toxicity, particularly neurotoxicity, and large episodes of human poisoning (Bakir et al., 1973), their use has since been banned. A discussion of organometal compounds is found in Chap. 23.

RODENTICIDES

Figure 22-25. Structures of the fungicides choroathalonil and benomyl.

absorbed and metabolized through glutathione conjugation. Chlorathalonil is not mutagenic in in vitro and in vivo tests (Parsons, 2001). Tumors in the forestomach and the kidney have been found in chronic toxicity studies in both rats and mice, but not in dogs. Tumors are believed to be due to regenerative hyperplasia, and it is assumed that a threshold can be established for carcinogenicity (Parsons, 2001). Chlorothalonil is not a reproductive or developmental toxicant. Known adverse effects in humans are limited to its irritant effects on the eye and the skin.

Benzimidazoles Benomyl is the main representative of this class of fungicides (Fig. 22-25). It inhibits fungal growth by binding to tubulin, and this mechanism also accounts for its toxic effects in mammals. Acute toxicity is low, whereas chronic studies have found effects in the liver, testes, bone marrow and gastrointestinal tract (Mull and Hershberger, 2001). Because of its ability to disrupt microtubule assembly during cell division, Benonyl causes chromosomal aberrations (aneuploidy) both in vitro and in vivo, but does not interact directly with DNA. Liver tumors have been observed in chronic oncogenicity studies in mice. The action on dividing cells has also raised concern for Benomyl’s potential teratogenicity and developmental toxicity. Teratogenic effects were observed following administration of high doses of Benomyl and Carbendazim (a metabolite of Benomyl, which is commercialized as a fungicide but not in the United States) to rats (Mull and Hershberger, 2001). However, such effects were not seen in feeding studies in rats or rabbits. Anecdotal evidence suggests that maternal exposure to Benomyl may result in anophthalmia in humans, but epidemiological studies did not demonstrate any convincing association (Spagnolo et al., 1994).

Inorganic and Organometal Fungicides Several inorganic and organic metal compounds are, or have been, used as fungicides (Clarkson, 2001). The fungicidal activity of

Rats and mice can cause health and economic damages to humans. Rodents are vectors for several human diseases, including plague, endemic rickettsiosis, spirochetosis, and several others; they can occasionally bite people; they can consume large quantities of postharvest stored foods, and can contaminate foodstuff with urine, feces, and hair, that may cause diseases. Hence, there is a need to control rodent population. Limiting their access to feed and harborage, and trapping, are two approaches; however, rodenticides still play and will likely continue to play an important role in rodent control. To be effective, yet safe, rodenticides must satisfy several criteria: (1) the poison must be very effective in the target species once incorporated into bait in small quantity; (2) baits containing the poison must not excite bait shyness, so that the animal will continue to eat it; (3) the manner of death must be such that survivors do not become suspicious of its cause; and (4) it should be species-specific, with considerable lower toxicity to other animals (Murphy, 1986; Ecobichon, 2001a). The compounds used as rodenticides comprise a diverse range of chemical structures having a variety of mechanisms of action. The ultimate goal is to obtain the highest species selectivity; in some cases (e.g., norbormide) advantage has been taken of the physiology and biochemistry unique to rodents. With other rodenticides, the sites of action are common to most mammals, but advantage is taken of the habits of the pest animal and/or the usage, thereby minimizing toxicity for nontarget species. Because rodenticides are used in baits which are often placed in inaccessible places, widespread exposures or contaminations are unlikely. However, toxicologic problems can arise from acute accidental ingestions or from suicidal/homicidal attempts. In particular, poison centers receive thousands of calls every year related to accidental ingestions of rodenticide baits by children, most of which are resolved without serious consequences.

Fluoroacetic Acid and Its Derivatives Sodium fluoroacetate (Compound 1080) and fluoroacetamide are the main representatives of this class of rodenticides. They are white in color and odorless, and due to their high mammalian toxicity, their use is restricted to trained personnel. Both compounds have indeed high acute toxicity (oral LD50 s in the rat = 2 and 13 mg/kg, respectively). The main targets of toxicity are the central nervous system and the heart. Fluoroacetate is incorporated into fluoracetyl-coenzyme A, which condenses with oxolacetate to form fluorocitrate, which inhibits mitochondrial aconitase. This results in

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inhibition of the Krebs cycle, leading to lowered energy production, reduced oxygen consumption, and reduced cellular concentration of ATP. Blockage of energy metabolism is believed to account for most signs of toxicity, though some may be due to accumulation of citrate, which is a potent chelator of calcium ions (Pelfrene, 2001). Since 1946, when sodium fluoroacetate was introduced in the United States, several cases of human poisoning have been reported. Initial gastrointestinal symptoms are followed by severe cardiovascular effects (ventricular tachycardia, fibrillation, hypotension), as well as CNS effects (agitation, convulsions, coma). The estimated lethal dose in humans ranges from 2–10 mg/kg. There is no specific antidote for sodium fluoroacetate. Monacetin (60% glycerol monoacetate) has proved beneficial in the treatment of poisoned primates. Use of procainamide (for cardiac arrhythmia) and barbiturates (to control seizures) are also indicated. Use of Compound 1080 in the United States is severely restricted primarily because of toxicity to nontarget animals, such as dogs.

Thioureas The discovery of α-naphtyl thiourea (ANTU) occurred fortuitously in the mid-1940s in the Psychobiological Laboratory of Curt Richter in Baltimore. While studying thioureas, favored by geneticists for taste tests, because they are so bitter to some people and tasteless to others, he discovered that ANTU was lethal yet tasteless to rodents, while being of low toxicity to humans (Keiner, 2005). A wide range of acute oral LD50 values has been reported for different species, the rat being the most sensitive at 6 mg/kg, and the monkey the least susceptible at 4 g/kg. The main target of toxicity is the lung, where ANTU causes marked edema of the subepithelial spaces of the alveolar walls. ANTU is believed to be biotransformed to a reactive intermediate which binds to lung macromolecules; however, the exact mechanism of its toxicity is unknown. Young rats are resistant to the chemical, whereas older rats become tolerant to it; both situations have been ascribed to developmentally low, or to ANTU-induced inhibition of, microsomal enzymes involved in its bioactivation (Boyd and Neal, 1976). There are no reports of human poisonings with ANTU. However, several cases involving a combination of chloralose and ANTU were reported in France; symptoms included motor agitation and coma, both characteristic of chloralose poisonings, and pulmonary effects, due to ANTU, but all patients recovered (Pelfrene, 2001). Suggestions that the presence of an impurity in ANTU, β-naphthylamine, may increase risk of bladder cancer, remain unsubstantiated (Case, 1966; Pelfrene, 2001).

Anticoagulants Following the report of an hemorrhagic disorder in cattle that resulted from the ingestion of spoiled sweet clover silage, the hemorrhagic agent was identified in 1939 as bishydroxycoumarin (dicoumarol). In 1948, a more potent synthetic congener was introduced as an extremely effective rodenticide; the compound was named warfarin, as an acronym derived from the name of the patent holder, Wisconsin Alumni Research Foundation (Majerus and Tollefsen, 2006). In addition to their use as rodenticides, coumarin derivatives, including warfarin itself, are used as anticoagulant drugs and have become a mainstay for prevention of thromboembolic disease (Majerus and Tollefsen, 2006). Coumarins antagonizes the action of vitamin K in the synthesis of clotting factors (factors II, VII, IX, and X). Their specific mechanism involves inhibition of the enzyme vitamin K epoxide reductase, which

regenerates reduced vitamin K necessary for sustained carboxylation and synthesis of relevant clotting factors (Fig. 22-26). The acute oral toxicity of warfarin in rats is approx. 50–100 mg/kg, whereas the 90-day dose LD50 has been reported as 0.077 mg/kg, indicating that multiple doses are required before toxicity develops. Human poisonings by these rodenticides are rare because they are dispersed in grain-based baits. However, there is a significant number of suicide or homicide attempts or of accidental consumption of warfarin. One often reported case involved a Korean family that consumed a diet of corn containing warfarin over a two-week period. Symptoms (massive bruises, hematomata, gum and nasal hemorrhage) appeared about 10 days after the beginning of the warfarin consumption. Consumption of warfarin in this episode was estimated to be in the order of 1–2 mg/kg/d (Lange and Terveer, 1954). Monitoring of anticoagulant therapy is done by measuring prothrombin time (PT) in comparison to normal pooled plasma. Values of INR (International Normatized Ratio) are then derived, with a target value of 2–3. In case of poisoning, PT is significantly longer, and leads to severe internal bleeding. When INR is above 5, vitamin K can be given as an antidote. The appearance of rats resistant to warfarin and to other early anticoagulant rodenticides, led to the development of “second generation” anticoagulants. Some are coumarins, such as the “superwarfarins” brodifacoum or difenacoum, whereas others are indan1,3 -dione derivatives (diphacinone, chlorophacinone). These compounds essentially act like warfarin, but have prolonged half-lives (e.g., brodifacoum 156 hours vs. warfarin 37 hours), and cause very long-lasting inhibition of coagulation. Some are extremely toxic to most mammalian species; for example the oral LD50 of brodifacoum is about 0.3 mg/kg in rat, rabbit, and dog (Pelfrene, 2001).

Other Compounds Norbormide This compound shows a remarkable selectivity in both toxicity and pharmacological effects. Oral LD50 in rat is about 5–10 mg/kg, whereas in dog and monkey 1000 mg/kg produces no effect. Such species difference in toxicity seems to be accounted for by differences in response of the peripheral blood vessels to norbormide-induced vasoconstriction. The exact mechanisms of this effect are not known. Zinc Phosphide The toxicity of this chemical can be accounted for by the phosphine gas (PH3 ) formed on ingestion following a hydrolytic reaction with water in the stomach. Phosphine causes widespread cellular toxicity with necrosis of the gastrointestinal tract and injury to liver and kidney. Cases of human poisoning have been reported (Ecobichon, 2001a). Additional inorganic compounds that have been used as rodenticides include aluminum phosphide, thallium sulfate, and arsenic salts. Thallium sulfate has the unusual feature of causing extensive alopecia (hair loss); because of its high acute toxicity in nontarget species, it was banned in the United States in 1972 (Clarkson, 2001). Other Some rodenticides used in the past that have become obsolete include strychnine, an extremely poisonous alkaloid derived from the seeds of Strychnos nux-vomica, which is a potent convulsant; red squill (sea onion) and its bioactive principle, scilliroside, which affect the cardiovascular and central nervous systems and cause emesis; the inability of rodents to vomit explains the rather selective action in these species; and pyriminyl, a substituted urea

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Figure 22-26. Site of action of the anticoagulant rodenticide warfarin. Reduced vitamin K (hydroquinone) serves as cofactor for the conversion of glutamic acid to γ-carboxyglutamic acid in the peptide chains of coagulation factors II, VII, IX, and X. During this reaction, vitamin K is oxidized to an epoxide that is then reduced to quinone and hydroquinone by vitamin K reductase, which is inhibited by warfarin.

introduced in 1975 but withdrawn in the United States a few years later. This compound targets complex I in the mitochondria, and there are many reports of human poisoning in the short period of its use (Pelfrene, 2001). As many other ureas, it has diabetogenic properties.

because of toxicological concerns. These include, for example, carbon disulfide, which is neurotoxic; carbon tetrachloride, a potent hepatotoxicant; 1,2-dibromo-3-chloropropane, a male reproductive toxicant; and ethylene dibromide, a carcinogen. Their toxicity is discussed in other sections of the book. Some of the most commonly used fumigants are discussed below.

FUMIGANTS A large number of compounds are used for soil fumigation or for fumigating postharvest commodities. They are active toward insects, mites, nematodes, weed seeds, fungi or rodents, and have in common the property of being in the gaseous form at the time they exert their pesticidal action. They can be liquids that readily vaporize (e.g., ethylene dibromide), solids that can release a toxic gas on reaction with water (e.g., phosphine released by aluminum phosphide), or gases (e.g., methyl bromide). For soil fumigation, the compound is injected directly into the soil, which is then covered with plastic sheeting, which is sealed. By eliminating unwanted pests, this treatment enhances the quality of the crops and increases yield. Fumigation of postharvest commodities, such as wheat, cereals, and fruits to eradicate pest infestations, typically occurs where the commodities are stored (e.g., warehouses, grain elevators, ship holds). Compounds used as fumigants are usually nonselective, highly reactive, and cytotoxic. They provide a potential hazard from the standpoint of inhalation exposure, and to a minor degree for dermal exposure or ingestion, in case of solids or liquids. Several fumigants used in the past are no longer marketed

Methyl Bromide Methyl bromide is a broad-spectrum pesticide, used for soil fumigation, commodity treatment, and structural fumigation. It has been used as a fumigant for over 50 years, and its use is strictly controlled and restricted to certified applicators. Since the mid-1990s, use of methyl bromide has substantially decreased, because of environmental and toxicological concerns (Ruzo, 2006). Methyl bromide is thought to contribute to ozone depletion in the stratosphere. In 1987, with the signing of the Montreal Protocol on Substances that Deplete the Ozone Layer, the international community initiated a series of steps to reduce emissions of ozone-depleting products, including methyl bromide. Concerns on certain toxicological aspects of methyl bromide, have also contributed to its decreasing use, and to the search of viable alternatives (Ruzo, 2006; Schneider et al., 2003). Yet, while several countries are substantially curtailing the use of methyl bromide, this compound still remains one of the most extensively used pesticides in the United States (Table 22-4; Ruzo, 2006), likely for convenience and economic reasons (Norman, 2005; McCook, 2006).

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The acute toxicity of methyl bromide relates to both its concentration and the duration of exposure. For example, LC50 values in rats were 2833 ppm for a 30 minute exposure, and 302 ppm for an 8 hour exposure (Piccirillo, 2001). Between 1953 and 1981, 301 cases of systemic poisoning and 60 fatalities resulted from use of methyl bromide as a fumigant (Alexeef and Kilgore, 1983). Additional cases of human intoxication have since been reported (Herzstein and Cullen, 1990). Acute exposure results in respiratory, gastrointestinal and neurologic symptoms; the latter include lethargy, headache, seizures, paresthesias, peripheral neuropathy, and ataxia, and are considered to be more relevant than other toxic effects for human risk assessment (Alexeef and Kilgore, 1983; Lifshitz and Gavrilov, 2000; Piccirillo, 2001). Acute and chronic neurotoxicity studies in rats have demonstrated behavioral effects and morphological lesions, which were concentration- and time-dependent (Piccirillo, 2001). Long-lasting behavioral and neuropsychiatric effects are also seen in humans (De Haro et al., 1997; Lifshitz and Gavrilov, 2000). The mechanism(s) underlying methyl bromide neurotoxicity are not known. Depletion of GSH in brain areas was observed following exposure of rats to methyl bromide (140 ppm for 6 h/d, for 5 days) (Davenport et al., 1992). This may be due to conjugation of methyl bromide with GSH. The role of GSH and the possible ensuing increase in oxidative stress in methyl bromide neurotoxicity remains, however, uncertain. In various subchronic toxicity studies, the NOEL for neurotoxicity ranges between 18 and 200 ppm (Piccirillo, 2001). Methyl bromide is positive in several genotoxicity tests in vitro and in vivo. Carcinogenicity studies produced carcinomas in the forestomach of rats following oral ingestion, and increased incidence of adenomas of the pituitary gland in male rats in an inhalation study. Other studies in rats and mice, however, provided no evidence of carcinogenicity. Methyl bromide is classified by IARC in Group 3 (not classifiable as to its carcinogenicity to humans), given the limited evidence in animals and the inadequate evidence in humans. As methyl bromide is an odorless and colorless gas, another fumigant, chloropicrin, which has a pungent odor and causes irritation of the eyes, is often used in conjunction with methyl bromide and other fumigant mixtures, to warn against potentially harmful exposures.

1,3–Dichloropropene 1,3–Dichloropropene, first introduced over fifty years ago, is a soil fumigant, extensively utilized (see Table 22-4) for its ability to control soil nematodes. It has a moderate to high acute toxicity in animals (oral LD50 in rats: 130–713 mg/kg; dermal LD50 :

>1200 mg/kg; inhalation LD50 : ∼ 1000 ppm). Human fatalities following oral exposure have been reported (Hernandez et al., 1994). 1,3–Dichloropropene is an irritant, and can cause redness and necrosis of the skin. It is extensively metabolized, with the mercapturic acid conjugate being the major urinary metabolite. Data on genotoxicity are contradictory, and carcinogenicity studies in rodents have found an increase in benign liver tumors in rats but not in mice, after oral administration (Stebbins et al., 2000), and of benign lung adenomas in mice following inhalation exposure (Lomax et al., 1989). The toxicology of 1,3 - dichloropropene has been recently reviewed (Stott et al., 2001).

Metam Sodium Metam sodium (C2 H4 NNaS2 ) is a widely used soil fumigant (Table 22-4), whose toxic action toward soil nematodes, fungi, and weed seeds is due to its hydrolysis product, methyl isothiocyanate (MITC). In mammals, metam sodium is metabolized in vivo to carbon disulfide and MITC (Pruett et al., 2001). In humans, metam sodium can act as a contact sensitizer, inducing allergic dermatitis, possibly due to MITC. Immunotoxicity is observed in rodents. In 1991, because of the derailment of a train car, approximately 19,000 gallons of metam sodium was spilled into the Sacramento River in California, causing a large kill of aquatic organisms. Symptoms reported by exposed individuals included headache, eye irritation, nausea, shortness of breath and dermatitis (Pruett et al., 2001). Metam sodium is increasingly being used as an alternative to methyl bromide (Ruzo, 2006).

Sulfur Elemental sulfur is considered the oldest of all pesticides, and its pesticidal properties were known to the ancient Greeks as early as 1000 b.c. (Tweedy, 1981). It is very effective as a fumigant for the control of many plant diseases, particularly fungal diseases, and still represents the most heavily used crop protection chemical in the United States (Table 22-4). Sulfur finds its major uses in grapes and tomatoes, and can be used in organic farming (Gammon et al., 2001). Though it is generally considered an environmentally and toxicologically safe compound, elemental sulfur used as a fungicide can make the soil too acidic for the continuous optimal growth of a particular crop (Gammon et al., 2001). The primary health effect in humans associated with the agricultural use of elemental sulfur is dermatitis (Gammon et al., 2001). In ruminants, excessive sulfur ingestion can cause cerebrocortical necrosis (polioencephalomalacia), possibly due to its conversion by microorganisms in the rumen to hydrogen sulfide (Gammon et al., 2001).

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Hasin Y, Avidan N, Bercovich D, et al.: A paradigm for single nucleotide polymorphism anlysis: The case of the acetylcholinesterase gene. Hum Mutat 24:408–416, 2004. Hayes, WJ: Pesticides Studied in Man. Baltimore: Williams & Wilkins, 1982, pp. 672. Hayes TB, Collins A, Lee M, et al.: Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc Natl Acad Sci USA 99:5476–5480, 2002. Hayes WJ, Laws ER (eds): Handbook of Pesticide Toxicology. San Diego: Academic Press, 1991 pp. 1576. He F, Wang S, Liu L, et al.: Clinical manifestations and diagnosis of acute pyrethroid poisoning. Arch Toxicol 63:54–58, 1989. Heikkila RE, Nicklas WJ, Vyas I, et al.: Dopaminergix toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their stereotaxic administration to rats: Implication for the mechanism of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine toxicity. Neurosci Lett 62:389–394, 1985. Hernandez AF, Martin-Rubi JC, Ballesteros JL, et al.: Clinical and pathological findings in fatal 1, 3-dichloropropene intoxication. Hum Exp Toxicol 13:303–306, 1994. Hertzstein J, Cullen MR: Methyl bromide intoxication in four field workers during removal of soil fumigation sheets. Am J Ind Med 17:321–326, 1990. Heudorf U, Angerer J, Drexler H: Current internal exposure to pesticides in children and adolescents in Germany: Urinary levels of metabolites of pyrethroid and organophosphorus insecticides. Int Arch Occup Environ Health 77:67–72, 2004. Heydens WF: Summary of toxicology studies with alachlor. J Pest Sci 24:75– 82, 1998. Heydens WF, Lamb IC, Wilson AGE: Chloroacetanilides, in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1543–1558. Heydens WF, Wilson AGE, Kier LD, et al.: An evaluation of the carcinogenic potential of the herbicide alachlor to man. Human Exp Toxicol 18:363–391, 1999. Hickey JP, Batterman SA, Chernyak SM: Trends of chlorinated organic contaminants in Great Lakes trout and walleye from 1970 to 1998. Arch Environ Contam Toxicol 50:97–110, 2006. Ho YS, Magnenat JL, Gargano M, et al.: The nature of antioxidant defense mechanisms: A lesson from transgenic mice. Environ Health Perspect 106:1219–1228, 1998. Hoar SK, Blair A, Holmes FF, et al.: Agricultural herbicide use and risk of lymphoma and soft-tissue sarcoma. JAMA 256:1141–1147, 1986. Hodgson E: In vitro human phase I metabolism of xenobiotics I: Pesticides and related compounds used in agriculture and public health, May 2003. J Biochem Mol Toxicol 17:201–206, 2003. Hollingworth RM: Chemistry, biological activity and uses of formamidine pesticides. Environ Health Perspect 14:57–69, 1976. Hollingworth RM: Inhibitors and uncouplers of mitochondrial oxidative phosphorylation, in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1169–1261. Hsu WH, Kakuk TJ: Effects of amitraz and chlordimeform on heart rate and pupil diameter in rats: Mediated by alpha2 -adrenoceptors. Toxicol Appl Pharmacol 73:411–415, 1984. Hsu WH, Lu ZH: Amitraz-induced delay of gastrointestinal transit in mice: Mediated by alpha2 -adrenergic receptors. Drug Dev Res 4:655–660, 1984. Hurt S, Ollinger J, Arce G, et al.: Dialkyldithiocarbamates (EBDCs), in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1759–1779. IARC (International Agency for Research on Cancer): Atrazine. IARC Monogr Eval Carcinog Risks Hum 73:59–113, 1999. IOM (Institute of Medicine): Veterans and Agent Orange. Update 1996. Washington DC: National Academy Press, 1996, pp. 365. IOM (Institute of Medicine): Gulf War and Health. Vol. I. Washington DC: National Academy Press, 2000, pp. 408. IPCS (International Programme on Chemical Safety): Chlordimeform. Environmental Health Criteria 1999. Geneva: World Health Organization, 1998, pp. 159.

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Singh S, Batra YK, Singh SM, et al.: Is atropine alone sufficient in acute severe organophosphorus poisoning? Experience of a North West Indian Hospital. Int J Clin Pharmacol Ther 33:628–630, 1995. Smith AG: Chlorinated hydrocarbon insecticides, in Hayes WJ, Laws ER (eds): Handbook of Pesticide Toxicology. San Diego: Academic Press, 1991, pp. 731–915. Smith AG: DDT and its analogs, in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1305–1355. Smith D: Worldwide trends in DDT levels in human breast milk. Int J Epidemiol 28:179–188, 1999. Smith LL: Mechanism of paraquat toxicity in the lung and its relevance to treatment. Hum Toxicol 6:31–36, 1987. Smith LL, Heath D: Paraquat. CRC Crit Rev Toxicol 4:411–445, 1976. Soderlund DM, Casida JE: Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pestic Biochem Physiol 7:391–401, 1977. Soderlund DM, Clark JM, Sheets LP, et al.: Mechanisms of pyrethroid neurotoxicity: Implications for cumulative risk assessment. Toxicology 171: 3–59, 2002. Song X, Seidler FJ, Saleh JL, et al.: Cellular mechanisms for developmental toxicity of chlorpyrifos: Targeting the adenylate cyclase signaling cascade. Toxicol Appl Pharmacol 145:158–174, 1997. Spagnolo A, Bianchi F, Calabro A, et al.: Anophthalmia and benomyl in Italy: A multicenter study based on 940,615 newborns. Reprod Toxicol 8:397–403, 1994. Stebbins KE, Johnson KA, Jeffries TK, et al.: Chronic toxicity and oncogenicity studies of ingested 1, 3-dichloropropene in rats and mice. Reg Toxicol Pharmacol 32:1–13, 2000. Stella J, Ryan M: Glyphosate herbicide formulation: A potentially lethal ingestion. Emerg Med Australia 16:235–239, 2004. Sterling TD, Arundel AV: Health effects of phenoxy herbicides. Scand J Work Environ Health 12:161–173, 1986. Stevens JT, Breckenridge CB: The avermectins: Insecticidal and antiparasitic agents, in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1157–1167. Stevens JT, Breckenridge CB, Simpkins J, et al.: Symmetrical and asymmetrical triazine herbicides,in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 1511–1519. Stevens JT, Breckenridge CB, Wetzel LT, et al.: A risk characterization for atrazine: Oncogenicity profile. J Toxicol Environ Health 56:69–109, 1999. Storm E, Rozman KK, Doull J: Occupational exposure limits for organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase. Toxicology 150:1–29, 2000. Stott WT, Gollapudi BB, Rao KS: Mammalian toxicity of 1,3 – Dichloropropene. Rev Environ Contam Toxicol 168:1–42, 2001. Stuetz W, Propamontol T, Erhardt JG, et al.: Organochlorine pesticide residues in human milk of a Hmong hill tribe living in Northern Thailand. Sci Total Environ 273:53–60, 2001. Sudakin DK: Biopesticides. Toxicol Rev 22:83–90, 2003. Sudakin DL, Trevathan WR: DEET: A review and update of safety and risk in the general population. J Toxicol Clin Toxicol 41:831–830, 2003. Sundwall A: Minimum concentrations of N-methylpyridinium -2-aldoxime methane sulphonate (P2S) which reverse neuromuscular block. Biochem Pharmacol 8:413–417, 1961. Taets C, Aref S, Rayburn AL: The clastogenic potential of triazine herbicide combinations found in potable water supplies. Environ Health Perspect 106:197–201, 1998. Talbot AR, Shiaw MH, Huang JS, et al.: Acute poisoning with a glyphosate surfactant herbicide (Round-up): A review of 93 cases. Hum Exp Toxicol 10:1–8, 1991. Tamura H, Yoshikawa H, Gaido KW, et al.: Interaction of organophosphate pesticides and related compounds with the androgen receptor. Environ Health Perspect 111:545–552, 2003. Tang J, Cao Y, Rose RL, et al.: Metabolism of chlorpyrifos by human cytochrome P450 isoforms and human, mouse and rat liver microsomes. Drug Metab Dispos 29:1201–1204, 2001.

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Tang J, Rose RL, Chambers JE: Metabolism of organophosphorus and carbamate pesticides, in Gupta RC (ed): Toxicology of Organophosphate and Carbamate Compounds. Amsterdam: Elsevier, 2006, pp. 127–143. Taylor JR, Selhorst JB, Houff SA, et al.: Chlordecone intoxication in man. I. Clinical observations. Neurology 28:626–630, 1978. Taylor P: Agents acting at the neuromuscular junction and autonomic ganglia, in Hardman JG, Limbird LE (ed): Goodman & Gilman’s The Pharmacological Basis of Therapeutics. New York: McGraw-Hill, 1996, pp. 177–197. Tenenbein M: Severe toxic reactions and death following the ingestion of diethyltoluamide-containing insect repellents. J Am Med Assoc 258:1509–1511, 1987. Thiruchelvam M, Richfield EK, Baggs RB, et al.: The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: Implications for Parkinson’s disease. J Neurosci 20:9207–9214, 2000. Tilson HA: Neurotoxicology risk assessment guidelines: Developmental neurotoxicology. Neurotoxicology 21:189–194, 2000. Tominack RL: Herbicide formulations. Clin Toxicol 38:129–135, 2000. Tomizawa M, Casida JE: Neonicotinoid insecticide toxicology: Mechanisms of selective action. Annu Rev Pharmacol Toxicol 45:247–268, 2005. Turosov V, Rakitsky V, Tomatis L: Dichlorodiphenyltrichloroethane (DDT): Ubiquity, persistence, and risks. Environ Health Perspect 110:125–128, 2002. Tweedy BW: Inorganic sulfur as a fungicide. Residue Rev 78:43–68, 1981. Ujvary I: Nicotine and other insecticidal alkaloids, in Yamamoto I, Casida JE (eds): Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor. Tokyo: Springer Verlag, 1999, pp. 29–69. Ujvary I: Pest control agents from natural products, in Krieger R (ed): Handbook of Pesticide Toxicology. San Diego: Academic Press, 2001, pp. 109–179. USEPA (U.S. Environmental Protection Agency): DDT: A review of Scientific and Economic Aspects of the Decision to Ban its Use as a Pesticide. Washington DC: USEPA, 1975, pp. 300. USEPA (U.S. Environmental Protection Agency): A SAB report: Assessment of potential 2,4-D carcinogenicity. Review of epidemiological and other data on potential carcinogenicity of 2,4-D. U.S. Environmental Protection Agency, Science Advisory Board, Washington, DC, 1994. USEPA (U.S. Environmental Protection Agency): Carcinogenicity peer review (4th) of 2, 4- dichlorophenoxyacetic acid (2,4-D). 1-29-1997. USEPA (U.S. Environmental Protection Agency): Exposure Factors Handbook, EPA/600/C-99/001, February. Washington DC: US EPA, Office of Research and Development, 1999. USEPA (U.S. Environmental Protection Agency): The use of data on cholinesterase inhibition for risk assessments of organophosphorus and carbamate pesticides. Washington, DC: USEPA, 2000, pp. 50 (accessible online at http://www.epa.gov/pesticides/trac/science/cholin.pdf). USEPA (U.S. Environmental Protection Agency): Potential developmental effects of atrazine in amphibians (www.epa.gov/oscpmont/sap/2003/ june/junemeetingreport.pdf). USEPA (U.S. Environmental Protection Agency): Picaridin, 2005 (http://www.epa. gov/opprd001/factsheets/picaridin.pdf). Van den Bercken J: The effect of DDT and dieldrin on myelinated nerve fibers. Eur J Pharmacol 20:205–214, 1972. Vanholder R, Colardyn F, De Reuck J, et al.: Diquat intoxication—Report of two cases and review of the literature. Am J Med 70:1267–1271, 1981. Veronesi B, Padilla S, Blackmon K, et al.: Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN). Toxicol Appl Pharmacol 107:311–324, 1991. Verschoyle RD, Aldridge WN: Structure-activity relationships of some pyrethroids in rats. Arch Toxicol 45:325–329, 1980. Vijverberg HPM, van der Zalm JM, van den Bercken J: Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves. Nature 295:601–603, 1982.

Villanueva CM, Durand G, Coutt´e MB, et al.: Atrazine in municipal drinking water and risk of low birth weight, pre-term delivery, and small-for-gestational-age status. Occup Environ Med 62:400–405, 2005. Wagner SL: Clinical Toxicology of Agricultural Chemicals. Park Ridge, NJ: Noyes Data Corporation, 1983, pp. 306. Waliszewski SM, Aguirre AA, Infanzon RM, et al.: Comparison of organochlorine pesticide levels in adipose tissue and human milk of mothers living in Veracruz, Mexico. Bull Environ Contem Toxicol 62:685–690, 1999. Watanabe T, Sano T: Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol 17:35–39, 1998. Weiss B, Amler S, Amler SW: Pesticides. Pediatrics 113:1030–1036, 2004. Wesseling C, Castillo L, Elinder CG: Pesticide poisonings in Costa Rica. Scand J Work Environ Health 19:227–235, 1993. Wesseling C, van Wendel De Joode B, Ruepert C, et al.: Paraquat in developing countries. Int J Occup Environ Health 7:275–286, 2001. Whalon ME, Wingend BA: Bt: Mode of action and use. Arch Insect Biochem Physiol 54:200–211, 2003. WHO (World Health Organization): DDT and its derivatives. Environmental Health Criteria 9. Geneva: WHO, 1979, pp. 194. WHO (World Health Organization): Public Health Impact of Pesticides Used in Agriculture. Geneva: WHO, 1990. Williams GM, Kroes R, Munro IC: Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Reg Pharmacol Toxicol 31:117–165, 2000. Wilson AGE, Lau H, Asbury KJ, et al.: Metabolism of alachlor by human nasal tissue. Fundam Appl Toxicol 15:1398, 1995. Wong CK, Leung KM, Poon BH, et al.: Organochlorine hydrocarbons in human breast milk collected in Hong Kong and Guangzhou. Arch Environ Contam Toxicol 43:364–372, 2002. Woods JS, Polissar L, Severson RK, et al.: Soft tissue sarcoma and nonHodgkin’s lymphoma in relation to phenoxyherbicide and chlorinated phenol exposure in western Washington. J Natl Cancer Inst 78:899– 910, 1987. Woolley DE: Neurotoxicity of DDT and possible mechanisms of action, in Prasad KN, Vernadakis A (eds): Mechanisms of Action of Neurotoxic Substances. New York: Raven Press, 1982, pp. 95–141. Worek F, Diebold C, Eyer P: Dimethylphosphoryl-inhibited human cholinesterases: Inhibition, reactivation and aging kinetics. Arch Toxicol 73:7–14, 1999a. Worek F, Mast U, Kiderlen D, et al.: Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta 288:73– 90, 1999b. Yang PY, Taso TC, Lin JL, et al.: Carbofuran-induced delayed neuropathy. J Toxicol Clin Toxicol 38:43–46, 2000. Yaramis A, Soker M, Bilici M: Amitraz poisoning in children. Hum Exp Toxicol 19:1–3, 2000. Yoon YJ, Kim ES, Hwang YS, et al.: Avermectin: Biochemical and molecular basis of its synthesis and regulation. Appl Microbiol Biotechnol 63:626–634, 2004. Zaccheo O, Dinsdale D, Meacock PA, et al.: Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells. J Biol Chem 279:24024–24033, 2004. Zadikoff CM: Toxic encephalopathy associated with use of insect repellents. J Pediatr 95:140–142, 1979. Zaki MM, Moran D, Harris D: Pesticide in ground-water: The aldicarb story in Suffolk County, New York. Am J Public Health 72:1391–1395, 1982. Zhang J, Fitsanakis VA, Gu G, et al.: Manganese ethylene-bisdithiocarbamate and selective dopaminergic neurodegeneration in rat: A link through mitochondrial dysfunction. J Neurochem 84:336–346, 2003. Zilker T, Fogt F, von Clarmann M: Kein Parkinsonsyndrom nach akuter paraquat intoxikation. Klin Wochenschr 66:1138–1141, 1988.

CHAPTER 23

TOXIC EFFECTS OF METALS Jie Liu, Robert A. Goyer, and Michael P. Waalkes

INTRODUCTION

ESSENTIAL METALS WITH POTENTIAL FOR TOXICITY

What is a Metal? Metals as Toxicants Movement of Metals in the Environment Chemical Mechanisms of Metal Toxicology Factors Impacting Metal Toxicity Biomarkers of Metal Exposure Metal-binding Proteins and Metal Transporters Pharmacology of Metals

Cobalt Toxicokinetics Essentiality Toxicity Copper Toxicokinetics Essentiality Toxicity Hereditary Disease of Copper Metabolism Treatment Iron Toxicokinetics Essentiality and Deficiency Toxicity Treatment Magnesium Toxicokinetics Essentiality and Deficiency Toxicity Manganese Toxicokinetics Essentiality and Deficiency Toxicity Molybdenum Toxicokinetics Essentiality and Deficiency Toxicity Selenium Toxicokinetics Essentiality and Deficiency Toxicity Trivalent Chromium Essentiality Zinc Toxicokinetics Essentiality and Deficiency Toxicity

MAJOR TOXIC METALS Arsenic Toxicokinetics Toxicity Carcinogenicity Treatment Beryllium Toxicokinetics Toxicity Carcinogenicity Cadmium Exposure Toxicokinetics Toxicity Carcinogenicity Treatment Chromium Toxicokinetics Toxicity Carcinogenicity Lead Exposure Toxicokinetics Toxicity Carcinogenicity Treatment Mercury Global Cycling and Ecotoxicology Exposure Toxicokinetics Toxicity Sensitive Sub-populations Treatment Nickel Toxicokinetics Toxicity Carcinogenicity Treatment of Nickel Toxicity

METALS RELATED TO MEDICAL THERAPY Aluminum Toxicokinetics Toxicity Treatment Bismuth Toxicokinetics Toxicity

931 Copyright © 2008 by The McGraw-Hill Companies, Inc. Click here for terms of use.

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Treatment Gallium Toxicokinetics Toxicity Gold Toxicokinetics Toxicity Lithium Toxicokinetics Toxicity Platinum Toxicokinetics Toxicity MINOR TOXIC METALS Antimony Toxicity Barium Toxicity Cesium Toxicity Fluorine

Toxicity Germanium Indium Toxicity Palladium Toxicity Silver Toxicity Tellurium Toxicity Thallium Toxicity Treatment Tin (Sn) Toxicity Titanium Toxicity Uranium Toxicity Vanadium Toxicity ACKNOWLEDGMENT

INTRODUCTION What is a Metal? The definition of a metal is not inherently obvious and the differences between metallic and nonmetallic elements can be subtle (Vouk, 1986a). Metals are typically defined by physical properties of the element in the solid state, but they vary widely with the metallic element. General metal properties include high reflectivity (luster); high electrical conductivity; high thermal conductivity; and mechanical ductility and strength. A toxicologically important characteristic of metals is that they may react in biological systems by losing one or more electrons to form cations (Vouk, 1986a). In the periodic table, within a group there is often a gradual transition from nonmetallic to metallic properties going from lighter to heavier atoms (e.g., Group IVa transitions from carbon to lead). Many metals exhibit variable oxidation states. Various names are applied to subsets of metallic elements including alkali metals (e.g., lithium and sodium), the alkaline earth metals (e.g., beryllium and magnesium), the transition (or heavy) metals (e.g., cadmium and zinc), and the metalloids (e.g., arsenic and antimony), which have characteristics between metal and nonmetals. In the periodic table, over 75% of the elements are regarded as metals and eight are considered metalloids. This chapter discusses metals that have been reported to produce significant toxicity in humans. This discussion will include major toxic metals (e.g., lead, cadmium), essential metals (e.g., zinc, copper), medicinal metals (e.g., platinum, bismuth), and minor toxic metals including metals in emerging technology (e.g., indium, uranium). Metal Toxicology will also discuss toxic metalloids (e.g., arsenic, antimony) and certain nonmetallic elemental toxicants (e.g., selenium, fluoride). An overview of Metal Toxicology is shown in Fig. 23-1.

Metals as Toxicants The use of metals has been critical to the progress and success of human civilization. It would be difficult to image an advanced society without extensive utilization of metallic compounds. Metals are unique among pollutant toxicants in that they are all naturally occurring and, in many cases, are ubiquitous within the human environment. Thus, regardless of how safely metals are used in industrial processes or consumer products, some level of human exposure is inevitable. In addition, all life has evolved in the presence of metals and organisms have been forced to deal with these potentially toxic, yet omnipresent, elements. In fact, many metals have become essential to various biological processes. Essentiality goes hand-in-hand with intentional accumulation and safe transport, storage, and usage mechanisms. Nonetheless, even essential metals will become toxic with increasing exposure. It is often the case that the nonessential toxicant metals mimic essential metals and thereby gain access to, and potentially disrupt, key cellular functions. This can also account for bioaccumulation of toxic metals. Metals differ from other toxic substances because, as elements, they are neither created nor destroyed by human endeavors. What human industry has accomplished is to concentrate metals in the biosphere. The anthropogenic contribution to the levels of metals in air, water, soil, and food is well recognized (Beijer and Jernelov, 1986). Human use of metals can also alter the chemical form or speciation of an element and thereby impact toxic potential. With a few notable exceptions, most metals are only sparingly recycled once used. These factors combine to make metals very persistent in the human environment. Metals are certainly one of the oldest toxicants known to humans due to their very early use. For instance, human use of lead

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Environmental Cycling Global distribution Biotransformation Biomagnification

Natural Anthropogenic

Occurrence

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Ecotoxicity Soil Plants Wildlife Domestic animals

Chemical form Speciation Essentiality

Occupational Environmental Dietary Medical

Dose Absorption Distribution Biotransformation Excretion

Chemistry

Human Exposure

Toxicokinetics

Adverse Health Effects

Adventitous bind, Mimicry Oxidative stress Enzyme inhibition DNA damage Gene expression

Mechanisms

Age, gender, etc. Adaptive mechanisms Metal transporters Metal-binding proteins

Host factors

Figure 23-1. Overview of Metal Toxicology.

probably started prior to 2000 bc, when abundant supplies were obtained from ores as a byproduct of smelting silver. The first description of abdominal colic in a man who extracted metals is credited to Hippocrates in 370 bc. Arsenic and mercury are discussed by Theophrastus of Erebus (370–287 bc), and Pliny and Elder (ad 23– 79). Arsenic was used early on for decoration in Egyptian tombs and as a “secret poison,” whereas mercury assumed almost a mystical stature in early science and was a large focus of alchemy. However, most of the use of the metals has occurred since the onset of the industrial revolution. In this regard, many of the metals of toxicological concern today were only relatively recently discovered. For instance, cadmium was first recognized in the early 1800s, and it was much later before the metal was widely used. The toxicological importance of some of the rarer or lesser used metals might well increase with new applications, such as chemotherapy and microelectronics, or other emerging technologies. Historically, metal toxicology largely concerned acute or overt, high-dose effects, such as abdominal colic from lead or the bloody diarrhea and uropenia after mercury ingestion. Because of advances in our understanding of potential toxicity of metals, and consequent improvements in industrial hygiene and stricter environmental standards, such acute high-dose effects are now uncommon. Focus has shifted to subtle, chronic, low-dose effects, in which cause-andeffect relationships may not be immediately clear. These might include a level of effect that causes a change in an important, but highly complex index of affected individual’s performance, such as lower than expected IQs due to childhood lead exposure. Other important chronic toxic effects include carcinogenesis, and several metals have emerged as human carcinogens. Assigning responsibility for such toxicological effects can often be difficult, particularly when the endpoint in question lacks specificity, in that it may be a

complex disease caused by a number of different chemicals or even combinations of chemicals. In addition, humans are never exposed to only a single metal, but rather to complex mixtures. The metals as a class of toxicants clearly present many challenges in toxicological research. The elemental nature of metals impacts their biotransformation and toxicity, as detoxification by destructive metabolism to subcomponents of lesser toxicity cannot occur with these atomic species. In essence, as elemental species metals are non-biodegradable. This indestructibility combined with bioaccumulation contributes to the high concern for metals as toxicants. Most elemental metals tend to form ionic bonds. However, biological conjugation to form organometallic compounds can occur for various metals (Dopp et al., 2004), particularly with metalloids, like arsenic, that show mixed carbonaceous and metallic qualities. The redox capacity of a given metal or metallic compound should also be considered as part of its metabolism. The metabolism of metals is intricate and subtle but can directly impact toxic potential.

Movement of Metals in the Environment Metals are redistributed naturally in the environment by both geologic and biologic cycles. Rainwater dissolves rocks and ores and transports materials, including metals, to rivers and underground water (e.g., arsenic), depositing and stripping materials from adjacent soil and eventually transporting these substances to the ocean to be precipitated as sediment or taken up into forming rainwater to be relocated elsewhere. Biological cycles moving metals include biomagnification by plants and animals resulting in incorporation into food cycles. In comparison, human activity often intentionally shortens the residence time of metals in ore deposits, and can result in

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the formation of new, non-naturally occurring metallic compounds. For instance, cadmium distribution mainly comes from human activities. Human industry greatly enhances metal distribution in the global environment by discharge to soil, water, and air, as exemplified by the 200-fold increase in lead content of Greenland ice since the onset of the industrial revolution. Mercury undergoes global cycling with elevated levels being found far from points of discharge, as, for example, with mercury in the Arctic Ocean. Mercury also undergoes biomethylation and biomagnification by aquatic organisms (see Fig. 23-6). Increased distribution of metals and metal compounds in the environment, especially through anthropogenic activities, raises increasing concern for ecotoxicological effects. Reports of metal intoxication are common in plants, aquatic organisms, invertebrates, fish, sea mammals, birds, and domestic animals. The ecotoxicity of various metals is discussed under each individual section. Mercury poisoning from consumption of fish containing high levels of methylmercury and cadmium poisoning from consumption of rice grown in soils contaminated with cadmium from industrial discharges are examples of human consequences from environmental pollution. Not all human toxicity occurs from metals deposited in the biosphere by human activity. For example, chronic arsenic poisoning from high levels of naturally occurring inorganic arsenic in drinking water is a major health issue in many parts of the world. Endemic intoxication from excess fluoride, selenium, or thallium can all occur from natural high environmental levels.

Chemical Mechanisms of Metal Toxicology The precise chemical basis of metal toxicology is inadequately understood but a uniform mechanism for all toxic metals is implausible because of the great variation in chemical properties and toxic endpoints. Chemically, metals in their ionic form can be very reactive and can interact with biological systems in a large variety of ways. In this regard, a cell presents numerous potential metal-binding ligands. For instance, metals like cadmium and mercury readily attach to sulfur in proteins as a preferred bio-ligand. Such adventitious binding is an important chemical mechanism by which exogenous metals exert toxic effects that can result in steric re-arrangement that impairs the function of biomolecules (Kasprzak, 2002). An example would be the inhibition of enzyme activity by metal interaction at sites other than the active center, such as the inhibition of heme synthesis enzymes by lead. The inhibition of biologically critical enzymes is an important molecular mechanism of metal toxicology. The metals can show more specific forms of chemical attack through mimicry. In this regard the toxic metals may act as mimics of essential metals, binding to physiological sites that normally are reserved for an essential element. Owing to their rich chemistry, essential metals control, or are involved in, a variety of key metabolic and signaling functions (Kasprzak, 2002; Cousins et al., 2006). Through mimicry, the toxic metals may gain access to, and potentially disrupt a variety of important or even critical metal-mediated cellular functions. For example, mimicry for, and replacement of zinc, is a mechanism of toxicity for cadmium, copper, and nickel. Thallium mimics potassium and manganese mimics iron as a critical factor in their toxicity. Mimicry of arsenate and vanadate for phosphate allows for cellular transport of these toxic elements whereas selenate, molybdate, and chromate mimic sulfate and can compete for sulfate carriers and in chemical sulfation reactions (Bridges and Zalpus, 2005). Organometallic compounds can also act as mimics of

biological chemicals, as, for example, with methylmercury, which is transported by amino acid or organic anion transporters (Bridges and Zalpus, 2005). Indeed, molecular or ionic mimicry at the level of transport is often a key event in metal toxicity. Another key chemical reaction in metal toxicology is metalmediated oxidative damage. Many metals can directly act as catalytic centers for redox reactions with molecular oxygen or other endogenous oxidants, producing oxidative modification of biomolecules such as proteins or DNA. This may be a key step in the carcinogenicity of certain metals (Kasprzak, 2002). Besides oxygen-based radicals, carbon- and sulfur-based radicals may also occur. Nickel and chromium are two examples of metals that act, at least in part, by generation of reactive oxygen species or other reactive intermediates (Kasprazak, 2002). Alternatively, metals may displace redox active essential elements from their normal cellular ligands, which, in turn, may result in oxidative cellular damage. For instance, cadmium, which is not redox active, may well cause oxidative stress through the release of endogenous iron, an element with high redox activity (Valko et al., 2006). Metals in their ionic form can be very reactive and form DNA and protein adducts in biological systems. For example, once hexavalent chromium enters the cell it is reduced by various intracellular reductants to give reactive trivalent chromium species that form DNA adducts or DNA-protein cross-links, events likely to be important in chromium genotoxicity (Zhitkovich, 2005). Metals can also induce an array of aberrant gene expression, which, in turn, produces adverse effects. For example, nickel can induce the expression of Cap43/NDRG1, under the control of the hypoxia-inducible transcription factor (HIF-1), which is thought to play a key role in nickel carcinogenesis (Costa et al., 2005). An array of aberrant hepatic gene expressions occurs in adult mice after in utero arsenic exposure, which could be an important molecular event in arsenic hepatocarcinogenesis (Liu et al., 2006).

Factors Impacting Metal Toxicity The standard factors that impact the toxic potential of all chemicals apply to the metals as well. Exposure-related factors include dose, route of exposure, duration, and frequency of exposure. Because metals can be quite reactive, and the portal of entry is often initially the organ most affected, as with the lung after inhalation. Host-based factors that can impact metal toxicity include age at exposure, gender, and capacity for biotransformation. For instance, it is quite clear that younger subjects are often more sensitive to metal intoxication, as, for example, with the neurotoxicity of lead in children. The major pathway of exposure to many toxic metals in children is food, and children consume more calories per pound of body weight than adults. Moreover, children have higher gastrointestinal absorption of metals, particularly lead. The rapid growth and proliferation in the perinate represent opportunities for toxic effects, including potentially carcinogenesis, of metallic agents, and several metals (e.g., arsenic, nickel, lead, and chromium) are transplacental carcinogens in rodents. Fetal-stage toxicity of metals is well documented, as with methylmercury, and many metals are teratogenic. For many inorganics there is no impediment to transplacental transport, as with lead or arsenic, and human fetal blood lead levels are similar to maternal levels. Elderly persons are also believed to be generally more susceptible to metal toxicity than younger adults. Recognition of factors that influence toxicity of a metal is important in determining risk, particularly in susceptible subpopulations.

CHAPTER 23

TOXIC EFFECTS OF METALS

Chemical-related factors directly impact the toxic potential of metals. This would include the precise metal compound and its valence state or speciation. For instance, methylmercury is a potent neurotoxin, whereas the inorganic mercurials primarily attack the kidney. Similarly, the oxidation state of chromium can differentiate the essential (naturally occurring trivalent chromium) from toxic species (hexavalent chromium). Lifestyle factors such as smoking or alcohol ingestion may have direct or indirect impacts on the level of metal intoxication. For instance, cigarette smoke by itself contains many toxic metals, such as cadmium, and it is thought that smoking will double the lifetime burden of cadmium in nonoccupationally exposed individuals. Other components of cigarette smoke may also influence pulmonary effects, as, for instance, with metals that are lung carcinogens. Alcohol ingestion may influence toxicity by altering diet, reducing essential mineral intake, and altering hepatic iron deposition. The composition of the diet can significantly alter gastrointestinal absorption of various dietary metals. The essentiality of metals has direct bearing on the toxic potential of a metal. Any “free” ionic metal would be potentially toxic due to reactive potential. The need to accumulate essential metals dictates the evolution of systems for the safe transport, storage, and utilization as well as, within limits, elimination of excess. For example, metallothionein is a metal-binding protein that may function in the homeostatic control of zinc (Cousins et al., 2006), and may represent a storage or transport form of this metal. Such factors imply that a threshold would exist for toxicity due to essential metal exposure. In this regard, the essential metallic elements would be expected to show a “U”-shaped dose–response curve in that, at very low exposure levels, toxic adverse effects would occur from deficiency, but at high exposure levels toxicity also occurs. The nonessential toxic metals can mimic essential elements and disrupt homeostasis, as with cadmium which will potentially displace zinc to bind to zinc-dependent transcription factors and enzymes (Waalkes, 2003). Adaptive mechanisms can be critical to the toxic effects of metals, and organisms have a variety of ways in which they can adapt to otherwise toxic metal insults. Typically, adaptation is acquired after the first few exposures and can be long lasting or transient after exposure ceases. Adaptation can be at the level of uptake or excretion, or with some metals, through long-term storage in a toxicological inert form. For instance, it appears enhanced arsenic efflux is involved in acquired tolerance to the metalloid on the cellular level (Liu et al., 2001). Conversely, intentional sequestration of toxic metals is another adaptive tactic and examples of such longterm storage include lead-inclusion bodies, which form in various organs and contain protein-immobilized lead in a distinct cellular aggresome. These bodies are thought to be protective by limiting the level of free, and therefore toxic, lead within the cell, and the inability to form such bodies clearly increases the chronic toxic effects of lead, including carcinogenesis (Waalkes et al., 2004c). Similarly, cadmium exposure causes the overexpression of metallothionein which will sequester cadmium and reduce its toxicity as an adaptive mechanism (Klaassen and Liu, 1998). Metal exposure can also induce a cascade of molecular/genetic responses that may, in turn, reduce toxicity, such as with metal-induced oxidative stress responses (Valko et al., 2006). It is clear that acquired metal adaptation, although allowing immediate cellular survival, may in fact be a potential contributing factor in long-term toxicity (Waalkes et al., 2000). For instance, acquired self-tolerance to cadmium- or arsenicinduced apoptosis may actually contribute to eventual carcinogene-

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sis by allowing survival of damaged cells that would otherwise have been eliminated (Hart et al., 2001; Pi et al., 2005).

Biomarkers of Metal Exposure Biomarkers of exposure, toxicity, and susceptibility are important in assessing the level of concern with metal intoxication. Exposure biomarkers, such as concentrations in blood or urine, have long been used with metals. Techniques in molecular toxicology have greatly expanded the possibilities for biomarkers. Thus, in the case of chromium, DNA–protein complexes may serve as a biomarker of both exposure and carcinogenic potential. The capacity for expression of genes that potentially play protective roles against metal toxicity, as, for example, with metallothionein and heme oxygenase, show promise as markers of both effect and susceptibility. The use of such biomarkers may well allow identification of particularly sensitive subpopulations. Estimates of the relationship of exposure level to toxic effects for a particular metal are in many ways a measure of the dose– response relationships discussed in great detail earlier in this book. The dose of a metal is a multidimensional concept and is a function of time as well as concentration. The most toxicologically relevant definition of dose is the amount of active metal within cells of target organs. The active form is often presumed to be the free metal, but it is technically difficult or impossible to precisely determine. A critical indicator of retention of a metal is its biological halflife, or the time it takes for the body or organ to excrete half of an accumulated amount. The biological half-life varies according to the metal as well as the organ or tissue. For example, the biological half-lives of cadmium in kidney and lead in bone are 20–30 years, whereas for some metals, such as arsenic or lithium, they are only a few hours to days. For many metals, more than one half-life is needed to fully describe the retention. The half-life of lead in blood is only a few weeks, as compared to the much longer half-life in bone. After inhalation of mercury vapor, at least two half-lives describe the retention in brain, one on the order of a few weeks and the other measured in years. Continued metal exposure clearly complicates retention kinetics. Blood, urine, and hair are the most accessible tissues for quantifying metal exposure. Results from single measurements may reflect recent exposure or long-term or past exposure, depending on retention time in the particular tissue. Blood and urine concentrations usually, but not always, are reflective of more recent exposures and correlate with acute adverse effects. An exception is urinary cadmium, which may reflect kidney damage related to a renal cadmium accumulation over several decades. Hair can be useful in assessing variations in exposure to metals over the period of its growth. Analyses can be performed on segments of the hair, so that metal content of the newest growth can be compared with past exposures. Hair levels of mercury have been found to be a reliable measure of exposure to methylmercury. For most other metals, however, hair is not a reliable tissue for measuring exposure because of metal deposits from external contamination that may complicate analysis.

Metal-binding Proteins and Metal Transporters Protein binding of metals is a critical aspect of essential and toxic metal metabolism (Zalpus and Koropatnick, 2000). Many different types of proteins play roles in the disposition of metals in the body. Nonspecific binding to proteins, like serum albumin or hemoglobin, act in metal transport and tissue distribution. Metals vary in their

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preferred site of proteinaceous binding, and can attack a variety of amino acid residues. For instance, cysteine sulfurs are preferred by cadmium and mercury, and these residues are commonly involved with overall protein structure. In addition, proteins with specific metal-binding properties play special roles in the trafficking of specific essential metals, and toxic metals may interact with these proteins through mimicry. Metal-binding proteins are an important, emerging issue in the physiology and toxicology of metals and only a few examples are highlighted here. The metallothioneins are a very important class of metalbinding proteins that function in essential metal homeostasis and metal detoxification (Klaassen et al., 1999). They are small (6000 Da), soluble, and rich in internally oriented thiol ligands. These thiol ligands provide the basis for high-affinity binding of several essential and toxic metals including zinc, cadmium, copper, and mercury. The metallothioneins are highly inducible by a variety of metals or other stimulants. Metallothioneins clearly play an important role in metal toxicity, as illustrated in the discussion of cadmium below. Transferrin is a glycoprotein that binds most of the ferric iron in plasma and helps transport iron across cell membranes. The protein also transports aluminum and manganese. Ferritin is primarily a storage protein for iron. It has been suggested that ferrin may serve as a general metal detoxicant protein, because it binds a variety of toxic metals including cadmium, zinc, beryllium, and aluminum. Ceruloplasmin is a copper-containing glycoprotein oxidase in plasma that converts ferrous iron to ferric iron, which then binds to transferrin. This protein also stimulates iron uptake by a transferrinindependent mechanism. In all cells there are mechanisms for metal ion homeostasis that frequently involve a balance between uptake and efflux systems. A rapidly increasing number of metal transport proteins are being discovered that transport metals across cell membranes and organelles inside the cells. Metal transporters are important for cellular resistance to metals or metalloids (Rosen, 2002). For instance, enhanced efflux via multidrug resistance protein pumps is involved in acquired tolerance to arsenic (Liu et al., 2001), wheraes decreased influx via reduced calcium G-type channels is involved in acquired tolerance to cadmium (Leslie et al., 2006). Over ten zinc transporters and four Zip family proteins are involved in cellular zinc transport, trafficking, and signaling (Cousins et al., 2006). The importance of metal transporters in human diseases is well illustrated by Menkes disease and Wilson disease, which are caused by genetic mutations in the copper-transport protein gene ATP7A, resulting in copper deficiency (Menkes disease), or ATP7B, resulting in copper overload (Wilson disease) (see Fig. 23-7).

Pharmacology of Metals Metal and metal compounds have a long history of pharmacological use. Metallic agents, largely because of their potential toxicity, have been often used in chemotherapeutic settings. For instance, mercury was used in the treatment of syphilis as early as the 16th century. Similarly, Ehrlich’s magic bullet (arsphenamine) was an organoarsenical. Today, many metallic chemicals remain valuable pharmacological tools in the treatment of human disease, as exemplified by the highly effective use of platinum compounds in cancer chemotherapy. In addition, gallium and titanium complexes are promising metal compounds in cancer chemotherapy. Other medicinal metals used today include aluminum (antacids and buffered analgesics), bismuth (peptic ulcer and Helicobacter pylori asso-

ciated gastritis), lithium (mania and bipolar disorders), and gold (arthritis). Treatment of metal poisoning is sometimes used to prevent, or even attempt to reverse, toxicity. The typical strategy is to give metal chelators that will complex the metal and enhance its excretion (Klaassen, 2001). Most chelators are not specific and will interact with a number of metals, eliminating more than the metal of concern. In addition, the vast array of biological metal ligands is a formidable barrier to chelator efficacy (Klaassen, 2001). Metal chelation therapy should be considered a secondary alternative to reduction or prevention of toxic metal exposures. Such therapy can be used for many different metals including lead, mercury, iron, and arsenic. For detailed discussion on the pharmacology of chelation therapy, see Klaassen (2001).

MAJOR TOXIC METALS Arsenic Arsenic (As) is a toxic and carcinogenic metalloid. The word arsenic is from the Persian word Zarnikh, as translated to the Greek arsenikon, meaning “yellow orpiment.” Arsenic has been known and used since ancient times as the Poison of Kings and the King of Poisons. The element was first isolated in about 1250. Arsenicals have been used since ancient times as drugs and even today are very effective against acute promyelocytic leukemia (Soignet et al., 2001). Arsenic exists in the trivalent and pentavalent forms and is widely distributed in nature. The most common inorganic trivalent arsenic compounds are arsenic trioxide and sodium arsenite, while common pentavalent inorganic compounds are sodium arsenate, arsenic pentoxide, and arsenic acid. Important organo-arsenicals include arsenilic acid, arsenosugars, and several methylated forms produced as a consequence of inorganic arsenic biotransformation in various organisms, including humans. Arsine (AsH3 ) is an important gaseous arsenical. Occupational exposure to arsenic occurs in the manufacture of pesticides, herbicides, and other agricultural products. High exposure to arsenic fumes and dusts may occur in smelting industries (ATSDR, 2005a). Environmental arsenic exposure mainly occurs from arsenic-contaminated drinking water. Arsenic in drinking water is often from natural sources. Although most U.S. drinking water contains arsenic at levels lower than 5 μg/L (ppb), it has been estimated that about 25 million people in Bangladesh alone drink water with arsenic levels above 50 ppb (IARC, 2004). Environmental exposure to arsenic also occurs from burning of coal containing naturally high levels of arsenic (Liu et al., 2002), and perhaps from wood treated with arsenical preservatives (Khan et al., 2006). It is not known, however, to what extent arsenic-treated wood products contribute to human exposure. Food, especially seafood, may contribute significantly to daily arsenic intake. Arsenic in seafood is largely in an organic form called arsenobetaine that is much less toxic than the inorganic forms (ATSDR, 2005a). Toxicokinetics Inorganic arsenic is well absorbed (80–90%) from the gastrointestinal tract, distributed throughout the body, often metabolized by methylation, and then excreted primarily in urine (NRC, 2001). Arsenic compounds of low solubility (e.g., arsenic trioxide, arsenic selenide, lead arsenide, and gallium arsenide) are absorbed less efficiently after oral exposure. Skin is a potential route of exposure to arsenic, and systemic toxicity has been reported in persons having dermal contact with solutions of inorganic arsenic

CHAPTER 23

Arsenite

Arsenate

OH O

5+

HO

MMA3+

OH

OH

SAH

3+

O

As OH

Arsenate reductase

937

MMA5+ SAM

OH

GSH

As OH O−

TOXIC EFFECTS OF METALS

5+

As CH3 O−

AS3MT or Arsenite methyltransferase

GSH

As

GSTO1 or AS3MT

3+

OH

CH3 SAM

AS3MT or Arsenite methyltransferase

O H3 C

O−

OH 5+

As CH3 CH3

As AS3MT

TMAO

3+

GSH

CH3

CH3 DMA3+

GSTO1 or AS3MT

O

SAH

5+

As CH3 CH3 DMA5+

Figure 23-2. Arsenic Metabolism. GSH, reduced glutathione; GSTO1, glutathione S-transferase omega-1; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; AS3MT, arsenic methyltransferase (Cyt19); MMA5+ , monomethylarsonic acid; MMA3+ , monomethylarsonous acid; DMA5+ , dimethylarsinic acid; DMA3+ , dimethylarsinous acid; TMAO, trimethylarsenic oxide.

(Hostynek et al., 1993). Airborne arsenic is largely trivalent arsenic oxide. Deposition in airways and absorption of arsenicals from lungs is dependent on particle size and chemical form. Excretion of absorbed arsenic is mainly via the urine. The whole-body biological half-life of ingested arsenic is about 10 hours, and 50–80% is excreted over 3 days. The biological half-life of methylated arsenicals is in the range of 30 hours. Arsenic has a predilection for skin and is excreted by desquamation of skin and in sweat, particularly during periods of profuse sweating. It also concentrates in forming fingernails and hair. Arsenic exposure produces characteristic transverse white bands across fingernails (Mees’ line), which appear about 6 weeks after the onset of symptoms of arsenic toxicity. Arsenic in the fingernails and hair has been used as a biomarker for exposure, including both current and past exposures, while urinary arsenic is a good indicator for current exposure. Methylation of inorganic arsenic species is no longer considered as a detoxication process, as recent work has identified the highly toxic trivalent methylated arsenicals. Some animal species even lack arsenic methylation capacity, perhaps as an adaptation mechanism. Figure 23-2 illustrates the biotransformation of arsenic. Arsenate (As5+ ) is rapidly reduced to arsenite (As3+ ) by arsenate reductase (presumably purine nucleoside phosphorylase). Arsenite is then sequentially methylated to form methylarsonate (MMA5+ ) and dimethylarsinic acid (DMA5+ ) by arsenic methyltransferase (AS3MT or Cyt19) or arsenite methyltransferase using S-adenosylmethionine (SAM) as a methyl group donor. The intermediate metabolites, methylarsonous acid (MMA3+ ) and dimethylarsinous acid (DMA3+ ), are generated during this process, and these trivalent methylated arsenicals are now thought to be more toxic than even the inorganic arsenic species (Aposhian and Aposhian, 2006; Thomas et al., 2007). In humans, urinary arsenicals are composed of 10–30% inorganic arsenicals, 10–20% MMA, and 55–76% DMA (NRC, 2001). However, large variations in arsenic methylation occur due to factors such as age and sex, and it is suspected that ge-

netic polymorphisms may exist. Arsenic metabolism also changes through the course of pregnancy, reflected in higher urinary excretion of DMA and lower urinary levels of inorganic arsenic and MMA, which may have toxicologic impact on the developing fetus (Hopenhayn et al., 2003).

Toxicity Acute Poisoning Ingestion of large doses (70–180 mg) of inorganic arsenic can be fatal. Symptoms of acute intoxication include fever, anorexia, hepatomegaly, melanosis, cardiac arrhythmia and, in fatal cases, eventual cardiac failure. Acute arsenic ingestion can damage mucous membranes of the gastrointestinal tract, causing irritation, vesicle formation, and even sloughing. Sensory loss in the peripheral nervous system is the most common neurologic effect, appearing at 1–2 weeks after large doses and consisting of Wallerian degeneration of axons, a condition that is reversible if exposure is stopped. Anemia and leucopenia, particularly granulocytopenia, occur a few days following high-dose arsenic exposure and are reversible. Intravenous arsenic infusion at clinical doses in the treatment of acute promyelocytic leukemia may be significantly or even fatally toxic in susceptible patients, and at least three sudden deaths have been reported (Westervelt et al., 2001). Acute exposure to a single high dose can produce encephalopathy, with signs and symptoms of headache, lethargy, mental confusion, hallucination, seizures, and even coma (ATSDR, 2005a). Arsine gas, generated by electrolytic or metallic reduction of arsenic in nonferrous metal production, is a potent hemolytic agent, producing acute symptoms of nausea, vomiting, shortness of breath, and headache accompanying the hemolytic reaction. Exposure toarsine is fatal in up to 25% of the reported human cases and may be accompanied by hemoglobinuria, renal failure, jaundice, and anemia in nonfatal cases when exposure persists (ATSDR, 2005a).

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Chronic Toxicity The skin is a major target organ in chronic inorganic arsenic exposure. In humans, chronic exposure to arsenic induces a series of characteristic changes in skin epithelium. Diffuse or spotted hyperpigmentation and, alternatively, hypopigmentation can first appear between 6 months to 3 years with chronic exposure to inorganic arsenic. Palmar-plantar hyperkeratosis usually follows the initial appearance of arsenic-induced pigmentation changes within a period of years (NRC, 2001). Skin cancer is common with protracted high-level arsenical exposure (see below). Liver injury, characteristic of long-term or chronic arsenic exposure, manifests itself initially as jaundice, abdominal pain, and hepatomegaly (NRC, 2001; Mazumder, 2005). Liver injury may progress to cirrhosis and ascites, even to hepatocellular carcinoma (Centeno et al., 2002; Liu et al., 2002). Repeated exposure to low levels of inorganic arsenic can produce peripheral neuropathy. This neuropathy usually begins with sensory changes, such as numbness in the hands and feet but later may develop into a painful “pins and needles” sensation. Both sensory and motor nerves can be affected, and muscle tenderness often develops, followed by weakness, progressing from proximal to distal muscle groups. Histological examination reveals a dying-back axonopathy with demyelination, and effects are dose-related (ATSDR, 2005a). An association between ingestion of inorganic arsenic in drinking water and cardiovascular disease has been shown (NRC, 2001; Chen et al., 2005; Navas-Acien et al., 2005). Peripheral vascular disease has been observed in persons with chronic exposure to inorganic arsenic in the drinking water in Taiwan. It is manifested by acrocyanosis and Raynaud’s phenomenon and may progress to endarteritis and gangrene of the lower extremities (Blackfoot disease). Arsenic-induced vascular effects have been reported in Chile, Mexico, India, and China, but these effects do not compare in magnitude or severity to Blackfoot disease in Taiwanese populations, indicating other environmental or dietary factors may be involved (Yu et al., 2002). Some studies have shown an association between high arsenic exposure in Taiwan and Bangladesh and an increased risk of diabetes mellitus, but the data for occupational exposure is inconsistent (Navas-Acien et al., 2006). Additional research is required to verify a link between inorganic arsenic exposure and diabetes. Immunotoxic effects of arsenic have been suggested (ATSDR, 2005a). The hematologic consequences of chronic exposure to arsenic may include interference with heme synthesis, with an increase in urinary porphyrin excretion, which has been proposed as a biomarker for arsenic exposure (Ng et al., 2005). Mechanisms of Toxicity The trivalent compounds of arsenic are thiol-reactive, and thereby inhibit enzymes or alter proteins by reacting with proteinaceous thiol groups. Pentavalent arsenate is an uncoupler of mitochondrial oxidative phosphorylation, by a mechanism likely related to competitive substitution (mimicry) of arsenate for inorganic phosphate in the formation of adenosine triphosphate. Arsine gas is formed by the reaction of hydrogen with arsenic, and is a potent hemolytic agent (NRC, 2001). In addition to these basic modes of action, several mechanisms have been proposed for arsenic toxicity and carcinogenicity. Arsenic and its metabolites have been shown to produce oxidants and oxidative DNA damage, alteration in DNA methylation status and genomic instability, impaired DNA damage repair, and enhanced cell proliferation (NRC, 2001; Rossman, 2003). Unlike many carcinogens, arsenic is not a mutagen in bacteria and acts weakly in

mammalian cells, but can induce chromosomal abnormalities, aneuploidy, and micronuclei formation. Arsenic can also act as a comutagen and/or co-carcinogen (Rossman, 2003; Chen et al., 2005). These mechanisms are not mutually exclusive and multiple mechanisms likely account for arsenic toxicity and carcinogenesis. Some mechanisms, however, may be organ specific. Carcinogenicity The carcinogenic potential of arsenic was recognized over 110 years ago by Hutchinson, who observed an unusual number of skin cancers occurring in patients treated for various diseases with medicinal arsenicals. IARC (2004) has classified arsenic as a known human carcinogen, associated with tumors of the skin, lung, and urinary bladder, and possibly kidney, liver, and prostate (NRC, 2001; IARC, 2004). Arsenic-induced skin cancers include basal cell carcinomas and squamous cell carcinomas, both arising in areas of arsenicinduced hyperkeratosis. The basal cell cancers are usually only locally invasive, but squamous cell carcinomas may have distant metastases. In humans, the skin cancers often, but not exclusively, occur on areas of the body not exposed to sunlight (e.g., on palms of hands and soles of feet). They also often occur as multiple malignant lesions. Animal models have shown that arsenic acts as a rodent skin tumor copromoter with 12-O-teradecanoyl phorbol-13acetate in v-Ha-ras mutant Tg.AC mice (Germolec et al., 1998) or as a co-carcinogen with UV irradiation in hairless mice (Rossman et al., 2001). The association of internal tumors in humans with arsenic exposure is well recognized (NRC, 2001). This includes arsenicinduced tumors of the urinary bladder, and lung, and potentially the liver, kidney, and prostate. In rats, the methylated arsenic species, DMA5+ , is a urinary bladder tumor initiator and promoter (Wei et al., 2002) and produces urothelial cytotoxicity and proliferative regeneration with continuous exposure (Cohen et al., 2001). However, the relevance of this finding to inorganic arsenic carcinogenesis must be extrapolated cautiously, due to the high dose of DMA required to produce these changes in rats (NRC, 2001). In contrast to most other human carcinogens, it has been difficult to confirm the carcinogenicity of inorganic arsenic in experimental animals. Recently, a transplacental arsenic carcinogenesis model has been established in mice. Short-term exposure of the pregnant rodents from gestation day 8 to day 18, a period of general sensitivity to chemical carcinogenesis, produces tumors in the liver, adrenal, ovary, and lung of offspring as adults (Waalkes et al., 2003, 2004a). The tumor spectrum after in utero arsenic exposure resembles estrogenic carcinogens and is associated with overexpression of estrogen-linked genes (Liu et al., 2006), and thus a hypothesis that arsenic may somehow act on estrogen signaling to produce hepatocarcinogenic effects has been proposed (Waalkes et al., 2004b). Indeed, when in utero arsenic exposure is combined with postnatal treatment with the synthetic estrogen diethylstilbestrol, synergistic increases in malignant urogenital system tumors, including urinary bladder tumors and liver tumors, are observed (Waalkes et al., 2006a,b). As a corollary in humans, increased mortality occurs from lung cancer in young adults following in utero exposure to arsenic (Smith et al., 2006). Thus, the developing fetus appears to be hypersensitive to arsenic carcinogenesis. Treatment For acute arsenic poisoning, treatment is symptomatic, with particular attention to fluid volume replacement and support of blood pressure. The oral chelator penicillamine or succimer

CHAPTER 23

TOXIC EFFECTS OF METALS

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(2,3-dimercaptosuccinic acid, DMSA) is effective in removing arsenic from the body. Dimercaptopropanesulfonic acid (DMPS) has also been used for acute arsenic poisoning with fewer side effects (Aposhian and Aposhain, 2006). However, for chronic poisoning, chelator therapy has not proven effective in relieving symptoms (Rahman et al., 2001; Liu et al., 2002) except for a limited preliminary trial with DMPS (Mazumder, 2005). The best strategy for preventing chronic arsenic poisoning is by reducing exposure.

Acute Chemical Pneumonitis Inhalation of beryllium can cause a fulminating inflammatory reaction of the entire respiratory tract, involving the nasal passages, pharynx, tracheobronchial airways, and the alveoli. In the most severe cases, it produces acute fulminating pneumonitis. This occurs almost immediately following inhalation of aerosols of soluble beryllium compounds, particularly the fluoride, during the ore extraction process. Fatalities have occurred, although recovery is generally complete after a period of several weeks or even months.

Beryllium

Chronic Granulomatous Disease Berylliosis, or chronic beryllium disease (CBD), was first described among fluorescent lamp workers exposed to insoluble beryllium compounds, particularly beryllium oxide. Granulomatous inflammation of the lung, along with dyspnea on exertion, cough, chest pain, weight loss, fatigue, and general weakness, are the most typical features. Impaired lung function and hypertrophy of the right heart are also common. Chest X-ray shows miliary mottling. Histologically, the alveoli contain small interstitial granulomas resembling those seen in sarcoidosis. In severe cases, CBD may be accompanied by cyanosis and hypertrophic osteoarthropathy (WHO, 1990; ATSDR, 2002). Beryllium sensitization following initial exposure can progress to CBD (Newman et al., 2005). As the lesions progress, interstitial fibrosis increases, with loss of functioning alveoli, impairment of effective air-capillary gas exchange, and increasing respiratory dysfunction. CBD involves an antigen-stimulated, cell-mediated immune response. Human leukocyte antigen, T cells, and proinflammatory cytokines (TNF-α and IL-6) are believed to be involved in the pathogenesis of CBD (Fontenot et al., 2002; Day et al., 2006).

Beryllium (Be), an alkaline earth metal, was discovered in 1798. The name beryllium comes from the Greek beryllos, a term used for the mineral beryl. Beryllium compounds are divalent. Beryllium alloys are used in automobiles, computers, sports equipment, and dental bridges. Pure beryllium metal is used in nuclear weapons, aircraft, X-ray machines, and mirrors. Human exposure to beryllium and its compounds occur primarily in beryllium manufacturing, fabricating, or reclaiming industries. Individuals may also be exposed to beryllium from implanted dental prostheses. The general population is exposed to trace amounts of beryllium through the air, food, and water, as well as from cigarette smoke (WHO, 1990; ATSDR, 2002). Toxicokinetics The primary route of exposure to beryllium compounds is through the lungs. After being deposited in the lung, beryllium is slowly absorbed into the blood. In patients accidentally exposed to beryllium dust, serum beryllium levels peak about 10 days after exposure with a biological half-life of 2–8 weeks (ATSDR, 2002). Gastrointestinal and dermal absorption of beryllium is low (3 mg Cu/L will produce gastrointestinal symptoms. Ingestion of large amounts of copper salts, most frequently copper sulfate, may produce hepatic necrosis and death. Epidemiological studies have not found any relation between copper exposure and cancer (WHO, 1998).

Blood Ceruloplasmin Cu Small Intestine

Cu

Portal Vein

Cu

Liver

Hereditary Disease of Copper Metabolism Menkes Disease This is a rare sex-linked genetic defect in copper metabolism resulting in copper deficiency in male infants. It is characterized by peculiar hair, failure to thrive, severe metal retardation, neurologic impairment, and death usually by 5 years of age. Bones are osteoporotic with flared metaphases of the long bones and bones of the skull. There is extensive degeneration of the cerebral cortex and of white matter. The gene responsible for Menkes disease, ATP7A, belongs to the family of ATPases and is a copper transporter (Fig. 23-8). Deficiency in this copper transporter in Menkes disease blocks copper transport across the basolateral membrane of intestinal cells into the portal circulation, resulting in accumulation of copper in the entrocytes and systemic copper deficiency in the body. The transport of copper to the brain is also blocked, causing severe neurological abnormalities (Mercer, 2001). Animal models for copper deficiency support the importance of adequate copper intake during embyrogenesis and early development (Shim and Harris, 2003). Wilson Disease This is an autosomal recessive genetic disorder of copper metabolism characterized by the excessive accumulation of copper in liver, brain, kidneys, and cornea. Serum ceruloplasmin is low and serum copper not bound to ceruloplasmin is elevated. Urinary excretion of copper is high. Clinical abnormalities of the nervous system, liver, kidneys, and cornea are related to copper accumulation. Patients with Wilson disease have impaired biliary excretion of copper, which is believed to be the fundamental cause of the copper overload. Genetic studies have identified the defect in copper transport as mutations of the Wilson disease locus (WND) on chromosome 13, encoding P-type ATPase (ATP7B) (WHO, 1998; Harris, 2000). There appears to be several polymorphisms of the defect, which may explain the clinical variability in the disorder. Diagnosis may be suspected with elevated serum copper but must be confirmed by liver biopsy and elevated liver copper (normally 15–55 μg/g versus > 250 μg/g in Wilson disease). Animal models of Wilson disease include the toxic milk (Tx) mouse and the Long–Evans Cinnamon (LEC) rats. Both rodent models develop hepatocellular damage from abnormal copper accumulation associated with mutations in ATP7B, but do not exhibit the neurological symptoms associated with the human disease (Shim and Harris, 2003). Hereditary Aceruloplasnaemia This is the autosomal recessive genetic disorder of copper-binding protein ceruloplasmin, associ-

Bile

Feces

Wilson Disease Menkes’ Disease

Figure 23-8. Pathways of Copper in the Body and Defects in Menkes’ and Wilson Diseases. Copper is absorbed by the enterocytes of the small intestine and transported across the basolateral membrane of enterocytes into the portal circulation. This later process is defective in Menkes’ disease patients and results in accumulation of copper in the enterocytes and overall copper deficiency in the body. Most of the newly absorbed copper is normally taken up by the liver. In cases of copper overload, excess copper is excreted in the bile and this process is blocked in Wilson disease, as is the delivery of copper to ceruloplasmin, the principal copper carrier in the blood. Other low molecular weight proteins such as Cu–metallothionein and Cu–histine are also proposed to be important sources of copper to tissues. The transport of copper to the brain is blocked in patients with Menkes’ disease, leading to the severe neurological abnormalities (Adapted from Mercer, 2001, with permission from Elsevier).

ated with the iron-overload syndrome. Clinical signs and symptoms include mental confusion, memory loss, dementia, cerebellar ataxia, altered motor function, retinal degeneration, and diabetes (WHO, 1998). Ceruloplasmin-null mice accumulate iron predominantly in organs of reticuloendothelial system. In these mice, hematologic indices and serum iron are abnormal by 10 weeks of age, with profound iron overload in the spleen and liver. Hepatic copper deposition is also approximately doubled in these mice. However, neurodegeneration and diabetes are not observed in these mice (Shim and Harris, 2003). Indian Childhood Cirrhosis (ICC) This is a disorder occurring in young children characterized by jaundice due to an insidious and progressive liver disease. Two distinguishing features are a widespread brown orcein staining (indicating copper) and intralobular hepatic fibrosis progressing to portal cirrhosis and chronic inflammation. The etiology is not known but it is suspected that bottle feeding of milk contaminated with copper from storage in brass vessels may be important. However, epidemiological studies also suggest an autosomal recessive genetic component because of strong familial occurrence and high consanguinity among affected children (WHO, 1998).

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Idopathic Copper Toxicosis or Non-Indian Childhood Cirrhosis This is a rare disorder in children similar to ICC occurring in some Western countries. The largest series of cases are reported from the Tyrol region of Austria. This population also used copper vessels to store milk, and the incidence of the disorder has declined since replacement of the copper vessels. A number of other cases have been reported from other parts of the world, some from increased amounts of copper in drinking water (WHO, 1998).

Treatment Clinical improvement can be achieved by chelation of copper with d-penicillamine, Trien [triethylene tetramine 2HCl)], zinc acetate, and tetrathiomolybdate. The combination of tetrathiomolybdate and zinc acetate is more effective (Brewer, 2005). N -Acetylcystein amide can cross the blood–brain barrier and was developed to help prevent neurodegenerative disorders (Cai et al., 2005).

Iron Iron (Fe) is a very abundant transition metal. Iron came into use around 4000 bc. An early source of iron was from fallen meteorites and the name may derive from the Ethruscan word aisar which means “the gods.” Iron is an essential metal for erythropoiesis and a key component of hemoglobin, myoglobin, heme enzymes, metalloflavoprotein enzymes, and mitochondrial enzymes. In biological systems, iron mainly exists as the ferrous (2+) and ferric (3+) forms. Toxicologic considerations are important in terms of iron deficiency, accidental acute exposures, and chronic iron overload due to idiopathic hemochromatosis or as a consequence of excess dietary iron or frequent blood transfusions (Yip and Dallman, 1996; IOM, 2001; Papanikolaou and Pantopoulos, 2005).

Toxicokinetics Iron metabolism is regulated by a complex series of events that maintain homeostasis, mainly involving absorption, storage, and excretion. Heme iron from meat, poultry, and fish is highly bioavailable. Nonheme iron absorption is influenced by its solubility and by other dietary factors, such as vitamin C (ascorbic acid) which enhances uptake. Absorption involves movement of ferrous ions from the intestinal lumen into the mucosal cells via the divalent metal transporter protein-1 (DMT-1) and then transfer from the mucosal cell to the plasma, where iron is bound to transferrin for transport and distribution. Transferrin is a β1-globulin with a molecular weight of 75,000 and is produced in the liver. Transferrin delivers iron to tissues by binding to transferrin receptor-1(TfR1) on the cell membrane, followed by endocytosis. Iron is then released in the acidic endosomal vesicle. The human body contains ∼3–5 g of iron. About two-thirds of body iron is in hemoglobin, 10% is in myoglobin and iron-containing enzymes, and the remainder is bound to the iron storage proteins like ferritin and hemosiderin, stored in liver and reticuloendothelial cells in the spleen and bone marrow. Iron stores serve as a reservoir to supply cellular iron needs, mainly for hemoglobin production. Erythrocyte destruction and production are responsible for most iron turnover. Hepcidin, a small peptide of liver origin, modulates iron absorption in response to erythropoiesis (Papanikolaou and Pantopoulos, 2005). The major route of excretion of iron is into the gastrointestinal tract and eventually the feces. Daily iron losses from urine, gastrointestinal tract, and skin are ∼0.08, 0.6, and 0.2 mg/day, respectively.

Essentiality and Deficiency Iron deficiency is the most common nutritional deficiency worldwide, affecting infants, young children, and women of child-bearing age. The critical period for iron deficiency in children is between the ages of 6 months and 2 years. The major manifestation of iron deficiency is anemia with microcytic hypochromic red blood cells. Other effects of iron deficiency include impaired psychomotor development and intellectual performance, decreased resistance to infection, adverse pregnancy outcomes, and possibly increased susceptibility to lead and cadmium toxicity. Oral ferrous sulfate is the treatment of choice for iron deficiency.

Toxicity Acute iron poisoning from accidental ingestion of ironcontaining dietary supplements is the most common cause of acute toxicity. It most often occurs in children. A decrease in occurrence of this type of poisoning followed the introduction of childproof lids on prescription medicines and vitamin supplements. Severe toxicity occurs after the ingestion of more than 0.5 g of iron or 2.5 g of ferrous sulfate. Toxicity occurs about 1–6 hours after ingestion. Symptoms include abdominal pain, diarrhea, and vomiting. Of particular concern are pallor or cyanosis, metabolic acidosis, and cardiac collapse. Death may occur in severely poisoned children within 24 hours. Supportive therapy and iron chelation with deferoxamine should be used as soon as possible. Inhalation of iron oxide fumes or dust may cause pneumoconiosis in occupational settings (Dotherty et al., 2004). Chronic iron toxicity from iron overload in adults is a relatively common problem. There are three basic ways in which excessive amounts of iron can accumulate in the body. The first is hereditary hemochromatosis due to abnormal absorption of iron from the intestinal tract. Hereditary hemochromatosis is an autosomal recessive disorder attributed to mutation in the hemochromatosis gene. About 90% of patients are homozygous for C282Y mutation, while a few patients are heterozygotes for C282Y with a second mutation for H63D. The frequency of homozygosity is ∼0.3–0.4% in populations of European ancestry. The second possible cause of iron overload is excess intake via the diet or from oral iron preparations. The third circumstance in which iron overload can occur is repeated blood transfusions for some form of refractory anemia and is referred to as transfusional siderosis. The pathologic consequences of iron overload are similar regardless of the basis. Hemosiderosis refers to increased iron stores in the form of hemosiderin. The body iron content can increase 20–40 g, up to 10 times higher than normal levels. Hemochromatosis refers to excessive deposition of iron that causes organ damage, often resulting in fibrosis. Inhalation of iron oxide fumes or dust by workers in hematic mines (mainly Fe2 O3 ), steel workers, and welders may produce siderosis (nonfibrotic), and in some cases silicosis (fibrotic) in the lung, with increases in total body iron (Doherty et al., 2006). Liver iron overload from hereditary hemochromatosis is associated with a high risk for hepatocellular carcinoma, as well as with other malignancies (Papanikolaou and Pantopoulos, 2005). Increased body iron may play a role in the development of cardiovascular disease. It is suspected that iron may act as a catalyst to produce free radical damage resulting in artherosclerosis and ischemic heart disease (Alpert, 2004). This iron hypothesis is controversial, but it is clear that mortality from cardiovascular disease is correlated with liver iron overload (Yuan and Li, 2003). Several neurodegenerative disorders are associated with aberrant iron metabolism in the brain, such as neuroferritinopathy,

CHAPTER 23

TOXIC EFFECTS OF METALS

aceruloplasminemia, and manganism (Aschner et al., 2005; Papanikolaou and Pantopoulos, 2005). Treatment Desferrioxamine is the chelator of choice for the treatment of acute iron intoxication and chronic iron overload. Iron chelators have also been proposed for the treatment of cancers with iron overload (Buss et al., 2004).

Magnesium Magnesium (Mg) was recognized as an element in 1755. The name originates from the Greek word for a district in Thessaly called Magnesia. Magnesium is a nutritionally essential metal that plays a key role in a wide range of important fundamental cellular reactions (Shils, 1996). Nuts, cereals, seafood, and meats are good dietary sources of magnesium. The drinking water content of magnesium increases with hardness of the water. Magnesium citrate, oxide, sulfate, hydroxide, and carbonate are widely taken as antacids or cathartics. Magnesium hydroxide, or milk of magnesia, is one of the universal antidotes for poisoning. Topically, the sulfate is also used to relieve inflammation. Parenteral administration of magnesium sulfate has been used in the treatment of seizures associated with eclampsia of pregnancy and acute nephritis. Toxicokinetics Oral magnesium is absorbed mainly in the small intestine. The colon also absorbs some magnesium. Calcium and magnesium are competitive with respect to absorption, and excess calcium will partially inhibit magnesium absorption. Serum magnesium levels are remarkably constant. Magnesium is excreted into the digestive tract by the bile and in pancreatic and intestinal juices. Approximately 60–65% of the total body magnesium is in the bone, 27% in muscle, 6–7% in other organs, and only 1% is in extracellular fluid. Of the magnesium filtered by the glomeruli about 95% is reabsorbed, an important factor in maintaining homeostasis. Essentiality and Deficiency Magnesium is a cofactor of many enzymes. In the glycolytic cycle, there are seven key enzymes that require divalent magnesium. Magnesium-containing enzymes are also involved in the citric acid cycle and in beta oxidation of fatty acids. Deficiency may occur as a complication of various disease states such as malabsorption syndromes, renal dysfunction, and endocrine disorders. Magnesium deficiency in humans causes neuromuscular irritability, frank tetany, and even convulsions. Magnesium deficiency induces an inflammatory syndrome (Mazur et al., 2007), and is a risk factor for diabetes mellitus, hypertension, hyperlipidemia, and ischemic heart diseases (Ueshima, 2005). Supplementation of magnesium, either by intravenous or oral administration, is beneficial. Toxicity In industrial exposures, no ill effects are produced with a twofold increase in serum magnesium, although concurrent increases occur in serum calcium. Inhaled freshly generated magnesium oxide can cause metal fume fever, similar to that caused by zinc oxide. In nonoccupationally exposed individuals, toxicity can occur when magnesium-containing drugs, usually antacids, are ingested chronically by persons with serious renal failure. The toxic effects may progress from nausea and vomiting to hypotension, electrocardiograph abnormalities, central nervous system effects, coma, and systolic cardiac arrest (Shils, 1996). Magnesium toxicity can sometimes be counteracted with calcium infusion.

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Manganese Manganese (Mn) was in use in prehistoric times. Paints that were pigmented with manganese dioxide can be traced back to ancient times. The pure element was isolated in 1774 and named after the Latin magnes, meaning “magnet.” Manganese is an essential metal required for many metabolic and cellular functions. Manganese metalloenzymes include arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and manganese superoxide dismutase (Aschner and Aschner, 2005). Manganese is also a cofactor for a number of enzymatic reactions. Manganese exists in many valences but the divalent cation is by far the predominant species within cells. Divalent manganese may be oxidized to the more reactive and toxic trivalent form. The major source of manganese intake is from the food. Vegetables, grains, fruits, nuts, and tea are rich in manganese. Daily manganese intake ranges from 2 to 9 mg (ATSDR, 2000). The Adequate Intake is 2.3 and 1.8 mg/day for adult men and women, respectively (IOM, 2002). Occupational exposures to high concentrations of manganese can occur in a number of settings, including manganese dioxide mines and smelters. Significant exposure can also occur in factories making manganese steel alloys, electrical coils, batteries, glass, welding rods, and during production of potassium permanganate (KMnO4 ). The industrial use of manganese has expanded in recent years as a ferroalloy in the iron industry and as a component of alloys used in welding (Crossgrove and Zheng, 2004). Environmental exposures are often associated with manganesebased organometallic pesticides, maneb and mancozeb. Manganese intoxication has also been reported after ingestion of contaminated water (ATSDR, 2000; Crossgrove and Zheng, 2004). There is current interest in the toxicology of manganese-containing fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT). In addition, manganese compounds, such as mangafodipir are increasingly used as MRI enhancers in clinical imaging techniques.

Toxicokinetics Approximately 1–5% of ingested manganese is normally absorbed. Interactions between manganese, and iron, as well as other divalent elements, occur and impact the toxicokinetics of manganese especially following oral exposure (Roth and Garrick, 2003). Iron and manganese can compete for the same binding protein in serum (transferrin) and the same transport systems (divalent metal transporter, DMT1). Inhalation of particulate manganese may result in direct transfer to brain tissue via the olfactory system (Tjalve and Henriksson, 1999). Within the plasma, manganese is largely bound to gamma globulin and albumin, with a small fraction bound to transferrin. Manganese concentrates in mitochondria, so that tissues rich in these organelles, like pancreas, liver, kidneys, and intestines, have the highest concentrations of manganese. Manganese readily crosses the blood–brain barrier and accumulates in specific brain regions (Crossgrove and Zheng, 2004). Manganese is eliminated in the bile and reabsorbed in the intestine. The principal route of manganese excretion is with the feces. Biliary excretion is poorly developed in neonates and exposure during this period may result in increased delivery of manganese to the brain and other tissues (Aschner and Aschner, 2005).

Essentiality and Deficiency Manganese deficiency has been produced in many species of animals, but questions remain about whether deficiency has actually been demonstrated in humans

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(WHO, 1996). Deficiency in animals results in impaired growth, skeletal abnormalities, and disturbed reproductive function.

Toxicity Chronic manganese-induced neurotoxicity (manganism) is of great concern and the brain is considered the most sensitive organ to manganese. Neurotoxicity due to inhalation of airbone manganese ranging from 0.027 to 1 mg Mn/m3 has been reported in a number of occupational settings. Overt manganism occurs in workers exposed to aerosols containing extremely high levels (>1–5 mg Mn/m3 ). Neurotoxicity also occurs following ingestion of manganese contaminated water (1.8 to 14 ppm; Aschner et al., 2005). Manganism is associated with elevated brain levels of manganese, primarily in those areas known to contain high concentrations of nonheme iron, such as the substantia nigra, basal ganglia, caudateputamen, globus pallidus, and subthalamic nuclei. Early manifestations of manganese neurotoxicity include headache, insomnia, memory loss, muscle cramps, and emotional instability. Initial outward symptoms progress gradually and are mainly psychiatric. As exposure continues and the disease progresses, patients may develop prolonged muscle contractions (dystonia), decreased muscle movement (hypokinesia), rigidity, hand tremor, speech disturbances, and festinating “cock-walk” gait. These signs are associated with damage to dopaminergic neurons that control muscle movement (Crossgrove and Zheng, 2004; Aschner et al., 2005). Specialized T1-weighted magnetic resonance brain imaging of manganism patients indicates high levels in the basal ganglia and especially in the globus pallidus. Inhalation of manganese-containing dust in certain occupational settings can lead to an inflammatory response in the lung. Symptoms of lung irritation and injury may include cough, bronchitis, pneumonitis, and occasionally, pneumonia (ATSDR, 2000). Men working in plants with high concentrations of manganese dust show an incidence of respiratory disease that is 30 times greater than normal. Manganese exposure also alters cardiovascular function in animals and humans, as evidenced by abnormal electrocardiogram and the inhibition of myocardial contraction. Manganese dilates blood vessels and induces hypotension (Jiang and Zheng, 2005). When manganese is combined with bilirubin, it produces intrahepatic cholestasis by acting on the synthesis and degradation of cholesterol and the inhibition of the transport pump Mrp2 (Akoume et al., 2003). Liver cirrhosis is a major contributing factor for hepatic encephalopathy, often associated with increased manganese levels in the brain (Mas, 2006). Interactions between manganese and iron play a role in manganese toxicity. The coaccumulation of iron with manganese in the globus pallidus raises the concern that iron may be a contributing factor facilitating neuronal cell loss during manganese intoxication. Chronic exposure to manganese alters iron concentrations in blood and cerebrospinal fluid, presumably due to manganese– iron interaction at certain iron–sulfur containing proteins, which regulate iron homeostasis. Manganese intoxication in monkeys causes elevated iron deposition in the globus pallidus and substantia nigra. The excess iron may produce oxidative stress via the Fenton reaction, leading to neuronal damage. Dysfunctional iron metabolism has also been seen in manganism patients. Serum parameters associated with iron metabolism, such as ferritin, transferrin, and total-iron-binding capacity are significantly altered (Roth and Garrick, 2003; Crossgrove and Zheng, 2004). High levels of total iron and iron-associated oxidative stress, decreased ferritin, and abnormal mitochondrial complex-1 have been repeatedly re-

ported in postmortem samples of substantia nigra from manganism patients. Available data indicate that inorganic manganese is not carcinogenic in humans or rodents, and negative in Ames test, but may cause DNA damage and chromosome aberrations in vitro in mammalian cells (Gerber et al., 2002).

Molybdenum Molybdenum (Mo) was first separated from lead and graphite in 1778. Molybdenum was derived from Greek molybdos meaning “lead-like.” As an essential element, molybdenum acts as a cofactor for at least three enzymes in humans: sulfite oxidase, xanthine oxidase, and aldehyde oxidase. Molybdenum exists in five oxidation states but the predominant species are Mo4+ and Mo6+ . Molybdenum concentration in food varies considerably depending on the local environment. Molybdenum is added in trace amounts to fertilizers to stimulate plant growth. The human requirement for molybdenum is low and easily provided by a common US diet. The RDA for molybdenum is 45 μg/day (IOM, 2001). The most important mineral source of molybdenum is molybdenite (MoS2 ). The industrial uses of this metal include the manufacture of high temperature-resistant steel alloys for gas turbines and jet aircraft engines and in the production of catalysts, lubricants, and dyes. Ammonium tetrathiomolybdate is used as a molybdenumdonating copper chelator in treatment of Wilson disease (Brewer, 2003). Toxicokinetics Water-soluble molybdenum compounds are readily absorbed when ingested. In laboratory animals, gastrointestinal absorption varies between 75% and 95%. In humans, absorption of molybdenum after oral intake varies from 28% to 77% (Vyskocil and Viau, 1999). Once absorbed, molybdenum rapidly appears in blood and most tissues. The highest molybdenum concentrations are found in kidneys, liver, and bones. Very little molybdenum appears to cross the placenta. When elevated exposure is ceased, tissue concentrations quickly return to normal levels. Molybdenum metabolism is related to copper and sulfur. Exposure to molybdenum decreases intestinal absorption of copper and sulfate, and impairs the sulfation of chemicals (Boles and Klaassen, 2000). Excretion, primarily via the urine, is rapid and 36–90% of a dose of molybdenum is excreted in urine in experimental animals. In humans, the urinary excretion ranges from 17% to 80% of the total dose. Very little ( 0.1 mg/kg), and presynaptically acting toxins (LD50 < 0.1 mg/kg) (Rosenberg, 1990). Although the sequences of these enzymes are homologous and their enzymatically active sites are identical, they differ widely in their pharmacologic properties. For example, taipoxin, a PLA2 enzyme from the venom of the Australian elapid Oxyuranus scutellatus, has an intravenous LD50 in mice of 2 μg/kg, whereas the neutral PLA2 from Naja nigricollis has an LD50 of 10,200 μg/kg, even though N. nigricollis PLA2 is enzymatically more active (Russell, 2001). Arginine ester hydrolase is one of a number of noncholinesterases found in snake venoms. The substrate specificities are directed to the hydrolysis of the ester or peptide linkage, to which an argine residue contributes the carboxyl group. This activity is found in many crotalid and viperid venoms and some sea snake venoms but is lacking in elapid venoms with the possible exception of Ophiophagus hannah. Some crotalid venoms contain at least three chromatographically separable arginine ester hydrolases. The bradykinin-releasing and perhaps bradykinin-clotting activities of some crotalid venoms may be related to esterase activity. Two distinct classes of fibrin(ogen)olytic enzymes, the metalloproteinases and the serine proteinases, have been isolated from venom of Viperidae, Elapidae, and Crotalidae snake families (Swenson and Markland, 2005). These two classes of proteinases differ in mechanism of action and their target in fibrin(ogen), but ultimately they break down fibrin-rich clots and help to prevent further clot formation. The properties of fibrolase, an α-chain fibrinolytic metalloproteinase from Agkistrodon contortrix contortrix venom, and β-fibrinogenase, a β-chain fibrinogenase from Vipera lebetina, are provided in Table 26-5. Properties of some newly characterized fibrin(ogen)ases are listed in Table 26-6. It is apparent that there are major differences in the properties of these enzymes from different snakes even though they have similar catalytic properties. An exciting development from the research on these enzymes is that one specific recombinant fibrinolytic enzyme derived from fibrolase called alfimeprase is progressing through clinical trials for the treatment of peripheral arterial occlusions. The snake venom hemorrhagic metalloproteinases (SVMP) are enzymes that disrupt the hemostatic system and they are characterized by their domain structure into four primary classes, PI– PIV. SMVPs are synthesized in vivo as multimodular proteins that

Table 26-6 Miscellaneous Properties of Some α-Chain and β-Chain Fibrin(ogen)ases properties

α-chain fibrinogenase

α-chain fibrinogenase

α-chain fibrinogenase

β-chain fibrinogenase

Genus species Common name Chain length Molecular weight pI Carbohydrate content pH optimum

Bothops neuwiedi Neuwiedase 198 amino acids 22.5 kDa 5.9 200 amino acids 24 kDa ND Glycosylated ND

Agkistrodon blomhoffi brevicadus Brevinase 233 amino acids 25.7 kDa 5.5 ND 5.5–8.5

ND, not determined. source: Data from Swenson and Markland (2005).

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Table 26-7 Comparison of Thrombin and Thrombin-like Snake Venom Enzyme Actions action on human fibrinogen

enzymes Thrombin Thrombin-like enzymes Agkistrodon C. contortrix Venom Bitis gabonica Venom

fibrinopeptides released

chain degradation

activation of factor xiii

prothrombin fragment cleavage

platelet aggregation and release

A-B A∗

α(A) α(A)† or β(B)‡

Yes No

Yes Yes or no§

Yes No

B

ND

Incomplete

ND

No

A+B

ND

Yes

ND

ND

∗

Includes ancrod, batroxobin, crotalase, and the enzyme from T. okinavensis. Ancrod [batroxobin degrades α(A) chain of bovine but not human fibrinogen]. ‡ Crotalase. §Fragment I released by crotalase and Agkistrodon contortrix venom, but not by ancrod or batroxobin. ND, not determined. source: Data from Russell (2001). †

Table 26-8 Comparison of Snake Venom Thrombin-Like Enzymes venom enzyme

molecular weight

carbohydrate content (%)

active site serine

Agkistrodon contortrix contortrix Bitis gabonica Bothrops marajoensis Bothrops moojeni Calloselasma rhodostoma Crotalus adamantus Crotalus horridus horridus Deinagkistrodon acutus Trimeresurus gramineus Trimeresurus okinavensis

100,000 32,500 31,400 36,000 59,000 32,700 19,400 33,500 27,000 34,000

ND ND High 5.8 36.0 8.3 Very low 13.0 25.0 6.0

+ ND + + + + ND + + +

ND, not determined. source: Data from Russell (2001).

comprise a signal peptide, a prodomain, and a metalloprotease domain (SVMP PI). SVMP potency tends to increase by class and those within the PIV class are larger and comprises additional disulfide bonds (Calvette et al., 2003, 2005). Class PII proteins exhibit a C-terminal disintegrin domain. The PII metalloproteases block the function of integrin receptors, a function that could alleviate a variety of pathological conditions such as inflammation, tumor angiogenesis and metastasis, and thrombosis. The integrin-blocking specificity of this class of metalloproteins is highly dependent on the conformation of the inhibitory loop, and thus the placement and bonding of cysteine residues. More specifically, within the inhibitory loops, RGD-containing disintegrins are specific to the class PII metalloproteases (Calvette et al., 2003, 2005). Class PIII SVMPs exhibit the disintegrin-like domain and the C-type lectin-like domain is present in PIV SVMPs. The metalloproteinase domain or catalytic domain is composed of about 215 amino acids and has metal-dependent endopeptidase activity (Calvette et al., 2005). SVMPs degrade proteins such as laminin, fibronectin, type IV collagen, and proteoglycans from the endothelial basal membrane; degrade fibrinogen and von Willebrand factor enhancing the hemorrhagic action; and inhibit

platelet aggregation and stimulate release of cytokines (Ramos and Selistre-de-Araujo, 2006). The proteolytic action of thrombin and thrombin-like snake venom enzymes is shown in Table 26-7. This table compares ancrod (from Calloselasma rhodostoma), batroxobin (from Bothrops moojeni), crotalase (from Crotalus adamanteus), gabonase (from Bitis gabonica), and venzyme (from A. contortrix). Table 26-8 shows the molecular size of some thrombin-like enzymes. A recent contribution on snake toxins, using mass spectrometric immunoassay and bioactive probe techniques, has been published by Ramirez et al. (1999). Considerable study has been given to the hemostatic properties of venoms (Markland, 1998). The hemostatically active components are summarized in Table 26-9. Phosphomonoesterase (phosphatase) is widely distributed in the venoms of all families of snakes except the colubrids. It has the properties of an orthophosphoric monoester phosphohydrolase. There are two nonspecific phosphomonoesterases, and they have optimal pH at 5.0 and 8.5. Many types of venom contain both acid and alkaline phosphatases, whereas others contain one or the other.

CHAPTER 26

PROPERTIES AND TOXICITIES OF ANIMAL VENOMS

Table 26-9 Snake Venom Proteins Active on the Hemostatic System general functional activity

specific biological activity

Procoagulant

Activates factors II, V, IX, X, and Protein C Fibrinogen clotting Factor IX/factor X-binding protein Thrombin inhibitor Phospholipase A Fibrin(ogen) degradation Plasminogen activation Hemorrhagic

Anticoagulant

Fibrinolytic Vessel wall interactive

sources: Data from Markland (1998) and Russell (2001).

Phosphodiesterase has been found in the venoms of all families of poisonous snakes. It is an orthophosphoric diester phosphohydrolase that releases 5-mononucleotide from the polynucleotide chain and thus acts as an exonucleotidase, attacking DNA and RNA. More recently, it has been found that it also attacks derivatives of arabinose. Acetylcholinesterase was first demonstrated in cobra venom and is widely distributed throughout the elapid venoms. It is also found in sea snake venoms but is totally lacking in viperid and crotalid venoms. It catalyzes the hydrolysis of acetylcholine to choline and acetic acid. The role of the enzyme in snake venoms is not clear. RNase is present in some snake venoms in small amounts as the endopolynucleotidase RNase. It appears to have specificity toward pyrimidine-containing pyrimidyladenyl bonds in DNA. The optimum pH is 7–9 when ribosomal RNA is used as the substrate. This enzyme in Naja oxiana venom has a molecular weight of 15,900. DNase acts on DNA to produce predominantly tri- or higher oligonucleotides that terminate in 3 monoesterified phosphate. C. adamanteus venom contains two DNases, with optimum pH at 5 and 9. 5 -Nucleotidase is a common constituent of all snake venoms; in most instances it is the most active phosphatase in snake venoms. It specifically hydrolyzes phosphate monoesters, which link with a 5 position of DNA and RNA. It is found in greater amounts in crotalid and viperid venoms than in elapid venoms. The molecular weight as determined from amino acid composition and gel filtration with Naja naja atra venom has been estimated at 10,000. The enzyme from N. naja venom is enhanced by Mg2+ , is inhibited by Zn2+ , is inactivated at 75◦ C at pH 7.0 or 8.4, and has an isoelectric point of about 8.6. That from Agkistrodon halys blomhoffi shows a pH optimum of 6.8–6.9, with activity being enhanced by Mg2+ and Mn2+ and inhibited by Zn2+ . The enzyme has a low order of lethality (Russell, 2001). Nicotinamide adenine dinucleotide (NAD) nucleotidase has been found in a number of snake venoms. This enzyme catalyzes the hydrolysis of the nicotinamide N -ribosidic linkage of NAD, yielding nicotinamide and adenosine diphosphate riboside. Its optimum pH is 6.5–8.5; it is heat labile, losing activity at 60◦ C. Nucleotidases function as ADP scavengers thereby acting as potent inhibitors of platelet aggregation. l-Amino acid oxidase has been found in all snake venoms examined so far. It gives a yellow color to the venom. This enzyme catalyzes the oxidation of l-α-amino and α-hydroxy acids. This activity results from a group of homologous enzymes with molec-

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ular weights ranging from 85,000 to 150,000. It has a high content of acidic amino acids. The mouse intravenous LD50 of the enzyme from C. adamanteus venom was 9.13 mg/kg body weight, approximately four times less than the lethal value of the crude venom, and this enzyme had no effect on nerve, muscle, or neuromuscular transmission (Russell, 2001).

Polypeptides Snake venom polypeptides are low-molecularweight proteins that do not have enzymatic activity. More than 80 polypeptides with pharmacologic activity have been isolated from snake venoms. Interested readers will find definitive reviews on these peptides in the works of Lee (1979), Eaker and Wadstr¨om (1980), and Gopalakrishnakone and Tan (1992). Most of the lethal activity of the poison of the sea snake Laticauda semifasciata was recovered as two toxins, erabutoxin-a and erabutoxin-b, using carboxymethylcellulose chromatography; 30% of the proteins were erabutoxins. More recently, erabutoxin-a, a short-chain curamimetic, has been crystallized in monomeric and dimeric forms (Nastopoulos et al., 1998). Erabutoxin-b is said to be relatively ineffective at the mammalian neuromuscular junction (Vincent et al., 1998). Another curamimetic, a long-chain polypeptide, is α-cobratoxin, while a novel “neurotoxin” from N. naja atra, having 61 amino acid residues and eight cystine residues, has been isolated by Chang et al. (1997). Disintegrins are a family of short cysteine-rich polypeptides and are divided into five subgroups based upon the combination of length and number of disulfide bonds of polypeptides. In general, the disintegrins comprise 40–100 amino acids. Their small size coupled with a relatively dense network of disulfide bonds contributes to the tertiary structure of these compounds and high potency of such small compounds. Disintegrins are released in venoms via proteolytic processes of PII metalloproteinases, whereas structures similar in form and function to disintegrins, or disintegrin like, are subject to PIII processes (Calvette et al., 2005). Monomeric disintegrins can vary from about 50 residues and four disulfide bonds as in echistatin and obtustatin, to around 70 amino acid residues and six disulfide bridges as in albolabrin, barbourin, and halysin, to over 84 amino acids and seven disulfide bonds for bitistatin and salmosin-3. Dimeric disintegrins are about 67 amino acids long and contain four intrachain disulfide linkages and two between-chain bonds. Examples include contortrostatin and acostatin. The monomeric disintegrin-like chemicals contain around 100 amino acids and eight disulfide bonds, and include trimelysin-I, bothropasin, and jararhagin (Calvette et al., 2003; Ramos and Selistre-de-Araujo, 2006). RGD (argine–glycine–asparagine) and non-RGD-containing disintegrins coexist in certain venoms and exhibit affinities for variable ligand receptors. In such cases, one copy of the gene encodes for the more conserved RGD function of platelet aggregation, whereas the duplicated genes have drifted toward facilitating other biological functions. Modeling and structure analysis of cyclic RGD peptides has implicated the importance of amino acid sequences on the C-terminal of the RGD sequence; furthermore, the physical features such as size of the integrin-binding loop contribute to the receptor-binding capabilities (Calvette et al., 2005). In general, the amino acid residues of this region of the RGD sequence are not well conserved and are believed to play a key role in determining integrin receptor-binding specificity. There are additional mechanisms within the C-terminal region, which include conformational epitopes that are utilized to alter receptor-binding capabilities.

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The small basic polypeptide myotoxins are widely distributed in Crotalus snake venoms. The specific agent crotamine from Crotalus durissus terrificus venom induces skeletal muscle spasms and paralysis by changing the inactivation process of sodium channels, which are inhibited by tetrodotoxin and potentiated by veratridine and grayanotoxin, leading to depolarization of the neuromuscular junction. Crotamine is composed of 42 amino acid residues and three disulfide bonds. The crotamine gene contains 1.8 kbp and has three exons that are separated by a long phase-1 and a short phase-2 intron and mapped to chromosome 2. In addition, the three-dimensional structure has been published, and the structural topology is similar to that of other three disulfide bridge containing peptides such as human β-defensins and scorpion sodium channel toxin. These structural properties enable crotamine to have a unique cell penetrating ability allowing the toxin to concentrate in the nucleus by means of a probable receptor-independent mechanism. It is interesting to note that topology and diversification of functional folds are common themes in animal venom peptides acting on ion channels and other targets (Menez, 1998; Mouhat et al., 2004; Oguiura et al., 2006). Toxicology In general, the venoms of rattlesnakes and other New World crotalids produce alterations in the resistances and often in the integrity of blood vessels, changes in blood cells and blood coagulation mechanisms, direct or indirect changes in cardiac and pulmonary dynamics, and—with crotalids like C. durrissus terrificus and C. scutulatus—serious alterations in the nervous system and changes in respiration. In humans, the course of the poisoning is determined by the kind and amount of venom injected; the site where it is deposited; the general health, size, and age of the patient; the kind of treatment; and those pharmacodynamic principles noted earlier in this chapter. Death in humans may occur within less than 1 hour or after several days, with most deaths occurring between 18 and 32 hours. Hypotension or shock is the major therapeutic problem in North American crotalid bites (Russell, 2001). Snakebite Treatment The treatment of bites by venomous snakes is now so highly specialized that almost every envenomation requires specific recommendations. However, three general principles for every bite should be kept in mind: (1) snake venom poisoning is a medical emergency requiring immediate attention and the exercise of considerable judgment; (2) the venom is a complex mixture of substances of which the proteins contribute the major deleterious properties, and the only adequate antidote is the use of specific or polyspecific antivenom; and (3) not every bite by a venomous snake ends in an envenomation. Venom may not be injected. In almost 1000 cases of crotalid bites, 24% did not end in a poisoning. The incidence with the bites of cobras and perhaps other elapids is probably higher. The reader is referred to other appropriate texts for appropriate treatment of snakebites (Russell, 2001; Dart, 2004; Sholl et al., 2004; Tintinalli et al., 2004; Singletary and Holstege, 2006). Snake Venom Evolution Considerable efforts are being expended to examine the complex process by which snake venom components are thought to have changed over the years. This evaluation involves tracing the ancestral roots of toxins, which is made even more cumbersome due to the distinct differences in the speed at which individual components of a venom evolve. The current assemblage of snake venoms with regard to functionality and ancestral protein

Table 26-10 Basal Bioactivities of Some Toxin Types toxin

activity

3FTx

α-Neurotoxicity, blocks nicotinic acetylcholine receptor In Viperidae venoms, proteolytic cleavage of C-terminal domains results in direct fibrinolytic activity, liberation of disintegrins, which inhibit platelet aggregation Causes unregulated activation of complement cascade, hemolysis, cytolysis Myonecrosis, modifies voltage-gated sodium channels In taipan and brown snake venom, combines with toxic form of factor X to convert prothrombin to thrombin Increases vascular permeability, stimulates inflammation, and reduces blood pressure Inhibits plasmin and thrombin and other serine proteases, blocks l-type calcium channels Induces apoptosis, decreases platelet aggregation, inhibits blood factor IX Releases arachadonic acid from phospholipids, resulting in inflammation and tissue destruction Increases permeability of vascular bed causing hypotension and shock Inhibit leukoproteinases

ADAM

Cobra venom factor

Crotamine Factor V

Kallikrein

Kunitz

l-Amino oxidase PLA2

VEGF Whey acidic proteins source: Data from Fry (2005).

activity is outlined in Table 26-10 (Fry, 2005). In general, the toxins from ancestral proteins that were constructed of dense networks of cysteine cross-linkages are considered among the most diverse today in terms of toxicological insult. Recent thinking suggests that the evolution of the PLA2 s appears to be directed toward modifying the molecular surface in order to maximize the diversity of the pharmacologic properties of each isozyme component (Kini and Chan, 1999; Li et al., 2005). As PLA2 s are not crucial for snake survival, mutations in some PLA2 isozymes may be ignored and allowed to progress, thereby expediting the evolution in comparison to other more critical enzymes in which mutations are monitored more closely and not allowed to progress. The functionality of PLA2 isozymes can be significantly hindered by increased rates of mutations within highly conserved regions of the amino acid sequence, which could in turn displace cysteine residues and alter the tertiary protein structure. However, increased mutation of a particular subset of enzymes, such as the PLA2 s, may be a result of a shift in ecological niche. A recent study (Li et al., 2005) reports a shift in the diet composition of the marbled sea snake as the root cause of decelerated evolution of the PLA2 enzymes. The diet change from live prey to fish eggs essentially negated the need for PLA2 enzymes to immobilize live prey for swallowing and digestion. Future research could focus on

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the declining need for PLA2 s, and on the altered digestive enzyme composition that has likely occurred as a result of diet shift.

ANTIVENOM Antivenoms have been produced against most medically important snake, spider, scorpion, and marine toxins. Animals immunized with venom develop a variety of antibodies to the many antigens in the venom. Antivenom consists of venom-specific antisera or antibodies concentrated from immune serum to the venom. Antisera contain neutralizing antibodies: one antigen (monospecific) or several antigens (polyspecific). Monovalent antivenoms have a high neutralization capacity, which is desirable against the venom of a specific animal. Polyvalent antisera are typically used to cover several venoms, such as snakes from a geographic region. Polyvalent preparations usually required higher doses or volumes than monovalent antivenoms. Neutralization capacity of antivenom is highly variable as there are no enforced international standards. Antivenom may cross-react with venoms from distantly related species and may not react with venom from the intended species. Nevertheless, in general, the antibodies bind to the venom molecules, rendering them ineffective. Antivenoms are available in several forms: intact IgG antibodies or fragments of IgG such as F(ab)2 and Fab. The molecular weight of the intact IgG is about 150,000, whereas that of Fab is approximately 50,000. The molecular size of IgG prevents its renal excretion and produces a volume of distribution much smaller than that of Fab. The elimination half-life of IgG in the blood is approximately 50 hours. Its ultimate fate is not known, but most IgG is probably taken up by the reticuloendothelial system and degraded with the antigen attached. Fab fragments have an elimination half-life of about 17 hours, and are small enough to permit renal excretion. All antivenom products may produce hypersensitivity reactions. Type I (immediate) hypersensitivity reactions are caused by antigen cross-linking of endogenous IgE bound to mast cells and basophils. Binding of antigen by a mast cell may cause the release of histamine and other mediators, producing an anaphylactic reaction. Once initiated, anaphylaxis may continue despite discontinuation of antivenom administration. Type III hypersensitivity (serum sickness) may develop several days after antivenom administration. In these cases, antigen–antibody complexes are deposited in different areas of the body, often producing inflammatory responses in the skin, joints, kidneys, and other tissues. Fortunately, these reactions are rarely serious. The risks of anaphylaxis should always be considered when one is deciding whether to administer antivenom, and thus antivenom should be given only by intravenous infusion under medical supervision (Heard et al., 1999; Russell, 2001; Mebs, 2002; Dart, 2004; Tintinalli et al., 2004).

POTENTIAL CLINICAL APPLICATION OF VENOMS Animal venoms are being used as research and clinical tools based upon their high affinity for specific targets and well-studied pharmacologic properties (Menez, 1998, 2003; Dimarcq and Hunneyball, 2003; Escoubas and Rash, 2004). Toxin specificities for receptors and channels that facilitate the interface and coordination of neuromuscular activity are utilized and manipulated to study, model, diagnose, and sometimes treat acute and degenerative conditions. Upon closer examination of α-bungarotoxin and candoxin nicotinic

1099

acetylcholine receptor specificity, plans are under way to utilize the reversible and irreversible receptor binding in muscular and neuronal tissues, respectively, in Alzheimer’s patients (Phui Yee et al., 2004). In addition to treating neurological diseases, specific α-toxins (longer chained) are also studied for their anti-angiogenic capabilities in treating malignant tumor growth in patients suffering from small cell lung carcinoma (Tsetlin and Hucho, 2004). In cases such as this, there is an inherent trade-off between promoting some degree of neurological deficit in light of combating tumor growth. Toxins such as the snake venom thrombin-like enzymes are valuable tools in both research and therapeutic applications. Similarly, fibrin(ogen)olytic enzymes break down fibrin-rich clots preventing further clot formation may be useful as controls in blood clotting research or to treat heart attacks and strokes (Castro et al., 2004; Marsh and Williams, 2005; Swenson and Markland, 2005). To facilitate research, the complement-activating cobra venom factor has been produced by recombinant techniques (Vogel et al., 2004). These pharmazooticals include reptilase-R, bothrocethin, stypven, ecarin, and protac. Other areas of active research indicate that animal venoms contain components that can reduce pain, can selectively kill specific cancers, may reduce the incidence of stroke via effects on blood coagulability, and function as antibiotics. For example, epibatidin comes from the skin of the South American frog, Epipedobates tricolor, and a synthetic derivative ABT-594 appears to be more effective than morphine without being addictive. TM 601 is derived from the Israeli yellow scorpion and attacks malignant brain tumors called glioma tumors responsible for two-thirds of the cases of brain cancer, without harming healthy cells. ET 743, which comes from sea squirts, is being tested for treatment of ovarian cancer and soft tissue sarcoma. Ancrod is an anticoagulant with potential to prevent cell damage and death when someone suffers a stroke. The active ingredient comes from the venom of the Malaysian pit viper. In Germany, where Ancrod has been marketed for a number of years, a specially built facility houses about 3000 snakes. Several other sources of anticoagulants are being examined. A substance called magainin 2, which comes from the skin of frogs, is an effective antibiotic to which bacteria do not appear to develop resistance. The clotting enzyme batroxobin is an ingredient in reptilase and has been used in the development of fibrin glue, which is used in surgery to stop diffuse bleeding from liver or lung by covering the surface with a thin layer of fibrin. Another major area of investigation and success involves the venom components that act as enzyme inhibitors. In particular, venom peptides from Bothrops jararaca were initially called bradykinin-potentiating peptides and lowered blood pressure. After further research, it became clear that these peptides were inhibitors of angiotensin I-converting enzyme, and chemical modification lead to orally active agents such as captopril. Venom toxins can also be used as a component of the toxin– receptor–antibody complex for diagnosis of autoimmune disorders (M´enez, 2003). In addition to providing a promising means for researching and treating muscular and neurological diseases and cancer, work is being conducted on designing methods by which to consistently reconstruct and conform the overall toxin structure to bind to a specific protein, such as HIV (M´enez, 2003). Additional work is being conducted on animals such as the mongoose, hedgehog, and opossum, which all embody a high level of resistance to snake bites. Blood from these animals contains proteins between 400 and 700 amino acids long that inhibit hemorrhagins. The exact mechanism of the many components in animal

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venoms that produce toxicity or resistance to certain toxins has yet to be determined. Further research will require a multidisciplinary approach involving techniques from parasitology, chemistry, molecular biology, genomics, proteomics, physiology, pharmacology, and toxicology.

ACKNOWLEDGMENTS The author gratefully acknowledges the assistance of Ruth A. Sanders and Ronnie L. Yeager in the preparation of this chapter.

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Tambourgi DV, Petricevich VL, Magnoli FC, et al.: Endotoxemic-like shock induced by Loxosceles spider venoms: Pathological changes and putative cytokine mediators. Toxicon 36:391–403, 1998. Terlau H, Olivera BM: Conus venoms: A rich source of novel ion channeltargeted peptides. Physiol Rev 84:41–68, 2004. Terlau H, Shon K, Grilley M, et al.: Strategy for rapid immobilization of prey by a fish-hunting cone snail. Nature 381:148–151, 1996. Thurn MJ, Gooley A, Broady KW: Identification of the neurotoxin from the Australian paralysis tick Ixodes holocyclus, in Tan P, et al. (eds.): Recent Advances in Toxicological Research. Singapore: National University of Singapore, 1992, pp. 243–256. Tintinalli JE, Kelen GD, Stapczynski JS (Eds): Emergency Medicine: A Comprehensive Study Guide, 6th ed. New York: McGraw-Hill, 2004. Tsetlin VI, Hucho F: Snake and snail toxins acting on nicotinic acetylcholine receptors: Fundamental aspects and medical applications. FEBS Lett 557:9–13, 2004. Uddman R, Goadsby PJ, Jansen-Olesen I, et al.: Helospectin-like peptides: Immunochemical localization and effects on isolated cerebral arteries and on local cerebral blood flow in the cat. J Cerebral Blood Flow Metabol 19:61–67, 1999. Ushkaryov YA, Volnski KE, Ashton AC: The multiple actions of black widow spider toxins and their selective use in neurosecretion studies. Toxicon 43:527–542, 2004. Valenzuela JG: Blood-feeding arthropod salivary glands and saliva, in Marquardt, WC (ed.): Biology of Disease Vectors. Amsterdam: Elsevier, 2004, Chapter 28. Valenzuela JG, Charlab R, Mather TN, et al.: Purification, cloning and expression of a novel salivary anticomplement protein from the tick Ixodes scapularis. J Biol Chem 275:18717–18723, 2000. Vincent A, Jacobson L, Curran L: Alpha-bungarotoxin binding to human muscle acetylcholine receptor. Neurochem Int 332:427–433, 1998. Vogel C-W, Fritsinger DC, Hew BE: Recombinant cobra venom factor. Mol Immunol 41:191–199, 2004. Volynski KE, Capogna M, Ashton AC, et al: Mutant α-latrotoxin (LTXN4C ) does not form pores and causes secretion by receptor stimulation. This action does not require neurexins. J Biol Chem 278:31058–31066, 2003. Wang X, Connor M, Smith R, et al.: Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge. Nat Struct Biol 7:505–513, 2000. Wang X, Connor M, Wilson D, et al.: Discovery and structure of a potent and highly specific blocker of insect calcium channels. J Biol Chem 276:40306–40312, 2001. White J: Snake venoms and coagulopathy. Toxicon 45:951–967, 2005. Wilson D, Alewood PF: Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques. Toxicon 47:614–627, 2006. Yellen G. The voltage-gated potassium channels and their relatives. Nature 419:35–42, 2002.

CHAPTER 27

TOXIC EFFECTS OF PLANTS Stata Norton Carcinogens Kidney Tubular Degeneration Blood and Bone Marrow Anticoagulants Bone Marrow Genotoxicity Cyanogens Nervous System Epileptiform Seizures Excitatory Amino Acids Motor Neuron Demyelination Cerebellar Neurons Parasympathetic Stimulation Parasympathetic Block Sensory Neuron Block Skeletal Muscle and Neuromuscular Junction Neuromuscular Junction Skeletal Muscle Bone and Tissue Calcification Bone and Soft Tissue Reproduction and Teratogenesis Abortifacients Teratogens

INTRODUCTION TOXIC EFFECTS BY ORGAN Skin Contact Dermatitis Allergic Dermatitis Photosensitivity Respiratory Tract Allergic Rhinitis Cough Reflex Toxin-Associated Pneumonia Gastrointestinal System Direct Irritant Effects Antimitotic Effects Protein Synthesis Inhibition Cardiovascular System Cardioactive Glycosides Actions on Cardiac Nerves Vasoactive Chemicals LIVER Hepatocyte Damage Mushroom toxins Mycotoxins Kidney and Bladder

SUMMARY

INTRODUCTION Plants may cause toxic effects as a result of inadvertent exposure on contact or accidental ingestion of the plant. Examples are “hay fever” (rhinitis) from exposure to airborne plant pollen and oral irritation, especially in children, from biting on a leaf of a plant such as dumb cane (Diffenbachia). Another source of toxicity may be from intentional ingestion of some herbs, especially when they are taken chronically. The possibility also exists for interactions of prescribed drugs with intake of herbal remedies. For example, the chemicals in some herbs affect hepatic cytochrome enzymes (Izzo and Ernst, 2001). In recent years, information on bioactive chemicals in plants has grown steadily, partly from increased interest in herbal remedies and from interest in identifying novel approaches to medical problems. One result of the latter interest is that toxic effects of plants are being examined for potential usefulness in cancers. This chapter will be restricted primarily to consideration of the toxic effects of plants from unintentional exposure and some intentional exposures, with only brief mention of possible value of toxic bioactive components. In the course of evolution, plants have been attacked by viruses, bacteria, and fungi, and have been eaten by animals of many kinds. In response, plants have developed various elegant defenses, including synthesis of antimicrobial chemicals and chemicals designed to repel animals by various means. Of the many species of plants that

contain toxic chemicals, only a few can be described here. Selection is based on three considerations: frequency with which exposure occurs; importance and seriousness of the exposure; and the scientific understanding of the nature of the action of the chemical. In considering any chemical synthesized by a plant it is important to note that there may be marked variability in the amount of a toxic chemical produced by a plant. The reasons for variability in concentration of toxic chemicals are several: 1. Different portions of the plant may contain different concentrations of a chemical. An example of localization of bioactive compounds is found in the bracken fern (Pteridium aquilinum) in which the carcinogenic terpene, ptaquiloside, is found in high concentrations in the fronds compared with the roots (Rasmussen et al., 2003). 2. The age of the plant contributes to variability. Peak concentrations of bioactive compounds often are found at different periods of growth. For example, in lettuce (Lactuca species) the concentration of lactucin and other sesquiterpenes increases with maturation, reaching a peak in the latex when the flower stalk is forming (Sessa et al., 2000). 3. Climate and soil influence the synthesis of some chemicals. For example, lichens produce carotenoids in direct relation to the amount of sunlight, with the advantage to the plant that carotenoids protect from excessive ultraviolet light.

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4. Genetic differences within a species alter the ability of plants to synthesize a chemical. Synthesis of related toxic chemicals is found in some plants as a characteristic of a genus and sometimes as a familial characteristic. For example, species of Raunculus (buttercup) produce an acrid juice that releases the irritating chemical, anemonin. Some other genera of the same family (Ranunculaceae) also release anemonin.

TOXIC EFFECTS BY ORGAN Skin Contact Dermatitis Several plants that are common to the temperate regions worldwide contain compounds that produce irritation on contact with the intact plant. The leaves of stinging nettles (Urtica species, Urticaceae) bear numerous trichomes, barb-like silicified hairs that puncture skin on contact, releasing an irritating sap. The sap has been reported to contain a mixture of formic acid, histamine, acetylcholine, and serotoin (Kavalali, 2003). Although there are about 100 species of Urtica worldwide, Urtica dioica and Urtica urens are the most common in the United States and Europe and Urtica ferox is widespread in New Zealand. Exposure to U. ferox (poisonous tree nettle) has caused death in humans and animals. A defense by the plant against ingestion by animals, similar to that of nettles, has been developed by Mucuna pruriens (cowhage), a legume with pods covered with barbed trichomes that cause pain, itching, erythema, and vesication. The trichomes contain mucinain, a proteinase responsible for the pruritis (Southcott and Haegi, 1992). Several species of Ranunculus (buttercup) cause contact dermatitis. These plants contain ranunculin, which releases the toxic principle protoanemonin, also present in Anemone, another genus of the buttercup family. Protoanemonin is readily converted to anemonin, which has marked irritant properties. In addition to contact dermatitis, ingestion of plants containing protoanemonin may result in severe irritation of the gastrointestinal tract (Kelch et al., 1992). The genus Euphorbia (Euphorbiaceae, spurge family) contains hundreds of species dispersed over temperate and tropical regions. Characteristically, the stems and leaves exude milky latex when damaged. The latex contains diterpene esters that are irritating to the skin. Euphorbia marginata (snow-on-the mountain) is a common plant in the United States, growing wild from Minnesota to Texas and cultivated for its attractive foliage. Individuals using the plant in flower arrangements may come in contact with the latex and develop skin irritation (Urushibata and Kase, 1991). Serious eye irritation has been reported (Frohn et al., 1993). The latex of cultivars of Euphorbia pulcherrima (poinsettia) may cause contact dermatitis (Massermanian, 1998). Allergic Dermatitis Most people are familiar with allergic dermatitis caused by contact with some plants, such as poison ivy. These allergens tend to be located in the outer cell layers of plant organs. In allergic dermatitis to chrysanthemums (Dendranthema species) the allergens are sesquiterpene lactones present in small hairs (trichomes) on the stems, undersides of leaves and in flowering heads (McGovern and Barkley, 1999). Rhus (Anacardiaceae, cashew family) and Philodendron (Araceae, arum family) are not closely related plants but both genera contain species causing contact dermatitis as an allergic reaction. Philodendron scandens is a common houseplant, while Rhus radicans (poison ivy) is widespread in North America. In addition to poi-

son ivy, the toxicodendron group of plants contains Rhus diversiloba (poison oak) and Rhus vernix (poison sumac). The active chemicals in P. scandens are resorcinols, especially 5-n-heptadecatrienyl resorcinol (Knight, 1991). In R. radicans the allergenic component is a mixture of catechols called urushiol. The most active compound in urushiol is 3-n-pentadecadienyl catechol, representing about 60% of urushiol (Johnson et al., 1972). As in allergies in general, there is no response to the initial exposure; even with repeated exposure, individuals show marked variation in severity of response. The allergens in usushiol will sensitize about 70% of persons exposed. Urushiol is fat soluble, penetrates the stratum corneum and binds to Langerhans cells in the epidermis. These haptenated cells then migrate to lymph nodes, where T-cells are activated (Kalish and Johnson, 1990). Ingestion of Rhus species has been reported to cause generalized dermatitis (Oh et al., 2003). Allergic dermatitis may also develop with repeated exposure to the sap of the mango fruit. The skin of the mango (Magnifera indica, Anacardiaceae) contains oleoresins that cross-react with allergens of poison ivy (Tucker and Swan, 1998). Flower growers and other individuals who handle bulbs and cut flowers of daffodils, hyacinths, and tulips sometimes develop dermatitis from contact with the sap. The rashes are due to irritation from alkaloids (masonin, lycorin, and several related alkaloids) or to needle-like crystals of calcium oxalate in the bulbs (Gude et al., 1998). Most of these chemicals do not act as allergens but one, tulipalin-A, that causes “tulip fingers” from sorting and peeling tulip bulbs, has allergenic properties. Tulipalin-A, alpha-methylenegamma-butyrolactone, is present in some cultivars in concentrations up to 2%. A safe threshold for this allergen is considered to be 0.01% (Hausen et al., 1983). The advantage of the tulipalins to the plant is that they are strong antifungal agents (Christensen and Kristiansen, 1999). An immunoglobulin-mediated hypersensitivity, called the “latex-fruit syndrome,” results in cross-sensitivity in some individuals to latex in rubber gloves and some fruits. The major allergen in natural rubber latex from the tree, Hevea brasiliensis, is prohevein, a chitin-binding polypeptide found in several plants. Hevein, a 43amino acid N-terminal fragment of prohevein is the major binding component. The structural analysis has been reported (Kolarich et al., 2005). Individuals sensitive to rubber latex may be sensitized to fruits containing a chitinase with a hevein-like domain, including banana, kiwi, tomato, and avocado (Blanco et al., 1999). Lichens, such as species of Usnea and Cladonia, are abundant on trees in areas away from major city smog. Dermatitis from contact with lichens is known in professions where individuals are repeatedly exposed to lichens, including workers in forestry and horticulture. The allergen is usnic acid (a benzofuran) and related acids in lichens (Aalto-Korte et al., 2005). Usnic acid is of current research interest as a non-genotoxic anticancer agent (Mayer et al., 2005b). Usnic acid has been implicated in hepatotoxicity following use of some nonprescription weight-loss supplements (Han et al., 2004).

Photosensitivity Not all cases of dermititis from plants are due to skin contact. Poisoning of livestock from Hypericum perforatum (St. John’s wort) has been reported from several countries. The toxic principle is hypericin (a bianthraquinone), present throughout the plant. Sheep are the most commonly affected animals, ingesting the plant in pasturage. The effect in sheep is development of edematous lesions of the skin in areas not well covered with hair, including the ears, nose, and eyes. Hypericin causes photosensitization and the lesions appear from exposure to sunlight (Sako et al., 1993).

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Although photosensitization in humans is a rare event after consumption of St. John’s wort, enhancement of response in humans to therapeutic exposure to ultraviolet therapy has been reported (Beattie et al., 2005).

Respiratory Tract Allergic Rhinitis Rhinitis from inhalation of plant pollens, also known as “hay fever” or pollinosis, is a seasonal problem for many individuals. A chromosomal association in individuals with seasonal pollen reactivity is under investigation (Blumenthal et al., 2006). Many plant species contribute to airborne pollen, especially grass pollens. Poa and Festuca species are major contributors to the allergic response. Pollen from several genera in the Asteraceae (e.g., mugwort, Artemisia vulgaris, in Europe, and ragweed, Ambrosia artemisiifolia, in North America) contains allergens causing summer rhinitis. Immunoglobulin antibodies produced by sensitized individuals from the two species cross react. The allergen has been identified as a highly conserved 14-kDa protein, profilin, found as well in birch pollen (Hirschwehr et al., 1998). Pollen from pellitory (Parietaria species, Urticaceae) is an important cause of pollinosis in the Mediterranean region (Arilla et al., 2006). Asthma and rhinitis associated with occupation are also recognized in individuals exposed to certain plants, such as cascara sagrada (Rhamnus purshiana) (Giavina-Bianchi et al, 1997), or workers in greenhouses in which bell peppers are growing (Groenewoud et al., 2006). Cough Reflex It has been found that workers who handle two types of peppers, Capsicum annuum (sweet pepper) and Capsicum frutescens (red pepper), have a significantly increased incidence of cough during exposure. The major irritants are capsaicin (trans-8-methyl-N -vanillyl-6-nonenamide) and dihydrocapsaicin (Surh et al., 1998). Capsaicin-sensitive nerves in the airway are involved in the irritation and cough (Blanc et al., 1991). Capsaicin activates a subtype of the vanilloid receptor found in the airway, spinal cord, dorsal root ganglion, bladder, urethra, and colon. Capsaicin can also be irritating to the skin, and individuals handling the peppers may experience irritation and vesication.

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chemicals may be responsible for this. Some have found a place in medicine as mild purgatives, such as cascara sagrada. Cascara is obtained from the bark of Rhamnus purshiana (California buckthorn). The active ingredient is an anthraquinone, emodin. Tung nut (Aleurites fordii) is grown widely in the world. The seeds, from which commercially useful oil is expressed, are the most toxic part. Ingestion of the ripe nuts causes abdominal pain, vomiting, and diarrhea. Outbreaks of poisoning are most common in children (Lin et al., 1996). Buffalo bean or buffalo pea (Thermopsis rhombifolia) is a legume growing wild in western United States. Loss of life in livestock has been reported from consumption of the mature plant with seeds. Children develop nausea, vomiting, dizziness, and abdominal pain from eating the beans (McGrath-Hill and Vicas, 1997). The toxic chemicals are quinolizidine alkaloids. Aesculus hippocastanum (horse chestnut) and Aesculus glabra (Ohio buckeye) are common trees with attractive panicles of flowers in the spring. Nuts of both trees contain a glucoside called esculin. When eaten by humans, the main effect is gastroenteritis, which may be severe if several nuts are consumed. Esculin is poorly absorbed from the gastrointestinal tract of humans and its systemic effects are usually limited. In cattle the glucoside may be hydrolyzed in the rumen, releasing the aglycone to cause systemic effects. Cattle develop signs of nervous system stimulation—a stiff-legged gait and, in severe poisoning, tonic seizures with opisthotonus (Casteel et al., 1992). While the most common poisoning of cattle occurs from ingestion of nuts, they may also be poisoned in pasture in spring from eating new leaves and buds. Beta-aescin, a triperpene saponin in horse chestnut seed extract, has been studied for use in chronic venous insufficiency in humans (Siebert et al., 2002).

Gastrointestinal System

Antimitotic Effects Podophyllum peltatum (May apple, Berberidaceae) contains the toxic purgative, podophyllotoxin, especially in foliage and roots. In low doses, mild purgation predominates. Overdose results in nausea and severe paroxysmal vomiting (Frasca et al., 1997). Podophyllotoxin inhibits mitosis by binding to microtubules, and this property has made the toxin of interest in treatment of cancer (Schacter, 1996). Colchicine is best known in western medicine for its antimitotic effect, resulting from block of formation of microtubules and failure of the mitotic spindle, for which it is useful in attacks of gout. Colchicine is the major alkaloid in the bulbs of Colchicum autumnale (autumn crocus, Liliaceae), native to Asia Minor. Severe gastroenteritis (nausea, vomiting, diarrhea, and dehydration) follows ingestion of the bulbs that may be mistaken for wild garlic. Systemic effects (confusion, hematuria, neuropathy, and renal failure) may develop in severe poisoning. Bone marrow aplasia results from block of mitosis in bone marrow. Additional toxic effects may be due to lectins in the plant. In southern Europe, hay for cattle may contain the wild autumn crocus, and deaths occur if contamination is heavy. Toxicity from human ingestion of tubers of Gloriosa superba (glory lily) similar to that from autumn crocus has been reported. This ornamental lily also contains colchicine. The plant grows wild in Sri Lanka and poisoning by Gloriosa tubers has been reported as the most common plant poisoning in that country. Poisoning has also been reported in India (Mendis, 1989).

Direct Irritant Effects The most common outcome of ingestion of a toxic plant is gastrointestinal disturbance (nausea, vomiting, and diarrhea) from irritation of the gastrointestinal tract. Many kinds of

Protein Synthesis Inhibition Some of the most toxic plant proteins act through inactivating protein systhesis. Ricinus communis

Toxin-Associated Pneumonia The formation of the pneumotoxin, 4-ipomeanol, in sweet potato roots (Ipomea batatas, Convolvulaceae) from the mold Fusarium solani has been known for some time. The toxin 4-ipomeanol is activated by human cytochrome P450s to a reactive intermediate that binds to DNA (Alvarez-Diez and Zheng, 2004). Differences in the pattern of organ toxicity are related to P450 isoforms. In cattle and rabbits, the major P450 activator is CYP4B1 in the lung. In the mouse, renal toxicity results from high levels of CYP4B1 in the kidney and in humans multiple subsets of liver P450 enzymes bioactivate 4-ipomeanol (Baer et al., 2005). Fungal pneumonia from Acremonium strictum has been reported (Pusterla et al., 2005). Pyrrolizidine alkaloids in Boraginaceae and Asteraceae families of plants have been implicated in lung toxicity and pneumonitis in humans (Altamirano et al., 2005).

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(castor bean) is a member of the family Euphorbiaceae, which contains several genera that produce toxic chemicals. The castor bean is an ornamental plant introduced from India. If the attractive, mottled seeds are eaten, children and adults experience no marked symptoms of poisoning for several days. In this interval there is some loss of appetite, with nausea, vomiting, and diarrhea developing gradually. With fatal doses the gastroenteritis becomes severe, with persistent vomiting, bloody diarrhea, and icterus. Death occurs in 6–8 days. The fatal dose for a child can be five to six seeds; it may be as low as 20 seeds for an adult. However, fatality is low— less than 10% when a “fatal” dose is consumed—because the toxic protein is largely destroyed in the intestine. Death from castor beans is caused by two lectins in the beans: ricin I and ricin II. The more toxic is ricin II. Ricin II consists of two amino acid chains. The A-chain (molecular weight 30,000) inactivates the 60s ribosomal subunit of cells by catalytic depurination of an adenosine residue within the 28s rRNA (Bantel et al., 1999) and blocks protein synthesis. The A-chain is endocytosed into the cell cytosol after the B-chain (molecular weight 30,000) binds to a terminal galactose residue on the cell membrane. The two chains are linked by disulfide bonds. Details of binding properties of ricin to glycoproteins have been investigated (Wu et al., 2006). Toxic lectins are found in the seeds of Abrus precatorius (jequirity bean, Leguminosae). They are attractive scarlet and black beans that are sometimes made into necklaces. Abrin-a, one of four isoabrins from the plant, has the highest inhibitory effect on protein synthesis and consists of an A-chain of 250 amino acids and a Bchain of 267 amino acids (Tahirov et al., 1994). The LD50 of abrin injected in mice is less than 0.1 μg/kg, making abrin one of the most toxic substances known. As with ricin, the A-chain inhibits protein synthesis and the B-chain is responsible for penetration of abrin-a molecules into cells (Ohba et al., 2004). Plants that produce only A-chains offer much less risk when ingested. Young shoots of pokeweed (Phytolacca americana, Phytolaccaceae) are sometimes used in the spring as a salad green without toxicity. Mature leaves and berries may cause gastrointestinal irritation with nausea and diarrhea. The plant produces three isozymes of single-chain lectins (PAP, PAPII, and PAP-S; molecular weight of about 30,000) that can inhibit protein synthesis in cells by inactivating rRNA. Single-chain, ribosome-inhibiting proteins do not enter intact cells readily, but if the cell membrane has been breached by a virus, they may enter the cell (Monzingo et al., 1993). Wisteria floribunda (Leguminosae) is a common ornamental climbing vine or small tree with lilac-colored flowers from which pods with seeds develop in the fall. The seeds of wisteria cause severe gastroenteritis when ingested. A few seeds can result in headache, nausea, and diarrhea within hours, followed by dizziness, confusion, and hematemesis (Rondeau, 1993). The seeds contain a lectin with affinity for N -acetylglucosamine on mammalian neurons.

Cardiovascular System Cardioactive Glycosides Several different families of plants contain species with cardioactive glycosides, the best known of which is Digitalis purpurea (foxglove, Scrophulariaceae). In the lily family, squill (Scilla maritima) contains scillaren, and lily of the valley (Convallaria majalis) contains convallatoxin in the bulbs. Both glycosides have actions resembling digitalis. Milkweeds (Asclepias species, Asclepiadaceae) are noted for the glycosides in the plants, which are consumed by monarch butterfly caterpillars during their development. The glycosides are retained into the adult stage and

help protect the butterflies from predators. The cardiac glycoside [6 -O-(E-4-hydroxycinnamoyl) desglucouzarin] in Asclepias asperula, like digitalis, inhibits Na+ , K+ -ATPase (Abbott et al., 1998). Other species of Asclepias also contain cardenolides (Roy et al., 2005). Two plants in the Apocynaceae (oleander family) contain cardioactive glycosides. Nereum oleander (bay laurel) is native to the Mediterranean area but is grown ornamentally in many regions. The major glycosides, oleandrin and nerium, may be present in concentrations as high as 0.5 mg/g plant material. The blood level of oleandrin estimated to be toxic to humans is 1–2 ng/mL (Pietsch et al., 2005). In addition to cardiac effects resembling digitalis toxicity, histopathological changes after fatal doses of N. oleander leaves to sheep include hemorrhages in several organs (Aslani et al., 2004). A related plant, Thevetia peruviana (yellow oleander), is a common ornamental plant in the United States. Human poisoning has also been reported in Australia, Melanesia, Thailand, and India. The seeds are the major source of the cardiac glycosides, the most active of which is thevetin A. The fatal dose to an adult is eight to 10 seeds (Prabhasankar et al., 1993).

Actions on Cardiac Nerves Veratrum viride (American hellebore, Liliaceae), native to eastern North America, produces several toxic alkaloids that are distributed in all parts of the plant. European hellebore (Veratrum album) and Veratrum californicum in western North America have similar alkaloids. V. album was one of the medicinal drugs used for centuries to “slow and soften the pulse.” These species of Veratrum contain a mixture of alkaloids, including protoveratrine, veratramine, and jervine. After ingestion the alkaloids cause nausea, emesis, hypotension, and bradycardia. The primary effect on the heart is to cause a repetitive response to a single stimulus resulting from prolongation of the sodium current (Jaffe et al., 1990). The bulbs of the wild camas (Zigadenus paniculatus and other species of Zigadenus, Liliaceae) have been ingested by mistake as wild onions. Severe gastrointestinal toxicity may result (Peterson and Rasmussen, 2003). Cattle are also poisoned in pastures where the plants are common. Several species of Zigadenus contain veratrum-like alkaloids. Aconitum species have been used in western and eastern medicine for centuries. The European plant, Aconitum napellus (monkshood, Ranunculaceae) is a perennial grown in gardens for its ornamental blue flowers. The roots of A. kusnezoffii (chuanwu) and A. carmichaeli (caowu) are in the Chinese materia medica. Poisoning may occur from intentional or accidental ingestion, and the concentration of the alkaloids (aconitine, mesaconitine, and hypoaconitine) varies depending on species, place of origin, time of harvest, and processing procedures (Chan et al., 1994). In addition to cardiac arrhythmias and hypotension, the alkaloids cause gastrointestinal upset and neurologic symptoms, especially numbness of the mouth and paresthesia in the extremities. The alkaloids cause a prolonged sodium current in cardiac muscle with slowed repolarization (Peper and Trautwein, 1967). The neurologic effects are due to a similar action on voltage-sensitive sodium currents in nerve fibers (Murai et al., 1990). In Greece about 2500 years ago, Xenophon described a serious condition called “mad honey poisoning” that developed in his soldiers after they had eaten honey contaminated with grayanotoxins. The poisoning resembles aconitine poisoning. In severe poisoning there is respiratory depression and loss of consciousness. Grayanotoxins bind to sodium channels in cardiac and muscle cells, increasing sodium conductance (Maejima et al., 2002). Grayanotoxins are

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produced exclusively by several genera of Ericaceae (heath family). They have been isolated from Rhododendron ponticum (Onat et al., 1991) and Kalmia angustifolia (Burke and Doskotch, 1990). The toxin gets into honey from nectar collected by bees. Grayanotoxins are present throughout the plants. The effect of the toxins is marked bradycardia, hypotension, oral paresthesia, weakness, and gastrointestinal upset, resembling aconitine poisoning. Grayanotoxins bind to sodium channels in cardiac and muscle cells, increasing sodium conductance (Maejima et al., 2002). Toxicity has been reported in goats and in sheep eating leaves of Rhododendron macrophyllum (Casteel and Wagstaff, 1989).

Vasoactive Chemicals Mistletoe is a parasitic plant on trees and has over the centuries been considered either holy or demonic. The poisonous qualities of mistletoe were recognized by John Gerard in his herbal in 1597. He described a case of poisoning from mistletoe berries in which the tongue was inflamed and swollen, the mind dostraught, and strength of heart and wits enfeebled. The American mistletoe, Phoradendron flavescens, is a member of the same family as the European mistletoe (Viscum album, Loranthaceae). The American mistletoe contains phoratoxin, a polypeptide with a molecular weight of about 13,000. Phoratoxin and the viscotoxins cause similar effects: hypotension, bradycardia with negative inotropic actions on heart muscle, and vasoconstriction of the vessels of skin and skeletal muscle. Viscotoxins are basic polypeptides (molecular weight of about 5000). Phoratoxin is only one-fifth as active as the viscotoxins (Rosell and Samuelsson, 1988). Serious poisoning from the plants is rare, and most poisonings include gastrointestinal distress and hypotension. An instance of anaphylaxis to repeated injections of mistletoe extract has been reported (Bauer et al., 2005). Vasoconstriction is the primary toxic effect of some plant chemicals. Of the various fungi that produce toxic principles, some develop on grains that are used as food. Claviceps purpurea (ergot) is a fungus parasitic on grains, especially on rye. The “ergot gene cluster” of several genes is required for production of ergot alkaloids (Haarmann et al., 2005). Ergot has caused outbreaks of poisonings in several European countries since the Middle Ages. The condition was called “St. Anthony’s fire” from the blackened appearance of the limbs of some sufferers. The main toxic effect of ergot alkaloids is vasoconstriction, primarily in the extremities, followed by gangrene. Abortion in pregnant women is also common after ingestion of contaminated rye flower. Ergot alkaloids are derivatives of lysergic acid. Some of the alkaloids have been used in therapeutics, especially ergotamine and ergonovine. Ergot alkaloids are produced by some other fungi not closely related to Claviceps, such as Aspergillus fumigatus, a common airborne fungus (Coyle and Panaccione, 2005). Another fungus, Acremonium coenophialum, grows symbiotically on the forage grass tall fescue (Festuca arundinacea) and produces some ergot alkaloids and other lysergic acid derivatives. The fungus causes “fescue toxicosis” in cattle grazing on infected plants (Hill et al., 1994). The condition in cattle includes decreased weight gain, decreased reproductive performance, and peripheral vasoconstriction. In southwestern United States, Stirpa robusta (sleepy grass) also may be infected with an Acremonium fungus. Horses grazing on infected grass become somnolent, presumably as a result of ingesting lysergic acid amide, ergonovine, and related alkaloids produced by the fungus (Petroski et al., 1992).

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LIVER Hepatocyte Damage Senecio (groundsel, Asteraceae) is a large genus of plants with worldwide distribution. Species containing significant concentrations of pyrrolizidine alkaloids are responsible for liver damage in the form of hepatic veno-occlusive disease associated with lipoperoxidation (Bondan et al., 2005). Hepatitis in cattle grazing on Senecio has been reported in Africa and Asia as well as in the United States. The condition is progressive and death occurs after weeks or months of grazing on contaminated pasture. Four genera of Boraginaceae, Echium (bugloss), Cynoglossum (hound’s tongue), Heliotropium (heliotrope), and Symphytum (comfrey) also contain pyrrolizidine alkaloids. Different animal species show marked differences in susceptibility to the alkaloids. Susceptible species are rats, cattle, horses, and chickens; resistant species are guinea pigs, rabbits, gerbils, hamsters, sheep, and Japanese quail. These differences are in general related to the rate of hepatic pyrrole formation, although other sources of differences must be present (Huan et al., 1998). Human deaths from pyrrolizidine alkaloids have been reported in several countries, including South Africa, Jamaica, and Barbados. In Afghanistan, there was an epidemic of hepatic venoocclusive disease from consumption of a wheat crop contaminated with seeds of a species of Heliotropium (Tandon et al., 1978). The clinical signs associated with the liver damage resemble those of cirrhosis and some hepatic tumors may be mistaken for the hepatotoxicity (McDermott and Ridker, 1990). The clinical condition is a form of the Budd–Chiari syndrome, with portal hypertension and obliteration of small hepatic veins. Human consumption also occurs from Symphytum in herbal preparations, such as “comfrey tea” (Rode, 2002). In addition to veno-occlusive damage to the liver, the alkaloids in Symphytum have been shown to be tumorigenic and mutagenic in rats (Mei et al., 2005). Lantana camara (Verbenaceae) has been called one of the 10 most noxious weeds in the world. It is an attractive shrub, native to Jamaica and commonly cultivated in greenhouses. L. camara thrives outdoors in hot, dry climates. An unusual property of the plant is that it inhibits the growth of neighboring plants. In India, livestock poisoning from L. camara is a serious problem. Cattle grazing on the plant develop cholestasis and hyperbilirubinemia. The leaves are toxic to some nonruminants, including rabbits and guinea pigs. Several triterpenoids have been isolated from the plant. One that has been shown to induce hepatotoxicity is lantadene A (22-betaangeloyloxy-3-oxo-olean-12-en-28-oic acid) (Sharma et al., 1991). Mushroom toxins Many of the nonedible mushrooms may cause gastrointestinal distress but most are not life threatening. Repeated consumption of the false morel, Gyromitra esculenta, has been reported to cause hepatitis as well as gastrointestinal disorders (Michelot and Toth, 1991). The toxic principle is gyromitrin (acetaldehyde-N -formylhydrazone). Several species of three genera of mushrooms are responsible for most fatal poisoning from consumption of wild mushrooms, Amanita, Galerina, and Lepiota (Karlson-Stiber and Persson, 2003). Amanita phalloides, is appropriately called “death cap.” Amanita ocreata (death angel) is equally dangerous. A. phalloides contains two types of toxins, phalloidin and amatoxins. Phalloidin is a cyclic heptapeptide that may be responsible for the diarrhea that develops 10–12 hours after ingestion of A. phalloides. Phalloidin combines with actin in muscle cells, but is not readily absorbed from the gastrointestinal tract (Cappell and Hassan, 1992). The alpha-, beta-, and gamma-amanitins are bicyclic peptides (mw 900) and are absorbed. The most toxic,

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alpha-amanitin, binds strongly to RNA polymerase II in hepatocytes, thus inhibiting protein synthesis (Jaeger et al., 1993). Intestinal mucosa and kidneys are also targets. Serious clinical signs develop slowly, beginning about the third day after ingestion. Treatment in severe cases may require liver transplant. There is a long history of use of the herb Silybum marianum (milk thistle, Asteraceae) for treatment of poisoning from amanitins. The active ingredient is silymarin, a flavanolignan. Silymarin and herbal preparations also have been tried in viral hepatitis and alcoholic liver disease (Ball and Kowdley, 2005; Mayer et al., 2005a). Mycotoxins Various fungi present during harvest, or subsequent storage of food, produce toxins. Exposure to low levels of the mycotoxins by way of ingestion of the moldy foods is common, depending on geographic location and type of food. Most notable toxins are aflatoxins from Aspergillus species in stored grain, ochratoxin A, an immunosuppressant chemical produced by Aspergillus and Penicillium molds, and zearalenone, a mycotoxin produced by Fusarium and Gibberella species (Al-Anati and Petzinger, 2006; Ding et al., 2006). Aflatoxin B1 from A. flavus and A. parasiticus has been associated with primary hepatocellular carcinoma in humans. The toxin forms guanine adducts and induces apoptotic cell death in human hepatocytes (Reddy et al., 2006). Fumonisins are toxins produced by the fungus Fusarium, primarily by Fusarium moniliforme and Fusarium proliferatum growing on corn. Ingestion by horses of corn contaminated with Fusarium mold causes “moldy corn poisoning” or equine leukoencephalomalacia. The signs in affected horses are lethargy, ataxia, convulsions, and death (Norred, 1993). There are several target organs but the liver is a primary target in every species; in horses, it is liver and brain; in pigs, liver and lung; in rats, liver and kidney; in chickens, liver (Riley et al., 1994). In humans, an association with esophageal cancer has been suggested (Yoshizawa et al., 1994). Fumonisins are diesters of propane-1,2,3-tricarboxylic acid and a pentahydroxyicosane containing a primary amino acid (Gurung et al., 1999). The structure of fumonisins is similar to that of sphingosine, and their toxicity has been related to block of enzymes in sphingolipid biosynthesis (Norred, 1993).

Kidney and Bladder Carcinogens Bracken fern (P. aquilinum) grows worldwide and, in the United States, primarily east of the Rocky Mountains. It has been called one of the five most common plants on the planet and is found locally in heavy concentrations. Bracken fern is the only higher plant known to cause cancer in animals under natural conditions of feeding. The commonest bladder tumors in cattle are epithelial and mesenchymal neoplasms (Kim and Lee, 1998). The major carcinogen is ptaquiloside, a norsesquiterpene glucoside present in high concentrations in the fern, especially in crosiers and young unfolding fronds. Ptaquiloside alkylates adenines, and guanines of DNA (Shakin et al., 1999). In the condition called “bovine enzootic hematuria,” caused by consumption of bracken fern, the number of chromosomal aberrations is significantly increased (Lioi et al., 2004). There is evidence that consumption of young bracken fern shoots by humans is associated with cancers of the upper alimentary tract (Alonso-Amelot and Avendano, 2002). Kidney Tubular Degeneration Species of Xanthium (cocklebur, Asteraceae) are annual plants found in several countries. Toxicosis

in livestock is most common in spring and early summer due to ingestion of seedlings and young plants of cocklebur. Pigs, sheep, cattle, horses, and fowl can be affected in pastures where two- and four-leaf seedlings are present. The signs are depression and dyspnea. Pathological findings include tubular degeneration and necrosis in the kidney and centrolobular necrosis in the liver (del Carmen Mendez et al., 1998). The active ingredient is a glycoside, carboxyatractyloside, which causes microvascular hemorrhages in multiple organs (Turgut et al., 2005). Acute renal failure is the cause of death in poisoning from Cortinarius species of mushroom. Cortinarius is a large genus of woodland fungi found especially in northern conifer forests. Species vary widely in habit and in edibility. In one series of poisonings from Cortinarius orellanus and related species, involving 135 cases in which the mushrooms were eaten, deaths secondary to acute renal failure occurred in almost 15% of the cases. Renal biopsy showed acute degenerative tubular lesions with inflammatory interstitial fibrosis (Bouget et al., 1990).

Blood and Bone Marrow Anticoagulants Fungal infections in sweet clover (Melilotus alba) silage and hay have caused serious toxicity and death in cattle in California and in the northern plains of the United States and Canada (Puschner et al., 1998). Deaths are from hemorrhages caused by dicumarol, a fungal metabolite. Dicumarol [3,3-methylene-bis(4hydroxycoumarin)] is an effective anticoagulant, causing prothrombin deficiency, and is used therapeutically for this purpose. Bone Marrow Genotoxicity Some species of poppy, such as Argemone (Papaveraceae) produce sanguinarine, a benzophenanthridine alkaloid, that intercalates DNA. Humans may be exposed to argemone oil as an adulterant of mustard oil. A single low dose has been shown to increase chromosomal aberrations in bone marrow cells in mice (Ansari et al., 2004). The term “epidemic dropsy” has been applied to the disease in humans and a link to gall bladder carcinoma has been suggested (Das et al., 2005). Cyanogens Cyanogens are constituents of several different kinds of plants. One that is present in the kernels of apples, cherries, peaches, and related genera in the rose family is amygdalin, found in the highest amounts in the seeds of the bitter almond, Prunus amygdalus, var. amara. Amygdalin is not present in the seeds of the sweet al mond, the nut used for food. The amount of cyanogen in peach (Prunus persica) kernels is enough to cause poisoning in small children if several kernels are eaten. The seeds of apples are unlikely to present a problem. In the stomach, amygdalin releases hydrocyanic acid that combines with ferric ion in cytochrome oxidase or methemoglobin. The result of ingestion of several bitter almond seeds is classic cyanide poisoning with death from asphyxia. Amygdalin is a component of some herbal remedies for cancer in some countries and unintentional cyanide poisoning has resulted (O’Brien et al., 2005). There may be an interaction with concurrent intake of large doses of vitamin C (Bromley et al., 2005). Cassava is a staple food starch from Manihot esculenta (Euphorbiaceae) grown extensively in some parts of Africa as a major food source. The untreated root contains linamarin, a cyanogenic glucoside. During processing of the root for human consumption, the cyanogen is removed. However, local processing may be inadequate. Chronic ingestion of linamarin in cassava has been proposed

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as the cause of epidemics of konzo, a form of tropical myelopathy with sudden onset of spastic paralysis (Tylleskar et al., 1992). Degeneration of the corticospinal motor pathway in affected individuals may be caused by production of thiocyanate from linamarin with stimulation of neuronal glutamate receptors by thiocyanate (Spencer, 1999). One important improvement in cassava as a food source is that it has been possible to produce transgenic cassava plants with marked reduction in linamarin content in the tubers (Jorgensen et al., 2005). Another plant containing linamarin is flax, Linum ustitatissumum, the seed of which is the source of linseed oil. In some European countries, as a domestic remedy, the flax seeds are soaked overnight and the extract is used as a laxative, possibly exposing these individuals to cyanide from the linamarin (Rosling, 1993).

Nervous System Historically, some of the most useful drugs have been plant-derived chemicals that act on the nervous system. However, some unintended serious neurotoxic syndromes may result from ingestion of certain plants. Epileptiform Seizures The parsley family of plants (Apiaceae) contains some of the most edible (e.g., carrots) and some of the most poisonous plants in the northern hemisphere. The fleshy tubers of Cicuta maculata (water hemlock) may be mistaken for other edible wild tubers. A single tuber may cause fatal poisoning, characterized by tonic–clonic convulsions. The toxic principle, cicutoxin (a C17polyacetylene), binds to GABA-gated chloride channels and this may play a role in the acute neurotoxicity (Uwai et al., 2000). Several members of the mint family (Labiatae) are noted for their essential oils, such as pennyroyal (Hedeoma), sage (Salvia), and hyssop (Hyssopus). These oils contain odorous monoterpenes. For example, sage contains thujone, camphor, and cineole. In high oral doses, well above amounts used for flavoring, these monoterpenes can cause tonic–clonic convulsions. Menthol is a selective modulator of inhibitory ligand-gated channels (Hall et al., 2004). Several species of Strychnos (Loganaceae) contain strychnine and brucine (dimethoxystrychnine). Both indoles cause marked CNS stimulation by blocking glycine-gated chloride channels. Strychnos nux vomixa is a small tree native to India. Cases of unintentional poisoning from seeds of Strychnos nux vomica have been reported (Wang et al., 2004). Seeds of Strychnos ignatii in the Phillipine islands have caused similar toxic effects. Excitatory Amino Acids Widely divergent species of plants produce amino acids that mimic the action of glutamate on the central nervous system. Most fast excitatory transmission in the mammalian brain is mediated by inotropic receptors for the amino acid, glutamate, on specialized neurons. Different types of glutamate receptors respond to different excitatory amino acids from plants, acting on one or more of these glutamate receptor subtypes. The consequence of ingestion of excitatory amino acids is excessive stimulation that may result in death of neurons. One of these acids, kainic acid, is present in the marine red alga Digenia simplex. Under some climatic conditions the algae reproduce rapidly, causing a “red tide.” Filter-feeding mussels eat the algae and humans may be poisoned by eating the mussels. A similar problem exists with the green alga Chondria aranta in northern oceans. The alga produces domoic acid, an analog of glutamate, as do several species of the marine diatom

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Nitzschia (Kotaki et al., 2005). Acute symptoms are gastrointestinal distress, headache, hemiparesis, confusion, and seizures. Prolonged effects are severe memory deficits and sensorimotor neuropathy (Teitelbaum et al., 1990). The fungus Amanita muscaria (fly agaric) got its name from its poisonous actions on flies. Poisoning from this woodland mushroom and from Amanita pantherian (panther agaric, common in western United States) is due to the content of the excitatory amino acid, ibotenic acid (isoxazole amino acid), and to its derivative, muscimol (Li and Oberlies, 2005). The effects are somewhat variable: central nervous system depression, ataxia, hysteria, and hallucinations. Myoclonic twitching and seizures sometimes develop (Benjamin, 1992). The content of ibotenic acid varies with the time of year; more has been reported in spring than in fall. Several other genera of fungi have hallucinogenic actions, notably Psilocybe, containing the indoles, psilocin, and psilocybin (Tsujikawa et al., 2003). Excitatory amino acids are also found in flowering plants. The pea family (Leguminosae) contains several species that produce excitatory amino acids in the seeds. Willardiine [1-(2-amino2-carboxyethyl)pyrimidine-2,4-dione] has been isolated from Acacia willardiana, Acacia lemmoni, Acacia millefolia, and Mimosa asperata (Gmelin, 1961). Willardiine acts as an agonist on glutamate receptors. Other important excitatory amino acids are present in species of Lathyrus. Lathyrus sylvestris (flat pea) is a perennial indigenous to Europe and central Asia and naturalized in Canada and northern United States. This plant is eaten by livestock in these areas. An acute neurologic condition in sheep begins with weakness and progresses to tremors and prostration, sometimes with clonic movements and seizures (Rasmussen et al., 1993). Seeds of Lathyrus sativus (grass pea) are used as food in several countries, including India and Ethiopia. The seeds contain the excitatory amino acid, beta-l-ODAP (beta-N -oxalyl-l-alpha, beta-diaminopropionic acid) (Warren et al., 2004). Lathyrism develops from consumption of seeds over periods of months or longer. Affected individuals have corticospinal motor neuron degeneration with severe spastic muscle weakness and atrophy but little sensory involvement (Spencer et al., 1986). Motor Neuron Demyelination Paralysis develops from some toxins without primary excitation of neurons. Karwinskia humboldtiana, family Rhamnaceae, is a shrub of southwestern United States, Mexico, and Central America. Common names are buckthorn, coyotillo, and tullidora. Anthracenones are found in the seeds, the amount varying with stage of growth; green fruit may be more toxic than ripe fruit (Bermudez et al., 1986). Both human and livestock poisonings occur, occasionally in epidemic proportions. The clinical syndrome that develops after a latency of several days is ascending flaccid paralysis, beginning with demyelination of large motor neurons in the legs and, in fatal cases, leading to bulbar paralysis (Martinez et al., 1998). Sensory fibers are largely spared. In addition to neurotoxicity, the anthracenones in Karwinskia, especially peroxisomicine A2 , [3,39-dimethyl-3,39,8,89,9,99-hexahydroxy-3,30,4,49-tetrahydro(7,10-bianthracene)-1,19-2H ,29H -dione], causes lung atelectasis and emphysema and massive liver necrosis. Inhibition of catalase in peroxisomes has been proposed as the mechanism of cell toxicity (Martinez et al., 1997). Cerebellar Neurons Swainsonine is an indolizidine alkaloid found in the legumes Swainsonia cansescens (an Australian plant),

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Astragalus lentiginosus (spotted locoweed) and Oxytropis sericea (locoweed) in western United States. Cattle consume these weeds in pasture. The common name comes from the most obvious consequence of ingestion of locoweeds: aberrant behavior with hyperexcitability and locomotor difficulty. In animals dying from locoweed poisoning, there is cytoplasmic foamy vacuolation of cerebellar neurons. The toxic ingredient, swainsonine, causes marked inhibition of liver lysosomal and cytosomal alpha-mannosidase and Golgi mannosidase II. Inhibition of the Golgi enzyme results in abnormal brain glycoproteins and accumulation of mannose-rich oligosaccharides (Tulsiani et al., 1988). The pathology is not limited to the nervous system and the effects of swainsonine poisoning are found in several tissues. A major effect in animals grazing at high altitudes is congestive heart failure. The condition has been called “high mountain disease” (James et al., 1991). A species of the fungus Embellisia, an endophyte on locoweeds, may also produce swainsonine (McLainRomero et al., 2004). Parasympathetic Stimulation Several plant alkaloids affect the autonomic nervous system, mimicking the transmitter acetylcholine at autonomic ganglia (nicotinic receptors) or the peripheral endings of the parasympathetic system (muscarinic receptors). The postsynaptic receptors at terminations of the parasympathetic nerve fibers are called “muscarinic” after the selective stimulation of these receptors by muscarine, a quaternary ammonium furan, that was first extracted from the mushroom A. muscaria. However, this mushroom contains only trace amounts of muscarine, and poisoning is due to its content of ibotenic acid. Some mushrooms of the genera Inocybe, Clitocybe, and Omphalatus contain significant amounts of muscarine, and consumption of toxic species causes diarrhea, sweating, salivation, and lacrimation, all referable to stimulation of parasympathetic receptors (de Haro et al., 1999). Parasympathetic Block The belladonna alkaloids (atropine, lhyoscyamine, and scopolamine), known for their block of muscarinic receptors, are found in several genera of Solanaceae, the nightshade family. The plants are widely distributed. Datura stramonium (jimson weed) is native to India and contains primarily scopolamine; Hyoscyamus niger (henbane) is native to Europe and contains primarily l-hyoscyamine; Atropa belladonna (deadly nightshade), also native to Europe, contains atropine; Duboisia myoporoides (pituri) in Australia contains l-hyoscyamine. Scopolamine has the greater action on the central nervous system. The effects of modest doses of l-hyoscyamine or atropine are referable to muscarinic receptor block: tachycardia, dry mouth, dilated pupils, and decreased gastrointestinal motility. Large doses of either or of scopolamine affect the central nervous system with confusion, bizarre behavior, hallucinations, and subsequent amnesia. In severe intoxication, tachycardia may be absent (Caksen et al., 2003). Deaths are rare, although recovery may take several days. Datura suaveolens (angel’s trumpet, Solanaceae) an ornamental houseplant, contains significant quantities of atropine and scopolamine (Smith et al., 1991). Seeds of Datura ferox contain belladonna alkaloids and are contaminants of animal feed in some parts of Europe. In areas where millet, wheat, rye, corn, and bean seeds are used for human consumption, and where D stramonium and Datura metel are common weeds, the grain sometimes has been contaminated with Datura seeds. Symptoms from eating bread made from contaminated flour are typical of poisoning from belladonna alkaloids (van Meurs et al., 1992). The amount needed to poison an

adult is about 20 seeds. An unusual source of belladonna poisoning has been reported from eating wasp honey. Polistes species of wasps store honey, and atropine-like poisoning has been reported from consuming the honey when the wasps have gathered nectar from Datura inoxia (Ramirez et al., 1999). The seeds of Solanum dulcamara (bittersweet) are brilliant red-orange and are gathered in the fall for flower arrangements. The seeds contain solanine, a glycoalkaloid, responsible for the acute toxicity from ingested seeds, including tachycardia, dilated pupils, and hot, dry skin, as in atropine poisoning (Ceha et al., 1997). Sensory Neuron Block Several plant toxins are being studied for agonist actions on sensory receptors of the vanilloid-type (VR1), especially capsaicin found in species of Capsicum (C. annuum, sweet pepper, and C. frutescens, red pepper, Solanaceae). Capsaicin produces a burning sensation on VR1 sensory endings, but also desensitizes the transient potential vanilloid 1 receptor (TRPV1) of sensory endings of C-fiber nociceptors to stimuli. This long-term sensory neuron blocking effect has therapeutic use in chronic pain (Szalcsany, 2002). The sensory desensitization produced by capsaicin is not due to acute cell death and long-lasting changes in mitochondria in cultured dorsal root ganglion cells have been demonstrated (Dedov et al., 2001). It has recently been suggested that capsaicin may also have an action on another ion channel, the 4-aminopyridine-sensitive K+ channel, and may inhibit contractile mechanisms by release of Ca2+ from intracellular storage sites (Fujimoto et al., 2006). Another plant toxin, polygodial, a sesquiterpene found in Polygonum hydropiper, has a capsaicin-like action on the TRPV1 (Andre et al., 2006). Resiniferatoxin, a homovanillic ester from Euphorbia resinifera (Euphorbiaceae), is a potent agonist of TRPV1 and causes sustained depolarization of the vanilloid receptor. The long-lasting effect of resiniferatoxin on nerve terminals is related to slow activation of TRPV1 with excessive influx of Ca2+ without generating action potentials (Raisinghani et al., 2005).

Skeletal Muscle and Neuromuscular Junction Neuromuscular Junction Block of the neuromuscular junction of skeletal muscle may result from either block of postsynaptic acetylcholine receptors (nicotinic receptors) by an antagonist or by an agonist causing excessive stimulation of the receptor followed by prolonged depolarization. Nicotine stimulates autonomic ganglia as well as the neuromuscular junction. An isomer of nicotine, anabasine, present in Nicotiana glauca (tree tobacco, Solanaceae), produces prolonged depolarization of the junction. Consumption of the leaves of the plant has caused flexor muscle spasm and gastrointestinal irritation, followed by severe, generalized weakness, and respiratory compromise (Mellick et al., 1999). Curare, the South American arrow poison is a potent neuromuscular blocking agent used clinically. Curare is obtained from tropical species of Strychnos and Chondrodendron. Not all plants blocking the neuromuscular junction are tropical in origin. In warm weather, blooms of bluegreen algae are not uncommon in farm ponds in temperate regions, particularly ponds enriched with fertilizer. Under these conditions, one species of alga, Anabaena flosaquae, produces a neurotoxin, anatoxin A, that depolarizes and blocks acetylcholine receptors, both nicotinic and muscarinic, sometimes causing death in animals that drink the pond water. The lethal effects develop rapidly, with death in minutes to hours from respiratory arrest (Short and

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Edwards, 1990). Anatoxin A is 2-acetyl-9-azabicyclo(4.2.10)non2-ene (Hyde and Carmichael, 1991). Methyllycaconitine is a norditerpenoid present in Delphinium barbeyi (tall larkspur, Ranunculaceae) and in some related species. The plant contaminates western pastures in the United States and causes death of livestock. Poisoned cattle show muscle tremors and ataxia followed by prostration and respiratory arrest in fatal cases. The compound has a high affinity for the acetylcholine receptor at the neuromuscular junction like curare. Physostigmine has been used successfully as an antagonist in some cases of methyllycaconitine poisoning (Pfister et al., 1994). Skeletal Muscle Direct damage to skeletal muscle fibers has been demonstrated in some plant poisonings. Species of Thermopsis (Leguminosae) are common in the foothills of the Rocky Mountains. Mature seeds of these plants form in attractive pods, as in many legumes. Seeds of the poisonous species of Thermopsis contain quinolizidine alkaloids, principally anagyrine and thermopsine. Human poisoning from eating the seeds is rare, but cases have been reported in young children (Spoerke et al., 1988). The symptoms are abdominal cramps, nausea, vomiting, and headache lasting up to 24 hours. Serious poisoning has occurred in livestock grazing on Thermopsis montana (false lupine). The animals develop locomotor depression and recumbancy. Microscopic areas of necrosis in skeletal muscle are found on autopsy (Keeler and Baker, 1990). Seeds of Cassia obtusifolia (sicklepod, Leguminosae) have been found as a contaminant of animal feeds. Consumption of the seeds in cattle, swine, and chickens causes a degenerative myopathy in cardiac and skeletal muscle. Extracts of C. obtusifolia inhibit NADH-oxidoreductase in bovine and porcine mitochondria in vitro (Lewis and Shibamoto, 1989) possibly related to the anthraquinone content of the seeds. Ingestion of white snakeroot (Eupatorium rugosum, Asteraceae), a common plant in central and western United States, causes tremors in cattle and “milk sickness” in humans after ingestion of milk from cows pasturing in fields infected with the plant (Beier et al., 1993). The toxic effect thought to be caused by tremetone, a benzofuran, that blocks gluconeogenesis from lactate, resulting in acidosis, tremor, and death (Polya, 2003).

Bone and Tissue Calcification Bone and Soft Tissue Worker and Carrillo (1967) proposed that a decrease in bone calcification and a wasting disease in cattle grazing along the eastern coastal plains of South America was due to the consumption of Solanum malacoxylon (Solanaceae). The disease, known in Argentina as “enteque seco” is characterized by calcification of the entire vascular system, especially the heart and aorta. Lungs, joint cartilage, and kidney are affected in the worst cases. Sheep and cows are both affected by ingestion of the plant. The general picture resembles vitamin D intoxication. A water-soluble vitamin D-like substance, a glycoside of 1,25-dihydroxycholecalciferol, has been isolated from the plant (Skliar et al., 1992). Cestrum diurnum (day-blooming jasmine, Solanaceae) causes hypercalcemia and extensive soft tissue calcification in grazing animals in Florida, resembling the action of S. malacoxylon. A dihydroxyvitamin D3 glycoside in the leaves is the toxic agent (Durand et al., 1999). Cestrum laevigatum causes a similar deposition of calcium in chickens (Mello and Habermehl, 1992). This species is occasionally a contaminant of hay in Europe. However, in

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cattle, marked hepatotoxicity occurs with centrolobular and midzonal necrosis (Peixato et al., 2000).

Reproduction and Teratogenesis Abortifacients The active alkaloid in the legumes Astragalus and Oxytropus is swainsonine. In addition to actions on the nervous system, swainsonine frequently causes abortions when locoweeds are ingested by pregnant livestock (Bunch et al., 1992). Two genera of tropical legumes, Leucaena and Mimosa, contain a toxic amino acid, mimosine [beta-N (3-hydroxy-4-pyridone)aalpha-aminopropionic acid]. Mimosine is found in large amounts in foliage and seeds of Leucaena leucocephala, Leucaena glauca, and Mimosa pudica. In cattle the amino acid causes incoordinated gait, goiter, and reproductive disturbances including infertility and fetal death (Kulp et al., 1996). Mimosine arrests the cell cycle in late G1-phase (Perry et al., 2005). Lectins that are ribosome-inactivating proteins may have many effects on reproduction when ingested, including antifertility, abortifacient, and embryotoxic actions. A lectin from bitter melon seeds (Momordia charantia, Curcurbitaceae) has been shown to have such effects. The lectins are alpha- and beta-momorcharins, single-chain glycoproteins with molecular weight of about 29,000. The momorcharins are known to induce midterm abortion in humans (Wang and Ng, 1998). Caulophylline (N -methylcytisine) is a quinolizidine from Caulophyllum thalictroides (blue cohosh, Berberidaceae). The plant is widely distributed in temperate North America and eastern Asia. Caulophylline is teratogenic in rats (Kennelly et al., 1999). Neonatal and maternal toxicity has been reported from maternal ingestion of herbal preparations of blue cohosh to terminate pregnancy (Jones and Lawson, 1998). The maternal signs resemble the action of an agonist to nicotinic acetylcholine receptors (Rao and Hoffman, 2002). Many pastures of tall fescue (Festuca arundinacea, Poaceae) are infected by the endophytic fungus Neotyphodium coenophialum. The presence of the endophyte is related to production in the fescue plant of ergovaline, an ergopeptine alkaloid that acts as an agonist to dopamine D2 receptors. Ergovaline inhibits prolactin release and acts as a vasoconstrictor. Pregnant mares are most susceptible toward the end of gestation, resulting in damage to the fetus and maternal loss of milk (Blodgett, 2001). Regulation of levels of different alkaloids in grasses as a result of Neotyphodium infestation is closely related to plant genotype as well as the presence of the endophyte (Spiering et al., 2005). Teratogens Veratrum californicum (Liliaceae) is native to the mountains of North America where sheep are grazed. An incidence of teratogenesis as high as 25% has been reported in pregnant sheep in these areas, along with early embryonic death as high as 75% (Keeler, 1990). The teratogenic manifestations are dependent on the developmental stage at the time of exposure, as with many teratogens. Malformations of the offspring involve cyclopia, exencephaly, and microphthalmia. During the 4th and 5th weeks of gestation, limb defects are common; on gestational days 31–33, the result of ingestion is fetal stenosis of the trachea (Omnell et al., 1990). The alkaloids in Veratrum that are responsible for the defects are jervine, 11deoxojervine and 3-O-glucosyl-11-deoxojervine. Although there is species difference in sensitivity, birth defects occur in cows and goats grazing on V. californicum. Birth defects have been produced experimentally in chickens, rabbits, rats, and mice (Omnell et al., 1990), hamsters (Gaffield and Keeler, 1993), and rainbow trout embryos

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(Crawford and Kocan, 1993). The Veratrum alkaloids cause teratogenesis by blocking cholesterol synthesis and thus the response of fetal target tissue to the sonic hedgehog gene (Shh). The Shh locus has a role in developmental patterning of head and brain, and block of cholesterol synthesis has been shown experimentally to result in loss of midline facial structures (Cooper et al., 1998). A cluster of fetal malformations characterized by deformation of limbs and spinal cord is found after maternal ingestion of related alkaloids from different species of plants during a sensitive gestational period. The syndrome has been found in cattle grazing on Lupinus caudatus and Lupinus formosus (lupines, Luguminosae), Nicotiana glauca (tree tobacco, Solanaceae), and Conium maculatum (poison hemlock, Solanaceae). Poison hemlock is known historically as the plant that Socrates drank when condemned to death in Athens. The fatal actions of the poison have been described by Plato in his Phaedo. The active alkaloids in these plants are anagyrine (L. caudatus), ammodendrine (L. formosus), anabasine (N. glauca), and coniine (C. maculatum). It has been proposed that these alkaloids depress fetal movements during susceptible gestational periods and in this way cause malformations (Lopez et al., 1999). Not all species are equally affected by coniine. Notably, rats and hamsters do not

show teratogenesis in response to coniine, but goat and chick embryos are susceptible (Forsyth et al., 1996).

SUMMARY A great variety of toxic chemicals have been produced by plants for their own protection from the environment and from predators, including pathogenic organisms. These defensive chemicals have been both deleterious and beneficial to humans. Throughout history a select number have been incorporated into therapy against disease and to combat morbidity, often with considerable success. Morphine from the latex of the opium poppy is an ancient historical example. If fungi are included with plants (as they are in this chapter), defensive chemicals of plants are responsible for some of our more successful therapies, such as antibiotics of the penicillin type and therapy for cancer. On balance, in spite of the long list of dangerous toxic chemicals from plants, it is fair to conclude that plants have proved therapeutically more useful than harmful to humans. Finally, there is simply the pleasure of scientific inquiry into the successful adaptations of plants to their complex and often hostile environments.

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Gude M, Hausen MD, Heitsch H, et al.: An investigation of the irritant and allergenic properties of daffodils (Narcissus pseudonarcissus L., Amaryllidaceae): A review of daffodil dermatitis. Contact Dermatitis 19:1–10, 1988. Gurung NK, Rankens DL, Shelby RA: In vitro ruminal disappearance of fumonisin B1 and its effects on in vitro dry matter disappearance. Vet Hum Toxicol 41:196–199, 1999. Haarmann T, Machado C, Lubbe Y, et al.: The ergot alkaloid gene cluster in Claviceps purpurea : Extension of the cluster sequence and intra species evolution. Phytochemistry 66:1312–1320, 2005. Hall AC, Turcotte CM, Betts BA, et al.: Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids. Eur J Pharmacol 506:9–16, 2004. Han D, Matsumaru K, Rettori D, et al.: Usnic acid-induced necrosis of cultured mouse hepatocytes: Inhibition of mitochondrial function and oxidative stress. Biochem Pharmacol 67:439–451, 2004. Hausen BM, Prater E, Shubert H: The sensitizing capacity of Alstroemeria cultivars in man and guinea pig. Contact Dermatitis 9:46–54, 1983. Hill NS, Thompson FN, Dawe DL, et al.: Antibody binding by circulating ergot alkaloids in cattle grazing tall fescue. Am J Vet Res 55:419–424, 1994. Hirschwehr R, Heppner C, Spitzauer S, et al.: Allergens, IgE mediators, inflammatory mechanisms. J Allergy Clin Immunol 101:196–206, 1998. Huan J-Y, Mironda CL, Buhler DR, et al.: Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids. Toxicol Lett 99:127–137, 1998. Hyde EG, Carmichael WW: Amatoxin-a(s), a naturally occurring organophosphate, is an irreversible active site-directed inhibitor of acetylcholinesterase (E.C.3.1.1.7). J Biochem Toxicol 6:195–201, 1991. Izzo AA, Ernst E: Interactions between herbal medicines and prescribed drugs: A systematic review. Drugs 61:2163–2175, 2001. Jaeger A, Fehl F, Flesch F, et al.: Kinetics of amatoxins in human poisoning: Therapeutic implications. Clin Toxicol 31:63–80, 1993. Jaffe AM, Gephardt D, Courtemanche L: Poisoning due to ingestion of Veratrum viride (false hellebore). J Emerg Med 8:161–167, 1990. James LF, Panter KE, Broquist HP, et al.: Swainsonine-induced high mountain disease in calves. Vet Hum Toxicol 33:217–219, 1991. Johnson RA, Baer H, Kirkpatrick CH, et al.: Comparison of the contact allergenicity of the four pentadecylcatechols derived from poison ivy urushiol in human subjects. J Allergy Clin Dermatol 49:27–35, 1972. Jones TK, Lawson BM: Profound neonotal congestive heart failure caused by maternal consumption of blue cohosh herbal medication. J Pediatr 132:550–552, 1998. Jorgensen K, Bak S, Busk PK, et al.: Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Plant Physiol 139:363–374, 2005. Kalish RS, Johnson KL: Enrichment and function of urushiol (poison ivy)specific T lymphocytes in lesions of allergic contact dermatitis to urushiol. J Immunol 145:3706–3713, 1990. Karlson-Stiber C, Persson H: Cytotoxic fungi—an overview. Toxicon 42:339–349, 2003. Kavalali G: The chemical and pharmacological aspects of Urtica, in Kavalali GM (ed.): Urtica. New York: Taylor and Francis, 2003, pp. 25–39. Keeler RF: Early embryonic death in lambs induced by Veratrum californicum. Cornell Vet 80:203–207, 1990. Keeler RF, Baker DC: Myopathy in cattle induced by alkaloid extracts from Thermopsis montana, Laburnum anagyroides and a Lupinus sp. J Comp Pathol 103:169–182, 1990. Kelch WJ, Kerr LA, Adair HS, et al.: Suspected buttercup (Ranunculus bulbosus) toxicosis with secondary photosensitization in a Charolais heifer. Vet Hum Toxicol 34:238–239, 1992. Kennelly EJ, Flynn TJ, Mazzola EP, et al.: Detecting potential teratogenic alkaloids from blue cohosh rhizomes using an in vitro rat embryo culture. J Nat Prod 62:1385–1389, 1999.

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Roy MC, Chang FR, Huang HC, et al. : Cytotoxic principles from the formosan milkweed, Asclepias curassavica. J Nat Prod 68:1494–1499, 2005. Sako MDN, Al-Sultan II, Saleem AN: Studies on sheep experimentally poisoned with Hypericum perforatum. Vet Hum Toxicol 35:298–300, 1993. Schacter L: Etopside phosphate: What, why, where and how? Semin Oncol 23 (suppl 13):1–7, 1996. Sessa RA, Bennett MH, Lewis MJ, et al.: Metabolite profiling of sesquiterpene lactones from Lactuca species. J Biol Chem 275:26877–26884, 2000. Shakin M, Smith BL, Prakash AS: Bracken carcinogens in the human diet. Mutat Res 443:69–79, 1999. Sharma OP, Dawra RK, Pattabhi V: Molecular structure, polymorphism, and toxicity of lantadene A, the pentacyclictriterpenoid from the hepatotoxic plant Latana camara. J Biochem Toxicol 6:57–63, 1991. Short SO, Edwards WC: Blue green algae toxicosis in Oklahoma. Vet Hum Toxicol 32:558–560, 1990. Siebert U, Brach M, Sroczynski G, et al.: Efficacy, routine effectiveness, and safety of horsechestnut seed extract in the treatment of chronic venous insufficiency. A meta-analysis of randomized controlled trials and large observational studies. Int Angiol 21:305–315, 2002. Skliar MI, Boland RL, Mourino A, et al.: Isolation and identification of vitamin D3 , 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24, 25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid. J Steroid Biochem Mol Biol 43:677–682, 1992. Smith EA, Meloan CE, Pickell JA, et al.: Scopolamine poisoning from homemade “moon flower” wine. J Anal Toxicol 15:216–219, 1991. Southcott RV, Haegi LAR: Plant hair dermatitis. Med J Aust 156:623–628, 1992. Spencer PS: Food toxins, AMPA receptors and motor neuron diseases. Drug Metab Rev 31:561–587, 1999. Spencer PS, Roy DN, Ludolph A, et al.: Lathyrism: Evidence for the role of the neuroexcitatory amino acid, BOAA. Lancet 2:1066–1067, 1986. Spiering MJ, Lane GA, Christensen MJ, et al.: Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants. Phytochemistry 66:195–202, 2005. Spoerke DG, Murphy MM, Wruk KM, et al.: Five cases of Thermopsis poisoning. J Toxicol Clin Toxicol 26:397–406, 1988. Surh Y-J, Lee E, Lee JM: Chemoprotective properties of some pungent ingredients present in red pepper and ginger. Mutat Res 404:259–267, 1998. Szalcsany J: Capsaiscin receptors as target molecules on nociceptors for development of novel analgesic agents, in Keri G and Toth I (eds.): Molecular Pathomechanisms and New Trends in Drug Research. London: Taylor & Francis, 2002, pp. 319–333.

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Tahirov THO, Lu T-H, Liaw Y-C, et al.: A new crystal form of abrina from the seeds of Abrus precatorius. J Mol Biol 235:1152–1153, 1994. Tandon HD, Tandon BN, Mattocks AR: An epidemic of veno-occlusive disease of the liver in Afghanistan. Am J Gastroenterol 70:607–613, 1978. Teitelbaum JS, Zatorre RJ, Carpenter S, et al.: Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med 322:1781–1787, 1990. Tsujikawa K, Kanamori T, Iwata Y, et al.: Morphological and chemical analysis of magic mushrooms in Japan. Forensic Sci Int 138:85–90, 2003. Tucker MO, Swan CR: The mango-poison ivy connection. N Engl J Med 339:235, 1998. Tulsiani DR, Broquest HP, James LF, et al.: Production of hybrid glycoprotein and accumulation of oligosaccharides in the brain of sheep and pigs administered swainsonine or locoweed. Arch Biochem Biophys 264:607–617, 1988. Turgut M, Alhan CC, Gurgoze M, et al.: Carboxyatractyloside poisoning in humans. Ann Trop Paediatr 25:125–134, 2005. Tylleskar T, Banca M, Bikongi N, et al.: Cassava cyanogens and konzo, an upper motoneuron disease found in Africa. Lancet 339:208–211, 1992. Urushibata O, Kase K: Irritant contact dermatitis from Euphorbia marginata. Contact Dermatitis 24:155–157, 1991. Uwai K, Ohashi K, Takaya Y, et al.: Exploring the structural basis of neurotoxicity in C(17)-polyacetylenes isolated from water hemlock. J Med Chem 43:4508–4515, 2000. van Meurs A, Cohen A, Edelbroek P: Atropine poisoning after eating chapattis contaminated with Datura stramonium (thorn apple). Trans R Soc Trop Med Hyg 86:221, 1992. Wang H, Ng TB: Ribosome inactivating protein and lectin from bitter melon (Momordica charantia) seeds: Sequence comparison with related proteins. Biochem Biophys Res Commun 253:143–146, 1998. Wang Z, Zhao J, Xing J, et al. : Analysis of strychnine and brucine in postmortem specimens by RP-HPLC: A case report of fatal intoxication. J Anal Toxicol 28:141–144, 2004. Warren BA, Patel SA, Nunn PB, et al.: The Lathyrus excitotoxin beta-N oxalyl-l-alpha, beta diaminopropionic acid is a substrate of the lcystine/l-glutamate exchanger system xc-. Toxicol Appl Pharmacol 200:83–92, 2004. Worker NA, Carrillo BJ: “Enteque seco,” calcification and wasting in grazing animals in the Argentine. Nature 215:72–74, 1967. Wu JH, Singh T, Herp A, et al.: Carbohydrate recognition factors of the lectin domains present in the Ricinus communis toxic protein (ricin). Biochemie 88:201–217, 2006. Yoshizawa T, Yamashita A, Luo Y: Fumonisin occurrence in corn from highand low-risk areas for human esophageal cancer in China. Appl Environ Microbiol 60:1626–1929, 1994.

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CHAPTER 28

AIR POLLUTION∗ Daniel L. Costa Sulfuric Acid and Related Sulfates Particulate Matter Metals Gas-Particle Interactions Ultrafine Carbonaceous Matter Photochemical Air Pollution Short-Term Exposures to Smog Chronic Exposures to Smog Ozone Nitrogen Dioxide Other Oxidants Aldehydes Formaldehyde Acrolein Carbon Monoxide Hazardous Air Pollutants Accidental versus “Fence-Line” Exposures

AIR POLLUTION IN PERSPECTIVE A Brief History of Air Pollution and Its Regulation ASSESSING RISKS ASSOCIATED WITH AIR POLLUTION Animal-to-Human Extrapolation: Issues and Mitigating Factors Air Pollution: Sources and Personal Exposure The Evolving Profile of Air Pollution Indoor Versus Outdoor Air Pollution EPIDEMIOLOGIC EVIDENCE OF HEALTH EFFECTS Outdoor Air Pollution Acute and Episodic Exposures Long-Term Exposures Indoor Air Pollution Sick-Building Syndromes Building-Related Illnesses

WHAT IS AN ADVERSE HEALTH EFFECT? POLLUTANTS OF OUTDOOR AMBIENT AIR CONCLUSIONS

Classic Reducing-Type Air Pollution Sulfur Dioxide

ACKNOWLEDGMENT

AIR POLLUTION IN PERSPECTIVE The second half of the twentieth century witnessed remarkable changes in environmental perspective. Until that time, national pride and prosperity were often visually depicted as an expanse of urban factories with smokestacks belching opaque clouds of industrial effluent into a seemingly neutral sky. The price of unchecked human progress through the first half of the century had led to a number of environmental catastrophes, which demonstrated the profoundly detrimental impact that such reckless prosperity could have on the natural environment. These images of “modern” prosperity gradually gave rise to public outcry for governmental action to protect environmental quality, wildlife, as well as public health. The ensuing fifty years of regulatory legislation in the United States and Western Europe have now made such industrial scenes rare in most technologically developed nations. But, ironically, even as regulatory control measures began to reduce much of the stationary sources of air pollution, large populations have fled the cities in pursuit of a cleaner, safer lifestyle. This change in demographics post World War II altered the composition and distribution of polluted air. The commute from suburban home to city workplace back to suburban home, led to increasingly congested vehicular thoroughfares, creating a photochemical cauldron of oxidant air pollution around these newly formed, rapidly expanding metropolitan areas. More∗

This article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency.

over, postwar population growth and rising expectations for a better (peace time) standard of living led to unrestrained consumption of inexpensive fossil fuels for energy production and recreational transportation needs. The result has been a world-wide destruction of limited environmental resources and an adverse effect on global climate, as energy-related polluted air masses drift and disperse across hundreds, even thousands of miles. And so, air pollution remains a twenty-first-century reality, and while great legislative strides have been made to reduce emissions from both stationary and mobile sources, nonetheless, unsatisfactory air quality now plagues much broader geographic areas. More than half the U.S. population resides in counties that are not in compliance with current National Ambient Air Quality Standards (NAAQS) (Fig. 28-1). These noncompliant areas correspond well with the spread of population growth from major urban and industrial centers. Episodes of extreme air pollution are rare in the Western world today. Occasionally, these areas experience unusual meteorological stagnations that coincide with “normal or expected” air pollution patterns that intensify the pollution over broad regions (e.g., 1995 in the United Kingdom and Western Europe). Conversely, specific locales dominated by a single, major pollutant source (e.g., Utah Valley smelter—through the 1980s and 1990s) can experience extremely poor air quality especially when exacerbated by meteorological inversions, with serious public health consequences. More typically, however, public exposures are characterized by prolonged periods of relatively low-levels of complex mixtures of photochemically transformed industrial and mobile emissions with periodic moderate excursions due to weather. This pattern of pollution now

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Eagle River Juneau North Star Fairbanks

AK

PR

GUAM Piti Power Plant Tanguisson Power Plant

CO Lead

Note: Incomplete data, not classified, and Section 185(A) areas are not shown, *Ozone nonattainment areas on map are based on the 1-hour ozone standard. **PM10 nonattainment areas on map are based on the existing PM10 standards.

Guaynabo

Ozone* PM10** SO2

Figure 28-1. Areas in the United States not in attainment for the NAAQS (September 2002) criteria pollutants: O3 , PM10 , SO2 , CO, and Pb. No area was in violation for NO2 . [Note: Incomplete data, and not classified or Section 185(A) areas are not shown. Ozone nonattainment areas are based on the 1-hour NAAQS and those for PM10 are based on the 1997 PM10 NAAQS. (Air Trends - Figure 4-1; http://epa.gov/airtrends/non.html).

extends even into remote wilderness areas, where considerable damage to flora and fauna has occurred. In contrast, many developing countries experiencing rapid population growth, industrialization, and economic expansion choose to ignore the lessons of Western industrial expansion and the consequences of inadequate air pollution control. Air pollution in some of these nations now rivals the atmospheric conditions that existed in Western Europe and America preceding the catastrophic air pollution episodes of the twentieth century. For example, two Hong Kong marathoners died in February 2006 while running during severe air pollution. In addition, industrial emissions from some developing nations and emerging economies, now contribute substantially to the global burden of many air pollutant constituents. A willingness to balance worldwide economic growth and industrialization using lessons learned from the misadventures of the Second Industrial Revolution and the Age of the Automobile will determine the impact of the “new” global economy on the health of both the earth and its inhabitants. Previously, the scientific information regarding the impacts– of air pollution on human health had largely been collected on the individual constituents that make-up air pollution’s complex mixture. In turn, this knowledge was used to develop both public health standards and to establish regulatory controls. Despite the obvious complexities of urban air pollution, it was felt that single pollutant regulations were the best path to success. Indeed, great strides have been made. However, research agendas have slowly been evolving

to include the study of the interaction and transformation of individual pollutant components within atmospheric systems—a truer reflection of real-world exposure complexities. This chapter will present an overview of the current state of knowledge regarding the production of air pollution and its subsequent impact on human health. Also, the complexities of regulatory decisions and risk assessment will be addressed. Hopefully, these topics will provide the reader with both a fundamental knowledge and appreciation of the nature of the problem, as well as a sense of the uncertainties in need of future investigation.

A Brief History of Air Pollution and Its Regulation For most of history, air pollution has been a problem of microenvironments and domestic congestion. The smoky fires of early cave and hut dwellers choked the air inside their homes. When the emissions were vented outdoors, they combined with those of the neighbors to settle around the village on damp cold nights. With urbanization and a concomitant decrease in forest wood as a source of fuel to heat and cooking, the need for energy led to the burning of cheap coal and ambient release of sulfurous, sooty smoke. Likewise, kilns to make quicklime for construction and metal smelters pushed smoke and chemical materials into the air of active “modern” cities. Unfortunately, the city dwellers had to endure the bad air, while those with wealth frequently had country homes to which they could escape from time to time. The poor quality of urban

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Table 28-1 U.S. National (Primary) Ambient Air Quality Standards∗ pollutant

unit

averaging time

concentration

statistic

Sulfur dioxide

μg/m3 (ppm)

Carbon monoxide

μg/m3 (ppm)

Ozone#

μg/m3 (ppm)

Nitrogen dioxide Particulates PM#10 PM#2.5 Lead#

μg/m3 (ppm) μg/m3 μg/m3 μg/m3

Annual 24 h 8h 1h 1h 8h Annual Annual and 24 h Annual and 24 h 3 months

80 (0.03) 365 (0.14) 10 (9) 40 (35) 235 (0.12) 157 (0.08) 100 (0.053) 150 and 50 65 and 15 1.5

Annual mean Maximum Maximum Maximum Maximum Maximum Annual mean Annual mean Quarterly average

∗

For detailed information regarding policy and precise statistical and time-based computations to achieve attainment, contact EPA Web site: www.epa.gov/airs/criteria.html. # NAAQS currently under review for potential revision in 2006. See noted Web site.

air was captured by many writers from Charles Dickens in his writings about London’s fogs to the ancients: Seneca, the Roman philosopher, in ad 61 wrote: “As soon as I had gotten out of the heavy air of Rome, and from the stink of the chimneys thereof, which being stirred, poured forth whatever pestilential vapors and soot they had enclosed in them, I felt an alteration to my disposition” (emphasis added: Miller and Miller, 1993). As in today’s world efforts to regulate air pollution competed with the industrial economies and as a result, they evolved slowly. Early on, in the time of Greece and Rome, individual civil suits could be levied against local polluters, although these were of marginal success. Beginning in the thirteenth century, community-based outcries received some recognition by governing officials, one example being the banning of “sea coal” from lime kilns and domestic heaters in London by Edward I. Enforcement, however, was not effective and people largely resigned themselves to polluted air as part of urban life. By the seventeenth century, England, in the middle of several decades some refer to as “the little ice age,” experienced further reductions in wood harvests, which only increased reliance on sea coal for domestic heating. Despite Percival Pott’s discovery that soot was related to the incidence of scrotal cancer in chimney sweeps, the health community offered only a simple recommendation: “Fly the city, shun its turbid air; breathe not the chaos of eternal smoke . . . ” (Brimblecombe, 1999)—advice hardly advanced from that of Seneca 1600 years earlier. In the late eighteenth century, the industrial revolution, which was powered by the burning of “cleaner” mined coal, added a second dimension to urban air pollution. These emissions were more acidic and hung in the air longer than the fluffy soot of the cheaper sea coal. Continued soiling of buildings and damage to nearby crops brought community boards to address sanitary reforms to cut the worse of the pollution peaks and episodes, but any gains were soon offset by growth. By the end of the nineteenth century and into the early twentieth century, power plants were being built to provide energy for factories and eventually to light homes. Steel mills and other industries proliferated along riverbanks and lakeshores, oil refineries rose in port cities and near oil fields and smelters roasted and refined metals in areas near large mineral deposits. By 1925, air pollution was common to all industrialized nations, but people grew less tolerant of the nuisance of acidic-soot corrosion of all exposed surfaces and the general discomfort that came with smoky air—this acidic, sooty form of air pollution has been termed “reducing” air pollution. Public surveys were initiated—as in

Salt Lake City in 1926, New York City in 1937, and Leicester, Great Britain, in 1939—to bring political attention to the problem and promote the implementation of controls (Miller and Miller, 1993). However, it was not until the great air pollution disasters in the Meuse Valley, Belgium, in 1930; Donora, Pennsylvania, in 1948; and the great London fog of 1952 that air pollution was indicted primarily as a health issue. In the United States, California was already leading the way with passage of the Air Pollution Control Act of 1947 to regulate the discharge of opaque smokes. Visibility problems in Pittsburgh during the 1940s had also prompted efforts to control smoke from local industries, but it was the initiative of President Truman that provided the federal impetus to deal with air pollution. This early effort culminated in congressional passage of a series of acts starting with the Air Pollution Control Act of 1955. The prosperity and suburban sprawl of the late 1950s provided the third and perhaps most chemically complex dimension of air pollution. The term smog, though originally coined to describe the mixture of smoke and fog that hung over large cities such as London, was curiously adopted for the eye-irritating photochemical reaction products of auto exhaust that blanketed cities like Los Angeles. Early federal legislation addressing stationary sources was soon expanded to include automobile-derived pollutants (the Clean Air Act of 1963, amended in 1967, and the Motor Vehicle Air Pollution Control Act of 1965). The landmark Clean Air Act (CAA) of 1970 evolved from the early legislation, and despite being only an amendment, it was revolutionary. It recognized the problem of air pollution as a national issue and set forth a plan to control it. The Act established the U.S. Environmental Protection Agency (USEPA) and charged it with the responsibility to protect the public from the hazards of polluted outdoor air. Seven “criteria” air pollutants—ozone (O3 ), sulfur dioxide (SO2 ), particulate matter (PM), nitrogen dioxide (NO2 ), carbon monoxide (CO), lead (Pb), and total hydrocarbons; the last now dropped from the list, leaving six criteria pollutants—were specified as significant health hazards in need of individual National Ambient Air Quality Standards (NAAQS). These NAAQS were mandated for review every 5 years as to the adequacy of the existent standard to protect human health (Table 28-1), although in many instances adherence to this schedule has not been achieved. The explosion in the literature databases for the criteria pollutants and the extensive review process has often led to delays in completing the process on schedule. For each of the criteria pollutants, a Criteria Document or science assessment is developed, which provides a

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Figure 28-2. Excess mortality due to outdoor and indoor particulate matter in various international economic groupings. Bottom and top of each bar represent the lower and upper estimates of mortality, respectively, computed using the methodology of Schwela (2000): Established Market Economics (EME); Eastern Europe (EE); China; India; South East Asia/Western Pacific (SEAWP); Eastern Mediterranean (EM); Latin America (AL); and sub-Saharan Africa (SSA). (Modified from Figure 2.6 by D. Schwela: Air Pollution in the Megacities of Asia - SEOUL WORKSHOP REPORT: Urban Air Pollution Management and Practice in Major and Megacities of Asia—http://www.asiairnet.org/seoulreport.asp).

detailed summary of the available literature on that pollutant. This assessment of the science is then integrated into a Staff Paper to develop a range of proposed standards based on risk analyses. The EPA Administrator considers proposals from both “evidence” and “risk” based assessments to establish policy and set the NAAQS (www.epa.gov/ttn/naaqs/naaqs process report march2006.pdf). In 2006, this process was changed with the steps in the process coming to be termed: Science Assessment, Risk Assessment and Policy Assessment to replace the Criteria Document and Staff Paper. With regard to the Primary NAAQS, only health criteria can be considered in the development of the standard, including safety considerations for potentially susceptible groups. The Secondary NAAQS considers agricultural and structural welfare. Economic impacts are not to be involved in standard setting itself—only in assessing the cost of the implementation procedures. Other hazardous air pollutants (HAPs), of which there were eight listed at the time, were to undergo health assessments to establish emission controls. The CAA of 1970 was by far the most far-reaching legislation to date. The accidental release of 30 tons of methyl isocyanate vapor into the air of the shanty village of Bhopal, India, on December 3, 1984, killed an estimated 3000 people within hours of the release, with several thousand delayed deaths, and 200,000 injured or permanently impaired. The tragedy shocked the world, and raised the issue of HAPs in the United States to a new level of concern. While such a disaster has never struck the United States, accidental industrial releases or spills of toxic chemicals are surprisingly common, with 4375 cases recorded between 1980 and 1987, inflicting 11,341 injuries and 309 deaths (Waxman, 1994). The HAPs, which had been the stepsister of the criteria pollutants for more than a decade after the passage of the 1970 CAA, have since garnered more public and policy attention. There is concern not only for accidental releases of

fugitive or secondary chemicals—such as phosgene, benzene, butadiene, and dioxin, into the air of populated industrial centers—but also for potential chronic health effects, with cancer often being the focus of attention. The slow progress of regulatory decisions on HAPs (only eight between 1970 and 1990) led to a mandated acceleration of the process under the CAA amendment of 1990. Section 112(b) currently lists 188 chemicals or classes of chemicals for which special standards and risk assessments are required. The chemicals listed are those of greatest concern on the basis of toxicity (including cancer) and estimated release volumes. Currently, there is a list of 33 HAPS from the list of 188 that are deemed to be of greatest concern, so-called a list of the “dirty thirty.” Emissions are of HAPS are mandated for control to the maximal achievable control technology (MACT), and any residual health risk after MACT is to be considered in a separate quantitative risk assessment. The database for this process utilizes existing knowledge or, if necessary, mandates further research by the emitter. While many of these chemicals are now better controlled than in the past, most residual risk estimates are yet to be completed. The database from which these assessments are made is called the Integrated Risk Information System (IRIS—www.epa.gov/iris/index.html). Emissions from motor vehicles are addressed primarily under the CAA Title II, Emission Standards for Mobile Sources. The reduction of emissions from mobile sources is complex and involves both fuel and engine/vehicle reengineering. Despite continued refinements in combustion engineering through the use of computerized ignition and timing, fuel properties have drawn recent attention for improvement. For example, to reduce wintertime CO, several oxygenates (including ethers and alcohols) have be formulated into fuels both to reduce cold-start emissions and enhance overall combustion. Perhaps the most prominent of the ethers is MTBE (methyl

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(A)

(B) Figure 28-3. Reduction in ambient particulate sulfate and nitrate concentrations between 1989 and 2004 in the eastern half of the United States (from CASTNet monitoring data). Sulfates and nitrates arising from industrial centers of the Midwest contribute to acid rain deposition (see Fig. 28-11). Sulfates are readily dispersed by toward the eastern half of the country. Nitrates arise from the industrial centers as well as metropolitan areas and show both a local and dispersed pattern. (Adapted from Clean Air Status and Trends Network—http://www.epa.gov/castnet/mapconc e.html).

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Figure 28-4. NRC risk assessment paradigm. Components of Risk Assessment within the left circle provide data to development of Risk Management as depicted in the right circle, modified to include an “Accountability” component as a means to address Air Quality Management impacts on the process risk reduction (National Research Council, 1983).

tertiary butyl ether), which became a controversial additive in the early 1990s, arising in part from odor and reports of asthma-like reactions by some individuals during auto refueling at service stations. Today, the controversy has taken an unexpected twist; MTBE has now been removed from fuel, not because health concerns associated with airborne exposure but rather due to leakage from service-station storage tanks into groundwater. Ironically, this prescribed remedy for an air problem has evolved into a new problem: groundwater contamination. This example illustrates the broad complexity of pollution control, measures that transcend engineering. Meanwhile, other fuel additives have been promoted or developed to boost octane ratings of fuels and/or improve engine performance and combustion (e.g., organic oxygenates, methylcyclopentadienyl manganese tricarbonyl (MMT), platinum compounds for diesel, etc.). These additives are being carefully reviewed under Title II because of concerns regarding the potential changes in combustion product reactivity or the introduction of metals into the environment, reminiscent of use lead in fuels from the 1930s to 1970s, when lead fuel additives were banned. Internationally, the magnitude and control of air pollution sources vary considerably, especially among developing nations, which often forgo concerns for health and welfare because of cost and the desire to achieve prosperity. Figure 28-2 illustrates the international variation in air pollution related mortality (outdoor and indoor) based on economic groupings. It is clear that there are wide differences reflecting economic imbalances—particularly prominent are the indoor particulate levels in developing nations where biomass combustion is used for heating and cooking. Likewise, these regions contain many of the megacities of the world with major air pollution problems. The political upheaval in Eastern Europe since 1990 has revealed the consequences of decades of uncontrolled in-

dustrial air pollution. While vast improvements are now becoming evident in this area, as industries are being modernized and emissions controlled, many Asian, African, and South American cities have virtually unchecked air pollution. Some nations as well as the World Health Organization (WHO) have adopted air quality standards as a rational basis for guiding control measures, but the lack of binding regulations and/or economic fortune has impeded significant controls and improvements (Lipfert, 1994). In addition to local socioeconomic and political concerns, emissions of air pollutants will, in all probability, spawn problems of “international pollution” as we enter the twenty-first century, when the impact of long-range transport of polluted air masses from one country to another fully matures as a global issue (Reuther, 2000). This was the subject of some controversy between Canada and the United States in the late 1980s and into the 1990s as a result of the air mass transport of acid sulfates from industrial centers of the Midwestern United States to southern Ontario. However, reduction in SO2 emissions has somewhat relieved the tension over the last several years (Fig. 28-3A). Improvement in NOx, also a product of stationary source fossil fuel burning, is also apparent (Fig. 28-3B).

ASSESSING RISKS ASSOCIATED WITH AIR POLLUTION “Risk Assessment” has become a formalized process, originally described in the landmark 1983 National Research Council Report, whereby toxicity, exposure, and dose-dependent outcome data can be systematically integrated to estimate risk to a population. Figure 28-4 provides a modified version of the paradigm of the NAS incorporating recent interest in providing evidence of “accountability” that the regulations indeed did have impacts. The health database

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Table 28-2 Strengths and Weaknesses of Disciplinary Approaches for Obtaining Health Information discipline

population

strengths

weaknesses

Epidemiology

Communities

Natural exposure

Difficult to quantify exposure Many covariates

Diseased groups

No extrapolation Isolates susceptibility trait Long-term, low-level effects Good exposure data Fewer covariates Focus on host traits Utilizes clinical evaluations Controlled exposures Few covariates Isolates susceptibility trait Cause–effect Maximum control Dose-response data Cause–effect Rapid data acquisition Mechanisms

Field/Panel groups

Clinical studies

Experimental Diseased subjects

Toxicology

Animals

In vitro systems

Minimal dose–response data Association vs. causation Usually short-term Volunteers Expensive Artificial exposures Acute effects only Hazards Volunteers Human extrapolation Realistic models of human disease? In vivo extrapolation

source: Data modified from Boubel et al., 1994.

for any air pollutant may comprise data from animal toxicology, controlled human studies, and/or epidemiology. But, because each of these research approaches has inherent strengths and limitations, an appropriate assessment of an air pollutant requires the careful integration and interpretation of data from all three methodologies. Thus, one should be aware of the attributes of each (Table 28-2). Epidemiologic studies reveal associations between exposure to a pollutant(s) and the health effect(s) in the community or population of interest. Because data are garnered directly under real-world exposure conditions and often involve large numbers of people, the data are of direct utility to regulators assessing pollutant impacts. With proper design and analysis, studies can explore either acute or long-term exposures and theoretically can examine trends in mortality and morbidity, accounting irreversible effects as well as responses in population subsets (i.e., sensitive groups). Why, then, is this approach to the study of air pollution not the exclusive choice of regulators in decision making? The problem is that it is difficult to control confounding personal variables in the population. Factors such as genetic diversity and lifestyle differences among individuals, and population mobility are difficult to control. Perhaps most problematic is the lack of adequate exposure data— especially on a personal basis. Exposure assessment is often one of the major weaknesses of an epidemiologic study, not only because of the difficulties of assessing exposure (as a measure of dose) to the pollutant of interest, but because it is difficult to segregate a single pollutant from correlated co-pollutants and other environmental influences, such as meteorology. Thus, only associations, and not causality, can be drawn between the broad-based exposure data and effects. Frequently, the effects are of mortality, hospitalizations, etc. Causal relationships are sometimes inferred in the presence of strong statistical significance, but such determinations are likely to be criticized. However, recent advances in exposure estimation and study design and analysis (e.g., time series) have allowed epidemiologists to examine relationships with greater confidence and specificity. These models limit the impact of covariates and longer timebased influences and thus allow epidemiologists to tease out effects

of short-term pollution not accessible formerly (Schwartz, 1991). Similarly, newer approaches that employ field studies—sometimes called panel studies—incorporate time-series design and multipleregression analyses of more focused and complete exposure data (ideally personal) and targeted clinical endpoints in the exposed population under study. The endpoints often derive from empirical human and animal studies and therefore have a priori conceptual ties. The advent of new genetic approaches for characterizing polymorphisms of potentially influential traits (e.g., GSTM1—glutathioneS-transferase M1—Tujague et al., 2006) opens the genomic door for assessing gene-environment interactions. These factors may well underlie much of human susceptibility to air pollution. Novel approaches such as this are evident in the most recent studies of PM air pollution (see below). Studies that involve controlled human exposures have been used extensively to evaluate the criteria air pollutants regulated by the USEPA. Because most people are exposed to these pollutants in their daily lives, human volunteers can be ethically exposed to them in a highly controlled fashion (with the exception of Pb, which has cumulative and irreversible effects). Exposures are conducted in a controlled environment (usually in a chamber or with a mask) and are generally of short or limited repeat durations, given assurances that all responses are reversible. Clearly, data of this type are very valuable in assessing potential human risk, because they are derived from the species of concern and are rooted in well-established clinical knowledge and experience. Suspected “susceptible or sensitive” individuals representing potential higher-risk groups can also be studied to better understand the breadth of response in the exposed public. However, clinical studies have several practical limitations. Ethical issues are involved in every aspect of a clinical test; potentially irreversible effects and carcinogenicity are also always of concern, along with the definition of an acceptable level of hyper-responsiveness in so-called sensitive individuals who volunteered to participate in the study. Likewise for any test subject, there are obvious restrictions on the invasiveness of biological procedures, although sophistication in medical

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technology has made accessible a large array of molecular biomarkers from peripheral blood and nasal, bronchial, and alveolar lavage fluids as well as biopsied cells from airway segments (Devlin et al., 1991; Salvi et al., 1999). As noted above, the advent of cutting-edge genomic and proteomic high-through-put technologies provide new tools to dissect human responses and their relationships to susceptibility. Obviously, the issue of cost, the limited numbers of subjects that can be practically evaluated, and the inability to address chronic exposure issues remain constraints on human testing. Where partnerships with animal toxicology studies have been established, studies in laboratory animal species can sometimes provide ethical justification for at least limited direct human exposure to address critical questions. Analogously, in vitro studies in both human and animal cell and tissue systems, often augmented with similar genomic tools, allow the elucidation of mechanisms of toxicity. These basic biological responses inform extrapolation models that link animal data to humans, and they support the feasibility and prescribe some of the ethical limitations of human study with some toxic air pollutants (see below). Animal toxicology is frequently used to predict or corroborate suspected effects in humans. In the absence of human data, animal toxicology constitutes the essential first step of risk assessment: hazard identification. Animal toxicology is often required before any controlled human exposure can be conducted. It is particularly useful in elucidating pathogenic mechanisms involved in toxic injury or disease, providing basic knowledge that is critical to extrapolating databases across species, to estimating uncertainties, and determining the relevance of information to humans. Knowledge of the toxic mechanism(s) provides the underpinnings to the “plausibility” of findings in the human context and, under carefully defined and highly controlled circumstances, may allow quantitative estimates of risk to human populations. Animal toxicology studies have been used to investigate all of the criteria air pollutants and many of the HAPs as well. The strength of this discipline is that it can involve methods that are not practical in human studies and can provide more rapid turnaround of essential toxicity data under diverse exposure concentrations and durations. The minimization of uncontrolled variables (e.g., genetic and environmental) may be the greatest strength of the animal bioassay. The clear limitation of animal studies in human risk assessment lies in the unknowns that weaken the extrapolation of findings in animals to the day-to-day human life scenario. Ideally, a test animal is selected with knowledge that it responds in a manner similar to that of the human (homology). Qualitative extrapolation of homologous effects is not unusual with many toxic inhalants, but quantitative extrapolation is frequently clouded by uncertainties of the relative sensitivity of the animal or specific target tissue compared with that of the human. Uncertainties about the target tissue dose also loom large, constituting the first obstacle to quantitative extrapolation (see below). With respect to the target tissue dose, however, most animal toxicologists make every effort to keep exposure concentrations at 5- to 10-fold that of the anticipated human exposure until appropriate dosimetric data can be ascertained. An often overlooked issue is that the dose to the target (lung region) for the test animal is less than that of the human under similar exposure conditions – especially when exposures are conducted during dormancy for the animals (Wichers et al., 2006). Most human studies involve exercise exposure paradigms. Additionally, higher doses may be needed to achieve a group response among a limited pool of genetically similar animals (maybe 6–10) to represent a large population effect, where perhaps only a few of hundreds or thousands may actually be

responsive if analyzed separately. Nevertheless, it must be appreciated that mechanisms may well differ at different dose levels and some responses may be misleading at the higher dose levels. Despite these limitations, however, animal studies have provided the largest database on a wide range of air toxicants and have proven utility in predicting human adverse responses to chemicals. To be effective, any health assessment should consider the strengths and weaknesses of the approaches selected to estimate actual toxic risk. In the larger picture, other scientific disciplines can be highly valuable to a more accurate assessment of the impact of air pollution on society. The atmospheric sciences (including the chemical and physical sciences) provide insight into actual exposures by characterizing what is in the air. Better pollutant characterization, linked to exposure assessment, can only strengthen epidemiological outcome associations. Similarly, these data support toxicity evaluations based on biological test systems. In the latter case, more controlled exposures provide better insights into biological outcomes, especially with defined pollutant physicochemical attributes and interactions. Recreating realistic exposure environments to the extent possible is invaluable to developing models to estimate human risk. Lastly, data derived from studies of botanical responses are now appreciated more than ever. Not only are commercial and native vegetation affected by pollution but some plant species are being exploited as sensitive “sentinels,” warning of the impacts of pollution on both human and environmental receptors. When considered collectively, economists can inform regulators and the public at large of the cumulative impact and adversity of pollution on our quality and standard of living (Maddison and Pierce, 1999). Interestingly, some basic mechanisms (e.g., the involvement of antioxidants) between plants and animals have remarkable parallels.

Animal-to-Human Extrapolation: Issues and Mitigating Factors The utility and value of animal toxicology is highly dependent on the ability to extrapolate or relate empirical findings to real-world scenarios. Several factors of study design play into the process of extrapolation (e.g., exposure concentration, duration, and patterns, etc.), but most important is the selection of the animal species that will serve as the toxicological model. Therefore, this selection should involve more than considerations of cost and convenience. Whenever possible, effects that are homologous and involve the same mode of action between the study species and the human should guide the decision of the most appropriate test species. For example, if upper airway irritant responses like bronchoconstriction are anticipated (e.g., SO2 or formaldehyde), then the guinea pig, with its humanlike labile and reactive airway reflexes, should be selected over the rat, which is not particularly responsive in this regard. However, if the underlying molecular events in tissue remodeling are of interest, the rat might better serve as model because of cellular mechanistic parallels with human tissue responses. In part, the availability of probes to aid in such studies would factor into the selection of the rat as well. As strains of rats differ in their neutrophilic responsiveness to deep lung inhalants (e.g., O3 ), contrasting the mode of action may be more revealing and support their selection for study (Costa et al., 1985). Other innate differences in sensitivity among species may also relate to differences in lung structure, specific regionality of cell metabolism, genetic polymorphisms, or antioxidant defenses (Paige and Plopper, 1999; Slade et al., 1985). Thus, ideally, when such nuances are unclear or unknown, the replication of responses in multiple species builds confidence in the finding as being

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Figure 28-5. Theoretical (normalized to the concentration in inspired air) uptake curves for the reactive gas ozone in a resting/exercising human and a rat (A). Likewise, the percent deposition in the airways of a 0.6 μm insoluble particle in the respiratory tracts of a resting/exercising human (B) and rat (C). Eight percent inspired CO2 in the rat augments ventilation up to threefold. Airway generation refers to that airway branch numbered from the trachea (0). [Panel (A) is from Overton and Miller, 1987, and panels (B) and (C) are from Martonen et al., 1992. Reproduced with permission.]

homologous or having species-conserved modes of action that are relevant to the human. An essential, but often overlooked, part of extrapolating responses from species to species is an accurate assessment of the relative dosimetry of the pollutant along the respiratory tract. Significant advances in studies of the distribution of gaseous and particulate pollutants have been made through the use of empirical and mathematical models, the latter of which incorporate parameters of respiratory anatomy and physiology, aerodynamics, and physical chemistry into predictions of deposition and retention. Empirical

models combined with theoretical models aid in relating animal toxicity data to humans and help refine the study of injury mechanisms with better estimates of the target dose. Figures 28-5A and B illustrate the application of such an approach to the reactive gas O3 and insoluble 0.6-μm spherical particles, respectively, as each is distributed along the respiratory tract of humans and rats. Anatomic differences between the species clearly affect the deposition of both gases and particles, but the qualitative and to a large extent quantitative similarities in deposition profiles are noticeable. This is not surprising if one argues teleologically that the lungs of each species

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evolved with similar functional demands (i.e., O2 –CO2 exchange, blood acid/base balance), mechanical impediments, and environmental stresses. One needs only a cursory review of the comparative lung physiology literature to appreciate the allometric consistency of the mammalian respiratory tract to meet the challenge of breathing air. This design coherency has provided the fundamental rationale for the use of animal models for the study of air pollutants. Susceptible subpopulations who may show exaggerated responsiveness to pollutants merit special mention. The existence of hyperresponsive individuals and groups is well accepted among those who conduct air pollution health assessments, but little is actually known about the host traits that make certain individuals responsive. This appreciation for sensitive populations is specifically noted in the CAA, where their protection is mandated in the promulgation of NAAQS. There are some definable subgroups that are considered inherently more susceptible, including children, the elderly, and those with a preexisting disease (e.g., asthma, cardiovascular disease, lung disease). The importance of susceptibility in air pollutant responses is gaining more and more attention as test subject responses that were once considered “outliers” may well be evidence of unusual responsiveness. In some cases susceptibility may simply reflect differences in dosimetry. Children who spend more time outdoors than adults, who are more active, and who have basal ventilation rates that exceed adults on a volume to body weight ratio, may experience overall greater dose to the lungs. Certainly, rapidly growing tissues may also factor in and may have contributed, perhaps with dosimetry factors, to recent findings that polluted urban air retards lung growth (Gauderman et al., 2004). Similarly, adult humans and animals with obstructive airway disease, for example, may have “hotspots” of particle deposition in the airways that exceed normal local tissue doses manyfold (Kim and Kang, 1997; Sweeney et al., 1995). Another often underappreciated aspect of susceptibility relates to the loss of functional reserve or compensation due to age or disease, perhaps altering a response threshold or impairing normal homeostasis or recovery. As already noted, there is also growing interest in gene-environment interactions where genetic differences determine responsiveness. In humans, GSTM1 polymorphisms affect ∼50% of the population and thus, may in theory undermine antioxidant defenses in some individuals (McCunney, 2005). In the end, susceptibility may be due to differences in dose, sensitivity, and/or compensation. Clearly, outcomes may be similar, but their underlying causes may be multifactoral. Because the study of susceptibility in compromised human subjects is limited, ethically, studies must confined to subjects of only modest suspected risk (e.g., mild asthmatics). However, inroads have been made in recent years, in part because of more thorough pre-study assessments of potential risk factors, allowing researchers to design studies that need not carry undue risk. Additionally, the development of more appropriate animal models of disease or dysfunction provides a useful adjunct to explore susceptibility factors prior to study in humans and in more depth. Hence, studies in animals and human subjects are now being better coordinated, to investigate specific questions regarding the roles of diet (e.g., antioxidant content), exercise (as it relates to dosimetry), age, gender, and race, as well as disease associated frailty. The goal is to elucidate patterns or common factors that may inform potential intervention or mitigation strategies as well as basic information to reduce the uncertainties regarding risk (Kodavanti et al., 1998). Recent advances in molecular biology have provided tools to assess traits in animal models that are under the control of identifiable genes that are homologous to humans. Natural variants in

mouse genetics and specially bioengineered transgenic and knockout strains (and in some cases rats), are now widely used to address mechanistic as well as risk hypotheses regarding responses to air pollutants. These new biological tools hold great promise in better understanding responses and establishing gene-environment interactions that may underlie variation in human responsiveness. Transgenic strains can be devised to express desired traits derived from humans as well as other animals, while knockout models can be made devoid of specific traits to isolate the impact of that trait on responsivity to a toxic challenge. These animal models add to the availability of natural mutants that have been inbred historically to purify a desired genotype to achieve a specific phenotype (Ho, 1994; Glasser et al., 1994). Current technology can also target genes for specific expression in the lung (e.g., linked to surfactant protein C), and in some cases control genes can be provided with which an investigator can switch the gene of interest on or off using a pharmacologic or chemical prechallenge. Such advances allow the dissection of underlying mechanisms under very controlled scenarios and avoid the problems of having a gene be inappropriately active or inactive through all life stages (Kistner et al., 1996). To date, the emphasis of studies using these genetically modified animal models have been on mechanisms associated with disease pathogenesis (Recio, 1995; Suga et al., 2000; Yoshida and Whitsett, 2006). Among the most popular uses of knockout and transgenic mice has been in the study of inflammatory cytokines and associated products in asthma, as the expression of many of these mediators are thought to be under the control of single genes (e.g., Kakuyama et al., 1999; Kuhn et al., 2000). Clearly these genetically modified mice are well suited for the study of mechanism of action where a specific mediator-based hypothesis can be tested as it relates to an impaired function, pathology, or altered inflammatory pattern. When these models are derived to exhibit a desired pathology or disease due to a genetic defect—for example involving lung structure or growth (e.g., emphysema or fibrosis), such that by adulthood the animal exhibits the disease—the model may serve as a surrogate of the human condition (e.g., O’Donnell et al., 1999). Analogous mouse models (e.g., ApoE −/− ) are currently being used to assess atherosclerosis and cardiac disease in long term air pollution studies (Sun et al., 2005). The use or genetically modified animal models in air pollution research has lagged behind that of basic science and toxicology in general. The reasons for this are unclear and may relate to the difficulties in incorporating such data into conventional riskassessment paradigms. However, with recent interest in susceptible groups, there has been a definitive upswing in the use of pharmacologically or naturally altered, as well as bioengineered animals (Kodavanti et al., 1998) to more closely link mechanistic profiles to basic human biology. Ozone has frequently been the test pollutant in these new studies, because more is known about O3 and its effects in humans than about any other air pollutant. Frequently, these studies address aspects of inflammation and antioxidant capacity relative to challenge by O3 and other oxidants (Johnston et al., 1999; Kleeberger et al., 2000). But with the current interest in PM health effects, these and other models are being redirected. Examples include: strain differences and acid coated PM (Ohtsuka et al., 2000); hypertransferrinemic mice and metal-rich PM (Ghio et al., 2000); and metallothionein-null mice and mercury vapor (Yoshida et al., 1999). Among rats, the Spontaneously Hypertensive Rat (SHR) has gained considerable popularity for use in studies of PM because of its resemblance to human hypertension with serum oxidant

CHAPTER 28

Figure 28-6. Emission trend for volatile organic compounds (VOC), nitrogen oxides (NOx ), sulfur dioxide (SO2 ), and particulate matter (PM < 10 μm) from 1900 (or when records began) to 1998. Note that since the passage of the Clean Air Act of 1970, most emissions have decreased or, in the case of nitrogen oxides, have leveled off (Reproduced from National Air Pollutant Emission Trends Report, 1998).

imbalances, heart disease, as well as its sensitivity to lung injury from inhalants (Kodavanti et al., 2005). The curious are directed to the rapidly evolving literature in this area of research.

Air Pollution: Sources and Personal Exposure In terms of tons of anthropogenic material emitted annually in the United States (as of 2002), five major air pollutants account for 98% of pollution: CO (112 thousand tons), SO2 (15.3 thousand tons), volatile organic compounds (VOCs; 16.5 thousand tons), PM (6.8 thousand tons), and NOx (21.1 thousand tons). The remainder consists of Pb, which is down >98% since 1973, when phase-out from gasoline began, and a myriad of other compounds considered under the category of HAPs. On a national basis, aggregate emissions since 1970 have been cut 48%, while at the same time the U.S. Gross National Product (GNP) has increased 164%. Energy consumption has increased 42%, and vehicle miles driven have increased 155% (National Air Quality and Emissions Trends Report, 2003). The changing profile of pollutant emissions since 1900 is reflected in Fig. 28-6. Since 1993, air quality has generally continued to improve with reductions in ambient SO2 being largest (−39%) but with O3 changing the least, if at all. Obviously, for any specific locality, air quality can vary depending on the emission profiles of local sources, geographic topography, and meteorology. In the vicinity of a smelter for example, SO2 , metals, and/or PM may dominate the pollutant profile, while a refinery air shed might be dominated by VOCs and other carbonaceous products. In suburban areas, where the automobile is the main source of pollution, CO, VOCs, and NO2 would prevail along with their primary photochemical product, O3 . NOx releases (including NO2 ) by stationary sources also contribute to the local O3 levels along with that derived secondarily from auto emissions. In all, about 146 million people in the United States live in counties that have violations of the NAAQS designed to minimize risks to the criteria pollutants. The Evolving Profile of Air Pollution Classically, air pollution has been distinguished on the basis of the chemical redox nature of its primary components. Dickens’s eighteenth-century “London’s

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particular,” was characterized by SO2 and smoke from incomplete combustion of coal accumulated during an inversion as a chilled, acidic fog. This acidic mix reacts with surfaces, corroding metal and eroding masonry, characteristic of reductive chemistry. Historically, reducing-type atmospheres have been associated with smelting and related combustion-based industries (as along the Meuse River in 1930 and Donora, Pennsylvania, in 1948) as well as large, coal-based urban centers such as London (1952) and New York (1962). In contrast, Los Angeles has always had a characteristically “oxidant-type” pollution consisting of NOx and many secondary photochemical oxidants, such as O3 , aldehydes, and electron-hungry hydrocarbon radicals. Los Angeles uniquely traps auto emissions by virtue of regional topography and summer meterology, while other areas, such as Atlanta, have stagnant summer air. The classic types of air pollution were implicitly seasonal. Reducing air pollution occurred during winter periods of oil and coal combustion and meteorological inversions, while the oxidant atmospheres occurred during the summertime, when sunlight is most intense and can catalyze reactions among the constituents of auto exhaust. Today the distinction between reducing and oxidant smogs is largely academic. Most urban areas have virtually eliminated smoky, sulfurous emissions but now have a proliferation of automobiles that contribute tons of oxidant precursors into the air. However, in the Midwest there remains heavy industry that continues to emit SO2 , albeit much less than decades ago. These emissions undergo complex cloud chemistry to form sulfate that disperses regionally – hence the term, regional haze. Metropolitan areas in the eastern half of the United States, now have atmospheres comprising regional reducing pollutants as well as local oxidant pollutants. Sulfates may still predominate over nitrates in the air, in contrast to the Southwest United States, but no longer is the Northeastern air pollution simply a sulfur-based problem. Not only has the composition of the haze changed, but the chemistry has affected it temporal patterns. Long extended periods of O3 now prevail rather than the prototypic spike patter of Los Angeles (although challenged by Houston for the number-one spot in 1999. Outside the U.S., however, many megacities remain plagued by the classic forms of air pollution. For example, uncontrolled industrial emissions surrounding cities like Beijing and the northern sectors of Mexico City are dominated by oil, coal, and industrial emissions, whereas southern Mexico City, Santiago, and Tokyo have substantially (but not so exclusively) automobile-derived oxidant smogs. As noted above, air pollution is a worldwide problem, where the estimate of people exposed to O3 at potentially harmful levels exceeds 480 million (Schwela, 1996), with WHO estimates of PM-related mortality at 500 thousand per year.

Indoor Versus Outdoor Air Pollution People in the United States (and in most industrialized nations) spend in excess of 80% of their time indoors at work, at school, and at home or between these places in an automobile (Robinson and Nelson, 1995). Generally, the time spent indoors is disproportionately higher for adults, who have relatively less time to participate in outdoor activities, especially during the day, when outdoor pollutants are usually at their highest levels. Children and outdoor workers, by contrast, are much more likely to encounter outdoor air pollution at its worst; in fact, because of the relatively high activity levels of these subgroups compared with inactive office workers, their lungs may incur a considerably larger dose of any given pollutant. Thus, whereas it is important to characterize and track pollution levels in outdoor air, the most

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Figure 28-7. Illustration of contributors to the total personal exposure paradigm showing how these indoor and outdoor factors interact.

appropriate measure for exposure should involve a paradigm that addresses the total personal exposure of the individual or group of concern, and taken one step further, also dose to the lungs. However, defining typical paradigms of personal exposure can be extremely difficult, as personal monitoring is tedious, expensive, and complex. Outdoor pollutants can be encountered, with total exposure being influence by the personal dynamics and lifestyle of the individual. Individual monitoring is often expensive and difficult, so whenever possible, groups of people are monitored to develop models to estimate more specific exposure values. The indoor environment has gained appreciation as a major contributor to total personal exposure. The energy crisis of the 1970s spurred efforts to increase home and building insulation, reduce infiltration of outside air, and minimize energy consumption. At the same time, indoor sources of air contaminants have been on the rise from household products and furnishings, which—when combined with poorly ventilated heating systems and overall reductions in air-exchange rates—give rise to potentially unhealthy indoor air environments. As people began to notice patterns of odors, microbiologic growth, and even ill health, measures of indoor air became a significant part of environmental assessment. Personal exposure has, therefore, come to include the myriad of potential sources, both outdoors and indoors. It is clear now that indoor air can at times be more complex than outdoor air. The national monitoring network for the criteria pollutants has been shown to reflect human exposure reasonably well for some pollutants. Indeed, outdoor air permeates the indoor environment in spite of the reduced air exchange in most buildings. However, many variables determine how well components of the outdoor air infiltrate. The current evidence suggests that the average insulated home has about one air change per hour, resulting in indoor concentrations of pollutants that range from 30 to 80% of those outdoors. For nonreactive gases (e.g., CO), there could likely be nearly a 1:1 indoor/outdoor ratio in the absence of a “sink” for that gas;

the ratio for fine PM (