Genotoxicity : Evaluation, Testing and Prediction [1 ed.] 9781617284267, 9781607417149

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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Genotoxicity : Evaluation, Testing and Prediction, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Genotoxicity : Evaluation, Testing and Prediction, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

GENETICS – RESEARCH AND ISSUES SERIES

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GENOTOXICITY: EVALUATION, TESTING AND PREDICTION

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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GENETICS – RESEARCH AND ISSUES SERIES Sex Chromosomes: Genetics, Abnormalities, and Disorders Cynthia N. Weingarten and Sally E. Jefferson 2009. ISBN: 978-1-60741-304-2 Genetic Diversity Conner L. Mahoney and Douglas A. Springer (Editors) 2009. ISBN: 978-1-60741-176-5 The Human Genome: Features, Variations and Genetic Disorders Akio Matsumoto and Mai Nakano (Editors) 2009. ISBN: 978-1-60741-695-1 Bacterial DNA, DNA Polymerase and DNA Helicases Walter D. Knudsen and Sam S. Bruns (Editors) 2009. ISBN: 978-1-60741-094-2

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Genotoxicity: Evaluation, Testing and Prediction Andor Kocsis and Hajna Molnar (Editors) 2009. ISBN: 978-1-60741-714-9

Genotoxicity : Evaluation, Testing and Prediction, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

GENETICS – RESEARCH AND ISSUES SERIES

GENOTOXICITY: EVALUATION, TESTING AND PREDICTION

ANDOR KOCSIS AND

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

HAJNA MOLNAR EDITORS

Nova Biomedical Books New York

Genotoxicity : Evaluation, Testing and Prediction, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Genotoxicity evaluation, testing, and prediction / [edited by] Andor Kocsis and Hajna Molnar. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61728-426-7 (E-Book) 1. Genetic toxicology. I. Kocsis, Andor. II. Molnar, Hajna. [DNLM: 1. Mutagenicity Tests. 2. DNA Adducts--diagnostic use. 3. Mutagens--adverse effects. 4. Toxicogenetics--methods. QU 450 G3354 2009] RA1224.3.G465 2009 616'.042--dc22 2009025762

Published by Nova Science Publishers, Inc.    New York

Genotoxicity : Evaluation, Testing and Prediction, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Contents Preface Chapter I

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Chapter II

vii The Importance of the In Vitro Genotoxicity Evaluation of Food Components: The Selenium Vanessa Valdiglesias, Josefina Méndez, Eduardo Pásaro and Blanca Laffon In Vitro High-Throughput Assays for Assessment of Genetic Toxicology Xuemei Liu, Jeffrey A. Kramer, Larry D. Kier and Alan G. E. Wilson

Chapter III

Genotoxicity Evaluation of Exposure to Lead Julia García-Lestón, Josefina Méndez, Eduardo Pásaro and Blanca Laffon

Chapter IV

Genotoxicity by Micronucleus Assay for Medical Devices Development Andrea Cecilia Dorion Rodas, Patricia Santos Lopes and Olda Zazuco Higa

1

41

79

111

Chapter V

Genotoxicity Produced by Disease and Drugs Guillermo M. Zúñiga-González, Belinda C. Gómez-Meda, Ana L. Zamora-Perez and Martha P. Gallegos-Arreola

127

Chapter VI

Modification of Chemical Mutagenesis A.D. Durnev, A.K. Zhanataev, E.S. Voronina, L.A. Oganesyantz and S.B. Seredenin

157

Chapter VII

Co-Mutagenic Effects of Nifedipine and Diltiazem in Vivo A.D. Durnev, A.K. Zhanataev, N.O. Daugel'-Dauge, A.V. Kulakova and E.A. Anisina

189

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vi Chapter VIII

Chapter IX

Chapter X

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Chapter XI

Contents The Usefulness of Bulky DNA Adduct Formation as a Biological Marker of Exposure to Airborne Particulate Matter (PM2.5) in In Vitro Cell Lung Models: A Comparative Study Sylvain Billet, Véronique Andre, Imane Abbas, Jeremie Le Goff, Anthony Verdin, François Sichel, Pirouz Shirali and Guillaume Garçon

201

Impact Assessment and Evaluation of Cadmium-Induced Genotoxicity and Clastogenicity in Mice Malay Chatterjee and Mary Chatterjee

225

Loss of Heterozygosity and/or Microsatellite Instability in Multiple Critical Regions of 3p and 9p Chromosomes in Human Epithelial Lung Cells (L132) Exposed to Air Pollution Particulate Matter (PM2.5) Françoise Saint-Georges, Guillaume Garçon, Fabienne Escande, Imane Abbas, Anthony Verdin, Pierre Gosset, Philippe Mulliez and Pirouz Shirali

253

Prediction of the Human Radiosensitivity: What is the Most Relevant Endpoint? Gene Expressions, Mutations or Functions? Catherine Massart, Aurélie Joubert, Adeline Granzotto, Muriel Viau, Fawzia Seghier, Jacques Balosso and Nicolas Foray

275

Chapter XII

Application of Genotoxicity to Dental Materials Research Daniel Araki Ribeiro

293

Chapter XIII

Use of the Comet Assay to Evaluate Pesticide Toxicity on NonTarget Microalgae R. Prado, R. Díaz, C. Rioboo, J. Abalde, C. Herrero and A. Cid

311

Numberless Drugs are Not Adequately Tested for the Potential Genotoxic-Carcinogenic Risk to Humans Giovanni Brambilla and Antonietta Martelli

321

Commentary

Index

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Preface This book looks at genotoxicity which is a deleterious action on cell genetic material affecting its integrity. Genotoxic substances are known to be potentially mutagenic or carcinogenic, specifically those capable of causing genetic mutation and of contributing to the development of tumors. In humans, DNA damage or genotoxicity may be caused by exposure to outside agents like radiation, pesticides, combustion of hydrocarbon products as well as antineoplastic drugs. DNA damage could also come from inside of the body, determined mainly by excessive free radical production generated by some disease process. The importance then, is to identify the genotoxity and try to protect the body, which may be as simple as removing the source of exposure or providing protection against such agents. This new important book gathers the latest research from around the globe in this dynamic field of study. Chapter 1 - Genotoxicity assays are used to assess the genetic damage associated with the exposure to different substances. When humans are directly exposed to a potentially genotoxic substance, as with food, there is an imperative need to evaluate diverse types of DNA alterations in order to thoroughly determine the health hazard. This evaluation requires the use of a battery of in vivo and in vitro tests. In vivo studies are useful but are also very expensive, require a high number of animals and raise important ethical concern. Moreover their results cannot always be extrapolated to humans. In these cases the use of in vitro assays becomes relevant and necessary. Oligoelements present at low levels in food, as selenium, frequently give rise to difficulties for regulators and food businesses to evaluate the potential risk of genotoxicity. In this concern, the use of in vitro assays gains importance since it allows controlling the features of the exposure and employing human cell lines that can provide a more real view of its effects on human organism. Selenium is an essential human micronutrient that participates in important cell processes and exerts different effects on the organism according to its chemical form and concentration. In the last decades, many studies have been performed to characterize these effects. Their findings indicate that selenium is a key player in cellular metabolism, is an essential component of antioxidant enzymes, and has important roles in thyroid metabolism, human fertility, and many other vital functions. Nevertheless, data also show that an excess of selenium in the diet can be toxic and a deficiency can result in chronic, and sometimes fatal, failure. Because of that, though selenium is probably the most widely investigated of all the

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viii

V. Valdiglesias, J. Méndez, E. Pásaro and B. Laffon

oligonutrients, it continues to be highly controversial and even health authorities have at times been confused. This chapter makes a review of the in vitro genotoxicity tests applied to evaluate the potential selenium-induced chromosomal alterations, mutagenicity, oxidative damage and influence on repair ability, among others, using different chemical forms and concentrations. Results from this kind of studies are expected to provide a suitable basis for the regulation of its use in nutrition and clinic. Chapter 2 - The potential genetic toxicity of drug candidates is a significant and potentially development limiting concern for most therapeutic indications. Currently, screening for genetic toxicity is achieved with a battery of in vitro and in vivo regulatory genetic toxicity assays. These assays have been selected based on the requirements that they can provide high precision and also predictivity of different mechanisms of genetic toxicity. These regulatory approaches provide prediction of human carcinogenic risk, but time and cost factors limit the number of chemicals that can be evaluated in these systems and most are still applied late in preclinical drug development. Therefore, predicting potential genetic toxicity of drug candidates earlier in the drug discovery process helps to prevent costly latestage failures. Although in recent years advances have been made in the development of in silico models for prediction of genetic toxicity, these in silico models are still not sufficiently global to robustly predict the diversity of chemotypes and mechanisms. Thus, there continues to be an important need for robust high-throughput in vitro genetic toxicity screens. In particular, cell-based reporter assays using gene-expression technologies to identify hazards and predict risks continue to be a promising area for improving the predictivity of in vitro genetic toxicity assays. These assays are able to predict genetic toxicity potential following relatively brief exposures using small amounts of test compound. The advantage of cell-based reporter assays is their high specificity, selectivity, and rapid reaction times. Expression of a reporter gene produces a measurable signal, which can be readily distinguished over the background. Such reporter gene assays have been developed for both eukaryotic and prokaryotic cells that are able to activate transcription of many genes following treatment with agents that cause stress and damage of DNA. However, despite their potential, reporter gene assays are limited in their capabilities as they are usually conducted in vitro and, therefore cannot predict the interactions that may occur in vivo. Moreover, many responses are tissue, species, and time-specific and therefore a single in vitro reporter gene assay may not accurately predict the responses observed in vivo. Despite the pitfalls of reporter gene assays, the advance of toxicogenomic technologies and understanding will continue to evolve, allowing development of more sensitive and simpler reporter gene vectors for application of early genetic toxicity determination. This review provides an overview of the current status of high-throughput techniques and presents strategies for the prediction of genotoxicities. Chapter 3 - Lead is a toxic and cumulative metal that exerts its harmful action in virtually all body organs and systems. Exposure to this metal may result in a wide variety of biological effects depending on the level and duration of the exposure. Despite the progressive reduction in its applications, there are many industry sectors in which it is still present. In addition, lead has been used by humans since ancient times so, since it is a non-biodegradable

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ix

element, environmental pollution caused is persistent and widespread, affecting the population at large. The International Agency for Research on Cancer has classified lead and its inorganic compounds as probable human carcinogens (group 2A). Nowadays there is broad evidence of the relationship between the interaction with genetic material and cancer development, and thus genotoxicity tests are applied as biomarkers of the early effects of most carcinogen agents. Although exposure to lead, both in environmental and occupational settings, has been frequently associated with an increase in genotoxic damage in humans, controversy is nurtured by several works reporting conflicting results. This chapter conducts a review of the studies on genotoxicity of exposure to lead, compiling in vitro, in vivo and epidemiological studies using different DNA damage parameters from mutagenicity to chromosomal alterations. Chapter 4 - Nowadays, there is a conscious work to diminish the number of animals used in new cosmetics, drugs or medical devices research and development. Concerning medical devices development, in vitro tests have being used as a screening for the final prototype, prior to the in vivo studies. For regulation purposes, some basal tests were standardized. The most well known standard is the ISO 10993, which recommends to any developed medical device three first biological evaluations, the acute toxicity, the genotoxicity and the cytotoxicity. The genotoxicity and cytotoxicity assays can be performed in vitro, and both hold a good correlation. According to the ISO recommendations, one evaluated the cytotoxicity through a vital dye, the MTS, a new generation of the MTT. The cytotoxic test is quick and reproducible, especially when fast growth cells are used. There are many genotoxic assays to be performed. In authors laboratory one standardized the micronucleus test, which can be tested in CHO cells instead of in the lymphocytes, as the original idea of the test. For medical devices development, the use of CHO cell line presents an advantage once it allows a large number of samples usage at the same time. In general, the result is expressed as Cell Proliferation Index (CPI) which is related to the score of the total cell number with mononuclear, binuclear and multinuclear characteristics. The test recommends a frequency of micronucleus estimation, but it does not lead to the result analysis, which represents a worth result. In bovine pericardium treated with different compounds it was observed that the CPI is similar to the negative control, but the micronucleus frequency is significantly altered, and depending on the sample exposition or not, to the metabolic activation system, the S9. The same was observed for liposomes as drug delivery system. Stain steel coated with TiCN showed to be safe as medical devices. The CPI and the micronucleus frequency correlation concerning the development of medical devices is the core of this work. Chapter 5 - In humans, DNA damage or genotoxicity may be caused by exposure to outside agents like radiation, pesticides, combustion of hydrocarbons products as well as antineoplastic drugs. But also DNA damage could be come from inside of the body, determined mainly for excessive free radicals production generated by some disease process and that their increase exceed the natural defense systems responsible for removing free radicals. In either case, the important thing is to identify the genotoxicity and try to protect the body, this may as simply as removing the source of exposure or provide protection against such agents.

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V. Valdiglesias, J. Méndez, E. Pásaro and B. Laffon

In this chapter, authors address some ways of how it has been identified genotoxic compounds by direct analyses, basically micronucleogenics ones expressed quantitatively by number of micronuclei in peripheral blood erythrocytes and micronucleated cells from oral mucosa, the genotoxicity of some antineoplastic drugs as well as the identification of some diseases that are ”genotoxic” to the individual that suffers it and that under certain conditions can also result in potentially teratogenic for children to mothers who had suffered some of these disorders. Chapter 6 - Having summarized mainly authors own data, methodological research problems in modification of chemical mutagenesis were considered, common recommendations on planning and performing experiments were given. Also possible ways of further research and perspective of practical use of obtained results were analyzed. Chapter 7 - Influence of calcium channel blockers nifedipine, diltiazem and verapamil on the clastogenic and DNA damaging effects of cyclophosphamide and dioxidine in mice bone marrow cells was studied by chromosome aberration test and comet assay. Nifedipine (0.1, 1 and 5 mg/kg, per os) depending upon the dose and mode of administration potentiated clastogenic effects of cyclophosphamide (20 mg/kg) 1.3-2.9 times and clastogenic effect of dioxidine (200 mg/kg) 1.5-2.9 times. Similarly diltiazem (0.5, 5 and 30 mg/kg, per os) potentiated clastogenic effects of cyclophosphamide 1.2-1.7 times and dioxidine effects - 1.5 times. It was shown by the comet assay that nifedipine (5 mg/kg) depending on the term of application (6 and 18 hours) reduced the level of DNA damage (%DNA in the tail) induced by cyclophosphamide by 36-70%, and dioxidine effects (1 and 3 hours) by 19-37%. Diltiazem (5 mg/kg) and verapamil (2.5 mg/kg) reduced cyclophosphamide induced DNA damages by 48-64% and 58-74%, respectively. Reduction of dioxidine induced DNA damages by 20-46% and 41-42% was observed respectively for diltiazem and nifedipine. Nifedipine and diltiazem potentiated in vivo clastogenic effects of mutagens with various mechanisms of action. Co-mutagenic actions of drugs were manifested in doses equal to daily ones for humans and were mostly expressed in case of multiple administrations. Comutagenic effects of calcium channel blockers were not connected with direct efforts of DNA damaging action of mutagens. Chapter 8 - Epidemiological evidence suggests the existence of a relationship between high environmental levels of Particulate Matter (PM) and increased incidence of both morbidity and mortality due to respiratory and/or cardiovascular diseases. The consistency among the findings of epidemiological studies argues for a causal association, but it is still difficult to attribute acute health effects to concentration levels in light of the current knowledge. The mechanisms underlying these adverse effects are also not well understood, and major questions still remain concerning the specific size fraction, chemical composition and causative toxicological mechanisms leading to the observed health effects. Hence, to improve the knowledge of air pollution PM-induced toxicity in human lungs, with a particular interest in the crucial role played by coated organic compounds, authors focused our attention on the metabolic activation of Polycyclic Aromatic Hydrocarbon (PAH)-coated onto air pollution PM and, thereafter, the formation of PAH-DNA adducts in three in vitro cell lung models: A549 cell line, L132 cell line and human Alveolar Macrophages (AM). PAH, PolyChlorinated Dibenzo-p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB) and PolyChlorinated Biphenyls (PCB) coated onto

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Preface

xi

collected PM were determined (i.e., GC/MS and HRGC/HRMS, respectively). Cells were exposed to Dunkerque City’s PM2.5 at its lethal concentrations at 10% and 50% (i.e., A549: LC10 = 6.33µg/cm²; and LC50 = 31.63µg/cm²; L132: LC10 = 5.02µg PM/cm²; and LC50 = 20.10µg PM/cm²; AM: LC10 = 5.97µg PM/cm²; and LC50 = 29.85µg PM/cm²). Cytochrome P450 (CYP) 1A1 gene expression (i.e., RT-PCR) and enzymatic activity (i.e., EROD activity) and the formation of PAH-DNA adducts (i.e., 32P-postlabelling) were investigated after 24, 48 and/or 72h. Particular (i.e., desorbed PM, dPM) and PAH-positive (i.e., Benzo[a]Pyrene, B[a]P; 1µM) controls were included in the experimental design. A statistically significant increase in CYP1A1 gene expression was observed in the three cell lung models under study, 24, 48 and/or 72h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Similarly, CYP1A1 gene expression was highly induced 24, 48 and/or 72h after lung cell exposure to PM or B[a]P. Accordingly, EROD activity was significantly increased only in A549 cells and AM, 24, 48 and/or 72h after their exposure to dPM, PM or B[a]P. Even if B[a]P exposure caused important bulky DNA adduct formation, only very low levels of PAH-DNA adducts, which could not be reliably quantified, were reported 72h after A549 cells and AM exposure to PM and dPM. The relatively low levels of PAH, together with the presence of PCDD/F, DLPCB and PCB coated onto Dunkerque City’s PM2.5 could contribute to explain this borderline detection of PAH-DNA adducts. Neither significant EROD activity nor PAH-DNA adduct formation could be measured in PM- or in B[a]P-exposed L132 cells. In contrast, A549 cells and AM seem to present higher sensibility to coated organic chemical-induced damage. The authors also concluded that, in the three lung cellular models authors used and in the experimental conditions authors chose, bulky DNA adduct formation only partly contribute to the lung toxicity arising from the exposure to Dunkerque City’s PM2.5. Chapter 9 - Cadmium is a heavy metal widely used in industry that affects human health through occupational and environmental exposures. In mammals, cadmium and cadmium compounds have been found to exert multiple toxic effects and have been classified as human carcinogens by the International Agency for Research on Cancer. The present study was designed to investigate the possible mechanisms of cadmium-induced genotoxicity and clastogenicity in vivo. The authors studied potential molecular markers of genotoxic DNA damage, viz. Cd2+DNA adducts, oxidative DNA lesions 8-hydroxy-2′-deoxyguanosines (8OHdGs), DNA single-strand breaks (SSBs), and DNA-protein crosslinks (DPCs) along with cytogenetic markers, viz. sister-chromatid exchanges (SCEs), and chromosomal aberration (CAs) in both a dose- and time-dependent manner in a murine model. Cadmium chloride (CdCl2) was administered subcutaneously in doses of 0.5, 1.5, 2.5, and 5.0 mg/kg body weight to Swiss albino Balb/c male mice. The animals were exposed for 8, 16, and 24 days following cadmium intoxications, respectively. Results showed that there was a significant induction of tissue-specific covalent Cd2+DNA adducts (86.54%; P Se dioxide >> selenic acid > sodium selenate. These results seem to be represented in other subsequent Se studies. Inorganic Se compounds, as sodium selenite, sodium selenate and sodium selenide, were reported to increase CA rates in different cell lines. Selenite induced CA in human fibroblasts (Lo et al., 1978) and lymphocytes (Whiting et al., 1980; Kalhil et al., 1989; Biswas et al., 2000; AbulHassan et al., 2004). DNA damage induced by sodium selenate (Whiting et al., 1980; Biswas et al., 2000) and sodium selenide (Whiting et al., 1980) was found in human cells. Furthermore, organic forms of Se, as SeMet (Kalhil et al., 1989) and other synthetic organoSe compounds (Kalhil et al., 1990) have shown their ability to induce CA in human lymphocytes. Nevertheless, many in vitro studies have proved that adequate levels of Se can reduce the CA induced by different mutagenic compounds. Se (as sodium selenite) was found to protect cells against sodium arsenite reducing the frequency of gaps and chromatid breaks induced by this compound (Sweins, 1983; Beckman and Nordenson, 1986). Another study concluded that sodium selenite under specific conditions reduces the percentage of cells with N-MethylN'-Nitro-N-Nitrosoguanidine (MNNG)-induced CA (An et al., 1988), but this protective effect is clearly time- and dose-dependent, resulting in toxic effects at high concentrations. Se (sodium selenite and SeMet) also protected mammalian cells against lead acetate- and sodium sulfite- (Beckman and Nordenson, 1986), carbon tetrachloride- (Siviková et al., 2001) and doxorubicin-induced damage (Santos and Takahashi, 2008).

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4.2. Sister Chromatid Exchanges The sister chromatid exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome (OECD, 1986c). These exchange process involves DNA breakage and reunion, originated from reciprocal DNA interchange in homologous loci of sister chromatids in the replication process (Latt and Schreck, 1980). Although they occur spontaneously at certain rates in all cells, some chemical and physical agents causing DNA damage may lead to increased SCE frequency, so induction of SCE represents a sensitive cytogenetic endpoint for detection of genotoxic activity of environmental mutagens and carcinogens (WHO, 1993). Furthermore, SCE can also be measured in mammals and in non-mammalian systems (OECD, 1986c). Unlike CA, the interchange does not involve morphological alteration and is detected only by differential labelling of the sister chromatids. Traditional methods using 3Hthymidine incorporation into newly transcribed DNA have been replaced by methods using 5bromo-2’-deoxyuridine (BrdU) labels combined with different stains (Barile, 2008). To perform this assay, mammalian cells are exposed in vitro to the test compound with and without an exogenous mammalian metabolic activation system, and cultured for two rounds of replication in a BrdU containing medium. After treatment with a spindle inhibitor (e.g.

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The Importance of the In Vitro Genotoxicity Evaluation of Food Components

15

colchicine) to accumulate cells in a metaphase-like stage of mitosis (c-metaphase), cells are harvested and chromosome preparations are made (OECD, 1986c). Some epidemiological studies have reported an inverse relationship between high intake of Se and SCE levels (Cheng et al., 1995), but in vitro studies have shown that those levels depend on Se concentration (Weitberg et al., 1985), chemical form (Ray and Altenburg, 1980; Sirianni et al., 1983), exposure time (Ray and Altenburg, 1978), and culture conditions employed (Ray et al., 1978). The different capabilities of the Se compounds to induce SCE were clearly demonstrated in the system employed by Sirianni et al. (1983). Their study was designed to investigate the effect of three Se compounds (sodium selenite, sodium selenide and sodium selenate) on the induction of SCE in the Chinese hamster V79 cell line, in presence and absence of S9 mixture. The results indicated that the most potent SCE inducer in the presence of S9 mixture was sodium selenite, followed by sodium selenide, while in the absence of S9 mixture the most effective SCE inducer was sodium selenide, followed by sodium selenite. For sodium selenate, the data showed no increase in SCE rate as compared to the control values both in the presence and absence of S9 mixture. Previously, Ray and Altenburg (1980) had shown that sodium selenide, Se dioxide, Se(0), sodium selenate, and sodium selenite have different abilities to induce SCE in human whole blood cultures. Sodium selenate did not induce SCE and, amongst the four SCEinducing Se compounds studied, Se(0) was the most potent inducer of SCE, and sodium selenite was the less effective SCE-inducing agent. The SCE-inducing abilities of the Se compounds in decreasing order of their effectiveness were: Se(0) > Se dioxide > solium selenide > sodium selenite > sodium selenate. In another experiment (Ray and Altenburg, 1982), sodium selenite was tested for its SCE-inducing ability in human whole-blood cultures. Relatively high sodium selenite concentrations resulted in a 3-fold increase in the SCE frequency over the background levels. Increases in SCE rates induced by sodium selenite in other in vitro studies were also found (Ray and Altenburg, 1978; Ray et al., 1978; Morimoto et al., 1982). Nevertheless, sodium selenite significantly reduced SCE frequencies induced by fluorescent light in human fibroblasts (Parshad et al., 1980), and also by arsenic (Hu et al., 1996), and by carbon tetrachloride (Siviková et al., 2001) in peripheral lymphocytes. Moreover, some studies demonstrated that sodium selenite can antagonize the ability of other compounds to cause DNA damage leading to the formation of SCE. This was the case of two mercury derivatives (Morimoto et al., 1982), and methyl methanesulfonate or N-hydroxy-2acetylaminofluorene (Ray and Altenburg, 1978), which cause an increase in SCE, but simultaneous addition of sodium selenite to the cultures resulted in SCE frequencies below the sum of the SCE frequencies produced by the individual compounds. Weitberg et al. (1985) concluded that these different Se effects were due to the dose applied, since it was shown that sodium selenite had variable effects on the number of SCE generated in mammalian cells depending on the concentration used. Low concentrations of sodium selenite protected target cells. However, intermediate concentrations had no effect on oxidant-induced SCE formation, and high concentrations increased the number of exchanges.

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4.3. Micronucleus Test Micronuclei (MN) are defined as small chromatinic bodies that appear in the cytoplasm by condensation of acentric chromosome fragments or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division (Martínez et al., 2005). As a consequence, to analyse the induction of MN it is essential that nuclear division has occurred in both treated and untreated cultures (OECD, 2007). The most informative stage for scoring MN is in cells that have completed one mitosis during or after treatment with the test substance. The development of the cytokinesis-block MN assay (Fenech and Morley, 1985) has allowed the identification of those cells which have divided only once after exposure by their binucleated appearance, therefore enhancing the sensitivity and accuracy of the traditional/classical MN test. MN test uses cultured human or rodent cells to measure the average frequency of MN in a population, and thus assess genotoxic effects (Berces et al., 1993). It provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected. There are extensive data to support the validity of the MN assay using various rodent cell lines (CHO, V79, CHL, and L5178Y) and human lymphocytes. Because of that, the in vitro MN test has been adequately validated as genotoxicity test, the respective OECD guideline has been already redacted (OECD, 2007), and is currently waiting for approval. Moreover, this cytogenetic biomarker has been suggested to provide a reasonable epidemiological evaluation of cancer predictivity (Tucker et al., 1996; Bonassi et al., 2007). MN test represents a sensitive indicator of chromosome damage that provides a measure of both chromosome breakage and chromosome loss, and has the advantages of relative ease of scoring and high statistical power, obtained from scoring larger number of cells than are typically used for metaphase analysis (Fenech et al., 1999). Furthermore, nucleoplasmic bridges (NPB) are an important biomarker of chromosome rearrangement originated from dicentric chromosomes that are pulled to opposite poles of the cell at anaphase (Fenech, 2000), and they may be also scored in binucleated cells. Tests designed for the measurement of MN are comparable in sensitivity and speed for detection of CA (Barile, 2008), although the former are able to detect both structural and numerical CA (Marzin, 1997). MN test proves to be reliable, easy to perform, and relatively quick for in vitro testing of genotoxic compounds. The method can be automated easily. Its advantages make it a valuable assay that can be performed routinely (Berces et al., 1993). One of the main advantages of the MN technique is its ability to combine with other molecular techniques, as fluorescence in situ hybridisation (FISH). The labelling and hybridisation procedures can be used when there is an increase in MN formation and the investigator wishes to determine if the increase was the result of clastogenic and/or aneugenic events (Parry and Sors, 1993; Kirsch-Volders, 1997). Some studies using MN test to evaluate the genotoxicity of Se have shown that this agent does not produce genotoxic effects. Sodium selenite and Se dioxide did not induce considerable increases of MN frequency in human lymphocytes at the concentrations tested (Berces et al., 1993), and sodium selenate was also negative for MN induction in mouse bone marrow cells (Itoh and Shimada, 1996). Hasgekar et al. (2006) did not find influence of

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selenite in arsenic toxicity in primary rat hepatocytes either. Moreover, the work of Ebert et al. (2006) with bone marrow stromal cells with low antioxidative capacity concluded that selenite supplementation of cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures. In contrast, many other studies reported genotoxic effects of several Se compounds in different cell lines. Treatment with diphenyl diselenide, for example, induced an increase in the number of MN in V79 Chinese hamster cells, showing mutagenic risk by this molecule at high concentrations (Rosa et al., 2007a). Selenous acid increased MN formation in mouse bone marrow cells (Itoh and Shimada, 1996), in human lymphocytes and in TK6 lymphoblastoid cell line (Cemeli et al., 2006); and sodium selenate and sodium selenite also showed genotoxicity in TK6 cells (Cemeli et al., 2006). Several studies designed to evaluate the antigenotoxic properties of different Se compounds by means of MN test are collected in the literature. Diphenyl diselenide at low concentrations showed antimutagenic properties against H2O2, methyl-methanesulphonate and UVC radiation in lung fibroblast cells (Rosa et al., 2007b); supplementation of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of sodium selenite protected these cells from ultraviolet-induced chromosome damage (Baliga et al., 2007); selenous acid and sodium selenate reduced the DNA damage induced by potassium dichromate in human lymphocytes and TK6 cells, respectively (Cemeli et al., 2006); sodium selenite decreased the MN rate induced by MNNG in children's foreskin fibroblasts (An et al., 1988); V79 cells showed diminished cadmium-induced MN frequency when treated with sodium selenite (Hurná et al., 1997) and its protective effect was also demonstrated in ovine peripheral lymphocytes cultured with carbon tetrachloride (Siviková et al., 2001). The results of these studies indicate that the genotoxic and antigenotoxic properties of Se compounds are highly dependent upon the conditions under which they are evaluated (Cemeli et al., 2006), and that the protection offered by Se compounds against damage induced in genetic material is time- and dose-dependent (An et al., 1988). 4.4. Comet Assay The alkaline single-cell gel electrophoresis (comet) assay is a rapid, simple and sensitive genotoxicity test for the measurement of induced DNA damage in individual cells, based on the principle of quantifying the amount of denatured DNA fragments migrating out of the cell nuclei during electrophoresis (McKelvey-Martin et al., 1993). This is a key point in the interpretation of the results provided by this assay, as these lesions are usually repaired quite fast and thus the reflected damage is relatively recent (Pérez-Cadahía, et al., 2008). The comet assay measures DNA strand breaks (including those coming from alkali-labile lesions such as apurinic sites, and from incomplete excision-repair processes). To perform this assay, the cells are embedded in agarose gels on microscope slides, lysed, and subjected to electrophoresis. Intact DNA, with very high molecular weight, remains in place, but damaged DNA, denatured in the alkaline buffer, migrates into the gel. After staining with a florescent dye, damaged cells take the appearance of a “comet”, with the head containing unbroken DNA and the tail, streaming away in the direction of electrophoresis, containing

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broken DNA (Laffon et al., 2002). DNA damage detected by the alkaline version of the comet assay can arise through various mechanisms, including DNA double- and single-strand breaks, DNA interstrand cross-linking, alkali-labile sites, and incompletely repaired excision sites (Rojas et al., 1999), so it gives a good reflect of a very early response of the organism to the effects of different chemical, physical and biological agents on DNA integrity, and constitutes a very useful tool for determining their genotoxicity (Cebulska-Wasilewska et al., 1998). This assay is not gathered in the OECD guides, but it is nowadays one of the most frequently employed genotoxicity tests. Tice et al. (2000) suggested several advantages of the in vitro comet assay compared to other genotoxicity assays: it has demonstrated sensitivity for detecting low levels of DNA damage; there is a requirement for small number of cells per sample; it has flexibility, low cost and ease of application; studies can be conducted using relatively small amounts of a test substance; and a relatively short time is needed to complete an experiment. Moreover, the comet assay can be virtually applied on any cell type as long as a single cell suspension is obtained (Cemeli et al., 2008). Thus, this assay represents an adequate complement to SCE and MN cytogenetic tests, since it reflects a more recent type of damage that could be repaired (Pérez-Cadahía et al., 2006). The study of different Se compounds by means of comet assay resulted in no significant genotoxic effect found for selenite, selenate, SeMet or Se-methylselenocysteine in C6 rat glial cells (Yeh et al., 2006), for SeMet in human lymphocytes (Laffon et al., 2009) and human fibroblasts (Seo et al., 2002), and for ebselen in HepG2 (Yang et al., 1999) and V79 cells (Miorelli et al., 2008). Nevertheless, prooxidant responses of Se compounds have been reported. DNA damage induced by sodium selenate, sodium selenite and selenous acid on their own was detected with comet assay in human lymphocytes (Cemeli et al., 2003). Results obtained with this test also showed that selenite induced oxidative stress and apoptosis, and these effects were significantly attenuated by superoxide-dismutase, catalase and deferoxamine (Shen et al., 1999). Methylseleninic acid induced apoptosis without induction of ROS in two prostate cancer cell lines, whereas selenite generated strand breaks in chromosomal DNA of LNCaP cells and induced apoptosis by producing superoxide to activate p53 (Li et al., 2007). At high doses, diphenyl diselenide also generated DNA strand breaks, as observed using the comet assay (Rosa et al., 2007a). The literature agrees with the protective effect of Se evaluated with the comet assay against a variety of chemical or physical toxic agents. In vitro investigations found that Se (sodium selenite and ebselen) prevented DNA damage from H2O2 in murine lymphoma cells (Bouzyk et al., 1997), in HepG2 cells (Yang et al., 1999), in Chinese hamster V79 cells (Miorelli et al., 2008), and in mouse hepatoma Hepa 1c1c7 cells (Keck and Finley, 2006). Sodium selenite also inhibited the DNA damage caused by cadmium chloride in rat hepatic cells (Yu et al., 2004). Sodium selenate avoided DNA damage mediated by UVA radiation in human skin fibroblasts (Emonet-Piccardi et al., 1998) and by quenched potassium dichromate in human lymphocytes (Cemeli et al., 2003). Sodium selenite and SeMet protected keratinocytes against UV-induced oxidative damage (Rafferty et al., 2003), as well as SeMet protected against genotoxicity induced by doxorubicin in human lymphocytes (Santos and Takahashi, 2008). Finally, low concentrations of diphenyl diselenide showed antimutagenic

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properties in V79 cells treated with H2O2, methyl-methanesulphonate and UVC radiation, probably due to the antioxidant diphenyl diselenide properties (Rosa et al., 2007a,b). The comet assay is usually employed in DNA repair studies because of its sensitivity for the measurement of radiation- or chemically-induced DNA damage and repair in viable cells (McKelvey-Martin et al., 1993). In this regard, Seo et al. (2002) confirmed by means of this assay that SeMet induces DNA repair in normal human fibroblasts in vitro after a challenge with UV-radiation, and Laffon et al. (2009) reported that bleomycin-induced DNA damage in human lymphocytes was repaired better in presence of SeMet.

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4.5. DNA Damage and Repair, Unscheduled DNA Synthesis As mammalian cells both in vivo and in vitro are continuously insulted with natural and experimental exposures to chemicals, they have by evolutionary selection developed defence systems that facilitate repair of the inflicted damage. The repair mechanism of the process of unscheduled DNA synthesis (UDS) is well characterized, consequently, this test is used to assay the influence of different chemical and physical agents on DNA synthesis and repair processes (Barile, 2008). The UDS test measures the DNA repair synthesis after excision and removal of a stretch of DNA containing the region damaged by chemical or physical agents. The principles of this toxicologic test rely on the incorporation of 3H-labelled thymidine into new synthesized DNA of cultured cells during or after treatment with the test chemical (Barile, 2008). The cells must be kept under conditions preventing normal semiconservative replication but allowing DNA repair (Madle et al., 1994). The assay generates semi-quantitative data by autoradiography or liquid scintillation counting, respectively. Mammalian cells in culture are treated with the test substance with and without exogenous mammalian metabolic activation, unless primary rat hepatocytes are used (OECD, 1986d; Püssa, 2008). The UDS test is commonly used to assay DNA repair in vitro because UDS estimation constitutes an indirect measurement of DNA damage, since the sequential enzymatic repair process is initiated once the cell recognizes the presence of DNA adducts formed as a result of chemical insult. The primary DNA structure is restored through excision of the adducts, DNA strand polymerization, and subsequent ligation. The intranuclear incorporation of the radioactive nucleotide during the repair process acts as a quantitative tracer for the repaired lesion during any period of anticipated or unanticipated (unscheduled) DNA synthesis (Barile, 2008). A role for Se in DNA repair was first noticed when Se treatment was shown to enhance host cell reactivation of a UV-damaged reporter plasmid template by enhancing DNA repair protein complexes (Seo et al., 2002). Enhancement of DNA repair could be a mechanism of chemoprevention and only very few compounds have been yet shown to act by this mechanism (Collins et al., 2003). Furthermore, Zhang et al. (2008) inferred that Se only enhances DNA repair of normal tissues as a consequence of the selective modulation of Se on Nrf2 in tumour and normal tissues (Kim et al., 2007). Studies of the UDS were conducted with mammalian cells to determine the effects of Se on cell proliferation and the stages of the cell cycle affected by this element (LeBoeuf et al., 1985). Despite of many of the in vitro studies concluded that Se (mainly as selenite form)

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induces an inhibition of DNA synthesis (reviewed in Frenkel and Falvey, 1988), Se has also been suggested to be a DNA repair promoter (Russell et al., 1980). Whiting et al. (1980) studied the induction of UDS in cultured human cells by different inorganic and organic Se compounds. They found that inorganic compounds (sodium selenate, sodium selenite and sodium selenide) induced low levels of UDS in absence of glutathione, but high levels of UDS were found in the presence of this peptide. Nevertheless, no UDS was detected in cells treated with organic compounds (selenocystamine or selenomethione), with or without added glutathione, and only selenocystine induced a low level of UDS, being also enhanced by glutathione. These differences between chemical forms of Se can also be due to the different concentrations of each compound assayed. Verma et al. (2004) for example, proved that gastric adenocarcinoma SNU-1 cells responded to SeMet with a biphasic proliferative curve: enhanced incorporation of 3H-thymidine into DNA within a very narrow range of SeMet concentrations, followed by decreased 3H-thymidine uptake at higher levels. This biphasic effect of Se on cell growth was also observed in another previous in vitro study (Medina and Oborn, 1984). Some Se concentrations stimulated cell growth whereas others were cytotoxic, and the inhibition of cell growth by Se resulted reversible when these doses were removed from the growth medium. The increased cell growth was reflected by an increased cell number, increased uptake of 3H-thymidine into DNA, increased DNA labelling index, and increased rate of DNA synthesis. The differential effects of Se were manifested by 48 h after addition of Se to the cell culture medium. DNA synthesis was evaluated in vitro by measuring incorporation of 3H-thymidine in rat lens following systemic delivery of a cataractogenic dose of selenite. UDS was found to be approximately 10% of the total DNA formed, but there was a 30% and 70% increase of this putative DNA repair in the lenses from selenite-treated animals at 6 and 24 h after the injection, respectively; 3H-thymidine incorporation in DNA remained elevated compared to controls through 96 h (Huang et al., 1990). In one recent study, different mammalian cells lines (rat gut epithelial cells, primary mouse bone marrow cells and human squamous cell carcinoma of the head and neck cells) were treated with SeMet and with a variety of DNA-damaging agents, and then UDS was determined. Data showed that SeMet pretreatment caused a DNA repair response, which protected from subsequent challenge with DNA-damaging agents (Fischer et al., 2007). As general conclusions from results of different assays to study the influence of Se on DNA synthesis, we know that it depends mainly on the cell line employed (Webber et al., 1985; Vadgama et al., 2000), chemical Se form (Whiting et al., 1980; Frenkel, 1985; Bansal and Sood, 1999) and concentration of Se assayed (Medina and Oborn, 1984; Morrison et al., 1988; Nano et al., 1989; Verma et al; 2004). 4.6. Flow Cytometry Despite of not being specifically recommended in any official guide for testing chemicals, the use of in vitro cytometry techniques has increased in the last decades providing, among other, interesting information about cell cycle alterations and apoptosis/necrosis indexes as indirect measurement of genotoxicity.

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Flow cytometry is a system for sensing cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area. The relative light-scattering and colour-discriminated fluorescence of the microscopic particles is measured. Analysis and differentiation of the cells is based on size, granularity, and whether the cell is carrying fluorescent molecules in the form of either antibodies or dyes (Macey et al., 2007). Flow cytometry is well suited to DNA analysis because dyes are available that bind DNA in a proportional and linear fashion. This allows the quantification of DNA content, enabling the identification of normal diploid cells at rest, those that are actively synthesizing DNA, and those that are either premitotic or actually in mitosis. Once these phases of the cell cycle have been identified, it is possible to relate protein expression with stages of the cell cycle and to evaluate the growing alterations and the number CA (ploidy or aneuploidy) (Macey et al., 2007). The effect of Se alone or in combination with other compounds on the growth and proliferation of different mammalian cells has been investigated by means of flow cytometry techniques. In an early study, Se, as sodium selenite, was shown to decrease the growth of fibroblasts and hepatoma cells in a dose-dependent manner, and this inhibition was reversible upon removal of Se from the growth medium (LeBoeuf et al., 1985). Later, it was reported that selenite inhibited cell growth by G2/M arrest in a mammary tumour cell line (Lu et al., 1995), in human oesophageal cancer cells when combined with zinc (Xiao et al., 2008), and in lymphoblastic leukaemia MT-4 cells (Philchenkov et al., 2007); however it promoted cell proliferation at high concentrations (Xiao et al., 2008). An increase of the S-phase fraction in the presence of Se was found in a human maxillary cancer cell line (Yamamoto et al., 1996). The effect of Se-garlic extract and Semethylselenocysteine on cell morphology, cell growth, and cell cycle progression was also studied in mammary epithelial cells, both agents inducing growth inhibition by G1-phase cell cycle arrest (Lu et al., 1996). SeMet also induced G2/M arrest in certain prostate and colon cancer cell lines (Venkateswaran et al., 2002; Goel et al., 2006; Zhao and Brooks, 2007), and methylseleninic acid caused G0/G1 arrest in prostate cancer cells (Zhao et al., 2004). A recent work investigated the variability of the effects on cell viability, redox modulation, and disruption of subcellular compartments by different selenocompounds (SeMet, methylseleninic acid, and selenazolidines) in several human lung cell lines (Poerschke et al., 2008). Results of this study demonstrated that all selenocompounds behave different and that the chemical form of the organic selenocompound is a major determinant in the expected cellular response. Apoptotic cells have many characteristics that can be measured by flow cytometry. These include cell plasma membrane permeability, caspase activation, and DNA cleavage. Determination of anyone or a combination of these changes by flow cytometry allows the identification and quantification of molecular pathways that cells take during cell death (Macey et al., 2007). From the results of Se apoptosis studies, it is known that several selenocompounds (mainly sodium selenite but also SeMet, Se dioxide and methylseleninic acid) induce cell death in different mammalian cell lines: human prostate cancer cells (Xiang et al., 2009), lymphoblastic leukemia MT-4 cells (Philchenkov et al., 2007), HepG2 cells (Zou et al.,

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2007), colon cancer cell lines (Goel et al., 2006), lymphoma cell lines and primary lymphoma cultures (Last et al., 2006), leukemia cell lines (Li et al., 2002; Wang et al., 2004), human pulmonary adenocarcinoma cells (Chen et al., 2003), and brain tumour cell lines (Rooprai et al., 2007). The precise mechanisms of apoptosis induced by the Se compounds are not well understood (Philchenkov et al., 2007); however it is believed that ROS may play a crucial role in Se-decreased cell viability and Se-induced apoptosis (Zou et al., 2007).

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4.7. Other Methodologies Genotoxicity tests previously exposed are the most frequently used, but there are many other assays to evaluate the direct o indirect DNA damage induced in different cell systems providing important additional information on the genotoxic potential of a specific agent. The mutation assays specified in the previous sections are capable of detecting different spectra of genetic damage. While the recommended battery consists of specific genetic toxicity tests, data from other systems that measure gene mutations, chromosomal effects, DNA damage, or cellular responses to DNA damage may be relevant to the overall genotoxicity evaluation of a chemical (FDA, 2000). There are a great variety of possible genotoxic in vitro tests according to the objective and characteristics of the study: evaluation of DNA adducts, microarrays studies, determination of oxidative damage, DNA ladders to evaluate strand breaks, gene expression assays, etc. All of them have contributed to increase our knowledge on the Se action mechanisms. Hurst et al. (2008) exposed two human prostate cell lines to nutritionally relevant doses of MeSeCys and selenite, ranging from deficient to the equivalent of Se supplementation in humans. Several Se-responsive genes were identified by means of two microarray platforms, many of which have been ascribed to cancer cell growth and progression. The study revealed that MeSeCys can alter the expression of several types of collagen and thus potentially modulate the extracellular matrix and stroma, which may at least partially explain the anticancer activity of MeSeCys. Shen et al. (2001) designed a study to investigate the interaction effects of selenite and SeMet plus vitamin C, trolox (a water-soluble vitamin E), and copper sulfate, on cell viability and induction of 8-OHdG adduct formation in DNA of primary human keratinocytes (NHK). The data showed that selenite, but not SeMet, induced oxidative DNA damage as 8-OHdG adducts, but coincubation with vitamin C or copper sulfate protected NHK cells against that selenite-induced cytotoxicity. However, synergistic effects were observed between selenite and trolox resulting in enhanced cytotoxicity. On the other hand, no effects on cell viability were observed when cells were treated with SeMet plus vitamin C, trolox or copper sulfate. Previous findings had already shown that high doses of selenite, acting as a prooxidant, induced cytotoxicity and DNA adducts in mouse skin cells whereas SeMet did not (Stewart et al., 1999). Furthermore, other studies reported that selenite and its metabolites at high doses resulted in cytotoxicity, DNA fragmentation (Wilson et al., 1992; Garberg et al., 1988), and cellular apoptosis (Lu et al., 1994; Davis et al., 1998; Stewart et al., 1996 and 1997). The effect of Se (as Se dioxide) on the accuracy of DNA synthesis in vitro was analyzed by means of the fidelity assay. Se did not alter fidelity under normal conditions of magnesium

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activation, nor affected the mutagenicity of manganese (Tkeshelashvili et al., 1980). However, several Se-derived compounds (dimethyl selenone, diphenyl selenone, sodium selenite and MeSeCys) reversed the proangiogenesis effect of arsenic, which is initiated at the endothelial cell plasma membrane by activation of the ERK1/2 signal transduction pathway (Mousa et al., 2007). The effect of two Se compounds and methyl mercury was also studied in cell cultures (Alexander et al., 1979). Selenite at low concentration and seleno-di-N-acetyl-glycine in thousandfold higher concentrations offered considerable protection against the growth inhibiting effect and the stimulation of glucose and lactate uptake caused by methyl mercury in rat Morris hepatoma cells. However, no protective effect of Se was observed in other cell types as human lymphocytes and human embryonic fibroblasts. The data obtained suggested that Se compounds exert their protective effect through cell-specific processes rather than by a direct chemical reaction between selenite and methyl mercury. Zhong and Oberley (2001) employed several methodologies (western blot, structural evaluation of mitochondria, cell growth analysis…) to study the effects of Se, as sodium selenite, in the LNCaP human prostate cell line. The data obtained enabled to conclude that the in vitro biological consequences of selenite exposure were different between acute and long-term exposure. In acute exposure, selenite caused cell death, mainly apoptosis attributable to oxidative stress; in chronic long-term exposure, selenite caused only minimal cell death but inhibited cell growth by modifying gene expression and cell cycle progression. Other studies using different techniques also contributed to enhance the knowledge on Se behaviour. Lu et al. (1995, 1996) observed by means of filter elution analyses that sodium selenite and sodium selenide induce single and double DNA strand breaks in a mouse mammary epithelial cell line, whereas MeSeCys and Se-garlic only induce single strand breaks and in lesser degree in the same cells. Morris et al. (2006) assayed the BrdU incorporation into DNA of primary epithelial prostate and LNCaP cells treated with SeMet or Se(0) to determine DNA synthesis. The results of the study demonstrated that both chemical Se forms can induce delay in DNA synthesis in a dose-dependent manner in both cell lines. Li et al. (2007) applied, among others, ELISA and western blot methodologies to two human prostate cancer cell lines after treatments with selenite and methylseleninic acid. Results showed that these Se forms induce ROS formation and apoptosis in both cell lines. In another work, the effects of methylseleninic acid on gene expression were evaluated by means of western blot and oligonucleotide array analysis in human prostate cancer cells (Dong et al., 2003). Data proved that Se alters the expression of different important genes inducing an increase in p21WAF1 and p19INK4d protein synthesis and a down-regulation of CDK1, CDK2 and cyclin A. This agrees with previous studies which reported that Se can upregulate or downregulate certain genes (El-Bayoumy and Sinha, 2005).

5. Concluding Remarks A number of guidelines for genotoxicity testing of several types of chemical entities have been published in the last few years (Kirkland et al., 2005); among others the UK Committee on Mutagenicity Guideline of Chemicals in Food, Consumer Products and Environment

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(COM, 2000), the Food and Drug Administration Redbook (FDA, 2000), and the OECD Guidelines for the testing of chemicals. All of these guidelines recommend performing several in vitro tests previously to identify potential genotoxic hazards. The in vitro tests include a wide variety of methods that are faster, cheaper and more convenient than whole animal tests for detection of mutations, breakage, and other genetic effects (Barile, 2008). In this chapter the most important in vitro assays to evaluate genotoxic effects induced by chemical, physical or biological agents have been described, and its applications to characterization of Se genotoxic properties have been reviewed. Most of these assays are already included in the OECD guidelines: the bacterial reverse mutation (Ames) test, the SCE assay, the CA test, the UDS test, and the S. cerevisiae-based assays; one, the MN test, has its guideline proposed and it is currently in consideration to be included in the OECD guidelines for testing chemicals; and other two, comet assay and flow cytometry techniques, have been included in this review for their importance and wide use in in vitro studies. Se is on of the oligoelements most studied because of its particular properties. Like some other trace elements, Se is bimodal in nature whereby its beneficial properties occur in a limited range of daily intake below which it cannot perform its essential functions, and above which it is toxic (Alaejos et al., 2000). This nutritional range between essentiality and toxicity in Se is fairly narrow in comparison to the other essential trace elements (Letavayová et al., 2008a), and this could explain, among other causes, the enormous variability in the Se studies. As a result of these properties, Se can be included in the class of “Janus compounds”, having two “faces” on the same head (Miorelli et al., 2008). At low concentrations, Se compounds are antimutagenic and anticarcinogenic, whereas at high concentrations they are mutagenic, toxic and possibly carcinogenic (Letavayová et al., 2008a). When the effects of different selenocompounds were evaluated by means of the different in vitro assays, results obtained varied highly showing a great controversy. As general conclusions, Se resulted in no mutagenic or weakly mutagenic effects in bacterial assays (Lofroth et al., 1978; Noda et al., 1979; Cemeli et al., 2003), but mutagenicity and genotoxicity of this element, mainly as sodium selenite, were observed in numerous studies with yeast (Rosin, 1981; Anjaria and Madhavanath, 1989; Berdicevsky et al., 1993; Letavayová 2008a). On the other hand, antigenotoxic properties of Se against a great variety of mutagenic agents were also detected in both cell systems (Martin et al., 1981; Longo et al., 1995; Bronzetti et al., 2001). This agrees with the results of different in vitro studies performed in mammalian cell systems. Positive results in some of these assays have been found for different selenocompounds. Data shown that Se induces CA (Nakamuro et al., 1976; Biswas et al., 2000) and SCE (Ray and Altenburg, 1980; Ray et al., 1982; Sirianni et al., 1983), inhibits DNA synthesis (Frenkel and Falvey; 1988) and cell growth (Lu et al., 1995; Goel et al., 2006; Philchenkov et al., 2007), and induces apoptosis (Chen et al., 2003; Last et al., 2006; Xiang et al., 2009). But also antigenotoxic and antimutagenic properties of adequate doses of Se against many chemical and physical agents have been described (Parshad et al., 1980; Sweins et al., 1983; Beckman and Nordenson, 1986; An et al., 1988; Hu et al., 1996; Bouzyk et al., 1997; Siviková et al., 2001; Cemeli et al., 2006; Rosa et al., 2007b; Santos and Takahashi, 2008). Nevertheless all these results are not constant in the literature and vary enormously even when the same in vitro tests are employed. Many factors contribute to this great variety of

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results, mainly its chemical form (Nakamuro et al., 1976; Whiting et al., 1980; Sirianni et al., 1983) and the concentration used (Weitberg et al., 1985, Biswas et al; 2000; Verma et al., 2004), but also the exposure time (Ray and Altenburg, 1978; An et al., 1988), the treatment conditions (Ray et al., 1978; Cemeli et al., 2006), the cell type or the target tissue (Webber et al., 1985; Vadgama et al., 2000), and other previous factors as method of administration, animal species, physiological status, interaction with other compounds, etc. (Burk and Levander, 2002). So, although it is common to speak of Se in the universal term of the element, just Se, the dose and form of the Se species actually determine its biological activity, be it the dietary essential nutrient, the cancer preventing agent, or the toxicant (Letavayová et al., 2008a). Se is an important element with beneficial properties as nutrient and its dietary deficiency is linked with decrease in glutathione-peroxidase activity, increase in lipid peroxidation (Colado-Megía et al., 2004), and also with some diseases, e.g. Keshan disease and Kashin-Beck disease (Thomson, 2004). Moreover, solid evidence based on epidemiological studies conducted in the last 50 years show an inverse relationship between Se intake and cancer mortality (Alaejos et al., 2000; Surai, 2006). For these reasons, today many people consume Se supplements on a regular basis to increase their intake and improve their nutritional status. They do this in the belief either that Se levels in the diet are inadequate or that the additional intake will provide protection against a variety of health problems. Much of current interest in Se as a supplement was triggered by the report by Clark et al. (1996). The use of dietary supplements is considerable in many countries and appears to be increasing. These products are tested in a battery of genotoxicity assays (Griffiths and Matulka, 2006), as those described in this chapter, before being commercially available, normally in tablet form, in quantities up to 200 µg, and sometimes more, per tablet (Reilly, 2006). Supplemental Se has been shown to have cancer-protective effects in a variety of experimental settings and clinical studies (reviewed in Whanger, 2004; Rayman, 2005), and to reduce the incidence and mortality of total cancer (Clark et al., 1996), prostate cancer (Duffield-Lillico et al., 2002), liver cancer (Yu et al., 1997), and stomach cancer (Blot et al., 1993) in human interventional trials. In general, the anticarcinogenic affect of Se against leukaemia and cancers of the colon, rectum, pancreas, breast, ovaries, prostate, bladder, lung and skin seems clear under at least some conditions (reviewed in Sunde, 2000), and is closely related to its role in selenoproteins reducing oxidative stress, to its ability to enhance the immune response or, more likely, to its ability to produce antitumourigenic metabolites (e.g. methylselenol or its precursors) that can perturb tumour-cell metabolism, inhibit angiogenesis and induce apoptosis in cancer cells (Rayman, 2000 and 2005; Whanger, 2004). But in spite of extensive literature describing the antimutagenic and anticarcinogenic effects of Se compounds, little is known about their mode of action (Miorelli et al., 2008), although the anticancer activity of Se seems to be also dose-dependent and species-specific (Hurst et al., 2008). The bulk of our knowledge on the mechanisms of cancer prevention by Se is based on animal data and from studies conducted in in vitro systems (El-Bayoumy and Sinha, 2005), and the modulation of certain in vitro markers may also be of value in predicting the effectiveness of novel forms of Se for cancer prevention. Thus there is a plausible correlation between the relevance of these in vitro markers and the consequence of

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V. Valdiglesias, J. Méndez, E. Pásaro and B. Laffon

in vivo cancer protection. Whether these markers apply only to the biology of Se chemoprevention or could be extended to other classes of anticancer agents remains to be investigated (Lu et al., 1996). In short, nowadays, besides the beneficial properties that Se has as nutrient and the fact that it seems to be effective in cancer prevention, the genotoxic effects of Se are currently being demonstrated in present studies. In this sense, the enormous variety of in vitro assays are allowing to describe, characterize and delimit these effects in order to provide important information on the correct use of Se supplements in human health and chemoprevention. These assays show several advantages, as allowing controlling the features of the exposure and employing human cell lines that can provide a more real view of its effects on human organism, what make them a perfect complement to in vivo assays when these can be used or an appropriate substitute when not.

Acknowledgments This work was supported (INCITE08PXIB106155PR).

by

a

grant

from

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Galicia

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Clark, L. C., Combs Jr, G. F., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow, J., Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad, A., Lesher Jr, J. L., Park, H. K., Sanders Jr, B. B., Smith, C. L. & Taylor, J. R. (1996). Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomized controlled trial. J. Am. Med. Assoc. 276, 1957-1963. Colado-Megía, M.I., Sánchez-Sánchez, V., Camarero-Jiménez, J., O’Shea-Gaya, E. 2004. Effect of dietary selenium on MDMA (“ecstasy”)-induced neurotoxicity in brain mouse (in Spanish). Mapfre Medicina 15, 53-62. Collins, A. R., Harrington, V., Drew, J., Melvin, R. (2003). Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis, 24, 511-515. COM, (2000). Guidance on a strategy for testing of chemicals for mutagenicity. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment. Combs, G. F. Jr. (1988). Selenium in foods. Adv. Food Res. 32, 85-113. Davis, R. L. Spallholz, J. E. & Pence, B. C. (1998). Inhibition of selenite-induced cytotoxicity and apoptosis in human colonic carcinoma (HT29) cells by copper. Nutr. Cancer 32, 181-189. Dilsiz, N., Celik S., Yilmaz. O. & Digrak, M. (1997). The effects of selenium, vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae. Cell Biochem. Funct. 15, 265-269. Dong, Y., Zhang, H., Hawthorn, L., Ganther, H.E. & Ip, C. (2003). Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer Res. 63, 52-59. Duffield-Lillico, A. J., Reid, M. E., Turnbull, B. W., Combs Jr, G. F., Slate, E. H., Fischbach, L. A., Marshall, J. R. & Clark, L. C. (2002). Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the nutritional prevention of cancer trial. Cancer Epidemiol. Biomarkers Prev. 11, 630-639. Duffield-Lillico, A. J., Slate, E. H., Reid, M. E., Turnbull, B. W., Wilkins, P. A., Combs Jr, G. F., Park, H. K., Gross, E. G., Graham, G. F., Stratton, M. S., Marshall, J. R. & Clark, L. C. (2003). Selenium supplementation and secondary prevention of nonmelanoma skin cancer in randomized trial. J. Natl. Cancer Inst. 95, 1477-1481. Ebert, R., Ulmer, M., Zeck, S., Meissner-Wiegl, J., Schneider, D., Stopper, H., Schupp, N., Kassem, M. & Jakob, F. (2006). Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro. Stem Cells 24, 1226-1235. El-Bayoumy, K. & Sinha, R. (2005). Molecular chemoprevention by selenium: A genomic approach. Mutat. Res. 591, 224-236. Emonet-Piccardi, N., Richard, M. J., Ravanat, J. L., Signorini, N., Cadet, J. & Beani, J. C. (1998). Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture. Free Radic. Res. 29, 307–313. Evans, H. J. (1976). Cytological methods for detecting chemical mutagens. In: Chemical mutagens, principles and methods for their detection, vol. 4. Plenum Press. New YorkLondon. Pp: 1-29.

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Chapter II

In Vitro High-Throughput Assays for Assessment of Genetic Toxicology

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Xuemei Liu*¶, Jeffrey A. Kramer¶, Larry D. Kier§ and Alan G. E. Wilson¶

Drug Metabolism, Pharmacokinetics, and Toxicology Lexicon Pharmaceuticals, Inc., The Woodlands, TX, 77381 § 16428 CR 356-8, Buena Vista, CO 81211

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Abstract The potential genetic toxicity of drug candidates is a significant and potentially development limiting concern for most therapeutic indications. Currently, screening for genetic toxicity is achieved with a battery of in vitro and in vivo regulatory genetic toxicity assays. These assays have been selected based on the requirements that they can provide high precision and also predictivity of different mechanisms of genetic toxicity. These regulatory approaches provide prediction of human carcinogenic risk, but time and cost factors limit the number of chemicals that can be evaluated in these systems and most are still applied late in preclinical drug development. Therefore, predicting potential genetic toxicity of drug candidates earlier in the drug discovery process helps to prevent costly late-stage failures. Although in recent years advances have been made in the development of in silico models for prediction of genetic toxicity, these in silico models are still not sufficiently global to robustly predict the diversity of chemotypes and mechanisms. Thus, there continues to be an important need for robust high-throughput in vitro genetic toxicity screens. In particular, cell-based reporter assays using geneexpression technologies to identify hazards and predict risks continue to be a promising area for improving the predictivity of in vitro genetic toxicity assays. These assays are able to predict genetic toxicity potential following relatively brief exposures using small *

Correspondence to: Xuemei Liu, Drug Metabolism, Pharmacokinetic, and Toxicology Lexicon Pharmaceuticals, Inc., 8800 Technology Forest Place, The Woodlands TX 77381, Phone: 281-8633626,Fax: 281-863-3564 E-mail: [email protected]

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Xuemei Liu, Jeffrey A. Kramer, Larry D. Kier et al. amounts of test compound. The advantage of cell-based reporter assays is their high specificity, selectivity, and rapid reaction times. Expression of a reporter gene produces a measurable signal, which can be readily distinguished over the background. Such reporter gene assays have been developed for both eukaryotic and prokaryotic cells that are able to activate transcription of many genes following treatment with agents that cause stress and damage of DNA. However, despite their potential, reporter gene assays are limited in their capabilities as they are usually conducted in vitro and, therefore cannot predict the interactions that may occur in vivo. Moreover, many responses are tissue, species, and time-specific and therefore a single in vitro reporter gene assay may not accurately predict the responses observed in vivo. Despite the pitfalls of reporter gene assays, the advance of toxicogenomic technologies and understanding will continue to evolve, allowing development of more sensitive and simpler reporter gene vectors for application of early genetic toxicity determination. This review provides an overview of the current status of high-throughput techniques and presents strategies for the prediction of genotoxicities.

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Introduction Genetic toxicology represents the study of the effects of chemical and physical agents on the deoxyribonucleic acid (DNA) and the genetic processes of living cells [1, 2]. Various forms of DNA alterations such as gene mutations, larger scale chromosomal damage, recombination, and numerical chromosome changes can lead to adverse heritable effects. In vitro genetic toxicity tests were designed to detect compounds that induce genetic damage either by directly measuring alterations in DNA or indirectly by the assessment of DNA repair. A compound is classified as genotoxic if it, or a metabolite, can modify DNA with resulting adverse effects [2]. The discipline of genetic toxicology is focused on employing different tests as short-term predictors of mutagenicity and carcinogenicity. Compounds that are positive in these tests , are considered to be potentially mutagenic or carcinogenic [3, 4]. Agents of genotoxicity include both chemical compounds and physical agents such as certain types of radiation [5]. Regulatory requirements for genetic toxicology testing were established in the early 1970's. They have been a part of the safety evaluation for all new chemical identities and the assay systems were based upon national regulatory guidelines [6, 7]. The in vitro genetic toxicology tests used for regulatory purposes measure the formation of gene mutations and chromosomal changes and the results are used to predict the carcinogenic potential [6, 7]. Considerable progress has been made to standardize protocol designs for genetic toxicity testing, particularly through the efforts of the International Conference on Harmonization (ICH) and the Organization of Economic Cooperation and Development (OECD). Biologic systems from single cells to complex plant and animal organisms have evolved many mechanisms to respond to and counter stressors in their environment. Many responses are mediated through coordinated changes between cells in specific patterns, which results in new operational characteristics of affected cells [8, 9]. Thus, it is clear that no single test is capable of detecting all relevant genotoxic agents or mechanisms. Therefore, the usual approach is to carry out a battery of in vitro and in vivo tests for genetic toxicity [10]. Such tests are complementary rather than representing different levels of hierarchy. The ICH process has resulted in the promulgation of guidelines for a “Standard Battery of Genetic toxicity Testing of Pharmaceuticals (ICH Harmonised Tripartite Guideline S2B)” [7] and

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“Guidance on Specific Aspects of Regulatory Genetic toxicity Test for Pharmaceuticals (ICH Harmonised Tripartite Guideline S2A)” [6]. These Guidelines have standardized genetic toxicity testing on pharmaceuticals between the United States, Europe, and Japan. The battery of in vitro and in vivo genetic toxicity tests has been a critical component of the safety assessment of drugs, pesticides, and chemicals for many years. The recommended ICH standard battery is (i) A test for gene mutation in bacteria, (ii) an in vitro mammalian cytogenetic test for chromosome damage or a mouse lymphoma tk locus test and (iii) An in vivo chromosome damage test. The bacterial gene mutation test is usually a bacterial reverse mutation assay (Ames test) that has been shown to detect frameshift and point mutations for the majority of rodent genotoxic carcinogens. The mammalian chromosome damage test is considered to be relevant for mammalian cells. Currently several mammalian cell systems are in use: systems that detect gross chromosomal damage (in vitro tests for structural and numerical chromosomal aberrations), systems that detect primarily gene mutations (tk locus using mouse lymphoma L5178Y cells or human lymphoblastoid TK6 cells, the hprt locus using CHO cells, V79 cells, or L5178Y cells, or the gpt locus using AS52 cells). The mouse lymphoma tk locus assay is believed to be able to detect both point mutations and chromosome effects. The in vivo genetic damage test should use an animal model in which additional relevant factors (absorption, distribution, metabolism, excretion) that may influence the genotoxic activity of a compound are included [10]. The in vivo test for chromosomal damage in rodents includes either an identification of chromosomal aberrations using bone marrow or the assessment of micronuclei formation using bone marrow or peripheral blood erythrocytes [10]. The most recent draft guidance from the U.S. FDA recommends completion of the first two in vitro assays prior to initiation of Phase-1 studies in human and completion of the in vivo test prior to IND submission [10]. The regulatory paradigm relies principally on qualitative interpretation of in vitro tests and limited in vivo testing due to the early findings of a strong correlation between mutagenicity in vitro and the outcome of rodent cancer bioassays [10]. However the species and individual animal variations confound the extrapolation of data to humans, particularly given the species differences in the extent of DNA repair and biochemical pathways [11]. Under certain circumstances the results of the bacterial assay may not be informative. For example, the test agents such as certain antibiotics may be excessively toxic to bacteria, or test agents may be specifically active in mammalian cells, such as topoisomerase inhibitors or nucleoside analogues [10]. Under such conditions, the FDA guidance suggests performing the in vitro cytogenetics and mouse lymphoma mammalian tests in addition to the bacterial mutation assay. Another circumstance in which both in vitro mammalian cell tests should be performed is in situations where the pharmacokinetic data demonstrate that sufficient exposure can not be achieved in vivo and the test agent is therefore not available to the target tissue(s), most often the bone marrow [10]. In general, if all the results in the standard battery are negative, it will usually be sufficient to demonstrate the absence of any significant genetic toxicity. If a compound shows a positive result in these basic tests, further in vitro mechanistic and in vivo genetic toxicity studies may be undertaken to assess the risk for humans [10]. Accumulated knowledge has shown that the rate of in vitro positive findings not confirmed in vivo as well as false positives for non-carcinogens has been high [12]. A recent analysis of nearly 1000 chemicals highlighted the strikingly imprecise nature of genetic toxicology tests in predicting carcinogenicity [13]. When the standard in vitro genetic toxicity tests were performed, at least 80% of compounds gave a false positive result [13]. For example, positive responses in the in vitro cytogenetics or the mouse lymphoma assay are

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not uncommon and in many cases these are not associated with in vivo activity. However, the ICH guidelines are unclear as to how to proceed with a positive genetox result. Data from the Physicians’ Desk Reference (PDR) suggests that positive results in the Ames assay and the in vivo micronucleus test are rare for approved drugs [10]. Snyder and Green reported that twenty-nine percent of the drugs in the 1999 PDR had at least one positive finding with a quarter of all in vitro cytogenetics and mouse lymphoma assays being positive [14]. Although approximately 70% of the Ames test positive compounds do turn out to be carcinogens when subjected to the standard rodent bioassay [15, 16], the high false positive rate of the current in vitro tests might increase the number of compounds that are required to be subjected to earlier and additional animal testing prior to first in human studies. This could increase the time and effort spent before the efficacy of the compound has been evaluated in phase II proof of concept trials. In some cases a positive in vitro genetic toxicity result may trigger the conduct of 2-year carcinogenicity studies on compounds that would not otherwise be subjected to such testing. Thus, there is a need to develop short-term tests that can more accurately predict the in vivo mutagenic and carcinogenic potential of chemicals. Development of more accurate in vitro tests for genetic toxicity with less false positives could significantly reduce animal usage and the need for data from in vivo tests.

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Section I - DNA Damage and Cell Stress Responses Although the current genetic toxicity testing battery has prevented the introduction of harmful genotoxic chemicals to patients and clinical subjects, the limited understanding of the underlying mechanisms resulting in false positive findings provide a challenge to industry and regulatory agencies. The development of methods using cellular stress mechanisms has potential for improving mechanistic interpretation [17]. Complex pathways have evolved in mammalian cells to maintain cellular health. Almost any change in cellular homeostasis, including damage to DNA, may be expected to cause stress, which in turn may initiate a complex cascade of stress-inducible enzymes and related transcription factors in an attempt to return the cell its original equilibrium [18]. Cellular rescue from stress is not achieved when the damage is too great, or when one or more components of the stress-activated cascade is impaired [18]. Commonly, cellular signal transduction networks encompass three tiers: (a) sensors that perceive a signal; (b) transducers that amplify signals; and (c) effectors that adjust cell function to signals [18-20]. In contrast, many aspects of the cellular stress response are not stressor specific because cells monitor stress based on macromolecular damage without regard to the type of stress that causes such damage [21, 22]. These represent the cell defense reaction to the damage to proteins, DNA, or other essential macromolecules [21, 22]. Cellular mechanisms activated by DNA damage and protein damage are interconnected and share common elements [23]. Thus stress sensors probably monitor macromolecular integrity in cells rather than an environmental signal per se. The cellular stress response depends on the proteins expressed in a cell at a particular time and is therefore species- and cell typedependent [18]. There are three types of stress-related sensors on the basis of their primary cellular localization: nuclear, cytoplasmic, and membrane/cell surface receptors. An important part of the mammalian stress response relates to the translocation of active components from the cytoplasm to the nucleus. Whereas the importance of nuclear localization for stress-responsive transcription factors is the key for their ability to elicit their

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activities, the mechanisms underlying import and export of transcription factors to and from the nucleus are better understood. Current knowledge of HOG-, AKT-, NFAT-, NFκB-, and p53-regulated nuclear localization points to the role of phosphorylation by stress-related kinases as a common mechanism for controlling interaction with specific nuclear import and export factors. Native DNA consists of long polymers of deoxyribonucleotides and each deoxyribonucleotide is composed of a deoxyribose, a phosphate, and a nitrogenous base [24]. All these constituents are potential targets of spontaneous alterations or genotoxic agents. Based on this, DNA damage is defined as “any modification of DNA that alters its coding properties or its normal function in replication or transcription” [24, 25]. DNA damage occurs in numerous ways, ranging from specific base modifications, e.g., 8-oxoguanine (8oxo-7,8-dihydroguanine) formation, to more stressor specific mechanisms, e.g., pyrimidine dimer formation during UV irradiation [24]. Despite numerous types of DNA adducts and base modifications, DNA damage effects can be grouped into a few major types, including DNA double-strand breaks (dsb), DNA nucleotide adduct formation and base modification, and DNA single-strand breaks (ssb) [26, 27]. Accordingly, the major classes of DNA repair are DNA dsb repair by homologous recombination (HR) or nonhomologous end-joining (NHEJ), nucleotide excision repair (NER), and nucleotide mismatch repair (MMR) [28]. DNA damage sensors probably recognize common intermediates of the major types of DNA damage. Candidate intermediates are DNA ssb that occur during all types of DNA damage [28] and recognition motifs that are common to different base mismatches and modifications [28-30]. Much work during the past decade has focused on DNA damage response proteins [31]. However, it is still nearly impossible to distinguish primary sensors from secondary transducers of DNA damage. The problem lies in complex circuits of feedback regulation of proteins involved in sensing DNA damage. For example, many candidate proteins are part of multiprotein complexes and, when activated, they become targets of further modification by their own substrates [18]. Most studies on DNA damage proteins have focused on responses of cells to damage induced by ionizing radiation or highly reactive chemicals [32, 33]. However, recent work has demonstrated that during other types of stress, including osmotic stress and heat shock, DNA damage occurs and key mechanisms involved in eukaryotic DNA damage sensing, transduction, and repair are activated [32, 34, 35]. These findings, in combination with extensive prior knowledge about the effects of many types of stress on protein folding and stability, led to the hypothesis that cellular stress response represents a universal reaction to macromolecular damage [36]. Much of the cellular machinery involved in DNA damage sensing is highly conserved in both eukaryotes and prokaryotes but differs considerably between these two major forms of life [37-39]. In E. coli, the single-stranded (ss) DNA binding protein RecA is an important DNA damage sensor. It plays a central role in the repair of stalled replication forks, doublestrand break repair, general recombination, induction of the SOS response, and SOS mutagenesis [40]. The eukaryotic homolog of RecA is Rad51, which catalyzes the central step of homologous recombination and is the key protein for repairing DNA dsb. The Mre11-Rad50-Nbs1 (MRN) complex is required for both homologous recombination and nonhomologous end-joining (NHEJ) [41, 42]. The most stable and deleterious base modification caused by reactive oxygen species (ROS) is the formation of 8-oxoguanine (8oxo-7,8-dihydroguanine; 8-oxoG). MutT/MTH protects cells from the mutagenic effects of 8-oxoG by degrading 8-oxo-dGTP to 8-oxo-dGMP [43]. Bacterial MutS and eukaryotic MSH proteins are involved in sensing DNA base mismatches. Another potential DNA

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damage biomarker is topoisomerase, which alters DNA topology by introducing transient ssb into DNA during replication and NER. Since DNA ssb might represent a common intermediate recognized by DNA damage sensors [44], topoisomerase may be a critical element of DNA damage sensing. Indeed, topoisomerase I is involved in NER during the bacterial SOS response to stress induced DNA damage [45, 46], and the homologous mammalian topoisomerase III is a sensor for the S phase DNA damage checkpoint [47]. One universal cellular stress effect is the impairment of growth and proliferation which represents an adaptive and integrated part of the stress response [18]. It allows for preservation of energy and reducing equivalents and redirects the utilization of these important metabolites toward macromolecular stabilization and repair [18]. In addition, dividing cells that actively undergo DNA replication and mitosis are more prone to suffer stress-induced damage to macromolecules than are cells in a resting state [48]. In bacteria, the ability to resist stress is greater in stationary phase than in the exponential growth phase, during which cells are rapidly dividing. Thus, rapidly dividing, metabolically active bacteria will experience growth arrest when exposed to stress [48]. When bacterial DNA replication is interrupted by stress, a component of the SOS response is induced and leads to transient inhibition of cell division [49]. Similarly, in eukaryotic cells, the activation of cell cycle checkpoints is a key aspect of the cellular stress response [50]. Cell cycle checkpoints monitor macromolecular integrity and the successful completion of cellular processes prior to initiating the next phase in the cell cycle [51]. The eukaryotic cell cycle proteins that control checkpoints maintain the fidelity of DNA replication, repair, and cell division in normal as well as stressed cells [52]. Checkpoints are built into every major transition in the cell cycle, including G1/S, intra-S phase, G2/M, mitotic spindle assembly, and cytokinesis. In mammalian cells such cell cycle checkpoints are controlled by a large number of proteins, such as ATM and ATR kinases, p53, GADD45 proteins, 14–3-3σ, CDC25, CDC2/cyclin B, p21, retinoblastoma protein (pRB), Chk1, Chk2, Polo kinases, and BRCA1 [50, 52, 53]. A striking feature of eukaryotic cell cycle checkpoints as well as DNA damage repair pathways is the central role of ATM and ATR kinases [54]. The p53 protein is also involved in the G2/M checkpoint by inhibition of CDC2 via its transcriptional targets GADD45, p21, and 14–3-3σ [55]. These proteins are critical intermediates between DNA damage sensors and effector protein complexes that control key features of the cell stress response, including cell cycle progression, DNA repair, and apoptosis [56]. In summary, cellular macromolecules, including DNA are subjected to constant attack, both by reactive species inside cells and by environmental agents. DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by exposure to carcinogens, cytotoxic compounds, and other environmental stressors [57]. The DNA damage signalling pathway is a core element of the cellular response to genotoxic insult, and its components play key roles in defending against neoplastic transformation. Using the DNA damage pathways and biomarkers can help predict carcinogens and thereby prevent the advancement of such agents into clinical trials [57].

Section II-In Silico Models Presently, pre-regulatory genetic toxicity tests on discovery compounds are frequently performed in pharmaceutical companies because of increased compound throughput and in order to avoid late stage termination of a cost-intensive drug development program due to unforeseen genetic toxicity [17, 58]. Such screening strategies rely primarily on in vitro

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assays, which either represent a scaled down version of the respective regulatory tests (e.g., the non-GLP Ames II screen), or make use of alternative assays (e.g., the in vitro micronucleus test for the detection of chromosomal damage) [17, 59-61]. In principle, the concordance between the screening assays and the regulatory tests is relatively high [60-62]. However, with respect to screening assays for chromosomal damage, the existing screens have only moderate throughput and their use in early discovery stages is restricted because of costs and compound availability. Additionally, genetic toxicity screens might be compromised by the possible presence of (genotoxic) impurities in early research compound batches, leading to potentially false positive results [63]. There is therefore an urgent need for techniques that are capable of identifying adverse effects at a very early stage of product development and providing toxicity estimates for a large number of discovery compounds [64]. As many of the genetic toxicity experiments can be too expensive and time consuming to be performed early in drug discovery, data mining techniques have gained much popularity during recent years as the earliest predictors of genetic toxicity risks [63, 65]. Computer based (in silico) techniques are an extremely fast and cost effective alternative or supplement to bioassays for the identification of toxic effects at an early stage of product development and can be applied even when compound availability is an issue [66]. The idea of employing computational approaches for predicting the mutagenicity of novel chemicals or drugs is attractive and, intuitively, likely to be successful [65]. This optimism is based on the understanding that while mutagenicity of pharmaceutical lead compounds can be the consequence of many different cellular processes, it is most often associated with covalent interactions between a chemical and cellular DNA [15]. Covalent interactions are dependent on the electrochemical properties of molecules, and most DNA-reactive moieties, identified through analysis of mutagenesis and carcinogenesis databases, are readily recognized and anticipated to have genotoxic potential [15, 63]. Computational models are currently being used by regulatory agencies and within the pharmaceutical industry to predict the mutagenic potential of new chemical entities [67]. The Commission of the European Communities proposes to increase the use of in silico and in vitro techniques [68] and many regulatory authorities (e.g. Danish Environmental Protection Agency, German Federal Institute for Risk Assessment, US Environmental Protection Agency, US Food and Drug Administration) use (Quantitative) Structure-Activity Relationship (Q)SAR models to support their decisions [67, 69]. If in silico predictions are used in a regulatory context, it is obvious that they have to meet rigorous quality standards. These standards have been drafted in the OECD Principles for (Q)SAR Validation (formerly Setubal Principles) which are currently under revision. In silico techniques include modeling biochemical events that are relevant for toxicity (Molecular Modeling), techniques that mimic human reasoning about toxicological phenomena (Expert Systems), and methods that derive predictions from a training set of experimentally determined data (Data Driven Systems) [70]. Molecular Modeling techniques assess the interaction of small molecules with biological macromolecules (predominately proteins), by fitting the ligand into the active site of the receptor [70, 71]. Although Molecular Modeling can be used to elucidate mechanisms and biotransformations and to predict receptor-mediated toxicity (e.g. estrogenicity), the prediction of toxicities with complex and partially unknown mechanisms is beyond their scope [71]. Expert Systems, such as Deductive Estimation of Risk from Existing Knowledge (DEREK) and Tissue Metabolism Simulator (TIMES), are intuitively appealing to most users, because they promise easy access to toxicological knowledge, and many of the most successful Predictive Toxicology software tools are Expert Systems [72]. The creation of a knowledge base for an Expert System requires extensive literature searches and the developer’s capability to create

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generally applicable knowledge from specific cases [71]. An inaccurate knowledge base (frequently inappropriate generalizations from a few examples) on the other hand can be a major pitfall of Expert Systems [72]. It is therefore necessary to update the knowledge base regularly and to integrate new scientific evidence as well as feedback from the users [72]. Data Driven Systems, such as Toxicity Prediction by Komputer Assisted Technology (TOPKAT) and Multi-Computer Automated Structure Evaluation (MCASE) are methods for the extraction of predictive models directly from experimental data. Classical (Q)SAR analysis uses regression techniques to derive equations from experimental data [71, 73]. These equations can be used for the prediction of further compounds with similar structure (i.e. congeneric compounds) and mechanisms. Prediction can be based on quantitative molecular properties (QSAR) as well as on the presence or absence of toxicity inducing substructures (SAR) [73, 74]. An important task for Data Driven Systems is the selection of chemical features that are relevant for the toxic effect under investigation. Currently, computational programs used for genetic toxicity prediction are mainly focused on the prediction of the outcome of the Ames test, and relatively good predictive accuracies (>70%) can be reached for this endpoint [71]. Each Predictive Toxicology technique has its own distinct advantages and weaknesses. In general, the inability to predict all Ames genetic toxicity using in silico approaches may be due to the incomplete understanding of genotoxic mechanisms, limited capability for predicting metabolites to mimic S9 system in the Ames test, and the necessarily insufficient structural coverage in these test system [75]. For example, some of the unpredicted genetic toxicity may arise from noncovalent DNA interaction, e.g., DNA intercalation [76]. Also, the end points of carcinogenesis are believed by most scientists to be a multiple step process that requires several different types of chemical toxicity effects, such as alteration of cellular mortality, cell-to-cell communication, and damage of genetic material [77]. Structural features conferring DNA intercalative ability are very poorly represented in current computational programs whose algorithms depend primarily on covalent DNA interaction, and this deficiency would be expected to result in lower predictivity [71]. Improving the sensitivity of these models through the augmentation of the learning set or the establishment of new rules will require pertinent new mechanistic insights. Another obvious approach to achieve optimal predictivity is to combine predictions from different models. A similar and equally beneficial strategy is the integration of bioassay results with in silico predictions [71, 75].

Section III - High Throughput Versions of Existing Regulatory Assays New synthesis strategies together with the establishment of high-throughput screening methods in HTS, ADME, and toxicology are increasing the number of compounds entering the “exploratory development” phase [16, 78]. Due to the need to optimally apply laboratory resources, compound prioritization must be performed in discovery and the early phases of development to minimize the risks of failure in later and more expensive phases of development. Emerging screens for detecting DNA damage and gene mutations enable the genetic toxicologist to provide crucial mutagenicity information for selection of the most promising candidates or in prioritization between several similar drug candidates [64, 79]. Additional considerations of a genetic toxicity screening strategy are compound requirements, cost, and throughput. Early genetic toxicity screening strategy should be

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directed by the choice of assays and endpoints, the protocol for conducting the selected assays, and the decision criteria for evaluating screen results [79, 80].

Ames Fluctuation Assay Bacterial gene mutation screens are principally the Ames reversion test adapted for moderate or higher throughput and with reduced test material requirement. One commercial high-throughput kit version of the Ames test is the Muta-chromoplate test. This is simply a kit version of the fluctuation test methodology that has been available for many years [8183]. In the regulatory Ames test treated bacteria (some 108 to 109 cells) are plated on agar plates and his+ revertants are detected as visible colonies each arising from a single bacterium mutated to restore histidine biosynthesis. In the fluctuation test small numbers of treated bacteria (e.g. 103 cells) are placed in multiple wells containing media depleted of histidine. If the well contains one or more bacterial mutated to restore histidine biosynthesis then growth will occur and the well is scored as positive by any of a number of growth markers (e.g. turbidity, pH change, and ATP formation). Counting the growth-positive wells provides an indication of mutation frequency.

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MiniAmes Miniaturized Ames plate incorporation methodologies can be successfully applied in moderate throughput scenarios (e.g. 10-20 compounds week) [84-86]. A primary purpose of miniaturization is not only to improve throughput but also to reduce test material requirements. Modifications from standard Ames test methodology include the use of concentrated small volume preincubation and plating on 24 or 48 well plates instead of individual large petri dishes. These modifications are relatively easy to implement in a laboratory experienced and equipped for standard Ames test methodology. They would require some development time and modest equipment investment in a laboratory that is not so experienced or equipped. Other modifications often include reducing the number of strains for screening. Two primary Ames strains, TA98 for frameshift mutagens and TA100 for base pair substitution mutagens, are capable of detecting most Ames positive compounds (estimated >80-85%), and these strains should be included in a core screen. The next highest priority strain might be TA102 (or an E. coli WP2 strain) which detects mutagens acting at AT sites such as some aldehydes. TA1537 (or TA97) detects certain specific frameshift mutagens and TA1535 uniquely detects a few base pair substitution mutagens. Selection of these other strains for screening might depend on chemical class being studied, availability of test material and desired economy for screening. The Ames II test is another version of the fluctuation assay using strains especially developed to include different point mutations [87, 88]. Because these strains can be combined into a single mix the assay has some definite advantages. The advantage of this would be the fact that a single mix of base-pair substitution strains replaces several original Ames test strains (TA100, TA102 and TA1535).

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Bioluminescent Ames Assay The standard Salmonella assay relies on the detection of colonies derived from cells with reversions of auxotrophic mutations in histidine synthesis pathway [89]. The treatment of histidine-dependent cells with mutagens leads to an increased number of histidineindependent colonies of revertant cells on plates containing only a trace amount of histidine [89]. In contrast, the bioluminescent Salmonella assay exploits bioluminescence for the detection of histidine-independent revertant colonies [90]. In this assay, histidineindependent revertant colonies are detected as producing measurable bioluminescence in the absence of histidine [90]. Since the size of revertant colonies in a microplate format is affected by the cytotoxicity of tested chemicals and likely also by bacterial quorum sensing mechanisms, the bioluminescent Salmonella assay requires measuring the incidence of revertant colonies instead of monitoring the overall luminescence output [90]. The application of image analysis algorithms would then enable detecting even small colonies of revertants cells in a high throughput format in an automated fashion.

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Yeast DEL Assay The second major in vitro regulatory assay category is an in vitro cytogenetic assay in mammalian cells (in vitro CA) or a mouse lymphoma tk locus gene mutation assay (ML), which is considered equivalent to the in vitro CA [10]. It is clearly prudent to consider a screening capability that will predict responses in a regulatory in vitro CA or ML assay [10]. These assays detect clastogenicity, which is the property of causing macroscopic chromosome alterations and damage (e.g. breaks, deletions, and rearrangements) in eukaryotic organisms. The use of this type of endpoint is supported by the fact that some agents are uniquely or preferentially detected in assays measuring a chromosome aberration endpoint rather than in assays detecting point mutations [28]. Also, because in vitro chromosome aberration assays employ mammalian cells they can detect genotoxic activity of some agents that are not detected by bacterial systems for reasons other than genotoxic endpoint specificity [28]. However, it is important to recognize that a high proportion of compounds are positive in this type of assay [14, 91-93]. There are many cases where positive responses are uniquely observed in in vitro CA or ML assays and negative responses are observed in bacterial reversion and in vivo assays [14, 94]. There is growing recognition that at least some of these unique positive responses may not be biologically relevant to the in vivo situation, and there are many examples of registered pharmaceuticals that have unique in vitro CA and/or ML responses [14, 94]. Such knowledge must be taken in account when interpreting the results for and clastogenicity screening test. The yeast DEL assay has been promoted as a potential screen for clastogenic activity and this assay has a reasonably extensive experience base [95-97]. The assay measures intrachromosomal recombination which restores a functional histidine synthesis capability. Induction of intrachromosomal recombination is believed to occur by clastogenic mechanisms such as the creation of DNA double-strand breaks; therefore, this assay is promoted as a screen for clastogens [95-97]. In practice, the yeast DEL assay methodologies are very similar to the Ames test. Cells which have undergone intrachromosomal recombination are detected as being able to grow in the absence of histidine, either as

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colonies each arising from a single recombinant or by growth in liquid medium [95-97]. Published studies show that the yeast DEL assay does detect a number of compounds that are clastogens and which are negative in the Ames test [95, 96, 98]. Some compounds positive in the yeast DEL assay such as safrole, benzene and urethane are difficult to detect as positive not only in the Ames test but also in in vitro CA or ML assays. Kirpnick et al. [98] compared an in vitro micronucleus assay in mammalian cells with yeast DEL for a series of clastogens. Although the concordance was reasonably good (70% with S9 activation and 80% without activation) it was clear that yeast DEL does fail to detect some clastogens that are active in mammalian assays. This may be due to failure of yeast to perform metabolic activation or to the lower permeability of yeast to large molecules [99].

DNA Alkaline Unwinding Assay Genotoxic effects may be detected by measuring different amounts of single- and double stranded DNA after separation and quantification following staining with fluorescent dye (picogreenR) [100]. With regard to the specific principle of the DNA unwinding assay, this test allows detection of primary DNA damage, which leads to increasing unwinding kinetics and subsequently amounts of detected DNA single strands in the analysed tissues [101, 102]. A disadvantage of the DNA unwinding assay is that in some cases cytotoxicity of compounds leads to an inhibitory effect on topoisomerases and DNA polymerases, whereby the number of natural unwinding points is reduced [103]. As a consequence, false negative results or underestimation of genetic toxicity are possible [103]. In general, the influence of cytotoxicity must be taken into account in most genetic toxicity tests [92, 102].

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Single Cell Gel Electrophoresis (Comet Assay) Detecting DNA damage in cells is not new; several techniques have been in use for many years. The Single Cell Gel Electrophoresis (SCGE) assay, commonly know as the comet assay, is a sensitive technique unique for its ability to measure DNA damage and repair in individual cells [104]. The comet assay was introduced by Ostling and Jonhanson when they embedded cells into agarose gels to immobilize them [105]. The cells undergo electrophoresis, which causes migration of DNA strands [105]. Subsequent DNA staining permits visualization of the nuclear DNA. If the DNA has been broken into small pieces this results in the formation of a “tail” extending from the large-size nuclear DNA. Since this image resembles a comet, this technique is now universally known as the comet assay [105]. The comet assay is unique because it can measure DNA damage at single cell level and requires only a small amount of cells [106]. The alkaline (pH 13) version of the comet assay enables the detection of a broad spectrum of DNA damage, which is measured as single and double strand breaks, and single strand breaks as a result of alkali-labile sites or nucleotide excision repair [104]. The alkaline comet assay can be used in in vitro and in vivo test systems. The in vitro comet assay is seen as a candidate for screening in early drug discovery/development and/or to complement the already existing cytogenetic methods. The

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major advantages of the in vitro comet assay are: 1) that cell proliferation is not needed for an assessment of genotoxic potential, 2) almost any mammalian cell type can be used for testing, 3) a small number of cells are needed so high-throughput methods may be used, 4) a small amount of test compound is needed for testing, and 5) results are obtained relatively quickly and in some instances can be automated [107, 108]. The in vivo (rodent) comet assay is increasingly being used to evaluate the genotoxic potential of industrial chemicals and pharmaceutical compounds [109]. The in vivo comet assay is also gaining popularity as part of the genotoxic hazard identification package, as a follow up or complementary test after an in vitro genetox positive [110, 111]. The comet assay in vivo can provide valuable information in organ tissues that were never tested before due to technical limitations [109, 110]. It is used as a supplemental assay for mechanistic and/or target organ specific toxicity testing [109]. This assay is also being discussed in the regulatory arena as a possible second in vivo assay in the newly proposed S2 (R1) ICH guidelines [109, 110]. It should be noted that DNA strand breakage effects may be secondary to toxic events such as oxidative damage and positive effects for this endpoint do not necessarily indicate a direct interaction with DNA.

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Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes The micronucleus test is widely used in research and regulatory safety assessment to evaluate the potential of chemical and physical agents to cause chromosomal damage [112, 113]. Erythroblasts in bone marrow undergo a final chromosome replication after which they divide and differentiate into polychromatic erythrocytes [112, 113]. Chromosomal breaks in the mitotic process that result in the lagging chromosomes during this division lead to the formation of micronuclei that are similar in appearance but much smaller than the nucleus in immature nucleated erythrocytes [113]. Litron laboratories have developed a patented, flow cytometric method to measure micronuclei in both the reticulocyte and normochromatic erythrocyte populations. Unlike the mature normochromatic erythrocytes, immature reticulocytes are still rich in RNA as well as certain surface proteins and can therefore be differentially stained based on these features. The increase in the frequency of micronucleated reticulocytes indicates genetic toxicity associated with a recent cell division [112]. The increase in the frequency of micronuclei in the normochromatic erythrocytes indicates accumulated DNA damage associated with a subchronic or chronic treatment regimen [112]. It has been shown that the Litron method has significant advantages over the traditional microscopic scoring techniques such as more cells examined, greater statistical power, and faster data acquisition [112, 113]. In summary, since the regulatory genetic toxicity tests must be performed to identify and characterize genetic toxicity prior to the initiation of clinical trials, the primary goal of a genetic toxicity screening program should be to provide results which will predict the outcome of these three principal regulatory assays [17]. This goal provides a clear standard for judging the performance of prospective screening assays. In general, early higherthroughput screens might be expected to have less predictive accuracy than later, lower throughput screens. The nature of the desired predictive accuracy should also be considered. One strategy would be to err on the side of “false positives,” which would eliminate compounds that would turn out negative in regulatory assays [80]. An alternate strategy

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would be to err on the side of “false negatives,” which would increase the probably of a compound passing into development that would ultimately turn out positive in regulatory genetic toxicity assays [28, 79, 80]. Another key potential problem with early genetic toxicity screens is compound impurities related to synthetic catalysts and source materials in the early drug discovery phase.

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Section IV-Reporter Assays Reporter genes have a wide variety of applications in modern science. Expression of a reporter gene produces a measurable signal, which can be readily distinguished over the background of endogenous expression [114, 115]. Cell culture as well as recent studies with transgenic animals and plants have employed reporter genes to identify transcription elements responsible for basal and tissue-specific gene expression and cis-acting elements involved in human diseases [115, 116]. Several characteristics are required for a gene to be useful as a reporter gene. First, quantification of reporter gene expression or activity must be conducted using a simple assay; second, the amount or activity of the reporter protein must be reflective of the xenobiotics being studied; and finally, similar endogenous proteins or enzyme activity should be absent or minimal in the target cells [116]. The identification of reporter genes (e.g., firefly luciferase and green fluorescent protein), which can be used as noninvasive markers of gene expression, and recent technological advancements in detection strategies employing charge-coupled device (CCD) imaging cameras and fluorescence microscopy have provided temporal as well as spatial information on gene expression at the single-cell level [115, 117]. A rapidly growing application of reporter genes is to study the effects of chemical exposure on gene regulation by monitoring expression of toxicity markers such as those associated with tumorigenesis, cytokine release, and transcriptional activation which relate to carcinogenicity, mutagenicity, inflammation, and endocrine disruption [114, 115]. In addition, reporter genes have also been used to study extracellular signaling mechanisms and their effects on gene regulation. This application of reporter genes has been incorporated in the development of biological screens for drug discovery [115]. Cell-based reporter assays have been developed to study a wide variety of structurally diverse endogenous and exogenous chemicals. Several unique reporter proteins have been employed in cell-based reporter systems, including chloramphenicol acetyltransferase, βgalactosidase, luciferases from bacteria, firefly, and Renilla, and fluorescent proteins such as aequorin and green fluorescent protein [118]. Each of these reporters has advantages and disadvantages based on the assay conditions and the detection method employed. The choice of reporter is dependent upon the background endogenous activities of the cell line used, gene expression and transfection efficiency, and the detection method as well as the analytical application of the system [116, 118].

Chloramphenicol Acetyltransferase Chloramphenicol acetyltransferase (CAT), identified in the 1960s and derived from Escherichia coli, was one of the first proteins used as a reporter and has been widely used to

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monitor gene transfer [119, 120]. The mechanism of chloramphenicol resistance resulted from CAT-mediated inactivation of chloramphenicol by O-acetylation of the C-3 hydroxyl position yielding 3-O-acetyl chloramphenicol [121, 122]. The CAT-Tox (L) assay has been developed to identify and measure molecular mechanisms of toxicity [123]. This assay, using human HepG2 cells, coupled the cat reporter gene with the promoter regions of 14 stress-related genes [123]. The system has been tested with a wide variety of agents, including genotoxins, heavy metals, and planar aromatic hydrocarbons [123]. In a recent study, CAT has been employed in a cell-based assay for nongenotoxic carcinogen peroxisome proliferators, in which a fusion construct of CAT with the promoter for rat acylCoA oxidase was transfected into a rat liver cell line [124]. The CAT-mediated acetylation reaction can be measured using either radio-labeled chloramphenicol or acetyl-CoA [114]. However, assays for CAT generally have a narrow linear range and, therefore, require testing of several sample dilutions to verify that the values obtained are within this range [115, 118]. Moreover, the health risks associated with the use of radioisotopes and the increasing restrictions on waste disposal of these agents are further disadvantages of this assay. Fluorescent CAT activity assays employ fluorescent chloramphenicol substrates, which can be quantified following their acetylation by CAT. The detection limits of the fluorescence-based assay are comparable to those using the radioisotope assay [118]. The requirement for separation of the substrates and products is the primary disadvantage of both the radioisotope and fluorescent assays for measuring CAT activity. Despite the limitations, CAT is still widely used as a reporter protein due to its stability and lack of endogenous expression in mammalian cells [115, 118].

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β-Galactosidase E. coli β-galactosidase (β-Gal), encoded by the lacZ gene, has been widely used to study transcriptional and translational gene regulation [125, 126]. Cell-based assays employing an inducible promoter and β-Gal as a reporter have been used to identify a wide variety of analytes including heavy metals, toxic salts, chlorocatechols, and viruses in controlled as well as natural environments [118, 127-129]. The detection strategies for β-Gal include colorimetric, histochemical, fluorescent, luminescent, and electrochemical dependent on the substrates used [116]. The most commonly used substrates for β-Gal are o-nitrophenyl β-D-galactopyranoside (ONPG) for colorimetric detection, 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-gal) for histochemical detection, 4-methylumbelliferyl-β-D-galactopyranoside (MUG) for fluorometry, 1,2dioxetane substrates for luminescence, and p-aminophenyl-β-D-galactopyranoside (PAPG) for electrochemical analysis [130-132]. The advantages of colorimetric and histochemical assays lie in their simplicity and rapidity; however, their low sensitivity and narrow dynamic range have led to their replacement by other methods of detection [130, 131]. Using fluorescein-di-β-D-galactopyranoside as the substrate and capillary electrophoresis laserinduced fluorescence detection strategies, Craig et al. were able to obtain detection limits as low as 6.5 × 10-14 M β-Gal in as little as 40 pL of the enzymatic mixture (2.6 × 10-24 mol) [132]. Luminescence-based assays not only rapid but extend over a dynamic range of more than 5-6 orders of magnitude [130]. The electrochemistry method for detection of β-Gal

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activity is promising [133], because it does not require lysis or permeabilization of the cells and thus could be ideal for on-line continuous measurement of enzymatic activity [133]. Also, this detection strategy can be performed in turbid solutions and under anaerobic conditions [133].

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Bacterial Luciferase Luciferase is a generic name for enzymes commonly used in nature for bioluminescence [134]. Bioluminescent organisms, including bacteria, algae, dinoflagellates, fungi, jellyfish, clams, fish, insects, shrimp, and squid, can be found in aquatic as well as terrestrial environments [135]. Among them, bacteria are the most abundant luminescent organisms and have been classified into three genera Vibrio, Photobacterium, and Xenorhabdus [135]. Bacterial luciferase catalyzes the oxidation of a reduced flavin mononucleotide (FMNH2) and a long-chain fatty aldehyde to FMN and the corresponding fatty acid in the presence of molecular oxygen [116, 136]. This reaction results in a blue-green light with a maximum intensity at 490 nm. Differences in the emitted light are believed to be the result of other proteins found in the organisms, which can induce shifts in wavelength [116]. The bacterial luxA, luxB, luxC, luxD, and luxE genes [137, 138] are conserved in all bioluminescent bacterial species identified to date. The expression of luxA and luxB in the host organism is sufficient for signal bioluminescence; however, expression of all five genes has the advantage of not requiring the addition of a substrate [138]. Microtox assay used of bacterial luciferase as a reporter to detect potentially toxic samples [137]. Decreases in luminescence of the exposed bacteria, upon comparison to control groups, suggested potential toxicity of the sample [137]. By fusing the lux operon to pertinent promoters, bacterial luciferase has been used as a marker of exposure to heavy metals, toxic organics, and nitrate in whole-cell bioassays [139]. Even though bacterial luciferases are useful for measurement of prokaryotic gene transcription, their applicability in mammalian systems is limited since these enzymes are heat labile (> 30oC) [116, 137] and the linear range of these assays is somewhat low as compared to that of other bioluminescent reporters [140, 141].

Firefly Luciferase Luciferase from the firefly Photinus pyralis is a peroximal protein found in the lightemitting organ known as the lantern within the abdomen of the insect [116]. Firefly luciferase requires ATP, molecular oxygen, and the heterocyclic compound firefly luciferin to generate light in a two-step process. The reaction has unusual kinetics in that firefly luciferase turns over very slowly [116, 136]. The maximum emissions of different firefly species occur between 550 and 575 nm and are attributed to different amino acid substitutions in the luciferase [141]. It has been shown that site-directed mutagenesis in single amino acid substitutions yielded emissions of light ranging from the green to the red spectrum [142]. Therefore, expression of these mutant reporters in host organisms can result in the generation of distinct signals, a property that can be exploited for multi-target detection [115, 116].

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In summary, the high sensitivity (subattomole level), broad dynamic range (7-8 orders of magnitude), simplicity, and no endogenous activity in mammalian cells are primary advantages of firefly luciferase reporter [143]. Moreover, it has been shown that the sensitivity of firefly luciferase has the potential to be greater than that of bacterial luciferase [143, 144]. Firefly luciferase, under the control of various promoters, has been employed in whole-cell biosensor assays for the detection of heavy metals, such as cadmium and lead, and aromatic organics [143, 144].

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Renilla Luciferase Renilla luciferase enzyme/protein, a 36kDa monomeric protein purified from sea pansy (Renilla reniformis), is a bioluminescent soft coral that displays blue-green bioluminescence upon mechanical stimulation [116, 145]. It is also widely distributed among coelenterates, fishes, squids, and shrimps [141]. It has been cloned and sequenced by Lorenz et al. and used as a marker of gene expression in bacteria, yeast, plant, and mammalian cells [146]. Like firefly luciferase, post-translational modification is not required for its activity, and the enzyme may function as a genetic reporter immediately following translation. The enzyme Renilla luciferase catalyzes coelenterazine oxidation leading to bioluminescence. Coelenterazine consists of an imidazolopyrazine structure {2-(phydroxybenzyl)-6-(phydroxyphenyl)-8-benzylimidazo [1,2-a]pyrazin-3-(7H)-one} that releases blue light across a broad range, peaking at 480 nm upon oxidation by Renilla luciferase in vitro [145]. Renilla luciferase and firefly luciferase have also been commonly used in cell culture with commercial substrate kits (Dual-Luciferase Reporter Assay System from Promega) to simultaneously monitor expression of two reporter genes [147]. In the Dual-Luciferase Reporter (DLR) Assay, the activities of firefly and Renilla luciferases are measured sequentially from a single sample [147]. Both reporters yield linear assay results with subattomole sensitivities and no endogenous activity of either reporter in the experimental host cells. Furthermore, the integrated format of the DLR Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions [147].

Aequorin Aequorin is a Ca2+-binding photoprotein cloned from the bioluminescent jellyfish Aequorea victoria. Following the addition of Ca2+, the substrate (coelenterazine) is oxidized to coelenteramide and blue light is emitted in the range of 460-470 nm [118, 148]. Aequorin can be regenerated by removal of Ca2+ followed by the addition of fresh coelenterazine in a reducing environment [148]. Although aequorin has been extensively used as an indicator of intracellular calcium [118], only one cell-based assay has used aequorin as a reporter. Rider et al. reported that the Cellular Analysis and Notification of Antigen Risks and Yields (CANARY) assay is a rapid (