Selenium: Its Molecular Biology and Role in Human Health [2nd ed.] 0387338268, 9780387338262

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SELENIUM Its Molecular Biology and Role in Human Health, Second Edition

SELENIUM Its Molecular Biology and Role in Human Health, Second Edition

Edited by Dolph L. Hatfield National Cancer Institute, USA Maria J. Berry University of Hawaii, USA and Vadim N. Gladyshev University of Nebraska, USA

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rineer Spri

Library of Congress Control Number: 2006924112 lSBN-10; 0-387-33826-8 ISBN-13: 978-0-387-33826-2

e-ISBN-!0: 0-387-33827-6

Printed on acid-free paper.

© 2006 Springer Science+Business Media, LLC. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science-t-Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed in the United States of America. 9 8 7 6 5 4 3 2 1 springer.com

TABLE OF CONTENTS Contributors

xi

Foreword Raymond F. Burk

xvii

Preface

xxi

Dolph L. Hatfield, Maria J. Berry and Vadim N. Gladyshev Acknowledgements

xxiii

Chapter 1 Selenium: A historical perspective James E. Oldfield

1

Part I. Biosynthesis of selenocysteine and its incorporation into protein Chapter 2 Selenium metabolism in prokaryotes August Bock, Michael Rother, Marc Leibundgut and Nenad Ban

9

Chapter 3 Mammalian and other eukaryotic selenocysteine tRNAs 29 Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Robert Irons, Nianxin Zhong, Dolph L. Hatfield, Byeong Jae Lee, Alexey V. Lobanov and Vadim N. Gladyshev Chapter 4 Evolution of selenocysteine decoding and the key role of selenophosphate synthetase in the pathway of selenium utilization 39 Gustavo Salinas, Hector Romero, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield and Vadim N. Gladyshev Chapter 5 SECIS RNAs and K-turn binding proteins. A survey of evolutionary conserved RNA and protein motifs Christine Allmang and Alain Krol

51

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Selenium: Its molecular biology and role in human health

Chapter 6 SECIS binding proteins and eukaryotic selenoprotein synthesis Donna M. Driscoll and Paul R. Copeland Chapter 7 The importance of subcellular localization of SBP2 and EFsec for selenoprotein synthesis Peter R. Hoffmann and Maria J. Berry Chapter 8 Selenocysteine biosynthesis and incorporation may require supramolecular complexes Andrea L. Small-Howard and Maria J. Berry

63

73

83

Part II. Selenium-containing proteins Chapter 9 Selenoproteins and selenoproteomes Vadim N. Gladyshev

99

Chapter 10 Deletion of selenoprotein P gene in the mouse Raymond F. Burk, Gary E. Olsen and Kristina E. Hill

Ill

Chapter 11 Selenium and methionine sulfoxide reduction Hwa-Young Kim and Vadim N. Gladyshev

123

Chapter 12 Selenoprotein W in development and oxidative stress Chrissa Kioussi and Philip D. Whanger

135

Chapter 13 The 15-kDa selenoprotein (SeplS): functional analysis and role in cancer 141 Vyacheslav M. Labunskyy, Vadim N. Gladyshev and Dolph L. Hatfield Chapter 14 Regulation of glutathione peroxidase-1 expression Roger A. Sunde

149

Table of Contents

Chapter 15 Selenoproteins of the glutathione system Leopold Flohe and Regina Brigelius-Flohe Chapter 16 New roles of glutathione peroxidase-1 in oxidative stress and diabetes Xin Gen Lei and Wen-Hsing Cheng Chapter 17 Selenoproteins of the thioredoxin system Arne Holmgren Chapter 18 Mitochrondrial and cytosolic tliioredoxin reductase loiocliout mice Marcus Conrad, Georg W. Bornkamm and Marcus Brielmeier

vii

161

173

183

195

Chapter 19 Selenium, deiodinases and endocrine function Antonio C. Bianco and P. Reed Larsen

207

Chapter 20 Biotechnology of selenium Linda Johansson and Elias S.J. Arner

221

Part III. Selenium and human health Chapter 21 Selenium, selenoproteins and brain function Ulrich Schweizer and Lutz Schomberg

233

Chapter 22 Selenium as a cancer preventive agent Gerald F. Combs, Jr. and Junxuan Lii

249

Chapter 23 Peering down the kaleidoscope of thiol proteomics and unfolded protein response in studying the anticancer action of selenium 265 Ke Zu, Yue Wu, Young-Mee Park and Clement Ip

viii

Selenium: Its molecular biology and role in human health

Chapter 24 Genetic variation among selenoprotein genes and cancer Alan M. Diamond and Rhonda L. Brown

277

Chapter 25 Selenium and viral infections MelindaA. Beck

287

Chapter 26 Role of selenium in HIV/AIDS Marianna K. Baum and Adriana Campa

299

Chapter 27 Effects of selenium on immunity and aging Roderick C. McKenzie, Geoffrey J. Becket and John R. Arthur

311

Chapter 28 Selenium and male reproduction 323 Matilde Maiorino, Antonella Roveri, Fulvio Ursini, Regina Brigelius-Flohe and Leopold Flohe Chapter 29 Mouse models for assessing the role of selenium in health and development 333 Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Nianxin Zhong, Dolph L Hatfield, Robert Irons, Cindy D. Davis, Byeong Jae Lee, Sergey V. Novoselov and Vadim N. Gladyshev Chapter 30 Drosophila as a tool for studying selenium metabolism and role of selenoproteins Cristina Pallares, Florenci Serras and Montserrat Corominas Chapter 31 Selenoproteins in parasites Gustavo Salinas, Alexey V. Lobanov and Vadim N. Gladyshev Chapter 32 Incorporating 'omics' approaches to elucidate the role of selenium and selenoproteins in cancer prevention Cindy D. Davis and John A. Milner

343

355

367

Table of Contents

ix

Chapter 33 Selenium-induced apoptosis Ick Young Kim, Tae Soo Kim, Youn Wook Chung and Daewon Jeong

379

Chapter 34 Selenoprotein mimics Junqiu Liu and Guimin Luo

387

Chapter 35 Update of human dietary standards for selenium Orville A. Levander and Raymond F. Burk

399

Index

411

Contributors Christine Allmang

Maria J. Berry

Architecture et Reactivite de I'arN UPR 9002 du CNRS-Universite Louis Pasteur Institut de Biologie Moleculaire et Cellulaire 67084 Strasbourg, France

Department of Cell and Molecular Biology John A. Bums School of Medicine University of Hawaii at Manoa Honolulu, HI 96813, USA

Antonio C. Bianco Elias S. J. Arner Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden

John R. Arthur Division of Vascular Health Rowett Research Institute Bucksbum, Aberdeen, Scotland AB219SB,UK

Nenad Ban Institute of Molecular Biology and Biophysics Swiss Federal Institute of Technology ETH Hfinggerberg, HPK Building CH-8093 Zurich, Switzerland

Marianna K. Baum

Thyroid Section, Division of Endocrinology Diabetes and Hypertension Department of Medicine Brigham and Women's Hospital and Harvard Medical School 77 Avenue Louis Pasteur Boston, MA 02115, USA

August Bdck Lehrstuhl flir Mikrobiologie der Universitat Munchen, D-80638 Munich, Germany

Georg W. Bornkamm Institute of Clinical Molecular Biology and Tumor Genetics GSF-Research Centre for Environment and Health 81377 Munich, Germany

Markus Brielmeier

Florida International University Stempel School of Public Health Department of Dietetics and Nutrition 11200 SW 8th Street Miami, FL 33199, USA

Department of Comparative Medicine GSF-Research Centre for Environment and Health 85764 Neuherberg, Germany

Melinda A. Beck

Regina Brigelius-Flohe

Department of Nutrition University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA

Department Biochemistry of Micronutrients German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) Arthur-Scheunert-Allee 114-116 D-14558 Nuthetal, Germany

Geoffrey J. Beckett Department of Clinical Biochemistry University of Edinburgh Combined Laboratories The Royal Infirmary of Edinburgh 51 Little France Cresdent Edinburgh, Scotland, EH 16 4SA, UK

Rhonda L. Brown Department of Human Nutrition University of Illinois at Chicago Chicago, IL 60612, USA

Xll

Selenium: Its molecular biology and role in human health

Raymond F. Burk

Paul R. Copeland

Division of Gastroenterology, Hepatology, and Nutrition Department of Medicine Vanderbilt University School of Medicine Nashville, TN 37232, USA

Department of Molecular Genetics Microbiology and Immunology UMDNJ - Robert Wood Johnson Medical School Piscataway, NJ 08854, USA

Bradley A. Carlson

Montserrat Corominas

Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Departament de Geneica Universitat de Barcelona, Diagonal 645 08028 Barcelona, Spain

Adriana Campa Florida International University Stempel School of Public Health Department of Dietetics and Nutrition 11200 SW 8th Street Miami, FL 33199, USA

Wen-Hsing Cheng Laboratory of Molecular Gerontology National Institute on Aging National Institutes of Health Bahimore, MD 21224, USA

Youn Wook Chung Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea

Gerald F. Combs, Jr. Grand Forks Human Nutrition Research Center, USDA-ARS Grand Forks, ND 58202, USA

Marcus Conrad Institute of Clinical Molecular Biology and Tumor Genetics GSF-Research Centre for Environment and Health 81377 Munich, Germany

Cindy D. Davis Nutritional Science Research Group Division of Cancer Prevention National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Alan M. Diamond Department of Human Nutrition University of Illinois at Chicago Chicago, IL 60612, USA

Donna M. DriscoU Department of Cell Biology Lemer Research Institute Cleveland Clinic Foundation Cleveland, OH 44195, USA

Leopold Flohe MOLISA GmbH Universitatsplatz 2 D-39106 Magdeburg, Germany

Vadim N. Gladyshev Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA

Dolph L. Hatfield Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Contributors

xiu

Kristina E. HiU

Hwa-Young Kim

Division of Gastroenterology, Hepatology, and Nutrition Department of Medicine Vanderbilt University School of Medicine Nashville, TN 37232, USA

Department of Biochemistry University of Nebraska Lincoln, Nebraska 68588, USA

Peter R. Hoffmann John A. Bums School of Medicine Department of Cell and Molecular Biology University of Hawaii at Manoa Honolulu, HI 96813, USA

Ick Young Kim Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea

Arne Holmgren Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden

Clement Ip Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA

Tae Soo Kim Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea

Chrissa Kioussi Robert Irons Nutritional Science Research Group Division of Cancer Prevention and Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Daewon Jeong BK2I HLS, Seoul National University 28 Yeonkun-Dong Chongno-Ku Seoul 110-749, Korea

Linda Johansson Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden

Department of Biochemistry and Biophysics Oregon State University Corvallis, OR 97331, USA

Alain Krol Architecture et Reactivite de I'arN UPR 9002 du CNRS-Universite Louis Pasteur Institut de Biologic Moleculaire et Cellulaire 67084 Strasbourg, France

Vyacheslav M. Labunskyy Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA

XIV

Selenium: Its molecular biology and role in human health

P. Reed Larsen

Junxuan Lii

Thyroid Section, Division of Endocrinology Diabetes and Hypertension Department of Medicine Brigham and Women's Hospital and Harvard Medical School 77 Avenue Louis Pasteur Boston, MA 02115, USA

Hormel Institute University of Minnesota Austin, MN 55912, USA

Guimin Luo Key Laboratory for Molecular Enzymology and Engineering Jilin University Changchun 130023, China

Byeong Jae Lee Laboratory of Molecular Genetics Institute of Molecular Biology and Genetics School of Biological Sciences Seoul National University Seoul 151-742, Korea

Matilde Maiorino

Xin Gen Lei

Roderick C. McKenzie

Department of Animal Science Cornell University Ithaca, NY 14853, USA

Marc Leibundgut Institute of Molecular Biology and Biophysics Swiss Federal Institute of Technology ETH Honggerberg, HPK Building CH-8093 Zflrich, Switzerland

Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy

Laboratory for Clinical and Molecular Virology Royal Dick Veterinary School University of Edinburgh Summerhall, Edinburgh EH9 IQH, UK

John A. Milner

Orville A. Levander

Nutritional Science Research Group Division of Cancer Prevention National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Beltsville Human Nutrition Research Center U. S. Department of Agriculture Agricultural Research Service Beltsville, MD 20705, USA

Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA

Junqiu Liu

James E. Oldfield

Key Laboratory for Supramolecular Structure and Materials Jilin University Changchun 130012, China

Alexey V. Lobanov Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA

Sergey V. Novoselov

Oregon State University Corvallis, OR 97331, USA

Gary £. Olson Division of Gastroenterology, Hepatology, and Nutrition Department of Cell and Developmental Biology Vanderbilt University School of Medicine Nashville, TN 37232, USA

Contributors

XV

Cristina Pallar^s

Ulrich Schweizer

Departament de Geneica Universitat de Barcelona Diagonal 645 08028 Barcelona, Spain

Neurobiology of Selenium Neuroscience Research Center and Institute for Experimental Endocrinology Charit^-Universitatsmedizin Berlin Charite Campus Mitte D-10117 Berlin, Germany

Young-Mee Park Department of Cellular Stress Biology Roswell Park Cancer Institute Buffalo, NY 14263, USA

Hector Romero Laboratorio de Organizacion y Evoluci6n del Genoma Dpto. de Biologia Celular y Molecular Instituto de Biologia Facultad de Ciencias Igua4225 Montevideo, CP 11400, Uruguay

Michael Rother Lehrstuhl fUr Mikrobiologie der Universitat Mflnchen D-80638 Munich, Germany

Antonella Roveri Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy

Aniruddlia Sengupta Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Florenci Serras Departament de Geneica Universitat de Barcelona Diagonal 645 08028 Barcelona, Spain

Rajeev Shrimali Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Andrea L Small-Howard Gustavo Salinas Cdtedra de Inmunologia Facultad de Quimica-Facultad de Ciencias Universidad de la Repiiblica Instituto de Higiene Avda. A. Navarro 3051 Montevideo, CP 11600, Uruguay

Lutz Sclioinburg Institute for Experimental Endocrinology Charit^-Universitatsmedizin Berlin Charity Campus Mitte D-10117 Berlin, Germany

Department of Cell and Molecular Biology John A. Bums School of Medicine University of Hawaii at Manoa Honolulu, HI 96813, USA

Roger A. Sunde 1415 Linden Drive University of Wisconsin Madison, WI53705, USA

Fulvio Ursini Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy

xvi

Selenium: Its molecular biology and role in human health

Philip D. Whanger Department of Environmental and Molecular Toxicology Oregon State University Corvallis, OR 97331, USA

YueWu Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA

Xue-Ming Xu Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Min-Hyuk Yoo Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

Nianxin Zhong Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA

KeZu Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA

Foreword The discovery of selenoproteins in 1973 was the starting point for today's flourishing selenium field [1,2]. It provided evidence that selenium had biochemical functions that could account for its nutritional effects [3,4]. Further, it opened the selenium field to investigation by the methods of biochemistry, which led to the identification of several more selenoproteins and showed that selenocysteine was the form of the element in animal selenoproteins and in most bacterial ones. Although noteworthy efforts were made to uncover the mechanism of selenocysteine and selenoprotein synthesis using biochemical methods, the problem yielded only when attacked with the methods of molecular biology [5,6]. The bacterial mechanism was characterized first; characterization of the animal mechanism is a work in progress. It is interesting to note that the only genes that are devoted to selenium metabolism are those that support selenoprotein synthesis and selenocysteine catabolism. Consequently, it seems likely that competition for selenium between selenoprotein synthesis and the production of selenium excretory metabolites [7] controls wholebody selenium homeostasis. The physiological functions of selenium derive fi-om the catalytic and physical properties of selenoproteins. Selenoproteins such as the glutathione peroxidases and the thioredoxin reductases have redox activities that allow them to serve in oxidant defense. The deiodinases use their redox activities to activate and inactivate thyroid hormones. From these two examples, it can be seen that selenoprotein functions are diverse while having in common a redox mechanism. Although a few of the biological functions of selenium have been identified, many have not. Application of bioinformatics techniques to genomic databases has identified 25 genes for selenoproteins in the human genome [8]. Most of the proteins represented by those genes have not been characterized to the point where their functions can be assessed. Thus, one of the major challenges in selenium research is to characterize all the selenoproteins so that their biological activities can be determined. The ultimate goal of selenium research is to improve human health. Veterinary and animal science investigators had already demonstrated that nutritional selenium deficiency occurred in animals fed plants firom areas with low soil selenium availability when, in 1979, Chinese researchers reported the existence of a selenium-responsive disease in such an area. Their study showed convincingly that the occurrence of Keshan disease, a childhood cardiomyopathy, could be prevented by selenium supplementation [9]. Although several other diseases have been postulated to be selenium deficiency conditions, studies to prove those claims have not appeared. Thus,

xviii

Selenium: Its molecular biology and role in human health

Keshan disease, which has almost disappeared from China as economic conditions have improved, remains the extreme example of pathology that can occur in selenium deficient human beings. While selenium deficiency severe enough to allow the occurrence of Keshan disease is rare, people in many areas of the world have selenium intakes that are not sufficient to allow full expression of all selenoproteins. New Zealand and many countries in Europe fall into this category. In response to learning that its selenium status was low, Finland chose in 1985 to add selenium to its fertilizer. It has thereby become a laboratory for studying the effects of supplementing a population with selenium. The selenium status of Firms rapidly became comparable to that of North Americans but without discemable effects on the incidences of major diseases [10]. This type of study without a control population would not be expected to detect subtle health effects or uncommon ones such as altered responses to drugs: so the question of whether full expression of selenoproteins is needed for optimum health must remain open. This issue needs attention from clinical investigators because of the large number of people affected and the implications it has for setting official dietary requirements for selenium. More directly related to basic selenium research, mutations and polymorphisms of selenoprotein genes and of genes involved in selenoprotein synthesis can cause human disease. An example of this is the congenital muscle disease that results from mutation of the gene for selenoprotein N, one of the selenoproteins of unknown function. Perhaps elucidation of the function of selenoprotein N will suggest a treatment for the muscle disease. Phenotypes of mice with deletion of a selenoprotein might be instructive in this respect. For example, deletion of selenoprotein P causes neurological dysfunction that can be prevented by selenium supplements above the nutritional requirement. If an analogous human condition were found, selenium supplements might be efficacious in its treatment. Examples of animal research that support understanding of human diseases stimulate basic selenium research. In addition to research on the physiological functions of selenium, considerable enthusiasm has been generated for studying the effects of pharmacological doses of the element. The results of numerous animal studies and limited human trials have suggested that administration of pharmacological doses of selenium can prevent some kinds of cancer [11]. Additional trials are underway to test this hypothesis. If such a chemopreventive effect of selenium can be proven, it would not likely be linked to the selenoproteins because the subjects in the trials were not selenium deficient before supplementation was started. This means that the selenoproteins would have been at their optimal levels initially and that selenium supplements would not have been expected to affect them. Other

Foreword

xix

metabolic effects of high selenium intake have been noted, however, and might account for its effects on cancer development. It will be important for public health reasons to determine whether selenium is an effective chemopreventive agent in human beings and, if it is, to determine the safety of pharmacological doses of selenium. Many tasks remain in the selenium field. Additional characterization of individual selenoproteins and elucidation of the mechanism of selenoprotein synthesis are needed to facilitate identification of pathological conditions involving selenium. Clinical studies are needed to determine the selenium intake needed to ensure full expression of all selenoproteins and to assess the health implications of selenium intakes that do not allow full expression of all selenoproteins. And, finally, whether selenium is efficacious as a chemopreventive agent needs to be determined. References 1. 2.

DC Turner, TC Stadtman 1973 Arch Biochem Biophys 154:366 JT Rotruck, AL Pope, HE Ganther, AB Swanson, D Hafeman WG Hoekstra 1973 Science 179:588 3. KE McCoy, PH Weswig 1969 JNutr 98:383 4. K Schwarz, CM Foltz 1957 J Amer Chem Soc 79:3292 5. A Bock, K Forchhammer, J Heider, C Baron 1991 Trends Biochem Sci 16:463 6. I Chambers, J Frampton, P Goldfarb, N Affara, W McBain, PR Harrison 1986 EMBO J 5:1221 7. Y Kobayashi, Y Ogra, K Ishiwata, H Takayama, N Aimi, KT Suzuki 2002 Proc Natl Acad Sci U S A 99: 15932 8. GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 Science 300:1439 9. Keshan Disease Research Group 1979 Chinese Medical Journal 92:471 10. M Eurola, G Alfthan, A Aro, P Ekholm, V Hietaniemi, H Rainio, R Rankanen, E-R Venalainen 2003 Agrifood Research Reports 36 Results of the Finnish selenium monitoring program MTT Agrifood Research Finland, FIN-31600 Jokioinen, Finland pp 42 11. LC Clark, GF Combs Jr, BW TumbuU, EH Slate, DK Chalker, J Chow, LS Davis, RA Glover, GF Graham, EG Gross, A Krongrad, JL Lesher, HK Park, BB Sanders, CL Smith, JR Taylor 1996 JAMA 276:1957

Raymond F. Burk

Preface Since the first edition of Selenium: Its Molecular Biology and Role in Human Health was published in 2001, many new insights into the biochemical, molecular, genetic and health aspects of this fascinating element have been elucidated. Several new human clinical trials have also been undertaken examining the role of selenium in protection against different cancers. For example, the National Cancer Institute initiated two new clinical trials involving selenium. One of these is called SELECT, Selenium and vitamin E Cancer Prevention Trial, and it involves examining the role of selenium and vitamin E in protecting against prostate cancer, with a goal of enrolling over 35,000 males in the study. The other trial involves examining the role of selenium in protection against lung cancer, a study incorporating 1960 individuals. The commitment of hundreds of millions of dollars to these trials for examining the role of selenium in protecting humans against different forms of cancer illustrates how highly important this element is regarded by the medical and scientific communities in health issues. What is of such significance to elucidating the role of selenium in health in these human clinical trials is that not only will the effect of selenium on prostate and lung cancers be assessed, but these trials will shed light on the role of many additional aspects of selenium in health such as aging, heart disease, viral inhibition and other forms of cancer including colon, liver and brain malignancies. Many exciting discoveries have occurred in the last five years which are described in the current edition. For example, the entire selenoprotein gene population, designated the selenoproteome, has been identified in humans and rodents. Furthermore, the various selenoproteins described in the last edition have been further characterized and their new features described. Numerous selenoprotein genes have been targeted for removal using standard or loxP-Cre technologies to further elucidate their functions in development and health. Selenoproteins have also been shown to be involved in different human genetic disorders. Many new and novel features have been uncovered on the biosynthesis of selenocysteine, the amino acid that contains selenium, and its incorporation into protein as the 21^' amino acid in the genetic code. Further studies on the various components involved in the biosynthesis of selenocysteine and its insertion into protein have determined that much of this vast selenoprotein machinery exists in supramolecular complexes. Finally, several mouse models that were specifically generated for examining the role of selenium and selenoproteins in health and development have been devised. The rapid expansion and many new discoveries in the selenium field in the last five years are reflected by the addition of many new chapters and a much longer current edition.

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Selenium: Its molecular biology and role in human health

The purpose of the new edition book is to inform the reader of these many new discoveries and to examine our present knowledge of the molecular biology of selenium, its incorporation into proteins as selenocysteine and the role that this element and selenium-containing proteins (selenoproteins) play in health. The book's emphasis is on our understanding of selenium metabolism in mammals and the role of this element in human health. The book begins with a brief history of selenium and how its face has changed through the years from one of a toxin and possible carcinogen to one of an essential micronutrient in the diets of humans and other animals. Indeed, selenium is now touted as an important cancer chemopreventative agent, as well as for its roles in inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, preventing heart disease and other cardiovascular and muscle disorders, slowing the aging process, and having roles in development, male reproduction and immune function. As more of the molecular biology of selenium is unraveled, we are understanding the manner in which this element does indeed have direct roles in each of these health issues. The present book, like the first edition, is divided into three parts with ten more chapters than the earlier edition. The chapters in Part I, which is entitled "Biosynthesis of selenocysteine and its incorporation into protein," define selenocysteine as the 21^' naturally occurring amino acid in the genetic code and describe how this amino acid is incorporated into protein. Interestingly, the inclusion of selenocysteine to the genetic code as its 21'' amino acid marks the first addition to the code since it was deciphered in the mid-1960s. Our current understanding of how selenoprotein expression is regulated and the nucleocytoplasmic shuttling of the selenocysteine biosynthesis and insertion machinery in eukaryotes is also discussed in Part I. Part II is entitled "Selenium-containing proteins" and it discusses our current understanding of selenoproteins, primarily in higher eukaryotes. Part III is entitled "Selenium and human health" and it covers our current understanding of the role of selenium in various diseases, including cancer and heart disease, in HIV infection and AIDS, in male reproduction, and as an antiviral agent. The role of small molecular weight, selenium-containing compounds (selenocompounds) in human health and the dietary selenium requirements for humans are also discussed. In summary, this book provides an up-to-date review of much of the ongoing research in the selenium field. It provides a resource for scientists working in the selenium field, as well as for physicians, other scientists and students who wish to learn more about this fascinating micronutrient.

Dolph L. Hatfield, Maria J. Berry and Vadim N. Gladyshev

Acknowledgements The support and generous help of Bradley A, Carlson throughout the preparation of this book is gratefully acknowledged. The editors also wish to thank Sergey V. Novoselov for his help with the book cover.

Chapter 1. Selenium: A historical perspective James E. Oldfield Oregon State University, Corvallis, Oregon 97331, USA

Summary: The path followed in the biochemistry of selenium has taken some sharp turns during its development. At first, feared as a poisoner of livestock and later impugned as a carcinogen, selenium has about-faced and is now recognized as an essential micronutrient with anti-carcinogenic properties. While early studies on selenium have focused on the role of this trace element in animal physiology and studies with microorganisms, the field has matured to employ molecular biology to explain and employ the protective effects of selenium against a number of human maladies, including cancer and heart disease. The emphasis of this chapter is an examination of selenium's early history as a toxin, its later recognition as an essential micronutrient in the diet of mammals and its impact in the livestock industry that provided the foundations for the vast amount of the current basic and health research on this fascinating element. Even before it had been discovered and named, there were reports of conditions occurring in animals that, in retrospect, must have been caused by an excess of selenium. The Venetian explorer, Marco Polo, wrote of problems encountered by travelers in a mountainous region of what is now Shaan-Xi province in China [1]. He noted that when horses or other beasts of burden grazed on some indigenous plants, their hooves would split and fall off In the light of present knowledge, it would seem that these plants were "selenium accumulators" that concentrate selenium fi'om the soil to levels that are toxic to grazing animals. Then, several hundred years later, Madison [2] an army surgeon stationed at Fort Randall in the Nebraska territory, described a similar condition among dragoon remount horses that had been newly introduced to the area. K.W. Franke, who was a State Chemist at South Dakota State College, headed much of the definitive work on local toxic plants [3]. Actual proof of selenium's involvement in this toxicity problem came when workers in South Dakota identified it as the toxic principle in plants causing what was locally called "alkali disease" in cattle, on range lands of the north-central United States [4].

Selenium: Its molecular biology and role in human health The discovery of selenium, as an element, was made in 1817 by a Swedish chemist, Jons Jakob Berzelius, through what was, at that time, an elegant analytical process [5]. Berzelius was investigating the cause of illnesses among workers at a sulfuric acid manufacturing plant that occurred when copper pyrites from a local mine were used as the source of sulfur. He scraped a red deposit from the walls of the lead chambers in which the pyrites were processed, anticipating that it might contain tellurium, an element he had recently discovered. Tellurium was not present but he isolated another new element which he named selenium, after Selene, the Greek goddess of the moon. Taken together, these three early indications of selenium toxicity were certainly an inauspicious beginning for what was eventually to be recognized as an essential micronutrient. At that time, if anyone thought of selenium at all, and few did, it was as a toxic element. The earliest organized research effort with selenium, then, was directed toward means of avoiding, or coping with its toxicity. It was recognized that certain areas in the United States had seleniferous soils and this, together with the identification of selenium-accumulating plants, spelled trouble for animal agriculture operations. Ranchers learned to identify and remove accumulator plants, to dilute their livestock's forage feed with nonseleniferous materials, and to move their animals around in cycles which included some time on low-selenium forage grazing areas. Then, in 1957, research by a German scientist, Klaus Schwarz, working at the U.S. National Institutes of Health in Bethesda, changed forever the way selenium was assessed by both the scientific community and the general public. Schwarz had been working in Germany on studies of brewers' yeast as a protein source, during World War II, and he continued these studies when he came to America. He found, when he fed torula yeast, rather than brewers' yeast to rats, that they developed necrotic livers and he concluded that the brewers' yeast contained some essential nutrient that the torula yeast did not. He named the unknown substance "factor 3," since two other substances that alleviated liver necrosis had already been identified: vitamin E and (mistakenly) L-cysteine, which were known as factors 1 and 2. In 1957, Schwarz and Foltz armounced that they had fractionated factor 3 and found it to contain selenium [6]. Although its toxicity remained a real and difficult problem, it was now evident that, at lower dietary levels, selenium was harmless and, indeed, was quickly recognized as a dietary essential. The response to this discovery was immediate, and surprisingly extensive, as selenium deficiency was shown to be implicated in a number of animal diseases beyond the original liver necrosis. Studies in Oregon [7] showed that it was the cause of "white muscle disease," a myopathy that affected hundreds of calves and lambs each year in the central part of the state. Then, in quick succession, selenium deficiency was linked to other diseases of domestic animals and birds, including exudative diathesis and pancreatic

Selenium: A historical perspective degeneration in poultry, hepatosis dietetica in pigs and "ill-thrift" in cattle and sheep [8]. Of these, white muscle disease is the most widespread and has the greatest economic impact - involving not only calves and lambs but also deer, goats, horses, poultry and rabbits and occurring in all the major sheepproducing countries in the world [9]. Questions naturally arose about the biochemical function of selenium: how such small amounts of it could produce such profound biological reactions. These were answered, at least in part, by research carried out simultaneously in America and Germany. At the University of Wisconsin, Rotruck and associates [10] discovered selenium's presence in the enzyme, glutathione peroxidase, while Flohe in Germany showed the precise placement of selenium in the enzyme molecule [11]. It seemed that this enzymic involvement might be one way in which selenium could perform its beneficial metabolic functions and would explain how so little selenium could accomplish so much. Farmers and ranchers are often accused of being slow in accepting and applying research results relevant to their operations but this was certainly not the case with selenium supplementation. Its benefits were so dramatic that it soon became an accepted husbandry practice in areas of selenium deficiency, worldwide. Research, too, developed a number of methods by which selenium might be made available to animals, including feed fortification, injection, and with ruminant animals, an ingenious heavy pellet that would remain in the forestomach and gradually make selenium available for periods as long as a year. Selenium was also added to fertilizer mixes used on range and pasture land to improve the selenium status of forage plants grown thereon [12]. So, early biological research with selenium was stimulated by the animal industries, which in countries like New Zealand, were major contributors to the nation's economy. At first thought, the possibility of a selenium deficiency occurring among humans seemed remote on the grounds that the great diversity of the human diet would make an overall selenium deficiency unlikely. Cases of a human selenium deficiency did emerge, however, in some rural areas in China, where the people lived almost entirely on food substances produced on their ovra (selenium-deficient) land. This led to a cardiac myopathy that was first reported in Keshan county, of Heilongjiang province in northeastern China, and was called Keshan disease [13]. It is interesting to compare these symptoms with those of white muscle disease among animals - they have much in common. One of the fascinations of selenium research has been the abrupt changes in direction that have taken place over the years as knowledge of selenium's functions developed. So it was in the latter years of the 20th century when research interest in selenium switched from animals to molecular research and an emphasis on the role of selenium in human health.. This change was

Selenium: Its molecular biology and role in human health fueled by observations that, in addition to its now accepted nutrient function, selenium could also exert beneficial effects on human health at dietary levels somewhat higher than those required for its purely-nutritional activity. In Finland, governmental agencies became concerned about the long-term effects of low-selenium diets in their country on the health of the human population. They authorized the addition of selenium, as selenate, to fertilizers applied in the production of animal and human foods and have shown that this process effectively raises the selenium content of the Finnish diet to levels consistent with good nutrition and human health. They have carefully monitored the situation since Se-fertilization began, in 1984 [14] and we can be grateful to the Finnish scientists for providing much useful information on this type of application of selenium. The Finnish experience, too, has led to studies of the selenium status of other populations where dietary levels have been decreasing, over time [15]. The health-preserving activities of selenium, at about double the dietary levels recommended by the U.S. National Research Council, have been reviewed in detail by Combs [16]. It is interesting that these studies drew on the findings of Clark and associates at the Arizona Cancer Center and this gives rise to another of selenium research's "about faces," since early research had proposed that selenium was a carcinogen [17]. The application of selenium supplementation of livestock feeds to overcome selenium deficiency was prohibited for a time by the U.S. Food and Drug Administration (FDA) because of concerns raised by studies in their own laboratories suggesting that selenium might be a carcinogen [18]. This ruling exasperated American livestock producers who pointed out that they were being denied application of research that their tax dollars had helped pay for, while their strong competitors in New Zealand and Australia were routinely applying selenium in diets of their livestock. This conflict was resolved in this country, and in fact, the use of selenium in livestock feeds has been estimated to have saved this industry hundreds of millions of dollars in preventing muscle disorders and numerous other anomalies including enhancing reproduction as discussed in detail by Combs and Combs [19]. So, to recapitulate, the trail of research with selenium has been a tortuous one, marked by sudden and sometimes dramatic changes in direction. Its discovery, by Berzelius, was serendipitous; he was expecting to find tellurium in the Swedish sulfuric acid vats, but instead, he isolated selenium. There was a corollary to this in the much later studies of its health-protecting properties. When the Oregon workers sought a cure against white muscle disease, they thought it would be vitamin E, which proved ineffective, but selenium worked. Most of the early research, done in the first half of the last century, focused on means of avoiding selenium's toxicity, but Schwarz's carefully controlled studies with yeast opened the door for investigation of its

Selenium: A historical perspective beneficial effects as a micronutrient. Commercial application of supplementary selenium in diets of farm animals was delayed for several years due to fear that it might be a carcinogen but then, in one if its most dramatic about-faces, selenium proved to be anti-carcinogenic. Interestingly, Clark's study aimed against skin cancer where selenium that proved ineffective, but it was found to have significant benefits with other types of cancer, including those of the prostate, lung and intestine/colon (see [17] and references therein). It is understandable, certainly, because of its dreaded consequences in human health that cancer should have received the major attention by investigators of this new area of selenium's activity. It is exciting, however, that it has been shown to be a useful strategy against a number of other human diseases, and the Antioxidant Vitamins newsletter published by Hoffinan La Roche company listed 50 diseases against which selenium may play a protective role [20]. These include diseases of the heart, long recognized as major killers of the world's human populations [21,22] and ADDS, which has been called the "greatest catastrophe in human history" [23]. But most importantly, these earlier studies showing the importance of selenium in the diets of laboratory animals and livestock and the finding of selenium in protein as the amino acid selenocysteine in the 1970s have provided the foundations for the remarkable transformation that this field witnessed in the last 20 years. Indeed, the basic research described in this edition specifies selenium as a preventative agent in cancer, heart disease and other cardiovascular and muscle disorders, as an inhibitor of viral expression and as a factor delaying the aging process and the progression of AIDS in HIV positive patients. Furthermore, selenium is identified as,an essential element in mammalian development, male reproduction and immune function. These many health benefits now attributed to selenium highlight the serrated road fi-om a toxin to what may now be designated as a magic bullet. References 1.

2. 3. 4. 5. 6. 7. 8.

Polo, Marco. 1967. The Travels of Marco Polo Translated by EW Marsden and revised by T Wright pp 100-101 Everymans Library, London (Cited in C Reilly 1996 Selenium in Food and Health Chapman & Hall London p 3) TC Madison 1860 Statistical Report on the Sickness and Mortality in the Army of the United States RH Cooledge ed Ex Doc 52:37 KW Franke 1934 J Nutrition 8:597-608 AL Moxon 1937 Bull. 311, S. Dakota AgExp Sta 81 pp JJ Berzelius 1818 Serie 2 7:194 (Cited in C Reilly 1996 Selenium in Food and Health Chapman & Hall London p 2) K Schwarz, CM Foltz \951JAm Chem Soc 78:3292 OH Muth, JE Oldfield, LP Remmert, JR Schubert 1958 Science 128:1090 C Reilly 1996 Selenium in Food and Health Chapman & Hall ed London 338

Selenium: Its molecular biology and role in human health

9. E Wolf, V Kollonitsch, CH Kline 1963 Agr & Food Chem 11:355 10. JT Rotruck, AL Pope, HE Ganther, AB Swanson, DG Hafeman, WG Hoekstra 1973 Science 179:588 11. L Flohe, WA Gunzler, HH Schock 1973 FEES Letters 32:132 12. JE Oldfield 1997 Biomed & Environ 10:280 13. B Gu 1993 Chinese Med J 96:25\ 14. P Koivistoinen, K Huttunen 1986 Ann Clin Res 18:13 15. MP Rayman 2000 Lancet 356:233 16. GF Combs Jr 2001 Nutrition and Cancer 40:6 17. LC Clark, GF Combs Jr, BW Tumbull, EH Slate, D Alberts, D Abele, R Allison, J Bradshaw, D Chalker, J Chow, D Curtis, J Dalen, L Davis, R Deal, M Dellasega 1996 J Am Med Assoc 216:1957 18. AA Nelson, OG Fitzhugh, HO Calvery 1943 Cancer Res 3:230 19. GF Combs Jr, SB Combs 1986 The Role of Selenium in Nutrition Academic Press Inc New York 20. 1993 Antioxidant Vitamins Newsletter Hoffinan LaRoche Co New York 7:12 21. AFM Kardinaal, FJ Kok, L Kohlmeier, M Martin-Moreno, J Ringstad, J Gomez-Aracena, VP Mazaer, M Thamm, BC Martin, P Van'tVeer, JK Huttunea 1997 Am J Epidemiology 145:373 22. JT Salonen 1985 Trace Elements in Health and Disease H Bostrom, N Ljungstedt ed Almquist and Wiksell International Stockholm 172 23. HD Foster 2002 What Really Causes AIDS? Trafford Publishing Victoria, Canada 197

Parti

Biosynthesis of selenocysteine and its incorporation into protein

Chapter 2. Selenium metabolism in prokaryotes August Bock and Michael Rother Lehrstuhlfiir Mikrobiologie der Universitat Munchen, D-80638 Munich, Germany

Marc Leibundgut and Nenad Ban Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, ETH Honggerberg, HPK building, CH-8093 Zurich, Switzerland

Summary: The biosynthesis and specific incorporation of selenocysteine into protein requires the function of a UGA codon determining the position of selenocysteine insertion and a secondary/tertiary structure within the mRNA, designated the SECIS element, following the UGA at its 3'side in bacteria and located in the 3 'non-translated region in archaea. Biosynthesis of selenocysteine takes place on a unique tRNA species, tRNA^**", which is charged by seryl-tRNA synthetase and serves as an adaptor for the conversion of the seryl moiety into the selenocysteyl product by selenocysteine synthase. Monoselenophosphate, provided by selenophosphate synthetase, is the selenium donor. Selenocysteyl-tRNA^**^ is bound by the special translation factor SelB, which in bacteria via its Cterminal extension interacts with the apical part of the SECIS stem-loop structure. Crystallographic and NMR structural analyses of this extension from Moorella thermoacetica SelB, either free or complexed with the SECIS element, showed that it is made up of four winged helix domains from which only the C-terminal one interacts with the RNA ligand. Structure of the entire SelB molecule from Methanococcus maripaludis in the apo- and GDP/GTP bound forms revealed that it is a chimera between elongation factor Tu and initiation factors. Comparison of the structures in the GDP and GTP forms and modelling of the interactions between selenocysteyl-tRNA and SelB provided information on how SelB may discriminate tRNA^**^ from canonical tRNAs and may differentiate between the selenocysteyl moiety and the serylresidue of the precursor. A scenario for the major steps in the decoding process is postulated and arguments are given why the interaction of SelB with the mRNA is crucial. Reasons are also presented for the necessity of a balanced ratio of the components of the selenocysteine insertion apparatus and how it is regulated in E. coli via translational repression implicating a SECIS-like element located at the ultimate 5 'end ofselAB mRNA.

Selenium: Its molecular biology and role in human health

10

Introduction When bacteria are challenged with low molecular weight selenium compounds in the medium, they can process selenium in a nonspecific or a specific manner. The nonspecific metabolism rests on the chemical similarity between selenium and its neighbor element in the periodic table, sulfijr. When present above a critical concentration in Escherichia coli, i.e., at selenite concentrations higher than 1 ^M, selenium intrudes the sulfur pathways and is metabolized along the routes of sulfur metabolism [1,2] (Figure 1). Thus, selenium in the form of selenate is taken up by the sulfate transport system and reduced to selenide via the assimilatory sulfate reduction system. When offered as selenite, reduction appears to proceed chemically by interaction with thiol compounds like glutathione (see [3] for review).

Sulfate/Selenate

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Sulfate/Selenate '

Selenite

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Selenite . R-SH

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Sulfide/Selenide O-Ac-Ser

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i I

ucu uco

S/Se-Methionine

S/Se-Met-tRNA'^^t

Se-Cysteyl-tRNASe

Scicnoprotcins — » I Selenylated Proteins AUG

Figure 1. Scheme for the specific and nonspecific metabolism and incorporation of selenium into macromolecules. The specific pathway is highlighted in bold. mnm's^U is the abbreviation for 5-methylamino-methyl-2-thiouridine and mnm'se^U for 5-methylaminomethyl-2-selenouridine. 0-Ac-Ser: 0-acetylserine, [Se] designates the reactive selenium species used by the selenophosphate synthetase as a substrate for the synthesis of selenophosphate; its possible metabolic origin is indicated by dashed arrows (see Chapter 4).

Selenium metabolism in prokaryotes

11

The first organic selenium compound formed is free selenocysteine, which can be converted to selenocystathionine and eventually to selenomethionine. On the other hand, selenocysteine has been shown to be a substrate for cysteyl-tRNA synthetase, which forms selenocysteyl-tRNA'^^^ and in this way incorporates selenocysteine at cysteine positions in proteins [4-6]. The decision whether selenium is incorporated nonspecifically as either selenocysteine or selenomethionine, therefore, should be dependent on the relative catalytic efficiencies of cysteyl-tRNA synthetase and cystathionine synthetase for the substrate cysteine and its analog selenocysteine. Nonspecific incorporation into macromolecules is drastically reduced when the cysteine biosynthetic pathway is interrupted by mutations or when it is fully repressed [6]. When selenomethionine is provided in the medium, it is almost indiscriminately incorporated into protein in place of methionine. This replacement is frequently used in x-ray analysis of protein crystals by multiwavelength anomalous dispersion [7] or in NMR spectroscopy [8]. Selenomethionine as the major selenium compound has also been detected when bacteria were grown on excessive amounts of selenite [9,10]. Free selenocysteine, on the other hand, is highly toxic and therefore growth inhibitory. Its incorporation in place of cysteine requires an overexpression system like the promoter-polymerase system of phage T7 to circumvent toxicity [11,12]. The specific incorporation of selenocysteine, on the other hand, is effective at much lower concentrations of selenite in the medium. With the aid of a fdhF-lacZ fusion reporter gene, in which readthrough into lacZ is dependent on the availability of selenium (see below), saturation has already occurred by 0.1 |iM selenite [13]. Specific incorporation does not involve free, low molecular weight selenocysteine since the biosynthesis of the molecule takes place from a precursor amino acid esterified with tRNA. It should be emphasized that the capacity to synthesize selenoproteins by the specific pathway is not ubiquitous. Actually, it is absent in the majority of microorganisms [14]. In this chapter we will discuss the specific incorporation of selenocysteine by bacteria, mainly E. coli and by members of archaea. Identification of the components involved in selenocysteine biosynthesis and specific insertion rests to a considerable degree on the early work of several groups studying the anaerobic formate metabolism oiE. coli [15-20]. Genes had been analyzed which, when mutated, abolished the ability of E. coli to synthesize active isoenzymes of formate dehydrogenase known as formate dehydrogenase N and formate dehydrogenase H which couple formate oxidation to the reduction of nifrate or protons, respectively. Thus, some mechanism must have been affected in the mutants that is required for generating activity of both enzymes. The genes had been mapped on the

12

Selenium: Its molecular biology and role in human health

chromosome oiE. coli and some of them (fdhAfdhB andfdhC) turned out to be involved in selenium metabolism [21]. Merits also go to two technical developments, namely the establishment of a plate overlay technique for screening large numbers of colonies for formate dehydrogenase activity [17] and the set-up of a procedure for specific incorporation of radioactive selenium into selenopolypeptides [22]. With the aid of these techniques, it was easy to differentiate between specific and nonspecific incorporation (see Figure 1). Specific incorporation of selenocysteine by bacteria The first genes discovered to contain an in-fi-ame UGA codon directing selenocysteine insertion were gpx, coding for glutathione peroxidase fi"om mouse [23], and fdhF fi-om E. coli, coding for the selenopolypeptide of formate dehydrogenase H [24]. Whereas an amino acid sequence was available for glutathione peroxidase showing colinearity between the UGA in the mRNA and selenocysteine in the protein, this was not the case for the bacterial enzyme. Evidence was obtained, however, by leading truncations from the 3'end into the gene and showing that removal of the segment containing the UGA also abolished selenium incorporation into the truncated gene product. Definite proof for the cotranslational insertion was then provided by fusion of the /acZ reporter gene upstream and downstream of the UGA in fdhF and the demonstration that readthrough of the UGA required the presence of selenium in the medium [13]. Analysis of mutations that affected readthrough led to the identification of the genetic elements involved in selenium metabolism in E. coli [21]. After the discovery that UGA also directs selenocysteine insertion into proteins in archaea [25], and with the results of the bioinformatic analysis of whole genome sequences from several hundred organisms, it has become an accepted notion that UGA is the universally conserved codon for selenocysteine [26]. tRNA^" The key element for specific selenocysteine insertion in E. coli was identified as the product of the fdhC gene, now designated as the selC gene [27]. It codes for a tRNA with unusual sequence and structural properties (Figure 2A). With 95 nucleotides, tRNA^'' is the largest tRNA in E. coli mainly because of an aminoacyl acceptor stem of eight possible base pairs and a 22 nucleotide long extra arm. There are also a number of deviations fi-om the consensus structure characteristic of canonical elongator tRNAs, namely a G at position 8, an A at position 14, a Y-R pair at the 10-25 sites and an R-Y base pair at positions 11-24. Moreover, the R-Y Levitt pair between the positions 15-48 is missing. As expected, extensive enzymatic and chemical

Selenium metabolism in prokaryotes

13

probing of the solution structure of tRNA^^*^ from E. colt, compared with that of canonical tRNA^^', showed that these deviations, plus the fact that the D stem is closed to a six base pair helix minimizing the D loop to four nucleotides, also restrict the types of tertiary interactions within the molecule [28]. Whereas the canonical G19-C56 interaction is still present, there are new interactions between CI6 of the D loop and C59 of the T loop and the canonical A21-(U8-A14) triple pair is substituted by a G8-(A21-U14) triple interaction. The extra arm is closed by a G45-A48 pair and connected to the anticodon coaxial helix only by interaction of A44 with U26. All these unusual sequence and structural properties are conserved in the sequences of other bacterial tRNA^^*^ species [29]. In view of the still open discussion on the structure of the eukaryal (see Chapter 3) and archaeal (Figure 2B) counterparts and of the lack of an x-ray structure, the conclusions can be concentrated on three characteristic features: (i) the acceptor-T stem stacked helix is extended to 13 base pairs made up of 8 plus 5 base pairs in bacteria and 9 plus 4 in archaea and eukarya, (ii) the closure of the D stem and the deviations from the sequence in canonical positions restrict the possibilities for tertiary interactions within the molecule, and (iii) the extra arm appears to be less well fixed to the body of the molecule than in classical elongator tRNAs. k

A C C i' G G—C 6—C A—U A—U

c

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C^G

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m u C U GM C C

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ll|f G A Dl, SelP, TRl > GPx4. The easiest hypothesis to explain this hierarchy is that GPx4 mRNA is best optimized to compete for selenium incorporation when selenium is in limited supply. A number of observations, however, indicate that additional and overlapping factors are likely to play a role. Selenium concentration clearly appears to be the preeminent factor modulating Sec translation. Without selenium and therefore with insufficient Sec-tRNA , UGA will be interpreted as a stop codon, thus limiting selenoprotein translation. Selenium status also affects the relative levels of two isoforms of the Sec-tRNA^^" in mice (containing mcm^U base modification versus the methylated mcm^Um modification at position 34 within the anticodon of the tRNA). High selenium status raises the concentration of the methylated form and appears to enhance selenium incorporation into GPxl and GPx3 relative to GPx4 and TR [32]. A second set of differences in relative translation of selenoproteins involve the selenocysteine insertion sequence (SECIS) elements in the 3'UTR. Berry and colleagues [33,34] used Dl chimeric constructs with 3'-UTRs from different selenoprotein mRNAs, and later used additional chimera, and showed that different SECIS elements can alter the translational efficiency of selenoproteins. Some of this effect may specifically be due to the differential affinity of SECIS-binding Protein-2 (SBP2) for different SECIS elements [35]. In addition, distances of I'DNA, ^RNA, >lprotein synthesis tapoptosis, 4'AP-l, >lNF-kB

NazSeO.

-GPXsJR • s —•• ^ROS, tredox control SeMet SeCys

t02", tH202, tDNA SSBs, S/G2-arrest, >lpolyamines, tapoptosis iPKC, i endothelial MMP, ^epithelial VEGF

CHjSeOjH CHjSeCN CHjSeCys

Gi arrest, tcaspase-mediated apoptosis (CH3)2Se (breath)

(w/o genotoxicity)

I. (CH3)3Se^ (urine) Figure 1. Se-metabolites apparently active in cancer prevention (after [92]). Abbreviations: SeMet, selenomethionine; SeCys, selenocyeteine; CH3Se02H, methyl-seleninic acid; CHsSeCN, methylselenocyanate; CHsSeCys, Se-methylseleno-cysteine; GPXs, glutathione peroxidases; TRs, thioredoxin reductases; ROS, reactive oxygen species; SSBs, DNA single strand breaks; PKC, protein kinase C; MMP-2, matrix metalloproteinase-2; VEGF, vascular endothelial growth factor; PSA, prostate specific antigen; AR, androgen receptor.

Hydrogen selenide appears to be an important player in Se-anticarcinogenesis by way of its further metabolism. Its oxidative metabolism produces superoxide anion (O2 ) and H2O2, the formation of which induces DNA single-strand breaks leading to S phase/G2 cycle arrest and cell death by

Selenium as a cancer preventive agent

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apoptosis [103-107]. This mechanism would appear to mediate seleniteinduced apoptosis, as the genotoxic and pro-apoptotic effects of selenite on leukemia, mammary or prostate cancer cells have been shown to be blocked by a superoxide dismutase or its mimetics [103,108,109], but not by an hydroxyl free radical scavenger [110]. Further, catalase added to the cell culture medium blocked the induction of cell death by selenite [111]. In addition, H2Se can be methylated to produce a string of metabolites that, although being readily excreted, include some that are anti-carcinogenic. Ip, Ganther and coworkers [112-120] have produced strong experimental evidence that the anti-tumorigenic effects of Se are mediated by methylselenol (CHsSeH) or its derivatives (see Figure 1). They found that the CHaSeH-precursors selenobetaine (CH3Se02H) and methyl-selenocysteine (CHsSeCys) are anti-carcinogenic in the 7,12-dimethylbenzanthracene (DMBA)-induced rat mammary tumor model, each being somewhat more efficacious than selenite. In contrast, dimethyl selenoxide, which is metabolized to dimethylselenide ([CH3]2Se) and very rapidly excreted in the breath, was very poorly chemo-preventive, and the rapidly excreted urinary metabolite trimethylselenonium ([CHsJsSe^) was completely ineffective. Further work has shown that the CHsSeH-precursors methylselenocyanate (CHsSeCN) and CHsSeCys can each inhibit mammary cell growth, arresting cells in the Gi or early S phase and inducing apoptosis [106,107,118-122], The latter effect is caspase-dependent [123], as methyl-Se induced apoptosis involves at least three caspase-dependent actions: mitochondrial release of cytochrome C, cleavage of poly(ADP-ribose), and DNA nucleosomal fragmentation. Selenite-induced cell death, in contrast, is independent of these death proteases [109,121,123,124]. That methyl-Se can cause caspasedependent apoptosis in cell lines that do not contain functional p53 [124] suggests that its pro-apoptotic action is independent of p53. This was also evident in a recent study in which methyl-Se induced apoptosis of p53positive, LNCaP cells was found not to involve a change in p53 activation [125]. Se-Methylselenocysteine has been shown to inhibit the cell cycle regulatory enzymes CDK2 and protein kinase C (PKC) [126,127]. Unlike the proximal H2Se-precursors, CHaSeH-precursors potently inhibit the expression of matrix metalloproteinase (MMP-2) in vascular endothelial cells and of vascular endothelial growth factor (VEGF) in cancer cells [121,122,128,129], critical components of the angiogenic response, suggesting that methyl-Se inhibits cellular proliferation and survival of activated endothelial cells by inhibiting neo-angiogenesis. Sub-apoptotic concentrations of methyl-Se have been shown to reduce androgen receptor protein expression [130] and to inhibit androgen-stimulated PSA promoter transcription [130-132], to reduce PSA expression and secretion [130], and to cause rapid PSA degradation [130]. These findings suggest a unique

258

Selenium: Its molecular biology and role in human health

mechanistic basis for the apparent sensitivity of the prostate to Seanticarcinogenesis [46,47]. Because methyl-Se compounds can be demethylated ultimately to feed the H2Se-exchangeable metabolic pool (see Figure 1), both CHsSeCys and dimethylselenoxide can support GPX expression [116]. Despite that phenomenon, evidence indicates that CHaSeH and its precursors have anticarcinogenic actions independent of those associated with the H2Se pool. Ip et al [112-117] found that arsenic, which competitively inhibits both the methylation of H2Se and the demethylation of CHsSeH (and the analogous di- and tri-methylated species) greatly reduced the anti-tumorigenic effects of selenite while enhancing those of selenobetaine or methylselenocysteine (CHjSeCys) which yields CHjSeH metabolically. Specifically, CHaSeHprecursors were shown to lack the genotoxic (DNA single-strand breaks [106,107,118,133] or DNA-oxidative damaging [134]) effects of selenite or selenide. The anti-carcinogenic activities of the methylated Se-metabolites and synthetic Se-compounds are likely related to reactions with critical proteins as well as to redox cycling, which effects may selectively impact the transformed phenotype. Ganther [128] described ways in which Secompounds may affect cellular proteins: through the formation of selenotrisulfide (-S-Se-S-) and selenylsulfide (-S-Se-) bonds and the catalysis of disulfide bonds formation/ dissolution, which would affect the activities of many enzymes with critical sulfhydryl groups; and through the formation of diselenide bonds (-Se-Se-) affecting the activities of selenoproteins which have SeCys residues at their active centers. Selenium-induced inhibition, presumably due to one or more of these reactions, has been demonstrated for a variety of relevant enzymes: ribonuclease [135], Na,K-ATPase [136], PKC [127,137,138]. Inhibition of PKC would be particularly important, as that enzyme system is known both to activate nuclear transcriptional factors and to bind phorbol ester-type tumor promoters. The inhibition of PKC by a Semetabolite such as CHsSeH would be expected to trigger a number of downstream effects including cell cycle arrest, apoptosis and angiogenic switch regulation. Evidence for at least some of these effects has been reported in response to the CHsSeH-precursors: decreased cdk2 kinase activity [126]; decreased DNA synthesis and elevated gadd gene expression [107]; inhibition of vascular endothelial MMPs and VEGF expression [139]. Thus, it appears that Se- doses large enough to support high, steady-state concentrations of CHsSeH can effect anti-carcinogenesis by inhibiting critical redox-sensitive factors including PKC and, probably, NF-kB [140] and AP-1, thus, impairing tumor cell metabolism and transformation. These effects would appear to be fairly targeted to certain factors, rather than involving wider perturbations in cellular redox control. After all, Semetabolites are typically present in tissues in much lower (nano- to micro-

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molar) concentrations than those (miUimolar) of thiols. In fact, susceptibility to redox modification by Se-attack seems to be limited to structures containing clustered cysteinyl residues [137,138]. Many of the effects of Se-compounds on cell proliferation may result from their abilities to form catalytically active, redox-cycling intermediates. Selenite, diselenides and the oxidation product of H2Se, selenium dioxide, for example, can each react with GSH to produce the selenolate ion (RSe) [141-143]. In the presence of GSH and molecular oxygen, RSe" can cycle continuously to generate Oi' and H2O2. This redox cycling is thought to be the basis of Se-toxicity, and it is possible that it may also contribute to anticarcinogenesis. Spallholz et al (144) found that dimethyldiselenide ([CH3Se]2) was the most catalytically active of a series of 19 Se-compounds^ in its ability to generate in vitro O2" in the presence of GSH and O2. They attributed this activity to CHsSeH produced by the reduction of ([CH3]Se)2 presumably generating the radical anion CHsSe'; however, it remains to be determined whether such catalytically active species can be generated intracellularly as the result of the metabolism of proximal (e.g., CHsSeCys, CH3Se02H) and/or upstream (e.g., SeMet, SeCys) precursors. There is no evidence that the common forms of Se in foods and feedstuffs, the selenoamino acids selenomethionine (SeMet) and selenocysteine (SeCys), are directly anticarinogenic. However, each can be metabolized first to H2Se and, then, to CHsSeH (see Figure 1). That conversion occurs directly for SeCys, which cannot be used directly in general protein synthesis; it is catabolized by a lyase to yield H2Se. The process is not direct for SeMet, which can enter the general protein pool as a mimic of methionine (Met). In fact, the conversion of SeMet from either dietary or proteinturnover sources necessarily involves its first being converted to SeCys by the Met-transsulfuration pathway. For this reason, most studies have found SeMet to be generally less anti-carcinogenically efficacious than SeCys or selenite [145-149], as would be expected in short-term studies and, particularly, under conditions of limiting Met supply. However, under steady-state conditions effected by long-term use, and particularly with highMet diets, one would expect the anti-tumorigenic efficacy of SeMet to approach that of SeCys and selenite. A number of synthetic Se-compounds have also been found to be anticarcinogenic. Ip et al [149] tested a series of alkylselenocyanates (H[CH2]xSeCN) using the DMBA-induced murine mammary carcinogenesis model, finding that anti-carcinogenic efficacy varied directly with increasing ^In addition to ([CH3]Se)2, these included nine other catalytically active Se-compounds: selenite, selenium dioxide, selenocystine, selenocystamine, diselenopropionic acid, diphenyldiselenide, dibenzyldiselenide, pXSC and 6-propylselenouracil; and nine Se-compounds that were not catalytically active: elemental Se, selenate, SeMet, CHaSeCys, selenobetaine, dimethylselenoxide, selenopyridine, TPSe and potassium selenocyante.

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chain length up to five carbons. The same group [150] also showed that allyl-selenocysteine, which is expected to yield allylselenol, a fairly hydrophobic metabolite, is more anti-carcinogenic than the corresponding alkylseleno-cysteine. Several aryl selenocyanates have also been found to be anti-tumorigenic. The more effective of these are benzylselenocyanate [151153], p-methoxybenzyl-selenocyate [152],/;-phenylselenocyanate [152-156]. These compoimds are thought to undergo initial metabolism through arylselenol, which may explain their similar responses to the alkylselenocyanates and other CHsSeH-precursors. Each induces apoptosis of cancer cells in vitro without inducing DNA single strand breaks. When compared to selenite on a molar basis, these forms are not only less effective in supporting GPX expression but also less toxic; yet, they offer comparable anti-tumorigenic efficacy [157,158]. It would appear that the anticarcinogenic efficacies of these synthetic Se-compounds are related to their relative lipophilicities and, thus, to uptake/retention by transformed cells. Accordingly, their anti-tumorigenic efficacies would appear to be affected by dietary fat intakes, being enhanced by the use of low-fat diets [159]. That anti-carcinogenicity need not involve selenoprotein expression is again evidenced, this time by triphenylselenonium chloride (TPSe), which is antitumorigenic at fairly high levels of exposure (dietary EC5o=15 ppm for preventing DMBA-induced mammary cancer [158]). The Se in TPSe is tightly bonded to three unsubstituted benzene rings rendering it unavailable to metabolism, ineffective in supporting GPX expression in the Se-deficient rat, and without adverse effects on rat growth at dietary levels as high as 200 ppm [160]. Conclusion Increasing evidence shows that Se-compounds can inhibit and/or delay carcinogenesis in animal models and reduce the risks for at least some kinds of cancer in humans. These effects may involve the protective, nutritional functions of Se as an essential constituent of a number of metabolically important selenoenzymes; such functions may be compromised in Sedeficient individuals. Recent evidence suggests that allelic variants of some selenoproteins may be related to cancer risk. In addition to such effects, certain Se-metabolites, notably methyl-Se compounds, appear to inhibit carcinogenesis through mechanisms unrelated to the nutritional functions of Se and at doses greater than necessary for such functions, i.e, at supranutritional levels of exposures. Thus, the emerging picture is of Se as a nutrient that functions in anti-carcinogenesis in two ways: as an essential constituent of metabolically important selenoproteins, and as a source of anticarcinogenic metabolites. Because selenoprotein expression appears to be optimized at lower levels of Se exposure than are necessary to support the

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latter functions, minimization of cancer risk appears to require Se intakes greater than those required for maximal selenoprotein expression. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

K Schwarz, CM Foltz 1957 J Am Chem Soc 79:3292 RJ Shamberger, DV Frost 1969 Can Med Assn 7104:82 J Shamberger, CE Willis 1971 Clin Lab Sci 2:211 GF Combs Jr, WP Gray 1998 Pharmacol Ther 79:179 GF Combs Jr, LC Clark 1999 in Nutritional Oncology D Heber, GL Blackburn VLW Go (eds) Academic Press Inc New York 215 P Kneckt 2002 in Handbook of Antioxidants E Cadenas, L Packer (eds) Marcel Dekker Inc New York 665 PDWhanger 2004 S/-yiV«/r 91:11 KJ Helzlsouer et al 1996 J Nat Cancer Inst 88:32 KJ Helzlsouer, GW Comstock, JS Morris 1989 Cancer Res 49:6144 FJ Kok, AM De Bruijn, A Hofman, R Vermeeren, HA Valkenburg 1987 Am J Epidemiol 125:12 JT Salonen et al 1985 fir Med J 290:417 J Nayini, K El-Bayoumy, S Sugie, LA Cohen, BS Reddy 1989 Carcinogen 10:509 W Willett et al 1983 Lancet 2:130 Van Den Brandt et al 1993 J Nat Cancer Inst 85:224 AMY Nomura, J Lee, GN Stemmemann, GF Combs Jr 2000 Cancer Epidemiol Biomarkers Prev 9:883 P Philipov, K Tzatchev 1988 Zentrabl Neurochir 49:344 K Jaskiewicz, WFO Marasas, JW Rossouw, FE Van Niekerk, EWP Heinetech 1988 Cancer 62:263 L. Geardsson, D Brune, IGF Nordberg, PO Wester 1985 Br J Industrial Med Al:(,\l H Miyamoto et al 1987 Cancer 60:1159 P Knekt et al 1990 J Nat Cancer Inst 82:864 T Westin et al 19S9 Arch Otolaryngol Head Neck Surg 115:1079 PGJ Bumey, GW Comstock, JS Morris 1989 J Clin Nutr 49:895 E Glattre et al 1989 Int J Epidemiol 18:45 CP Caygill, K Lavery, PA Judd, MJ Hill, AT Diplock 1989 Food Addit Contam 6:359 P Knekt et al 1988 Int J Cancer 42:846 U Reinhold, H Blitz, W Bayer, KH Schmidt 1989 Acta Derm Venerol 69:132 K Yoshizawa et al 1998 J Nat Cancer Inst 90:1219 LC Clark et al 1993 Cancer Epidemiol Biomarkers Prev 2:41 GF Combs Jr 1989 Nutrition and Cancer Prevention T Moon, M Micozzi (eds) Marcel Dekker New York, 389 C l p 1998 yA^M/r 128:1845 L Yan, J A Yee, MH McGuire, GL Graef 1997 Nutr Cancer 28:165 L Yan, JA Yee, D Li, MH McGuire, GL Graef 1999 Anticancer Res 19:1327 J Ankerst, H Sjogren \9S2 Int J Cancer 29:701 RD Dorado, EA Porta, TM Aquino 1985 Hepatol 5:1201 H Nakadaira, T Ishizu, M Yamamoto 1996 Cancer Lett 106:279 JP Perchellet, NL Abney, RM Thomas, YL Guislan, EM Perchellet 1987 Cancer Res 47:477 SY Yu, YJ Zhu, WG Li 1997 Biol Trace Elem Res 56:1 \7 SY Yu, YJ Zhu, QS Huang, CZ Wang, QN Zhang 1991 Biol Trace Elem Res 29:289 JY Li et al 1993 J Nat Cancer Inst 85:1492 PR Taylor, B Li, S Dawsey 1994 Cancer Res 54:2029s

262 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.

Selenium: Its molecular biology and role in human health WJ Blot, JY Li, PR Taylor, W Guo, SM Dawsey, B Li 1995 Am J Clin Nutr 62:14248 WJ Blot et al 1993 J Nat Cancer Inst 85:1483 WJ Blot 1997 Proc Soc Exp Biol Med 216:291 K Krishnaswamy, MP Prasad, TP Krishna, VV Annapuma, GA Reddy 1995 Eur J Cancer 31:41 MP Prasad, MA Makunda, K Krishnawamy 1995 Eur J Cancer 31B:155 LCClarketal 1996y.4/«iWe^^i5oc 276:1957 LC Clark et al 1998 Brit J Urol 81:730 AJ Duffield-Lillico, et al 2002 Cancer Epidem Biomarkers Prev 11:630 ME Reid et al 2002 Cancer Epidem Biomarkers Prev 11:1285 AJ Duffield-Lillico et al 2003 Br J Urol 91:608 AJ Duffield-Lillico et al 2003 J Nat Cancer Inst 95:1477 J N^ve 1995 J Trace Elements Med Biol 9:65 Panel on Dietary Antioxidants and Related Compounds 2000 Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Beta-Carotene and other Carotenoids. National Academy Press Washington DC KE Burke, GF Combs Jr, EG Gross, KC Bhuyan, H Abu-Libdeh 1992 Nutr Cancer 17:123 BC Pence, E Pelier, DM Dunn 1994 J Invest Dermatol 102:759 AM Diamond, P Dale, JL Murray, DJ Grdina 1996 Mutat Res 356:147 Y Kise et al 1991 Nutr Cancer 16:153 GF Combs Jr, LC Clark, BW Tumbull 2001 Proc 7'* Internat Symp Selenium Biol Med 152 J Lii, C Jiang 2005 Antioxidants Redox Signaling 7:1715 M Berggren et al 1996 Anticancer Res 16:3459 GPowisetal 1996^nricancerZ)n.001)

The immune response to influenza is characterized by 2 stages, an innate stage and a cell mediated stage. The initial, innate response, involving natural killer (NK) cells, dendritic cells and macrophages is essential for directing the subsequent cell-mediated response. We examined key aspects of the innate response in Se-deficient mice. Specifically, interferon (IFN)-a and IFN-P mRNA, which help control viral replication and activate a host of anti-viral genes, were reduced in Se-deficient influenza infected mice (Figure 3). IFN-y, which is produced by NK cells early in infection, was also reduced in Se-deficient infected mice (Figure 3). Interestingly, NK cell activity is unaffected in Se-deficient mice (data not shown). Together, these

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Selenium: Its molecular biology and role in human health

data indicate that Se-deficient mice have impaired, early anti-viral cytokine responses.

IFN-a

IFN-P

IFN-Y

Figure 3. Quantitative RT-PCR was performed for the cytokines and normalized to G3PDH. The data are expressed as arbitrary units +/- SEM. Se deficiency resulted in 2-4 fold decrease in production of IFN-a, IFN-P and IFN-y, at 24h p.i.

In order for the lung inflammation to occur in the infected influenza mice, a co-ordinate production of chemokines must occur. This process was altered in the Se-deficient mice. Chemokine mRNA levels for RANTES, MlP-la, MIP-P and MCP-1 were highest on days 4 and 5 post infection for the Se-adequate mice, and then began to decrease, whereas these chemokines were highest at later time points for the Se-deficient mice [19]. Clearly, Se deficiency leads to an increase in influenza-induced histopathology which is associated in part with altered chemokine and IFN expression. Because host Se deficiency induced changes in the coxsackievirus genome, we reasoned that the increased virulence of the influenza virus in the Sedeficient mice may also be due to changes in the viral genome. Virus was recovered from the lungs of Se-deficient and Se-adequate mice and all 8 viral RNA segments were sequenced and compared with the sequence of the input strain. Surprisingly, few changes were found in the HA and NA segments of the virus, which are associated with a high mutation rate. Changes in the HA and NA segment were random, and found in viruses obtained from both Seadequate and Se-deficient mice. In stark contrast, however, the M gene

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contained multiple mutations [20]. As shown in Table 1, three separate isolates from 3 individual Se-deficient mice all had identical mutations in 29 positions. One of the 3 isolates from a Se-deficient mouse had an additional 6 mutations. None of these changes were seen in viruses obtained from the Se-adequate mice. Thus, as for coxsackievirus, influenza virus replicating in a Se-deficient host undergoes rapid genetic change, resulting in a more virulent virus which can now cause disease even in a host with normal Se status. Poliovirus and Se deficiency A recent study has reported that subjects in the United Kingdom with low Se status (< 1 i^Mol/L) have a decreased immune response to poliovirus vaccination [21]. Of particular note, they found an increased mutation rate of the vaccine strain of vaccine virus which had been shed in the feces. Supplementation with Se of the low Se status population enhanced the immune response and lowered the number of mutations found in the shed vaccine strain of the virus. Thus, low Se status was associated with increased mutation rate of the live attenuated poliovirus vaccine strain when compared with vaccinated individuals supplemented with Se. To assess the mutation rates, the investigators utilized temporal temperature gradient electrophoresis (TTGE). Although this technique can identify mutations occurring in the genome, it does not provide information on which specific nucleotides were altered. The Broome et al. study supports the hypothesis that polio vaccination of individuals with low Se status may lead to increased mutations in the vaccine strain of virus. This area of research is particularly relevant in view of recent findings that attenuated poliovirus vaccine sfrains have circulated and reverted to virulence in several areas of the world where undernutrition is prevalent [22]. Selenium and other viruses Infection with human immunodeficiency virus (HIV) results in a loss of CD4+ helper T cells and subsequent immune dysfunction leading to increased opportunistic infections. In addition, oxidative stress increases during an HIV infection. A number of studies have examined the relationship between specific nutritional factors and disease progression and survival of HIV infected individuals. Se status of HIV infected individuals has also been studied. In developed countries (France and the US), 3 studies have demonstrated that lower serum Se levels are associated with an increased risk of mortality from HIV [23-25]. In a study of HFV infected pregnant women in Tanzania, low selenium status was found to be associated with accelerated HIV disease progression [26].

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Selenium: Its molecular biology and role in human health

Table 1. Comparison of nucleotide sequences of influenza A/Bangkok/1/79 M gene of the infecting virus and of virus isolated from Se-adequate (Se+) and Se-deficient (Se-) mice.

Nucleotide Position 136 205 238 309 322 325 328 331 334 370 371 406 439 454 455 502 503 524 525 544 566 567 568 610 619 652 655 667 669 670 677

Infecting I Virus A G G G A C A A T A G C A C C

cA G G A C C G A G C G G G A G

Se status of host virus isolated from: Se± Se+ Se+ Se; Soz Sez A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G

A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G

A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G

C A G A C T G C C C T T G A C T C A G C T C A G A T A A G G A

C A G A C T G C C C T T G A C T C A G C T C A G A T A A G G A

C A A A C T G C C C T T G A A T C A A C T T A G A T A A A G A

AA Change

RtoK

AtoS

AtoT TtoA

AtoT

AtoT

Se levels have also been inversely correlated with hepatitis B virus infection. Lifection with hepatitis B virus is a major health problem throughout the world. In addition, chronic hepatitis B infection is thought to

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be a significant factor in most hepatocellular carcinomas, a highly malignant neoplasm with a high mortality rate. A study from Taiwan [27] demonstrated that mean Se plasma levels were significantly lower in hepatocellular carcinoma patients, as compared with individuals testing positive for hepatitis B virus. A further study from Qidong county in China [28] demonstrated a protective effect of Se supplementation in a population at high risk of developing primary liver cancer due to a high prevalence of hepatitis positive individuals. Conclusion Low host selenium status has been shown to be important in driving viral mutations. This increase in viral mutations in a Se-deficient host may be due to an increase in oxidative stress status, as virus which replicated in GPX-1 knockout mice also mutated. Emerging viruses are either newly arisen viruses or are viruses that are rapidly expanding their range. Understanding the mechanisms underlying the evolution of emerging viruses is critical to predicting new viral outbreaks and devising new strategies to limit the emergence and spread of these new pathogenic forms. Data from the Se studies demonsfrates that host Se status is a driving force for emergence of new viral variants. These observations suggest a new area for research, namely the interaction of host nufrition and viral evolutionary processes. The precise mechanism(s) by which a deficiency in Se leads to mutations in a viral genome remains to be determined. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

BQ Gu 1983 Chin Med J 96:251 C Su, C Gong, J Li, L Chen, D Zhou, Q Jin 1979 Chin MedJ59:466 LQ Ren, XJ Li, GS Li, ZT Zhao, B Sun, F Sun 2004 World J Gastroenterol 10:3299 JF Woodruff 1980 Am J Pathol 101:427 J Bai, S Wu, K Ge, X Deng, C Su 1980 Acta Acad Med Sin 2:29 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander \994 J Infect Dis 170:351 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander 1994 J Med Virol 43:166 JK Reffett, JW Spears, TT Brown Jr 1988 J Animal Sci 66:1520 JF Reffett, JW Spears, TT Brown Jr 1988 JNutr 118:229 DN Cook, MA Beck, T Coffman, SL Kirby, JF Sheridan, IB Pragnell, O Smithies 1995 5«e«ce 269:1583 MA Beck, CC Matthews 2000 Proc Nutr Soc 59:1 MA Beck, Q Shi, VC Morris, OA Levander 1995 Nat Med 1:433 MA Beck, RS Esworthy, Y-S Ho, F-F Chu \99SFASEBJ \2:l\43 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander 1993 J Nutr 124:345 MA Beck, Q Shi, VC Morris, OA Levander 2005 Free Radic Biol Med 38:112 CB Bridges, SA Harper, K Fukuda, TM Uyeki, NJ Cox, JA Singleton 2003 Morb Mortal Wkly Rep 52:1 BR Murphy, RG Webster 1996 Fields Virology BN Fields (ed) Lippincott-Raven Philadelphia PA pi 397

298 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Selenium: Its molecular biology and role in human health AC Ward 1997 Virus Genes 14:187 MA Beck, HK Nelson, Q Shi, P Van Dael, EJ Schiffrin, S Blum, D Barclay, OA Levander 2001 F^SESy 10.1096/fj.00-072ige HK Nelson, Q Shi, P Van Dael, EJ Schriffrin, S Blum, D Barclay, OA Levander, MA Beck. 2001 FASEBJ\0M9m].Q\-Q\\5f)e CS Broome, F McArdle, J A Kyle, F Andrews, NM Lowe, CA Hart, JR Arthur 2004 J Clin Nutr m:\54 OM Kew, RW Sutter, EM de Gourville, WR Dowdle, MA Pallansch 2005 Ann Rev Microbiol 59:5S7 MK Baum, G Shor-Posner, S Lai, G Zhang, H Lai, MA Fletcher, H Sauberlich, JB Page 1997 y.4/D5 15:370 A Campa, MK Shor-Posner, F Indacochea, G Zhang, H Lai, D Asthana, GB Scott, MK Baum 1999 y^/DS 20:508 J Constans, JL Pellegrin, C Sergeant, M Simonoff, L Pelegrin, H Fleury, B Leng, C Conri 1995 y4/DS 10:392 R Kupka, GI Msamanga, D Spiegelman, S Morris, F Mugusi, DJ Hunter, WW Fawzi 2004 yiVMfr 134:2556 M-W Yu, I-S Homg, K-H Hsu, Y-C Chiang, Y-F Liaw, C-J Chen 1999 Am J Epidemiol 150:367 SY Yu, YJ Zhu, WG Li 1997 Biol TraceElem Res5(,:\\l

Chapter 26. Role of selenium in HIV/AIDS Marianna K. Baum and Adriana Campa Florida International University, Stempel School of Public Health, Department of Dietetics and Nutrition, U200SW8th Street, Miami, Florida 33199, USA

The advent of Highly Active Antiretroviral Therapy (HAART) in the late 90s has transformed HIV infection from a deadly condition into a chronic, manageable viral infection in developed countries [1]. The developing world, however, accounts for 96% of the global HIV-l infections, and in most of these countries, antiretrovirals are not yet widely available. The number of persons living with Human Immuno-Deficiency Virus (HIV) infection and Acquired Immuno-deficiency Syndrome (AIDS) worldwide has been estimated to be approximately 40 million [2], and this figure includes approximately 5 million people who acquired HIV in 2004. In the same period, approximately 3.1 million adults and children died from AIDS, and 14,000 new individuals are still infected daily, a number that lessens hopes for a rapid solution to this pandemic [2]. The gap between developed and developing countries in the control of the pandemic and treatment of infected persons is growing, and one of the factors fueling the epidemic in poor countries is malnufrition. Moreover, protein-energy malnutrition (PEM), and the accompanying and aggravating micronufrient deficiencies, are already an overwhelming health problem and still the main cause for immune disturbances in poor countries [3,4]. Sub-Saharan Africa, where the greatest growth in severe and generalized malnutrition has occurred in the last two decades [5], is also the region in which 12 out of 44 countries have more than 10% prevalence of HIV in the adult population [6]. Numerous studies have demonstrated that nutritional deficiencies accelerate HIV disease progression and decrease survival [7-16]. Moreover, nutrient deficits interfere with the effectiveness of antiretrovirals by delaying the recuperation of the immune system and aggravating side-effects attributed to treatment [17-20]. Selenium appears to have a multifactorial role in HIV-l infection. Selenium status affects HIV disease progression and mortality [14-16] through various potential mechanisms. In two recent studies, deficiency of selenium has been associated with elevated measures of HIV infectivity [21,22], and therefore, with increased potential to transfer the infection.

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Selenium is required for the function of gluthatione peroxidase, a biological antioxidant that protects against oxidative stress. Other selenoproteins may also act as antioxidants by the incorporation of selenocysteine in their molecules [23]. In HIV-infected persons, dietary selenium intake was strongly associated with reduced measures of oxidative stress [24]. Adequate selenium status may also be essential for controlling viral emergence and evolution [25,26]. In addition, selenium may enhance resistance to infection through modulation of both cellular and humoral immunity. Plasma selenium levels are associated with interleukin production and subsequent changes in Thl/Th2 cytokine responses [27,28]. Other nutritional factors interact with selenium status and are important in HIV-1 disease progression and mortality. These factors include disease stage, nutritional status at the onset of the disease, types of treatment and compliance, and secondary infections that may act independently or in combination. Treatment of malnutrition, and the accompanying micronutrient deficiencies, thus, requires a carefully individualized approach. This chapter will review the role of selenium in HFV-l disease progression, morbidity and mortality, as well as the factors that may affect these relationships. Selenium and immunity in HIV Selenium has been shown to affect the immune process [29]. In vivo and in vitro studies suggest that selenium may act at different levels of immune function. In animal models, selenium deficiency was shown to impair the ability of phagocytic neutrophils and macrophages to destroy antigens, and selenium status was associated with humoral immune response [30]. In humans, Broome and colleagues [31] found that in a population of sixty-six healthy participants who were marginally deficient in selenium (A1

seliy""^ mutation selectively modulates the RAS/MAPK pathway through alteration of the redox balance is further supported by the finding that an increase in ROS caused by the amorphic catalase allele Caf', one of the main enzymes of the Drosophila antioxidant system, also reduces RAS/MAPK signaling [29]. The results of these experiments strongly suggest that accumulation of ROS should be substantially different between heterozygous flies for those mutations and wild-type organisms. Although haplo-insufficient selDf""^fliesdo not have an apparent phenotype when kept under normal laboratory conditions, a significant decrease in life span is observed when they are treated with oxidants [30]. In contrast, while increased amounts of superoxide dismutase (SOD) extend longevity [31], overexpression of spsl in motomeurons leads to a reduction in life span, possibly due to an accumulation of toxic precursors [30]. Finally, it is clear that selLf"^ causes an impairment of selenoprotein synthesis, as revealed by the failure to detect selenoproteins in protein extracts of mutant larvae grown in fly medium containing ^^Se [25]. Therefore, it is tempting to speculate that selenoproteins may be instrumental in maintaining a certain redox state in the cell, as has already been shown for several mammalian selenoproteins [9,32]. Despite the findings mentioned above, it should be noted that purified Drosophila selD/spsl expressed in E. coli does not catalyze selenidedependent ATP hydrolysis in vitro and does not complement a selD deficiency in bacteria [11]. Although at first sight these results appear to disagree with those of other studies using the human spsl gene, in which weak complementation of an E. coli mutation was observed [33], if we take into account the fact that organisms that possess one variant also contain the other we can consider the possibility that SelD/Spsl may have a different function in Se metabolism. It is possible that SelD/Spsl is only efficient in using Se delivered fi-om Sec recycling pathways, as seems to be the case in the human spsl gene fi-om human-lung adenocarcinoma cells [14] or in the E. coli selD(C17S) [15]. The other selenophosphate synthetase, Sps2, contains a Sec residue at the position equivalent to E. coli Cys-17, suggesting a possible autoregulatory role in Sec synthesis; this gene also carries a mammalian-type SECIS. Transgenic embryos expressing a luciferase reporter containing the 3'UTR of the sps2 gene showed significantly higher reporter activity than those lacking the sps2 3'UTR, demonstrating the functionality of the SECIS element present in this 3' terminal region [12]. The spsl gene, located at 2L/31D, is expressed as at least two transcripts: sps2-RA (1348 bp), which was described initially [12], and sps2-RB (1295bp), which is the product of an alternative splicing event in the 4rt exon that, according to FlyBase (www.flybase.org), generates a change in the open reading fi-ame. Both transcripts could encode putative selenoproteins due to

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Selenium: Its molecular biology and role in human health

an in-frame TGA codon and the SECIS element. Both proteins contain the ATP/GTP binding site required for ATP hydrolysis during selenophosphate production [12]. Expression of the different transcripts has been assessed by both Northern blot analysis and in situ hybridization [12]. Northern blot analyses at different developmental stages revealed the presence of at least the larger transcript throughout development. In situ hybridization studies show a strong accumulation of maternal transcripts produced by nurse cells during oogenesis; these remain present at high levels up to the blastoderm stage (maternal effect). During the remainder of embryogenesis, zygotic expression occurs in a more restricted pattern and is first detected in the embryonic midgut primordium and later in a variety of tissues and organs including the gut and nervous system. A regulatory element that is thought to regulate cell-proliferation-related genes in Drosophila has been identified in the sps2 gene [34,35]. This DNA replication-related element (DRE) located downstream of the initiation site of the gene is essential for its transcription [35]. A transcription factor that specifically binds the DRE sequence has also been isolated in flies and ablation of this factor by double-stranded RNA interference (dsRNAi) experiments shows a significant decrease in dsps2 promoter activity [35]. Sec translational machinery Although much progress has been made in resolving the machinery associated with Sec translation in prokaryotes and vertebrates, less is known about the particular features of the system in Drosophila. Nevertheless, the conservation of some of the elements that have been identified suggests that the basic machinery would be the same. Briefly, the model proposed for eukaryotes includes a requirement for cis and trans factors that form a ribonucleoprotein complex known as a selenosome that functions to incorporate Sec at a UGA codon and thereby prevents translation being stopped. This selenosome complex will consist of at least a SECIS element in the 3 'UTR of the selenoprotein mRNA as a cis factor and a SECIS binding protein 2 (SBP2), a Sec-elongation factor (SelB/eEFsec), a Sec-tRNA '^''^ ^^ and the ribosome itself as trans factors. SBP2 is thought to interact with a conserved region of the SECIS element known as the quartet, with the 28S ribosomal RNA, and with SelB/eEFsec, which will also specifically bind the Sec-tRNA^^'^'^'''^, leading to co-translational incorporation of a Sec when a UGA triplet is encountered [36]. In the fruit fly. Sec insertion is directed, like in other eukaryotes, by the presence of a SECIS in the 3'UTR of the selenoproteins [8]. The Drosophila SECIS element contains the canonical characteristics found mostly in eukaryotic SECIS: the core structure with the quartet of non-Watson-Crick interacting base pairs, and the unpaired adenosines in the apical loop.

Drosophila selenium metabolism and selenoproteins

349

Moreover, this SECIS appears to belong to form 2 in all the identified selenoproteins because of the presence of an additional small stem-loop at the top of the SECIS element [5,12,38]. The trans factors that are known to be required include the Sec-tRNA gene [10] and SelB/eEFsec [37]. Drosophila selB/eEFsec, located at position 57E on Chromosome 2, was found by sequence homology in an in silico analysis of the genome [37]. Its single transcript seems to be expressed throughout development, at high levels during early embryogenesis, lower levels during larval stages, and then at increasing levels in pupae and adults. Because SelB/eEFsec is essential for selenoprotein biosynthesis, it is a suitable target to mutate as a model in which to study the effects of selenoprotein deficiency. selB/eEFsec knockout mutants have been generated by homologous recombination, giving rise to flies that are unable to synthesize SelB/eEFsec and consequently fail to decode the UGA codon as Sec. Moreover, in spite of the impairment of the Sec UGA-decoding mechanism, selB/eEFsec mutant animals develop into fertile flies. In addition, although most known selenoproteins in eukaryotes seem to be involved in antioxidative defense and redox metabolism, life-span studies in the selB/eEFsec mutant do not reveal a role in viability and the mutants do not show sensitivity to induced oxidative stress. These findings challenge the view that a Sec-based oxidative stress defense system was responsible for conserving the selenoprotein biosynthesis system over the course of evolution [37]. Selenoproteins In recent years, genome projects have become an extremely powerful tool through which to identify protein-coding genes. However, because of the non-standard use of the UGA codon, computational gene prediction methods were unable to identify selenoproteins in the sequence of eukaryotic genomes until recently. Only the identification of members of the synthesis machinery was possible based on homology with known genes, as mentioned earlier, for example, with Sps2, the first selenoprotein identified in flies [12]. Using a biochemical approach it was shown that metabolic labeling of flies with '^Se revealed three clear major bands [5,25]. According to the predicted molecular weight, the 42-43 kDa band could correspond to the Sps2 selenoprotein itself, but those experiments did not provide any information on the nature of the other bands. Two different in silico approaches have been used in an attempt to solve this problem. Briefly, one approach combined and improved existing gene prediction programs and developed a method that relies on the prediction of SECIS elements alongside the prediction of genes in which a strong codon bias characteristic of protein-coding regions extends beyond a TGA codon that interrupts the open reading fi-ame [38]. The other approach involved a computational screen to search for SECIS elements followed by

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Selenium: Its molecular biology and role in human health

selenoprotein gene signature analyses [5]. Both screens led to the identification of three selenoprotein genes in fruit flies: the previously identified sps2 and two new selenoproteins named SelM (also known as SelH or BthD) and SelG (SelK or G-rich). Both proteins incorporate ^*Se when transfected in mammalian cells [38] and all of them were detected after metabolic labeling of flies with ^^Se [5]. These findings indicate that the genes encode true selenoproteins. Furthermore, both of the newly identified genes have paralogs that employ Cys instead of Sec as well as orthologs that use Sec or Cys in other organisms, including vertebrates [38]. SelM/BthD/SelH Selenoprotein SelM/BthD was the first component identified [5,38] in a new family that currently includes several Cys or Sec ortholog representatives in eukaryotes, including the uncharacterized human selenoprotein H [39]. This gene maps to position 12A8 on the X chromosome and has two distant paralogs, CG13186 and CG15147, the latter containing Cys instead of Sec [38]. The selM/BthD gene encodes a 30 kDa protein containing 249 amino acids and a Sec residue belonging to the CXXU motif near the N-terminus [5]. This motif, which is also found in both bacteria and animals, including the mammalian selenoproteins SelT, SelW, and SelH, is similar to the redox motif CXXC [22], suggesting a redox function, with the Sec possibly forming a selenenylsulfide bridge. SelM/BthD shows a dynamic expression pattern. High levels of transcript are detected in adult females, with abundant expression in the developing ovary. In contrast, the expression in males is very weak [5,40]. During early embryogenesis, both transcript and protein seem to display abundant ubiquitous expression, especially in the blastoderm, suggesting that there is a strong maternal contribution [38,40]. At late stages of embryogenesis selM/BthD expression accumulates in the developing salivary gland [40]. Finally, although selM/BthD mRNA appears to be more weakly expressed during larval stages [5,40], in situ hybridization reveals that the transcript is ubiquitously distributed in imaginal disc and larval brain [38]. A dynamic subcellular distribution has been detected using a specific antibody against SelM/BthD [40]. The protein distribution in various Drosophila tissues is cytoplasmic, with a particulate pattern observed in salivary glands. Immunolocalization studies in SL2 cells reveal a colocalization with a Golgi marker, suggesting a possible role in protein secretion or processing [40]. Two different RNAi strategies for silencing selM/BthD expression have been employed to show that loss of selM/BthD reduces viability, although with differing penetrance. Hypomorphic mutants generated by dsRNA injection in embryos exhibit dramatically reduced embryonic viability [41]. Moreover, the use of inducible duplex RNAi under the control of Gal4 drivers revealed that loss of selM/BthD interferes with salivary gland

Drosophila selenium metabolism and selenoproteins

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morphogenesis and reduces animal viability [40]. SelM/BthD silencing decreases total anti-oxidant capacity in embryos and Schneider cells and increases lipid peroxidation. On the other hand, transient expression of selM/BthD in the cell line decreases lipid peroxidation, suggesting that this protein may have antioxidant functions [41]. SelG/SelK/G-rich Selenoprotein G/G-rich—^named on the basis of its 28% glycine residues—is a less well-known selenoprotein with a mass of 12 kDa. Although homologs of SelG/SelK containing Cys or Sec can be found in vertebrates [5,38,39], its function remains to be elucidated. Located at position 10F4 on Chromosome X, the selG/selK gene gives rise to a single transcript that encodes a 110amino acid selenoprotein, with a Sec residue at the C-terminal penultimate position, similar to some mammalian thioredoxin reductases [22]. SelG/SelK has a cysteine paralog, SelG-like [38]. The two genes appear in tandem, separated by only 320 bp, have the same exonic structure, and share 65% identity at the protein level. Northern blot analysis shows that it is expressed at all stages of fly development [5], while in situ hybridization reveals the distribution to be ubiquitous during embryonic stages [38]. One approach to elucidating selG/selK function has been the characterization of RNAi hypomorphic mutants in cells and embryos. Embryos microinjected with dsRNA corresponding to selG/selK display decreased viability, considered as a percentage of hatched larvae, in addition to morphological defects or developmental retardation [41]. On the other hand, studies in S2 cells have not revealed an effect of SelG/SelK on the redox system. Concluding remarks We would like to conclude this chapter by addressing some of the challenging questions that remain to be resolved. First, only three selenoproteins have been identified in Drosophila so far and it remains possible that the true number of selenoproteins will prove to be higher. Moreover, while interfering with mRNA encoding selG/selK and selM/BthD seems to reduce viability [39,40], the precise cellular function of these fly selenoproteins is unknown except in the case of Sps2, an enzyme involved in the synthesis of selenoproteins [12]. Second, it will be essential to elucidate the role of the various Cyscontaining paralog genes. Do they act as a functional backup? If so, to what extent are the selenoproteins and their paralog genes redundant? Also, it remains to be determined whether or not the two variants of selenophosphate synthetase, SelD/Spsl and Sps2, are redundant. Third, loss of function mutations in selD/spsl are lethal and result in a lack of selenoproteins [1,25]. However, eEFsec mutants, which lack

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Selenium: Its molecular biology and role in human health

selenoproteins, are viable [37]. This raises the question of whether or not selenoproteins are essential for insect viability and to what extent fly selenoproteins contribute to redox balance or have adopted other functions. It is possible that, in addition to its role in Se metabolism, selD/spsl is also involved in processes that are essential for viability. Alternatively, the accumulation of Se compounds due to the lack of enzymatic activity could account for reduced viability. Drosophila genetics provides an opportunity to approach these questions using a variety of tools to create alleles for those genes and, beyond that, to study their role in metabolism, proliferation, growth and development. Transposable P-elements are still widely used as mutagenesis reagents and form the backbone of projects that seek to generate mutant insertions in every predicted gene in the fly genome. Molecularly mapped deletions have been generated at both a genome-wide and a custom-made level using genetically engineered vectors based on the FLP/FRT system [42]. Moreover, elements have been developed for a wide range of transgenic applications, including enhancer trapping, gene tagging, targeted misexpression, RNA interference delivered by the Gal4AJAS system and homologous recombination. Further genetic experiments will be required to reconcile these issues. References 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

B Alsina, F Serras, J Bagufta, M Corominas 1998 Mol Gen Genet 151-.WZ F Roch, F Serras, FJ Cifuentes, M Corominas, B Alsina, M Amoros, A Lopez-Varea, R Hernandez, D Guerra, S Cavicchi, J Bagutia, A Garcia-Bellido 1998 Mol Gen Genet 257:103 K Schwarz, CM Foltz 1957 J Am Chem Soc 79:3292 OF Combs Jr, SB Combs 1986 The Role of Selenium in Nutrition Academic Press Inc New York pp 532 FJ Martin-Romero, GV Kryukov, AV Lobanov, BA Carlson, B J Lee, VN Gladyshev, DL Hatfield 2001 J Biol Chem 276:29798 DM DriscoU, PR Copeland 2003 Annu Rev Nutr 23:17 MJ Berry, L Banu, YY Chen, SJ Mandel, JD Kieffer, JW Harney, PR Larsen 1991 Nature 353:273 A Krol 2002 Biochimie 84:765 D Behne, A Kyriakopoulos 2001 Annu Rev Nutr 2\:453 BJ Lee, M Rajagopalan, YS Kim, KH You, KB Jacobson, DL Hatfield 1990 Mol Cell fi/o/10:1940 BC Persson, A Bock, H Jackie, G Vorbruggen 1997 J Mol Biol 274:174 M Hirosawa-Takamori, H Jackie, G Vorbruggen 2000 EMBO Rep 1:441 CB Allan, GM Lacourciere, TC Stadtman 1999 Annu Rev Nutr 19:1 T Tamura, S Yamamoto, M Takahata, H Sakaguchi, H Tanaka, TC Stadtman, K Inagaki 2004 Proc Natl Acad Sci USA 101:16162 GM Lacourciere, H Mihara, T Kurihara, N Esaki, TC Stadtman 2000 J Biol Chem 275:23769 GM Lacourciere, TC Stadtman 2001 Biofactors 14:69 H Mihara, T Kurihara, T Watanabe, T Yoshimura, N Esaki 2000 J Biol Chem 275:6195

Drosophila selenium metabolism and selenoproteins 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

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PM Moriarty, CC Reddy, LE Maquat 1998 Mol Cell Biol 18:2932 TC Stadtman 1996 Amu Rev Biochem 65:83 X Zhou, SI Park, ME Moustafa, BA Carlson, PF Grain, AM Diamond, DL Hatfield, BJ Lee, 1999 J Biol Chem llA:mi9 H Romero, Y Zhang, VN Gladyshev, G Salinas 2005 Genome Biol 6: R66 DL Hatfield, VN Gladyshev 2002 Mol Cell Biol 22:3565 Z Veres, lY Kim, TD Scholz, TC Stadtman 1994 J Biol Chem 269:10597 lY Kim, Z Veres, TC Stadtman 1992 J Biol Chem 161:19650 B Alsina, M Corominas, MJ Berry, J Bagufla, F Serras 1999 J Cell Sci 112:2875 M Morey, M Corominas, F Serras 2003 J Cell Sci 116:4597 FJ Diaz-Benjumea, E Hafen 1994 Development 120:569 T Finkel 1998 Curr Opin Cell Biol 10:248 M Morey, F Serras, J Bagufla, E Hafen, M Corominas 2001 Dev Biol 238:145 M Morey, F Serras, M Corominas 2003 FEBS Lett 534:111 TL Parkes, A J Elia, D Dickinson, AJ Hilliker, JP Phillips, GL Boulianne, 1998 Nat Genet 19:171 S Gromer, JK Eubel, BL Lee, J Jacob 2005 Cell Mol Life Sci 61:1A\A SC Low, JW Harney, MJ Berry 1995 J Biol Chem 270:21659 A Matsukage, F Hirose, Y Hayashi, K Hamada, M Yamaguchi 1995 Gene 166:233 JS Jin, S Back, H Lee, MY Oh, YE Koo, MS Shim, SY Kwon, I Jeon, SY Park, K Back, MA Yoo, DL Hatfield, BJ Lee 2004 Nucleic Acids Res 32:2482 A Lescure, D Fagegaltier, P Carbon, A Krol 2002 Curr Protein Pept Sci 3:143 M Hirosawa-Takamori, HR Chung, H Jackie 2004 EMBO Rep 5:317 S Castellano, N Morozova, M Morey, M J Berry, F Serras, M Corominas, R Guigo 2001 EMBO Rep imi GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 &ie«ce 300:1439 SY Kwon, P Badenhorst, FJ Martin-Romero, BA Carlson, BM Paterson, VN Gladyshev, BJ Lee, DL Hatfield 2003 Mol Cell Biol 23:8495 N Morozova, EP Forry, E Shahid, AM Zavacki, JW Harney, Y Kraytsberg, MJ Berry, 2003 Genes Cells 8:963 E Ryder 2004 F Blows, M Ashbumer, R Bautista-Llacer, D Coulson, J Drummond, J Webster, D Gubb, N Gunton, G Johnson, CJ O'Kane, D Huen, P Sharma, Z Asztalos, H Baisch, J Schulze, M Kube, K Kittlaus, G Renter, P Maroy, J Szidonya, A RasmusonLestander, K Ekstrom, B Dickson, C Hugentobler, H Stocker, E Hafen, JA Lepesant, G Pflugfelder, M Heisenberg, B Mechler, F Serras, M Corominas, S Schneuwly, T Preat, J Roote, S Russell Genetics 167:797

Chapter 31. Selenoproteins in parasites Gustavo Salinas Catedra de Inmunologia, Facultad de Quimica-Facultad de Ciencias, Universidad de la Republica. Instituto de Higiene, Avda. A. Navarro 3051, Montevideo, CP 11600, Uruguay

Alexey V. Lobanov and Vadim N. Gladyshev Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA

Summary: Parasites, which cause an enormous burden in the population of the third world, are a diverse group of organisms, many of which are sensitive to oxidative stress imposed by their hosts. In recent years, several selenoprotein families, some with antioxidant properties, have been described and characterized in metazoan parasites. Glutathione peroxidase and thioredoxin glutathione reductase (TGR) appear to be essential selenoproteins in flatworms (phylum Platyhelminthes). TGR is the single enzyme that provides reducing equivalents to both thioredoxin and glutathione pathways, in contrast to hosts, which evolve parallel pathways. In roundworms (phylum Nematoda), selenoproteins have recently been described, revealing species differences in the Sec/Cys protein sets and the presence of an unusual SECIS element. Plasmodium sp, one of the most important protozoan parasites that affect humans, also decode Sec. The selenoprotein families encoded by Plasmodial genomes have neither Sec nor Cys homologs in their hosts, raising the possibility that targeting their selenoproteomes may provide new treatment strategies. Introduction Although significant research efforts have been made to study selenoproteins and selenocysteine insertion systems in humans and various model organisms, little has been reported in the literature regarding the utilization of selenium in eukaryotic parasitic organisms. This chapter focuses on the progress made in the characterization of selenoenzyme families in flatworms, the recent advances in the synthesis and utilization of selenoproteins in roundworms and protozoan parasites, and discusses why selenoproteins of platyhelminths and Plasmodia may represent interesting targets for chemo- or immune-prophylaxis.

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Parasites: diverse organisms that face similar oxidative stress challenges Parasites live at least part of their lifecycle inside another organism (the host), which they exploit for their own survival and reproduction. This definition includes different types of infectious agents (viruses, bacteria, fungi, protozoa, helminths). However, for historical reasons, the term is most often reserved for 'protozoa' and 'helminths' organisms. Indeed, parasitology was identified as a separate research field during the exploration of the tropics and the establishment of 'tropical medicine' [1]. Both 'protozoa' and 'helminths' also include free-living organisms, and neither 'protozoa' nor 'helminths' are monophyletic; on the contrary, both groups are represented by highly divergent phyla. Nonetheless, this historical classification is not useless. These two groups of parasites are very different: protozoan are unicellular protists, which multiply quickly within the host, and are, in most cases, intracellular in habitat; in contrast, helminths are metazoan organisms with complex multicellular organization (with nervous system and reproductive organs), which undergo complex metamorphoses and migrations within the host. Table 1 presents the main features of the major human parasitic infections. Table 1. Major human parasites (Source: [2])

Protozoan parasites'* Species (Disease) Plasmodium sp (Malaria) Trypanosoma brucei (sleeping sickness ) Trypanosoma cruzi (Chagas disease*) Leishmania sp (Leishmaniasis)

Helminths parasites^ Species/Disease Schistosoma sp (Schistosomiasis or bilharzia^) Onchocerca volvulus (Onchocerciasis or river blindness ) Filariidae family (Lymphatic filariasis )

Phylum Apicomplexa

Death per year/DALYs' 1,124,000/42,280,000

Kinetoplastida

50,000/1,590,000

Kinetoplastida

13,000/649,000

Kinetoplastida

59,000/2,357,000

Phylum Plathyhelminthes

Death per year/DALYs 15,000/1,760,000

Nematoda

0/987,000

Nematoda

0/5,644,000

"DALYs: DisabiUty Adjusted Life Years (the number of healthy years of life lost due to premature death and disabiUty). Protozoan parasites include many diverse phyla, among them Apicomplexa and Kinetoplastida.

Selenoproteins in parasites

357

Distribution: mainly confined to poorer tropical areas of Africa, Asia and Latin America. More than 90% of malaria cases and the great majority of malaria deaths occur in ttopical Africa. Plasmodium falciparum is the main cause of severe clinical malaria and death. Distribution: 36 countries in sub-Saharan Africa *Distribution: Latin America f Distribution: Endemic in 88 countries on 4 continents. Two forms of the disease: cutaneous (caused by Leishmania major), and visceral (caused by L. donovani) Helminth parasites are contained in three phyla: Nematoda (roundworms), Platyhelminthes (flatworms) and Acantocephala (spiny-headed worms). Helminth infections are rarely fatal, but pose an enormous burden to human population in the tiopics Distribution: endemic in 74 developing countries with more than 80% of infected people living in sub-Saharan Africa Distribution: 35 countries in total. 28 in tropical Africa, where 99% of infected people live. Isolated foci in Latin America and Yemen. Distribution: Endemic in over 80 countries in Africa, Asia, South and Central America and the Pacific Islands. Three species are of significance, Wuchereria bancrofti, Brugia malayi and Brugia timori.

In Spite of the diversity of parasites, all face similar biological problems that relate to their parasitic lifestyle. Among them, the neutralization of the effector mechanisms deployed by the host immune system is of paramount importance. Resident macrophages and inflammatory-site phagocytic leukocytes (mostly neutrophils, but also monocytes and eosinophils, depending of the type of infection) are cells equipped to kill foreign organisms. They possess an oxidase system located in their plasma membrane, which becomes activated upon certain stimuli, for example, by interaction of cell receptors with antibodies bound to the foreign organism or with parasite molecular motifs (Figure la) [3]. Subsequently, 'respiratory burst' (increase in oxygen uptake not linked to respiration) takes place and produces superoxide anion and additional reactive oxygen species (ROS) [4]. Large amounts of nitric oxide (NO) are also produced by macrophages (and to a lesser extent by neutrophils) activated by a variety of immunological stimuli, such as y-interferon and tumor necrosis factor. NO reacts with superoxide to produce peroxynitrite and other reactive nitrogen species (RNS) (Figure lb) [5]. In addition, activated neutrophils and eosinophils release myeloperoxidase and eosinophil peroxidase, respectively, that catalyze the conversion of hydrogen peroxide and halides into hypohalous acids that are powerful oxidants and can form further damaging species [4]. Collectively, ROS and RNS are powerful oxidants and nitrating species: they can inactivate enzymes and initiate the process of lipid peroxidation and nitration, which leads to radical chain reactions that further damage membranes, nucleic acids and proteins (Figure Ic). These processes (and an additional arsenal of the host effector cells, such as hydrolytic enzymes) may

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Selenium: Its molecular biology and role in human health

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R 9 , RSS?, R3DK R 3 0 0 H nittotyrosine , inactivatlon of Fe/Sclusteis (metal OMdation) • Lipid peroxidation and nitration: LDOK LNO,, IDONO^, propagation of radical ctiain reactions • DMA strand breaks

(d) Bizymatic defenses SOD GR< •
FEH ONOOH-+NO, FSff?-* R3H R90OH^.R33H

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359

Figure 1. Reactive oxygen and nitrogen species generated by the host immune response and antioxidant defenses, (a) Recognition of parasites by host leukocytes (such as macrophages, neutrophils and eosinophils) occurs by pattern recognition receptors (PRR) that bind to pathogen-associated molecular patterns (PAMPs), or through antibodies (Ig), and leads to activation of host immune cells. Upon activation, these cells produce superoxide ("02") and nitric oxide ('NO) radicals. 'NO is produced in the cytosol (but can cross membranes) by inducible nitric oxide synthase (iNOS); 02" is produced by a multi-component, membraneassociated NADPH oxidase. Superoxide is released towards the extracellular space in the case of non-phagocytosable parasites {e.g., worms), or towards the phagosome (topologically equivalent to the extracellular space) in the case of intracellular parasites {e.g., protozoans), (b) 'NO and 'O2" react at diffusible controlled rate to produce peroxynitrite (ONOO"). Peroxynitrite can react in one-electron oxidations {e.g., with transition metal centers), two electrons oxidations (of a given target), or with CO2, redirecting its reactivity. It also decomposes spontaneously into other ROS and RNS such as 'OH and NO2 • . In addition, activated neutrophils and eosinophils release myeloperoxidase and eosinophil peroxidases, respectively, which catalyze the conversion of hydrogen peroxide and halides into hj^sohalous acids, (c) Collectively, these products can inactivate enzymes, damage membranes and nucleic acids, and ultimately kill the parasitic organisms. (D) Parasites' defenses include antioxidant enzymes that directly scavenge superoxide, decreasing peroxynitrite formation (superoxide dismutases), and hydrogen and organic peroxide reductases (GPx and TPx). Some TPx have also been shown to reduce peroxynitrite catalytically. Repair mechanisms include methionine sulfoxide reductase, thioredoxin, and sulfiredoxin among others. *R'H denotes a hydrocarbon chain, or alcohol (R'H=ROH), or a thiol R'H=RSH)

ultimately lead to killing parasitic organisms. Yet, well-adapted parasites cope with the oxidative stress imposed by the host's immune response by a series of cellular chemicals and antioxidant enzymes that directly neutralize ROS and RNS (Figure Id), and constitute important model organisms to study antioxidant defense. Several antioxidant enzymes found in parasites belong to selenoprotein families. Glutathione peroxidase: tlie first selenoenzyme described in parasites Glutathione peroxidase was the first selenoenzyme to be characterized from a parasite. A cDNA from the platyhelminth Schistosoma mansoni encoding a GPx with a TGA in-frame at the active site was cloned in the early 1990s [6]. The protein encoded by this gene has biochemical properties similar to mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPx); its activity being higher with phosphatidyl choline hydroperoxide and other phospholipid hydroperoxides than with hydroperoxide substrates, such as cumene hydroperoxide and hydrogen peroxide [7]. GPx and superoxide dismutase, another antioxidant enzyme, co-localize in the tegument and gut epithelium of adult worms, which are the exposed interfaces of the parasite towards the host [8]. Additional evidence suggests that antioxidant enzymes, and GPx in particular, are vital for ROS neutralization and parasite survival within the host. Indeed, expression of GPx is developmentally regulated, with the highest levels present in the adult worm [8], the stage most resistant

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Selenium: Its molecular biology and role in human health

to oxidative stress and immune elimination [9]. In addition, GPx expression is upregulated by hydrogen peroxide and xanthine/xanthine oxidase generated ROS [10]. Recently, a search for GPx in Expressed Sequence Tag databases (dbEST) of platyhelminths identified a second GPx (GPx2) in S. mansoni and S. japonicum [11]. GPx2 also encodes a Sec residue at the active site and possesses an N-terminal signal peptide, which targets this isoform to the extracellular compartment, suggesting that this secreted variant would be important for extracellular hydroperoxide removal, helping to protect the parasite in its immediate environment. In this study, a GPxl ortholog whose 3 '-untranslated region revealed the presence of a SECIS element was also identified in Echinococcus granulosus (another flatworm) transcriptome using the SECISearch algorithm (Chapter 9 and http://genome.unl.edu/SECISearch.html) [12]. In contrast to platyhelminths, the corresponding Cys-containing enzymes appear to occur in nematodes [13], as reviewed in [14]. Nevertheless, recent datamining of nematode dbEST revealed some exceptions (see below) [15]. Free-living nematode Caenorhabditis elegans has no Sec-containing GPx encoded in its genome [15]. GSH- and Trx-reduction pathways in platyhelminth parasites are controlled by a single selenoenzyme In most living organisms, there are two analogous and mutually supporting enzymic systems that provide antioxidant defense to cells: the glutathione (GSH) and the thioredoxin (Trx) systems (Figure 2) [16,17]. These systems have overlapping yet distinct targets. GSH, due to its reactivity and intracellular concentration, is one of the most important cellular antioxidants, being efficient in rescuing small disulfide molecules and in reacting directly with ROS. The major function of Trx is to maintain cysteine residues in substrate proteins in the reduced form. In addition to their direct function as antioxidants, GSH and Trx provide electrons to GPx and Trx peroxidase (TPx), respectively, which reduce hydrogen peroxide and organic hydroperoxides, and to methionine sulfoxide reductase, which is also an important antioxidant repair enzyme. GSH and Trx are usually reduced by GSH and Trx reductases (GR and TR), respectively, at the expense of NADPH oxidation. Recent characterization of these systems in platyhelminth parasites has shown that 'conventional' GR and TR are absent; instead, the GSH and Trx systems are intermingled with the enzyme thioredoxin glutathione reductase (TGR), which provides reducing equivalents to both pathways (Figure 2).

Selenoproteins in parasites

361

(a) Comparison of the SSH, Tnc and llntod Tnc43SH systems

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362

Selenium: Its molecular biology and role in human health

Figure 2. Linked thioredoxin-glutathione systems, (a) Comparison of thioredoxin, glutathione and linked thioredoxin-glutathione systems. The glutathione system comprises (i) GR, GSH and Grx, whereas the thioredoxin system consists of (ii) TR and Trx. In linked Trx-GSH systems (iii), TGR functionally replaces TR, GR and Grx, providing reducing equivalents to targets of both systems. In all systems, NADPH is the upstream donor of reducing equivalents, (b) Components of the thioredoxin and glutathione systems. Redox centers of GR, TR, TGR, Grx and Trx are indicated, as well as the FAD prosthetic group and the ligands NADPH and GSH. TR and TGR possess a C-terminal extension missing in GR, which contains the Cterminal GCUG redox-active motif TGR possesses an N-terminal Grx domain that is absent in TR and GR. The Grx and Trx domains contain the CXXC redox center. Grx, unlike Trx, binds GSH. (c) Schematic representation of electron flow in TGR. TGR, like GR and TR, is a homodimer, with monomers oriented in a head-to-tail manner. Electrons flow from NADPH to FAD, to the CX4C redox center, to the C-terminal GCUG redox center of the second subunit, to the CX2C redox center of the Grx domain of the first subunit, and to targets, including GSSG (left scheme). Alternatively, electrons can flow, presumably directly, from the GCUG redox center to Trx (right scheme). The model proposes a flexible hinge, which connects the TR and Grx domains. This organization allows electrons to flow to the "in built' Grx domain or to Trx. Parts (a) and (b) in the figure reprinted with modifications from [11] with copyright with permission from Elsevier.

This protein is a second selenoenzyme family that has been characterized in platyhelminth parasites (reviewed in [11]). TGR is an oxidoreductase shown to possess TR, GR and Grx activities, achieving its broad substrate specificity by a fusion between Grx and TR domains (Figure 2b); this domain fusion was originally described in a mouse testis TGR [18]. Experimental and in silico data support the proposition that TGR is the single enzyme responsible for recycling both oxidized Trx and GSH in platyhelminth parasites. Treatment of S. mansoni adult worm extracts with auranofin, a known inhibitor of Sec-containing TRs, resulted in complete inhibition of TR and GR activities [19]. In addition, TGR was the single protein isolated from Taenia crassiceps (also a flatworm) extracts as a result of tracing GR and TR activities [20]. Examination of EST databases from Schistosoma species, which covers more than 90% of the gene content of this organism [21], revealed cDNAs encoding TGR, but not conventional TR or GR [11]. The biochemical characterization of E. granulosus and T. crassiceps TGR indicated that the native enzyme shuttles elecfrons from NADPH to oxidized Trx (TR activity), GSSG (GR activity) and glutathionemixed disulfides (Grx activity). The stoichiometric inhibitory effect of auranofin on both GR and TR activities of TGR indicates that the Seccontaining C-terminal redox center participates in elecfron fransfer to GSSG and oxidized Trx [20,22]. In addition, TR and Grrx domains can function either in coupled reactions or independently. Conventional TRs neither bind GSH nor possess GR activity; thus, the N-terminal Grx domain of TGR would reduce GSSG, accepting elecfrons from the Sec-containing C-terminal redox center. The idea that the C-terminal redox center donates elecfrons to the fused Grx domain implies that the Grx domain of TGR would be linked

Selenoproteins in parasites

363

to the TR domains by a flexible hinge to allow reduction of the oxidized Trx (Figure 2c). It is interesting to note that T. crassiceps TGR showed a hysteretic behavior in enzymatic assays with GSSG at high concentrations; this observation led the authors to propose a model in which TGR would possess high and low affinity sites for glutathione [20]. Clearly, further biochemical characterization and structural data on this multifunctional enzyme are needed that will shed light on the mechanism of catalysis, hi addition, molecular characterization of the corresponding gene could also provide clues regarding the mechanism of generation of isoforms. hideed, the analysis of TGR in E. granulosus revealed two trans-spliced cDNAs derived from a single gene [22]. These variants code for mitochondrial (mt) and cytosolic (c) TGRs, containing identical Grx and TrxR domains, but differing in their N-termini. These variants derive from alternative initiation of transcription, followed by trans-splicing. Similarly, mtTGR and cTGR variants also derived fi"om a single gene have been identified in S. mansoni [11]. Collectively, the results from platyhelminth studies strongly suggest that TGR is the main pyridine-nucleotide thiol-disulfide oxidoreductase in these organisms, in contrast to their hosts, where there is some redundancy of mechanisms for recycling oxidized Trx and GSH. Very little has been published about these pathways in the other phylum of helminth parasites (Nematoda), and to the best of our knowledge, nothing is known about Sec/Cys-containing TR or TGR in parasitic nematodes. However, no single genome has yet been completed from metazoan parasites. Selenoproteins of nematode parasites: old families, unusual SECIS An in silica analysis of Caenorhabditis elegans and Caenorhabditis briggsae (free-living nematodes) genomes revealed that these organisms encode a single a selenoprotein, TR [15], corroborating earlier experimental data [23]. However, no experimental studies have yet been performed with selenoproteins from parasitic nematodes. Nevertheless, in a recent study [15], the existing nematode ESTs were searched for selenoprotein genes using SECISearch and by screening for homologs of known selenoproteins. These analysis identified selenoprotein homologs of selK, selT, selW, Sepl5, selenophosphate synthetase and GPx. Two interesting points were noted from these analyses. First, various nematodes encode different selenoproteins, and the distribution of selenoprotein families within this phylum is mosaic. Second, it was found that all detected nematode selenoprotein genes contained an unusual form of SECIS element, with G rather than a canonical A at the conserved position preceding the quartet of non-Watson-Crick base pairs [15].

364

Selenium: Its molecular biology and role in human health

Selenoproteins of protozoan parasites: waiting for surprises? Very little is known about selenoproteins from protozoan parasites. Recently, the presence of tRNA^^' was described in several species of the phylum Apicomplexa [24] (Lobanov et al., submitted). Plasmodium falciparum, which is the causative agent of malaria - the most overwhelming human parasitic infection, belongs to this phylum. The finding of tRNA^'" was consistent with the presence of putative EFsec and selenophosphate synthetase in P. falciparum and other Plasmodia. In addition, tRNA^*'' was observed in Toxoplasma, but not in Cryptosporidium parasites. Genomewide searches for SECIS elements in the six Plasmodium genomes revealed four selenoprotein genes. Interestingly, homology analyses of these proteins identified no hits outside Apicomplexa, suggesting that these selenoproteins do not exist in the apicomplexan hosts. These properties make the new selenoproteins attractive targets for anti-malaria drug development. The other reference in the literature to a parasite Sec-decoding protozoan is the description of a Cys-containing selenophosphate synthetase from Leishmania major [25]. Leishmania belongs to trypanosomatidae family, which also includes Trypanosoma brucei, and T. cruzi (Table 1), which are causative agents of disabling and fatal diseases in the poorest rural population of the third world [26]. Consistent with the finding of selenophosphate synthetase, recent bioinformatics analyses revealed three selenoprotein genes in several Trypanosoma genomes (Lobanov and Gladyshev, unpublished). Finally, no single reference could be found in the literature regarding a Sec-decoding amoebae, a traditional group of protozoa that include the parasitic amoebae of humans, Entamoebae histolytica. Parasite selenoproteins: drug or vaccine candidates? From a global perspective, the confrol of parasitic infections, which are a major cause of disability and mortality in many developing countries, remains as one of the most important challenges for medicine in the 21^' century [2]. Although there are safe and effective drugs to control some parasitic diseases, parasites can develop resistance to drugs rendering them ineffective, as it has been the case of certain antimalarial drugs [27]. Thus, effective vaccines and new drugs against parasitic organisms are needed. The task ahead is enormous considering that parasite and hosts are eukaryotic organisms; as yet, there is not a single vaccine for a human parasitic infection. Whether selenoproteins can be drug targets or generate immunity depends on premises that are not necessarily different from those for any other target protein: the validity of a drug target would rely on it being an essential protein, and sufficiently different from the host homolog(s) as to be selectively inhibited. Likewise, a good vaccine candidate should generate an

Selenoproteins in parasites

365

appropriate and selective immune response against the parasite, without inducing pathology to the host. In platyhelminths, TGR is an attractive pharmacological target because of the lack of redundant mechanisms (i.e., TR and GR) to provide reducing equivalents to essential enzymes. Inhibition of this enzyme could lead to impaired synthesis of DNA and antioxidant defenses, compromising parasite survival. TGR may also be a good vaccine candidate, since it is a large protein with a degree of identity to host enzymes below 60%. However, there are no studies regarding TGR as an immunogen. Contrary to TGR, there are promising studies on the use of GPx as a vaccine candidate. Vaccination of mice (not a natural host) against the platyhelminth S. mansoni with naked DNA constructs containing Sec-containing GPx showed significant levels of protection compared to a control group [28]. In this context, it is important to emphasize not only the fact that GPx appears to be important at the host parasite interface, but also that platyhelminth lack catalase and rely exclusively on GSH and Trx peroxidases for hydrogen peroxide removal. In the case of protozoan parasites, further studies are needed to identify and functionally characterize their selenoproteins. Nevertheless, it is highly significant that the four selenoproteins identified in Plasmodium sp have neither Sec nor Cys homologs in humans. Considering that Sec is usually located at the redox-active sites of enzymes, the selenol- and thiol-based redox systems may play vital an important role in the survival of protozoan parasites [29]. Finally, selenoproteins may be different to other proteins in one respect: electrophilic drugs, such as gold or platinum compounds, or alkylating agents that react preferentially with Sec over Cys may affect the parasite and the host to a different extent, depending on the relative importance of selenoproteins for the two organisms, and the presence/absence of Cyscontaining enzymatic back up systems. Acknowledgements This work has been supported by Fogarty International Research Collaboration Award TW006959 and Ministry of Education, Uruguay, PDT 29/171. References 1. 2. 3. 4. 5. 6.

K Warren 1988 The Biology of Parasitism PT Englund, A Sher (Ed) Alan R. Riss Inc New York 3 WHO The world health report -changing history. 2004 (http://www.who.int/whr/2004/en/report04_en.pdf) DH McGuinness, PK Dehal, RJ Pleass 2003 Trends Parasitol 19:312 BG Halliwell, JMC Gutteridge 1999 Free Radicals in Biology and Medicine Oxford University Press Inc New York R Radi, G Peluffo, MN Alvarez, M Naviliat, A Cayota 2001 Free Radic Biol Med 30:463 DL Williams, RJ Pierce, E Cookson, A Capron 1992 Mol Biochem Parasitol 52:127

366 I. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26.

27. 28. 29.

Selenium: Its molecular biology and role in human health M Maiorino, C Roche, M Kiess, K Koenig, D Gawlik, M Matthes, E Naldini, R Pierce, L Flohe 1996 EurJBiochem 238:838 H Mei, PT LoVerde 1997 Exp Parasitol 86:69 GM Mkoji, JM Smith, RK Prichard 1988 Int J Parasitol 18:661 UE Zelck, B Von Janowsky 2004 Parasitology 128:493 G Salinas, ME Selkirk, C Chalar, RM Maizels, C Fernandez 2004 Trends Parasitol 20:340 GV Kryukov, VM Kryukov, VN Gladyshev \999 J Biol Chem 274:33888 L Tang, K Gounaris, C Griffiths, ME Selkirk 1995 J Biol Chem 270:18313 K Henkle-Duhrsen, A Kampkotter 2001 MolBiochem Parasitol 114:129 K Taskov, C Chappie, GV Kryukov, S Castellano, AV Lobanov, KV Korotkov, R Guigo, VN Gladyshev 2005 Nucleic Acids Res 2005 ZTi-.llll A Holmgren 2000 Antioxid Redox Signal 2:811 PG Winyard, CJ Moody, C Jacob 2005 Trends Biochem Sci 30:453 QA Sun, L Kimarsky, S Sherman, VN Gladyshev 2001 Proc Natl Acad Sci USA 2001 98:3673 HM Alger, AA Sayed, MJ Stadecker, DL Williams 2002 Int J Parasitol 32:1285 JL Rendon, IP del Arenal, A Guevara-Flores, A Uribe, A Plancarte, G MendozaHemandez 2004 Mol Biochem Parasitol 133:61 S Verjovski-Almeida, R DeMarco, EA Martins, PE Guimaraes, EP Ojopi, AC Paquola, JP Piazza, MY Nishiyama, Jr., JP Kitajima, RE Adamson, PD Ashton, MF Bonaldo, PS Coulson, GP Dillon, LP Farias, SP Gregorio, PL Ho, RA Leite, LC Malaquias, RC Marques, PA Miyasato, AL Nascimento, FP Ohlweiler, EM Reis, MA Ribeiro, RG Sa, GC Stukart, MB Soares, C Gargioni, T Kawano, V Rodrigues, AM Madeira, RA Wilson, CF Menck, JC Setubal, LC Leite, E Dias-Neto 2003 Nat Genet 2003 35:148 A Agorio, C Chalar, S Cardozo, G Salinas 2003 J Biol Chem 2003 Apr 1111%: 12920 VN Gladyshev, M Krause, XM Xu, KV Korotkov, GV Kryukov, QA Sun, BJ Lee, JC Wootton, DL Hatfield 1999 Biochem Biophys Res Commm 259:244 T Mourier, A Pain, B Barrell, S Griffiths-Jones 2005 RNA 11:119 PC Jayakumar, VV Musande, YS Shouche, MS Patole 2004 DNA Seq 15:66 CM Morel, T Acharya, D Broun, A Dangi, C Elias, NK Ganguly, CA Gardner, RK Gupta, J Haycock, AD Heher, PJ Hotez, HE Kettler, GT Keusch, AF Krattiger, FT Kreutz, S Lall, K Lee, R Mahoney, A Martinez-Palomo, RA Mashelkar, SA Matlin, M Mzimba, J Oehler, RG Ridley, P Senanayake, P Singer, M Yun 2005 Science 309:401 TE Mansour Chemotherapeutic Targets in Parasites: Contemporary strategies 2002 T Mansour (ed) Cambridge University Press Cambridge 4 KA Shalaby, L Yin, A Thakur, L Christen, EG Niles, PT Lo Verde 2003 Vaccine 22:130 S MuUer, E Liebau, RD Walter, RL Krauth-Siegel 2003 Trends Parasitol 19:320

Chapter 32. Incorporating 'omics' approaches to elucidate the role of selenium and selenoproteins in cancer prevention Cindy D. Davis and John A. Milner Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

Summary: Epidemiologic and preclinical studies provide evidence that increasing dietary selenium (Se) may have cancer protective properties. However, variation in cancer incidence among and within populations with similar Se intake suggests that an individual's response may reflect interactions with genetic and/or environmental factors. The "omics" of nutrition (nutrigenomics, nutrigenetics, nutritional epigenomics, nutritional transcriptomics, proteomics and metabolomics) may assist in understanding the cellular and molecular events associated with the cancer protective effects of Se, as well as in identifying responders and non-responders. Approaches, that utilize transgenic and knockout mice with altered selenoprotein expression offer models to evaluate the importance of selenoproteins or small molecular selenocompounds in mediating the cancer protective effects of Se. While the challenges will be enormous, the potential rewards in terms of both cancer morbidity and mortality will be of equally great magnitude. Introduction Extensive evidence indicates that dietary selenium (Se) supplementation reduces the incidence of cancer in experimental animals. Adding Se to the diet or drinking water inhibits initiation and/or post-initiation stages of liver, esophageal, pancreatic, colon and mammary carcinogenesis and spontaneous liver and mammary tumorigenesis in several rodent models [reviewed in 1,2]. Similarly, ecological studies have usually found an inverse relationship between Se status and mortality from cancer of the large intestine, rectum, prostate, breast, ovary, lung, and leukemia [3]. Data from most case-control and cohort studies indicate its possible protective relationship with lung and prostate cancer, but data is not overly convincing for other cancer sites, including breast and colon/rectum [4]. A recent meta-analysis suggests that Se supplementation may afford some protection against lung cancer in populations where average Se levels are traditionally low [5]. Evidence

368

Selenium: Its molecular biology and role in human health

suggests that toenail Se may be a useful predictor of status [5]. Cohort studies also have identified low baseline serum or toenail Se concentrations as a risk factor for prostate cancer [6,7]. A recent intervention study provides the most compelling evidence for the protective effects of Se against cancer [8]. This randomized controlled trial was designed to test Se as a deterrent to the development of basal or squamous carcinomas. Secondary end-point analyses showed that the mineral resulted in a significant reduction in total cancer mortality (RR = 0.5), total cancer incidence (RR = 0.63), and incidence of lung (RR = 0.54), colorectal (RR = 0.42), and prostate (RR = 0.37) cancer [8]. Participants with baseline plasma Se concentration in the lowest two tertiles (^NMe,

^

^

^

20

Singh et al reported the GPx-like activity of a series of diaryl diselenides having intramolecular Se—N interactions [42]. It was found that the diselenides that have quite strong Se—N intramolecular interactions were less active, whereas the diselenides that contain a built-in basic amino group but no Se—N interactions showed excellent GPx activity [43]. The ferrocene-based diselenides (18-20 in Scheme 3) were reported to display much higher activities than those of the phenyl-based diselenides, and the enhancement in catalytic activity could be ascribed to the synergistic effect of redox-active ferrocenyl and internally chelating amino groups.

Selenoprotein mimics

391

By analogy to diselenides, ditellurides and related compounds were proposed as GPx mimics. Engman et al [44] was first to report that some of these compounds (e.g. 21-22) show GPx-like activity. Modifying the basic structure of diphenyl ditelluride based on substituent effects and isosteric replacements further impacts the reactivity of diphenyl ditelluride. More recently, Mugesh et al [45] directly compared the thiol peroxidase activity of several ditellurides (e.g., 23-26 in Scheme 4) with that of their selenium analogues. All ditellurides were found to be much more efficient catalysts than the corresponding diselenides in reducing H2O2 with PhSH as cosubstrate. Scheme 4 ^R

"O'^'K!}"' "O'^'-^'O'' 0-^=-'^^ 22

21

oci

^ 24

23

22-1

NMej

Teh

25

26

Scheme 5 X-X

0

0)_ I ' f 27,28 X = Se, Te

32

0

29-31 X =Se,Te,Sec o HNCH2(CH2NHCH2)„CH2-N^

Se-S(

SeOgH

Q

j[

J

SeOgH

33-35 n = 1-3

Cyclodextrins (CDs) are cyclic oligosaccharides containing a hydrophobic cavity, in which many complexes can be formed via host-guest chemistry [46]. They have extensively been exploited as enzyme models and molecular receptors [47]. To elucidate the effect of substrate recognition on catalysis, a series of cyclodextrin-derived organoselenium and organotellurium compounds (e.g. 27-35) were developed as GPx models (Scheme 5) [48-51].

392

Selenium: Its molecular biology and role in human health

The first model compound 29 was prepared by attaching a diselenide group to the CD primary face [48]. Attachment of a ditelluride group onto cyclodextrin resuhed in GPx models 28 (2-TeCD) and 30 (6-TeCD) [50]. The catalytic efficiency of 2-TeCD-catalyzed reduction of hydroperoxides by GSH was found to be 350,000-fold higher than that involving diphenyl diselenide (PhSeSePh). Selenoenzyme transformation by chemical modification Transformation of natural enzymes into selenoenzymes Although many low molecular weight GPx mimics are known, they possess serious disadvantages: low activity, low solubility in water, and in some cases, toxicity. In this regard, natural proteins may have advantage as protein macromolecules carry molecular information for both substrate recognition and efficient catalysis. Engineering proteins by genetic and chemical methods is a valuable strategy for introducing new functions into protein scaffolds. So far, three corresponding protein design methods have been described: site-directed mutagenesis, chemical modification and the combination of both. By using these strategies, natural enzymes, proteins and antibodies have been used successfully to construct efficient selenoenzyme models [16]. Scheme 6 ICH2—Q_'^!^L_/2|l/4(H

"^°2

/||^^XxO;H

The first example in the field of selenoenzyme design is the chemical conversion of the active site serine residue of the bacterial serine protease subtilisin (EC 3.4.21.14) into selenocysteine [52]. The hydroxyl group of Ser221 could be selectively modified to introduce distinct functional groups into the active site of subtilisin. Inspired by the earlier work on the first semisynthetic enzyme, thiolsubtilisin [53], Wu and Hilvert prepared selenosubtilisin by using a similar method [54] (Scheme 6). The semisynthetic selenoenzyme exhibited significant GPx-like redox activity. It catalyzed the reduction of a variety of hydroperoxides by 3-carboxy-4nitrobenzenthiol (ArSH). The reduction of ter^butyl hydroperoxide (^ BuOOH) by ArSH was at least 70,000-fold faster than the reaction catalyzed by diphenyl diselenide, a well-studied antioxidant [54,55]. Since selenosubtilisin has the same substrate binding pocket as subtilisin, it was possible to rationalize and even predict its substrate selectivity. Thus, a series of different racemic hydroperoxides was chemically synthesized by the Schreier group and subjected to selenosubtlisin-catalyzed reactions [56,57].

Selenoprotein mimics

393

All alkyl aryl hydroperoxides showed an enrichment of enantiomers. In a similar fashion, Liu et al prepared a selenotrypsin by converting the active site serine into Sec [58]. The study revealed that GSH is not a particularly good substrate for selenotrypsin. Nevertheless, the data showed that it was possible to convert an active site serine into Sec in various serine proteases. Recent studies showed that tellurium is an excellent alternative element for the construction of GPx models [14-16]. However, introducing tellurium into proteins is currently a challenge. Following selenosubtilisin, Liu and coworkers developed a methodology to introduce tellurium into the binding pocket of subtilisin and yielded a first semisynthetic telluroen2yme tellurosubtilisin (Scheme 6) [59]. Like natural GPx, tellurosubtilisin can catalyze the reduction of ROOH by thiols efficiently and acts as an excellent GPx mimic.

Table 1. Catalytic activities of selenoprotein GPx mimics (Data from ref. 21). selenoenzyme mimic

GPx activity (U/nmol)

ebselen

1

printed protein

100-800

catalytic antibody

1100-24300

selenoGST

2000-6200

natural GPX

5780

Although seleno/tellurosubtilisin and selenotrypsin were generated via covalent modification of naturally occurring enzymes [54,58,59], it is a great challenge to prepare highly efficient semisynthetic enzymes that can rival natural selenoenzymes. Recently, Luo et al developed a method to mimic the action of GPx by chemically modifying a naturally occurring enzyme glutathione transferase (GST, EC.2.5.1.18) [60]. Taking advantage of the highly specific GSH binding site of GST, seleno-GST(Se-GST) was generated by chemical mutation using a method described for preparation of selenosubtilisin [54]. The selenium-containing Se-GST can efficiently catalyze the reduction of hydrogen peroxide with an activity that is greater than that for some natural counterparts (Table 1) [60].

394

Selenium: Its molecular biology and role in human health

Transformation of natural proteins into selenoenzymes To create an efficient artificial enzyme, the affinity for the substrate in the enzjmie-substrate complex must be reasonably high, and the catalytic groups should be adjacent to the reactive group of the substrate. An alternative approach to artificially creating such binding sites is the molecular imprinting technique [61]. Biopolymers can also be used as an alternative backbone for the imprinting procedure. This innovation has led to the development of the bioimprinting technique for the synthesis of proteinbased binding and catalytic sites. An imprinted enzyme model with GPx activity has been developed by a combination of bioimprinting and chemical mutation [62], A';,S-bis-2,4dinitrophenyl-glutathione (GSH-2DNP), a GSH derivative, was synthesized and acted as a template molecule (36 in Scheme 7). In the bioimprinting process, the imprinted molecule was allowed to interact with denatured proteins (e.g., egg albumin) to form a new conformation via hydrogen bonds, ion pairing and hydrophobic interactions. Scheme 7 N02 N02

--

H^

'>N02 '

O - < 0 > - "COOH - ^ ^ N J YO" ^ COOH 36

The new conformation was then fixed using the cross-linker glutaraldehyde. After removal of the imprinting molecule by dialysis, the serine residues located at the binding sites of the inprinted proteins were activated using phenylmethene sulfonyl fluoride and then converted into Sec in the presence of NaHSe. The imprinted protein exhibits GPx activity and is 100-800 fold more active than ebselen (Table 1). Transformation of antibodies into selenoenzymes One important way for designing binding sites is the use of antibodies. A recognition site for enzyme substrate is easy to generate by using a standard monoclonal antibody (McAb) preparation technique. This strategy was widely applied in the design of catalytic antibodies using transition state analogs as haptens. Recently, Luo et al employed this strategy in the design of GPx mimics [63-65]. The authors used substrate analogs instead of transition state analogs as haptens in order to generate monoclonal antibodies with the substrate binding site. In the design of catalytic antibodies, the polar groups of substrate GSH were modified by different hydrophobic

Selenoprotein mimics

395

groups and the modified substrates were used as a series of haptens (Scheme 8). Scheme 8 NH2

o

H

6

O

S

ISIH-,

"

o

o

HO

os-^^^o

6^°^

O H ,-^0 O

NO2

H3CO-^^'^V^N'^^V"OCH3 O H NH2 40

37-39 R = H, CHj, CHjCHjCHjCHj

The substrate binding sites were first made by monoclonal antibody preparation technique using hydrophobically modified GSH and GSSG as haptens (37-40 in Scheme 8). Thus, not only is the hydrophobic cavity of the antibody similar to that of the active site of native enzymes, but the affinity of the antibody active site for the substrate can also be adjusted to that of the native enzyme. The catalytic Sec was then incorporated into the McAb by chemical modification of the serine residue (Scheme 9) [63]. Surprisingly, these catalytic antibodies exhibited remarkably high catalytic efficiency which could rival the natural enzyme (e.g., rabbit liver GPx) (Table 1). In order to produce pharmaceutical proteins and elucidate the reason why this novel catalyst exhibited high catalytic efficiency, a selenium-containing single chain antibody was prepared (Se-ScFv) [65]. Scheme 9

MaAb production

y - y I 'S-CHjOH

PMSF

f - y " lS-CH20S02CH2-/>

NaHSe

(^ " ljS-CH2SeH

Similarly, Ding and coworkers [19] prepared a selenium-containing catalytic antibody (Se-4C5) by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T4) into Sec. Se-4C5 catalyzes the deiodination of T4 to 3,5,3'-triiodothyronine (T3) in the presence of dithiothreitol via a ping-pong mechanism, with a Vmax value of 270 pmol mg" ' min"'. Thus, Se-4C5 acted as a deiodinase mimic.

396

Selenium: Its molecular biology and role in human health

Transformation of proteins into selenoenzymes by genetic engineering Since Sec is encoded by a stop codon UGA, it is difficult to prepare selenoproteins by traditional recombinant DNA technology. The most suitable approach to bioincorporating selenium is the auxotrophic expression technique. In 1975, the first use of selenium in sulfur pathways in E.coli was reported by Cowie & Cohen [66]. Following this early work, there was considerable interest in the insertion of selenium analogs of sulfur-containing amino acids into proteins. Moroder and Budisa further developed this strategy and incorporated selenomethionine, telluromethionine and their isosteric analogs into proteins in order to solve the phase problem in protein X-ray crystallography [67,68]. Furthermore, Bock et al used a similar auxotrophic expression system and incorporated Sec into thioredoxin [69]. The biosynthetic substitution of the catalytically essential cysteine (Cysl49) of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by Sec led to selenoGAPDH that displayed GPx-like properties [70]. This selenoenzyme catalyzed the reduction of hydroperoxides with aryl thiols instead of GSH as this enzyme lacked a GSH-binding site. Similarly to the studies on seleno-GAPDH, Liu et al converted glutathione transferase (Lucilia cuprina LuGSTl-1) into selenoenzyme (seleno-LuGSTl-1) by means of auxotrophic expression [71]. The Ser9 in the GSH-binding site was mutated to cysteine and then biosynthetically substituted to selenocysteine in an auxotrophic expression system. This novel selenium-dependent enzyme exhibited high catalytic activity toward H2O2 in the presence of GSH, which was similar to that of the native GPx. For the first time, a seleniumcontaining enzyme with such remarkable GPx activity was generated by genetic engineering. It is now clear that the design of selenoprotein mimics plays important roles in understanding biochemical functions and reaction mechanisms. It is becoming apparent that selenoprotein mimics possess therapeutic potential against various diseases and that their fiinctions range from antioxidants to anticancer and antiviral agents. It can be anticipated that as our understanding of the basic biology and biochemistry of selenoproteins increases, future efforts will result in even more sophisticated approaches to the rational development of new selenoprotein mimics. References 1. 2. 3. 4. 5. 6. 7. 8.

JR Andreesen, L Ljungdahl 1973 J Bacterial 116:867 DC Turner, TC Stadtman 1973 Arch Biochem Biophys 154:366 L Flohe, EA GOnzler, HH Schock 1973 FEBSLett 32:132 JT Rotruck, AL Pope, HE Ganther, et al 1973 Science 179:588 GV Kryukov, S Castellano, SV Novoselov et al 2003 Science 300:1439 VN Gladyshev, GV Kryukov, DE Fomenko, DL Hatfield 2004 Annu Rev Nutr 24:579 T Tamura, TC Stadtman 1996 Proc Natl Acad Sci U.S.A. 93:1006 JR Arthur, F Nicol, GJBeckett 1990 Biochem J 111:52,1

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D Mustacich, G Powis 2000 Biochem y 346:1 MA Motsenbocker, AL Tappel 1984 J Nut 114:279 MA Beilstein, SC Vendeland, E Barofsky et al 1996 Inorg Biochem 61:117 TC Stadtman 1996 Ann Rev Biochem 65:83 L Floh6, 1989 in Glutathione: Chemical, Biochemical, and Medical Aspects, D Dolphin, R Poulson, O Avamovic,(eds) Wiley, New York, p644 GMugesh,MWWdu,HSies2001C/ieOT7?ev 101:2125 GMugesh,HB Singh 2002 ^ccC/iem/fes 35:226 G Luo, X Ren, J Liu, Y Mu, J Shen 2003 Curr Med Chem 10:1151 TC Stadtman 1991 J Biol Chem 266:16257 F Ursini, 1994 In Oxidative Processes and Antioxidants; R Paoletti (ed) Raven Press New York p25 G Lian, L Ding, M Chen et al IQQXBiochem Biophys Res Commun 283:1007 AY Sun, YM J Chen 1998 Swmaf&j 5:401 JM Mates, C Peter-Gomez, I Nunez de Castro 1999 Clin Biochem 32:595 S Cuzzocrea, DP Riley, A? Caputi, D Salvemini 2001 Pharmacol Rev 53:135 H Sies, 1985 In Oxidative Stress, H Sies (ed) Academic Press, London, pi L Benov, I Fridovich 1998 J Biol Chem 273:10313 H Aebi, 1974 In: Methods of enzymatic analyses, HU Bergmeyer (ed) Academic Press New York, p673 GC Mills 1957 J Biol Chem 229:189 A Roveri, M Maiorino, C Nissii, F Ursini 1994 Biochem Biophys Acta 1208:211 TR Pushpa-Rekha, AL Burdsall, LM Oleksa et al 1995 J Biol Chem 270:26993 RS Esworthy, KM Swiderek, YS Ho, FF Chu 199 SBiochem Biophys .4cto 1381:213 F Ursini, M Maiorino, M Valente et al 1982 Biochem Biophys Acta 710:197 Y Saito, T Hayashi, A Tanaka et al 1999 J Biol Chem 29:2866 MA Beilstein, SC Vendeland, E Barofsky et al \9% J Inorg Biochem 61:117 O Epp, R Ladenstein, A Wendel 1983 Eur J Biochem 733:51 B Ren, W Huang, B Akesson, RJ Ladenstein 1991 Mol Biol 268:869 AEP MUller, E Cadenas, P Graf H, Sies 1984 Biochem Pharmacol 33:3235 A Wendel, M Fausel, H Safayhi, G Tiegs, RA Otter 1984 Biochem Pharmacol 33:3241 C Lambert, R Cantineau, L Christiaens et al 1986 Bull Soc Chim Belg 23:59 L Engman, A Hallberg 1989 J Org Chem 54:2964 TG Back, BP Dyck 1997 J Am Chem Soc 119:2079 SR Wilson, PA Zucker, RRC Huang, A Spector 1989 J Am Chem SocWX :5936 M Iwaoka, S Tomoda \99AJAm Chem Soc 116:2557 G Mugesh, A Panda, HB Singh, NS Punekar, RJ Butcher 1998 Chem Commun 227 G Mugesh, A Panda, HB Singh et al 2001 J Am Chem Soc 123:839 L Engman, D Stem, lA Cotgreave, CM Andersson 1992 J Am Chem Soc 114:9737 G Mugesh, A Panda, S Kumar et al 2002 Organometallics 21:884 G Wenz 1994 Angew Chem Int Ed Engl 33:803 R Breslow, SD Dong 1998 Chem Rev 98:1997 JQ Liu, SJ Gao, GM Luo et al 1998 Biochem Biophys Res Common 247:397 JQ Liu, GM Luo, XJ Ren et al 2000 Biochim Biophys Acta 1481:222 ZY Dong, JQ Liu, SZ Mao et al 2004 J Am Chem Soc 126:16395 Y Liu, B Li, L Li, HY Zhang 2002 Helv Chim Acta 85:9 FSJr Markland, E Smith, 1971 In The Enzymes, PD Boyer (ed) Academic Press New York Vol III p 561 T Nakatsuka, T Sasaki, ET Kaiser 1987 J Am Chem Soc 109:3808 ZP Wu, D Hilvert 1990 J Am Chem Soc 112:5647 EB Peterson, D Hilvert, 1995Biochemistry 34:6616 D Haring, M Herderich, E Schuler, et al 1997 Tetrahedron Asymmetry 8:853 D Haring, E Schueler, W Adam, CR Saha-Moeller, P Schreier \999 J Org Chem 64:832

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Selenium: Its molecular biology and role in human health JQ Liu, MS Jiang, GM Luo, GL Yan, JC Shen 1998 Biotechnol Lett 20:693 SZ Mao, ZY Dong, JQ Liu et al 2005 J Am Chem Soc 127:11588 XJ Ren, P Jemth, PG Board et al 2002 Chem Biol 97:89 G Wulff 1995 Angew Chem Int Ed Engl 34:1812 J Liu, G Luo, S Gao, K Zhang, X Chen, J Shen 1999 Chem Commun 199 GM Luo, ZQ Zhu, L Ding et al 1994 Biochem Biophys Res Commun 198:1240 L Ding, Z Liu, ZQ Zhu, GM Luo, DQ Zhao, JZ Ni 1998 Biochem J 2,2,2:251 XJ Ren, SJ Gao, DL You et al 2001 Biochem J 359:369 DB Cowie, GN Cohen, 1957 Biochim Biophys Acta 26:252 N Budisa, B Steipe, P Demange et al 1995 Eur J Biochem 230-JS& N Budisa, C Minks, FJ Medrano et al 1998 Proc Natl Acad Sci USA 95:455 S MuUer, H Senn, B Gsell, W Vetter, C Baron, A Bock 1994 Biochemistry 33:3404 S Boschi-Muller, S Muller, AV Dorsselaer et al 1998 FEBS Letters 439:241 HJ Yu, JQ Liu, A Bock, J Li, GM Luo, JC Shen 2005 J Biol Chem 280:11930

Chapter 35. Update of human dietary standards for selenium Orville A. Levander Beltsville Human Nutrition Research Center, U. S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

Raymond F. Burk Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

Summary: An update of the human dietary standards for selenium is presented, including the year 2000 Dietary Reference Intakes (U.S.A.), the 1996 standards of the World Health Organization (WHO), and a recent relevant intervention trial carried out in China. Two criteria have been used by official bodies to set recommendations. One is the prevention of Keshan disease, the only proven selenium-responsive disease. The other is full expression of all selenoproteins as indicated by optimization of a plasma biomarker, either glutathione peroxidase activity or selenoprotein P concentration. An average per capita intake of 20 |xg selenium/day will prevent Keshan disease in a population but will not allow optimization (full expression) of selenoproteins. Using plasma glutathione peroxidase activity as the selenium biomarker to be optimized, the RDA for adults in the U.S.A. was set at 55 )ig in 2000. The recent trial in China utilized selenoprotein P as a biomarker and its results suggest that an upward revision of the current RDA will be needed. Even higher intakes of selenium have been postulated to prevent cancer. Intervention trials now underway in the U.S.A. are evaluating that possibility and the safety of large selenium supplements. Introduction In South Dakota during the 1930s, selenium was identified as the toxic agent in animal feeds and forages that caused the livestock poisoning known as "alkali disease" [1]. Plants that grew in certain areas of the Great Plains of the United States took up so much selenium from the selenium-rich soils that they became toxic to poultry and livestock. In cattle the disease is characterized by hair and hoof loss and a generalized emaciated appearance. For an extensive description of selenosis in farm animals, consult the monograph by Rosenfeld and Beath [2].

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There were suggestions in the literature that selenium might have a beneficial effect under certain conditions (e.g., see the work of Pinsent with bacteria [3]), but the prevailing opinion was that selenium was a toxin that played no positive role in metabolism. Moreover, selenium compounds were thought to be carcinogenic. Then in 1957 Schwarz and Foltz [4] announced their discovery that traces of dietary selenium could protect vitamin Edeficient rats from developing liver necrosis. Very soon thereafter, seleniumresponsive diseases were found in a variety of economically important farm animals including turkeys, chickens, sheep, swine, and cattle [5]. The need for selenium in human nutrition was shown by Chinese scientists who demonstrated in 1979 that this essential trace mineral protected against Keshan disease, a cardiomyopathy affecting young children and women of child bearing age residing in low-selenium areas in China. This finding increased greatly the interest in the human selenium requirement [6]. The current and previous dietary standard for selenium, the year 2000 Dietary Reference hitake (DRI) [7] and the 1989 RDA [8], respectively, were both based on maximization (or optimization) of plasma glutathione peroxidase activity so their values were rather similar. A proposal to base the next selenium standard on the full expression of selenoprotein P would lead to a higher value for the standard and is evaluated below. This review concludes with a discussion about the possible use of selenium as a cancer chemoprevention agent. Such a practice, if justified by studies that are in progress and planned, might result in substantially elevated recommendations of selenium intake. RDAs - tenth edition (1989) hiterest in the possible beneficial effects of selenium in human health continued to grow well into the 1980s and this was reflected in the Tenth Edition of the RDAs [8]. Literature citations increased six-fold. This was due not only to the increasing number of papers dealing with selenium but also to the expressed desire of the RDA Committee to make the RDA book more "scientific" and one in which every step of the derivation of the RDA was "transparent" so that the logic and reasoning behind the derivation of the dietary standard was clear and open for everybody to see. Fortunately for selenium researchers, several studies from China allowed the RDA Committee to pinpoint human selenium requirements with increased precision such that it was possible to advance selenium to full RDA status for the first time. One group of studies examined the dietary selenium intake needed to prevent Keshan disease in regions of China where it was endemic [9]. The disease did not occur in those areas in which the selenium intake by adults was 17 jxg/day or more. Thus, 17 ^g/day was suggested as a minimum daily requirement based on disease prevention.

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In another study, a "physiological" selenium requirement was determined by following increases in glutathione peroxidase activity in the plasma of men living in a Keshan disease area who were given graded doses of selenomethionine as a supplement over a period of several months [10]. At a total intake of 41 jig/day or more (11 p.g from diet), the plasma glutathione peroxidase activity became optimized. Therefore, it was concluded that Chinese men had a physiological requirement of 41 fig/day. In order to convert this figure to an RDA for North American males, it was necessary to apply a correction factor for differences in body weight (79/60) and to apply a safety factor (1.3) to allow for individual differences in requirement. Thus, the calculation for adult males became: 41 X 79/60 X 1.3 = 70^g/day For adult North American females, the calculation was: 41 X 63/60 X 1.3 = 55ng/day A more detailed explanation of the RDA calculations for adults was presented elsewhere [11]. Because of the lack of data, the RDAs for young adults also served as the basis of RDAs for the elderly. Likewise, because of the lack of data, RDAs for infants and children were based on adult values with extrapolations downward on the basis of body weight plus a factor arbitrarily allowed for growth. The RDA during pregnancy was calculated using a factorial technique based on the fetal accretion of selenium. The RDA during lactation provided sufficient selenium to avoid depletion of the mother and permit a satisfactory selenium content in the breast milk. The Tenth Edition discussed selenium toxicity only in general terms [8]. An episode of human selenosis in China was described in which hair loss and fingernail changes were observed on intakes approximating 5000 jig selenium/day. It was pointed out that sensitive and specific biochemical indices of selenium overexposure were not available and no attempt was made to establish a safe upper limit of selenium intake. World Health Organization (1996) In 1996, the World Health Organization (WHO) published its dietary standards for several trace elements, including selenium [12]. WHO has the responsibiUty for setting recommendations that apply to many different countries around the globe (United Nations member states) that have highly varied national diets. For that reason, the Organization tends to suggest nutrient intakes that are often somewhat lower than those set in the U.S.A. This also turned out to be true for selenium since large parts of the U. S. Great Plains, a major wheat production area, have soils that are rich in

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Selenium: Its molecular biology and role in human health

selenium and relatively generous amounts of the trace element are incorporated into the food chain. Intakes of selenium exceeding 100 i^g/day are not uncommon in the U.S. and so meeting an RDA of 55 to 70 }ig/day is not difficult. On the other hand, meeting the 1989 RDA for selenium could be quite a challenge for some other coimtries. Many parts of China, for example, routinely consume much lower amounts of selenium in their diet [9] and New Zealanders rarely ingest such RDA levels [13]. Likewise, Finland had a low-selenium food supply before deciding in 1985 to add selenium to its fertilizers [14]. In fact, dietary surveys indicate that several European countries would have problems achieving intakes as high as the 1989 RDA, including Belgium, Denmark, France, Germany, United Kingdom, Slovakia, and Sweden [reviewed in [15]]. The selenium intake in Switzerland was somewhat higher because of the common use of North American wheat rich in selenium. So it is not surprising that WHO was reluctant to set a dietary standard that so many of its member states could not attain, especially in the absence of any evidence of signs of human selenium deficiency outside of China. The reader will recall that the rationale used by the 1989 RDA Committee for its selenium recommendation was full expression of plasma glutathione peroxidase activity. The WHO Committee decided that such full activity was probably not necessary for human health and that only two-thirds full activity of plasma glutathione peroxidase still afforded sufficient protection against oxidative stress. This conclusion was based on observations that blood cells metabolized hydrogen peroxide satisfactorily until their glutathione peroxidase activity fell to one-quarter or less of normal. Of course, if one selects a lower target glutathione peroxidase activity for the biochemical criterion of adequate nutriture, this allows a lower dietary standard to be proposed also. In this case, the WHO Committee (formal designation: Joint FAO/IAEAAVHO Expert Consultation on Trace Elements in Human Nutrition) came up with 40 and 30 |J.g/day for the lower limit of the safe range of population mean dietary selenium intake that would meet the normative requirement of most adult males and females, respectively. As defined by WHO, the normative requirement referred to the "level of intake that serves to maintain a level of tissue storage or other reserve that is judged by the Expert Consultation to be desirable" [12]. WHO also defined a basal requirement that referred to the "intake needed to prevent pathologically relevant and clinically detectable signs of impaired function attributable to inadequacy of the nutrient." For selenium, the basal requirement was taken from the quantity needed to protect against Keshan disease. The lower limit of the safe range of population mean dietary selenium intake that would meet the basal requirement of most adult males

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403

and females was calculated to be 21 and 16 p-g/day, respectively, after adjusting for body weight.

Table 1. The 1996 WHO Lower Limits of the Safe Ranges (Basal and Normative) of Population Mean Intakes of Dietary Selenium (ng/day)

Life Stage

Aee fvears)

Infants

Basal

Normative

0-0.25 0.25-0.5 0.5-1.0

3 5 6

6 9 12

1-3 3-6 6-10

10 12 14

20 24 25

Males

10-12 12-15 15-18 18+

16 19 21 21

30 36 40 40

Females

10-12 12-15 15-18 18+

16 16 16 16

30 30 30 30

Pregnancy

18

39

Lactation 0-3 months 3-6 months 6-12 months

21 25 26

42 46 52

Children

Adapted from [12].

The WHO Committee also attempted to deal with the question of tolerances of high dietary selenium intakes. On the basis of considerable fieldwork with human selenosis in China, Yang and associates proposed 750850 ^g as a marginal level of daily safe dietary selenium intake [16], defined as "the level of selenium intake at which few individuals have functional signs of excessive intake and above which the tendency to exhibit functional signs is apparent and symptoms may first appear among ... susceptible individuals [whose] selenium intake [is] further increased". The Committee

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Selenium: Its molecular biology and role in human health

took one-half of the average of this range because of the uncertainty surrounding the harmful dose of selenium for people to suggest a maximal daily safe dietary selenium intake of 400 |ig. Dietary reference intakes (2000) The new millennium saw a host of changes in the way that dietary standards for selenium (and many other nutrients) were handled in the U. S. A. [7]. First of all, selenium was grouped with a variety of so-called "dietary antioxidants" (vitamins C and E and the carotenoids) instead of with the trace elements where it had traditionally been put. This change made sense because selenium, due to its multitude of roles protecting against oxidative stress, really had more in common with the nutritional antioxidants than it did with a collection of various microminerals. Another substantial change was in the dietary standards themselves [7]. The general term "Dietary Reference Intakes" was used to describe not only the RDA, but also Adequate Intake (AI), Tolerable Upper Intake Level (UL), and Estimated Average Requirement (EAR). Each of these terms has a particular role in describing the dietary standards of a nutrient and it might be worthwhile to repeat here their meanings as presented by the Panel on Dietary Antioxidants and Related Compounds: Recommended Dietary Allowance {RDA): the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 to 98 percent) healthy individuals in a particular life stage (which considers age, and when applicable, pregnancy and lactation) and gender group. Adequate Intake (AI): a value based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of healthy people that are assumed to be adequate—used when an RDA cannot be determined. Tolerable Upper Intake Level (UL): the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the risk of adverse effects increases. Estimated Average Requirement (EAR): a daily average nutrient intake value that is estimated to meet the requirement of half the healthy individuals in a life stage and gender group." Thus, the redefinition of the RDA echoes the 1989 version [8], which states that they are "... the levels of intake of essential nutrients that... are judged ... to be adequate to meet the known nutrient needs of practically all healthy persons." The AI is reminiscent of the "Estimated Safe and Adequate Daily Dietary Intake" which was a dietary standard to be used when insufficient data were available to posit an RDA. The UL represents the first formal attempt by an "RDA Committee" to establish a ceiling of intake for the nutrients being considered by the group. In the "Dietary Reference Intakes"

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(what the handbook on dietary standards in North America is now called— the title is no longer "Recommended Dietary Allowances") an entire chapter is devoted to describing a model for the development of ULs for nutrients. The EAR occupies a critical place in the new dietary standards, for without it, there can be no RDA. These two entities are related by the equation: RDA = EAR + 2 SD where SD is the standard deviation of the EAR. If the SD was unknown, the 2000 Committee generally assumed a coefficient of variation of 10% for the EAR so that RDA = 1.2 X EAR The 2000 Committee based its EAR on 2 intervention trials designed to estimate selenium requirements by determining the intake needed to optimize plasma glutathione peroxidase activity. The first trial was carried out in China [10] and in fact was the same study that served as the basis for the 1989 RDA [8]. The selenium intake needed to optimize plasma glutathione peroxidase in that work was 41 (ig/day, which came to 52 j^g/day after adjustment for Western body weight. The second intervention trial was from New Zealand [13] and the 2000 Committee interpreted that research as suggesting an EAR of 38 ^ig/day. Although other interpretations of the New Zealand trial may be possible [15], the average of both the New Zealand and Chinese trials, 45 |ig/day, was selected as the EAR. The RDA for adult males then was calculated as 45 X 1.2 to yield 55 ng/day. Thus, by using a lower base requirement figure than the 1989 Committee (45 vs. 52 (ig/day after adjustment for body weight) and a smaller correction factor for individual variation (1.2 vs. 1.3), the 2000 Committee arrived at a lower RDA figure for adult males than the 1989 Committee (55 vs. 70 |ig/day). Given the reported greater susceptibility of women to develop Keshan disease, their RDA was also set at 55 |ig/day despite their smaller body weight. The 2000 Committee could find no data available to calculate an EAR for children or adolescents, so the RDAs for them were extrapolated from young adult values. Similarly, there were no data that specifically addressed the selenium requirement for elderly persons and the 2000 Committee found no information that suggested that the aging process impaired selenium absorption or utilization, so their RDA was the same as young adults. A major philosophical shift occurred in the way that requirements were presented for infants up to one year if age. Because "No functional criteria of selenium status have been demonstrated that reflect response to dietary intake in infants", the 2000 Committee rescinded the 1989 RDA, so to speak.

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Selenium: Its molecular biology and role in human health

and replaced it with an AI based on the "mean selenium intake of infants fed principally with human milk." This fundamental change in viewing infant requirements was not limited to selenium. In fact, other nutrients have also been accorded only AI status for infants, including calcium, magnesium, vitamin D, thiamin, and riboflavin. The 2000 Committee selenium AI for infants for the first and second 6 months of life are 15 and 20 ng/day, respectively, up 50% and 33% from their 1989 counterparts, respectively. Using somewhat different assumptions, the 2000 Committee came up with RDAs for pregnancy and lactation that were slightly less than those set by the 1989 Committee (60 vs. 65 fxg/day and 70 vs. 75 |ig/day, respectively). Table 2. The 2000 Dietary Reference Intakes (DRI) for Selenium (^g/day)

Life Stage

Age

Infants

0-6 mo 7-12 mo

15* 20*

Children

1-3 y 4-8 y

20 30

Males

9-13 y 14-70 y >70y

40 55 55

Females

9-13 y 14-70 y >70y

40 55 55

DRI (ng/day)

Pregnancy

60

Lactation

70

Adapted from [7]; values with asterisk are AI, others are RDA.

Another innovation in the 2000 DRI was the establishment of an upper limit of intake. The UL for selenium was based on the criteria of h^ir and nail brittleness and loss due to dietary overexposure in a high-selenium region in China. Intakes of selenium from food sources were inferred from blood levels. A No-Observed-Adverse-Effect-Level (NOAEL) was calculated to be 800 |ig/day. An imcertainty factor of 2 was chosen to protect sensitive individuals, thereby leading to a UL of 400 fig/day for adults 19 years of age

Update of human dietary standards for selenium

407

and older, a figure in agreement with upper limits set by others [12]. Finding no evidence of teratogenicity or selenosis in infants of mothers consuming high but not toxic amounts of selenium, the 2000 Committee kept to 400 Jig/day UL for pregnant and lactating women. The UL for infants 0 to 6 months old consuming human breast milk exclusively was set at 45 |ig/day based on the lack of any adverse effects (NOAEL) reported in such infants consuming breast milk containing 60 |ig selenium/L. The ULs for older infants, children, and adolescents were extrapolated on the basis of body weights. Recent trial using Selenoprotein P and glutathione peroxidase as biomarkers A report appeared in 2005 that described a selenium intervention trial carried out in 2001 in a low-selenium area of China [17]. The trial was designed to determine the selenium intake needed to 'optimize' the plasma selenium biomarkers, glutathione peroxidase and selenoprotein P. These 2 selenoproteins are accessible representatives (biomarkers) of the entire family of selenoproteins [18]. Optimization of them is used as an indicator of optimization (full expression) of all the selenoproteins in the body. A given selenoprotein is 'optimized' when providing additional selenium does not result in an increase in its concentration. There is a 'hierarchy' of selenoproteins with respect to their claim on available selenium. This means that when the selenium available will not allow optimization (full expression) of all selenoproteins, the selenoproteins most essential to the organism receive selenium and selenoproteins that are less essential do not. Thus, the "least essential" selenoprotein will be the last to be optimized when selenium availability is increased from inadequate to adequate, according to this concept. Animal experiments have suggested that liver glutathione peroxidase is the lowest selenoprotein in the hierarchy [19]. Because human tissues cannot be sampled for routine studies and blood can, plasma selenoproteins have been used as the best available representatives of the selenoproteins in the body. The subjects studied were farmers in a low-selenium region of Sichuan Province. Their average dietary selenium intake was 10 |j,g per day. hiitial plasma glutathione peroxidase activity was 40% of tj^ical U.S. values and selenoprotein P concentration was 23% of typical U.S. values. Subjects were supplemented for 20 weeks with placebo or several dose levels of selenite or L-selenomethionine (henceforth selenomethionine). Glutathione peroxidase became optimized with a supplement of 37 i^g of selenium per day as selenomethionine. The same level of glutathione peroxidase was achieved with 66 |j,g per day as selenite. The selenomethionine results are close to those of the 1983 study in China [10] and the 1995 study in New Zealand [13] and essentially confirm their results.

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Selenium: Its molecular biology and role in human health

Selenite has not been evaluated previously in this type of study and its lower bioavailability than that of selenomethionine is noteworthy. Selenoprotein P did not become optimized in this trial, even when 61 ng selenium per day was administered as selenomethionine. Thus, selenoprotein P is lower in the hierarchy of human selenoproteins than plasma glutathione peroxidase and is therefore the better biomarker for optimization of the selenoproteins in the body. It seems likely that use of selenoprotein P as a selenium biomarker will lead to an increase in the selenium dietary intake recommendations. However, the results in this study do not allow an estimation of the amount of selenium needed because optimization of selenoprotein P was not achieved. Moreover, it is possible that supplementation at the dose levels used in this study for more than 20 weeks will optimize selenoprotein P. Thus, a study that includes higher supplemental dose levels and a longer supplementation period is needed. This study has pointed out that different biomarkers are optimized at different selenium intakes and that therefore the choice of biomarker is important. Presently, selenoprotein P appears to be a better biomarker than plasma glutathione peroxidase activity. The study has confirmed the results of the 2 earlier studies that used plasma glutathione peroxidase. It indicates, however, that the current selenium recommendations, based on glutathione peroxidase, are likely to be too low. Another important finding of this study is the greater bioavailability of selenium in the form of L-selenomethionine than in the form of selenite. This has implications for formulation of selenium supplements and addition of selenium to foods. Selenium as a possible cancer chemoprevention agent In total, almost 150,000 individuals have participated in phase III nutritional intervention studies to prevent cancer [20]. Besides selenium, nutritional intervention agents have included beta-carotene, alpha-tocopherol, retinal, and various vitamin and mineral combinations. Several lines of evidence have suggested that selenium might be a suitable candidate for testing as a chemoprevention agent. First, experiments with laboratory animal models indicated that various selenium compounds protect against tumorigenesis under a variety of conditions [21]. Second, about half of the 36 epidemiological studies evaluated by the FDA implied some value of selenium against cancer [22]. Finally, the selenium intervention trial of Clark and colleagues [23] found that subjects given 200 jxg selenium/day in yeast form to prevent skin cancer had lower incidences of several cancers than did the placebo group. However, no effect on skin cancer was observed. Because of the positive results in these studies, the U.S. National Cancer Institute is sponsoring a large trial (32,400 men) called SELECT (the

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409

Selenium and Vitamin E Cancer Prevention Trial) [24]. SELECT is a phase III randomized, placebo-controlled test of selenium (200 (j.g/day as Lselenomethionine) and/or vitamin E (400 lU/day) to prevent prostate cancer in U.S. men. The subjects in SELECT are presumed to be selenium replete, with full expression of their selenoproteins. Thus, if SELECT demonstrates a preventive effect of selenium and/or vitamin E on prostate cancer, it will indicate that there is a health-related function of selenium independent of selenoproteins. Studies to characterize the dose of selenium needed to achieve the chemopreventive effect would then be desirable to inform strategies of supplementation. SELECT will also evaluate safety. Further consideration of the use of selenium to prevent cancer will depend on its being safe. The Food and Nutrition Board set an Upper Limit of selenium intake in their 2000 DRJ's at 400 Jig/day [7]. Intakes of subjects in the SELECT trial will be in the 300+ Hg/day range and determination of the safety of that intake over years will be important. Orderly progression of selenium chemoprevention studies is important. Only when data are produced that show selenium to be safe and effective in cancer prevention can recommendations to the public be made. References 1. 2. 3. 4. 5.

6. 7. 8.

9. 10.

11. 12. 13. 14. 15. 16. 17.

AL Moxon, M Rhian 1943 Physiol Rev 23:305 I Rosenfeld, O Beath 1964 Selenium. Geobotany, biochemistry, toxicity, and nutrition Academic Press New York J Pinsent 1954 Biochem J 57:10 K Schwarz, CM Foltz 1957 JAmer Chem Soc 79:3292 Subcommittee on Selenium, Committee on Animal Nutrition, Board on Agriculture, National Research Council 1983 Selenium in Nutrition Revised Edition National Academy Press Washington p 174 Keshan Disease Research Group 1979 Chinese Medical Journal 92:471 Institute of Medicine 2000 Selenium. In: Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids National Academy Press Washington pp 284-324 Subcommittee on the Tenth Edition of the RDAs, Food and Nutrition Board, National Research Council 1989 Recommended Dietary Allowances lO"" Edition National Academy Press Washington GQ Yang, KY Ge, JS Chen, XS Chen 1988 World Rev Nutr Diet 55:98 G-Q Yang, L-Z Zhu, S-J Liu, L-Z Gu, P-C Qian, J-H Huang, M-D Lu 1987 In: Selenium in biology and medicine.. Part B (eds. GF Combs Jr, JE Spallholz, OA Levander, JE Oldfield) AVI New York pp 589-607 OA Levander 1991 J Am Diet Assoc 91:1572 Trace Elements in Human Nutrition and Health. Report of a Joint FAO/IAEA/WHO Expert Consultation 1996 World Health Organization, Geneva pp 343 AJ Duffield, CD Thomson, KE Hill, S Williams 1999 Am J Clin Nutr 70:896 A Aro, G Alfthan, P Varo 1995 Analyst 120:841 MP Rayman 2000 Lancet 356:233 G Yang, S Yin, R Zhou, L Gu, B Yan, Y Liu, Y Liu 1989 J. Trace Elem. Electrolyes Health Dis y.\22 Y Xia, KE Hill, DW Byrne, J Xu, RF Burk 2005 Am J Clin Nutr 81:829

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18. GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 Science 300:1439 19. J-G Yang, KE Hill, RF Burk \9S9 JNutr 119:1010 20. PR Taylor, P Greenwald 2005 J Clin Oncol 23:333 21. CIp 1998JiV«/r 128:1845 22. PR Trumbo 2005 JNutr 135:354 23. LC Clark, GF Combs Jr, BW Tumbull, EH Slate, DK Chalker, J Chow, LS Davis, RA Glover, GF Graham, EG Gross, A Krongrad, JL Lesher, HK Park, BB Sanders, CL Smith, JR Taylor 1996 JAMA 276:1957 24. SM Lippman, PJ Goodman, EA Klein, HL Pames, IM Thompson Jr., AR Kristal, RM Santella, JL Probstfield, CM Moinpour, D Albanes, PR Taylor, LM Minasian, A Hoque, SM Thomas, JJ Crowley, JM Gaziano, JL Stanford, ED Cook, NE Fleshner, MM Lieber, PJ Walther, FR Khuri, DD Karp, GG Schwartz, LG Ford, CA Coltman Jr 2005 J Natl Cancer Inst 97-M

Index

Acquired immunodeficiency syndrome, 299-310 immunity, 300-302 metabolic syndrome, 302-304 micronutrient deficiencies, 304-305 selenium deficiency, 305-306 selenium supplementation, 306-308 wasting, 302-304 Adhesion molecules, cytokines, 314-315 Aging methionine sulfoxide reduction, 126-127 selenium in, 318-320 AIDS. See Acquired immunodeficiency syndrome Aminoacyl-tRNA recycling, 83-95 Antibodies, mimics of selenoprotein, selenoenzyme transformation, 394-395 Apoptosis, selenium-induced, 379-385 mitochondrial dysfunction, selenium-induced, 382-384 oxidative stress, selenium-induced, 381-382 safe levels of exposure, 380-381 selenium toxicity, 380 thiol modification, selenium-induced, molecules targeted for, 382 Atom detection, selenium, 224-225 Bacteria biosynthesis of selenocysteine, 14-15 decoding UGA with selenocysteine, 24-25 E. coli SECIS element, in gene expression, 16-11 incorporation of selenocysteine by, 12 SECIS element interaction, 18-21 SelB, domain structure, 18-21 termination vs. readthrough, 11-14 translation factor, SelB, 15-18 tRNAS'^'', 12-13 Bioinformatics tools for, selenoprotein identification, 99-102 Bios)mthesis of selenocysteine, 14—15 Brain function

effects of selenoprotein P deletion, 115 epilepsy, 238-239 neurodegenerative disorders, 239-241 Parkinson's disease, 236-237 selenium selenoproteins, 233-248 stroke, 234-236 transgenic selenoprotein-deficient mouse models, 241-245 Cancer prevention, 249-264, 367-368 animal models, 250 clinical trials, 250-252 epidemiological evidence, 250 15-kDa selenoprotein, 141-148 in cancer prevention, 142-143 dietary selenium, 146 glycoprotein glucosyltransferase, 143-142 thiol-disulfide oxidoreductase function, 145-146 mechanisms, 252-254 metabolic bases, 254-260 metabolomics, 373-374 nutrigenetics, 369-370 nutritional epigenetics, 370-371 nutritional transcriptomics, 371-372 proteomics, 373-374 Se-metabolities, 255-260 selenium dietary standards, 408^09 selenoenzymes, 254—255 selenoprotein gene variation, 277-286 GPx-1 in cancer etiology, 279-282 polymorphisms, 283-284 selenoproteins, 279 Sepl5, 282-283 thiol proteomics, 265-276 BiP/GRP78 over-expression, 272-273 methylseleninic acid, 266 monomethylated selenium, as protein redox modulator, 266-269 redox-modified proteins, 269 unfolded protein response, ER stress and, 269-271

412

Selenium: Its molecular biology and role in human health

UPR signaling, 271-272 Catalytic mechanisms, methionine sulfoxide reduction, 128 Cell-mediated immunity, 315-316 Conditional knockout mouse models, 339-340 Conformation-specific SEClS-binding activities, SBP2, eukaryotic selenoprotein synthesis, 69-70 Coxsackievirus, 287-290 Cys-containing counterparts, methionine sulfoxide reduction. Sec-containing proteins compared, 131 Cytoplasmic supramolecular complex, supramolecular complexes, selenocysteine biosynthesis, isolation, 89-91 Cytosolic, mitochondrial thioredoxin reductase knockout mice, 195-206 phenotype Txnrdl knockout embryos, 199-200 Txnrd2 knockout embryos, 201-202 Txnrdl/Txnrd2 embryonic expression profile, 198-199 embryonic lethality, 198 heart-specific inactivation of, 202-204 mouse models with conditional alleles for, 197-198 Decoding selenocysteine, 39-50 genetic code, 4 8 ^ 9 phenotype, dynamic process of evolution, 45^6 Sec decoding common origin, 40-41 lost trait, 41-42 Sec-tRNA^'^'' synthesis, non-canonical mechanism, 46-48 selenophosphate synthetase, 42-45 Decoding selenophosphate synthetase trait, 41^2 Decoding UGA with selenocysteine, 24-25 Deiodinases, endocrine function, 207-219 adaptive thermogenesis, 212-214 Dl overexpression, h5fperthyroidism, 217 D3 overexpression in hemangiomas, 216 deiodinases conservation, 3D structure, 208 fasting, changes in iodothyronine deiodination, 214-215 illness, changes in iodothyronine deiodination, 214-215 thyroid hormone homeostasis, 211

tissue-specific control of thyroid hormone action, 211-212 ubiquitination pathway, D2 inactivation, 209-211 Detection of selenium atom, 224-225 Diabetes, 173-182 early research, 175-176 glutathione peroxidase-1 in, 173-182 early research, 175-176 insulin function, 180 metabolic impact, 175 selenoprotein expression, 174-175 insulin function, 180 metabolic impact, 175 Dietary standards, selenium, 399^10 cancer chemoprevention, 408^09 dietary reference intakes, 404-407 glutathione peroxidase, as biomarker, 407-408 RDAs, 400-401 selenoprotein P, as biomarker, 407^08 World Health Organization, 401-404 Domain structure, SelB, 18-21 Drosophila, selenoproteins, 343-353 SelG/SelK/G-rich,351 SelM/BthD/SelH, 350-351 synthesis machinery, 344-348 intake, 344 selenocysteyl-tRNA, 344-345 selenophosphate synthetase, 345-348 translational machinery, 348-349 Drug development, parasite selenoproteins, 364-365 E. coll SECIS element, in gene expression, 26-27 EEFSec, SBP2 interactions, eukaryotic selenoprotein synthesis, 66-67 Eicosanoid metabolism, 313-314 Endocrine function, deiodinases and, 207-219 adaptive thermogenesis, 212-214 Dl overexpression, hyperthyroidism, 217 D3 overexpression, hemangiomas, 216 deiodinases conservation, 3D structure, 208 fasting, changes, iodothyronine deiodination, 214—215 illness, changes, iodothyronine deiodination, 214-215 thyroid hormone homeostasis, 211 tissue-specific control of thyroid hormone action, 211-212

Index ubiquitination pathway, D2 inactivation, 209-211 Endogenous Sec factors, supramolecular complexes, selenocysteine biosynthesis, supramolecular complexes composed of, 92 En2ymes, natural, mimics of selenoprotein, selenoenzyme transformation, 392-393 Epilepsy, selenium selenoproteins, 238-239 Eukaryotic Sec bios}Tithesis, supramolecular complexes, selenocysteine biosynthesis, protein factors, 86-87 Eukaryotic selenocysteine tRNAs, 29-37 biosynthesis, 32-34 evolution, insertion machinery, 34—36 insertion machinery, 34-36 mammalian Sec tRNA[s«]s