The Comet Assay in Toxicology (Issues in Toxicology)

The Comet Assay in Toxicology Issues in Toxicology Series Editors: Professor Diana Anderson, University of Bradford, ...

Author: Alok Dhawan | Diana Anderson

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The Comet Assay in Toxicology

Issues in Toxicology

Series Editors: Professor Diana Anderson, University of Bradford, UK Dr Michael D Waters, Integrated Laboratory Systems, Inc, N Carolina, USA Dr Timothy C Marrs, Edentox Associates, Kent, UK

Titles in the Series: 1: Hair in Toxicology: An Important Bio-Monitor 2: Male-mediated Developmental Toxicity 3: Cytochrome P450: Role in the Metabolism and Toxicity of Drugs and other Xenobiotics 4: Bile Acids: Toxicology and Bioactivity 5: The Comet Assay in Toxicology

How to obtain future titles on publication: A standing order plan is available for this series. A standing order will bring delivery of each new volume immediately on publication.

For further information please contact: Sales and Customer Care, Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge, CB4 0WF, UK Telephone: +44 (0)1223 432360, Fax: +44 (0)1223 420247, Email: [email protected] Visit our website at http://www.rsc.org/Shop/Books

The Comet Assay in Toxicology Edited by Alok Dhawan Developmental Toxicology Division, Indian Institute of Toxicology Research, Lucknow, India

Diana Anderson Division of Biomedical Sciences, University of Bradford, Bradford, UK

The cover image shows photomicrographs of comets from (A) Escherichia coli (B) Bacopa monerii L. (C) Drosophila melanogaster (D) differential DNA damage lymphocytes (E) human lymphocytes (F) irradiated diploid human lymphocyte with FISH showing double hybridisation signals indicating strand breakage (G) comets in human sperm (H) human sperm showing double breaks (I) haploid human sperm with FISH showing single hybridisation signal.

Issues in Toxicology No 5 ISBN: 978-0-85404-199-2 ISSN: 1757-7179 A catalogue record for this book is available from the British Library r The Royal Society of Chemistry, 2009 All rights reserved Apart from fair dealing for the purposes of research for non-commercial purposes or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry or the copyright owner, or in the case of reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 0WF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org

Preface This book is the first of its kind to be devoted exclusively to the Comet assay and its applications as an important tool in current toxicology. This multiauthor book will serve as both a reference and a guide for investigators in the biomedical, biochemical and pharmaceutical sciences. Specialists from the fields of genetic toxicology and human epidemiology, with first-hand knowledge of their chosen subspecialities, have contributed to this peer-reviewed scientific venture. Simplicity, rapidity, versatility and ease of application of the Comet assay have made it a favourite amongst researchers and it is now also gaining acceptance amongst regulators. It can be used in all single cells from prokaryotes and eukaryotes, in plants and animals including humans, involving both somatic and germ cells. It is also a relatively inexpensive assay to perform. The book is divided into different sections, reflecting the range of interest in the exploitation of this assay. It begins with an introductory section reviewing the genesis of the assay for those new to the technique, and details the various fields in which it finds wide acceptance. This sets the scene by explaining why the assay has become the most sensitive and sought after assay in modern toxicology. There is a section that describes the protocols being followed to assess various types of DNA damage in different cell types. The third section brings together the specific applications of the assay in diverse areas ranging from genetic toxicity testing to human monitoring, and environmental toxicology. The last section considers strategies for the conduct of the assay using in vitro and in vivo systems, based on internationally accepted guidelines. The book draws to a close with an assessment of image-analysis principles and the statistics used for evaluating the data generated by the assay. This book is a culmination of over fifteen years of active collaboration and friendship between the editors and provides a good basic understanding of issues relating to the assay. The Editors Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

v

Contents Section I: Genesis of Comet Assay Chapter 1

The Comet Assay: A Versatile Tool for Assessing DNA Damage Alok Dhawan, Mahima Bajpayee and Devendra Parmar 1.1 1.2 1.3

Introduction Bacteria Plant Models 1.3.1 The Comet Assay in Lower Plants 1.3.2 The Comet Assay in Higher Plants 1.4 Animal Models 1.4.1 Lower Animals 1.5 Higher Animals 1.5.1 Vertebrates 1.6 The Specificity, Sensitivity and Limitations of the Comet Assay 1.7 Conclusions Acknowledgements References

3

3 5 5 5 18 19 19 24 24 28 30 30 30

Section II: Various Procedures for the Comet Assay Chapter 2

Detection of Oxidised DNA Using DNA Repair Enzymes Amaya Azqueta, Sergey Shaposhnikov and Andrew R. Collins

57

2.1 2.2

57 60

Introduction Methods for Measuring DNA Oxidation Damage

Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

vii

viii

Chapter 3

Chapter 4

Contents

2.3 2.4 2.5

Enzyme Specificity Applications Protocol 2.5.1 Equipment 2.5.2 Supplies 2.5.3 Reagents, Buffers and Enzymes 2.5.4 Procedure Acknowledgment References

61 62 64 64 65 65 66 74 75

Microplate-Based Comet Assay Elizabeth D. Wagner and Michael J. Plewa

79

3.1 3.2 3.3 3.4 3.5

Introduction Microplate Comet Assay Drinking-Water Disinfection Byproducts Chinese Hamster Ovary Cells CHO Cell Microplate Comet Assay Protocol 3.5.1 CHO Cell Treatment 3.5.2 Preparation of Comet Microgels 3.5.3 Comet Microscopic Examination 3.5.4 Normalisation of CHO Cell Comet Data and Statistical Analysis 3.6 Utility of the Microplate Comet Assay in Comparing Classes of DBPs 3.6.1 Microplate Comet Analysis of the Haloacetonitriles 3.6.2 Microplate Comet Analysis of the Haloacetamides 3.6.3 Comparison of SCGE Genotoxic Potency Values of the Haloacetonitriles and Haloacetamides 3.7 Advantages of the Mammalian Cell Microplate Comet Assay Acknowledgements References

79 80 80 81 81 81 83 84

The Use of Higher Plants in the Comet Assay Tomas Gichner, Irena Znidar, Elizabeth D. Wagner and Michael J. Plewa

98

4.1 4.2

Introduction Differences between the Animal and Plant Comet Assay

85 87 89 90

91 92 92 93

98 99

ix

Contents

4.3

Cultivation and Treatment of Plants for the Comet Assay 4.3.1 Onion (Allium cepa) 4.3.2 Tobacco (Nicotiana tabacum) 4.3.3 Broad Bean (Vicia faba) 4.3.4 Plants used for In-Situ Studies 4.4 Isolation of Nuclei from Plant Tissues 4.4.1 Isolation of Nuclei via Protoplast Formation 4.4.2 Isolation of Nuclei by Mechanical Destruction of the Cell Wall 4.5 Preparation of Comet Assay Slides 4.6 DNA Unwinding and Electrophoresis 4.7 DNA Staining 4.8 Reading the Slides, Expressing DNA Damage, Statistics 4.9 Comet Assay Procedure 4.10 Reagents, Media, Buffers 4.11 Equipment and Software 4.12 Determination of Toxicity 4.13 Correlation between the DNA Damage Evaluated by the Comet Assay and Other Genetic Endpoints in Plants 4.14 The Utility of the Comet Assay for Genotoxic Studies in the Laboratory 4.15 The Utility of the Comet Assay as an In Situ Marker 4.16 Comet Assay with Irradiated Food of Plant Origin 4.17 Recommendations for Plant Comet Assay Users Abbreviations References Chapter 5

Methods for Freezing Blood Samples at 80 1C for DNA Damage Analysis in Human Leukocytes Narendra P. Singh and Henry C. Lai 5.1 5.2

Introduction Materials and Methods 5.2.1 Protocol I 5.2.2 Protocol II 5.2.3 Fresh Blood 5.2.4 Fresh Blood Stored on Ice Prior to Freezing 5.2.5 Image and Data Analysis 5.3 Results and Discussion References

99 99 100 100 100 101 101 101 101 102 103 103 104 105 107 107

108 109 109 110 110 114 115

120

120 121 121 122 122 122 123 123 127

x

Chapter 6

Contents

Development and Applications of the Comet-FISH Assay for the Study of DNA Damage and Repair Valerie J. McKelvey-Martin and Declan J. McKenna

129

6.1 6.2 6.3

129 130 135

Introduction The Comet-FISH Assay Procedure Applications of the Comet-FISH Assay 6.3.1 Discovery of the Comet-FISH Assay 6.3.2 Using Comet-FISH to Measure DNA Damage 6.3.3 Using Comet-FISH to Quantify DNA Repair 6.3.4 Summary of Studies 6.4 Limitations of Comet-FISH Assay 6.4.1 Practical Difficulties 6.4.2 Imaging Difficulties 6.4.3 Interpretation of Results 6.5 Conclusion References

Chapter 7

Detection of DNA Damage in Drosophila and Mouse Alok Dhawan, Mahima Bajpayee and Devendra Parmar General Protocol for the Assessment of DNA Damage Using the Alkaline Comet Assay 7.1.1 Chemicals and Materials 7.1.2 Preparation of Reagents 7.1.3 Preparation of Agarose-Coated (Base) Slides for the Comet Assay 7.1.4 Preparation of Microgel Slides for the Comet Assay 7.1.5 Electrophoresis of Microgel Slides 7.1.6 Evaluation of DNA Damage 7.2 The Alkaline Comet Assay in Multiple Organs of Mouse 7.2.1 Chemicals and Materials 7.2.2 Methodology 7.3 The Alkaline Comet Assay in Drosophila melanogaster 7.3.1 Chemicals and Materials 7.3.2 Methodology 7.4 Conclusions Acknowledgements References

135 135 142 143 144 144 144 145 146 146 151

7.1

153 153 153 155 155 156 157 157 158 158 161 162 162 165 166 166

xi

Contents

Section III: Applications of Comet Assay Chapter 8

Chapter 9

Clinical Applications of the Comet Assay S. M. Piperakis, K. Kontogianni, G. Karanastasi and M. M. Piperakis

173

8.1 Introduction 8.2 The Comet Assay Methodology 8.3 Clinical Studies 8.4 Discussion and Conclusions References

173 174 175 195 196

Applications of the Comet Assay in Human Biomonitoring Andrew R. Collins and Maria Dusinska

201

9.1 9.2 9.3 9.4

201 202 202

9.5 9.6 9.7

9.8

Biomonitoring and Biomarkers – An Introduction The (Modified) Comet Assay Guidelines for Biomonitoring Studies Biomonitoring with the Comet Assay: Special Considerations 9.4.1 Surrogate and Target Cells; The Use of White Blood Cells 9.4.2 Sampling Time and Transport 9.4.3 Reference Standards 9.4.4 What Affects the Background Level of DNA Damage? DNA Damage as a Marker of Environmental Exposure and Risk DNA Repair as a Biomarker of Individual Susceptibility Protocols 9.7.1 Protocol for Blood Sample Collection and Long-Term Storage of Lymphocytes for the Measurement of DNA Damage and Repair 9.7.2 Comet Assay – Determination of DNA Damage (Strand Breaks and Oxidised Bases) 9.7.3 In Vitro Assays for DNA Repair Solutions, etc. 9.8.1. Lysis Solution 9.8.2. Buffer F (Enzyme Reaction Buffer for FPG, Endonuclease III, and In Vitro BER Assay) 9.8.3 Buffer F+Mg (Used for In Vitro NER Assay) 9.8.4 Buffer A (Used in In Vitro Repair Assays) 9.8.5 Triton Solution 9.8.6 Ro 19-8022 (Photosensitiser)

204 204 205 206 206 207 207 208

208 211 214 216 216 217 217 217 217 217

xii

Contents

9.8.7 Electrophoresis Solution 9.8.8 Neutralising Buffer 9.8.9 Agarose 9.8.10 Enzymes 9.9 Analysis and Interpretation of Results 9.9.1 Quantitation 9.9.2 Calculation of Net Enzyme-Sensitive Sites 9.9.3 Calibration 9.9.4 How to Deal with Comet Assay Data Statistically 9.10 Conclusions Acknowledgements References Chapter 10 The Comet Assay in Human Biomonitoring Mahara Valverde and Emilio Rojas 10.1 10.2 10.3 10.4 10.5 10.6 10.7

Introduction Human Monitoring Environmental Exposure Lifestyle Exposure Occupational Exposure Reviews Usefulness of the Comet Assay in Human Monitoring 10.8 Conclusions References Chapter 11 Comet Assays in Dietary Intervention Trials Armen Nersesyan, Christine Hoelzl, Franziska Ferk, Miroslav Misˇı´k and Siegfried Knasmueller 11.1 11.2 11.3 11.4

11.5 11.6 11.7

Introduction Experimental Design of Human Studies Indicator Cells and Media Conventional SCGE Trials with Complex Foods and Individual Components – The Current State of Knowledge Use of SCGE Trials to Detect Protection Against DNA-Reactive Carcinogens Use of SCGE Experiments to Monitor Alterations of the DNA-Repair Capacity What Have We Learned from Intervention Studies so Far?

217 218 218 218 218 218 219 219 219 220 221 221 227

227 228 230 234 237 248 249 251 252 267

267 268 269

270 275 279 281

xiii

Contents

11.8 Future Perspectives References Chapter 12 The Comet Assay for the Evaluation of Genotoxic Exposure in Aquatic Species G. Frenzilli and B. P. Lyons 12.1 12.2 12.3

Introduction Protocols, Cell Types and Target Organs Application of the Comet Assay to Invertebrate Species 12.3.1 Freshwater Invertebrates 12.3.2 Marine Invertebrates 12.4 Application of the Comet Assay to Vertebrate Species 12.4.1 Freshwater Vertebrates 12.4.2 Marine Vertebrates 12.5 Conclusions References Chapter 13 The Alkaline Comet Assay in Prognostic Tests for Male Infertility and Assisted Reproductive Technology Outcomes Sheena E. M. Lewis and Ishola M. Agbaje 13.1 13.2

13.3

13.4

13.5 13.6

Introduction Sites of DNA Damage in Sperm 13.2.1 Oxidative Stress, a Major Cause of DNA Damage 13.2.2 Oxidative Stress, Antioxidant Therapies 13.2.3 Sperm DNA Damage Tests 13.2.4 Modifications to the Alkaline Comet Assay for Use with Sperm 13.2.5 Sperm DNA Adducts and their Relationship with DNA Fragmentation Can Sperm DNA Integrity Predict Success? Relationships with Assisted Conception Outcomes Clinically Induced DNA Damage 13.4.1 Cryopreservation 13.4.2 Vasectomy A Major Barrier to Progress Opportunities and Challenges – The Establishment of Clinical Thresholds and the Integration of DNA Testing into Clinical Practice

282 284

297

297 298 299 299 300 301 301 302 303 303

310

310 311 312 312 313 314 316

317 318 319 319 320

320

xiv

Contents

Acknowledgements References

Chapter 14 The Comet Assay in Sperm – Assessing Genotoxins in Male Germ Cells Adolf Baumgartner, Eduardo Cemeli, Julian Laubenthal and Diana Anderson 14.1 14.2 14.3

Introduction Single-Cell Gel Electrophoresis The Use of Sperm with the Comet Assay 14.3.1 Human Sperm 14.3.2 Modifying Existing Comet Protocols for the Use of Sperm 14.3.3 Sperm DNA and the Comet Assay 14.3.4 The Sperm Comet Assay and the Use of Repair Enzymes 14.3.5 Assessing the Sperm Comet 14.3.6 Comet-FISH on Sperm 14.3.7 Cryopreserved versus Fresh Sperm 14.3.8 Viability Considerations 14.3.9 Statistical Analysis 14.4 Utilising Male Germ Cells with the Comet Assay 14.4.1 In Vivo Comet Assay 14.4.2 In Vitro Comet Assay 14.5 The Sperm Comet Assay versus Other Assays Used in Reproductive Toxicology 14.6 Conclusions Acknowledgements References

321 321

331

331 332 333 333 333 334 335 336 337 338 338 339 339 348 349 350 351 351 351

Section IV: Regulatory, Imaging and Statistical Considerations Chapter 15 Comet Assay – Protocols and Testing Strategies Andreas Hartmann and Gu¨nter Speit 15.1 15.2 15.3

Introduction Applications of the In Vivo Comet Assay for Regulatory Purposes Recommendations for Test Performance 15.3.1 Genetic Endpoint of the Comet Assay 15.3.2 Basic Considerations for Test Protocol 15.3.3 Selection of Tissues and Cell Preparation 15.3.4 Image Analysis

373

373 374 375 375 376 377 378

xv

Contents

15.3.5

Assessment of Cytotoxicity – A Potential Confounding Factor 15.3.6 Ongoing Validation Exercises 15.4 Applications of the In Vivo Comet Assay for Regulatory Purposes 15.4.1 Follow-Up Testing of Positive In Vitro Cytogenetics Assays 15.4.2 Follow-Up Testing of Tumourigenic Compounds 15.4.3 Assessment of Local Genotoxicity 15.4.4 Assessment of Germ Cell Genotoxicity 15.4.5 Assessment of Photogenotoxicity 15.4.6 Genotoxicity Testing of Chemicals 15.5 Conclusions References

Chapter 16 Imaging and Image Analysis in the Comet Assay Mark Browne 16.1 16.2 16.3 16.4

16.5

16.6

16.7

Introduction 16.1.1 Experimental Design and Applications Comet Sample Preparation Comet Fluorescence Staining and Visualisation Fluorescence Microscopy for Comet Imaging 16.4.1 Light Sources 16.4.2 Epifluorescence Light Path 16.4.3 Fluorescence Filter Sets 16.4.4 Microscope Objectives 16.4.5 Beam-Splitter and C-Mount Adapter Image Detection – CCD, EMCCD and CMOS Cameras 16.5.1 Practical Matters Image Processing and Comet Scoring 16.6.1 Image Analysis 16.6.2 Segmentation 16.6.3 Further Segmentation – Identifying Head and Tail of the Comet 16.6.4 Analysis of the Comet, Head and Tail Distributions 16.6.5 Comet Analysis – Other Approaches How Many Cells, How Many Replicates? 16.7.1 Data Presentation and Preparation for Analysis 16.7.2 Statistical Analyses 16.7.3 Data Storage and Management

378 379 380 380 381 382 382 382 384 384 385

390

390 390 391 392 395 396 398 399 401 402 403 407 408 409 410 413 413 416 417 418 419 420

xvi

Contents

16.8 Conclusions References Chapter 17 Statistical Analysis of Comet Assay Data David P. Lovell 17.1 Introduction 17.2 Experimental Design and Statistical Analysis 17.3 Study Design 17.4 Endpoints 17.5 The Experimental Unit and Experimental Design 17.6 Statistical Methods 17.7 Use of Control Groups 17.8 Assessment of Results 17.9 Multiple Comparison Issues 17.10 Power and Sample Size 17.11 Human Studies 17.12 Standardisation and Interlaboratory Comparisons References Subject Index

421 421 424

424 425 425 427 431 432 436 437 438 441 443 445 446 451

SECTION I: GENESIS OF COMET ASSAY

CHAPTER 1

The Comet Assay: A Versatile Tool for Assessing DNA Damage ALOK DHAWAN*, MAHIMA BAJPAYEE AND DEVENDRA PARMAR Developmental Toxicology Division, Indian Institute of Toxicology Research (Formerly Industrial Toxicology Research Centre), P.O. Box 80, M.G. Marg, Lucknow, 226 001, India

1.1 Introduction New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g. pesticides and heavy metals, may be genotoxic to the sentinel species and/or to nontarget species, causing deleterious effects in somatic or germ cells. Test systems that help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or for commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive and rapid tool for assessing DNA * Corresponding author Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

3

4

Chapter 1

damage and repair in individual eukaryotic as well as some prokaryotic cells, and it has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of the Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. Ostling and Johanson1 were the first to quantify DNA damage in cells using a microgel electrophoresis technique, known as the single-cell gel electrophoresis (SCGE) or Comet assay. However, the neutral conditions that they used allowed the detection of only double strand breaks in the DNA. Later, the assay was adapted under alkaline conditions by Singh et al.,2 which led to a sensitive version of the assay that could assess both double- and single-strand DNA breaks as well as alkali-labile sites expressed as frank strand breaks in the DNA. Since its inception, however, the assay has been modified at various steps (lysis, electrophoresis) to make it suitable for various kinds of damage in different cells.3,4 The assay is now a well-established, simple, versatile, rapid, visual, and a sensitive, extensively used tool to assess DNA damage and repair, quantitatively as well qualitatively in individual cell populations.5 Some other lesions of DNA damage such as DNA crosslinking (e.g. thymidine dimers) and oxidative DNA damage may also be assessed using lesion specific antibodies or specific DNA repair enzymes in the Comet assay. It has gained wide acceptance as a valuable tool in fundamental DNA damage and repair studies,4 genotoxicity testing6 and human biomonitoring.7,8 Relative to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution and the micronucleus assays, the advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage (one break per 1010 Daltons of DNA9), requirement for small number of cells (B10 000) per sample, flexibility to use proliferating as well as nonproliferating cells, low cost, ease of application, and the short time needed to complete a study. It can be conducted on cells that are the first site of contact with mutagenic/carcinogenic substances (e.g. oral and nasal mucosal cells). The data generated at the single-cell level allow for robust types of statistical analysis. A limitation of the Comet assay is that aneugenic effects, which may be a possible mechanism for carcinogenicity,10 and epigenetic mechanisms (indirect) of DNA damage such as effects on cell-cycle checkpoints are not detected. The other drawbacks such as single cell data (which may be rate limiting), small cell sample (leading to sample bias), technical variability and interpretation are some of its disadvantages. However, its advantages far outnumber the

The Comet Assay in Different Models

5

disadvantages and hence it has been widely used in fields ranging from molecular epidemiology to genetic toxicology. The present review deals with various models ranging from bacteria to man used in the Comet assay for assessing DNA damage (Figure 1.1).

1.2 Bacteria The first study to assess the genetic damage in bacteria treated with 12.5–100 rad of X-rays, using the Comet assay was conducted by Singh et al.11 In the study, the neutral Comet assay was used for direct (visual) determination of DNA double-strand breaks in the single electrostretched DNA molecule of Escherichia coli JM101. A significant increase in DNA breaks was induced by a dose as low as 25 rad, which was directly correlated to X-ray dosage. The study supported a hypothesis that the strands of the electrostretched human DNA in the Comet assay represented individual chromosomes.

1.3 Plant Models Plant bioassays are important tests that help detect genotoxic contamination in the environment.12 Plant systems can provide information about a wide range of genetic damage, including gene mutations and chromosome aberrations. The mitotic cells of plant roots have been used for the detection of clastogenicity of environmental pollutants, especially for in situ monitoring of water contaminants. Roots of Vicia faba and Allium cepa have long been used for assessment of chromosome aberrations13 and micronuclei.14 During the last decade, the Comet assay has been extensively applied to plants (leaves, shoots, and roots) to detect DNA damage arising due to chemicals and heavy metals in polluted soil (Table 1.1).

1.3.1 1.3.1.1

The Comet Assay in Lower Plants Fungi

Schizosaccharomyces pombe has been used as a model organism to investigate DNA damage due to chlorinated disinfectant, alum and polymeric coagulant mixture in drinking-water samples.15 The authors observed a significantly higher (Po0.001) DNA damage in chlorinated water (i.e. tap water) when compared to untreated (negative control) or distilled water (laboratory control). Hahn and Hock16 used mycelia of Sordaria macrospora grown and treated with a variety of DNA-damaging agents directly on agarose minigels for the assessment of genotoxicity using the Comet assay. DNA-strand breaks were detected by an increase in the DNA migration from the nucleus. This model allowed for the rapid and sensitive detection of DNA damage by a number of chemicals simultaneously. Saccharomyces cerevisiae has also been employed for successful investigation of DNA damage at low concentrations of chemicals.202

Sea

Water

Figure 1.1

Wet land plant (Bacopa)

Freshwater mussel (Unio/ Dreissena)

Earthworms (Eisenia)

blood, liver, spleen, brain, bone marrow, sperm

Soil

blood, nasal, buccal cells, sperm

Humans

Schematic diagram of the use of the Comet assay in assessing DNA damage in different models from bacteria to humans.

hemocytes, gills

Oyster (Crassostrea) Clam (Mya/ Tapes)

Marine mussel (Mytilus)

erythrocytes, gills, hepatocytes

hemocytes, gills Fishes (goldfish-Carassius carp-Cyprinus)

Pond, lakes, rivers

gut, brain

Rodents (mouse, rat)

leaf and root nuclei

Terrestrial plant (Tabacum)

Fruitfly (Drosophila)

Amphibians coelomocytes (Toad-Bufo/ FrogXenopus) erythrocytes

leaves, stem and Algae roots (Chalamydomonas Euglena) in vivo

Blood, sperm, spleen cells

Birds Stork (Ciconia) Kite (Milvus)

erythrocytes, gills, hepatocytes

Marine Fishes (flounder-Paralichthys)

coelomocyte

Sea urchin (Strongylocentrotus)

In vivo

Bacterium (Escherichia coli)

Air

6 Chapter 1

1-Methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene, mitomycin C and actinomycin D. 4-Nitroquinoline-1-oxide (4-NQO), N-nitrosodimethylamine, and hydrogen peroxide UV (UVA+UVB) radiation N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) Ethyl methanesulfonate Age Kinetics of DNA repair Ethyl methanesulfonate (EMS) and N-ethyl-Nnitrosourea (ENU), maleic hydrazide (MH) O-phenylenediamine (o-PDA), hydrogen peroxide and ethyl methanesulfonate (EMS). Heavy metal (Cd, Cu, Pb, and Zn) Polychlorinated biphenyls Heavy metal (Cd, Cu, Pb, and Zn)

Euglena gracilis

Tetrahymena thermophila

Potato plants (Solanum tuberosum var. Korela) Phaeseolus vulgaris Impatiens balsamina Bacopa monnieri L.

Tobacco (Nicotiana tabacum I)

Chlamydomonas reinhardtii Rhodomonas Vicia faba

X-rays

Escherichia coli JM101

Cell used

18 17 19 21 22 23 24 25,26

m m m m m

Whole organism in vivo Whole organism in vivo Root tip meristematic cells

28 29 28

– m m m

Isolated root nuclei Leaf nuclei

33

m

Phenol, hydrogen peroxide, and formaldehyde, influent and effluent water samples

Whole animal in vivo

30 31 32

– m m dose- and time-dependent roots 4 leaves

Uranium Root or shoot cells Stem, root and leaves Cr61 and airborne particulate Ethyl methanesulfonate, methyl methanesulfoNuclei isolated from roots nate, cadmium and leaves Animal models

Nuclei from leaf tissue

27

m

Nuclei from leaf tissue Leaf nuclei Leaf nuclei Whole roots in vivo

Whole organism in vivo Plant Models Whole organism in vivo

Ref.

11

DNA damage

m

Bacteria

Agent tested

Comet assay for assessment of DNA damage – bacteria to humans.

Model

Table 1.1

The Comet Assay in Different Models 7

Gill and haemocytes Haemocytes Digestive gland cells Haemocytes

Polycyclic aromatic hydrocarbons Seasonal variation Polyphenols

Guaı´ ba Basin water

Organotin compounds (MBTC, DBTC and TBTC) Environmental stress Heavy oil spill Cadmium Hydrostatic pressure change

Vent mussels (Bathymodiolus azoricus) Green-lipped mussel (Perna viridis) Freshwater mussel (Utterbackia imbecillis) Oyster (Crassostrea gigas)

Freshwater mussels (Unio tumidus) Golden mussel (Limnoperna fortunei) Bivalve mollusc (Scapharca inaequivalvis) Mytilus galloprovincialis

Chemicals used in lawn care (atrazine, glyphosate, carbaryl, and copper) Cryopreservation

Benzo[a]pyrene

Gills Haemolymph cells Haemocytes Gill and haemolymph

Mytilus edulis

Polybrominated diphenyl ethers (pbdes) Sodium hypochlorite, chlorine dioxide and peracetic acid Pentachlorophenol Varying temperatures Polluted waters Cadmium (Cd) and chromium (Cr) Styrene Tritium Marine waters (Denmark), French Atlantic Coast

48 49 50 51 52,53 54 55 56

m m

m m m

Erythrocytes Haemocytes Gills Digestive gland cells Haemocytes and gill tissues Haemocytes Glochidia Spermatozoa

m

47

m

36 37 38 39 40 41 42

m m m – m m m m m

44 45 46

34 35

Ref.

mm m

DNA damage

m

Haemocytes

Invertebrates – Bivalves

Freshwater bivalve zebra mussel (Dreissena polymorpha)

Cell used

Agent tested

(Continued ).

Model

Table 1.1

8 Chapter 1

Sea anemone (Anthopleura elegantissima)

Sea urchins (Strongylocentrotus droebachiensis) Grass shrimp, (Paleomonetes pugio)

Fruit fly (Drosophila melanogaster)

Aporrectodea longa (Ude)

Eisenia foetida

Manila clam (Tapes semidecussatus) Clams (Mya arenaria)

Haemolymph, gill and digestive gland Haemocytes and digestive gland cells Invertebrates – Earthworms

m dose response

Hepatopancreas Blood cells

76 77 78

75

Estuarine sediments Coal combustion residues Hydrogen peroxide ethylmethanesulfonate (EMS) or benzo[a]pyrene (B[a]P)

72 71 73 74

m m m m concentration-dependent m damage and decreased repair m

70,71

m

Embryos

68 69

m m intestine 4 crop

UV, benzo[a]pyrene, and cadmium

60 61 62 63 64 65 66 67

mdose dependent m m m m m m m

Coelomocytes

59

57,58

–

m

Ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP) Cypermethrin Lechates of industrial waste Cisplatin Dispersed crude oil

Gut and brain cells of first instar larvae

Chemical-treated soil Coelomocytes Soil from coke ovens Coelomocytes Soil from industrialised contaminated areas Coelomocytes Sediment from polluted river Coelomocytes Wastewater-irrigated soil Coelomocytes Commercial parathion Coelomocytes Imidacloprid and RH-5849 Sperm cells PAH-contaminated soil and hydrogen peroxide, Eleocytes cadmium (in vitro) Nickel chloride Coelomocytes Soil samples spiked with benzo[a]pyrene (B[a]P) Intestine and crop/gizzard and/or lindane cells Other Invertebrates

Petroleum hydrocarbons

Sediment-bound contaminants


Trout (Oncorhynchus mykiss)

Brown trout (Salmo trutta fario) Marine flatfish

Turbot (Scophthalmus maximus L.) Brazilian flounder (Paralichthys orbignyanus) Bullheads (Ameiurus nebulosus) Carp (Cyprinus carpio)

Estuarine mullet (Mugil sp.) and sea catfish (Netuma sp.) Fresh water teleost fish (Mystus vittatus) Eastern mudminnow (Umbra pygmaea L.) Neotropical fish (Prochilodus lineatus) Freshwater goldfish (Carassius auratus)

Cryopreservation (freeze–thawing)

Ethyl methanesulfate

Polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) polluted waters Polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) polluted waters PCB77 (3,3 0 ,4,4 0 -tetrachlorobiphenyl)

Diesel water soluble fraction acute (6, 24 and 96 h) and subchronic (15 days) exposures, Technical herbicide Roundup containing glyphosphate salt ADDB and PBTA-6 Sediment collected from polluted sites in Cork Harbour (Ireland) Contaminated estuary waters

Rhine water for 11 days

Endosulfan

PAHs, PCBs, organochlorine pesticides (OCPs), as well as heavy metals Exhaustive exercise Organochlorine pesticides and heavy metals High temperature

Chub (Leuciscus cephalus)

Cell used

80 81,82 83 84 85 86 87 88 89 90 90 91 92 93

m m m in all cells m m mm dose dependent m mm m m – m in all tissues Slight m

Erythrocytes Erythrocytes

Erythrocytes Erythrocytes Hepatocytes Blood cells Erythrocytes Erythrocytes Erythrocytes Blood, gill, liver and kidney Spermatozoa

Gill, kidney, and erythrocytes Blood erythrocytes

79

Ref.

m

DNA damage

Hepatocytes

Vertebrates – Fishes

Agent tested

(Continued ).

Model

Table 1.1

10 Chapter 1

Amphibian larvae (Xenopus laevis and Pleurodeles waltl) Amphibian larva (Xenopus laevis)

Zebrafish (Danio rerio) Rainbow trout hepatoma cell line (RTH-149) Rainbow trout gonad (RTG-2) cell line liver (RTL-W1) cell line

Trout (Oncorhynchus mykiss)

Carp (Cyprius carpio)

In vitro

Hornyhead turbot (Pleuronichthys verticalis)

Eelpout (Zoarces viviparus) Gilthead sea bream (Sparus aurata) Dab (Limanda limanda)

European eel (Anguilla anguilla)

43 97

m in adults and males m

Blood cells

PAHs and PCBs polluted waters of English Channel Gender and age Sediments collected from a natural petroleum seep (pahs)

Cadmium (CdCl2) Captan (N-trichloromethylthio-4-cyclohexene1,2-dicarboximide) Benzo[a]pyrene, ethyl methanesulfonate methyl methanesulfonate, aqueous extracts of five sediments from French channels

99 100 101 102 103 104 105

106,107

m m k k m m m dose-dependent response

mconcentration and time dependent – m

Erythrocytes Erythrocytes

108,109

98

m

Organic sediment extracts from the North Sea Leukocytes (Scotland) Cadmium Hepatocytes Oxidative stress and its prevention by Erythrocytes indolinic and quinolinic nitroxide radicals Tannins Diaryl tellurides and ebselen (organoselenium) Surface waters of German rivers, Rhine and Elbe Hepatocytes and gill cells Water samples from the polluted Kishon river Liver (Israel) Gonad 4-Nitroquinoline-N-oxide N-methyl-N 0 -nitro-Nnitrosoguanidine, benzo[a]pyrene, nitrofurantoin, 2-acetylaminofluorene, and dimethylniEpitheloid liver trosamine, and surface waters Vertebrates – Amphibians

Liver cells

96

mm

Erythrocytes

95

m

Copper

94

m

Nucleated erythrocytes

Erythrocytes

Benzo[a]pyrene, Arochlor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and betanaphthoflavone Oil spill (PAH)


Sperm

Toxic acid mining waste rich in heavy metals Short-term storage

Turkey

Blood cells

Heavy metals and arsenic Toxic acid mining waste rich in heavy metals Heavy metals and arsenic

Wild nestling white storks (Ciconia ciconia) Black kites (Milvus migrans)

Blood cells

High peak-power pulsed electromagnetic field Erythrocytes Vertebrates – Birds

Erythrocytes

Erythrocytes

Erythrocytes

Erythrocytes

Liver cells and erythrocytes Splenic lymphocytes

Cell used

Xenopus laevis

In vitro

Tadpole Rana clamitans Rana pipiens

Bullfrog (Rana catesbeiana) tadpoles

Rana hexadactyla tadpoles

Tadpoles of Rana N. Hallowell

Imidacloprid [ 1-(6-chloro-3-pyridylmethyl)-Nnitro-imidazolidin-2-ylideneamine] and RH5849 [ 2 0 -benzoyl-l 0 -tert-butylbenzoylhydrazinel] Sulfur dyes (Sandopel Basic Black BHLN, Negrosine, Dermapel Black FNI, and Turquoise Blue) used in the textile and tannery industries Herbicides AAtrex Nine-O (atrazine), Dual-960E (metalochlor), Roundup (glyphosate), Sencor500F (metribuzin), and Amsol (2,4-D amine) Agricultural regions, Industrial regions

Petrochemical (mainly oil and phenol) polluted area Bleomycin-induced DNA damage and repair

Toad (Bufo raddei)

Toad (Xenopus laevis, and Xenopus tropicalis)

Agent tested

(Continued ).

Model

Table 1.1

113

114 115

mm

mm m industrial regions 4 agricultural regions

118,119,120 121

117 118,119,120 117

112

m correlated with arsenic mm m correlated with copper and cadmium m (2-10 fold) m

111

m DNA damage X. tropicalis 4 X. laevis DNA repair in X. laevis 4 X. tropicalis m

116

110

m

m due to rise in temperature

Ref.

DNA damage

12 Chapter 1

UV A+Fluoroquinolones (clinafloxacin, lomefloxacin, ciprofloxacin) UVA+ 8-methoxypsoralene (8-MOP) Ageing Diesel exhaust particles Trypanosoma cruzi infection

Lead acetate

Sanguinarine alkaloid, argemone oil

SKH-1 mice

Dyslipidemic ApoE(–/–) mice Balb/c mice

CD-1 mice

Swiss albino mice

Sulfonamide, protozoan parasite Toxoplasma gondii Rutin and quercetin

Isogenic mice

Cirrhotic rats

Pesticide formulations (Bravo and Gesaprim)

Male CBA mice

Apomorphine, 8-oxo-apomorphine-semiquinone Ethanol, grape seed oligomer and polymer procyanidin fractions

Steviol

Cypermethrin

Ethanol Melphalan

Aldh2 knockout mice P53(+/–) mice

Hepatic cells, bone marrow cells, spleen cells Peripheral blood cells, liver cells and brain cells Bone marrow cells

Peripheral blood, liver, heart and spleen cells Nasal epithelial cells, lung, whole blood, liver, kidney, bone marrow, brain and testes Blood, bone marrow cells and liver Brain, liver, kidney, bone marrow, blood, spleen Stomach cells, hepatocytes, kidney and testicle cells Brain cells Brain cells

Aorta, liver, and lung

Hepatic cells Liver, bone marrow, peripheral blood and the distal intestine Epidermal cells

Spleen leukocytes Liver and breast muscle cells Vertebrates – Rodents

T-2 toxin and deoxynivalenol (DON) Storage conditions (4 1C)

Chicken

126

mm for fluoroquinolones

138 139

m in peripheral blood cells mm

134

m

137

133

135 136

131,132

m dose dependent in blood and bone marrow m

– m k ethanol-induced protection by grape seed mm

130

m in all organs on prolonged exposure – in testes

129

127,128

124 125

m oxidative damage DNA cross-links in all cells tested

k for MOP m Oxidative damage in liver – in lung or aorta m in heart and spleen

122 123

m m liver cells 4 breast muscle cells


(Continued ).

Transitional cell carcinoma patients and controls Aaxia telangiectasia heterozygote

Breast cancer patients and controls Breast cancer patients and controls Normal individuals

Murine primary cultures of brain cells and a continuous cell line of astrocytes Chinese hamster ovary cell line (CHO)

FE1 muta mouse lung epithelial cell line L5178Y mouse lymphoma cells

In vitro

Model

Table 1.1

Lymphoma cells

Ketoprofen, promazine, chlorpromazine, dacarbazine, acridine, lomefloxacin, 8-methoxypsoralen, chlorhexidine, titanium dioxide, octylmethoxycinnamate Xanthine/xanthine oxidase, hydrogen peroxide superoxide dismutase, catalase, or ascorbic acid

X-irradiation

DNA-strand breaks

Chlorhexidine

Radiosensitivity

Radiosensitivity

Peripheral leukocytes

Peripheral blood mononuclear cells Peripheral blood mononuclear cells Buccal epithelial cells and peripheral blood lymphocytes Exfoliated cells extracted from bladder washing

Endosulfan Ovary cells Cypermethrin, pendimethalin, dichlorovous Humans – Clinical

Brain cells

Lung epithelial cell line

Cell used

Carbon black

Agent tested

141

142

143 144

145 146 147 148 149

k by antioxidants

m

m m and reduced DNA repair m m in patients m (B3 times high) in patients

140

Ref.

Positive with phototoxic compound

m

DNA damage

14 Chapter 1

–

Oxidative DNA damage DNA integrity

Tomato drink Green vegetables Grape juice Vitamin C supplementation Vitamin E and vitamin C

Breast cancer patients

Type 2 diabetes mellitus Cancer (testicular cancer, lymphoma and leukemia) patients

Healthy subjects

cells

mono-

mono-

mono-

Benzene in printing Lead (Pb) and cadmium (Cd) Asbestos cement plant Fenvalerate (FE) exposure Organic solvents

Sperm Peripheral blood

"

"

Exhaustive exercise Ionising radiation

Peripheral lymphocytes

Lymphocytes Peripheral blood leukocytes Human T- and B-lymphocytes, and granulocytes

Substances used in the rubber industry Air pollutants

Rubber factory workers Outdoor workers in Mexico cities Rickshaw pullers Nuclear medicine personnel Workers

Polycyclic aromatic hydrocarbons (PAH)

Peripheral blood Blood lymphocytes

Pesticides

Agricultural workers

Exfoliated buccal cells and lymphocytes Lymphocytes

Jet fuel vapours, jet fuel combustion products

Humans – Occupational

Blood lymphocytes Blood lymphocytes

Blood lymphocytes

Humans – Dietary intervention

Peripheral blood nuclear cells Peripheral blood nuclear cells Peripheral blood nuclear cells Peripheral blood Spermatozoa

Airport personnel

Smokers Technical anesthesiology staff

–

X-irradiation

Nijmegen breakage syndrome (NBS) patients Alzheimer disease patients 152 153 154

m in patients m Decreased DNA integrity

166 167 168

m m m B-lymphocytes 4 T-lymphocytes 4 granulocytes m m m m m

169 170 171 172 173

161 162,163 164 165

160 – m k in exposed population m

m

k k in oxidative damage

155 156 157 158 159

151

m in patients

k

150

m in patients


(Continued ).

Human keratinocytes MCF-7 cells JM1 cells HepG2 cells

Sperms Prostate tissues primary culture

Episkin

In vitro

Rural Indian women Normal individuals

Normal individuals Active and passive smokers Normal individuals

Nurses

Model

Table 1.1 Cell used

UV, Lomefloxacin and UV or 4-nitroquinolineN-oxide (4NQO) and protection by Mexoryl Reproductive toxins 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), its N-hydroxy metabolite (N-OHPhIP) and benzo[a]pyrene (B[a]P) UVA or UVB Estradiol Estradiol Endosulfan Indirect acting genotoxins (cyclophosphamide)

Lymphocytes

Smoking Diet (vegetarian or non-vegetarian) Biomass fuels Benzo[a]pyrene, beta-naphthoflavone (BNF)

Skin cells Breast cells Lymphoblast cells Liver cells

Male germ cells Prostate cells

Skin fibroblast cells

Lymphocytes Human umbilical vein endothelial cells (HUVEC)

Lymphocytes Lymphocytes

Endurance exercise Smoking

Coke oven emissions (coe) Blood lymphocytes Welders (Cd, Co, Cr, Ni, and Pb) Lymphocytes Pesticide formulators (organophosphorus Lymphocytes pesticides) Copper smelters (inorganic arsenic) Leukocytes Chrome-plating workers (chromium VI) Lymphocytes Workers in foundry and pottery (silica) Lymphocytes 5-Fluorouracil, cytarabine, gemcitabine, cycloLymphocytes phosphamide, and ifosfamide Humans – Lifestyle

Agent tested

193 194 194 195 196

m m concentration dependent –

m m

190,191 192

187 188

m

m m dose related

183–186

m m

189

181 182

m mm m Slight m

m reduced by Mexoryl

177 178 179 180

m

Ref. 174 175 176

DNA damage

16 Chapter 1

Sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl-N-nitro-N-nitroso-guanidine (MNNG) Mono(2-ethylhexyl) phthalate (MEHP), benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), or N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG). Heterocyclic amine and prevention by monomeric and dimeric flavanols and black tea polyphenols C60 Fullerenes Municipal sludge leachates Lymphocyte

Nasal cells

199 200 201

m m

198

197

k in oxidative damage

m with sodium dichromate and MNNG – with NDEA m with BPDE and MNNG – with MEHP

m significant increase in DNA damage; mm highly significant increase in DNA damage; k decrease in DNA damage; – no DNA damage reported

Human lymphocytes

Miniorgan cultures of human inferior nasal turbinate epithelia


18

1.3.1.2

Chapter 1

Algae

Aquatic unicellular plants like algae provide information on the potential genotoxicity of the water in which they grow. Being single celled they can be used as a model for assessment of DNA damage and monitoring of environmental pollution utilising the Comet assay. Unicellular green alga Chlamydomonas reinhardtii was used for evaluation of DNA damage due to known genotoxic chemicals and also demonstrated that oxidative stress was better managed by the algal cells under light rather than dark conditions.17 The Comet assay was found to be useful for evaluating chemically induced DNA damage and repair in Euglena gracilis and responses were more sensitive than those of human lymphocytes under the same treatment conditions.18 The ease of culturing and handling E. gracilis as well as its sensitivity, makes it a useful tool for testing the genotoxicity of chemicals and monitoring environmental pollution. A modified version of the Comet assay was used as an alternative technique to assess DNA damage due to UV radiation in Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate.19

1.3.2

The Comet Assay in Higher Plants

Vicia faba has been widely used for the assessment of DNA damage using the Comet assay. Strand breaks and abasic (AP) sites in meristematic nuclei of V. faba root tips were studied by the neutral and alkaline Comet assay.20,21 The alkaline electrophoresis procedure was found to be most sensitive at low doses, while the neutral electrophoresis procedure yielded an optimal dose– response curve within a wider dose range. Angelis et al.20 also suggested that the Comet assay was able to detect a phenomenon resembling clastogenic adaptation at the molecular level. Gichner and Plewa22 developed a sensitive method for isolation of nuclei from leaf tissue of Nicotiana tabacum. The method resulted in high resolution and constant low tail moment values for negative controls, and hence it could be incorporated as a test for in situ plant environmental monitoring.22 The Comet assay has also been used to study the effect of age of plant on DNA integrity23 as well as the kinetics of DNA repair24 in isolated nuclei from leaves of tobacco plants. A small but significant increase in DNA damage compared to controls was noted in heterezygous tobacco and potato plants grown on soil contaminated with heavy metals.28 The tobacco and potato plants with increased DNA damage were also found to be severely injured (inhibited growth, distorted leaves), which may be associated with necrotic or apoptotic DNA fragmentation. No DNA damage was observed in the root or shoot cells of Phaeseolus vulgaris treated with different concentrations of uranium.30 The ornamental plant Impatiens balsamina was used as a model to understand the genotoxic effect of Cr61 and airborne particulate matter,31 which produced increased strand breaks in plant parts (stem, root and leaves). Thus, this plant could be used for environmental biomonitoring studies involving air pollution and heavy metals.


19

The major drawback with plant models was the fact that exposure needs to be given in the soil and it is difficult to say whether the result demonstrates synergies with other chemicals in the soil or nonavailability of the toxicant due to its soil binding affinity. Therefore, Vajpayee et al.32 used Bacopa monnieri L., a wetland plant, as a model for the assessment of ecogenotoxicity using the Comet assay. In vivo exposure to cadmium (0.01–500 mM) for 2, 4, and 18 h resulted in doseand time-dependent increases in DNA damage in the isolated roots and leaf nuclei, with roots showing greater DNA damage than leaves. In vitro (acellular) exposure of nuclei from leaves of B. monnieri to 0.001–200 mM cadmium resulted in significant (Po0.05) levels of DNA damage. These studies revealed that DNA damage measured in plants using the Comet assay is a good model for assessment of genotoxicity of polluted environments since in situ monitoring and screening can be accomplished. Higher plants can be used as an alternative first-tier assay system for the detection of possible genetic damage resulting from polluted waters/effluents due to industrial activity or agricultural run offs.

1.4 Animal Models To assess safety/toxicity of chemicals/finished products, animal models have long been used. With the advancements in technology, knockouts and transgenic models have become common to mimic the effects in humans. The Comet assay has globally been used for assessment of DNA damage in various animal models (Table 1.1).

1.4.1

Lower Animals

Tetrahymena thermophila is a unicellular protozoan, widely used for genetic studies due to its well-characterised genome. Its uniqueness lies in the fact that it has a somatic and a germ nucleus in the same cell. Therefore it has been validated as a model organism for assessing DNA damage using a modified Comet assay protocol standardised with known mutagens such as phenol, hydrogen peroxide, and formaldehyde.33 The method was then used for the assessment of genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant.33 The method provided an excellent, low-level detection of genotoxicants and proved to be a cost-effective and reliable tool for genotoxicity screening of wastewater.

1.4.1.1

Invertebrates

Studies have been carried out on various aquatic (marine and freshwater) and terrestrial invertebrates (Table 1.1). The genotoxicity assessment in marine and freshwater invertebrates using the assay has been reviewed.203–205 Cells from haemolymph, embryos, gills, digestive glands and coelomocytes from mussels (Mytilus edulis42), zebra mussel (Dreissena polymorpha), clams

20

Chapter 1

(Mya arenaria), and polychaetes (Nereis virens), have been used for ecogenotoxicity studies using the Comet assay. DNA damage has also been assessed in earthworms61,63 and fruit flies, Drosophila.72,206 The Comet assay has been employed to assess the extent of DNA damage in organisms at polluted sites in comparison to those at reference sites in the environment. In the laboratory it has been widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage.78

1.4.1.2

The Comet Assay in Mussels

Freshwater and marine mussels have been used to study the adverse effect of contaminants in the aquatic environment as they are important pollutionindicator organisms. These sentinel species are adversely affected by the pollution of the water bodies and thus provide the potential for environmental biomonitoring. The Comet assay in mussels has been used to detect a reduction in water quality caused by chemical pollution.41,42,49,207 Mytilus edulis has been widely used for Comet assay studies to evaluate DNA-strand breaks in gill and digestive gland nuclei due to polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P),44 and oil spills with petroleum hydrocarbons.59 The DNA damage was found to be elevated in the exposed mussels. However, the damage returned to normal levels, after continued exposure to a high dose (20 ppb-exposed diet) of B[a]P for 14 days. This was attributed to an adaptive response in mussels to prevent the adverse effects of DNA damage.44 The green lipped mussels (Perna viridis) also showed a similar result on exposure to B[a]P in water.54 Significant levels of interindividual variability, including seasonal variations in DNA damage have been reported from some studies, both laboratory and field.45,49,208,209 Baseline monitoring thus has to be carried out over long time intervals. Temperature-dependent DNA damage was observed in haemocytes of freshwater mussel Dreissena polymorpha37 showing that the mussels are sensitive towards change in water temperatures. Thus, monitoring ecogenotoxicity with these species should take into account variations in temperatures. Findings have also suggested that antioxidant supplementation can improve the sensitivity of the Comet assay by lowering the baseline damage in untreated animals.208 Villela et al.210 used the golden mussel (Limnoperna fortunei) as a potential indicator organism for freshwater ecosystems due to its sensitivity to water contaminants. The Comet assay in haemocytes of freshwater Zebra mussel, D. polymorpha Pallas, was used as a tool in determining the potential genotoxicity of water pollutants.34–36,38 Klobucar et al.38 suggested the use of the Comet assay in haemocytes from caged, nonindigenous mussels as a sensitive tool for monitoring genotoxicity of freshwater. DNA damage and repair studies in vent mussels, Bathymodiolus azoricus, have been carried out to study the genotoxicity of a naturally contaminated deep-sea environment.52,53 The vent mussels demonstrated similar sensitivity to environmental mutagens as that of coastal mussels and thus could be used for ecogenotoxicity studies of deep sea waters using the Comet assay.


21

In vitro Comet assay has also been used in cells of mussels. Dose–response increases in DNA-strand breakages were recorded in digestive gland cells211 haemocytes212 and gill cells208,212 of M. edulis exposed to both direct (hydrogen peroxide and 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone) and indirect (B[a]P, 1-nitropyrene, nitrofurantoin and N-nitrosodimethylamine) acting genotoxicants. Digestive gland cells of Unio tumidus were also used for in vitro studies of DNA damage and repair due to pro-oxidative effect of polyphenolic compounds.46,213 Wilson et al.208 demonstrated the potential application of the Comet assay to the gill cells of M. edulis as a potential in vitro screen for agents destined for release or disposal into the marine environment.

1.4.1.3

The Comet Assay in Other Bivalves

Coughlan et al.57 showed that the Comet assay could be used as a tool for the detection of DNA damage in clams (Tapes semidecussatus) as biomonitor organisms for sediments. Significant DNA-strand breaks were observed in cells isolated from haemolymph, gill and digestive gland from clams exposed to polluted sediment.57,58 The Comet assay was used for the assessment of sperm DNA quality of cryopreserved semen in Pacific oysters (Crassostrea gigas) as it is widely used for artificial fertilisation.56 Gielazyn et al.214 demonstrated the use of lesion-specific DNA repair enzyme formamidopyrimidine glycosylase (Fpg) to enhance the usefulness and sensitivity of the Comet assay in studying oxidative DNA damage in isolated haemocytes from oysters (Crassostrea virginica) and clams (Mercenaria mercenaria). The studies in mussels have shown the Comet assay to be a sensitive, but nonspecific, molecular biomarker of genotoxicity. One of the drawbacks when applying single-cell gel electrophoresis to field populations may be the adapatability of the animals to high concentrations of contaminants (e.g. B[a]P), which may pose a major problem.44 Also, seasonal variation and temperature altered both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants under in vitro conditions.37,58 The Comet assay detecting DNA-strand breaks has demonstrated that higher basal levels of DNA damage are observed in marine invertebrates, hence the protocol followed in these animals should be considered for biomonitoring the ecogenotoxicity of a region.215

1.4.1.4

The Comet Assay in Earthworms

The Comet assay applied to earthworms is a valuable tool for monitoring and detection of genotoxic compounds in terrestrial ecosystems61,66 (Table 1.1). Since the worms feed on the soil they live in, they are a good indicator of the genotoxic potential of the contaminants present in the soil and thus used as a sentinel species. Verschaeve et al.60 demonstrated a dose–response effect with the extent of DNA damage in coelomic leucocytes (coelomocytes) of

22

Chapter 1

earthworms (Eisenia foetida) from soil treated with different chemicals as an indicator of soil pollution. Coelomocytes from E. foetida demonstrated increased DNA damage when worms were exposed to soil samples from polluted coke oven sites,61 or industrialised contaminated areas62 and even sediment samples from polluted river system.63 An insecticide, parathion, produced DNA-strand breaks at all time points and doses in the sperm cells of E. foetida65 while dose-effect relationships were displayed by two pesticides, Imidacloprid and RH-5849 in the same species,66 showing that pesticides could also have adverse effects on nontarget species. In vitro exposure of coelomocytes primary cultures to nickel chloride as well as whole animals either in spiked artificial soil water or in spiked cattle manure substrates exhibited increased DNA-strand breaks due to the heavy metal.68 The eleocytes cells, a subset of coelomocytes produced increased DNA-strand breaks under both in vitro and in vivo conditions and could be used a sensitive biomarker for genotoxicity in earthworms.67 Another earthworm, Aporrectodea longa (Ude), when exposed to soil samples spiked with B[a]P and/or lindane demonstrated genotoxicity in the intestinal cells to be more sensitive to the effect of the toxicants than the crop/gizzard cells.69 Fourie et al.216 used five earthworm species (Amynthas diffringens, Aporrectodea caliginosa, Dendrodrilus rubidus, Eisenia foetida and Microchaetus benhami) to study genotoxicity of sublethal concentrations of cadmium sulfate, with significant DNA damage being detected in E. foetida followed by D. rubidus and A. caliginosa. The study showed the difference in sensitivity of species present in an environment and its influence on the genotoxicity risk assessment. Hence, for environmental biomonitoring, specific species have to be kept in mind to reduce false-negative results.

1.4.1.5

The Comet Assay in Drosophila

The simple genetics and developmental biology of Drosophila melanogaster has made it the most widely used insect model and has been recommended as an alternate animal model by the European Centre for the Validation of Alternative Methods.217 Recently, Drosophila has evolved into a model organism in toxicological studies.218,219 D. melanogaster has also been used as an in vivo model for assessment of genotoxicity using the Comet assay70–72,206 (Table 1.1). Neuroblast cells of third instar larvae, DNA repair deficient in nucleotide excision repair (mus201) and a mechanism of damage bypass (mus308), have been used for mechanistic studies.206 Third instar larvae of D. melanogaster (Oregon R+) were validated for genotoxicity assessment using a modified Comet assay.70,71 Since the cells of Drosophila are smaller than mammalian cells, modifications in the Comet assay were done, e.g. higher concentration of agarose (for the smaller size of Drosophila cells), removal of DMSO from lysing solution (DMSO is toxic to the cells) and lower electrophoresis time (for improved performance of the assay). This modified protocol was validated in gut and brain cells using


23

well-known alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP) that were mixed in standard Drosophila diet and produced a significant dose-dependent response.70,71 Cypermethrin, a synthetic pyrethroid, even at low concentrations (at 0.002 ppm) and leachates of industrial waste produced significant dose-dependent increases in DNA damage in the brain ganglia and anterior mid gut of D. melanogaster.71,72 Results from the Comet assay have also shown a direct correlation between the concentrations of cisplatin adducts and DNA damage in somatic cells of D. melanogaster.73 In vitro studies using Drosophila S2 cells demonstrated that the ectopically expressed DNA glycosylases (dOgg1 and RpS3) reduced the oxidised guanosine (8-OxoG), but contributed to increased DNA degradation due to one of the constituents of the DNA repair system.220 The studies in Drosophila have shown it to be a good alternative to animal models for the assessment of in vivo genotoxicity of chemicals using the Comet assay.

1.4.1.6

The Comet Assay in Other Invertebrates

Nereis virensa, a polychaete, plays an important role in the distribution of pollutants in sediments due to their unique property of bioturbation. These worms are similar to earthworms in soil and can be used for genotoxicity assessment of sediments. Intracoelomic injection of B[a]P was given to the worms and the Comet assay was conducted on coelomocytes.221 Nereis species was, however, not found to be suitable for assessing PAH genotoxicity probably due to its lack of metabolic capability to convert B[a]P to its toxic metabolite.221 DNA damage was assessed in neuroblast cells of brains of 1st instars of grasshoppers (Chorthippus brunneus) exposed to various doses of zinc from a polluted site to understand the mechanism of toxicity in insects due to industrial pollutants.222 The estuarine grass shrimp, Palaemonetes pugio, exposed to coal-combustion residues from coal-fired electrical generation, were studied for DNA damage using the Comet assay. Chronic exposure caused DNA damage in hepatopancreatic cells of adult shrimps as compared to the reference shrimp.77 The Comet assay in planarians is an important test for environmental monitoring studies since these are simple organisms with high sensitivity, low cost and a high proliferative rate.223 The genotoxic potential of water from Diluvio’s Basin was evaluated in planarians, where an increase in pollutants towards the basin led to an increase in the DNA damage in these species.223 A significant increase of primary DNA damage was observed in planarian cells due to a Norflurazon, a bleaching herbicide224 and copper sulfate,225 when compared to the control animals. These studies have also shown the use of the Comet assay in biomonitoring diverse environmental conditions utilising sentinel species.

24

Chapter 1

1.5 Higher Animals 1.5.1

Vertebrates

Studies of vertebrate species where the Comet assay is used include fishes, amphibians, birds and mammals. Cells (blood, gills, kidneys and livers) of different fishes, tadpoles and adult frogs, as well as rodents have been used for assessing in vivo and in vitro genotoxicity of chemicals, and human biomonitoring has also been carried out employing the Comet assay (Table 1.1).

1.5.1.1

The Comet Assay in Fishes

Various fishes (freshwater and marine) have been used for environmental biomonitoring, as they are endemic organisms, which serve as sentinel species for a particular aquatic region to the adverse effects of chemicals and environmental conditions. The Comet assay has found wide application as a simple and sensitive method for evaluating in vivo as well as in vitro DNA damage in different tissues (gills, liver, blood) of fishes exposed to various xenobiotics in the aquatic environment (Table 1.1). Environmental biomonitoring to assess the water quality in rivers has been carried out in hepatocytes of chub,79 erythrocytes of mullet (Mugil sp.), sea catfish (Netuma sp.81,82), bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio90,226). The basal level of DNA damage has been shown to be influenced by various factors, such as the temperature of water in erythrocytes of mullet and sea catfish,81,82 age and gender in dab (Limanda limanda43), and exhaustive exercise in chub.80 Therefore, these factors should be accounted for during environmental biomonitoring studies. The sensitivity of the assay may be affected by high intraindividual variability.43 The protocol and experimental conditions used for the Comet assay for monitoring marine ecosystems may lead to differences in the results obtained.92 The use of chemical and mechanical procedures to obtain cell suspensions may also lead to DNA damage.227 Anesthesia did not contribute towards DNA damage in vivo in methyl methanesulfonate (MMS) treated fishes and the anesthetic benzocaine did not alter the DNA damage in erythrocytes after in vitro exposure to MMS or H2O2.228 Hence keeping in mind animal welfare, multi sampling in the same fish can be conducted. In vitro studies on fish hepatocytes,99 primary hepatocytes and gill cells103 as well as established cell lines (with metabolic competence229) using the Comet assay have also been conducted to assess the genotoxicity of chemicals in water samples. The antioxidant potential of indolinic and quinolinic nitroxide radicals,100 tannins101 and low concentrations (o10 mM) of diaryl tellurides and ebselen – an organoselenium compound102 – in oxidative DNA damage has been studied in nucleated trout (Oncorhynchus mykiss) erythrocytes for use of these compounds in biological systems. Kammann et al.98 demonstrated the Comet assay in isolated leukocytes of carp as an in vitro model for evaluating genotoxicity of marine sediment extracts and increased sensitivity of the


25

method with use of the DNA repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara C). The Comet assay with fish cell lines may be a suitable tool for in vitro screening of environmental genotoxicity, however, the metabolising capabilities of the cell line need to be taken into account. Cryopreservation has been shown to induce DNA-strand breaks in spermatozoa of trout,93,230 sea bass (Dicentrarchus labrax231) and gilthead sea bream (Sparus aurata230). The DNA damage was prevented by the addition of cryopreservants such as BSA and dimethyl sulfoxide.231 These studies have demonstrated the sperm Comet assay as a useful model in determining the DNA integrity in frozen samples for commercially cultured species. The above studies have shown the usefulness of the Comet assay in fishes as a model for monitoring genotoxicity of aquatic habitats.

1.5.1.2

The Comet Assay in Amphibians

The Comet assay in amphibians has been carried out at adult and larval stages for ecogenotoxicity of aquatic environments and studies since 1999 have been well reviewed by Cotelle and Ferard.203 The animals chosen for the Comet assay act as sensitive bioindicators of aquatic and agricultural ecosystems (Table 1.1). The animals were either collected from the site (in situ) or exposed to chemicals under laboratory/natural conditions. Erythrocytes from tadpoles of two species Rana clamitans and Rana pipiens have been used for the assessment of genotoxicity of water bodies as in situ sentinel organisms for environmental biomonitoring.115 R. clamitans tadpoles collected from agricultural regions showed significantly higher (Po0.001) DNA damage than tadpoles collected from sites of little or no agriculture. Similarly R. pipiens tadpoles collected from industrial sites showed significantly higher (Po0.001) DNA-strand breaks than samples from agricultural areas. The higher levels of DNA damage may be due to the pesticides used in the agricultural region. Variation in DNA damage due to sampling time115 and during various metamorphosis states232 was also observed. Hence, for biomonitoring environmental genotoxicity using the Comet assay, pooling of early tadpole phases could be helpful. Studies have also been conducted on caged tadpoles in areas where the indigenous population is not present, due to ecological imbalance from pollution. Rana clamitans and the American toad (Bufo americanus) tadpoles were caged at the polluted reference site and demonstrated significant (Po0.05) increases in DNA damage, relative to control tadpoles in the laboratory.233 These results demonstrated that caged tadpoles could be used for monitoring genotoxicity of water habitats that do not support the survival of tadpoles, e.g. large lakes and aquatic areas near high industrial activity. Huang et al.110 have shown the genotoxicity of petrochemicals in liver and erythrocytes of toad Bufo raddeis. DNA damage was found to be positively correlated to the concentration of petrochemicals in liver, pointing to the fact that liver is the site for metabolism and may be a good marker for studying genotoxicity of compounds that require metabolic activation. The effect of

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Chapter 1

polyploidy on bleomycin-induced DNA damage and repair in X. laevis (pseudotetraploid) and Xenopus tropicalis (diploid) was studied using the Comet assay.111 The X. tropicalis was more sensitive with a lower capacity for repair than X. laevis, showing that polyploidy protects DNA damage and allows rapid repair, and hence these species may be used as a good model for DNA damage and repair studies.

1.5.1.3

The Comet Assay in Birds

There are few studies involving the Comet assay in birds (Table 1.1). Genetic damage due to a mining accident involving heavy metals has been reported in free-living, nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) from southwestern Spain,117–120 however, species-specific and intraspecies differences were observed. Faullimel et al.123 showed that the neutral Comet assay could be used to study the impact of freezing and thawing on DNA integrity in breast fillets and liver cells of frozen chicken. Frankic et al.122 reported that T-2 toxin and deoxynivalenol (DON) induced DNA fragmentation in chicken spleen leukocytes that was abrograted by dietary nucleotides. Kotlowska et al.121 have demonstrated increased DNA fragmentation in turkey sperm after 48 h of liquid storage which might be helpful in evaluating the DNA integrity for artificial insemination.

1.5.1.4

The Comet Assay in Rodents

Mice and rats have been widely used as animal models for the assessment of in vivo genotoxicity of chemicals using the Comet assay (Table 1.1). The in vivo Comet assay has been accepted by the UK Committee on Mutagenicity Testing of Chemicals in Food, Consumer Products and Environment10 as a test for assessing DNA damage, and is recommended for follow-up testing of positive in vitro findings. A positive result in the in vivo Comet assay assumes significance if mutagenic potential of a chemical has already been demonstrated in vitro. Within a battery of tests, the Comet assay finds a place as a supplemental in vivo test that has been accepted by international guidelines.234 There are specific guidelines for the performance of the Comet assay in vivo for reliable results.235–237 Multiple organs of mouse/rat including brain, blood, kidney, lungs, liver, bone marrow have been utilised for the comprehensive understanding of the systemic genotoxicity of chemicals.133,134,238,239 The most important advantage of the use of Comet assay is that DNA damage in any organ can be evaluated without the need for mitotic activity and DNA damage in target as well as nontarget organs can also be seen.239 Comprehensive data on chemicals representing different classes, e.g. PAHs, alkylating compounds, nitroso compounds, food additives, etc. that caused DNA-strand breaks in various organs of mice was compiled by Sasaki et al.239,240 The mouse or rat organs exhibiting


27

increased levels of DNA damage were not necessarily the target organs for carcinogenicity. Therefore, for the prediction of carcinogenicity of a chemical, organ-specific genotoxicity was necessary but not sufficient.240 The Comet assay can be used as an in vivo test apart from the cytogenetic assays in haematopoietic cells and also for those compounds that have poor systemic bioavailability. Different routes of exposure in rodents have been used, e.g. intraperitoneal,131,133 oral241,242 and inhalation130,243 to study the genotoxicity of different chemicals. The route of exposure is an important determinant of the genotoxicity of a chemical due to its mode of action.134 The in vivo Comet assay helps in hazard identification and assessment of dose–response relationships as well as the mechanistic understanding of a substance’s mode of action. Besides being used for testing the genotoxicity of chemicals in laboratory-reared animals, the Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation.244 The in vivo Comet assay in rodents is an important test model for genotoxicity studies, since many rodent carcinogens are also human carcinogens, and hence this model not only provides an insight into the genotoxicity of human carcinogens but is also suited for studying their underlying mechanisms.

1.5.1.5

The Comet Assay in Humans

The Comet assay is a valuable method for detection of occupational and environmental exposures to genotoxicants in humans and can be used as a tool in risk assessment for hazard characterisation6,8,245,246 (Table 1.1). The DNAdamage assessed by the Comet assay gives an indication of recent exposure and at an early stage where it could also undergo repair247 and thus it provides an opportunity for intervention strategies to be implemented in a timely manner. The assay can be conducted in the same population after removal of genotoxicant/dietary intervention to detect the extent of reduction in DNA damage. The assay is a noninvasive technique compared to other DNA-damage techniques (chromosomal aberrations, micronucleus), which require larger samples (B2–3 ml) as well as a proliferating cell population (or cell culture). Human biomonitoring using the Comet assay is advantageous since it is rapid, cost effective, with easy compilation of data and concordance with cytogenetic assays.248 The assay has been widely used in studying DNA damage and repair in healthy individuals,3,194,249,250 in clinical studies31,251,252 as well as in dietary intervention studies,155,158,253–255 and in monitoring the risk of DNA damage resulting from occupational,161,256–258 environmental,187,259 oxidative DNA damage,177,260 exposures or lifestyle.185,261 White blood cells or lymphocytes are the most frequently used cell type for the Comet assay in human biomonitoring studies.248,262,263 However, other cells have also been used, e.g. buccal cells,264 nasal,265 sperm,191,266–268 epithelial269–271 and placental cells.272

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The Comet assay has been used as a test to predict the risk for development of diseases (renal cell carcinoma, cancers of the bladder, oesophagus and lung) due to susceptibility of the individual to DNA damage.149,273–275 The in vitro Comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates276 as well for neurotoxicity. Certain factors like age, diet, lifestyle (alcohol and smoking) as well as diseases have been shown to influence the Comet assay parameters and for interpretation of responses these factors need to be accounted for during monitoring human genotoxicity.277,278 Human biomonitoring studies using the Comet assay provide an efficient tool for measuring human exposure to genotoxicants, thus helping in risk assessment and hazard identification.

1.6 The Specificity, Sensitivity and Limitations of the Comet Assay The Comet assay has found worldwide acceptance for detecting DNA damage and repair in prokaryotic and eukaryotic cells. However, there are issues relating to the specificity, sensitivity and limitations of the assay that need to be addressed by genetic toxicologists before it gets accepted in the regulatory framework including interlaboratory validation of in vitro and in vivo Comet assay. The variability in the results of the Comet assay is largely due to its sensitivity and minor differences in the conditions of various laboratories as well as the effect of confounding factors in human studies (lifestyle, age, diet, interindividual and seasonal variation). Prospective cohort studies have not been conducted to find the predictive value of the Comet assay in human biomonitoring, further limiting its application.8 Cell to cell, gel to gel, culture to culture, animal to animal variability as well as use of various image-analysis systems or visual scoring279 and use of different Comet parameters, e.g. Olive tail moment and tail (%) DNA, are the other factors contributing to interlaboratory differences in the results. The limitation of the Comet assay is that it only detects DNA damage in the form of strand breaks. The alkaline (pH413) version of the assay assesses direct DNA damage or alkali-labile sites, while specific classes of DNA damage including base oxidation DNA adduct formation cannot be measured. The specific and sensitive detection of these lesions requires the use of lesion-specific enzymes.3 These enzymes are bacterial glycosylase/endonuclease enzymes, which recognise a particular type of damage and convert it into a break that can then be measured in the Comet assay. Hence, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts can be detected as an increased amount of DNA in the tail.8 Oxidised pyrimidines are detected with use of endonuclease III, while oxidised purines are detected with formamidopyrimidine DNA glycosylase (FPG). Modifications have been made in the protocol to specifically detect double-strand breaks (neutral Comet

29


281

assay ), single-strand breaks (at pH 12.1, ), DNA crosslinking (decrease in DNA migration due to crosslinks280) and apoptosis.280 The neutral Comet assay also helps to distinguish apoptosis from necrosis as evidenced by the increased Comet score in apoptotic cells and the almost zero Comet score in necrotic cells.282 An adaptation of the Comet assay was also developed that enables the discrimination of viable, apoptotic and necrotic single cells.283 Use of proteinase-K specifically removes DNA–protein crosslinking, leading to increased migration but would not affect the DNA–DNA crosslinking, thereby indicating a specific type of lesion.280 Tail (%) DNA and Olive tail moment give a good correlation in genotoxicity studies and since most studies have reported these Comet parameters, it has been recommended that both these parameters should be applied for routine use. Since the OTM is reported as arbitrary units and different image-analysis systems give different values, tail (%) DNA is a considered a better parameter.285 It is therefore required that the in vitro and in vivo testing be conducted according to the Comet assay guidelines, and appropriately designed multilaboratory international validation studies be carried out. Guidelines for the in vitro as well as in vivo Comet assay have been formulated.235,236 Recently, issues relating to study design and data analysis in the Comet assay were discussed by the International Workgroup on Genotoxicity Testing (IWGT), where particular attention was given to the alkaline version (pH413) of the in vivo Comet assay and recommendations were made for a standardised protocol, which would be acceptable to international agencies.237 It was decided that a single dose should be replaced with multiple dosing to avoid misinterpretation of data, isolated cells or nuclei could be used for the studies, cytotoxicity should be tested in the cells to prevent mechanisms of apoptosis/necrosis from interfering with the results, and scoring of comets could be carried out both manually as well as with image-analysis systems. Consensus was also reached on the need for an international validation study to stringently evaluate the reliability and accuracy of the in vivo Comet assay (as well as in vitro versions). These recommendations are also aimed at reducing the variability arising in interlaboratory studies. Since in vivo Comet assay has been accepted as the first tier screening assay for assessment of DNA damage in rodents by the Committee on Mutagenicity, UK,10 international validation studies are underway supported by the European Centre for Validation of Alternative Methods (ECVAM), Japanese Centre for Validation of Alternative Methods (JaCVAM), US Interagency Coordinating Committee on Validation of Alternative Methods (ICCVAM), US National Toxicology Program Interagency Centre for Evaluation of Alternative Toxicological Methods (NICEATM) and Japanese Environmental Mutagen Society.237 There has been only one multilaboratory validation study in the European countries that has been conducted to study the FPG sensitive sites and background level of base oxidation in DNA using the Comet assay, in human lymphocytes.284 It was found that half of the laboratories demonstrated a

30

Chapter 1

dose–response effect. However, many laboratories have carried out their own validation studies for DNA damage to optimise their research work.8 Moller263 has critically evaluated the published Comet assay data on human biomonitoring studies using blood cells from 22 countries and has established reference values for DNA damage. The large number of biomonitoring studies has indicated that the Comet assay is a useful tool for detecting exposure and its validation status as a biomarker in biomonitoring is dependent on its performance in cohort studies.8

1.7 Conclusions The Comet assay is now well established and its versatility has imparted a sensitive tool to the toxicologists for assessing DNA damage. This has been demonstrated with its wide applications in assessing genotoxicity in plant and animal models, both aquatic as well as terrestrial, in a variety of organisms, tissues and cell types. In vitro, in vivo, in situ and biomonitoring studies using the Comet assay have proved it to be a ‘‘Rossetta Stone’’ in the garden of genetic toxicology.

Acknowledgments The authors wish to thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India for funding through the Networked Projects (CMM0018 and NWP34) as well as the support from the UK-India Education and Research Initiative (UKIERI).

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257. V. Garaj-Vrhovac and D. Zeljezic, Assessment of genome damage in a population of Croatian workers employed in pesticide production by chromosomal aberration analysis, micronucleus assay and Comet assay, J. Appl. Toxicol., 2002, 22(4), 249–55. 258. R. J. Sra´m and B. Binkova´, Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens, 1997– 1999, Environ. Health Perspect., 2000, 108, 57–70. 259. M. E. Gutie´rrez-Castillo, D. A. Roubicek, M. E. Cebriań-Garcı´ a, A. De Vizcaya-Ruı´ z, M. Sordo-Cedenõ and P. Ostrosky-Wegman, Effect of chemical composition on the induction of DNA damage by urban airborne particulate matter, Environ. Mol. Mutagen., 2006, 47(3), 199–211. 260. D. Cavallo, C. L. Ursini, P. Bavazzano, C. Cassinelli, A. Frattini and B. Perniconi, et al., Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs, Ann. Occup. Hyg., 2006, 50(3), 211–8. 261. P. H. Avogbe, L. Ayi-Fanou, H. Autrup, S. Loft, B. Fayomi and A. Sanni, et al., Ultrafine particulate matter and high-level benzene urban air pollution in relation to oxidative DNA damage, Carcinogenesis, 2005, 26(3), 613–20. 262. J. Angerer, U. Ewers and M. Wilhelm, Human biomonitoring: state-ofthe-art, Int. J. Hyg. Environ. Health, 2007, 210(3–4), 201–28. 263. P. Moller, Assessment of reference values for DNA damage detected by the Comet assay in human blood cell DNA, Mutat. Res., 2006b, 612(2), 84–104. 264. Y. T. Szeto, I. F. Benzie, A. R. Collins, S. W. Choi, C. Y. Cheng and C. M. Yow, et al., A buccal cell model Comet assay: development and evaluation for human biomonitoring and nutritional studies, Mutat. Res., 2005, 578(1–2), 371–81. 265. P. Mussali-Galante, M. R. Avila-Costa, G. Pinõń-Zarate, G. Martı´ nezLevy, V. Rodrı´ guez-Lara and M. Rojas-Lemus, et al., DNA damage as an early biomarker of effect in human health, Toxicol. Ind. Health, 2005, 21(7–8), 155–66. 266. G. Delbes, B. F. Hales and B. Robaire, Effects of the chemotherapy cocktail used to treat testicular cancer on sperm chromatin integrity, J. Androl., 2007, 28(2), 241–51. 267. T. E. Schmid, B. Eskenazi, A. Baumgartner, F. Marchetti, S. Young and R. Weldon, et al., The effects of male age on sperm DNA damage in healthy non-smokers, Hum. Reprod., 2007, 22(1), 180–7. 268. N. P. Singh, C. H. Muller and R. E. Berger, Effects of age on DNA double-strand breaks and apoptosis in human sperm, Ferti. Steril., 2003, 80(6), 1420–30. 269. B. Graham-Evans, H. H. Cohly, H. Yu and P. B. Tchounwou, Arsenicinduced genotoxic and cytotoxic effects in human keratinocytes, melanocytes and dendritic cells, Int. J. Environ. Res. Public. Health, 2004, 1(2), 83–9.

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270. H. Emri, D. Schaefer, B. Held, C. Herbst, W. Zieger and I. Horkay, et al., Low concentrations of formaldehyde induce DNA damage and delay DNA repair after UV irradiation in human skin cells, Exp. Dermatol., 2004, 13(5), 305–15. 271. E. Rojas, M. Valverde, M. C. Lopez, I. Naufal, I. Sanchez and P. Bizarro, et al., Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single-cell gel electrophoresis assay, Mutat. Res., 2000, 468(1), 11–7. 272. K. Augustowska, Z. Magnowska, M. Kapiszewska and E. L. Gregoraszczuk, Is the natural PCDD/PCDF mixture toxic for human placental JEG-3 cell line? The action of the toxicants on hormonal profile, CYP1A1 activity, DNA damage and cell apoptosis, Human Exp. Toxicol., 2007, 26(5), 407–17. 273. X. Lin, C. G. Wood, L. Shao, M. Huang, H. Yang and C. P. Dinney, et al., Risk assessment of renal cell carcinoma using alkaline Comet assay, Cancer, 2007, 110(2), 282–8. 274. M. B. Schabath, M. R. Spitz, H. B. Grossman, K. Zhang, C. P. Dinney and P. J. Zheng, et al., Genetic instability in bladder cancer assessed by the Comet assay, J. Natl. Cancer Inst., 2003, 95(7), 540–7. 275. L. Shao, J. Lin, M. Huang, J. A. Ajani and X. Wu, Predictors of esophageal cancer risk: assessment of susceptibility to DNA damage using Comet assay, Genes, Chromosomes Cancer, 2005, 44(4), 415–22. 276. I. Witte, U. Plappert, H. de Wall and A. Hartmann, Genetic toxicity assessment: employing the best science for human safety evaluation part III: the Comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection, Toxicol. Sci., 2007, 97(1), 21–6. 277. D. Anderson, Factors that contribute to biomarker responses in humans including a study in individuals taking vitamin C supplementation, Mutat. Res., 2001, 480–481, 337–47. 278. P. Moller, L. E. Knudsen, S. Loft and H. Wallin, The Comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors, Cancer Epidemiol. Biomarkers Prev., 2000, 9(10), 1005–15. 279. L. Forchhammer, E. V. Brauner, J. K. Folkmann, P. H. Danielsen, C. Nielsen, A. Jensen, et al., Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the Comet assay with visual scoring. Mutagenesis. 2008, 23, 223–231. 280. N. P. Singh, Microgels for estimation of DNA-strand breaks, DNA protein crosslinks and apoptosis, Mutat. Res., 2000, 455(1–2), 111–27. 281. Y. Miyamae, K. Iwasaki, N. Kinae, S. Tsuda, M. Murakami and M. Tanaka, et al., Detection of DNA lesions induced by chemical mutagens by the single-cell gel electrophoresis (Comet) assay: relationship between DNA migration and alkaline conditions, Mutat. Res., 1997, 393, 107–13. 282. S. Yasuhara, Y. Zhu, T. Matsui, N. Tipirneni, Y. Yasuhara and M. Kaneki, et al., Comparison of Comet assay, electron microscopy, and


53

flow cytometry for detection of apoptosis, J. Histochem. Cytochem., 2003, 51(7), 873–85. 283. N. Morley, A. Rapp, H. Dittmar, L. Salter, D. Gould and K. O. Greulich, et al., UVA-induced apoptosis studied by the new apo/necro-Comet-assay which distinguishes viable, apoptotic and necrotic cells, Mutagenesis, 2006, 21(2), 105–14. 284. T. S. Kumaravel and A. N. Jha, Reliable Comet assay measurements for detecting DNA damage induced by ionizing radiation and chemicals, Mutat. Res., 2006, 605, 7–16. 285. C. M. Gedik and A. Collins, ESCODD (European Standards Committee on Oxidative DNA Damage). Establishing the background level of base oxidation in human lymphocytes DNA: results of an interlaboratory validation study, FASEB J., 2005, 19(1), 82–4.

SECTION II: VARIOUS PROCEDURES FOR THE COMET ASSAY

CHAPTER 2

Detection of Oxidised DNA Using DNA Repair Enzymes AMAYA AZQUETA*, SERGEY SHAPOSHNIKOV AND ANDREW R. COLLINS Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046 Blindern, 0316, Oslo, Norway

2.1 Introduction Cells are continuously exposed to endogenous and exogenous agents that damage DNA. One of the most common kinds of damage is oxidation. Normal cellular metabolism produces reactive oxygen species (ROS) that interact with the DNA creating oxidised bases. These ROS include hydrogen peroxide, superoxide anion radical, singlet oxygen, hydroxyl radical and nitric oxide. ROS are produced in several metabolic pathways related with cellular respiration, biosynthetic and biodegradation processes of normal intermediary metabolism, biotransformation of xenobiotics and activation of phagocytic cells by natural stimuli. There are about 60 enzymatic reactions that use O2 as a substrate where ROS are formed. Ionising or UV radiation and exogenous chemicals can also give rise to oxidised bases. UV together with certain chemicals can generate ROS or produce electron abstraction from bases creating oxidised bases in DNA and UVA causes a photodynamic generation of active oxygen species. A great variety of oxidised bases have been identified in nuclear DNA but 8-oxo-7,8-dihydroguanine (8-oxo-G) is one of the most abundant and readily formed oxidised DNA lesions. * Corresponding author Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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The oxidation of DNA is not only a consequence of the production of ROS; decreases in antioxidant defence and inhibition of repair of oxidative damage should also be taken into account. Xenobiotics can produce ROS, decrease antioxidant defences or inhibit the repair of oxidative damage. The presence of 8-oxo-G in DNA may lead to misincorporation of A unless repair occurs prior to DNA replication. Therefore, the presence of 8-oxo-G in cells may lead to transversion mutations. Mutations are a crucial step in carcinogenesis, and so elevated levels of DNA oxidation damage can increase the risk of developing malignancies. Since elevated levels of oxidised DNA bases have been detected in many tumours, it has been suggested that this kind of lesion plays an important role in the initiation, promotion and progression of tumours.1 However, we cannot be sure that the high levels of oxidised bases found in tumours are not simply a consequence of the tumour. Many of the data implicating ROS as a tumour initiator have been derived indirectly. On the other hand, the treatment of laboratory animals with carcinogenic agents causes oxidised base modifications in their target organs before the tumour is formed.2 So, the potential role of oxidative stress in carcinogenesis appears well established but the extent of the contribution has not been well defined. DNA oxidation damage has been associated with other diseases. Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have oxidative stress implicated in their pathogenesis.1 Hepatitis, atopic dermatitis, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.) as well as other diseases where inflammation is present are also associated with an oxidative state.1 The association between inflammation and oxidative stress is well documented. The high levels of oxidised bases present in patients infected with the human immunodeficiency virus (HIV) might influence the progression of the infection into acquired immunodeficiency syndrome (AIDS).2 Also, high levels of 8-oxo-G have been found in lesions of the aorta wall in atherosclerosis patients.2 ROS play a critical role in the etiology of defective sperm function and male infertility.3 Finally, one long-standing theory of aging proposes that aging occurs through the gradual accumulation of free-radical damage to biomolecules.4 Levels of oxidised bases in DNA are the consequence of a balance between lesion induction and repair. Oxidised bases are mostly repaired by the base excision repair pathway (BER; the removal of a single damaged base by a glycosylase), although the nucleotide excision repair pathway (NER; the removal of an oligonucleotide containing the lesion) may also play a role in the repair of some oxidised bases in DNA.5 The fact that there is more than one route to deal with 8-oxo-G is one more indication of the importance of the removal of this lesion that constitutes a serious threat to the integrity of the genome. The most important enzymes in the repair of purine-derived DNA lesions are the 8-oxoguanine-DNA glycosylases OGG1 and OGG2.6 There are also two more glycosylases that have been studied during recent years: MutY homologue (MYH) that removes misincorporated A and MutT homologue (MTH1) that removes dGTP that has become 8-oxo-dGTP.7,8 Several proteins, such as NTH-1 and NEIL glycosylases (Nei-like), are known to be involved in the repair of pyrimidine-derived DNA lesions.9

Detection of Oxidised DNA Using DNA Repair Enzymes

59

As a protection against the deleterious effects of ROS the organism produces antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase. These enzymes catalyse reactions that eliminate ROS. There are also some chemical compounds that induce a decrease of the levels of oxidised bases by scavenging ROS. Some of these compounds are synthesised in the organism (for example glutathione) and some are taken from food (e.g. vitamin C, carotenoids, flavonoids). The Comet assay is a simple, sensitive method for detecting DNA-strand breaks. Cells embedded in agarose on a microscope slide are lysed with detergent and 2.5 M NaCl to remove membranes and soluble cell constituents, including most histones, leaving nucleoids, in which supercoiled loops of DNA are attached to a nuclear matrix. A break in one strand of a DNA loop is enough to release the supercoiling, and during electrophoresis the relaxed loops are able to extend towards the anode. Fluorescence microscopy reveals comet-like images, where the relative tail intensity reflects the number of loops and therefore the break frequency. This assay (see Figure 2.1) was adapted to measure oxidised purines and oxidised pyrimidines by the incubation of the nucleoids with bacterial DNA repair enzymes.10 Formamidopyrimidine glycosylase (FPG) is used to detect oxidised purines, mostly 8-oxo-G, and endonuclease III (EndoIII; also known as Nth) is used to detect oxidised pyrimidines. In this chapter we will discuss the use of these enzymes in the Comet assay.

Figure 2.1

Schematic representation of the standard Comet assay (left), and including digestion with lesion-specific enzymes (right).

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Chapter 2

2.2 Methods for Measuring DNA Oxidation Damage Oxidation of DNA in eukaryotic cells is measured by a variety of laboratory approaches that are described as either direct or indirect. Direct approaches depend on advanced analytical methods, such as high-performance liquid chromatography (HPLC) coupled to electrochemical detection, gas chromatography-mass spectrometry (GC-MS), and HPLC linked to tandem mass spectrometry (HPLC-MS/MS). These methods require isolation of DNA for subsequent hydrolysis, fractionation by chromatography and detection of compounds. The indirect methods include those based on the detection of lesions in intact DNA structures. The conversion of base lesion to break in otherwise intact DNA is performed by bacterial lesion-specific repair enzymes. The methods include the Comet assay, alkaline elution and alkaline unwinding. A number of drawbacks associated with the application of the available ‘‘direct’’ analytical approaches are now identified.11,12 The HPLC approach with electrochemical detection allows accurate measurement of 8-oxo-G, but is limited to compounds having a low oxidation potential.13 In addition to the lack of versatility, the assay suffers from low sensitivity that means that large amounts of DNA are required for measuring low levels of DNA lesions. GC-MS14–16 is much more versatile and allows measurement of a large number of DNA lesions. However, significant DNA oxidation occurring during the assay has been reported.17,18 An improvement in the measurement of oxidative damage to DNA is expected with the HPLC-MS/MS technique, which combines the efficiency of HPLC separation together with the accuracy and versatility of tandem mass spectrometry.19 However, a significant level of DNA oxidation during the preparation of samples is still a problem. The alternative indirect approaches, based on the use of lesion-specific repair endonucleases that introduce strand breaks into DNA, have shown no evidence of artifactual DNA oxidation. The alkaline elution procedure20–22 involves lysis of the cells at high pH followed by elution of single-strand DNA through filters. Small DNA fragments elute differently from large fragments. The alkaline unwinding procedure involves adding to DNA an alkaline solution for a limited time, allowing partial DNA unwinding that is dependent on the frequency of breaks. The DNA is then neutralised, sonicated and single- and doublestranded DNA are separated on hydroxyl apatite columns followed by fluorescence detection.23 These two methods are noted for sensitivity, but on the other hand, they are labour intensive and time consuming. The Comet assay approach combined with repair enzymes is also labour intensive, particularly in the scoring, but it has become the method of choice, being simple and economical to perform, and having the advantage that damage is assessed at the level of individual cells. In the carefully controlled interlaboratory trials undertaken by ESCODD (the European Standards Committee on Oxidative DNA Damage), large differences in the measured levels of cellular 8-oxo-G were observed depending on the methods used. It was found that values for background levels of DNA


Figure 2.2

61

Illustration of the difference between a method with high precision but low accuracy, and a method with low precision but high accuracy (on average).

oxidation in human cells obtained with HPLC-based methods were about ten times higher than those obtained by the enzymatic assays.24,25 The difference is explained by high and variable background DNA oxidation that is occurring during the sample preparation process for chromatographic (direct) approaches. However, it would be misleading to draw the conclusion that these approaches are useless. Their advantage over enzymatic assays is their precision in identifying and measuring experimentally induced lesions and elucidating the mechanisms of DNA damage. For determining low levels of background damage, however, it is clearly preferable to use the imprecise but, on average, accurate enzymatic approach, rather than a chromatographic method that is very precise at measuring an artefact. This is illustrated figuratively in Figure 2.2.

2.3 Enzyme Specificity The conversion of base lesions to breaks in the Comet assay and other indirect enzymatic methods is normally performed by FPG and EndoIII, which are bacterial repair endonucleases that recognise different types of oxidative damage. FPG acts on oxidised purines, including 8-oxo-G, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FaPyG) and 4,6-diamino-5-formamidopyrimidine (FaPyA) and other ring-opened purines. EndoIII recognises oxidised pyrimidines, including thymine glycol and uracil glycol.26 A further complication arises because both FPG and EndoIII are able to break DNA at apurinic/apyrimidinic- (AP-)sites – the baseless sugars left as intermediates during base excision repair after glycosylases have removed the damaged base. AP-sites are alkali-labile, so in principle they are expected to appear among the strand breaks detected in the standard (alkaline) Comet

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assay. But it has not been convincingly demonstrated that all AP-sites are converted under these conditions. Recently, the mammalian analogue of FPG, OGG1, has been applied in the Comet assay. Substrate specificity of the three enzymes was compared by Smith et al.27 Human cells were treated with either methyl methanesulfonate (MMS) or ethylnitrosourea (ENU), to induce alkylation damage in DNA. After incubation with MMS, FPG revealed enzyme-sensitive sites in the DNA of cells; EndoIII produced about a quarter of the number of breaks seen with FPG, while OGG1 was ineffective. Treating the cells with ENU resulted in a similar increase in strand breaks with both FPG and EndoIII, but no increase in OGG1-recognised break sites, further supporting the suggestion that OGG1, in contrast to FPG and EndoIII, does not recognise damage – either alkylated bases or AP-sites – arising from treatment with alkylating agents. These results imply that even if additional strand breaks are induced by digestion with either FPG or EndoIII after treatment with an agent that has an unknown mode of action, they cannot necessarily be ascribed to oxidative damage. Thus, care must be taken when interpreting the results obtained with FPG and EndoIII. OGG1 might be more specific and might give more reliable estimates of oxidation damage; but a systematic comparison of OGG1 and FPG applied to measuring basal damage in, for instance, lymphocyte samples from a biomonitoring trial, has yet to be carried out. Meanwhile, FPG continues to be the enzyme of choice for oxidised purines, if only because it is readily obtained at high yield from an overproducing bacterial strain. When using these enzymes to measure oxidative DNA damage, the usual practice is to include a control slide (which is incubated with buffer alone) in parallel with the +enzyme slide, and to subtract the mean Comet score of the control from the mean score of the +enzyme slide. Net enzyme-sensitive sites are then the measure of the oxidised bases concerned.

2.4 Applications Applications of the enzyme-modified Comet assay include: studying the mechanisms of action of genotoxic chemicals; investigating oxidative damage as a factor in disease; monitoring oxidative stress in animals or human subjects resulting from exercise, or diet, or exposure to environmental agents; studying the effects of dietary antioxidants; and monitoring environmental pollution by studying sentinel organisms. Tests for genotoxicity of novel chemicals generally involve animal experiments and analysis of DNA-strand breaks in various tissues. It is unusual for such trials to include lesion-specific enzymes, which would in all probability show up additional damaging effects. However, as an alternative to in vivo animal experiments, cell culture model systems are increasingly employed – often making use of cells with a semblance of the xenobiotic metabolising activity of the corresponding normal cells in the organism. CaCo-2 cells, derived from a human colon carcinoma are a good example, resembling colon


63

epithelial cells in morphology and in their expression of cytochrome P450 (Cyp450) enzymes. Azqueta et al.28 recently tested a chemotherapy drug, a quinoxaline 1,4-di-Noxide derivative, with particularly strong activity in the hypoxic conditions of many tumours. Under both normal and hypoxic conditions, FPG- and EndoIII-sensitive sites in the DNA of CaCo-2 cells were increased by low concentrations of the drug. At the highest concentration tested, so many strand breaks were produced that the additional enzyme-sensitive sites were underestimated, since the assay was close to saturation. This is a potential problem that should be noted whenever total damage exceeds about 70% DNA in tail and the dose–response curve becomes nonlinear. The problem can be dealt with by using a calibration curve (see Section 2.5.4.12), but this is rarely done. Reeves et al.29 tested titanium dioxide nanoparticles for genotoxic effects in fish cells and found significant induction of FPG-sensitive sites but no increase in EndoIII-sites over control levels. Oxidised bases are found at elevated levels in the lymphocyte DNA from patients with various human diseases; whether as cause or effect is often not clear. We first used the Comet assay to investigate patients with insulin-dependent diabetes mellitus (type 1 diabetes).30 EndoIIIsensitive sites were significantly higher in diabetic patients compared with normal controls. FPG-sensitive sites significantly correlated with blood glucose concentration, implicating hyperglycemia as a possible cause of the high damage level. In type 2 diabetes patients, Pitozzi et al.31 found increased levels of FPG-sensitive sites in total leukocytes, but not in isolated lymphocytes, while Blasiak et al.32 found significantly elevated levels of oxidised bases in lymphocytes of type 2 diabetics. FPG-sensitive sites were elevated in lymphocytes of patients with ankylosing spondylitis (a rheumatoid disease associated with inflammation-induced oxidative stress).33 In hyperlipidemic patients (at risk of cardiovascular disease), although strand breaks were elevated compared with controls, no increase in base oxidation was seen.33 Mutlu-Tu¨rkog˘lu et al.34 compared patients with angiographically defined coronary artery disease with healthy controls, and claimed to find increased levels of oxidised bases. However, the data are presented as overall Comet scores with FPG or with EndoIII – without subtracting the DNA-strand breaks as measured in the incubation with buffer alone. The increases in overall damage are accounted for by an increase in strand breaks; net FPG- and EndoIII-sensitive sites are the same in controls and patients. Oxidised purines (FPG-sensitive sites)35 or both oxidised purines and pyrimidines36 were found to be elevated in the lymphocyte DNA of patients with Alzheimer’s disease. Morawiec et al.37 report elevated levels of oxidised bases in lymphocytes of children with Down’s Syndrome compared with normal children. FPG and EndoIII have been employed in several studies to investigate environmental or occupational exposure of humans to agents associated with oxidative stress. Male former asbestos factory workers had significantly higher levels of oxidised pyrimidines in lymphocyte DNA than nonexposed controls,38 while exposure to substitute man-made fibres (mineral wool) had no such

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effect. On taking 12 healthy subjects to high altitude (4559 m, for 3 days) an increase in EndoIII-sensitive sites was seen,40 but there was no induction of base oxidation by strenuous exercise, either at high altitude or at sea level. The enzyme-modified Comet assay has been widely used in human nutritional studies, particularly to investigate effects of antioxidant supplements or of antioxidant-rich foods. Generally, effects on endogenous base oxidation in lymphocyte DNA are seen only after several days or weeks of supplementation (whereas protection against H2O2-induced strand breakage is often seen after a single dose). The first such study tested a combined supplement of vitamin C, b-carotene and vitamin E, in groups of smokers and nonsmokers, and found, after 20 weeks, a significant decrease in EndoIII-sensitive sites in supplemented compared with placebo groups.41 EndoIII- and FPG-sensitive sites were measured by Møller et al.42 in a 4-week placebo-controlled trial of normal or slow release vitamin C capsules (plus vitamin E); oxidised bases were decreased only after the slow-release capsules. Three ‘‘doses’’ of a whole fruit (1, 2 or 3 kiwi fruits per day for 3 weeks) were tested in a randomised crossover trial,43 and significant decreases in both oxidised purines and oxidised pyrimidines were seen, though with no dependence on dose. In contrast, no effects on EndoIII- or FPG-sensitive sites were found in a parallel 3-week intervention trial with blackcurrant juice, an anthocyanin drink, or control.44 These are just a few examples: the topic is thoroughly reviewed by Møller and Loft.45 Several potential problems should be borne in mind when interpreting results or examining publications describing experiments with enzymes in the Comet assay, some of which have been mentioned above: the inability to measure additional enzyme-sensitive sites above a high level of DNA breaks; the fact that results are variously expressed as total damage (strand breaks plus enzyme-sensitive sites) or as net enzyme-sensitive sites; and doubts over the effectiveness of certain batches of enzyme. As a general rule, we find – in human lymphocytes – roughly equal numbers of strand breaks and of oxidised purines or pyrimidines (i.e. the enzyme digestion doubles the yield of breaks). If enzyme digestion only marginally increases the level of damage over that found in nonenzyme-treated controls, then it may be that the enzyme is ineffectual. Making sure that the enzymes are optimally active requires a simple titration experiment, as described below (Section 2.5.3.3).

2.5 Protocol 2.5.1 – – – – –

Equipment

Staining jars (vertical or horizontal) Water bath Microwave oven Incubator (37 1C/55 1C) Moist chamber (e.g. glass or plastic box with platform for slides above a layer of water). – Eletrophoresis tank (horizontal)


– Electrophoresis power supply – Fluorescence microscope – Equipment for cell culture and lymphocyte isolation

2.5.2

Supplies

Glass slides (frosted end) Coverslips (20 20 mm and 22 22 mm) Parafilm Metal plate Ice bucket Image analysis software (optional) Standard laboratory material (tubes, tips . . . ) Standard material for cell culture

2.5.3 2.5.3.1

Reagents, Buffers and Enzymes Reagents

Normal melting point agarose (NMP agarose) Low melting point agarose (LMP agarose) Phosphate-buffered saline (PBS) NaCl EDTA Tris base HEPES KCl Bovine serum albumin (BSA) NaOH KOH Glycerol Enzymes: FPG and EndoIII Reagents for cell culture and lymphocyte isolation

2.5.3.2

Solutions

NMP agarose: 1% in H2O Prepare a few hundred ml – enough for several hundred slides LMP agarose: 1% in PBS Store at 4 1C in small aliquots (10 ml) Lysis solution: 2.5 M NaCl 0.1 M EDTA 10 mM Tris base

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pH 10 (Adjust with a solution of NaOH) Store at 4 1C. Add 1% Triton X-100 just before use. Enzyme buffer: 40 mM HEPES 0.1 M KCl 0.5 mM EDTA 0.2 mg/ml BSA pH 8 (Adjust with a solution of KOH) Prepare at 10 times concentration and store in aliquots at 20 1C. For use, thaw, dilute in H2O and store at 4 1C. Electrophoresis solution: 0.3 M NaOH 1 mM EDTA Store at 4 1C. Can be used twice. Prepare it fresh each 2 weeks. DAPI (4 0 ,6-diamidino-2-phenylindole): 1 mg/ml in water Store in 1 ml aliquots at 20 1C Enzyme storage solution: Enzyme buffer with the addition of 10% glycerol just before use

2.5.3.3

Enzymes

FPG and EndoIII are commercially available for use in the Comet assay. If you get the enzymes from a company you should follow its protocol to use and store them. Nevertheless, it is always good practice to calibrate the enzymes in your experimental system by carrying out some dose–response experiments using different concentrations and different times of incubation. We work with crude extract from bacteria containing overproducing plasmid. So for each batch we look for the optimal concentration by carrying out titration experiments (see Figure 2.3). The enzymes should be stored as small aliquots in the enzyme storage solution to minimise thaw–freeze cycles. They are kept at 80 1C.

2.5.4 2.5.4.1

Procedure Cells

The Comet assay using enzymes has been applied to measure both endogenous DNA damage and damage induced by genotoxic agents in a wide range of cells including established cell lines, primary cultures and tissues. During recent years the alkaline Comet assay has been used on lymphocytes to monitor human exposure to genotoxic agents as a result of occupation, drug treatment, diseases or environmental pollution. Lymphocytes can be easily collected in a noninvasive way and are assumed to be representative of the overall status of the body. We have worked extensively with this type of cell and also with some tumour and nontumour cell lines such as HeLa cells (derived from human


Figure 2.3

67

Experiment to determine optimal conditions for digestion with FPG. HeLa cells were treated with 1 mM Ro 19-8022 and irradiated with visible light (500 W, 33 cm, 4.5 min on ice) to induce 8-oxo-G. They were embedded in agarose and lysed to produce substrate nucleoids for FPG. FPG was diluted 1 in 300 (), 1 in 3000 () and 1 in 30 000 (’) from the crude extract, and incubated with the substrate nucleoids for 15, 30 or 45 min before electrophoresis. FPG buffer was used as control (E). Comets were scored visually. Broken lines indicate the results of incubation with control nucleoids (without Ro 19-8022 treatment). Mean values are shown from two independent experiments.

cervical cancer cells), CaCo-2 cells (from colorectal adenocarcinoma), K562 (from human chronic myeloid leukaemia), HEF (human embryonic fibroblasts), HK-2 cells (a human renal proximal tubular epithelial cell line), V-79 cells (established from Chinese hamster lung fibroblasts), Vero cells (African green monkey kidney cells) and CHO (Chinese hamster ovary) cells. The number of cells needed for carrying out an experiment is calculated taking into account that B30 000 cells are used per slide (see below). After treatment with a damaging agent, cells should be maintained at 4 1C until the cells are embedded in agarose. Our experience with tissues is limited but we have been working successfully with normal prostate tissues and prostate tumour tissues. Once the tissue is disaggregated, the procedure is the same as for cultured cells or lymphocytes.

2.5.4.2

Precoating Slides

Carefully heat the 1% NMP agarose in the microwave oven until it is melted. Then place it in a water bath previously heated at 55 1C. When the agarose is at

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55 1C, dip a glass slide vertically in the solution. Cover the slide with agarose to half-way up the frosted part, drain off excess agarose and clean one side of the slide with tissue. Do not forget to label the side of the slide that is covered with the agarose for future reference, as when dry the agarose is difficult to see. Let slides dry at room temperature overnight. Slides can be stacked in boxes and stored indefinitely at room temperature. Special slides for performing the Comet assay can be bought from different companies. You can also work with GelBondTM plastic film instead of glass slides.

2.5.4.3

Preparation of Gels

Carefully heat the 1% LMP agarose in the microwave oven until it is melted. Then place it in a water bath previously heated at 37 1C for 10 min (to be sure the agarose has cooled). Add 140 ml of 1% LMP agarose and 30 ml of cell suspension (1 000 000 cells/ml in PBS) to a microcentrifuge tube. Mix gently (once up and down in a pipettor tip) and quickly place 2 drops of 70 ml on the precoated side of a slide; cover them with two coverslips of 20 20 mm and leave slides in the fridge for 5 min to allow the agarose to set. After this time remove the coverslips carefully (with the thumb gently press the coverslip and move it to one side). Prepare 4 slides per cell suspension and label them as: ‘‘lysis’’, ‘‘buffer’’, ‘‘FPG’’ and ‘‘EndoIII’’. 1% LMP agarose can be heated several times (eventually, by evaporation, it becomes too thick to use).

2.5.4.4

Lysis

Add 1 ml of Triton X-100 per 100 ml of lysis solution and mix thoroughly. The amount of lysis solution prepared depends on the number of slides and design of staining jar in which the lysis is done. Triton is viscous, so to dispense it accurately, use a pipette with a wide opening. Place the slide in the lysis solution (it does not matter if it is vertical or horizontal). Leave the slides in the lysis solution at 4 1C for at least 1 h. Lysis of the cells can be for between 1 and 24 h. We have not found any difference in DNA damage detected with different times of lysis but certain cell types may require more or less extended lysis.

2.5.4.5

Enzyme Treatment

Wash the slides labelled as ‘‘buffer’’, ‘‘FPG’’ and ‘‘EndoIII’’ 3 times at 4 1C, 5 min each time, with enzyme buffer. We use staining jars to carry out this step. Meanwhile, the enzyme should be prepared. Remove slides from the last wash, drain the excess with tissue and put them on a cold plate (such as a metal plate on ice). Place 30–50 ml of enzyme buffer, FPG enzyme solution or EndoIII enzyme solution onto the gels of the slides labelled as ‘‘buffer’’, ‘‘FPG’’ and


69

‘‘EndoIII’’, respectively. Cover with a coverslip of 22 22 mm. Then transfer the slides to a moist box and incubate them at 37 1C for 30 min. The concentration of enzyme on each gel and the time of incubation will depend on the protocol provided by the commercial source or the results of the titration experiments. Parafilm squares can be used instead of coverslips. They are not as heavy as coverslips so they will spread buffer or enzymes to a lesser extent when put on top of the gel. On the other hand, they are quite difficult to handle and bubbles are formed easily. Alternatively, slides can be immersed in enzyme solution in a shallow dish. The slide labelled as ‘‘lysis’’ is in the lysis solution during the incubation of the rest of the slides.

2.5.4.6

Alkaline Treatment

At the end of the incubation period, remove the coverslips and place the slides in an electrophoresis tank. If you are working with many slides, this step should be done at 4 1C to avoid different times of incubation from slide to slide. The slides should be covered with electrophoresis solution and left for 20 min at 4 1C. Do not forget the ‘‘lysis’’ slides!

2.5.4.7

Electrophoresis

Run the electrophoresis for 30 min with constant voltage setting, at around 0.8 V/cm (measured between the electrodes). The current is not critical, but about 300 mA is common. To adjust the amperage electrophoresis buffer should be added (to increase it) or removed (to decrease it).46

2.5.4.8

Neutralisation

Wash the slides for 10 min at 4 1C with PBS. After that wash for 10 min with water. We use staining jars to carry out this step. You can stain the slides at this time but the stain will be diluted in the remaining water. We recommend placing the slides to dry at room temperature. A further advantage of drying the slides is that the depth of nucleoids or Comets varies much less than in a wet gel so less refocusing is needed.

2.5.4.9

Staining

Place 20 ml of 1 mg/ml DAPI onto each gel and cover with a coverslip of 22 22 mm. Leave the slide a few min before viewing in a fluorescence microscope. We generally use DAPI but other DNA dyes such as Sybr Green, Sybr Gold, ethidium bromide, acridine orange or propidium iodide can be used.

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2.5.4.10

Quantitation

After electrophoresis, the image of a nucleoid with damage is similar to the stellar comets. The comet tail is formed by the broken DNA loops extending toward the anode. Quantitation is done comet by comet. There are two ways of quantitation: using analysis software or visually. The analysis software will calculate different parameters for each comet. The investigator should select each comet to be analysed. The most common parameters are described below: – Tail length is the distance of DNA migration from the centre or the edge of the head, to the end of the tail. – Percentage of DNA in head is the intensity of the head compared with the intensity of the whole comet – Percentage of DNA in tail is the intensity of the tail compared with the intensity of the whole comet. – Olive tail moment is defined as the product of the tail length and the fraction of total DNA in the tail. Olive tail moment and percentage of DNA in tail are the most used. Although, in theory, tail moment combines information on tail length and tail intensity, in practice information is lost since we are unable to visualise the comets being referred to. On the other hand, to use the percentage of DNA in the tail is more informative and very easy to interpret.47 Comets can be scored visually without using analysis software, classifying them as belonging to one of five classes according to the tail intensity. Each comet class is given a value between 0 and 4: (0) ¼ undamaged and (4) ¼ maximum damage. Typical comets of each class are shown in Figure 2.4. The parameter ‘‘total comet score’’ (TCS) is calculated from this classification and measured in arbitrary units. The TCS is calculated by the following equation: ð% of cells in class 0Þ 0 þ ð% of cells in class 1Þ 1 þ ð% of cells in class 2Þ 2 þ ð% of cells in class 3Þ 3 þ ð% of cells in class 4Þ 4:

Consequently, the total score is in the range from 0 to 400 arbitrary units. A TCS of 0 means that all comets have been classified in class 0 (undamaged cells) and a TCS of 400 means that all comets have been classified in class 4 (maximum damage). Nevertheless, a TCS of, say, 250 can have different interpretations depending on the frequencies obtained in different classes, so it is important to specify if there is homogeneity or not in the classification of the comets. In some cases it is useful to show the actual distribution of comet classes. Up to 100 comets per slide should be scored using analysis software or visual scoring. Both methods have their advantages and disadvantages. Software is quite expensive and it takes a long time to analyse each slide. On the other hand it is an objective method in so far as the results obtained in different laboratories


Figure 2.4

71

Typical comets from lymphocytes treated with H2O2 to induce breaks. Damage categories are indicated.

can readily be compared. However, in practice human intervention is often necessary when comets are misrecognised. Visual scoring does not need any software and for a trained person it is extremely fast, but it is difficult to compare the results obtained by different people unless the scoring is calibrated. In any case, the correlation between the analysis with software and the visual scoring is very good (see Figure 2.5). Fully automatic scoring systems are available commercially, in which the computer will analyse all the comets in a defined zone without any kind of manipulation. But they are very expensive and not yet fully validated.

2.5.4.11

Interpretation of Results

Different things are measured with the 4 slides. The slide labelled as ‘‘lysis’’ reveals the strand breaks (SBs) (including alkali-labile sites) in cell DNA. The one labelled as ‘‘buffer’’ reveals these SBs plus any further breaks induced by incubation with the reaction buffer for 30 min at 37 1C (normally only a small increase is seen). This slide is the control slide for the ones incubated with enzymes that, in addition, reveal the oxidised purines or pyrimidines detected by FPG or EndoIII, respectively. So, to determine the SBs induced by the enzymes, i.e. the net enzyme-sensitive sites, the difference between the value obtained after the digestion with each enzyme and with the enzyme buffer should be calculated. As an example: HeLa cells were treated on ice with different concentrations of the photosensitiser Ro 19-8022 plus 500 W visible light (at 30 cm) for 9 min to induce 8-oxo-G.48 Then, the Comet assay was performed using FPG as described

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Figure 2.5

Chapter 2

Comparison of visual scoring and image analysis. Slides prepared from HeLa cells and lymphocytes with varied amounts of damage were analysed in parallel using visual scoring and Comet IV software (Perceptive Instruments). Total comet score (TCS) is shown as arbitrary units (AU).

above, so 3 slides were prepared per condition: ‘‘lysis’’, ‘‘buffer’’ and ‘‘FPG’’. 100 comets per condition were scored visually and the TCS was calculated. To calculate the FPG-sensitive sites we subtracted the TCS obtained in the slides incubated with buffer from the slides incubated with FPG (Table 2.1).

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Table 2.1

C0.2 0.5 1

Figure 2.6

TCS of HeLa cells treated with Ro plus light. C- represents cells irradiated for 9 min with light; 0.2, 0.5 and 1 represent cells treated with 0.2, 0.5 and 1 mM of Ro plus 9 min of light. FPG-sensitive sites are calculated as FPG minus Buffer. Lysis

Buffer

FPG

FPG-sensitive sites

37 30 46 90

27 20 67 92

116 226 332 390

89 206 265 298

SBs and FPG-sensitive sites induced in HeLa cells by different concentrations of Ro 19-8022 plus light.

Figure 2.6 shows that there is a clear dose-response in net FPG-sensitive sites and also – though to much less an extent – in SBs. Note, however, the evident saturation effect at the highest dose of Ro 19-8022, where the level of damage is apparently only slightly higher than with a dose half as great.

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Figure 2.7

Comet assay calibration curves obtained with human lymphocytes and HeLa cells using ionising radiation to induce DNA breaks. Cells were X-irradiated on ice after embedding in agarose. (From Collins et al.46)

2.5.4.12

Calibration of the Assay

Sometimes it is necessary, or informative, to know the actual frequency of enzyme-sensitive sites per 109 Da of DNA or per cell. In that case it is necessary to calibrate the assay. Cells treated with different doses of ionising radiation have been used to calibrate the assay. Ahnstro¨m and Erixon,49 using alkaline sucrose sedimentation, showed that 1 Gy of X- or g-irradiation introduces 0.31 breaks per 109 Da of cellular DNA, which is close to 1000 breaks per diploid mammalian cell. Figure 2.7 shows an example of calibration curves for the alkaline Comet assay with damage dose expressed in terms of Gy. Although calibration curves from different laboratories are not all in agreement (probably because of differences in protocols) it is accepted that the range of damage detectable with the Comet assay is, roughly, from 0.2 to 10 Gy equivalents, or from 0.06 to 3 breaks per 109 Da.46 So, we can detect from about one hundred to several thousand breaks per cell.

Acknowledgment We acknowledge the support of EC contract LSHB-CT-2006-037575 (COMICS).


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References 1. M. S. Cooke, M. D. Evans, M. Dizdaroglu and J. Lunec, Oxidative DNA damage: mechanisms, mutation, and disease, FASEB. J., 2003, 17, 1195. 2. R. Olinski, D. Gackowski, M. Foksinski, R. Rozalski, K. Roszkowski and P. Jaruga, Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome, Free Radic. Biol. Med., 2002, 33, 192. 3. H.-M. Shen and C.-H. Ong, Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility, Free Radic. Biol. Med., 2000, 28, 529. 4. D. Harman, Aging: a theory based on free radical and radiation chemistry, J. Gerontol., 1956, 11, 298. 5. J. T. Reardon, T. Bessho, H. C. Kung, P. H. Bolton and A. Sancar, In vitro repair of oxidative DNA damage by human nucleotide excision repair system: possible explanation for neurodegeneration in xeroderma pigmentosum patients, Proc. Natl. Acad. Sci. USA, 1997, 94, 9463. 6. T. K. Hazra, J. W. Hill, T. Izumi and S. Mitra, Multiple DNA glycosylases for repair of 8-oxoguanine and their potential in vivo functions, Prog. Nucleic Acid Res. Mol. Biol., 2001, 68, 193. 7. H. Bai, S. Grist, J. Gardner, G. Suthers, T. M. Wilson and A.-L. Lu, Functional characterization of human mutY homolog (hMYH) missense mutation (R231l) that is linked with hMYH-associated polyposis, Cancer Lett., 2007, 250, 74. 8. Y. Nakabeppu, Molecular genetics and structural biology of human MutT homolog, MTH1, Mutat. Res., 2001, 477, 59. 9. T. K. Hazra, A. Das, S. Das, S. Choudhury, Y. W. Kow and R. Roy, Oxidative DNA damage repair in mammalian cells: a new perspective, DNA Repair (Amst.), 2007, 6, 470. 10. A. R. Collins, S. J. Duthie and V. L. Dobson, Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis, 1993, 14, 1733. 11. J. Cadet, T. Douki and J. L. Ravanat, Artifacts associated with the measurement of oxidized DNA bases, Environ. Health Perspect, 1997, 105, 1034. 12. A. Collins, J. Cadet, B. Epe and C. Gedik, Problems in the measurement of 8-oxoguanine in human DNA, Report of a Workshop, DNA oxidation, Aberdeen, UK, 19–21 January, 1997, Carcinogenesis, 1997, 18, 1833. 13. H. Kasai, P. F. Crain, Y. Kuchino, S. Nishimura, A. Ootsuyama and H. Tanooka, Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair, Carcinogenesis, 1986, 7, 1849. 14. M. Dizdaroglu, Application of capillary gas chromatography-mass spectrometry to chemical characterization of radiation-induced base damage of DNA: implications for assessing DNA repair processes, Anal. Biochem., 1985, 144, 593.

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15. M. Dizdaroglu, Gas chromatography–mass spectrometry of free radicalinduced products of pyrimidines and purines in DNA, Meth. Enzymol., 1990, 193, 842. 16. M. Dizdaroglu, Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry, FEBS Lett., 1993, 315, 1. 17. J. L. Ravanat, R. J. Turesky, E. Gremaud, L. J. Trudel and R. H. Stadler, Determination of 8-oxoguanine in DNA by gas chromatography–mass spectrometry and HPLC–electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography–mass spectrometry assay, Chem. Res. Toxicol., 1995, 8, 1039. 18. T. Douki, T. Delatour, F. Bianchini and J. Cadet, Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EIMS method, Carcinogenesis, 1996, 17, 347. 19. J. Cadet, T. Douki, S. Frelon, S. Sauvaigo, J. P. Pouget and J. L. Ravanat, Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement, Free Radic. Biol. Med., 2002, 33, 441. 20. B. Epe, DNA damage profiles induced by oxidizing agents, Rev. Physiol. Biochem. Pharmacol., 1996, 127, 223. 21. M. Pflaum, O. Will and B. Epe, Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases, Carcinogenesis, 1997, 18, 2225. 22. M. Pflaum, O. Will, H. C. Mahler and B. Epe, DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation, Free Radic. Res., 1998, 29, 585. 23. A. Hartwig, H. Dally and R. Schlepegrell, Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding, Toxicol. Lett., 1996, 88, 85. 24. ESCODD, Measurement of DNA oxidation in human cells by chromatographic and enzymic methods, Free Radic. Biol. Med., 2003, 34, 1089. 25. ESCODD, Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study, FASEB J., 2005, 19, 82. 26. S. S. David and S. D. Williams, Chemistry of glycosylases and endonucleases involved in base-excision repair, Chem. Rev., 1998, 98, 1221. 27. C. C. Smith, M. R. O’Donovan and E. A. Martin, hOGG1 Recognizes oxidative damage using the Comet assay with greater specificity than FPG or ENDOIII, Mutagenesis, 2006, 21, 185. 28. A. Azqueta, L. Arbillaga, G. Pachon, M. Cascante, E. E. Creppy and A. Lopez de Cerain, A Quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment, Chem. Biol. Interact., 2007, 168, 95. 29. J. F. Reeves, S. J. Davies, N. J. Dodd and A. N. Jha, Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells, Mutat. Res., 2008, 640, 113.


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30. A. R. Collins, K. Raslova, M. Somorovska, H. Petrovska, A. Ondrusova, B. Vohnout, R. Fabry and M. Dusinska, DNA damage in diabetes: correlation with a clinical marker, Free Radic. Biol. Med., 1998, 25, 373. 31. V. Pitozzi, L. Giovannelli, G. Bardini, C. M. Rotella and P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes, Mutat. Res., 2003, 529, 129. 32. J. Blasiak, M. Arabski, R. Krupa, K. Wozniak, M. Z. Zadrozny, J. Kasznicki, M. Zurawska and J. Drzewoski, DNA damage and repair in type 2 diabetes mellitus, Mutat. Res., 2004, 554, 297. 33. M. Dusinska, J. Lietava, B. Olmedilla, K. Raslova, S. Southon, A. R. Collins, Indicators of oxidative stress, antioxidants and human health in Antioxidants in Human Health, eds. T. K. Basu, N. J. Temple, M. L. Garg, CAB International, 1999, p. 411. 34. U. Mutlu-Tu¨rkog˘lu, Z. Akalin, E. Ilhan, E. Yilmaz, A. Bilge, Y. NiSanci and M. Uysal, Increased plasma malondialdehyde and protein carbonyl levels and lymphocyte DNA damage in patients with angiographically defined coronary artery disease, Clin. Biochem., 2005, 38, 1059. 35. M. Morocz, J. Kalman, A. Juhasz, I. Sinko, A. P. McGlynn, C. S. Downes, Z. Janka and I. Rasko, Elevated levels of oxidative DNA damage in lymphocytes from patients with Alzheimer’s disease, Neurobiol. Aging, 2002, 23, 47. 36. E. Kadioglu, S. Sardas, S. Aslan, E. Isik and A. Esat Karakaya, Detection of oxidative DNA damage in lymphocytes of patients with Alzheimer’s disease, Biomarkers, 2004, 9, 203. 37. A. Morawiec, K. Janik, M. Kowalski, T. Stetkiewicz, J. Szaflik, A. MorawiecBajda, A. Sobczuk and J. Blasiak, DNA damage and repair in children with Down’s syndrome, Mutat. Res., 2008, 637, 118. 38. M. Dusinska, A. R. Collins, A. Kazimirova, M. Barancokova, V. Harrington, K. Volkovova, M. Staruchova, A. Horska, L. Wsolova and A. Kocan, Genotoxic effects of asbestos in humans, Mutat. Res., 2004, 553, 91. 39. M. Dusinska, M. Barancokova, A. Kazimirova, V. Harrington, K. Volkovova, M. Staruchova, A. Horska, L. Wsolova and A. R. Collins, Does occupational exposure to mineral fibres cause DNA or chromosome damage?, Mutat. Res., 2004, 553, 103. 40. P. Møller, S. Loft, C. Lundby and N. V. Olsen, Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans, FASEB J., 2001, 15, 1181. 41. S. J. Duthie, A. Ma, M. A. Ross and A. R. Collins, Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes, Cancer Res., 1996, 56, 1291. 42. P. Møller, M. Viscovich, J. Lykkesfeldt, S. Loft, A. Jensen and H. E. Poulsen, Vitamin C supplementation decreases oxidative DNA damage in mononuclear blood cells of smokers, Eur. J. Nutr., 2004, 43, 267.

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43. A. R. Collins, V. Harrington, J. Drew and R. Melvin, Nutritional modulation of DNA repair in a human intervention study, Carcinogenesis, 2003, 24, 511–515. 44. P. Møller, S. Loft, G. Alfthan and R. Freese, Oxidative DNA damage in circulating mononuclear blood cells after ingestion of blackcurrant juice or anthocyanin-rich drink, Mutat. Res., 2004, 551, 119. 45. P. Møller and S. Loft, Dietary antioxidants and beneficial effect on oxidatively damaged DNA, Free Radic. Biol. Med., 2006, 41, 388. 46. A. R. Collins, A. Azqueta, G. Brunborg, I. Gaivao˜, L. Giovannelli, M. Kruszewski, C. C. Smith and R. Sˇtı` tina, The Comet assay: topical issues, Mutagenesis, 2008, 23, 143. 47. A. R. Collins, The Comet assay for DNA damage and repair: principles, applications, and limitations, Biotechnol., 2004, 26, 249. 48. O. Will, E. Gocke, I. Eckert, I. Schulz, M. Pflaum, H. C. Mahler and B. Epe, Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022, Mutat. Res., 1999, 435, 89. 49. G. Ahnstro¨m and K. Erixon, Measurement of strand breaks by alkaline denaturation and hydroxyl apatite chromatography, in: DNA Repair A Laboratory Manual of Research Procedures, eds. E. C. Friedberg, P. C. Hanawalt, Marcel Dekker, New York, 1981, p. 403.

CHAPTER 3

Microplate-Based Comet Assay ELIZABETH D. WAGNER AND MICHAEL J. PLEWA Department of Crop Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, 366 NSRC; MC-635, 1101 West Peabody Drive, Urbana, IL 61801, USA

3.1 Introduction The Comet assay, also known as the single-cell gel electrophoresis or SCGE assay, is a molecular genetic assay that can quantitatively measure the level of genomic DNA damage induced in individual nuclei of cells.1,2 The Comet assay was first introduced by O¨stling and Johanson in 1984 as a microelectrophoretic technique to directly visualise DNA damage in the nuclei of single cells.3 In 1988 a significant improvement of the assay was developed by Singh and his colleagues in that the electrophoresis was conducted under alkaline (pH413) conditions.4 Under these high alkaline conditions increased DNA migration was directly correlated with increased levels of DNA single-strand breaks (SSB), single-strand breaks associated with incomplete excision repair sites, and alkalilabile sites (ALS). Because almost all genotoxic agents induce orders of magnitude more SSB and/or ALS than DNA double-strand breaks (DSB), this version of the assay offered greatly increased sensitivity for identifying genotoxins. Questions have arisen whether the Comet assay can differentiate between apoptosis-induced DNA fragmentation and induced genotoxic damage.5 However, studies have supported the general consensus that the Comet assay, under proper conditions, accurately measures DNA-strand breaks associated with genotoxic insult.6,7


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3.2 Microplate Comet Assay Among the manifold advantages of the Comet assay is the ability to evaluate very limited amounts of test agents. In many cases compounds for a research study are not commercially available and the chemicals must be individually synthesised at great expense and with large amounts of time involved. In this chapter we shall describe some of our work with a series of high-priority nitrogen-containing drinking-water disinfection byproducts (DBPs) that were synthesised as highpurity chemical analytical standards. The use of 96-well microplates allowed us to analyse the genotoxicity of these DBPs in mammalian cells.

3.3 Drinking-Water Disinfection Byproducts The drinking-water community provides an important public health service by its generation of high-quality, safe and palatable tap water using chemical disinfectants such as chlorine, chloramines, ozone and chlorine dioxide.8 These disinfectants are oxidants that convert naturally occurring and synthetic organic material, bromide, and iodide in the source water into chemical disinfection byproducts (DBPs). DBPs, first discovered over 30 years ago, are an unintended consequence of water disinfection.9,10 DBPs represent an important class of environmentally hazardous chemicals that carry long-term human health implications; some of the over 600 DBPs identified to date are regulated by the US Environmental Protection Agency (US EPA) and regulatory agencies throughout the world.8,11–16 Although drinking-water disinfection was a major public health advancement of the twentieth century, epidemiological studies demonstrated that individuals who consume disinfected drinking water have an elevated risk of cancer.17–24 DBPs have been linked to adverse reproductive and developmental effects, including the induction of spontaneous abortions in humans.25–32 Two earlier pioneering studies, under the auspices of the US EPA, established the need for a quantitative, comparative analysis of the cytotoxicity and genotoxicity of emerging DBPs. The US EPA reported a mechanism-based structure–activity relationships (SAR) analysis for the carcinogenic potential ranking of DBPs.33 A list of priority DBPs was generated based on this ranking that met the criteria of (i) being detected in actual drinking water samples, (ii) having insufficient cancer bioassay data for risk assessment, and, (iii) having structural features/alerts or short-term predictive assays indicative of carcinogenic potential. The priority DBPs include iodinated trihalomethanes and other halomethanes, haloacids, haloacetonitriles, haloketones, halonitromethanes, haloaldehydes, halogenated furanones, haloamides, and nonhalogenated carbonyls. The US EPA Nationwide Occurrence Study included this list of more than 50 priority DBPs as well as currently regulated DBPs.34 This landmark study generated quantitative occurrence information for new and emerging DBPs for prioritising future health effects studies. The decreasing availability of pristine source waters fostered by population growth is encouraging utilities to exploit waters impaired by agricultural runoff

Microplate-Based Comet Assay

81

or wastewater effluents. Whether via direct dissolved organic nitrogen (DON) inputs from wastewater effluents or via algal blooms fostered by inorganic nitrogen loadings from agricultural runoff, such waters often feature higher DON concentrations that serve as precursors for nitrogen-based DBPs (N-DBPs). With the increased occurrence of N-DBPs, a comparative investigation of the genotoxicity of N-DBP chemical classes is warranted.35 As an example of the utility of the microplate Comet assay we chose to investigate and quantitatively compare the N-DBP classes of the haloacetonitriles36 and the haloacetamides.37 Most of these agents were not commercially available and small amounts of the compounds were synthesised for the research project by contracting with individual chemical companies (Table 3.1).

3.4 Chinese Hamster Ovary Cells Chinese hamster ovary (CHO) cells are widely used in toxicology. The transgenic CHO cell line AS5238,39 was derived from the parental CHO line, K1BH4.40,41 We isolated clone 11-4-8 from AS52; it expresses a stable chromosome complement, a consistent cell-doubling time as well as functional p53 protein.42–44 Stock cultures of the CHO cells were frozen in a solution of 90% fetal bovine serum (FBS):10% dimethylsulfoxide (DMSO) (v/v) and stored at 80 1C. Cells were grown on glass culture plates in Hams F12 medium plus 5% FBS at 37 1C in a humidified atmosphere of 5% CO2 (Figure 3.1). The cells exhibited normal morphology, expressed cell-contact inhibition and grew as a monolayer without expression of neoplastic foci. The CHO cells were transferred when cultures became confluent.

3.5 CHO Cell Microplate Comet Assay Protocol 3.5.1

CHO Cell Treatment

The day before treatment, 4 104 CHO cells were added to each microplate well in 200 mL of F12+5% FBS and incubated overnight. The next day the cells were washed with Hank’s balanced salt solution (HBSS) and treated with a series of concentrations of a specific DBP in F12 medium without FBS in a total volume of 25 mL for 4 h at 37 1C, 5% CO2. By using cells that grow as a monolayer attached to the bottom of the microplate well and by using a treatment volume of 25 mL, the absolute amount of the test compound consumed by the experiment is small. This is an important benefit of using microplates with adherent cell cultures. The wells were covered with sterile AlumnaSealt to prevent evaporation of the medium or the dispersal of volatile DBPs. Within each experiment a negative control, a positive control (3.8 mM ethylmethanesulfonate, EMS) and 9 concentrations of a specific DBP were conducted concurrently. After incubation the cells were washed twice with HBSS and harvested with 50 mL of 0.01% trypsin+53 mM EDTA at 37 1C. The trypsin solution was inactivated with 70 mL of F12+FBS. To measure acute

CAS number 590-17-0 83463-62-1 107-14-2 3252-43-5 3018-12-0 624-75-9 545-06-2 683-57-8 62872-34-8 98137-00-9 62872-36-0 79-07-2 62872-35-9 598-70-9 855878-13-6 683-72-7 5875-23-0 144-48-9 594-47-8 594-65-0

Chemical formula

C2H2NBr C2HNBrCl C2H2NCl C2HNBr2 C2HNCl2 C2H2NI C2NCl3 C2H4BrNO C2H3BrClNO C2H2BrCl2NO C2H3BrINO C2H4ClNO C2H3ClINO C2H3Br2NO C2H2Br2ClNO C2H3Cl2NO C2H3I2NO C2H4INO C2H2Br3NO C2H2Cl3NO

Bromoacetonitrile Bromochloroacetonitrile Chloroacetonitrile Dibromoacetonitrile Dichloroacetonitrile Iodoacetonitrile Trichloroacetonitrile Bromoacetamide Bromochloroacetamide Bromodichloroacetamide Bromoiodoacetamide Chloroacetamide Chloroiodoacetamide Dibromoacetamide Dibromochloroacetamide Dichloroacetamide Diiodoacetamide Iodoacetamide Tribromoacetamide Trichloroacetamide

N-DBPs analysed in the microplate Comet assay.

Compound

Table 3.1

119.95 154.39 75.50 198.84 109.94 166.95 144.39 137.96 172.41 206.85 263.86 93.51 219.41 216.86 251.31 127.96 310.85 184.96 295.75 162.40

Mol.weight 97 4 95 4 99 97 4 99 98 98 98 4 99 4 95 85 4 95 4 95 4 95 4 95 98 4 99 4 97 4 95 99

Purity(%)

Chem. Service Chem. Service Chem. Service Chem. Service Chem. Service CanSyn Chem. Co. Aldrich Chem. Co. US EPA CanSyn Chem. Co. CanSyn Chem. Co. CanSyn Chem. Co. US EPA CanSyn Chem. Co. CanSyn Chem. Co. CanSyn Chem. Co. US EPA CanSyn Chem. Co. Sigma Chem. Co. CanSyn Chem. Co. US EPA

Source

82 Chapter 3


Figure 3.1

83

Chinese hamster ovary (CHO) cells, line K1-BH4, AS52, clone 11-4-8 growing as a monolayer in Hams F12 medium plus 5% fetal bovine serum at 37 1C in a humidified atmosphere of 5% CO2.

cytotoxicity, a 10 mL aliquot of cell suspension was added to individual wells of a second 96-well microplate. This cell suspension was mixed with 10 mL of 0.05% trypan blue vital dye in phosphate-buffered saline (PBS).45 Approximately 100–200 cells of each suspension were analysed immediately with a microscope. The per cent survival for each treatment group was determined by counting the dead cells (dark) and the live cells (clear) (Figure 3.2). The Comet data were not used if the acute cytotoxicity for a specific treatment group exceeded 30%.

3.5.2

Preparation of Comet Microgels

Prior to the experiment, clear microscope slides were coated with a layer of molten 1% normal melting point agarose prepared with deionised water and dried overnight. When the treatment period was completed, the cell suspension from each microplate well was mixed with an equal volume of a molten (42 1C) solution of 1% low melting point agarose (LMA) prepared with PBS. The microplate was kept at 42 1C; 90 mL from the microplate well were removed and placed on a coated microscope slide, and a 24 50 mm cover glass was placed on the cell suspension. Two microgels were prepared from each microplate well. The microgels were placed on a metal surface at 4 1C for 5 min. After the microgels solidified, the cover glasses were carefully removed and a final layer of 0.5% LMA solution prepared in PBS was placed upon the previous layers. The microgels were cooled for 5 min on a metal surface at 4 1C after which the cover glasses were removed. The membranes of the embedded cells were removed by overnight immersion in lysing solution at 4 1C (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% sodium sarcosinate, 1% Triton X-100,

84

Chapter 3

Figure 3.2

Measurement of acute cytotoxicity using trypan blue vital dye after treatment of CHO cells in the microplate Comet assay. The per cent survival for each treatment group was determined by counting the dead cells (dark) and the live cells (clear).

and 10% DMSO). The slides were placed in an alkaline buffer (1 mM Na2EDTA, 300 mM NaOH, pH413.5) in an electrophoresis tank and the DNA was denatured for 20 min at 4 1C. The microgels were electrophoresed at 25 V, 300 mA (0.72 V/cm) for 40 min at 4 1C. The microgels were removed from the tank, neutralised with Tris buffer, pH 7.5, rinsed in cold water, dehydrated in cold methanol, dried at 50 1C and stored at room temperature in a covered slide box until microscopically examined.

3.5.3

Comet Microscopic Examination

For microscopic examination, the microgels were hydrated in cold water for 20–30 min and stained with 65 mL of ethidium bromide (20 mg/mL) for 3 min. The microgels were rinsed in cold water; a cover glass was placed on the gel and the nuclei were analysed with a Zeiss fluorescence microscope with an excitation filter of 546/10 nm and a barrier filter of 590 nm. For each experiment 2 microgels were prepared per treatment group. Twenty-five randomly chosen nuclei were analysed in each microgel using a charged coupled device camera. A computerised image-analysis system (Komet version 3.1, Kinetic Imaging Ltd., Liverpool, UK) was employed to determine the tail moment of the nuclei (integrated value of migrated DNA density multiplied by the migration distance) and the % tail DNA (the amount of DNA that migrated into the gel from the nucleus) as measures of DNA damage. The digitised data were automatically transferred to a computer-based spreadsheet for subsequent

85


statistical analysis. In general, the experiments were repeated three times for each DBP.

3.5.4

Normalisation of CHO Cell Comet Data and Statistical Analysis

The Comet tail moment and the % tail DNA for each nucleus analysed was generated using the Komet 3.1 software. The median tail moment value for each microgel was calculated and transferred to a data spreadsheet. The mean % tail DNA value for each microgel was also calculated and transferred to a spreadsheet. In addition, the acute cytotoxicity of the treated cells was entered into the same data spreadsheet. A summary sheet of all the microgels for a specific DBP was prepared with the average median tail moment values and the average mean % tail DNA values. An example for bromoiodoacetamide is presented in Table 3.2. A positive control of 3.8 mM ethyl methanesulfonate was included with each experiment (data not shown). Within the bromoiodoacetamide concentration range that allowed for 70% or greater viable cells, a concentration–response curve was generated (Figure 3.3). The data were plotted and a regression analysis was used to fit the curve (r2 ¼ 0.98). The SCGE genotoxic potency value was calculated as the midpoint of the curve within the concentration range that expressed above 70% cell viability. In the case of bromoiodoacetamide, the SCGE genotoxic potency value is 72.1 mM (Figure 3.3). The SCGE genotoxic potency value is useful in that it expresses the relative genotoxicity of a test compound as a single value. This allows for direct comparisons of the genotoxicity of a series of compounds that have been analysed under identical conditions. The data were transferred to the SigmaStat 3.1 program spreadsheet for a one-way analysis of variance (ANOVA) statistical test. The tail moment values

Table 3.2

CHO cell microplate Comet results for bromoiodoacetamide.

Concentration (mM)

Number of microgels

Average median TM value

0 5 10 25 50 75 100 125 150 200 250

8 4 4 6 8 5 8 2 8 7 5

0.29 0.41 1.37 28.22 44.11 58.18 60.75 72.10 82.31 98.99 105.65

TMSE

Average mean % tail DNA

% tail DNA SE

0.04 0.07 0.30 2.19 6.87 5.05 7.88 0.84 7.27 8.77 11.50

5.00 5.75 13.36 44.21 64.78 74.15 77.50 90.45 86.36 93.29 92.88

0.69 0.61 1.60 3.03 5.15 0.84 4.94 0.39 2.63 1.20 1.35

86

Chapter 3

80

CHO Cell Genomic DNA Damage as the Average Median SCGE Tail Moment Value (±SE)

60 120

100

I

Br

O

C

C

% Viable Cells

100

NH2

H 80

60

40

20 SCGE Genotoxic Potency Value = 72.1 µM 0

0

50

100

150

200

250

Bromoiodoacetamide (µM)

Figure 3.3

Concentration–response curve of the induction of genomic DNA damage by the recently identified N-DBP, bromoiodoacetamide, as measured by the microplate Comet assay. The SCGE genotoxic potency value is calculated as the concentration of the test agent that is the midpoint in the concentration–response curve.

in the Comet assay are not normally distributed, and the distributions of the tail moment values differ for the control and each concentration of the test agent. The lack of normal distributions violates the requirements for analysis by parametric statistics. To address this problem the data were normalised with the unit of measure as the microgel. The median tail moment value for each microgel was calculated and the medians were averaged amongst all of the microgels for each DBP concentration. Averaged median or averaged mean values express a normal distribution according to the central limit theorem.46 The averaged median tail moment values obtained from repeated experiments were analysed with a one-way ANOVA test.47 If a significant F value of P r 0.05 was obtained, a Holm–Sidak multiple comparison versus the control group analysis was conducted. The power of the test statistic was maintained as Z 0.8 at a ¼ 0.05. An example of the statistical analysis of the Comet tail moment values for bromoiodoacetamide is presented in Table 3.3. The data demonstrate that for the Comet tail moments induced by bromoiodoacetamide, a significant F value was obtained (F10,54 ¼ 29.379; P r 0.001). Employing the Holm–Sidak multiple comparison test, concentrations above 10 mM were significantly different from the control value.

87


Table 3.3

One-way analysis of variance for Comet tail moment values induced by bromoiodoacetamide. The tail moment data are presented in Table 3.2.

Source of variation

DF

SS

MS

F

Between groups Residual Total

10 54 64

84955.827 15615.444 100571.270

8495.583 289.175

29.379

Power of the performed test with a ¼ 0.050: 1.000. P r 0.001 Multiple comparisons versus control group (Holm–Sidak method)

Diff. of means

BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm BIAcAm

0.112 1.072 27.926 43.813 57.885 60.450 71.809 82.014 98.702 105.350

0 mM 0 mM 0 mM 0 mM 0 mM 0 mM 0 mM 0 mM 0 mM 0 mM

vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.

5 mM 10 mM 25 mM 50 mM 75 mM 100 mM 125 mM 150 mM 200 mM 250 mM

t

0.011 0.103 3.041 5.153 5.971 7.110 5.341 9.646 11.215 10.867

Unadjusted P Significant

0.991 0.918 0.004 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001

No No Yes Yes Yes Yes Yes Yes Yes Yes

Definitions: DF ¼ degrees of freedom, which represent a measure of the sample size that affects the power or sensitivity of the ANOVA test. SS ¼ sum of squares, the sum of the squared deviations from the mean, a measure of the variability of the average differences of the sample groups (between groups), the underlying variability of all individual samples (residual) and the total variability of the observations about the grand mean (total). MS ¼ mean squares, a measure of the estimates of the population variances between groups and within groups.

3.6 Utility of the Microplate Comet Assay in Comparing Classes of DBPs Recently, a new iodinated N-DBP, bromoiodoacetamide, was identified (with EI mass spectroscopy analysis) in drinking water from 12 treatment plants located in 6 US states. The haloacetonitriles36 and the haloacetamides37 are two important emerging classes of N-DBPs; their occurrence is associated with increased use of chloramines for water disinfection. We conducted a comparative analysis of genomic DNA damage induced by these emerging N-DBP classes using the microplate Comet assay with CHO cells. As indicated in Table 3.1, most of the haloacetonitriles and haloacetamides had to be chemically synthesised for the studies and the amount of sample was highly limited. The utility of the microplate Comet assay is well demonstrated by these studies. In Table 3.4, the lowest concentration for each haloacetonitrile or haloacetamide was identified by the ANOVA test statistic that induced significant genomic DNA-strand breakage (as measured by SCGE median tail moment values) as compared to their concurrent negative controls. The SCGE genotoxic potency

88

Table 3.4

Chapter 3

CHO cell microplate Comet analysis of the haloacetonitriles and haloacetamides.

Compound

Lowest genotox. conc. (M)

R2

SCGE gen. potency (M)

ANOVA test statistic

Bromoacetonitrile

4.00 105

0.99

3.85 105

Bromochloroacetonitrile

2.50 104

0.98

3.24 104

Chloroacetonitrile

2.50 104

0.99

6.01 104

Dibromoacetonitrile

3.00 105

0.95

2.97 105

Dichloroacetonitrile

2.40 103

0.98

2.75 103

Iodoacetonitrile

3.00 105

0.98

3.71 105

Trichloroacetonitrile

1.00 103

0.98

1.01 103

Bromoacetamide

2.50 105

0.99

3.68 105

Bromochloroacetamide

4.00 104

0.99

5.83 104

Bromodichloroacetamide

7.50 105

0.99

1.46 104

Bromoiodoacetamide

2.50 105

0.99

7.21 105

Chloroacetamide

7.50 104

0.99

1.38 103

Chloroiodoacetamide

2.00 104

0.99

3.02 104

Dibromoacetamide

5.00 104

0.99

7.44 104

Dibromochloroacetamide

2.50 105

0.98

6.94 105

Dichloroacetamide

NA

NA

NS 4 1 102

Diiodoacetamide

2.50 105

0.98

3.39 105

Iodoacetamide

3.00 105

0.99

3.41 105

Tribromoacetamide

3.00 105

0.97

3.25 105

Trichloroacetamide

5.00 103

0.98

6.54 103

F6,36 ¼ 32.7; P r 0.001 F10,41 ¼ 19.1; P r 0.001 F11,42 ¼ 28.9; P r 0.001 F9,46 ¼ 46.1; P r 0.001 F17,62 ¼ 14.2; P r 0.001 F10,53 ¼ 46.6; P r 0.001 F7,32 ¼ 30.5; P r 0.001 F9,38 ¼ 29.77; P r 0.001 F9,48 ¼ 53.86; P r 0.001 F9,39 ¼ 58.41; P r 0.001 F10,54 ¼ 29.38; P r 0.001 F11,46 ¼ 25.02; P r 0.001 F17,62 ¼ 35.19; P r 0.001 F10,47 ¼ 21.09; P r 0.001 F8,37 ¼ 185.59; P r 0.001 F11,34 ¼ 1.026; P ¼ 0.417 F11,60 ¼ 29.12; P r 0.001 F15,43 ¼ 13.11; P r 0.001 F17,62 ¼ 35.19; P r 0.001 F9,50 ¼ 5.75; P r 0.001

value was calculated for each chemical from the concentration–response curve. It represents the midpoint of the curve within the concentration range that expressed above 70% cell viability of the treated cells. Finally, the R2 refers to the fit of the regression analysis from which the SCGE genotoxic potency value was calculated. All concentrations are presented in molar (M) units of measure.

89


3.6.1

Microplate Comet Analysis of the Haloacetonitriles

CHO Cell Genomic DNA Damage as the Average Median SCGE Tail Moment Value

Using the microplate Comet assay for genomic DNA damage in CHO cells we analysed seven haloacetonitriles. The concentration–response curves for these haloacetonitriles are presented in Figure 3.4. Using the SCGE genotoxic potency values, the rank order in declining genotoxicity was dibromoacetonitrile 4 iodoacetonitrile E bromoacetonitrile 4 bromochloroacetonitrile 4 chloroacetonitrile 4 trichloroacetonitrile 4 dichloroacetonitrile. Structure–activity relationship (SAR) analysis may be used to further investigate possible mechanisms of action. The SAR analysis of these seven haloacetonitriles was in general agreement with the genotoxicity rank order. Haloacetonitriles have two potential electrophilic reactive centres: (i) displacement of a halogen atom at the a carbon by SN2 reaction, and (ii) addition at the partially positively charged carbon of the cyano group.48 Both reactions could contribute to the genotoxicity of the haloacetonitriles. The SN2 reactivity of the haloacetonitriles is dependent on the leaving tendency of the halogen and the degree of halogenation. The SN2 reactivity of an alkyl iodide is 3–5 greater than that of alkyl bromide, which is 50 greater than alkyl chloride,49 a similar relative order was observed for iodoacetonitrile 4 bromoacetonitrile 4 chloroacetonitrile. The leaving tendency of a halogen is expected to decrease with increasing halogenation; therefore, the alkylating potential of the haloacetonitriles is also expected to decrease. The potential of the haloacetonitriles to undergo nucleophilic addition at the partially positively charged carbon of the cyano group is dependent on the degree of halogenation. Polyhalogenation at

100

Iodoacetonitrile Bromoacetonitrile Dibromoacetonitrile Bromochloroacetonitrile Chloroacetonitrile Dichloroacetonitrile Trichloroacetonitrile

80

60

40

20

0

1

10

100

1000

Haloacetonitrile (µM)

Figure 3.4

Comparison of the microplate Comet genotoxicity concentration– response curves of seven haloacetonitriles.

90

Chapter 3

the a carbon provides the ideal situation because (i) the halogens withdraw electrons away from the cyano carbon, making it more electrophilic, and (ii) the halogens have a lower tendency to leave. The SN2 reactivity would be expected to significantly contribute to the genotoxic potential of the haloacetonitriles. The observed relative order of the 7 haloacetonitriles is generally in agreement with the SN2 SAR expectation. The higher activity of trichloroacetonitrile than dichloroacetonitrile may suggest that nucleophilic addition at the cyano carbon could also make some contribution to the genotoxicity.

3.6.2

Microplate Comet Analysis of the Haloacetamides

We analysed 13 haloacetamides for their ability to induce genomic DNA damage with the microplate Comet assay; the results are presented in Table 3.4. The concentration–response curves for these haloacetamides are presented in Figure 3.5. The rank order of genotoxic potency from most potent to least was tribromoacetamide 4 diiodoacetamide E iodoacetamide 4 bromoacetamide 4 dibromochloroacetamide 4 bromoiodoacetamide 4 bromodichloroacetamide 4 chloroiodoacetamide 4 bromochloroacetamide 4 dibromoacetamide 4 chloroacetamide 4 trichloroacetamide. Dichloroacetamide was not genotoxic. For SAR analysis the haloacetamides have or may generate a number of electrophilic reactivities: (i) for monohaloacetamides, alkylation by the SN2 reaction, inducing the displacement of a halogen atom at the a carbon, (ii) for dihaloacetamides, the potential generation of highly reactive a-halothioether electrophilic intermediates by cellular glutathione GSH or –SH compounds,

CHO Cell Genomic DNA Damge as the Average Median SCGE Tail Moment Value

120

100

80

60

40

Iodoacetamide Diiodoacetamide Bromoiodoacetamide Chloroiodoacetamide Bromoacetamide Dibromoacetamide Tribromoacetamide Bromochloroacetamide Dibromochloroacetamide Bromodichloroacetamide Chloroacetamide Dichloroacetamide Trichloroacetamide

20

0

Figure 3.5

10−6

10−5

10−4 Haloacetamide (M)

10−3

10−2

Comparison of the microplate Comet genotoxicity concentration– response curves of 13 haloacetamides.


91

(iii) for trihaloacetamides, nucleophilic attack at the electrophilic carbonyl carbon to yield trihalomethyl carbanions, that may lead to trihalomethanes as well as electrophilic dihalocarbene intermediates. In addition to chemical reactivity, the capacity to cross cell membranes is an important factor for toxicity. The logarithm of the octanol–water partition coefficient (log P) is a measure of lipophilicity that correlates with cell permeability. Log P increased with the degree of halogenation and with the size of the halogen.37 The rank order and relative genotoxic activities for the monohaloacetamides was iodoacetamide 4 bromoacetamide 44 chloroacetamide. The genotoxicity of the dihaloacetamides followed the order of diiodoacetamide 4 bromoiodoacetamide 4 chloroiodoacetamide 4 bromochloroacetamide 4 dibromoacetamide; dichloroacetamide was inactive. The genotoxicity rank order of the trihaloacetamides was tribromoacetamide 4 dibromochloroacetamide 4 bromodichloroacetamide 44 trichloroacetamide. The rank order and relative genotoxic activity of the monohaloacetamides are related to their SN2 reactivity. Owing to increasing bond length and decreasing dissociation energy, the leaving tendency of the halogen in alkyl halides followed the order, I 4 Br 44 Cl. The SN2 reactivity of an alkyl iodide was 3–5 greater than an alkyl bromide, which was 50 greater than an alkyl chloride.49,50 Iodoacetamide was more genotoxic than bromoacetamide, which was 38 more potent than chloroacetamide. The data suggest that log P does not play a major role for the monohaloacetamides. There is a small difference in the log P of bromoacetamide versus chloroacetamide, whereas there is a large difference in their relative activities.37 Consistent with SAR expectations are the relative leaving tendencies of the halogen dihaloacetamides containing one or two iodo group(s) expressed increased genotoxicity, followed by bromo group(s) and chloro group(s). Dichloroacetamide was not genotoxic. The estimated log P values of the dihaloacetamides followed the order: I2 4 IBr 4 ICl 4 Br2 4 BrCl 4 Cl2. This relative order is similar to their genotoxicity. Log P may play a more important role in the activity of dihaloacetamides by affecting cellular uptake. The genotoxicity of the trihaloacetamides decreased with a decrease in the number of bromo groups. The genotoxicity of trihaloacetamides could be partially explained by electrophilic reactivity at the carbonyl carbon as well as the possible release of electrophilic dihalocarbene intermediates (see discussion above). Alternatively, it is possible that reductive dehalogenation may yield cytotoxic free radicals; this pathway and the metabolic competency of the CHO cells have only been partially defined.51 A more complete discussion of the SAR analyses of the haloacetonitriles and haloacetamides has been published.36,37

3.6.3

Comparison of SCGE Genotoxic Potency Values of the Haloacetonitriles and Haloacetamides

By using the SCGE genotoxic potency value for each compound we can directly compare genotoxicity within a chemical class as well as compare the

92

Chapter 3

distribution of the genotoxicity between two or more DBP chemical classes. The distribution of the individual SCGE genotoxic potency values derived from the CHO cell microplate Comet analyses of the haloacetonitriles and the haloacetamides are presented in Table 3.4. Each SCGE genotoxic potency value for each chemical was calculated from a concentration–response curve derived from, in general, 8 concentrations with 6 replicate microgels per concentration. These comparisons are useful in that they can be used to prioritise specific hazardous chemicals for further study and their relative positions for genotoxicity can be employed for SAR studies.

3.7 Advantages of the Mammalian Cell Microplate Comet Assay The mammalian cell microplate Comet assay has several advantages especially when working with limited amounts of sample. We have used the microplate Comet assay to investigate regulated and emerging drinking-water DBPs,15,35–37,50,52–56 pesticides and their degradation products,57 antimutagens,58,59 DNA repair,7 sulfide,60,61 and complex mixtures.62,63 Using mammalian cells that attach as a monolayer to the bottom of a 96-well microplate ensures uniform exposure to the test compound. With equal numbers of cells plated, the amount of test agent available per cell within a specific chemical concentration is constant. A small volume of test solution (25–50 mL) consumes a very small amount of test agent per experiment. This permits increased numbers of concentrations per experiment and allows for more experimental replicates. With adherent cells washing after treatment is very easy and eliminates the need for centrifugation before and after treatment. Acute cytotoxicity is readily analysed with a very small aliquot (10 mL) of the treated cell suspensions. The primary advantages of the microplate Comet assay include: (i) it allows for the use of specialised cells (primary or nontransformed cells) that may be expensive to procure and maintain, (ii) very small treatment volumes are employed, which conserves valuable or limited test agents, (iii) it generates less toxic waste and less consumption of medium, serum and growth factors, and (iv) it allows for more concentration replicates and thus more robust data.

Acknowledgements This research was funded in part by AwwaRF Grant 3089, and a MTAC Grant. We appreciate the support by the Center of Advanced Materials for the Purification of Water with Systems, a National Science Foundation Science and Technology Center, under Award No. CTS-0120978. This chapter is dedicated to the memory of Dr. Jir˘ ı´ Velemı´ nsky´ (Academy of Sciences of the Czech Republic), scientist, scholar and dear friend.


93

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16.

17.

18.

19.

20.

21.

22. 23. 24.

25.

26.

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disinfection byproducts, Am. Water Works Res. Foundation, Denver, CO, 2009 (in press). U. S. Environmental Protection Agency, National primary drinking water regulations: Stage 2 disinfectants and disinfection byproducts rule, Federal Register, 2006, 71, 387. G. E. Bove Jr, P. A. Rogerson and J. E. Vena, Case control study of the geographic variability of exposure to disinfectant byproducts and risk for rectal cancer, Int. J. Health Geogr., 2007, 6, 18. D. S. Michaud, M. Kogevinas, K. P. Cantor, C. M. Villanueva, M. GarciaClosas, N. Rothman, N. Malats, F. X. Real, C. Serra, R. Garcia-Closas, A. Tardon, A. Carrato, M. Dosemeci and D. T. Silverman, Total fluid and water consumption and the joint effect of exposure to disinfection byproducts on risk of bladder cancer, Environ. Health Perspect., 2007, 115, 1569. C. M. Villanueva, K. P. Cantor, J. O. Grimalt, N. Malats, D. Silverman, A. Tardon, R. Garcia-Closas, C. Serra, A. Carrato, G. Castano-Vinyals, R. Marcos, N. Rothman, F. X. Real, M. Dosemeci and M. Kogevinas, Bladder cancer and exposure to water disinfection byproducts through ingestion, bathing, showering, and swimming in pools, Am. J. Epidemiol., 2007, 165, 148. K. P. Cantor, C. Villanueva, M. Garcia-Closas, D. Silverman, F. X. Real, M. Dosemeci, N. Malats, M. Yeager, R. Welch and S. Chanock, Bladder cancer, disinfection byproducts, and markers of genetic susceptibility in a case-control study from Spain, Epidemiology, 2006, 17, S150. C. M. Villanueva, K. P. Cantor, S. Cordier, J. J. Jaakkola, W. D. King, C. F. Lynch, S. Porru and M. Kogevinas, Disinfection byproducts and bladder cancer: a pooled analysis, Epidemiology, 2004, 15, 357. K. P. Cantor, Drinking water and cancer, Cancer Causes Control, 1997, 8, 292. T. Umemura and Y. Kurokawa, Etiology of bromate-induced cancer and possible modes of action-studies in Japan, Toxicology, 2006, 221, 154. M. Koivusalo, J. J. Jaakkola, T. Vartiainen, T. Hakulinen, S. Karjalainen, E. Pukkala and J. Tuomisto, Drinking water mutagenicity and gastrointestinal and urinary tract cancers: an ecological study in Finland, Am. J. Public Health, 1994, 84, 1223. J. E. Andrews, H. P. Nichols, J. E. Schmid, L. M. Mole, E. S. Hunter III and G. R. Klinefelter, Developmental toxicity of mixtures: the water disinfection byproducts dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture, Reprod. Toxicol., 2004, 19, 111. E. S. Hunter 3rd, E. H. Rogers, J. E. Schmid and A. Richard, Comparative effects of haloacetic acids in whole embryo culture, Teratology, 1996, 54, 57. K. Waller, S. H. Swan, G. C. Windham and L. Fenster, Influence of exposure assessment methods on risk estimates in an epidemiologic study of total trihalomethane exposure and spontaneous abortion, J. Expo. Anal. Environ. Epidemiol., 2001, 11, 522.


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28. S. H. Swan, K. Waller, B. Hopkins, G. Windham, L. Fenster, C. Schaefer and R. R. Neutra, A prospective study of spontaneous abortion: relation to amount and source of drinking water consumed in early pregnancy, Epidemiology, 1998, 9, 126. 29. K. Waller, S. H. Swan, G. DeLorenze and B. Hopkins, Trihalomethanes in drinking water and spontaneous abortion, Epidemiology, 1998, 9, 134. 30. F. Bove, Y. Shim and P. Zeitz, Drinking water contaminants and adverse pregnancy outcomes: a review, Environ. Health Perspect., 2002, 110(Suppl 1), 61. 31. M. J. Nieuwenhuijsen, M. B. Toledano, N. E. Eaton, J. Fawell and P. Elliott, Chlorination disinfection byproducts in water and their association with adverse reproductive outcomes: a review, Occup. Environ. Med., 2000, 57, 73. 32. D. A. Savitz, P. C. Singer, K. E. Hartmann, A. H. Herring, H. S. Weinberg, C. Makarushka, C. Hoffman, R. Chan and R. Maclehose, Drinking Water Disinfection By-products and Pregnancy Outcome, Awwa Research Foundation, Denver, CO, 2005. 33. Y. T. Woo, D. Lai, J. L. McLain, M. K. Manibusan and V. Dellarco, Use of mechanism-based structure-activity relationships analysis in carcinogenic potential ranking for drinking water disinfection byproducts, Environ. Health Perspect., 2002, 110(Suppl 1), 75. 34. S. W. Krasner, H. S. Weinberg, S. D. Richardson, S. J. Pastor, R. Chinn, M. J. Sclimenti, G. D. Onstad and A. D. Thruston Jr., The occurrence of a new generation of disinfection byproducts, Environ. Sci. Technol., 2006, 40, 7175. 35. M. J. Plewa, E. D. Wagner, M. G. Muellner, K. M. Hsu and S. D. Richardson, Comparative mammalian cell toxicity of N-DBPs and C-DBPs, in disinfection byproducts in Drinking Water, Occurrence, Formation, Health Effects and Control, eds. T. Karanfil, S. W. Krasner, P. Westerhoff and Y. Xie, American Chemical Society, Washington, D.C., Symposium Series No. 995, 2008, 36. 36. M. G. Muellner, E. D. Wagner, K. McCalla, S. D. Richardson, Y. T. Woo and M. J. Plewa, Haloacetonitriles vs. regulated haloacetic acids: are nitrogen containing DBPs more toxic?, Environ. Sci. Technol., 2007, 41, 645. 37. M. J. Plewa, M. G. Muellner, S. D. Richardson, F. Fasano, K. M. Buettner, Y. T. Woo, A. B. McKague and E. D. Wagner, Occurrence, synthesis and mammalian cell cytotoxicity and genotoxicity of haloacetamides: an emerging class of nitrogenous drinking water disinfection byproducts, Environ. Sci. Technol., 2008, 42, 955. 38. K. R. Tindall, L. F. Stankowski Jr, R. Machanoff and A. W. Hsie, Detection of deletion mutations in pSV2gpt-transformed cells, Mol. Cell Biol., 1984, 4, 1411. 39. K. R. Tindall and L. F. Stankowski Jr., Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells, Mutat. Res., 1989, 220, 241.

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40. A. W. Hsie, P. A. Brimer, T. J. Mitchell and D. G. Gosslee, The doseresponse relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somatic Cell Genet., 1975, 1, 247. 41. A. W. Hsie, P. A. Brimer, T. J. Mitchell and D. G. Gosslee, The doseresponse relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells, Somatic Cell Genet., 1975, 1, 383. 42. E. D. Wagner, A. L. Rayburn, D. Anderson and M. J. Plewa, Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and forward mutation assays in an isolated clone of Chinese hamster ovary cells, Environ. Mol. Mutagen., 1998, 32, 360. 43. E. D. Wagner, A. L. Rayburn, D. Anderson and M. J. Plewa, Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells, Mutagenesis, 1998, 13, 81. 44. B. S. Tzang, Y. C. Lai, M. Hsu, H. W. Chang, C. C. Chang, P. C. Huang and Y. C. Liu, Function and sequence analyses of tumor suppressor gene p53 of CHO.K1 cells, DNA Cell Biol., 1999, 18, 315. 45. H. J. Phillips, Dye exclusion tests for cell viability, in Tissue Culture: Methods and Applications, eds. P. F. Kruse and M. J. Patterson, Academic Press, New York, 1973, p. 406. 46. G. E. P. Box, W. G. Hunter and J. S. Hunter, Statistics for Experimenters: An Introduction to Design, Data Analysis, and Model Building, Wiley & Sons Inc., New York, NY, 1978. 47. D. P. Lovell, G. Thomas and R. Dubow, Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro Comet studies, Teratogen. Carcinogen. Mutagen., 1999, 19, 109. 48. E. L. Lin and C. W. Guion, Interaction of haloacetonitriles with glutathione and glutathione-s-transferase, Biochemic. Pharmacol., 1989, 38, 685. 49. G. M. Loudon, Organic Chemistry, 3rd edn., Benjamin/Cummings Publ. Co., Redwood, CA, 1995. 50. M. J. Plewa, E. D. Wagner, S. D. Richardson, A. D. Thruston Jr., Y. T. Woo and A. B. McKague, Chemical and biological characterization of newly discovered iodoacid drinking water disinfection byproducts, Environ. Sci. Technol., 2004, 38, 4713. 51. D. B. McGregor, I. Edwards, C. R. Wolf, L. M. Forrester and W. J. Caspary, Endogenous xenobiotic enzyme levels in mammalian cells, Mutat. Res., 1991, 261, 29. 52. E. Cemeli, E. D. Wagner, D. Anderson, S. D. Richardson and M. J. Plewa, Modulation of the cytotoxicity and genotoxicity of the drinking water disinfection byproduct iodoacetic acid by suppressors of oxidative stress, Environ. Sci. Technol., 2006, 40, 1878. 53. M. J. Plewa, E. D. Wagner, P. Jazwierska, S. D. Richardson, P. H. Chen and A. B. McKague, Halonitromethane drinking water disinfection


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byproducts: chemical characterization and mammalian cell cytotoxicity and genotoxicity, Environ. Sci. Technol., 2004, 38, 62. S. D. Richardson, A. D. Thruston Jr., C. Rav-Acha, L. Groisman, I. Popilevsky, O. Juraev, V. Glezer, A. B. McKague, M. J. Plewa and E. D. Wagner, Tribromopyrrole, brominated acids, and other disinfection byproducts produced by disinfection of drinking water rich in bromide, Environ. Sci. Technol., 2003, 37, 3782. M. J. Plewa, Y. Kargalioglu, D. Vankerk, R. A. Minear and E. D. Wagner, Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection byproducts, Environ. Mol. Mutagen., 2002, 40, 134. M. J. Plewa, Y. Kargalioglu, D. Vankerk, R. A. Minear and E. D. Wagner, Development of quantitative comparative cytotoxicity and genotoxicity assays for environmental hazardous chemicals, Water Sci. Technol., 2000, 42, 109. K. C. Sorensen, J. W. Stucki, R. E. Warner, E. D. Wagner and M. J. Plewa, Modulation of the genotoxicity of pesticides reacted with redox-modified smectite clay, Environ. Mol. Mutagen., 2005, 46, 174. M. J. Plewa, M. A. Berhow, S. F. Vaughn, E. J. Woods, M. Rundell, K. Naschansky, S. Bartolini and E. D. Wagner, Isolating antigenotoxic components and cancer cell growth suppressors from agricultural byproducts, Mutat. Res., 2001, 480–481, 109. M. A. Berhow, E. D. Wagner, S. F. Vaughn and M. J. Plewa, Characterization and antimutagenic activity of soybean saponins, Mutat. Res., 2000, 448, 11. M. S. Attene-Ramos, E. D. Wagner, H. R. Gaskins and M. J. Plewa, Hydrogen sulfide induces direct radical associated DNA damage, Mol. Cancer Res., 2007, 5, 455. M. S. Attene-Ramos, E. D. Wagner, M. J. Plewa and H. R. Gaskins, Evidence that hydrogen sulfide is a genotoxic agent, Mol. Cancer Res., 2006, 4, 9. L. G. Stork, C. Gennings, W. H. Carter, R. E. Johnson, D. P. Mays, J. E. Simmons, E. D. Wagner and M. J. Plewa, Testing for additivity in chemical mixtures using a fixed-ratio ray design and statistical equivalence testing methods, J. Agric. Biol. Environ. Statistics, 2007, 21, 514. S. D. Richardson, F. G. Crumley, F. Fasano, M. J. Plewa, E. D. Wagner, L. Williamson, M. Bartlett, P. Angel and R. Orlando, The 19th Annual Mass Spectrometry and Toxicity Characterization of Drinking Water Fractions: Tandem Mass Spectrometry Workshop, Lake Louise, Canada, 2006.

CHAPTER 4

The Use of Higher Plants in the Comet Assay TOMAS GICHNER,a,* IRENA ZNIDAR,b ELIZABETH D. WAGNERc AND MICHAEL J. PLEWAc a

Institute of Experimental Botany, Academy of Sciences of Czech Republic, Na Karlovce 1a, 16 000, Prague 6, Czech Republic; b Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, SI-1000, Ljubljana, Slovenia; c Department of Crop Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at UrbanaChampaign, 1101 West Peabody Drive, Urbana, IL 61801, USA

4.1 Introduction The global plant biomass represents over 90% of the total mass of the living biota. The food and feed chain of the biosphere begins with plants, however, the genotoxic effects of chemical pollutants on plant systems have often been overlooked. Plants are exposed to many environmental pollutants that are globally dispersed through aerial or aqueous pathways. In addition, agronomic crops as well as other plants are deliberately exposed to pesticides and other chemicals applied in modern agriculture. These pollutants may be not only a serious hazard for the plants themselves, but also for animals, including people who use these plants for food or feed. Assays to detect the genotoxicity of these pollutants are at present not available for most plant species. This limitation hampers or prevents the detection of the genotoxicity of xenobiotics in plants growing, for example, on *

Corresponding author


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polluted soil. To overcome the limitation, the Comet assay can be applied to detect DNA damage in plants. Although this technique has been primarily applied to animal cells, the incorporation of the Comet assay with plant tissues1–4 significantly extends the utility of plants in basic and applied studies in environmental mutagenesis and toxicology. In theory, the Comet assay can be used with every plant species. This review focuses on the Comet assay in higher plants. Data on the Comet assay in lower plants (fungi and algae) are presented in a recent review.5

4.2 Differences between the Animal and Plant Comet Assay The Comet assay protocols for plants and animals differ due to the presence of the plant cell wall formed of cellulose, while animal cells have only a membrane. Nuclei from human and animal cells may be isolated by lysing with high salt concentrations and detergents, that generates nucleus-like structures (nucleoids) with presumably loss of protective histone proteins and non-DNA-associated nuclear components.6 Plant nuclei cannot be treated in this manner, but the nuclei must be isolated via the formation of protoplasts or mechanically.

4.3 Cultivation and Treatment of Plants for the Comet Assay For laboratory studies three plant species have been primarily employed: onion (Allium cepa), tobacco (Nicotiana tabacum) and broad bean (Vicia faba). They are easily cultivated under controlled conditions in plant growth rooms or chambers. The protocols for cultivation and treatment are in the following sections.

4.3.1

Onion (Allium cepa)

The outer scales of onion bulbs are removed and the root primordia at the base of each bulb are scraped off to remove the dead, old roots. The bulbs are placed on the mouth of glass vials containing water. To speed up the sprouting of roots, the water can be aerated by using aquarium bubblers. The water is changed every day and the entire growth apparatus is maintained in the dark. When the roots grow to a suitable size (3 to 5 cm) they are rinsed with water and placed on the mouth of a vial containing the test agent for 1–24 h.7 A different treatment method consists of onion roots longer than 2 cm being excised from the bulb and dipped (with the apex at the bottom) for 3 h in plastic microtubes containing different concentrations of the agents to be tested.8 The advantage of using onion roots is that in the same test system induced DNA damage can be measured by the Comet assay as well as the frequency of induced chromosome aberrations and micronuclei. The roots are of sufficient

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size for treatment in approximately 1 week after placing the base of the bulbs in water. However, well-sprouting bulbs may not be available in several countries year round.

4.3.2

Tobacco (Nicotiana tabacum)

For cultivation of tobacco seedlings in vitro, seeds are sterilised in 70% ethanol for 2 min followed by a 20 min immersion in a sterilising solution (see Section 4.10.8). The sterilising solution is aspirated and the seeds are washed 5 times in sterile distilled water. Each seed is placed in a sterile container with 50 ml of sterile, solid growth medium (see Section 4.10.9). The seedlings are grown in vented containers in a plant growth chamber at 26 1C with a 16-h photoperiod to the 4 to 5 leaf stage. When tobacco seedlings are cultivated under these conditions, both leaves and roots can be used for the Comet assay. For studies on DNA damage in root nuclei, the roots of seedlings are immersed for 1–48 h into the test agent dissolved in water and cultivated in the dark at 26 1C. For longer treatments for 2–7 days, the test agent is dissolved in a nutrient solution. For studies on DNA damage in leaf nuclei, the roots of seedlings must be immersed in the test agent for at least 18 h to enable the agent to be evenly distributed in all of the leaves.3 When tobacco plants are cultivated in soil, it is difficult to use the roots for analysis as soil particles cling to the roots.

4.3.3

Broad Bean (Vicia faba)

Broad bean seeds are surface-sterilised with 10% H2O2 for 10 min or disinfected for 3–10 min with 5% sodium hypochlorite solution. The seeds are thoroughly rinsed 3–4 times with tap water and soaked for 6–24 h in distilled water at room temperature. It is recommended to remove the seed coat. The seeds are maintained for about 4–6 days at 20–22 1C on filter paper or moist Perlite beads regularly moistened with distilled water. At this time, the primary roots are 3– 5 cm long. The excised roots can then be treated with the test agent in a Petri dish for 2–6 h.9 As with onion roots, in the same test system the level of induced DNA damage can be compared with the frequency of induced chromosome aberrations and micronuclei. Broad bean seeds are available for experiments year round.

4.3.4

Plants used for In Situ Studies

For applying the Comet assay to plants growing in the field, garden or in the wild, the following preliminary method can be used.10,11 Nuclei are isolated (see Section 4.4.2.2 ) from a small piece of a leaf of the plant growing in a nonpolluted area to determine whether a sufficient number of intact, undamaged nuclei can be obtained (see Section 4.17.1). If so, a dose–response test is performed using ethyl methanesulfonate (EMS). Small young leaves are immersed into 2-ml plastic microtubes containing 1 ml of 0 (control), 2, 4, 6, 8, and 10 mM EMS for 18 h (see Section 4.10.2). After the treatment, the leaves are


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rinsed and nuclei are isolated from the part of the leaf that was not immersed in EMS solution. The Comet assay must be calibrated by modifying the DNA unwinding and electrophoresis time (see Section 4.6). For further studies, the conditions that ensure the minimal DNA migration in negative control samples and at the same time maximum sensitivity of treated samples should be used.

4.4 Isolation of Nuclei from Plant Tissues In plants, the nuclei can be isolated from the cells by either the formation of protoplasts (i.e. plant cells with cell wall eliminated) or by mechanical procedures.

4.4.1

Isolation of Nuclei via Protoplast Formation

With enzymes such as cellulases and pectinases, the plant cell wall is digested and the protoplasts are embedded in agarose on the Comet slides. The slides are then subjected to a lysis solution.12–17 The isolation of nuclei via the formation of protoplasts is costly, time consuming and currently seldom used. It is not recommended for toxicity studies.

4.4.2 4.4.2.1

Isolation of Nuclei by Mechanical Destruction of the Cell Wall Isolation of Nuclei from Plant Cell Suspensions

Nuclei from plant cell suspensions may be isolated by gently agitating 400 mg of rinsed cells with 100 mg washed sea sand and 500 mL cold 0.4 M phosphate buffer (pH ¼ 10) in a cold microfuge tube. After the sand settles to the bottom of the tube, the mixture is poured into a cold microtube that was previously modified by slicing the bottom off and fusing it with a 53-mm mesh nylon filter. The cell/nuclei suspension is agitated for 20 to 30 s with a flat-blade metal spatula; the nuclei pass through the filter and are collected in an attached microtube on ice.18,19

4.4.2.2

Isolation of Nuclei from Intact Roots or Leaves

Nuclei from roots or leaves may be isolated by gently slicing a small piece of leaf (approximately 2 cm 2 cm) or tufts of roots with a fresh razor blade (Figure 4.1). This procedure is conducted on ice in a plastic 60 mm Petri dish with 320 mL of 0.4 M Tris-HCl buffer, pH ¼ 7.5 (see Section 4.10.10). The Petri dish is kept tilted in the ice so that the isolated nuclei will collect in the buffer.

4.5 Preparation of Comet Assay Slides Microscope slides (the best are those with one-fourth frosted ends) are kept in ethanol for approximately 1 week. Individual slides are then removed from the ethanol and held over a burning wick of a burner. The slides are then dipped into 1% normal melting point agarose (NMPA) at 50 1C (see Section 4.10.5).

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Figure 4.1

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Isolation of nuclei from tobacco leaves. A small piece of leaf is placed onto a drop of isolation buffer in a Petri dish, and gently sliced with the edge of a fresh razor blade. The isolated nuclei are collected in the buffer.

The back of the slides are wiped to remove excess agarose and placed horizontally on a level surface for drying at room temperature. These slides serve as the agarose base-coated slides and are kept dry in slide boxes until further use. The first layer of agarose should be firmly attached to the slide, so the other layers can be added. The second layer to be placed upon these slides is a mixture of 1% low melting point agarose (LMPA) (see Section 4.10.6) and a suspension of freshly isolated nuclei. A suspension of nuclei is gently mixed with LMPA at 40 1C, in a ratio of 1:1 in a microtube; 100 mL of the mixture is placed on each slide. A coverslip (24 mm 50 mm) is placed on the mixture. The slides are placed on an iced surface for a minimum of 5 min after which the coverslips are removed and the slides are ready for the DNA unwinding step. A third layer of 0.5% agarose was used previously, but it was recently found that it is not necessary.20

4.6 DNA Unwinding and Electrophoresis During the DNA unwinding step the supercoiled DNA loops are relaxed.21 Predominantly DNA unwinding and electrophoresis are performed at the same pH. DNA unwinding and electrophoresis at neutral pH (pH 7–8) facilitates the detection of double-strand breaks and crosslinks and the total DNA damage is much less pronounced than at alkaline conditions.4 This can be an advantage


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when studying cells that have a high level of background damage, or when induced damage is high.22 DNA unwinding and electrophoresis at alkaline pH (pH 12.1–12.4) facilitates the detection of single- and double-strand breaks, incomplete excision-repair sites and crosslinks; DNA unwinding and electrophoresis at a pH higher than 12.6 expresses alkali-labile sites in addition to all types of lesions listed above.23 A combination of alkaline DNA unwinding and a neutral electrophoresis in root tips of broad bean plants was also reported.22 For toxicity studies, the alkaline version of the Comet protocol with DNA unwinding and electrophoresis at pH 4 13 is performed. The DNA unwinding step can be performed by immersing the slides into the electrophoresis solution in a Coplin jar that is placed on ice or by placing the slides into an electrophoresis tank with the same solution at 4–8 1C. The duration of DNA unwinding needs to be optimised for each plant species. Optimisation of the Comet assay refers to the most appropriate time of DNA unwinding and electrophoresis. Optimisation ensures the minimal DNA migration in control samples and at the same time maximum sensitivity of treated samples. The electrophoresis time depends on the temperature, voltage per the distance between anode and cathode (V cm1), amperage, and pH of the electrophoresis solution. For example, in the alkaline Comet assay calibrated for potato (Solanum tuberosum) plants, the optimal DNA unwinding time was 10 min, followed by 15 min electrophoresis at 0.74 V cm1 (26 V, 300 mA) and 4– 8 1C.24 In other plant species at the same electrophoresis voltage, different DNA unwinding and electrophoresis times have to be used, e.g. for tobacco plants 15 min DNA unwinding and 25 min electrophoresis,20 for durum wheat (Triticum durum) 5 min DNA unwinding and 15 min electrophoresis,11 and for dandelion (Taraxacum officinale) 30 min DNA unwinding and 30 min electrophoresis.10

4.7 DNA Staining A variety of fluorochromes have been applied in the Comet assay for DNA staining, e.g. acridine orange, DAPI, EtBr, propidium iodide.23,25,26 Developments in the synthesis of DNA-binding dyes have led to a new family of asymmetric cyanine dyes with improved fluorescence properties upon binding to DNA (Molecular Probes, Inc.). Several of these dyes were tested for DNA imaging applications and it was reported that YOYO-1 in particular, improves the image quality.27 For the alkaline version of the Comet assay in plants, the fluorochromes EtBr, DAPI, and YOYO-1 may be used with the same efficiency.28 However, YOYO-1 is very expensive and unstable. The most widely used dye in the Comet assay is EtBr, and to a lesser extent DAPI.

4.8 Reading the Slides, Expressing DNA Damage, Statistics After DNA staining, the slides should be evaluated within 6 h. Several companies supply software that, linked to a charged coupled digital camera

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mounted on the microscope, automatically analyses individual Comet images. The primary parameters of DNA-damage extent are the % of tail DNA (the % of total fluorescence in the tail) or the tail moment (the tail length multiplied by the fluorescence intensity in the tail divided by 100, expressed in mm).29 It is also possible to analyse comets quantitatively without imageanalysis software. In this visual scoring method, comets are classified as belonging to one of five classes according to tail intensity and are given a value of 0, 1, 2, 3, or 4 (from undamaged ¼ class 0, to maximally damaged ¼ class 4).30 The tail moment (TM) values and the % tail DNA values are not normally distributed and violate the requirements for analysis by parametric statistics. To resolve this problem the microgel on the slide is used as the unit of measure rather than the cell.31 When comparing the averaged mean or the averaged median TM values, the use of the median TM value was proposed.3 The mean value is affected by a few highly damaged nuclei in the sample that may have been caused during isolation of the nuclei, while the median value is less affected by such outliers. The averaged median TM values or averaged mean % tail DNA obtained from repeated experiments are used in a one-way analysis of variance test. If a significant F-value of P r 0.05 is obtained, a Dunnett’s multiple comparison test is conducted. The difference between two groups is statistically evaluated by the paired t-test.

4.9 Comet Assay Procedure 4.9.1 Prepare slides with one layer of 1% NMPA (see 4.5).

4.9.2 Treat plants or cell suspension culture in the laboratory with potential genotoxic agents or collect plants growing in polluted areas (see 4.3.).

4.9.3 Isolate nuclei from treated plant tissues (see 4.4.).

4.9.4 In a 1:1 ratio mix nuclear suspension and 1% LMPA at 40 1C in a microtube. Place 100 mL of this mixture on a slide previously coated with 1% NMPA. For each treatment group, prepare 3 slides. Place a 24 mm 50 mm coverslip on the mixture to obtain a uniform layer. Allow the agarose to solidify by keeping the slides on a metal tray on ice for a minimum of 5 min. It is not necessary to apply an additional layer of 0.5% LMPA.


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4.9.5 Carefully remove the coverslip and place the slides in a horizontal electrophoresis tank containing freshly prepared cold electrophoresis solution (see 4.10.1), allow the DNA to unwind prior to electrophoresis. If possible, DNA unwinding and electrophoresis should be performed in a cold room.

4.9.6 After electrophoresis rinse the slides three times with 0.4 M Tris-HCl buffer, pH 7.5 (see 4.10.10) and air-dry overnight.

4.9.7 Slides may be stored in slide boxes and at room temperature for several months.

4.9.8 Immerse the slides for 10 min in cold water and then stain for 5 min with 100 mL EtBr (20 mg/mL) (see 4.10.4). Carefully dip the slides in water to remove excess stain and cover with a coverslip.

4.9.9 Analyse the Comet images using a fluorescence microscope. With EtBr staining, an excitation filter of BP 546/10 nm and an emission filter of 590 nm is used.

4.9.10 Assess DNA-damage extent quantitatively by visual scoring or using imageanalysis software.

4.9.11 Repeat each experiment two or three times.

4.10 Reagents, Media, Buffers 4.10.1 Alkaline electrophoresis solution (pH 4 13). For 2000 mL solution, add 60 mL of 10 M NaOH (see 4.10.11) and 10 mL of 200 mM EDTA (see 4.10.3) to 1930 mL dH2O. Make fresh before each electrophoresis run.

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4.10.2 10 mM Ethyl methanesulfonate (EMS; Sigma M-0880). For 100 mL solution, add 106 mL EMS. Make fresh before use.

4.10.3 200 mM Ethylenediaminetetraacetic acid, disodium salt : dihydrate (EDTA; Sigma ED2SS). Dissolve 14.89 g in 175 mL dH2O, warm slightly while stirring, adjust pH to 10 with 10 M NaOH, add dH2O to the final volume of 200 mL. Store at room temperature.

4.10.4 20 lg mL1 Ethidium bromide (EtBr; Sigma E-8751): Stock solution: dissolve 10 mg ethidium bromide in 50 mL dH2O. For staining: mix 1 mL stock solution with 9 mL dH2O and filter with a Millipore filter (0.22 mm) to remove crystals. Store solutions in brown vials at room temperature under dark conditions.

4.10.5 1% Normal melting point agarose (NMPA) (Roth 2267, Sigma A9539). Dissolve 1 g NMPA in 100 mL dH2O, microwave or heat until near boiling. Store in a refrigerator.

4.10.6 1% Low melting point agarose (LMPA) (Roth 6351, Sigma, A9414). Prepare 1% LMPA. Dissolve 250 mg LMPA in 25 mL PBS (see 4.10.7), microwave or heat until near boiling. Prepare 2 to 5 mL aliquots in small vials and store in the refrigerator. The best way to melt the LMPA is to place the vials in a heater block adjusted to 70 1C.

4.10.7 Phosphate-buffered saline (PBS). Dissolve 50 mg KCl, 50 mg KH2PO4, 2 g NaCl, and 720 mg Na2HPO4.12 H2O in 200 mL dH2O, add dH2O to a final volume of 250 mL, adjust pH to 7.4, and filter sterilise. Store in a refrigerator.

4.10.8 Tobacco seed sterilising solution. Add 0.5 mL sodium hypochlorite (5.25% solution) and 5 mL 10% Triton-X to 4.5 mL dH2O.


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4.10.9 Tobacco solid growth medium. For 1 L medium: dissolve 4.6 g Murashige and Skoog salts (Sigma M-5524), 15 g sucrose, 10 ml vitamin stock solution (500 mL of vitamin stock solution contains 5 g myo-inositol (Sigma I-5125) and 500 mg thiamine hydrochloride (Sigma T-3902), 500 mg MES (Sigma M-3671), and 2.2 g Phytagel (Sigma P-8169) in 1L dH2O. Adjust to pH 5.6 and autoclave 50 mL medium per sterile Magenta box at 120 1C for 20 min.

4.10.10 0.4 M Tris-HCl buffer. Dissolve 9.7 g Tris in 180 mL dH2O, adjust pH to 7.5 with concentrated hydrochloric acid, add dH2O to a final volume of 200 mL. Store at room temperature.

4.10.11 10 M NaOH. Carefully dissolve 200 g NaOH in 500 mL dH2O. Store at room temperature.

4.11 Equipment and Software Several types of electrophoresis tanks, power supplies, fluorescence microscopes and computerised image-analysis systems are available. We present a set of equipment that has provided reliable results. (1) Horizontal gel electrophoresis tank (e.g. Horizon 20.25 GIBCO-BRL). (2) Power supply (e.g. BioRad Power Pac 300). (3) Fluorescence microscope (e.g. Olympus BX 60) with filters specific to the DNA stain, e.g. for EtBr an excitation filter of BP 546/10 nm and an emission filter of 590 nm is used. (4) Computerised image-analysis system (e.g. Komet, Kinetic Imaging Ltd., Liverpool, UK), linked to a CCD camera.

4.12 Determination of Toxicity Treatment of plants with very high concentrations of test agents or unfavourable cultivation conditions such as high temperatures or poor nutrient conditions of the soil may induce detrimental effects in the plants that lead to necrotic DNA fragmentation manifested by the formation of Comet images. To be sure that the demonstrated DNA damage is not of necrotic origin, toxicity evaluations should be performed. When seedlings (e.g. tobacco) are treated and only one leaf is excised for the isolation of nuclei, the seedling may be further cultivated in water or in a

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nutrient solution for 1 to 2 weeks to observe plant survival and growth. When root nuclei are used for the Comet assay, additional plants may be treated in parallel, and checked for survival. In broad bean or onion, plants treated in parallel may be cultivated for survival studies and the root length of treated plants may be compared with the root length of the control. In tobacco, the leaf area of treated plants and in potato the plant height can be used as a measure of the toxicity of the test agent. In onion, the loss of cell viability or cell death was evaluated using the Evan’s blue staining method.32 Freshly harvested roots were stained with 0.25% (w/v) aqueous solution of Evans blue for 15 min. After washing with distilled water for 30 min, 10 root tips of equal length (10 mm) were excised and soaked with 4 mL of N,N-dimethylformamide for 1 h at room temperature. The absorbance of Evan’s blue released was measured at 600 nm. In plant cell suspension cultures the cell viability after treatment can be determined using the phenosafranine dye exclusion method.33

4.13 Correlation between the DNA Damage Evaluated by the Comet Assay and Other Genetic Endpoints in Plants The DNA-damaging effect of the monofunctional alkylating agents EMS, MMS, ENU and MNU is highly correlated with the frequency of somatic mutations in tobacco leaves.34 In addition, the level of EMS- and ENU-induced DNA damage is also correlated with the recombination activity measured by GUS gene reactivation assay and by the twin sectors assay.35 MNU-induced DNA damage detected by the Comet assay in Arabidopsis thaliana4 and in barley (Hordeum vulgare)36 is correlated with a high frequency of chlorophyll and embryonic mutations,37 and with the formation of chromosome aberrations and micronuclei.36 The aromatic amine o-PDA induced both DNAdamage measured by the Comet assay and somatic mutations, whereas m-PDA and p-PDA induced neither DNA damage nor somatic mutations in tobacco plants.38 However, with the pesticide and plant-growth regulator maleic hydrazide (MH), there was no correlation between DNA damage detected by the Comet assay and other genetic endpoints. After tobacco seedlings or roots of broad bean were treated with MH, a very high frequency of somatic mutations in tobacco leaves and chromosome aberrations in broad bean were induced, but DNA-damage measured by the Comet assay did not differ from the negative control.39 Thus, most, but not all genotoxic agents, induce DNA damage detectable by the Comet assay as well as damage measurable by different genetic endpoints. In the case of gamma irradiation of tobacco seedlings, a complete repair of DNA-damage measurable by the Comet assay was observed 24 h after treatment, whereas the yield of somatic mutations manifested in the newly formed leaves increased with the increased dose of irradiation.40,41 Mutational events


109

may be the result of misrepaired DNA lesions, which may not be detected by the Comet assay, as the DNA chain is not disrupted.

4.14 The Utility of the Comet Assay for Genotoxic Studies in the Laboratory The plant Comet assay is a very suitable method to assess DNA damage induced after treating plants with known doses of genotoxins. With this method, plants may be cultivated and treated under stringent experimental conditions. Several agronomic (Table 4.A1) and wild (Table 4.A2) plant species have been employed in the Comet assay. Nuclei were isolated from leaves, roots, or cell cultures. Agents examined included different chemicals, pollutants, or irradiation. DNA unwinding and electrophoresis were mainly performed at alkaline pH and the most widely used DNA dye was EtBr. The extent of induced DNA damage was primarily dose dependent. With the Comet assay, the DNA-damaging effects of a test agent can be evaluated and compared between different plant tissues (e.g. leaves and roots). Dose- and time-dependent effects can also be measured. Using both cellular and acellular (cytoplasm free) methods, the difference between direct and indirect acting agents can be observed.42 For a fuller understanding of the genotoxic properties of a test agent, not only the Comet assay, but also other assays and endpoints (somatic mutations, chromosome aberrations, or micronuclei) should be performed. Studies in the laboratory are usually performed under fully controlled conditions. This is one reason why the results of the Comet assay can only partly be used to predict the effects under environmental conditions, which are much more complex and unstable.

4.15 The Utility of the Comet Assay as an In Situ Marker Few studies have been performed using the Comet assay with plants grown in soil from polluted areas. In general, the polluted soil was transferred to pots in which the plants were cultivated in a greenhouse or in a cultivation chamber. DNA damage was assessed in leaves of wood small-reed (Calamagrostis epigejos) growing in sediment reservoir substrates from uranium mining. The average specific activity of natural radionuclides measured in the substrate was for 226Ra ¼ 11 800 kg1 soil. Using the Comet assay no significant increase in DNA damage was observed in plants grown in the sediment substrate.43 When tobacco plants were cultivated for 8 weeks in soil heavily polluted with heavy metals (Cd ¼ 11, Cu ¼ 556, Pb ¼ 12 190, and Zn ¼ 132 mg kg1 soil), DNAdamage extent in leaf nuclei slightly but significantly increased compared to the control. However, the plants with slightly increased DNA damage were severely damaged with stunted leaves and a much smaller leaf area.44 No increase in the frequency of somatic mutations was reported. Similarly, tobacco

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plants growing for eight weeks in soil heavily polluted with polychlorinated biphenyls (PCBs ¼ 165 to 265 mg kg1 soil) were severely damaged, with reduced growth and distorted leaves. DNA-damage extent in the leaves of those plants increased significantly versus the control. As no increase in the frequency of somatic mutations was observed, the increased DNA migration in the severely injured plants may be associated with necrotic DNA fragmentation.45 A question has been raised, whether the Comet assay is a useful method for monitoring the genotoxic effects of environmental pollutants in plants growing in situ. The increased DNA damage in leaf nuclei was associated with high toxic effects (leaf growth inhibition and brownish, distorted leaves). Root nuclei could be more sensitive to environmental pollutants. However, when plants were grown for longer periods in polluted soil, soil clings to the roots and it is difficult to isolate a sufficient number of nuclei.

4.16 Comet Assay with Irradiated Food of Plant Origin Different types of foodstuff are irradiated to reduce microbial contamination and insect pests.46 A special Comet assay protocol was developed to detect irradiated foodstuffs, also of plant origin. ‘‘DNA Comet assay’’ has been adopted as a European Standard by the European Committee for Standardisation.47 The Comet assay has already been applied successfully to seeds of several species: almond, fig, lentil, linseed, rose´ pepper, sesame, soybean, sunflower,48,49 orange, lemon, apple, watermelon, tomato,50 different species of beans,51 spices,52 papaya, melon,53 buckwheat, maize, millet, oat, peanut, walnut, hazelnut, pine nut,54 and kiwi fruit.55 The protocol of Cerda et al.49 is primarily followed, with minor changes for each plant sample. Cell suspensions prepared from seeds are mixed with agarose and the mixture is spread onto microscope slides. Lysis and electrophoresis are performed at neutral pH. The tail length is measured visually or with image-analysis systems. Irradiated seeds have a longer tail than nonirradiated seeds. However, there are some limitations of the Comet assay to detect irradiated food, so this method is not appropriate for all samples.56

4.17 Recommendations for Plant Comet Assay Users 4.17.1 The initial step in employing higher plants in the Comet assay is to be sure that you are able to isolate nuclei without causing their damage. We recommend the following procedure: isolate nuclei from leaves, roots, or cells of the plant species you intend to work with (see 4.4), prepare comet slides (see 4.5) and immerse them for 10 min in the alkaline electrophoresis solution (see 4.10.1). However, do not subject the slides with the embedded nuclei to electrophoresis.

A/A; EtBr

o-PDA, m-PDA, p-PDA Pb(NO3)2 EMS H2O2 EMS, DNase-1, hyperthermia MNU, ENU, MMS, EMS MH Gamma rays Soil polluted with PCB Soil polluted with heavy metals

Leaves

Roots, leaves

Cell culture Cell culture Leaves

Leaves Roots Roots, leaves Leaves Roots, leaves

Nicotiana tabacum

Nicotiana tabacum

Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum

Nicotiana Nicotiana Nicotiana Nicotiana Nicotiana

tabacum tabacum tabacum tabacum tabacum

A/A; EtBr

Gamma rays AlCl3 Atrazine, CdCl2 H2O2. Gamma rays EMS TCB, CB, HCB MNU Bleomycin EMS EMS H2O2 CdCl2

Roots Roots Roots Roots Leaves Roots Roots, leaves Roots Leaves Leaves Leaves Roots, leaves

Allium cepa Allium cepa Allium cepa Allium cepa Beta vulgaris Glycine max Hordeum vulgare Hordeum vulgare Lens esculenta Medicago sativa Nicotiana tabacum Nicotiana tabacum

A/A; EtBr A/A; EtBr A/A; EtBr, DAPI, YOYO-1 A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr

A/A; EtBr A/A; Silver staining A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/N; EtBr A/A, N/N; AO A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr

Agent tested

Nuclei from

DNA unwinding/electrophoresis; staining

Dose-response No response Dose-response Weak-response Weak-response

Dose-response Dose-response Weak-response Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response, No response Dose-response, No response, No response Dose-response, No response Dose-response Dose-response Dose-response

Response

34 39 41 45 44

18 19 28

20

38

1 32 57 58 11 59 36 60 11 11 61 62

Ref.

Overview of agronomic plant species used in the Comet assay to assess DNA damage induced by various agents.

Plant species

Table 4.A1

The Use of Higher Plants in the Comet Assay 111

Soil polluted with heavy metals EMS, gamma rays EMS EMS Gamma rays MMS, Menadione Na2HAsO4 MH Quinoline Yellow, Brilliant Black EMS

Leaves

Roots, leaves Leaves Leaves Roots

Roots

Roots, leaves Roots Roots

Leaves

Solanum tuberosum

Solanum tuberosum Triticum aestivum Triticum durum Vicia faba

Vicia faba

Vicia faba Vicia faba Vicia faba

Zea mays

A/A; EtBr

A/A; EtBr A/A; EtBr A/A; EtBr A/A, A/N; EtBr, N/N; EtBr A/A, A/N; EtBr, N/N; EtBr A/A; EtBr A/A; EtBr A/A; DAPI, EtBr

A/A; EtBr


Dose-response

Dose-response No response Dose-response

Dose-response No response

Dose-response Dose-response Dose-response Dose-response No response

Weak-response

Response

11

64 39 65

22

24 11 11 63

44

Ref.

A/A, alkaline DNA unwinding/alkaline electrophoresis; A/N, alkaline DNA unwinding/neutral electrophoresis; N/N, neutral DNA unwinding/neutral electrophoresis. AO, acridine orange; CB, chlorobenzene; DAPI, 4 0 ,6-diamidino-2-phenylindole, dilactate; EMS, ethyl methanesulfonate; ENU, N-ethyl-N-nitrosourea; EtBr, ethidium bromide; HCB, hexachlorobenzene; MH, maleic hydrazide; MMS, methyl methanesulfonate; MNU, N-methyl-N-nitrosourea; PCB, polychlorinated biphenyls; m-PDA, m-phenylenediamine; o-PDA, o-phenylenediamine; p-PDA, p-phenylenediamine; TCB, 1,2,4-trichlorobenzene. Dose-response: At least 2–3 doses significantly higher than control; Weak-response: 1 or 2 doses only slightly higher than control.

Agent tested

Nuclei from

(continued ).

Plant species

Table 4.A1

112 Chapter 4

Fenarimol EMS Pb(NO3)2 EMS EMS EMS EMS, MH, NDEA EMS Road-side pollutants

Leaves Roots, leaves Leaves Leaves Leaves Leaves Leaves Roots, stems, leaves Leaves Leaves Roots Leaves Leaves Leaves Stamen hairs

Leaves Leaves

Arabidopsis thaliana Bacopa monnieri Bellis perennis Calamagrostis epigejos Chenopodium rubrum Convolvulus arvensis Epipremnum aureum Impatiens balsamina

Dose-response Weak-response

Dose-response Dose-response Weak-response Dose-response Dose-response Dose-response Dose-response

Dose-response Dose-response, No response, Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response Dose-response Weak-response Weak-response

Response

10 67

68 10 69 10 10 10 70

10 66 10 43 10 10 67 8

10 4

Ref.

A/A, alkaline DNA unwinding/alkaline electrophoresis; A/N, alkaline DNA unwinding/neutral electrophoresis; N/N, neutral DNA unwinding/neutral electrophoresis. EMS, ethyl methanesulfonate; ENU, N-ethyl-N-nitrosourea; EtBr, ethidium bromide; MH, maleic hydrazide; MMS, methyl methanesulfonate; MNU, Nmethyl-N-nitrosourea; NDEA, N-nitrosodiethylamine. Dose-response: At least 2–3 doses significantly higher than control; Weak-response: 1 or 2 doses only slightly higher than control.

A/A; EtBr A/A; EtBr

EtBr EtBr Silver staining EtBr EtBr EtBr EtBr

A/A; A/A; A/A; A/A; A/A; A/A; A/A;

EMS MNU, MMS, MNU, MMS, Bleomycin EMS EMS, MMS, CdCl2 EMS EMS, gamma rays EMS EMS Road-side pollutants K2Cr2O7

Leaves Whole plantlets

Agropyron repens Arabidopsis thaliana

Impatiens balsamina Lamium album Lupinus luteus Plantago media Poa annua Taraxacum officinale Tradescantia clone 4430 Urtica dioica Vinca rosea

A/A; EtBr A/N; EtBr N/N; EtBr A/N, N/N; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr A/A; EtBr

Agent tested

Nuclei from


Overview of selected wild plant species used in the Comet assay to assess DNA damage induced by various agents.

Plant species

Table 4.A2

The Use of Higher Plants in the Comet Assay 113

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Rinse the slides three times (see 4.9.6) and stain with EtBr (see 4.9.8). Check if at least 50 to 100 undamaged nuclei are isolated per slide.

4.17.2 The alkaline version of the Comet assay is the best for toxicology and genotoxicity studies as it detects most types of induced DNA damage.

4.17.3 For staining use ethidium bromide. It is inexpensive compared to other fluorescence dyes, the substance is stable if properly stored, and provides very good comet images.

4.17.4 Toxicity evaluation, e.g. plant, leaf, or root-growth inhibition, and survival should also be included in all the studies. Toxic and lethal effects induce necrotic DNA fragmentation that may be detected with the Comet assay as increased DNA-damage extent, which simulates genotoxin-induced DNA migration.

4.17.5 As a positive control use EMS. It is a classical mutagen, genotoxic for all plant species and induces DNA damage in roots, leaves, and plant cell suspension cultures.

4.17.6 Storing mutagen-treated Arabidopsis thaliana plants at 80 1C did not significantly influence the extent of DNA migration.4 Perhaps this method can be used also for other plant species.

4.17.7 When handling mutagens, pesticides, and other toxic substances, observe all rules that meet the International Agency for Research on Cancer (Lyon) specifications. The electrophoresis solution is highly alkaline and one should be careful when working with the solution.

Abbreviations DAPI ENU

4 0 ,6-diamidino-2-phenyindole, dilactate N-ethyl-N-nitrosourea


EMS EtBr MMS MH MNU m-PDA o-PDA p-PDA TM

115

ethyl methanesulfonate ethidium bromide methyl methanesulfonate maleic hydrazide N-methyl-N-nitrosourea m-phenylenediamine o-phenylenediamine p-phenylenediamine tail moment

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43. O. Ptacek, Z. Muhlfeldova, J. Dostalek, T. Cechak and T. Gichner, Monitoring DNA damage in wood small-reed (Calamagrostis epigejos) plants growing in a sediment reservoir with substrates from uranium mining, J. Environ. Monit., 2002, 4, 592. 44. T. Gichner, Z. Patkova, J. Szakova and K. Demnerova, Toxicity and DNA damage in tobacco and potato plants growing on soil polluted with heavy metals, Ecotoxicol. Environ. Saf., 2006, 65, 420. 45. T. Gichner, P. Lovecka, L. Kochankova, M. Mackova and K. Demnerova, Monitoring toxicity, DNA damage, and somatic mutations in tobacco plants growing in soil heavily polluted with polychlorinated biphenyls, Mutat. Res., 2007, 629, 1. 46. H. Delincee, Analytical methods to identify irradiated food – a review, Radiat. Phys. Chem., 2002, 63, 455. 47. CEN, EN 13784 Foodstuffs – DNA Comet assay for the detection of irradiated foodstuffs – Screening method. European Committee for Standardization, 2001, Brussels. 48. G. Koppen and H. Cerda, Identification of Low-dose Irradiated Seeds using the Neutral Comet assay, Lebensm.-Wiss. u.-Technol., 1997, 30, 452. 49. H. Cerda, H. Delincee, H. Haine and H. Rupp, The DNA ‘Comet assay’ as a rapid screening technique to control irradiated food, Mutat. Res., 1997, 375, 167. 50. N. S. Marin-Huachaca, M. T. Lamy-Freund, J. Mancini-Filho, H. Delincee and A. L. C. H. Villavicencio, Detection of irradiated fresh fruits treated by e-beam or gamma rays, Radiat. Phys. Chem., 2002, 63, 419. 51. A. A. Khan, H. M. Khan and H. Delincee, Detection of radiation treatment of beans using DNA Comet assay, Radiat. Phys. Chem., 2002, 63, 407. 52. A. A. Khan, H. M. Khan and H. Delincee, Identification of irradiated spices using the novel technique of DNA Comet assay, J. Food Sci., 2002, 67, 493. 53. N. S. Marin-Huachaca, J. Mancini-Filho, H. Delincee and A. L. C. H. Villavicencio, Identification of gamma-irradiated papaya, melon and watermelon, Radiat. Phys. Chem., 2004, 71, 191. 54. A. A. Khan, H. M. Khan and H. Delincee, DNA Comet assay – a rapid screening method for detection of irradiated cereals and tree nuts, Food Contr., 2005, 16, 141. 55. D. Jo and J.-H. Kwon, Detection of radiation-induced markers from parts of irradiated kiwi fruits, Food Contr., 2006, 17, 617. 56. H. Delincee, A. A. Khan and H. Cerda, Some limitations of the Comet assay to detect the treatment of seeds with ionising radiation, Eur. Food Res. Technol., 2003, 216, 343. 57. A. J. Lin, Y. G. Zhu, Y. P. Tong and C. N. Geng, Evaluation of genotoxicity of combined pollution by cadmium and atrazine, Bull. Environ. Contam. Toxicol., 2005, 74, 589.


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58. M. Saghirzadeh, M. R. Gharaati, Sh. Mohammadi and M. Ghiassi-Nejad, Evaluation of DNA damage in the root cells of Allium cepa seeds growing in soil of high background radiation areas of Ramsar – Iran, J. Environ. Radioact., 2008, 99, 1698. 59. W. Liu, Y. S. Yang, P. Li, Q. Zhou and T. Sun, Root growth inhibition and induction of DNA damage in soybean (Glycine max) by chlorobenzenes in contaminated soil, Chemosphere, 2004, 57, 101. 60. M. Georgieva and L. Stoilov, Assessment of DNA-strand breaks induced by bleomycin in barley by the Comet assay, Environ. Mol. Mutagen., 2008, 49, 381. 61. A. Mancini, A. Buschini, F. M. Restivo, C. Rossi and P. Poli, Oxidative stress as DNA damage in different transgenic tobacco plants, Plant Sci., 2006, 170, 845. 62. T. Gichner, Z. Patkova, J. Szakova and K. Demnerova, Cadmium induces DNA damage in roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves, Mutat. Res., 2004, 559, 49. 63. G. Koppen and K. J. Angelis, Repair of X-ray induced DNA-damage measured by the Comet assay in roots of Vicia faba, Environ. Mol. Mutagen., 1998, 32, 281. 64. A. Lin, X. Zhang, Y.-G. Zhu and F.-J. Zhao, Arsenate-induced toxicity: effects on antioxidative enzymes and DNA damage in Vicia faba, Environ. Toxicol. Chem., 2008, 27, 413. 65. V. K. Macioszek and A. K. Kononowicz, The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151), Cell. Mol. Biol. Lett., 2004, 9, 107. 66. P. Vajpayee, A. Dhawan and R. Shanker, Evaluation of the alkaline Comet assay conducted with the wetlands plant Bacopa monnieri L. as a model for ecogenotoxicity assessment, Environ. Mol. Mutagen., 2006, 47, 483. 67. C. Sriussadaporn, K. Yamamoto, K. Fukushi and D. Simazaki, Comparison of DNA damage detected by plant Comet assay in roadside and non-roadside environments, Mutat. Res., 2003, 541, 31. 68. P. Poli, M. A. de Mello, A. Buschini, V. L. de Castro, F. M. Restivo, C. Rossi and T. M. Zucchi, Evaluation of the genotoxicity induced by the fungicide fenarimol in mammalian and plant cells by use of the single-cell gel electrophoresis assay, Mutat. Res., 2003, 540, 57. 69. R. Rucinska, R. Sobkowiak and E. A. Gwozdz, Genotoxicity of lead in lupin root cells as evaluated by the Comet assay, Cell. Mol. Biol. Lett., 2004, 9, 519. 70. C. Alvarez-Moya, A. Santerre-Lucas, G. Zuniga-Gonzalez, O. TorresBugarin, E. Padilla-Camberos and A. Feria-Velasco, Evaluation of genotoxic activity of maleic hydrazide, ethyl methane sulfonate, and N-nitroso diethylamine in Tradescantia, Salud Publica Mex., 2001, 43, 563.

CHAPTER 5

Methods for Freezing Blood Samples at 80 1C for DNA Damage Analysis in Human Leukocytes NARENDRA P. SINGH* AND HENRY C. LAI Department of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195-5061, USA

5.1 Introduction Large-scale studies assessing DNA damage in the human population often have increased variability in results due to variations in handling and logistical restrictions of field work.1–2 In these studies, it is necessary to minimise DNA damage caused during collection, shipping, storage and analysis of human leukocytes in order to evaluate the toxicological effects of exposures to various chemicals. Often in such studies, large numbers of samples are collected in a short time and it is not feasible to process all samples at once, necessitating the use of long-term cryopreservation (conveniently at 80 1C). Preservation of whole blood at 80 1C is used primarily for assays involving DNA extraction, where levels of DNA damage are not as critical as in microgel electrophoresis [also known as single-cell gel electrophoresis (SCGE) or the Comet assay]. Because of the high sensitivity of the Comet assay in detecting

*



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Methods for Freezing Blood Samples

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low levels of DNA-strand breaks, special steps must be taken when collecting, transporting, freezing, thawing and processing blood samples. While there are several reports of using isolated human leukocytes and lymphocytes frozen at 70 to 80 1C for the assessment of DNA damage by the Comet assay,3–6 there are only few7,8 using human whole blood. It was hypothesised that certain freezing protocols would more fully reveal DNA damage induced by chemical or physical agents. X-Ray induced DNA damage was used as a model for DNA damage induced by exposure to genotoxic agents. Here, we report two new protocols for the assessment of DNAdamage levels in human whole blood nucleated cells.

5.2 Materials and Methods All chemicals were purchased from Sigma Chemical Co. (Saint Louis, MO) unless stated otherwise. Venous blood was drawn from a healthy adult male volunteer for all experiments. The blood was divided into 4 aliquots. Two of these aliquots served as controls and the other two were irradiated with 2 Gy of X-rays at a rate of 1 Gy/min using a Kelley-Koett X-ray machine (Covington, CT). Aliquots, one control and one irradiated each, were frozen for a month at 80 1C using Protocol I and II. DNA damage was assessed as described below. All experiments were performed three times.

5.2.1

Protocol I

One hundred mL of whole heparinised blood was pipetted into a 2-mL cryovial and was mixed well with 900 mL of freshly prepared freezing solution having 10% bovine serum albumin (BSA, Amresco Solon, OH) and 15% DMSO (Fisher Scientific, Fair Lawn, NJ) in RPMI 1640 (final concentrations: 9% BSA and 13.25% DMSO). Cryovials were then stored at 80 1C for 1 month. Frozen samples were then taken from 80 1C and transferred to a 37 1C water bath for rapid thawing to avoid damage to membranes and DNA by ice crystals.9–11 Samples were processed for the Comet assay as described previously2 with some modifications. Briefly, a first layer of microgel was prepared in two steps. First, 50 mL of 0.5% high-resolution agarose (high-resolution agarose 3:1 from Amresco, Solon, OH) was pipetted on top of the frosted part of an MGE slide (Erie Scientific Co., New Haven, CT), near the lower margin of the marking area. Then, using a pipette tip, agarose was smeared over the rest of the slide, coating both frosted and clear window areas. The slides, thus made, were airdried. Second, 200 mL of 0.7% agarose was pipetted onto the centre of the slide and a cover glass (24 50 mm2, Corning Glass Works, Corning, NY) was placed over it. The cover glass was removed and a second layer of microgel was made by pipetting 100 mL of an agarose-cell mixture on to the centre of the slide. The mixture was composed of 50 mL of the thawed blood sample and 50 mL of 0.7% agarose (cells were not centrifuged and washed to avoid their clumping and to minimise procedural and time-dependent DNA damage). A cover glass was

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placed on top and the slide was placed on an ice-cold plate for 1 min. The cover glass was removed and a third layer of microgel was made using 200 mL of 0.7% agarose in PBS. After removing the cover glass, slides were immersed in a cold lysing solution (0.01% sodium lauroyl sarcosine, 1.25 M NaCl, 10 mM EDTA, glutathione 1 mg/mL and 100 mM Tris, pH 10) for 1 h at 4 1C. The microgels were then treated with proteinase K in 1.25 M NaCl, 5 mM EDTA and 5 mM Tris (pH 10) at 37 1C for 1 h. The slides were placed in a special electrophoresis unit (Ellard Instrumentation, Monroe, WA) containing 1 L of 300 mM NaOH, 1 mM EDTA, 0.2% DMSO and 0.01% 8-hydroxyquinoline (pH413.5). The DNA from the lysed cells in the microgels was allowed to unwind for 20 min in this alkaline solution and then electrophoresed at 12 V (0.4 V/cm), 250 mA for 20 min at room temperature in indirect, incandescent low light. During electrophoresis, the solution was recirculated at a rate of 100 mL/min. The slides were immersed in 50% ethanol having 1 mg/mL of spermine and 20 mM of Tris HCl (pH 7.4) for 10 min at room temperature. This step was repeated once more. The slides were then transferred to 75% ethanol having 20 mM Tris HCl and this step was repeated twice more. The slides were dried in air and were stained with 100 mL of 0.25 mM YOYO in 2.5% DMSO and 0.5% sucrose and 100 images per slide were captured for image analysis, as described in Section 5.2.5.

5.2.2

Protocol II

One hundred mL of whole heparinised blood samples, control and irradiated, were pipetted into 2-mL cryovials and each was mixed well with 900 mL of freshly prepared freezing solution having 20% DMSO in RPMI 1640 (final concentration: 18% DMSO). Cryovials were then stored at 80 1C for 1 month. The Comet assay on these samples was performed as described in Section 5.2.1.

5.2.3

Fresh Blood

For comparison between DNA-damage levels in frozen and thawed blood and in fresh blood, the samples were divided into four aliquots. Two of these served as controls and two were irradiated with 2 Gy of X-rays. Five mL of blood from each of these aliquots, one control and one irradiated, was mixed with 45 mL of freshly made freezing solutions (Protocol I, RPMI 1640 having 10% bovine serum albumin and 15% DMSO and Protocol II, RPMI 1640 medium with 20% DMSO). 50 mL of the mixture, having blood and freezing medium, was mixed with 50 mL of 0.7% high-resolution agarose 3:1 and microgels were made. The Comet assay was performed on these samples as described in Section 5.2.1.

5.2.4

Fresh Blood Stored on Ice Prior to Freezing

In another experiment DNA damage was assessed in leukocytes from heparinised whole blood, stored on regular ice for various time points before freezing


123

at 80 1C. These conditions were designed to simulate the storage and transportation of blood drawn in the field. In these experiments, the blood sample was divided into three aliquots. These aliquots were stored in a 2 mL cryovial on regular ice (without dilution) for various time points (1, 8, and 24 h). Samples were then frozen at 80 1C (100 mL blood was mixed with 900 mL of RPMI 1640 having 20% DMSO). The samples were then thawed after 1 month. A 50-mL thawed blood sample was mixed with 50 mL of 0.7% high-resolution agarose and microgels were made. The Comet assay was performed on these samples as described in Section 5.2.1.

5.2.5

Image and Data Analysis

Images of 100 cells from each blood sample were captured at 400 magnification using a charge-coupled device (CCD) camera, GW525x (Genwac Inc., Orangeburg, NY) attached to a DMLB epifluorescence microscope (Leica, Germany) with an excitation filter of 490 nm, a 500-nm dichroic filter, and an emission filter of 515 nm. Images were analysed using the VisComet imageanalysis software (Impulse Bildanalyse GmbH, Gilching, Germany). Tail and head were distinguished from each other by a vertical boundary calculated by the imaging software from an integrated horizontal profile. Comet tail moment was calculated by multiplying tail distance in pixels by the fraction of DNA in the tail. One hundred images per slide were analysed for tail moment and averages from 3 slides per data point were used to calculate means and standard deviations that were used in subsequent statistical analysis. Data were analysed by one-way ANOVA and difference between groups compared by the Newman–Keuls multiple range test. Difference at Po 0.05 was considered statistically significant.

5.3 Results and Discussion Levels of DNA damage in nonirradiated samples frozen using the two protocols were not significantly different from each other, and were comparable with those found in fresh blood samples (Figure 5.1). There were significant differences between controls and their respective X-ray irradiated samples, (Po0.001 for Protocol I, II and fresh blood). Typical DNA migration patterns in control and X-ray irradiated samples are shown in Figures 5.2 and 5.3, respectively (both images were from frozen samples using Protocol II). There were also significant differences in X-ray induced levels of DNA damage between the two protocols tested (Po0.001). Samples frozen using Protocol I revealed higher levels of X-ray-induced DNA breaks (tail moment ¼ 4897 527) than those frozen using Protocol II (tail moment ¼ 3459 439). Also, DNA-damage levels were significantly higher in samples irradiated and processed using Protocol I compared to any other samples. These differences are likely to be due to BSA used in Protocol I as it

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Figure 5.1

Chapter 5

Bar graph comparison of DNA single-strand breaks (tail moment) in control samples and samples irradiated with 2 Gy of X-rays and then frozen at 80 1C using two different freezing protocols and fresh blood. Fresh blood sample labels include a roman numeral in parentheses to indicate the corresponding freezing solution used for diluting blood in agarose to make microgels. Each bar represents a mean of averages from 100 cells and the error bar represents standard deviation of means. All experiments were done in triplicate.

may have changed the gel matrix of the second layer of microgel allowing retention of smaller pieces of DNA lost during lysis step. Using Protocol I, we were able to retain very small pieces of DNA (including from highly damaged and apoptotic cells, data not shown) in microgels. In studies where blood is drawn from several subjects on the same day, samples are usually stored on ice for various times prior to freezing. Table 5.1 shows that, using Protocol II, blood samples can be stored on regular ice for at least 1 h and then, frozen and maintained at 80 1C for up to 30 days without a significant difference in results. One-way ANOVA showed a significant time effect (i.e. time of storing on ice before freezing) F3,8 ¼ 68.03, po0.0001. A Newman–Keuls test comparing time points showed that DNA damage was significantly higher in the samples stored on ice for 8 and 24 h before freezing, compared to samples frozen immediately. There was no significant difference between samples stored on ice for an hour and the samples frozen immediately. Our results indicate that Protocol I and Protocol II may be useful for longterm storage of a variety of cells at 80 1C. These protocols differ from reported protocols7,8 for freezing whole human blood at 80 1C for DNA-damage


125

Figure 5.2

Photomicrograph of DNA migration pattern from normal human leukocytes frozen using Protocol II and assessed for DNA damage by alkaline microgel electrophoresis (Comet assay). Magnification: 400. Dye: YOYO 1.

Figure 5.3

Photomicrograph of DNA migration pattern from irradiated (2 Gy) human leukocytes frozen using Protocol II and assessed for DNA damage by alkaline microgel electrophoresis (Comet assay). Magnification: 400. Dye: YOYO 1.

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Table 5.1

Chapter 5

DNA single-strand breaks in blood samples stored on ice and frozen using Protocol II. Tail moment

Hours on ice Mean SD

0

1 a

2026 101

2019 46

8 a

24 b

2243 95

2850c 75

Table shows means and standard deviation (SD) of averages from 3 slides per time point. One hundred cells per slide were analysed for tail moment and their average was calculated. Means with a letter in common are not significantly different (Newman–Keuls multiple comparison test).

assessment, mainly in dilution of whole blood and concentrations of DMSO and BSA. Determination of the ideal concentrations of cryoprotectants was made after extensive experimentation. Freezing solutions having a DMSO concentration from 5 to 20% were tried before determining the ideal DMSO final concentrations. For Protocol I, 13.25% was used and for Protocol II, 18.0% was used. For Protocol I, these concentrations of DMSO were used with various concentrations of BSA (1, 5, and 10%) and since lower BSA concentrations resulted in higher levels of DNA damage, a final concentration of 9% BSA was determined to be ideal in conjunction with 13.25% DMSO. Use of DMSO concentrations of up to 10%12 and BSA concentrations of up to 12.5%13 have been reported previously for freezing cells. However, the combination of high concentrations of DMSO and BSA used in Protocol I have not been reported. These high concentrations of BSA and DMSO provide a strong antioxidant effect. BSA is used in Protocol I because it is a cryoprotective agent13 and has antioxidant activity.14 DMSO is used in the protocols because it is a known membrane stabiliser, cryoprotective agent and antioxidant. Both of these provide good preservation of whole blood at 80 1C.7 Most commonly, DMSO is used in a 5%7,15 to 10%8,16 final concentration for the freezing of human whole blood for DNA-damage assessment. However, in our laboratory, the 10% DMSO concentration in freezing cocktails used in the above-mentioned studies was found to be inadequate for the detection of low levels of DNA-strand breaks in whole blood leukocytes stored at 80 1C. This led us to develop a freezing protocol for studies where blood from a large number of subjects is collected in the field and shipped to a distant laboratory for DNA-damage analysis in leukocytes. We also experimented using various dilutions of whole blood (1:1, 1:10 and 1:100). There was no significant difference between the concentrations and for convenience we used only 100 mL of blood per mL of freezing solution. Thus, cell concentration does not seem to affect the level of damage due to cryopreservation, in agreement with earlier reports.13,17 Additionally, in thawing the frozen samples, temperatures lower than 37 1C and a thawing duration of more than 65 s resulted in higher levels of DNA damage, particularly in irradiated samples (data not shown). Both Protocols I and II may allow the storage of a large number of blood samples at 80 1C for assessment of DNA damage/repair in a


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population for periods of at least 1 month. Both protocols are highly precise and reproducible: no significant day-to-day variations were observed in the Comet assay results when aliquots from the same sample were processed on different days (intrasample variations) or samples drawn from the same person on different days (intersample variations) [data not shown]. Additionally, the freezing solutions used in either protocol can be prepared ahead of time and stored for up to 7 days without any significant difference in the results [data not shown]. Although Protocol II may be more suitable for larger scale studies where ease of use and a minimum of steps are important, Protocol I offers slightly greater sensitivity in detection of DNA damage and may provide a more accurate assessment of subtle genotoxic effects.

References 1. G. Frenzilli, C. Betti, T. Davini, M. Desideri, E. Fornai, L. Giannessi, F. Maggiorelli, P. Paoletti and R. Barale, Evaluation of DNA damage in leukocytes of ex-smokers by single-cell gel electrophoresis, Mutat. Res., 1997, 375, 117. 2. M. Toraason, D. W. Lynch, D. G. DeBord, N. P. Singh, E. Krieg, M. A. Butler, C. A. Toennis and J. B. Nemhauser, DNA damage in leukocytes of workers occupationally exposed to 1-bromopropane, Mutat. Res., 2006, 603, 1. 3. D. Anderson, A. Yardley-Jones, C. Vives-Bauza, W. Chua-Anusorn, C. Cole and J. Webb, Effect of iron salts, haemosiderins, and chelating agents on the lymphocytes of a thalassaemia patient without chelation therapy as measured in the Comet assay, Teratog. Carcinog. Mutagen., 2000, 20, 251. 4. S. J. Duthie, L. Pirie, A. M. Jenkinson and S. Narayanan, Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability, Mutagenesis, 2002, 17, 211. 5. S. I. Tsilimigaki, N. Messini-Nikolaki, M. Kanariou and S. M. Piperakis, A study on the effects of seasonal solar radiation on exposed populations, Mutagenesis, 2003, 18, 139. 6. L. E. Knudsen, M. Gaskell, E. A. Martin, P. T. Poole, P. T. Scheepers, A. Jensen, H. Autrup and P. B. Farmer, Genotoxic damage in mine workers exposed to diesel exhaust, and the effects of glutathione transferase genotypes, Mutat. Res., 2005, 583, 120. 7. C. H. Chuang and M. L. Hu, Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay, Mutat. Res., 2004, 564, 75. 8. I. Hininger, A. Chollat-Narny, S. Sauvaigo, M. Osman, H. Faure, J. Cadet, A. Favier and A. M. Roussel, Assessment of DNA damage by Comet assay on frozen total blood: method and evaluation in smokers and non-smokers, Mutat. Res., 2004, 558, 75.

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9. P. Mazur, Cryobiology: the freezing of biological systems, Science, 1970, 168, 939. 10. G. J. Morris, E. Acton and S. Avery, A novel approach to sperm cryopreservation, Hum. Reprod., 1999, 14, 1013. 11. J. A. Gilmore, J. Liu, E. J. Woods, A. T. Peter and J. K. Critser, Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods, Hum. Reprod., 2000, 15, 335. 12. C. M. Celuzzi and C. Welbon, A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment, Transfusion, 2003, 4, 3488. 13. M. L. Disis, C. de la Rosa, V. Goodell, L. Y. Kuan, J. C. Chang, K. KuusReichel, T. M. Clay, H. Kim Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino and H. T. Maecker, Maximizing the retention of antigen specific lymphocyte function after cryopreservation, J. Immunol. Methods, 2006, 308, 13. 14. B. Chalidis, N. Kanakaris and P. V. Giannoudis, Safety and efficacy of albumin administration in trauma, Expert Opin. Drug Saf., 2007, 6, 407. 15. E. W. Fiebig, D. K. Johnson, D. F. Hirschkorn, C. C. Knape, H. K. Webster, J. Lowder and M. P. Busch, Lymphocyte subset analysis on frozen whole blood, Cytometry, 1997, 29, 340. 16. M. L. Hu, C. H. Chuang, H. M. Sio and S. L. Yeh, Simple cryoprotection and cell dissociation techniques for application of the Comet assay to fresh and frozen rat tissues, Free Radic. Res., 2002, 36, 203. 17. B. Feuerstein, T. G. Berger, C. Maczek, C. Ro¨der, D. Schreiner, U. Hirsch, I. Haendle, W. Leisgang, A. Glaser, O. Kuss, T. L. Diepgen, G. Schuler and B. Schuler-Thurner, A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use, J. Immunol. Methods, 2000, 245, 15.

CHAPTER 6

Development and Applications of the Comet-FISH Assay for the Study of DNA Damage and Repair VALERIE J. MCKELVEY-MARTIN AND DECLAN J. MCKENNA Biomedical Sciences Research Institute, University of Ulster, Coleraine, Northern Ireland, BT52 1SA

6.1 Introduction The single-cell gel electrophoresis (SCGE) assay was first reported by O¨stling and Johanson1 in 1984 as a technique for visualising the migration of DNA containing strand breaks in individual agarose-embedded cells under electrophoretic conditions. A few years later, Singh et al.2 used a similar method, but modified it slightly to use highly alkaline (pH413) conditions that encourage unwinding of DNA around a strand break. In both studies, the underlying principle of the assay is that when DNA is subjected to an electric current, DNA containing strand breaks will migrate through an agarose gel due to relaxation of the DNA supercoils, whilst unbroken DNA remains immobile. Following the staining of DNA with a fluorescent DNA-specific dye, the resulting image can be visualised and resembles a comet, with undamaged DNA forming a ‘‘head’’ and damaged DNA forming a ‘‘tail’’, an observation that has led this technique to be more commonly called the Comet assay.3


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Since then, it has become a widely accepted and versatile method for measuring a variety of DNA lesions in individual cells.4–6 Its versatility stems from various modifications to the original Comet assay that have been developed in order to measure different types of DNA damage, including single- and doublestrand breaks, crosslinking and oxidative damage, as well as DNA breaks associated with replicating DNA and DNA repair (reviewed in Collins6). One further modification is the combination of fluorescent in situ hybridisation (FISH) with the Comet assay, in which fluorescently labelled probes are hybridised to a specific gene sequence, region or chromosome. Thus, the localisation of a particular DNA sequence within an individual comet can be visualised, thereby providing information about DNA damage in and around the probed region, compared to overall DNA. Further information about whether or not DNA breakage occurs within the probed region is obtained by observing changes in the number of hybridisation signals in the comet. This review discusses the methods for employing the Comet-FISH assay in the laboratory and summarises the various studies that have successfully used this approach, examining the different applications and uses of this versatile technique. The potential and limitations of the assay are also considered and the importance of these limitations on accurate data interpretation is emphasised.

6.2 The Comet-FISH Assay Procedure Several slightly different protocols exist for the Comet assay, but all follow the same basic premise.7 Cells under investigation are embedded in an agarose gel on microscope slides and are subsequently lysed by placing the slides in a highsalt solution (usually containing detergent). Following this lysis step, which typically occurs for 1 to 24 h at 4 1C, the slides are then placed into an electrophoresis tank filled with buffer and are subjected to DNA unwinding and electrophoresis. The lysis and electrophoresis may be carried out under alkaline8 or neutral9 pH conditions depending on whether the investigator wishes to examine single-strand breaks, alkali-labile sites and/or double-strand breaks. Modifications that allow analysis of crosslinking DNA damage10,11 or that employ restriction enzymes12,13 can also be incorporated as these depend on the production of DNA breaks for indirect measurement. Depending on the damaging agent being used, cells may be treated in culture (e.g. with chemicals14) prior to collection for slide preparation, or alternatively can be treated on the slides immediately after they are embedded in agarose (e.g. with radiation15). In the standard Comet assay, the cells are finally stained with a DNA-specific dye such as ethidium bromide or propidium iodide to allow visualisation under the fluorescent microscope. However, in the Comet-FISH assay, this staining step is omitted and instead the cells on the prepared slides are subjected to an extra hybridisation protocol. This can be performed following either the alkaline or neutral version of the Comet assay16 and the steps involved in this procedure are discussed below and summarised in Table 6.1.

Conditions

Standard Comet assay conditions Slide preparation : 1–2 h Lysis : 1–24 h Unwinding & Electrophoresis : 1 h Neutralisation : 20 min

Immerse slides in 0.5 M NaOH/1 M NaCl for 30 min at room temperature.

Sequential immersion of slides in 70%, 85%, 100% v/v ethanol for 5 min each. Leave slides to air dry on tissue paper.

Probe mixture prepared according to manufacturer’s protocol for in situ hybridisation. Probe is denatured at specified temperature in heating block. Alternatively, heat codenaturation can be used.

Probe is applied to agarose gel and coverslip applied on top. Hybridisation occurs in a humidified dark box overnight at 37–42 1C.

Comet assay

Alkaline denaturation

Slide dehydration

Probe preparation

Hybridisation

Cells may be treated prior to slide preparation or on slides if protocol allows. Specific times for each step may vary from protocol to protocol, but should be kept constant between experiments. This step can be omitted if a heat codenaturation step is used for hybridisation (see hybridisation step below) Slides can be stored until ready for hybridisation. Dehydrated slides can be stored for several weeks at room temperature. Rapid drying (e.g. at 37 1C) may cause gels to crack Approximately 10 mL total hybridisation volume is needed for a 22 22 mm gel. Probe should be labelled with suitable fluorophore for visualisation on user’s imaging system. If heat codenaturation hybridisation is being performed place slide on hotplate at correct temperature (typically 75–80 1C) and add probe followed quickly by coverslip. Leave for 2 mins, then remove slide and place in humidified dark box overnight at 37–42 1C. Slides should be prewarmed prior to probe application to aid efficient hybridisation.

Notes

Steps in the Comet-FISH assay. This gives an overview of the sequential steps involved in the Comet-FISH assay. For further reading and information, a detailed account of the Comet assay procedure is provided by Olive and Bana´th,7 whilst a discussion of the Comet-FISH assay procedure can be found in Rapp et al.16

Step

Table 6.1

Development and Applications of the Comet-FISH 131

Slides washed in sequential washing solutions. Typical wash solutions would be :- (i) 2X SSC/50% formamide, pH 7.0 (3 5 mins at 45 1C) (ii) 2X SSC, pH 7.0 (1 5 min at 45 1C) (iii) 2X SSC/0.1% Igepal pH 7.0 (1 5 min at 45 1C) Add suitable counterstain and place coverslip on top. Store slides in dark box at 4 1C until viewing.

View images using appropriate filter set and microscope.

Count 25 or 50 cells per slide using standard comet analysis. Record number and location of hybridisation signals

Posthybridisation washes

Imaging

Analysing

Counterstain

Conditions

(Continued ).

Step

Table 6.1

Counterstain should have clearly separate excitation/ emission spectra from probe fluorophore. Slides can also be stored overnight in the dark at 20 1C. However, viewing immediately after staining is preferable as fluorescent signals may fade. To aid visualisation of hybridisation signals, 60 magnification or more is preferable. Care should be taken to select suitable fluorophores for probes/counterstain, ensuring a minimum of overlap in the excitation/emission spectra. Likewise, the microscope and filter settings should be calibrated to minimise bleed-through of signals Triplicate experiments are recommended. Several published studies have analysed between 25 and 50 cells per slide. However, it is recommended that not less that 50 cells are counted without statistical verification to justify using fewer numbers.

Slides should be delicately treated to ensure gels do not dislodge from slide. Excessive shaking should be avoided.

Notes

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Development and Applications of the Comet-FISH

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Fluorescent probes are hybridised to the agarose-embedded cells using standard in situ hybridisation techniques. Typically, the probe is mixed with a hybridisation buffer and denatured by heating, before being added to the slides. The DNA of cells contained on the slides is also denatured, either by alkaline treatment prior to the hybridisation15or by heat.8 If heat denaturation is preferred, it is usual to carry out codenaturation of the probe and target cellular DNA simultaneously on a hotplate. The slides are then placed in a humidified box overnight at the appropriate temperature (typically 37–42 1C) to allow hybridisation to occur. Following this, posthybridisation washing steps are carried out, whereby the slides are moved through a series of wash buffers to remove any unbound probe and minimise nonspecific binding. Finally, the overall cellular DNA is counterstained with a suitable fluorescent dye. During microscopic examination, specific hybridisation signals need to be clearly visible against the counterstained total genomic DNA, therefore care should be taken to ensure there is minimum overlap in the excitation and emission spectra for the different fluorophores used. Likewise, it is of utmost importance that the microscope and filter settings being used for analysis are calibrated correctly to allow clear visualisation of cells and minimise bleed-through of fluorescent signals, which can otherwise interfere with analysis of cells. Analysis of cells involves examining both the distribution of total genomic DNA in the comet, together with the number of hybridisation signals, and the location of each signal. Overall DNA distribution is measured using standard Comet assay analysis software, whereby the amount of DNA in the head and tail of each selected cell is assessed using a number of parameters, including % tail DNA and Olive tail moment, the two preferred measurements of DNA damage in Comet assay experiments. Then, in the same selected cells, the number of signals and the position of each hybridisation signal in the head or tail of the comet is recorded, thereby giving an indication of whether it lies in, or close to, a region of damaged DNA. The appearance of hybridisation signals in the comet tail generally indicates that the region of DNA within, or around, the probe contains strand breakage. Information about where exactly the DNA breakage occurs in relation to the probed region is obtained by counting the frequency distribution of signals in each comet. Increase in signal number would suggest the probed region itself contains strand breakage, since the probe will bind to each broken DNA fragment from the target region. Of course, control cells must always be included to give an indication of baseline damage for both overall DNA and hybridisation signals. Figure 6.1 shows representative examples of images from Comet-FISH experiments, demonstrating the differences between an unirradiated, control cell (Figure 6.1(a)) and an irradiated, damaged cell (Figure 6.1(b)). Similar images can be obtained using a variety of different DNA-damaging agents to generate comets that are visualised using selected fluorescent stains/probes. Both cells have been hybridised with Spectrum Orange-labelled TP53 probe, which fluoresces with a pink/orange colour, and total genomic DNA counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI), which fluoresces blue. Visualisation was performed using an epifluorescence microscope (Nikon

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Figure 6.1

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Representative images of cells processed using the Comet-FISH protocol. (a) Unirradiated control cell showing little DNA damage as evidenced by the absence of a comet tail. Two hybridisation signals (bright spots) are clearly visible in the intact head. (b) Immediately after exposure to g-radiation a large comet tail is visible, reflecting the extent of DNA damage. Several p53 hybridisation signals are located in the comet tail, indicating that strand breakage has occurred near and/or within the probed region.

Eclipse E400) fitted with a Nikon X60 fluor lens (Plan Apo 60:1 N.A. 1.4) and equipped with a Hamamatsu Orca digital CCD camera. A double bandpass filter set (Chroma HiQ) tuned for DAPI (excitation 370 nm, emission 450 nm) and spectrum orange (excitation 560 nm, emission 590 nm) was utilised, allowing simultaneous detection of DAPI and Spectrum Orange labels.


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Using this novel approach, several interesting papers have been published over the past decade in which the Comet-FISH assay has been utilised to investigate the cellular response following DNA damage.

6.3 Applications of the Comet-FISH Assay 6.3.1

Discovery of the Comet-FISH Assay

Two independent laboratories were the first to report the Comet-FISH assay as a new technique. In 1997, Santos et al.17 used the technique to detect centromeres and telomeres of chromosomes, as well as regions of the O6-methylguanine DNA methlytransferase (MGMT) gene, in human lymphocytes and concluded that the method offered potential for studying chromatin ultrastructure and spatial organisation in the nucleus. At the same time, another study, by McKelvey-Martin et al.,8 successfully hybridised a locus-specific identifier (LSI) probe for TP53 to both human leukocytes and bladder cancer cells and proposed the Comet-FISH assay as a powerful tool for investigating damage and repair in specific DNA sequences in individual cells. Together, these two studies provided a springboard for many other studies to investigate DNA damage and repair and nuclear chromatin arrangement. These are discussed below and details of each experiment presented in Table 6.2.

6.3.2

Using Comet-FISH to Measure DNA Damage

Comet-FISH studies using whole chromosome paint (WCP) probes have generated interesting results about the susceptibility of different chromosome regions to DNA damage. Differences in DNA damage have been detected between chromosomes in human lymphocytes exposed to UV-A, with telomeres shown to be more sensitive than centromeres.18 In a similar experiment on human lymphocytes, an inverse correlation was shown between density of active genes and sensitivity to UV-A damage, which the authors proposed was due to the higher repair enzyme activity located at active gene loci.19 In a further experiment, exposure of healthy mucosal cells taken from patients with oropharyngeal carcinoma to benzo(a)pyrene-diolepoxide (BPDE) resulted in higher strand breakage in chromosomes 3, 5 and 8 compared to chromosome 1.20 Since chromosomes 3, 5 and 8 are known to show alterations in carcinomas of the upper aerodigestive tract, this led to the suggestion that the Comet-FISH assay was a method that could allow the detection of gross chromosomal aberrations and selected genetic alterations in cells.20 Hence, it may prove useful for investigating other chromosomes and large DNA regions for susceptibility to DNA damage in response to a wide variety of DNA-damaging agents. Furthermore, it offers potential as a predictive test for detecting specific endogenous risk factors or genetic biomarkers for disease, an extension of an idea already postulated for the Comet assay alone since it is a method

Primary human colon cells and colon adenoma cells (LT97) Primary human colon cells and colon adenoma cells (LT97)

Primary human colon cells and colon adenoma cells (LT97)

Texas Red-labelled probe to TP53 (10.6 Mb) Texas Red-labelled probe to TP53 (10.6 Mb)

Cells treated in vitro with uranyl-nitrilotriacetate. (UNTA)(0–1000 mM)

Digoxygenin- or Texas Red-labelled probes to APC, KRAS & TP53

Chromosome band probes 5q31 (200 kB) and 11q23 (350 kB)

Texas Red-labelled probe to TP53 (10.6 Mb)

Probe

Cells treated in vitro with FeNTA(1–1000 mM)

Cells treated in vitro with ferric-nitrilotriacetate. (Fe-NTA) (1–500 mM) [Alkaline Comet assay using Endo III; Fpg] Cells treated in vitro with melphalan, etoposide or hydroquinone (HQ) (final concentration for all compounds ¼ 0.25%) Cells treated in vitro with hydrogen peroxide (H2O2) (0–150 mM), trans-2-hexenal (0–1600 mM) & 4-hydroxy-2nonenal (HNE)(0–250 mM)

Human leukocytes

Human lymphoblastoid cell line (TK6)

Treatment

All 3 compounds induced DNA breaks at both probed regions. HQ caused more DNA damage at 5q31 than 11q23 All 3 regions showed increased dose-dependent migration of signals into tails for all 3 compounds. TP53 more sensitive to damage than KRAS, APC and overall DNA in H2O2-treated primary colon cells and in all cells treated with HNE & trans-2-hexenal Fe-NTA enhanced migration of TP53 signals into comet tail in both cell types U-NTA enhanced migration of TP53 signals into comet tail in both cell types

Fe-NTA enhanced migration of TP53 signals into comet tail

Results

32

33

29

25

36

Ref.

Summary of Comet-FISH studies.a This summarises the various Comet-FISH experiments published to date. Where known, details of probe label and size are given, as well as treatment conditions for cells and chemical concentrations used. All experiments utilised the alkaline version of the Comet assay, unless otherwise indicated in the Treatment column

Cell type

Table 6.2

136 Chapter 6

Cells treated in vitro with UVC (1 J/m2) OR with H2O2 (0.2 mM) OR with photosensitiser Ro 19-8022 f/b irradiation at 0.33 m with 1000 W halogen lamp.

DHFR gene, Exon 1-biotinylated Exon 6-fluorescein; MGMT gene, bases 8–33 – biotinylated, bases 463–488 – fluorescein; TP53 gene, exon 2 – biotinylated, exon 11-fluorescein (all probes 26bp)

Whole chromosome paint (WCP) probes to Chr 1, 3, 5&8

Cells treated in vitro with benzo(a)pyrene-diolepoxide (BPDE) (9 mM)

CHO cell line

Telomere-specific PNA probes

Cells treated in vitro with bleomycin (0–100 IU/ml) or mitomycin C (MMC) (0– 100 mg/ml)

Chinese hamster ovary (CHO) cell line, human fibrosarcoma cell line (HT1080), acute lymphoblastic leukemia cell line (CCRF-CEM) Healthy oropharyngeal mucosal cells biopsied during surgery of oropharyngeal carcinoma (10 patients)

Both TP53 and HER-2 loci more susceptible than ZNF217 loci to damage by irradiation and H2O2

TP53 probe (145 kB); HER-2 probe (190 kB); ZNF217 probe (320 kB) – all labelled with either Spectrum Orange or Spectrum Green Telomere-specific peptide nucleic acid (PNA) probes Cisplatin reduces telomere signal migration more than total DNA, indicating crosslinking by cisplatin is preferentially telomeric Dose-dependent detection of telomeric signals in comet tail observed for CHO, CCRF-CEM, but less so for HT1080 Significantly higher damage in Chr 3, 5, & 8 compared to Chr 1 in healthy mucosa of patients with oropharyngeal carcinoma Signals from both TP53 probes located in tail of damaged cells. However, DHFR probe signals were rarely observed in comet tail and only one of MGMT signals appeared consistently in tail. Preferential repair of strand breaks in TP53 gene and oxidised bases in MGMT gene observed

All 3 regions showed increased dose-dependent fragmentation after irradiation

Biotinylated probes to Abl (127 kB), TP53 (127 kB), Ret(150 kB)

Cells treated in vitro with bleomycin (0–100 IU/ml) and/or cisplatin

Mice irradiated with 0–4 Gy X-rays. Cells collected 30 min, 24 h and 30 days after exposure Cells irradiated in vitro with 2 and 10 Gy g-radiation OR exposed to H2O2 (100 mM) [Neutral Comet assay]

Human peripheral blood cells

Normal mammary epithelium (AG11134) and breast cancer cell line (MDA-MB468)

Mouse peripheral leukocytes

35

20

24

23

34

37


Treatment

Cells treated in vitro with bleomycin (0–100 IU/ml) or MMC (0–100 mg/ml)

Cells treated in vitro with H2O2 (0–150 mM) OR trans2-hexenal (0–1600 mM)

Cells treated in vitro with H2O2 (18.8–150 mM) OR HNE (100–250 mM)

Cells treated in vitro with g-radiation (0–10 Gy)

Cells treated in vitro with MMC (0–200 mg/ml)

Cells irradiated in vitro with X-rays (0–52 Gy) [Alkaline and neutral Comet assay both used]

Human peripheral blood cells

Primary human colon cells

Primary human colon cells & human colon adenoma cell line (LT97)

Bladder cancer cell lines (RT4 & RT112)

Bladder cancer cell line (RT4)

Human peripheral blood leukocytes, double-strandbreak repair deficient fibroblast cell line (MO59J) and parental fibroblast cell line (MO59 K)

(Continued ).

Cell type

Table 6.2

Biotinylated whole human genome probe

Spectrum Orange-labelled probe to TP53 (B145 kB)

Spectrum Orange-labelled probe to TP53 (B200 kB)

Digoxygenin-labelled probe to TP53 (B80 kB)

Digoxygenin-labelled probe to TP53 (B80 kB)

Telomere-specific PNA probes

Probe Breakage frequency for DNA in vicinity of telomeres found to be proportional to overall DNA TP53 gene region was more sensitive than global DNA to damage caused by H2O2 and trans-2-hexenal TP53 gene region was more sensitive than global DNA to damage caused by HNE in both primary cells and cell line Repair of strand breaks in TP53 gene region was faster than overall DNA during first 15 min following 5 Gy irradiation Repair of MMC-induced crosslinks in TP53 gene region was faster than overall DNA during first 4 h following treatment DNA breakage detection (DBD)-FISH successfully distinguished between double- and single-strand breaks

Results

38

14

15

30

31

22

Ref.

138 Chapter 6

Cells treated in vitro with endonculeases DNase I, FokI or EcoRI [Alkaline & Neutral Comet assay both used]

Cells irradiated in vitro with UV-A (500 kJ/m2)

Cells irradiated in vitro with UV-A (500 kJ/m2)

HT1376 cells treated in vitro with g-radiation (10 Gy)

Vicia faba plant cells

Human lymphocytes

Human lymphocytes

Human leukocytes & bladder cancer cell line (HT1376)

Spectrum Orange-labelled TP53 probe (B200 kB)

Various digoxygenin-labelled probes to centromeres, asatellites, telomeres & whole chromosomes, c-myc, TP53 and TP58

WCP probes to Chr 1, 2, 3, 8, 9, 11, 14, 18, 19, 21, X & Y

Digoxgenin-labelled probes to FokI repeat (B59 bp), 25SrDNA gene, intergenic space and telomere repeat. Increased FokI signals in tails of cells treated with FokI. rDNA signals were randomly distributed for FokI and DNase treated cells. rDNA and telomere signals rarely found in tail in EcoRI treated cells Inverse correlation found between density of active genes and sensitivity to UV-A Telomeres more sensitive than centromeres to UV-A induced damage. c-myc locus more sensitive to chromosome breakage than TP53 and TP58. Chr X more sensitive than Chr 1 to UV-A Successful visualisation of TP53 hybridisation spots in head and tail of comets 8

18

19

27


No treatment [Neutral Comet assay]

Human lymphocytes

Biotinylated SO-aAllCen probes to all centromeres (B86 bp); biotinylated probes to all telomeres; biotinylated probe to Chr 7 centromere ; 3 biotinylated probes to segments of MGMT ; digoxygenin-labelled probe to Chr 3 long arm telomere

Probe Centromeres demonstrate dispersed localisation along migrated DNA. Telomeres localized as concise nodules near nuclear membrane MGMT signals detected in both comet head and tail

Results 17

Ref.

Abbreviations. Fe-NTA: ferric-nitrilotriacetate, Endo III: endonuclease III, Fpg: formamidopyrimidine-DNA glycosylase, TP53: tumour protein 53, HQ: hydroquinone, H2O2: hydrogen peroxide, HNE: hydroxyl-2-nonenal, APC: adenomatous polyposis coli, U-NTA: uranyl-nitrilotriacetate, MMC: mitomycin C, PNA: peptide nucleic acid, BPDE: benzo(a)pyrene-diolepoxide, WCP: Whole chromosome paint, Chr: chromosome, DHFR: dihydrofolate reductase, MGMT: O6-methylguanine-DNA methyltransferase, CHO: Chinese hamster ovary, DBD: DNA breakage detection, SO-aAllCen: synthetic oligomer-a all centromeres.

a

Treatment

(Continued ).

Cell type

Table 6.2

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that is reasonably fast, sensitive and requires only a few cells to generate results.21 The potential use of the Comet-FISH assay in understanding disease progression is further underlined by a number of studies that have used telomerespecific peptide nucleic acid (PNA) probes to investigate DNA breakage in, and adjacent to, telomeres. These studies have demonstrated that telomeric DNA damage can be detected in human blood leukocytes exposed to bleomycin, mitomycin C (MMC) and cisplatin.22–24 Thus, an insight can be gained into telomere fragility, which is of great importance in aging and malignant transformation of cells. More recent experiments using chromosome band probes have also demonstrated breakage at 5q31 and 11q23 loci in human lymphoblastoid TK6 cells following in vitro exposure to melphalan, etoposide or hydroquinone (HQ), with HQ causing increased damage at 5q31 in particular.25 Since many malignant cells demonstrate large-scale chromosomal abnormalities, the Comet-FISH assay could prove to be a useful approach for detecting such damage. Moreover, these studies also lend credence to the suggestion that DNA damage is not random throughout the genome and is likely to be affected by nuclear architecture and ultrastructure.26 Thus, the Comet-FISH assay also shows promise as a valuable technique for investigating the role that higher-order chromatin structure has in influencing the susceptibility of different DNA regions to damage. Comet-FISH studies are not just restricted to mammalian cells. A study on Vicia faba plant cells used DNA probes to specific chromosomal domains, such as FokI element-containing heterochromatin, nucleolus-organising regions (NORs) and telomeres, following treatment of the cells with various endonucleases.27 This study demonstrated that the distribution of FISH signals between comet head and tail reflected the distribution of restriction endonuclease cleavage sites within these domains and proposed that the technique would allow localisation of various DNA-damage endpoints in genotoxicity studies. Since the Comet assay is already widely used in genotoxicity testing, employing the Comet-FISH assay as well affords the extra opportunity to gain genetic information about DNA damage in specific regions of the genome. With respect to specific gene loci, many studies have used the Comet-FISH assay to detect DNA damage and repair in response to different DNAdamaging agents. In particular, the TP53 gene has been investigated by several studies, since damage to this crucial gene plays a major role in the development of cancer. Although an early Comet-FISH study showed little damage in the TP53 gene in human lymphocytes exposed to UV-A,18 subsequent studies have demonstrated that damage to this region occurs in human bladder cancer cells in response to both g-radiation15,28 and MMC.14 Similarly, damage to this gene region has also been detected by the Comet-FISH assay in primary human colon cells and colon adenoma cells following treatment with hydrogen peroxide (H2O2), trans-2-hexenal and 4-hydroxy-2-nonenal (HNE).29–31 This damage reflects the fact that these toxic compounds are produced in vivo via oxidative stress mechanisms, such as lipid peroxidation that produces

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the compounds trans-2-hexenal and HNE, and may contribute to cancer development by reacting with DNA bases. Moreover, Glei et al.29 also demonstrated significantly different sensitivities between TP53, APC and KRAS genes in colon cells. In their experiments, dose-dependent migration of hybridisation signals into comet tails was observed for all 3 loci in both primary colon cells and colon adenoma cells in response to H2O2, trans-2hexenal and HNE. However, the TP53 region was more sensitive to damage than KRAS, APC and overall DNA in H2O2-treated primary colon cells and in all cells treated with HNE & trans-2-hexenal, particularly in HNEtreated adenoma cells. Since alterations in APC and KRAS are early events in colon carcinogenesis, they concluded that all three compounds tested could potentially initiate cancer, but only HNE was important for cancer progression. The Comet-FISH assay has also detected damage to the TP53 gene region in primary human colon cells and colon adenoma cells following treatment with uranyl nitrilotriacetate (U-NTA)32 and ferric nitrilotriacetate (Fe-NTA)33 which emphasised the potentially genotoxic effects of iron and uranium in vivo through their interactions with DNA. Damage to both TP53 and HER-2/neu gene regions has been shown in g-irradiated breast cancer cells,34 whilst H2O2treated human lymphocytes35 and Fe-NTA-treated human leukocytes also demonstrate damage in the TP53 gene.36 In leukocytes from X-ray-irradiated mice, the Comet-FISH assay has been used to detect damage in TP53, Ret and Abl1,37 whilst increased susceptibility of the c-myc gene was detected in human lymphocytes exposed to UV-A.18 Taken together, these studies demonstrate the ability of the Comet-FISH assay to measure the extent of DNA damage in specific genes and DNA regions, particularly those related to disease progression. It could therefore prove to be a valuable tool in understanding the cellular response to damage and its biological effects and may also prove to be useful in deriving appropriate and effective interventions in disease pathology.

6.3.3

Using Comet-FISH to Quantify DNA Repair

DNA repair of specific genes/gene regions can also be measured using the Comet-FISH assay. Fernandez et al. performed FISH using a whole genome probe following both neutral and alkaline versions of the Comet assay, calling their technique DNA-breakage detection-FISH (DBD-FISH).38 They demonstrated that damage and repair of both double-strand breaks and single-strand breaks could be analysed simultaneously in irradiated leukocyte cells by analysis of fluorescence intensity and surface area of each comet and concluded that the use of different probes would allow similar analysis of repair in specific DNA sequences. Indeed, several studies have shown some intriguing results for the TP53 gene. In our laboratory we have demonstrated that the actively transcribed TP53 gene region in bladder cancer cell lines is preferentially repaired in comparison to the overall genome following treatment with both

Development and Applications of the Comet-FISH 15,28

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g-irradiation and MMC. These findings have been corroborated by other studies that have shown that damage in the TP53 gene is repaired more rapidly than total DNA in human lymphocytes treated with H2O235 and g-irradiated breast cancer cell lines.34 Given these observations, it is tempting to speculate that this preferential repair is a reflection of the occurrence of transcriptioncoupled repair (TCR) in the cell. Preferential repair has been shown to occur in TP5339,40 and other transcriptionally active genes41–44 in comparison to the overall genome in mammalian cells following UV damage using other methods and it is likely a similar repair hierarchy exists in response to other DNAdamaging agents. Indeed, recent investigations in our laboratory have demonstrated that in g-irradiated normal fibroblasts, the TP53 gene region was preferentially repaired compared to both the transcriptionally inactive hTERT gene region and the overall genome, whereas in TCR-deficient Cockayne syndrome (CS) fibroblasts, this preferential repair was not observed (personal communication from B.A. Doherty, D.J. McKenna, C.S. Downes, G. McKerr, S.R. McKeown and V.J. McKelvey–Martin).

6.3.4

Summary of Studies

Taken together, the studies above have demonstrated that the CometFISH assay can detect DNA damage and repair in a number of genes, gene regions and loci. In theory, any gene could be detected if a suitable probe is available. Along with the assay’s relative speed and sensitivity, this means that it has vast potential as a laboratory technique for studying the cellular response to damage, with the added advantage of being able to study specific genes and gene regions of interest, particularly those associated with disease. This also makes it an attractive candidate for use in a clinical setting whereby data can be quickly obtained from patient cell samples, thereby helping to inform the development of treatment that is tailored to the individual needs of the patient. This would prove particularly beneficial in cancer management, where increasing emphasis is being placed on personalised medicine. However, a recent review of the potential of the Comet assay for use in the management of cancer raised concerns about reproducibility and validation of the assay, uncertainties that would also apply to the Comet-FISH assay.45 Furthermore, it is worth remembering that the nature of the Comet assay precludes us from stating categorically that damage and repair is occurring within a given gene. Rather, examining the position of each hybridisation spot enables us to conclude whether or not the damage and repair is occurring in the vicinity of the gene of interest, whilst counting changes in spot number allows us to estimate if damage and repair is within the probed region. These are important subtleties to grasp in analysing Comet-FISH data and, as many authors of the studies above have acknowledged, the Comet-FISH assay has certain limitations and therefore caution must be taken in the interpretation of results to ensure the correct conclusions are arrived at.

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6.4 Limitations of Comet-FISH Assay 6.4.1

Practical Difficulties

It is worth stating that the development and optimisation of the Comet-FISH assay has not been without its problems and investigators have encountered several practical difficulties with this technique. In situ hybridisation to cells embedded in agarose is not ideal as the gel matrix can affect the ability of probes to hybridise effectively to their target, especially if hybridisation is reliant on temperatures above which the agarose melts. Although this can be overcome to some extent by dehydrating the agarose prior to hybridisation and by using alkaline denaturation instead, larger probe fragments can still become trapped in the gel matrix itself, resulting in inefficient and/or nonspecific binding. Researchers have also found that standard hybridisation washing steps may not be sufficient to remove all unhybridised probe, whilst excessive washing can result in dislodging the delicate gel from the slide, with the loss of all cells into the wash buffer. For the same reason, use of signal amplification steps is also undesirable as extra washing steps increase the likelihood of gel loss. Although several of the published studies listed in Table 6.2 have used probes that require secondary antibodies, many of them acknowledge the difficulties that this presents in obtaining slides with sufficiently low background for accurate analysis. It is also worth remembering that many of the fluorescent probes described in the Comet-FISH studies above can only be purchased commercially and are often expensive to buy, which increases the pressure on researchers to generate good-quality comet slides each time for analysis. One alternative is to design specifically labelled probes against coding sequences in the gene under investigation, which has the added advantage of not being restricted to what is commercially available in terms of probe target or probe size. One study has successfully used this approach35 and it may be a viable alternative for researchers working on a tight budget or where the analysis of large numbers of samples or time points are required.

6.4.2

Imaging Difficulties

Even if gels remain intact, microscopic examination can often reveal high background and nonspecific fluorescence that can mask true signals and make accurate analysis difficult. Unfortunately, despite collaborations and dialogue with several software companies, no research group has yet reported the successful use of a reliable software package for accurately counting hybridisation signals from comet slides, preferring instead to manually count signals. As well as being time consuming and laborious, this can also result in user subjectivity, leading to problems with data collection during image analysis. Hybridisation signals close to the head/tail boundary in a comet may be considered by one user to lie in the head, but in the tail by another user. Similarly, two signals close together may be recorded as one signal by some users, perhaps


145

due to poor eyesight or simple fatigue. Hence, the development of computer imaging technology that is capable of detecting and counting hybridisation spots accurately is a priority if this problem is to be resolved. Once a suitable technology becomes available, this will go a long way to helping to generate data that are free from user bias.

6.4.3

Interpretation of Results

In order to interpret Comet-FISH results, it is imperative that there is a clear understanding of what occurs to DNA during the Comet assay process. The essential basis of the Comet assay recalls the experiments of Cook et al.46 who investigated nuclear structure by lysis of cells with nonionic detergent and highmolarity sodium chloride in order to remove the membrane and cytoplasm and disrupt the nucleosomes. This left a nucleoid structure in which negatively supercoiled DNA, RNA and proteins form a nuclear matrix. Cook et al. proposed that the DNA was attached at intervals to the matrix in a series of loops, thereby preventing free rotation of the DNA. However, using ionising radiation or the intercalating agent ethidium bromide was enough to damage the DNA and therefore relax the supercoiling in these loops. The Comet assay works on a similar basis, with the issue of DNA loops and matrix attachment still fundamental to the understanding of the results. Strand break(s) in a loop of DNA will result in that region of DNA being pulled to one side by electrophoresis to form the comet tail. However, if the DNA loop is attached to the nuclear matrix it will reach a point whereby it can stretch no further, unless a further break in this region produces an unattached fragment of DNA. With regard to Comet-FISH experiments, this means that it is not possible to state conclusively that the position of the hybridisation signal in the head or tail shows damage in the probed region. Instead, we can only surmise that a break must have occurred in the vicinity of the probed region, unless the number of spots has increased. A signal may simply be detected in the tail due to electro-stretching effects as a strand of broken DNA migrates into the tail. Likewise, a signal in the head may actually be from damaged DNA but is restricted from migrating due to close attachment to the matrix. Furthermore, an extremely damaged region of DNA may generate fragments that migrate so far from the head that they are not considered part of that cell, or may be so small as to be completely undetectable. Therefore, counting the number of hybridisation signals is crucial as an increase in spot number in cells can only occur from breaks within the probed region. However, this counting must take into account the ploidy of the cells, particularly with regards to cancer cells, and if they are at various stages of the cell cycle, and clear baseline data with regard to hybridisation signal number are also essential. It is also crucial to take into account the size of the probe used as the probability that a probed region of DNA will contain a strand break will depend to some extent upon how large the targeted area is. For example, if we accept the long-held assumption that 1 Gy of irradiation randomly introduces

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47

0.31 breaks per 10 Da of cellular DNA, it is clear that a large probe has more likelihood of targeting a region that happens to contain strand breakage than a smaller probe. Added to this, it is clear that there are distinct differences in the sensitivity of different DNA regions to damage, caused by factors such as chromatin structure,26 DNA repair efficiency41 and transcriptional activity,48 therefore DNA damage and strand breakage throughout the genome is unlikely to be absolutely uniform. Furthermore, even if strand breakage does occur, the DNA may be prevented from migrating due to attachment to the nuclear matrix as discussed above, meaning hybridisation signals may remain in the comet head. Hence, it is important to have an appreciation of all these issues with regards to each individual study performed in order to draw the correct conclusions and subsequent implications from the data recorded.

6.5 Conclusion Over the past decade, the Comet-FISH assay has proven a rapid and relatively simple procedure for measuring DNA damage and repair in both gene-specific loci and whole chromosomes in a variety of different cell types. The versatility of the assay means it offers great potential as a method for assessing DNA damage in specific gene regions, as well as the overall genome, in individual cells in response to many damaging agents, with clear implications for both basic science research and clinical application. However, the usefulness of this assay relies heavily on the correct analysis of results and on an accurate understanding of the dynamics of DNA movement under the conditions of the assay. Without due appreciation of these aspects, data generated from this assay can be misinterpreted and unreliable. Hence, there is a clear need for experiments that investigate fundamental issues relating to validation and standardisation of the Comet-FISH assay, ideally by comparison with other techniques used for measuring gene- and region-specific DNA damage and repair and nuclear architecture. Only by carrying out such experiments can the Comet-FISH assay gain widespread acceptance as a valuable and reliable method and thereby deliver on its potential for investigating DNA damage and repair in specific gene regions.

References 1. O¨. Ostling and K. J. Johanson, Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun., 1984, 123, 291–298. 2. N. P. Singh, M. T. McCoy, R. R. Tice and E. L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 1988, 175, 184–191. 3. P. L. Olive, J. P. Bana´th and R. E. Durand, Heterogeneity in radiationinduced DNA damage and repair in tumor and normal cells measured using the ‘‘comet’’ assay, Radiat. Res., 1990, 122, 86–94.


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4. D. W. Fairbairn, P. L. Olive and K. L. O’Neill, The Comet assay: a comprehensive review, Mutat. Res., 1995, 339, 37–59. 5. R. R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu and Y. F. Sasaki, Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen., 2000, 35, 206–221. 6. A. R. Collins, The Comet assay for DNA damage and repair: principles, applications, and limitations, Mol. Biotechnol., 2004, 26, 249–61. 7. P. L. Olive and J. P. Bana´th, The Comet assay: a method to measure DNA damage in individual cells, Nat. Protoc., 2006, 1, 23–29. 8. V. J. McKelvey-Martin, E. T. S. Ho, S. R. McKeown, S. R. Johnston, P. J. McCarthy, N. F. Rajab and C. S. Downes, Emerging applications of the single-cell gel electrophoresis (Comet) assay. 1. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization comets for the identification of damaged and repaired DNA sequences in individual cells, Mutagenesis, 1998, 13, 1–8. 9. P. L. Olive, D. Wlodek and J. P. Bana´th, DNA double-strand breaks measured in individual cells subjected to gel electrophoresis, Cancer Res., 1991, 51, 4671–4676. 10. O. Merk and G. Speit, Detection of crosslinks with the Comet assay in relationship to genotoxicity and cytotoxicity, Environ. Mol. Mutagen., 1999, 33, 167–172. 11. S. Pfuhler and H. U. Wolf, Detection of DNA-crosslinking agents with the alkaline Comet assay, Environ. Mol. Mutatgen., 1996, 27, 196–201. 12. A. R. Collins, S. J. Duthie and V. L. Dobson, Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis, 1993, 14, 1733–1735. 13. M. Dusinska´ and A. Collins, Detection of oxidised purines and UVinduced photoproducts in DNA of single cells by inclusion of lesion-specific enzymes in the Comet assay, Altern. Lab. Anim., 1996, 24, 405–411. 14. D. J. McKenna, M. Gallus, S. R. McKeown, C. S. Downes and V. J. McKelvey-Martin, Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells, DNA Repair, 2003, 2, 879–890. 15. D. J. McKenna, N. F. Rajab, S. R. McKeown, G. McKerr and V. J. McKelvey-Martin, Use of the Comet-FISH assay to demonstrate repair of the p53 gene region in two human bladder carcinoma cell lines, Radiat. Res., 2003, 159, 49–56. 16. A. Rapp, M. Haussmann and K. O. Greulich, The Comet-FISH technique: a tool for detection of specific DNA damage and repair, Methods Mol. Biol., 2004, 291, 107–119. 17. S. J. Santos, N. P. Singh and A. T. Natarajan, Fluorescence in situ hybridisation with comets, Exp. Cell Res., 1997, 232, 407–411. 18. C. Bock, A. Rapp, H. Dittmer, S. Monajembashi and K. O. Greulich, Localisation of specific sequences and DNA single-strand breaks in

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20.

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

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individual UV-A irradiated human lymphocytes by Comet-FISH, Prog. Biomed. Opt., SPIE., 1999, 3568, 207–217. A. Rapp, C. Bock, H. Dittmar and K. O. Greulich, UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size, J. Photochem. Photobiol. B., 2000, 56, 109–117. U. A. Harreus, N. H. Kleinsasser, S. Zieger, M. R. Wallner, P. Schuller and A. Berghaus, Sensitivity to DNA-damage induction and chromosomal alterations in mucosa cells from patients with and without cancer of the oropharynx detected by a combination of Comet assay and fluorescent insitu hybridisation, Mutat. Res., 2004, 563, 131–138. S. R. McKeown, T. Robson, M. E. Price, E. T. Ho, D. G. Hirst and V. J. McKelvey-Martin, Potential use of the alkaline Comet assay as a predictor of bladder tumour response to radiation, Br. J. Cancer, 2003, 89, 2264–2270. R. Arutyunyan, E. Gebhart, G. Hovhannisyan, K. O. Greulich and A. Rapp, Comet-FISH using peptide nucleic acid probes detects telomere repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage, Mutagenesis, 2004, 19, 403–408. R. Arutyunyan, A. Rapp, K. O. Greulich, G. Hovhannisyan, S. Haroutiunian and E. Gebhart, Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique, Exp. Oncol., 2005, 27, 38–42. G. Hovhannisyan, A. Rapp, R. Arutyunyan, K. O. Greulich and E. Gebhart, Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells, Int. J. Mol. Med., 2005, 15, 437–442. P. A. Escobar, M. T. Smith, A. Vasishta, A. E. Hubbard and L. Zhang, Leukaemia-specific chromosome damage detected by comet with fluorescence in situ hybridization (Comet-FISH), Mutagenesis, 2007, 22, 321–7. A. Groth, W. Rocha, A. Verreault and G. Almouzni, Chromatin challenges during DNA replication and repair, Cell, 2007, 128, 721–733. M. Menke, K. J. Angelis and I. Schubert, Detection of specific DNA lesions by a combination of Comet assay and FISH in plants, Environ. Mol. Mutagen., 2000, 35, 132–138. N. F. Rajab and V. J. McKelvey-Martin, Preferential rejoining of gradiation induced DNA-strand breaks in the p53 domain of J82 bladder carcinoma cells, Mutagenesis, 1999, 14, 649–650. M. Glei, A. Schaeferhenrich, U. Claussen, A. Kuechler, T. Liehr, A. Weise, B. Marian, W. Sendt and B. L. Pool-Zobel, Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes, Toxicol. Sci., 2007, 96, 279–284. A. Schaeferhenrich, G. Beyer-Sehlmeyer, G. Festag, A. Kuechler, N. Haag, A. Weise, T. Liehr, U. Claussen, B. Marian, W. Sendt, J. Scheele and B. L. Pool-Zobel, Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal, Mutat. Res., 2003, 526, 19–32.


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31. A. Schaeferhenrich, W. Sendt, J. Scheele, A. Kuechler, T. Liehr, U. Claussen, A. Rapp, K. O. Greulich and B. L. Pool-Zobel, Putative colon cancer risk factors damage global DNA and Tp53 in primary human colon cells isolated from surgical samples, Food Chem. Toxicol., 2003, 41, 655–664. 32. Y. Kno¨bel, M. Glei, A. Weise, K. Osswald, A. Scha¨ferhenrich, K. K. Richter, U. Claussen and B. L. Pool-Zobel, Uranyl nitrilotriacetate, a stabilized salt of uranium, is genotoxic in nontransformed human colon cells and in the human colon adenoma cell line LT97, Toxicol. Sci., 2006, 93, 286–297. 33. Y. Kno¨bel, A. Weise, M. Glei, W. Sendt, U. Claussen and B. L. PoolZobel, Ferric iron is genotoxic in non-transformed and preneoplastic human colon cells, Food Chem. Toxicol., 2007, 45, 804–811. 34. T. S. Kumaravel and R. G. Bristow, Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells using Comet-FISH, Breast Cancer Res. Treat., 2005, 91, 89–93. 35. E. Horva´thova´, M. Dusinska´, S. Shaposhnikov and A. R. Collins, DNA damage and repair measured in different genomic regions using the Comet assay with fluorescent in situ hybridization, Mutagenesis, 2004, 19, 269–276. 36. E. Park, M. Glei, Y. Kno¨bel and B. L. Pool-Zobel, Blood mononucleocytes are sensitive to the DNA-damaging effects of iron overload – in vitro and ex-vivo results with human and rat cells, Mutat. Res., 2007, 619, 59–67. 37. R. Amendola, E. Basso, P. G. Pacifici, E. Piras, A. Giovanetti, C. Volpato and G. Romeo, Ret, Abl1 (cAbl) and Trp53 gene fragmentations in CometFISH assay act as in vivo biomarkers of radiation exposure in C57BL/6 and CBA/J mice, Radiat. Res., 2006, 165, 553–61. 38. J. L. Fernańdez, F. Va´zquez-Gundı´ n, M. T. Rivero, A. Genesca´, J. Gosa´lvez and V. Goyanes, DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells, Exp. Cell Res., 2001, 270, 102–109. 39. M. K. Evans, B. G. Taffe, C. C. Harris and V. A. Bohr, DNA strand bias in the repair of the p53 gene in normal human and xeroderma pigmentosum group C fibroblasts, Cancer Res., 1993, 53, 5377–5381. 40. J. M. Ford, L. Lommel and P. C. Hanawalt, Preferential repair of ultraviolet light-induced DNA damage in the transcribed strand of the human p53 gene, Mol. Carcinog., 1994, 10, 105–109. 41. V. A. Bohr, C. A. Smith, D. S. Okumoto and P. C. Hanawalt, DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall, Cell, 1985, 40, 359–369. 42. I. Mellon, V. A. Bohr, C. A. Smith and P. C. Hanawalt, DNA repair of an active gene in human cells, Proc. Natl. Acad. Sci. USA, 1986, 83, 8878–8882. 43. J. Venema, Z. Bartosova, A. T. Natarajan, A. A. van Zeeland and L. H. Mullenders, Transcription affects the rate but not the extent of repair of

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cyclobutane pyrimidine dimers in the human adenosine deaminase gene, J. Biol. Chem., 1992, 267, 8852–8856. H. J. Ruven, C. M. Seelen, P. H. Lohman, H. van Kranen, A. A. van Zeeland and L. H. Mullenders, Strand-specific removal of cyclobutane pyrimidine dimers from the p53 gene in the epidermis of UVB-irradiated hairless mice, Oncogene, 1994, 9, 3427–3432. D. J. McKenna, S. R. McKeown and V. J. McKelvey-Martin, Potential use of the Comet assay in the clinical management of cancer, Mutagenesis, 2008, 23, 183–190. P. R. Cook, I. A. Brazell and E. Jost, Characterization of nuclear structures containing superhelical DNA, J. Cell Sci., 1976, 22, 303–24. G. Ahnstro¨m and K. Erixon, The measurement of strand breaks by DNA unwinding in alkali and hydroxyl apatite chromatography, in DNA Repair. A Laboratory Manual of Research Procedures, eds. E. C. Friedberg and P. C. Hanawalt, Marcel Dekker, New York, 1981, p. 403–418. T. Boulikas, The non-uniform repair of active and inactive chromatin domains (review), Int. J. Oncol., 1996, 8, 65–75.

CHAPTER 7

Detection of DNA Damage in Drosophila and Mouse ALOK DHAWAN*, MAHIMA BAJPAYEE AND DEVENDRA PARMAR Developmental Toxicology Division, Indian Institute of Toxicology Research (Formerly – Industrial Toxicology Research Centre), P.O. Box – 80, M.G. Marg, Lucknow – 226 001, India

The single-cell gel electrophoresis (SCGE)/Comet assay has, since its inception, been widely used for the simple, sensitive and rapid determination of DNA damage and repair, quantitatively as well as qualitatively in individual cell populations.1 Comet is the perfect acronym for credible observation and measurement of exposure to toxicants. The assay combines the simplicity of biochemical techniques for detecting DNA single-strand breaks with the single-cell approach of cytogenetic assays. The advantages of the assay include its need for small numbers of cells per sample (o10 000), collection of data at the level of the individual cell, allowing for robust statistical analyses, its sensitivity for detecting quantitative and qualitative DNA damage. The assay has versatility in detecting DNA single- and double-strand breaks, oxidative DNA damage, crosslinks as well as apoptosis and necrosis in proliferating or nonproliferating cells, and has thus gained popularity as a test for genetic toxicology. Single cells obtained from various organisms ranging from simple bacteria (prokaryotes) to complex humans (eukaryotes) have been used to monitor in vitro or in vivo genotoxicity of chemicals.2 It has also been used

*



151

Figure 7.1

EtBr

Wet land plant (Bacopa) leaves, stem and roots

Alkaline Electrophoresis

pH > 13

Alkaline Unwinding Nucleoid with dsDNA

Nucleus

Cell Nucleus

Prepared slide

High Salt concentration

pH 10

Lysis

Lysed Cell

Marine Fishes (flounder- Paralichthys) erythrocytes, gills, hepatocytes

Sea urchin Marine mussel (Mytilus) (Strongylocentrotus) Oyster (Crassostrea) coelomocyte Clam (Mya/ Tapes) hemocytes, gills

Normal melting agarose precoated slide

Single cells + Low Melting Point Agarose

Rodents (mouse, rat) blood, liver, spleen, brain, bone marrow, sperm Soil Earthworms (Eisenia) coelomocytes Algae (Chalamydomonas; Euglena) in vivo

Humans blood, nasal, buccal cells, sperm

Terresterial plants (Tabacum) leaf and root nuclei

Freshwater mussel Amphibians (Toad –Bufo; Frog- (Unio/ Dreissena) hemocytes, gills Xenopus) rs erythrocytes ive &r Fishes s ke (goldfish-Carassius , la carp-Cyprinus ) a ds n Se erythrocytes, gills, Po hepatocytes

Water

Bacterium (Escherichia coli) in vivo

Fruitfly (Drosophila) gut, brain

Birds (Stork- Ciconia; Kite Milvus) Blood, sperm, spleen cells

Air

Single cell suspension of cells of interest

Schematic diagram of the use of the Comet assay in assessing DNA damage in different models from bacteria to humans.

pH > 13

Single DNA strands

pH 7.5

Hair pin loops

Neutralizing (Tris buffer)

Staining

Scoring

Image Processed

152 Chapter 7

Detection of DNA Damage in Drosophila and Mouse

153

for ecogenotoxicology studies for assessing the genotoxicity of environmental conditions on the sentinel species.3 In this chapter, the general protocol for the assessment of DNA damage in any cell type as well as protocols utilised in mouse and Drosophila melanogaster for evaluation of in vivo genotoxicity are discussed.

7.1 General Protocol for the Assessment of DNA Damage Using the Alkaline Comet Assay The Comet assay may be used in any cell type that can be obtained as a singlecell suspension. The cells may be of animal, plant or human origin. The white blood cells are the most frequently used cell type for the Comet assay in human biomonitoring studies,4,5 however, other cells have also been used, e.g. buccal cells,6 nasal,7 sperm,8–10 epithelial11 as well as placental cells.12 The Comet assay has also been used for detecting the genotoxicity in plant models13,14 with cells from leaves,15 stems and roots.16 From animals, blood lymphocytes, bone marrow cells, and cells from organ/tissues such as liver, brain, and spleen have also been used.17–19 Guidelines for conducting the assay have been formulated and recommendations have been published.20,21 Detailed protocols for performing the assay in different samples and for different types of DNA damage are also available on the Comet assay website (www.cometassayindia.org). The general protocol for conducting the Comet assay in different models has been depicted in Figure 7.1.

7.1.1

Chemicals and Materials

Low melting point agarose (LMPA), normal melting agarose (NMA), phosphate buffered saline-PBS (Ca21, Mg21 free), ethylene diamine tetraacetic acid disodium salt (EDTA), ethidium bromide, sodium chloride (NaCl), sodium hydroxide (NaOH), Triton X-100, trizma base. Microscope slides (end frosted conventional microscope slides, 75 mm 25 mm, with 19 mm frosted end), coverslips (No. 1, 24 60 mm), frozen ice packs, microcentrifuge tubes, micropipettors and tips, Coplin jars (opaque), microscope slide tray (aluminium).

7.1.2

Preparation of Reagents

1. PBS (Ca21, Mg21 free): 1 L packet of Dulbecco’s PBS is added to 990 mL distilled water (dH2O), pH adjusted to 7.4, and volume made up to 1000 mL. Stored at room temperature. 2. Low melting point agarose: Prepare 1% (500 mg per 50 mL PBS) and 0.5% LMPA (250 mg per 50 mL PBS). Microwave or heat until near boiling and the agarose dissolves. Aliquot 5 mL samples into scintillation vials (or other suitable containers) and refrigerate until needed.

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When required, briefly melt agarose (60–701C) in a microwave or by another appropriate method. Place LMPA vial in a 37 1C dry/water bath to cool and stabilise the temperature. Do not keep heating the agarose or the concentration will change. 3. Normal melting agarose NMA (1.0%): 500 mg per 50 mL in Milli Q water. Microwave or heat until near boiling and the agarose dissolves. Maintain temperature B 60 1C in a dry bath for use. 4. Lysing solution: Ingredients per 1000 mL: 2.5 M NaCl 146.1 g 100 mM EDTA 37.2 g 10 mM Trizma base 1.2 g Add all the above ingredients to about 700 mL dH2O with B8 g NaOH and allow the mixture to dissolve on a stirrer. pH is adjusted to 10 using concentrated HCl or NaOH. q.s. to 1000 mL with dH2O and stored the stock at room temperature. NaOH is used for dissolving EDTA. Final lysing solution is prepared fresh before each experiment. Add 10% DMSO (in the case of haeme-containing cells) and/or 1% Triton X100 to the stock lysing solution and then refrigerate for at least 30 min prior to slide addition. NOTE: The purpose of the DMSO in the lysing solution is to scavenge radicals generated by the iron released from haemoglobin when blood or animal tissues are used. It is not needed for other situations. 5. Electrophoresis buffer (1X solution: 300 mM NaOH/1 mM EDTA) Stock solutions: 1. 10 N NaOH (200 g/500 mL dH2O) 2. 200 mM EDTA (14.89 g/200 mL dH2O, pH 10) Stored at room temperature, and fresh stock solutions of NaOH and EDTA can be prepared every B2 weeks. (Dissolve the EDTA with help of NaOH pellets or concentrated NaOH.) For use, 1X Buffer is made fresh before each electrophoresis run. Mix 30 mL NaOH and 5 mL EDTA stock solutions and make up to 1000 mL with chilled dH2O. The total volume depends on the gel box capacity. Prior to use, the pH of the buffer has to be ensured to be 413. 6. Neutralisation buffer (0.4 M Tris): 48.5 g is added to B800 mL dH2O, adjusted pH to 7.5 with concentrated (410 M) HCl made up to 1000 mL with dH2O, store at room temperature. Use chilled. 7. Staining solution: ethidium bromide (EtBr; 10X Stock – 200 mg/mL): add 10 mg in 50 mL dH2O, and store at room temperature. For use, 1X solution is prepared with 1 mL stock and 9 mL dH2O. CAUTION: carcinogen.

Handle EtBr with adequate precautions as it is a known


7.1.3

155

Preparation of Agarose-Coated (Base) Slides for the Comet Assay

1. The conventional microscope slides (with 19 mm frosted end) are dipped in methanol and burnt over a blue flame to remove the machine oil and dust. (If precleaned slides are available then this step can be omitted). 2. While NMA (1%) is hot (601C), the slides are dipped up to one-third the frosted area and gently removed. The underside of the slide is wiped to remove agarose and the slides laid on a flat surface to dry. Do not touch the slide to the side of the beaker or agarose runs off. 3. The slides may be air dried or warmed at 50 1C for quicker drying, and stored at room temperature until needed, avoiding high-humidity conditions. Generally, slides are prepared a day before use. NOTE: Slides should be labelled on the agarose side before storage.

7.1.4

Preparation of Microgel Slides for the Comet Assay

1. Cells of interest (whole blood, lymphocytes, cells from various tissues) are diluted with PBS and equal volumes of diluted cells (100 mL) and 1% LMPA (100 mL) are mixed. 80 mL of this mixture are placed onto two duplicate slides. Alternatively, to each of the coated slides 75 mL of LMPA (0.5%; at 37 1C) mixed with B10 000 cells in B5–10 mL (do not use more than 10 mL) are added. 2. Coverslips are placed on the slides to evenly spread the gel. 3. The slides are placed on a slide tray resting on ice packs until the agarose layer hardens (B5 to 10 min). 4. Coverslips are gently removed and a third agarose layer (80 mL LMPA; 0.5%) is added to the slide. Note: The final concentration of LMPA in the second and third layers should be the same to prevent uneven migration of DNA in the two layers. 5. Replace the coverslip to evenly spread the gel and return to the slide tray until the agarose layer hardens (B5 to 10 min). 6. The coverslips are finally removed and the slides carefully put into Coplin jars containing chilled, freshly prepared final lysing solution. 7. The slides are protected from light and refrigerated for a minimum of 1 h. The slides may be stored for at least 4 weeks in cold lysing solution without affecting the results. (The Lysing time may depend on the cell type and should be standardised.) NOTE: The amounts indicated are based on using No. 1, 24 60 mm coverslips. Proportional volumes can be used for coverslips differing in size. If the gels are not sticking to the slides properly, avoiding humidity and/or increasing the concentration of NMA agarose in the lower layer to 1.5% should eliminate the problem. Steps 4 to 6 should be performed under dim yellow lights to prevent additional DNA damage.

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7.1.5

Electrophoresis of Microgel Slides

The procedure described here is for electrophoresis under pH413 alkaline conditions. 1. After lysis at B4 1C, slides are gently removed from the lysing solution and placed side by side in the horizontal gel box near one end, sliding them as close together as possible. 2. The buffer reservoirs are filled with freshly made, chilled 1X electrophoresis buffer (pH413) until the liquid level completely covers the slides (avoid bubbles over the agarose). 3. Let slides sit in the alkaline buffer for 20 min to allow for unwinding of the DNA and the expression of alkali-labile damage. NOTE: The longer the exposure to alkali, the greater the expression of alkali-labile damage. 4. The power supply is turned on to 24 V (B0.7 V/cm) and the current adjusted to 300 milliamperes by raising or lowering the buffer level. Depending on the purpose of the study and on the extent of migration in control samples, electrophoresis is carried out for 10 to 40 min. NOTE: The goal is to obtain migration among the control cells without it being excessive. The optimal electrophoresis duration differs for different cell types. If crosslinking is one of the endpoints being assessed then having controls with about 25% migrated DNA is useful. A lower voltage, amperage and a longer electrophoresis time may allow for increased sensitivity. Different gel boxes will require different voltage settings to correct for the distance between the anode and the cathode. The electrophoresis should be carried out between 0.7–1V/cm. 5. After electrophoresis, the slides are lifted from the buffer and placed on a drainage tray. Neutralising buffer (pH 7.5) is added dropwise to coat the slides and allowed to sit for at least 5 min. The slides are drained and this step is repeated two more times. 6. These slides may be either stained and scored immediately or dried for later processing. a. Slides are stained with 80 mL 1X EtBr for 5 min. Then, the slides are dipped in chilled distilled water to remove excess stain, a coverslip is placed over it and the slides are stored in a humidified slide box until scoring. b. For drying, the slides are kept in cold 100% ethanol/methanol for dehydration for 20 min. The slides are air dried and then placed in an oven at 50 1C for 30 min. The slides are then stored in a dry box. When convenient, the slides are rehydrated with chilled distilled water for 30 min and stained with EtBr as above and covered with a fresh


157

coverslip. For archival purposes, the slides after scoring, are destained with 100% alcohol, dried and stored dry. NOTE: Perform steps 1 through 6 under yellow light; the premise is that normal lighting will cause DNA damage.

7.1.6

Evaluation of DNA Damage

1. EtBr-stained DNA damage is visualised using a 40 objective on a fluorescence microscope. 2. Although any image-analysis system may be suitable for the quantitation of SCGE data, we use Komet 5 image-analysis software developed by Kinetic Imaging, Ltd. (Liverpool, UK). The software is linked to a CCD camera to assess the quantitative and qualitative extent of DNA damage in the cells by measuring the length of DNA migration and the percentage of migrated DNA. Generally, 50 to 100 randomly selected cells are analysed per sample. Finally, the program calculates and automatically generates the values for tail (%) DNA, tail length, and tail moment. The tail moment is defined as the distance between the centre of mass of the tail and the centre of mass of the head, in micrometres, multiplied by the percentage of DNA in the tail and is considered to be the most sensitive as both the quality and quantity of DNA damage are taken into account. 3. The amount of migration per cell, the number of cells with increased migration, the extent of migration among damaged cells is then compared.

7.2 The Alkaline Comet Assay in Multiple Organs of Mouse The Comet assay is now a well-established supportive assay to the standard battery of genotoxicity tests and recommended as an in vivo test in the second stage of genotoxicity testing.22 It can be used to investigate the potential mechanisms of tumorigenic responses and to evaluate genotoxicity of chemicals that are positive in other in vivo mutagenicity tests. Guidelines and recommendations for performing the in vivo assay have been developed.21,23 The most important advantage provided by the Comet assay for assessing the genotoxicity in vivo is that the DNA damage can be measured in cells of any organ, regardless of the extent of mitotic activity. Cytogenetic techniques like the micronucleus assay, chromosomal aberration test and the sister chromatid exchange assay, require a proliferating cell population for assessing genotoxicity in cells, e.g. cells of the haematopoietic system. However, several chemicals pass the blood–organ barrier, reach the organs and elicit their toxic response including genotoxicity. Hence, genotoxicity in organs cannot be assessed using conventional cytogenetic techniques unless the cells are made to undergo

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Chapter 7 24,25

mitosis. Therefore, Sasaki et al. devised a method to assess multiorgan genotoxicity in the mouse using the alkaline Comet assay using a homogenisation technique to detect the genotoxicity of chemicals in vivo in their target organs. Using this method, each organ was minced, suspended in chilled homogenising buffer containing NaCl and Na2EDTA, gently homogenised using a Potter-type homogeniser set in ice, and the centrifuged nuclei were used for the alkaline Comet assay.24 Later, Sasaki et al. also compiled the data of genotoxicity of 208 carcinogenic chemicals from the IARC database using the alkaline Comet assay.17 The assay showed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens.17 The findings suggest that the alkaline Comet assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.17 The assay has been used to determine the threshold dose at which it has beneficial or toxic effects.26 Ueno et al. carried out DNA damage and repair studies in multiple organs of whole-body X-irradiated mice that suggested differences in the radiosensitivity of nuclear DNA and DNA-repair capacity among organs.27 A comparative investigation of species differences in genotoxicity in multiple organs of mice and rats using the Comet assay was conducted by Sekihashi et al.28 since sensitivity to xenobiotics is different for different species and species differences in carcinogenicity for mice and/or rats is known. Here, we describe the methodology followed in our laboratory for performing the in vivo Comet assay in multiple organs of mouse (Figure 7.2).19

7.2.1


Chemicals and materials for the Comet assay were as described earlier in 7.1. However, Heparin Hank’s balanced salt solution (HBSS), Histopaque-1077, and RPMI-1640 medium are also required in studies involving mice.

7.2.2 7.2.2.1

Methodology Animals

Male Swiss albino mice (o6-weeks old, 20 2 g) obtained from the breeding colony at our institute were raised on a commercial pellet diet and water ad libitum. Animals were cared for in accordance with the policy laid down by the Animal Ethics Committee of our Institute.

7.2.2.2

Treatment

Experiments were planned according to the Comet assay guidelines.20,21 4 or 5 mice were included in each treatment group and caged separately. They were treated intraperitoneally for 5 consecutive days with the test sample (e.g. cypermethrin19).

159


Mouse Orbital sinus

Cervical dislocation and dissection

Blood

Femurs

20 µl Blood +1 ml RPMI layered over 100 µl Histopaque-1077

Cleaned thoroughly in PBS

Centrifuge

Flushed in 1ml PBS

Chopped in mincing solution (HBBS + 20mM EDTA + 10% DMSO)

Bone marrow

Single cell suspension

500 x g; 5 mins

Medium/ Histopaque interface added to 1ml RPMI Centrifuge

Liver

Brain

Spleen

Kidney

Washed in HBSS buffer

500 x g; 5 mins Viability checked by 5, 6-carboxyfluorescein Viability Checked by Trypan blue dye

Isolated Lymphocytes COMET ASSAY PROTOCOL 1% LMPA + Cells

Alkaline Lysis Base slide 5 min. on ice

overnight

pH 10 (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, 10% DMSO

pH >13 Alkaline Unwinding 20 min Electrophoresis 30 min (300 mM NaOH, 1 mM EDTA

5 min. on ice

0.5% LMPA

Neutralize (0.4 M Tris, pH 7.5)

5 min

Prepared slide

Staining (EtBr, 20µg/ml)

5 min., Thrice

Image analysis

Figure 7.2

7.2.2.3

Schematic representation of alkaline Comet assay for assessing DNA damage in multiple organs of mouse

Sacrifice and Collection of Tissue Samples

Blood was drawn from the orbital sinus and 20–50 mL collected into heparinised Eppendorffs tubes. Animals were sacrificed by cervical dislocation. Organs (brain, liver, kidney and spleen) were dissected out and collected immediately in cold HBSS medium.

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Both the femurs were dissected out and cleaned thoroughly to remove muscles and other tissue. Bone marrow cells were flushed in 1 mL PBS using a syringe.

7.2.2.4

Single-Cell Preparation

i) Blood lymphocytes: the lymphocytes were isolated from whole blood using Histopaque-1077 by the method of Boyum29 with slight modifications. Briefly, 20 mL of blood was added to 1 mL RPMI-1640 and layered over 100 mL Histopaque. This was centrifuged at 500g for 3 min. The interface of medium/Histopaque containing the lymphocytes was taken and added to 1 mL medium (RPMI-1640). It was then centrifuged at 500g for 3 min to pellet the lymphocytes, which were resuspended in PBS for the Comet assay. ii) Preparation of a single-cell suspension from organs was done according to the method of refs. 18 and 20. Briefly,o0.2 g of each organ was placed in 1 mL of freshly prepared chilled mincing solution (Hank’s balanced salt solution, with 20 mM EDTA and 10% DMSO) in a Petri dish and chopped into pieces with a pair of scissors. The pieces were allowed to settle and the supernatant containing the single cells was taken.18 Cell counting was done and a cell suspension of B20 000 cells in 100 mL PBS was used for the studies.

7.2.2.5

Cell Count and Cell Viability Assay

Cells from organs and tissues were counted using a haemocytometer and diluted with PBS to achieve a concentration of 0.2106 cells/ml. The viability of cells isolated from liver, spleen, kidney and brain was checked by 5,6-carboxyfluorescein dye,30 while trypan blue was used for the blood and bone marrow cells.31

7.2.2.6

Single-Cell Gel Electrophoresis/Comet Assay

i) Preparation of slides and lysis: The base slides were prepared with 1% NMA using conventional end frosted slides as discussed earlier. On this base layer, 80 mL of sample (containing 100 mL cell suspension mixed with 100 mL of 1% LMPA) was added to form the second layer. A coverslip was placed gently to evenly spread the cells in the agarose. After the gel solidified, a third layer of 0.5% LMPA (90 mL) was added onto the slide and placed over ice for 10 min. Finally, the coverslips were removed and the slides immersed in freshly prepared chilled lysing solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) with 10% DMSO and 1% Triton X-100 being added just before use. The slides remained in the lysing solution overnight at 4 1C. ii) Electrophoresis: Electrophoresis was carried out according to the method of Singh et al.32 as described earlier. The slides were placed in a


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horizontal gel electrophoresis tank (Life Technologies, Gaithersburg, USA) side-by-side and avoiding spaces, with the agarose ends nearest to the anode. Fresh and chilled electrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH, pH413) was poured into the tank to cover the slides. The slides were left in this solution for 25 min to allow DNA unwinding and expression of alkali-labile sites as DNA-strand breaks. Electrophoresis was conducted at 24 V (0.7 V/cm) and a current of 330 mA using a power supply (Electra Comet III from Techno Source India Pvt. Ltd., Mumbai, India) for 30 min at 4 1C. All steps were performed under dimmed light to avoid additional DNA damage due to stray light. iii) Neutralisation and staining: After electrophoresis, the slides were drained and placed horizontally in a tray. Tris buffer (0.4 M; pH 7.5) was added dropwise and left for 5 min to neutralise excess alkali. The procedure was repeated thrice. The slides were stained with 75 mL EtBr (20 mg/mL) for 5 min and dipped in chilled distilled water to wash off excess EtBr, and a coverslip placed over them. Slides were placed in a dark humidified slide box to prevent drying of the gel. The slides were scored within 24 h. iv) Scoring of slides: Slides were scored using an image-analysis system (Kinetic Imaging, Liverpool, UK) attached to a fluorescence microscope (Leica, Germany) equipped with appropriate filters (N2.1, excitation wavelength of 515–560 nm and emission wavelength of 590 nm). The microscope was connected to a computer through a charge-coupled device (CCD) camera to transport images to software (Komet 3.1) for analysis. The final magnification was 400. The comet parameters recorded were Olive tail moment (OTM, arbitrary units), tail DNA (%) and tail length (TL; mm). Images from 100 cells (50 from each replicate slide) were analysed.

7.2.2.7

Statistics

Homogeneity of variance and normality assumption of data were tested and was found to be normally distributed. The mean values of OTM, tail % DNA, and TL at each concentration of the test sample were compared with the negative control using one-way ANOVA18 at Po0.05 level of statistical significance.

7.3 The Alkaline Comet Assay in Drosophila melanogaster Drosophila melanogaster, or the fruit fly, has been intensely studied for almost 100 years. It is a complex multicellular organism with many aspects of its development and behaviour parallel to those in human beings. Unique genetic and molecular tools have evolved for analysis of gene function in this organism. These advantages have allowed the use of Drosophila for the understanding of fundamental biological processes and provide unique insights in the genomic era. Drosophila has hence found extensive use as a model organism in various fields including

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toxicology testing. Concerns have been raised about the ethics and use of animals for toxicology research and testing, and emphasis now is given to the use of alternatives to mammals in research as well as education. The European Centre for the Validation of Alternative Methods (ECVAM) promotes scientific and regulatory acceptance of alternative methods for reducing, refining or replacing the use of laboratory animals39,40 and recommends D. melanogaster as an alternative to animal models for testing and research. Here, we describe the usefulness of D. melanogaster as an in vivo model for assessment of genotoxicity using the alkaline Comet assay (Figure 7.3).

7.3.1


All chemicals and materials for the Comet assay were as described earlier in 7.1. However, Poel’s salt solution, sodium phosphate buffer, and collagenase are also required in studies involving D. melanogaster.

7.3.2 7.3.2.1

Methodology Drosophila melanogaster

The wild-type (Oregon R+) fly and larvae of D. melanogaster were cultured at 24 1 1C and grown on a standard diet of agar, corn meal, brown sugar and yeast. Freshly emerged first or third instar larvae (22 2 h) were then used for the genotoxicity studies. The larvae were fed on a diet of standard Drosophila food containing different concentrations of genotoxicants, e.g. cypermethrin41 or industrial leachates,42,43 and allowed to grow on it. Larvae grown only on standard Drosophila food constitute the negative control, while those fed ethyl methanesulfonate, a known mutagen44 constitute the positive control.

7.3.2.2

Preparation of Cell Suspension

10–50 larvae are used for preparing cell suspensions for each concentration. At 96 2 h, the larvae were removed from the food and washed with 50 mM sodium phosphate buffer. Brain ganglia and the anterior region of the midgut are dissected and explanted in Poels’ salt solution (PSS).45 A single-cell suspension of the issues was then prepared by the modified method of Howell and Taylor.46 PSS was replaced with collagenase (0.5 mg/mL in PBS, pH 7.4) and cells incubated for 15 min at 24 1C. The cells were then passed through nylon mesh (60 mm). Collagenase was removed by washing the cell suspension three times with PBS. The cells were finally suspended in 80 mL of PBS.

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Detection of DNA Damage in Drosophila and Mouse Treatment in food

Midgut/ Brain ganglia in Poel’s salt solution

48 ± 2 hrs First / Third instar larvae

Single cell suspension 15 mins

Drosophila melanogaster

Collagenase (0.5mg/ mL)

Passed through Nylon mesh (60 µm) and checked for cell viability

1.5% LMPA + Cells 5 min. on ice

Preparation of slides Base slide

0.75% LMPA

5 min. on ice

Prepared slide

Alkaline Lysis (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10) DMSO was omitted in the solution 2hrs Alkaline Unwinding (300 mM NaOH, 1 mM EDTA, pH >13) 10 min. Alkaline Electrophoresis (~0.7V/cm, 300mA) 15 min. Image analysis

Neutralize (0.4 M Tris, pH 7.5) 5 min., Thrice 5 min. Staining (EtBr, 20µg/mL)

Figure 7.3

Schematic representation of the alkaline Comet assay for assessing DNA damage in Drosophila melanogaster.

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7.3.2.3

The Alkaline Comet Assay

The Comet assay technique still requires some modification and standardisation under different experimental conditions and/or using different experimental materials. The modifications and their reasons are discussed below: i) Slide preparation: Slides were prepared in duplicate for each concentration as discussed earlier in the chapter. The base slides were prepared with NMA (1%) as discussed earlier. However, owing to the difference in the size of the cells, modification in the concentration of LMPA was made. Generally, 1% LMPA is mixed with the cells in equal volumes (final concentration 0.5%) and is recommended for use in the second layer.20,44 Since the midgut and brain ganglia cells of Drosophila are smaller than mammalian cells, for our studies equal volumes of 1.5% LMPA (0.75% final concentration) and cell suspension were mixed. Similarly, the third layer consisted of LMPA (0.75%). ii) Lysing: A major modification was made in the composition of the lysing solution as compared with that used by Bilbao et al.44 This was removal of DMSO from the final lysing solution. DMSO is recommended at 10% and is usually added to scavenge radicals generated by the iron released from haemoglobin.32 However, no such heme groups are present in Drosophila. No scorable cells could be detected when slides were placed in lysing solution containing DMSO as used conventionally. Also, an earlier study had shown that a dietary concentration of over 0.3% DMSO was cytotoxic to D. melanogaster.47 Thus, in the final lysing solution DMSO was not added. The slides were finally immersed in freshly prepared chilled lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH 10.0 and 1% Triton X-100, pH 10) for 2 h. iii) Electrophoresis: After lysis, the slides were placed in a horizontal gel electrophoresis tank (Life Technologies, Gaithersburg, MD) filled with fresh, chilled electrophoresis solution (1 mM Na2EDTA and 300 mM NaOH, pH413). Although Bilbao et al.44 in their study used 20 min unwinding and electrophoresis of neuroblast cells of Drosophila, in our laboratory, no scorable cells could be observed when the time was maintained at 20 min. The experimental conditions were optimised in our laboratory and the times of unwinding and electrophoresis were reduced to 10 and 15 min, respectively, resulting in an improvement in performance of the assay. Electrophoresis was conducted at 0.7 V/cm and 300 mA at 4 1C using a power supply. iv) Neutralisation and staining of slides: Tris buffer (0.4 M Tris pH 7.5) was added dropwise to neutralise excess alkali and procedure repeated thrice. Slides were then stained with EtBr (20 mg/mL, 75 mL per slide) for 10 min in the dark. They were dipped in chilled distilled water to remove excess stain and subsequently coverslips were placed on


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them. The prepared slides were kept in a humidified slide box until scoring. v) Scoring: The slides were scored as discussed earlier using a fluorescent microscope attached to a CCD camera. 25 cells per slide were randomly captured, avoiding the cells present in the edges of the gel and superimposed comets. Each experiment was performed in triplicate with 10–50 larvae.

7.3.2.4

Statistics

Prior to analysis, homogeneity of variance and normality assumptions concerning the data were tested. The mean values of the Comet parameters were compared using the Student’s t-test.

7.4 Conclusions The Comet assay has gained wide acceptance in genotoxicity testing due to its simplicity, and sensitivity. The added advantage of being simple and able to detect different types of DNA damage in any cell, regardless of its proliferating status makes the assay more versatile. This has resulted in widespread progression of this technique in many areas, e.g. environmental monitoring,3 human monitoring,5,48–50 genetic toxicology.51 The assay has been accepted by international guidelines as an in vivo test52 and guidelines as well as recommendations have been published.20,21,23 In this chapter, the use of the Comet assay in assessing DNA damage in two in vivo models, i.e. an animal model, mouse, and an alternate to the animal model, Drosophila, have been discussed. The Comet assay in rodents is an important test model for genotoxicity studies, since it provides an insight into the genotoxicity and its underlying mechanisms of human carcinogens (since many rodent carcinogens are known to be human carcinogens). The mouse organs exhibiting increased levels of DNA damage may not be the target organs for carcinogenicity. Therefore, for the prediction of carcinogenicity of a chemical, organ-specific genotoxicity was necessary but not sufficient.17 The Comet assay can also be used as an in vivo test for assessing DNA damage for those compounds which have poor systemic bioavailability. Multiple organs of mouse/rat including brain, blood, kidney, lungs, liver, bone marrow have been utilised for the comprehensive understanding of the systemic genotoxicity of chemicals.17,18,28,53,54 Also, since the scientific world is moving towards a reduction in the use of animals in toxicity testing, alternatives to animal models become important. One such organism is the fruit fly Drosophila melanogaster. This model was earlier used mostly for germ cell mutagenicity studies, however, recently, it has gained importance in studying somatic cell genotoxicity of chemicals.41–44

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Although the Comet assay is a useful technique, the variability in results from different laboratories, interpretation of the results and lack of validated studies are some of its disadvantages. Care should be taken during preparation of the single-cell suspension so that viable cells are obtained for analysis. Also, viability of the cells should be checked before proceeding with the Comet assay so that the DNA damage observed is due to the genotoxicity of the compound and not due to DNA fragmentation of damaged/dead cells. Each step of the assay should be conducted under the proper conditions (pH, temperature) to reduce ambiguous results. The International Workgroup on Genotoxicity Testing (IWGT) has discussed study design and data analysis in the Comet assay and emphasis was given to the alkaline version (pH413) of the in vivo Comet assay and recommendations were made for a standardised protocol, which would be acceptable to international agencies.23 Scoring manually or with the help of automated software are both allowed, however, user bias can be reduced by scoring random cells. The statistical analysis in the Comet assay takes into account the study design and has been well reviewed.55,56 Both univariate and multivariate analyses can be conducted on the results obtained in the assay. The Comet assay in in vivo models such as mice and Drosophila allows the assessment of genotoxicity of chemicals that can mimic the responses in humans. These models thus provide an understanding of the mechanism of genotoxicity57 as well as the response of biological systems to these chemicals.

Acknowledgements The authors wish to thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India for funding through the Networked Projects (CMM0018 and NWP34) as well as the support from UK-India Education and Research Initiative (UKIERI).

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19. S. Patel, A. K. Pandey, M. Bajpayee, D. Parmar and A. Dhawan, Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the Comet assay, Mutat. Res., 2006, 607(2), 176. 20. R. Tice, E. Agurell, D. Anderson, B. Durlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu and Y. F. Sasaki, Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing Environ. Mol. Mutagen., 2000, 35, 206. 21. A. Hartmann, E. Agurell and C. Beevers, et al. Recommendations for conducting the in vivo alkaline Comet assay, Mutagenesis, 2003, 18, 45. 22. Guidance on a strategy for testing of chemicals for mutagenicity. December 2000. http://www.advisorybodies.doh.gov.uk/com/guidance.pdf. 23. B. Burlinson, R. R. Tice, G. Speit, E. Agurell, S. Y. Brendler-Schwaab, A. R. Collins, P. Escobar, M. Honma, T. S. Kumaravel, M. Nakajima, Y. F. Sasaki, V. Thybaud, Y. Uno, M. Vasquez and A. Hartmann, In vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing. Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup, Mutat. Res., 2007, 627, 31. 24. Y. F. Sasaki, F. Izumiyama, E. Nishidate, N. Matsusaka and S. Tsuda, Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow), Mutat. Res., 1997, 391, 201. 25. Y. F. Sasaki, S. Tsuda, F. Izumiyama and E. Nishidate, Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single-cell gel electrophoresis (Comet) assay, Mutat. Res., 1997, 388, 33. 26. R. M. Rosa, NC. Hoch, GV. Furtado, J. Saffi and JA. Henriques, DNA damage in tissues and organs of mice treated with diphenyl diselenide, Mutat. Res., 2007, 633(1), 35. 27. S. Ueno, T. Kashimoto, N. Susa, H. Natsume, M. Toya, N. Ito, S. TakedaHomma, Y. Nishimura, Y. F. Sasaki and M. Sugiyama, Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the Comet assay, Mutat. Res., 2007, 634(1–2), 135. 28. K. Sekihashi, A. Yamamoto, Y. Matsumura, S. Ueno, M. WatanabeAkanuma, F. Kassie, S. Knasmu¨ller, S. Tsuda and Y. F. Sasaki, Comparative investigation of multiple organs of mice and rats in the Comet assay, Mutat. Res., 2002, 517(1–2), 53. 29. A. Boyum, Seperation of leukocytes from blood and bone marrow introduction, Scand J. Clin. Lab. Invest., 1968, 97(Suppl), 7. 30. M. Provinciali, G. Di Stefano and N. Fabris, Optimization of cytotoxic assay by target cell retention of the fluorescent dye carboxyfluorescein diacetate (CFDA) and comparison with conventional 51CR release assay J. Immunol. Methods, 1992, 155, 19. 31. H. J. Phillips, Dye exclusion tests for cell viability, in Tissue Culture Methods and Applications, eds. P.F. Kruse and M.J. Patterson, Academic Press, New York, 1973, p. 406.


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32. N. P. Singh, M. T. McCoy, R. R. Tice and E. L. Schneideer, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 1988, 175, 184. 33. I. Gaivao, L. M. Sierra and M. A. Comendator, The w/w1 SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds, Mutat. Res., 1999, 440, 139. 34. D. Kar Chowdhuri, A. Nazir and D. K. Saxena, Effect of three chlorinated pesticides on hsrw stress gene in transgenic Drosophila melanogaster J. Biochem. Mol. Toxicol., 2001, 15, 173. 35. A. Nazir, D. K. Saxena and D. Kar Chowdhuri, Induction of hsp70 in transgenic Drosophila: biomarker of exposure against phthalimide group of chemicals Biochem. Biophys. Acta, 2003, 1621, 218. 36. I. Mukhopadhyay, A. Nazir, K. Mahmood, D. K. Saxena, M. Das, S. K. Khanna and D. Kar Chowdhuri, Toxicity of argemone oil: effect on hsp70 expression and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ)Bg9 Cell Biol. Toxicol., 2002, 18, 1. 37. I. Mukhopadhyay, D. K. Saxena, V. K. Bajpai and D. Kar Chowdhuri, Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: protective role of 70 kDa heat shock protein, J. Biochem. Mol. Toxicol., 2003, 17, 223. 38. H. R. Siddique, S. C. Gupta, K. Mitra, V. K. Bajpai, N. Mathur, R. C. Murthy, D. K. Saxena, D. K. Chowdhuri, Adverse effect of tannery waste leachates in transgenic Drosophila melanogaster: role of ROS in modulation of Hsp70, oxidative stress and apoptosis. J. Appl. Toxicol. 2008, 28, 734. 39. M. F. W. Festing, V. Baumans, D. R. Combes, M. Hadler, F. M. Hendriksen, B. R. Howard, D. P. Lovell, G. J. Moore, P. Overend and M. S. Wilson, Reducing the use of laboratory animals in biomedical research: problems and possible solutions, Altern. Lab. Anim., 1998, 26, 283. 40. D. J. Benford, B. A. Hanley, K. Bottrill, S. Oehlschlager, M. Balls, F. Branca, J. J. Castengnaro, J. Descotes, K. Hemminiki, D. Lindsay and B. Schitter, Biomarkers as predictive tools in toxicity testing, Altern. Lab. Anim., 2000, 28, 119. 41. I. Mukhopadhyay, D. K. Chowdhuri, M. Bajpayee and A. Dhawan, Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using alkaline Comet assay, Mutagenesis, 2004, 19, 85–90. 42. H. R. Siddique, D. K. Chowdhuri, D. K. Saxena and A. Dhawan, Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay, Mutagenesis, 2005, 20(4), 285. 43. H. R. Siddique, A. Sharma, S. C. Gupta, R. C. Murthy, A. Dhawan, D. K. Saxena and D. K. Chowdhuri, DNA damage induced by industrial solid waste leachates in Drosophila melanogaster: a mechanistic approach Environ. Mol. Mutagen., 2008, 49(3), 206.

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SECTION III: APPLICATIONS OF COMET ASSAY

CHAPTER 8

Clinical Applications of the Comet Assay S. M. PIPERAKIS*, K. KONTOGIANNI, G. KARANASTASI AND M. M. PIPERAKIS Biology Unit, Department of Pre-School Education, Faculty of Human Sciences, University of Thessaly, Volos, Greece

8.1 Introduction The Single Cell Gel Electrophoresis (SCGE) or Comet assay is a very sensitive method for measuring DNA-strand breaks in individual cells. It is widely used among others in environmental toxicology, radiation toxicology and cancer research to assess DNA damage and repair. This technique was developed in 1984 when Ostling and Johanson1 proposed a method for detecting genotoxic damage in single cells. In recent years the Comet assay has become a new tool in the area of assessing genetic damage in vitro and in vivo in a variety of cells. Although most of the reports on the Comet assay measure the genotoxic effects induced in human, in animal and in plant cells in vitro, the number of human studies in vivo has also increased. In particular, reports on the application of Comet assay in monitoring DNA damage from disease conditions or treatment with genotoxic drugs, environmental pollution, occupational exposure and dietary studies have increased substantially. In the present review we focus on studies investigating the clinical applications of the Comet assay. In particular, we attempted to analyse the uses of the Comet assay in clinical medicine. * Corresponding author Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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Chapter 8

8.2 The Comet Assay Methodology In 1976 Cook et al.2 published a paper investigating the nuclear structure based on the lysis of cells with nonionic detergent and high-molarity sodium chloride. The earliest attempt to quantify DNA-strand breaks directly was made by Rydberg and Johanson3 in 1978 with cells embedded in agarose on slides and lysed under mild alkaline conditions. Staining of the nucleoid with acridine orange showed a higher ratio of red fluorescence, indicating single-stranded DNA, to green, indicating double-stranded DNA, in cells with DNA damage. In 1984 Ostling and Johanson1 based on the approach described above developed the Comet assay, also called Single Cell Gel Electrophoresis (SCGE). This was an assay in which the lysis and electrophoresis were performed under neutral conditions. The staining of the DNA was done with acridine orange. The image obtained looked like a ‘‘comet’’ with a distinct head, comprising of intact DNA and a tail, consisting of damaged DNA. As a consequence the name ‘‘Comet assay’’ was given. The amount of DNA liberated from the head of the comet depends on the dose of mutagen used. However, in this procedure, only double-strand breaks could be analysed. This assay was later modified by two groups, Singh et al.4 in 1988 and Olive et al.5 in 1990. The first group performed electrophoresis under highly alkaline conditions (pH413). This enables the DNA supercoils to relax and unwind and makes possible the detection of alkali-labile sites and single-strand breaks in DNA during electrophoresis. This method measures low levels of strand breaks with high sensitivity. The second group conducted electrophoresis under neutral or mild alkaline conditions to detect single-strand breaks. This method was optimised to detect a subpopulation of cells with varying sensitivity to drugs or radiation. The version of the Comet assay developed by Singh et al.4 was found to be up to two orders of magnitude more sensitive. The simplest types of DNA damage detected by the Comet assay are doublestrand breaks (DSBs). DSBs result in DNA fragments and can be detected by merely subjecting them to electrophoretic mobility at neutral pH. Single-strand breaks (SSBs) do not produce DNA fragments unless the two strands of the DNA are separated/denatured. This is accomplished by unwinding the DNA at pH 12.1. It is also possible that single-strand breaks can relax the DNA and hence can also be detected with the Comet assay at neutral pH. Other types of DNA damage broadly termed alkali-labile sites (ALS) are expressed when the DNA is treated with alkali at pH greater than pH 13. Breaks can also be introduced at the sites of DNA base modifications by treating the DNA with lesion-specific glycosylases/endonucleases and the fragments thus produced can also be detected by the Comet assay. By controlling the conditions that produce nicks at the sites of specific DNA lesions the Comet assay can be used to detect various classes of DNA damage. While breaks increase DNA migration, DNA binding and crosslinks can retard DNA migration and can also be detected by the Comet assay.

Clinical Applications of the Comet Assay

175

Therefore, increased migration in the Comet assay can be attributed to strand breaks, alkali-labile sites and incomplete excision repair sites, while decreased DNA migration could be attributed to crosslinks, DNA–DNA or DNA–protein interactions.

8.3 Clinical Studies The Comet assay has been applied in a large number of clinical studies to investigate mainly the consequence of therapeutical exposure to certain chemicals or to study certain pathological conditions at the cellular level (Table 8.1). Elevated levels of DNA damage were found in irradiated cells isolated from ataxia telangiectasia patients. In addition, the DNA-repair process was found to be almost three times slower than the controls.6 In another study with four xeroderma pigmentosum donors Green et al.7 found that few strand breaks appear after UV-irradiation if compared to controls. Bu¨rger et al.8 in a study of six Nijmegen breakage syndrome families using peripheral blood mononuclear cells found that two out of the six families were more sensitive to X-irradiation than the controls. DNA repair was also reported to take longer in four out of the six families, while cells from five families with the syndrome had significantly increased residual DNA damage following repair. In an assessment of genome instability between 30 Down’s Syndrome and 14 Fanconi anaemia individuals9 increased DNA damage was observed. In a study of the level of primary and oxidative DNA damage in a group of mild cognitive impairment (MCI) and another of Alzheimer’s disease (AD) a significantly higher level of primary DNA damage in leukocytes of AD and also MCI patients if compared to controls, was found. Moreover, the amount of oxidised DNA bases (both purines and pyrimidines) was significantly higher in these two groups of patients.10 Malnourished infected children treated with antibiotics11 revealed that the observed DNA damage in lymphocytes was associated with malnourishment and antibiotic therapy. Elevated DNA damage was found in persons subjected to abortion12 and malnutrition and parasite infection.13 The daily use of substances for personal hygiene, i.e. chlorhexidine digluconate, a mouth rinse, used as an antiplaque agent, caused a significant increase in DNA damage in buccal and peripheral blood cells.14 Higher level of DNA damage was also reported in leprosy patients undergoing treatment,15 chronic renal failure patients on haemodialysis16 and patients who underwent anaesthesia.17 Sardas et al.18 reported the induction of DNA damage in lymphocytes of operation room personnel occupationally exposed to the anaesthetic agent isofurane. Biri et al.19 reported that women who used oral contraceptives had higher levels of DNA-strand breakage than the control group. Investigations with lymphocytes of head and neck squamous cell carcinoma patients20 and urothelial cells of urinary bladder cancer patients21 revealed that

Mean comet length (mm)

Mean comet length (mm)

Tail moment

pH¼13.5; 20 min; 25 V (0.83 V cm1)

Alkaline, 20 V (3000 mA), (1 V/cm), 24 min

pH¼13.5, 22 V (0.83 V/cm), 20 min

i) N¼10 healthy donors ii) N¼10 patients with breast cancer showing a normal reaction to radiotherapy iii) N¼20 Ataxia Telangiectasia carriers and iv) N¼4 Ataxia Telangiectasia homozygotes

N¼4 healthy controls N¼4 xeroderma pigmentosum patients

i) N¼10 controls ii) N¼13 Nijmegen breakage syndrome (NBS) patients iii) N¼10 breast cancer patients with normal sensitivity to radiotherapy

Study subject

Parameter of DNA damage/indicator cells

NBS patients and breast cancer patients

Xeroderma pigmentosum

Ataxia telangiectasia patients

Clinical case

Few strand breaks appear after UV-irradiation if compared to controls. DNA damage was higher in patients.

Radiosensitivity

Increased DNA damage (about 3 times high) in patients.

Results and remarks

UVC sensitivity

Radiosensitivity

Genotoxic factor

Clinical application of the Single Cell Gel Electrophoresis assay.

Alkali unwinding and electrophoresis

Table 8.1

8

7

6

Ref.

176 Chapter 8

Tail length (percentage DNA damage)

pH413, 20 min, 25 V, 300 mA.

i) N¼20 patients affected by Alzheimer’s Disease (AD), 14F; 6M age range¼53–82 years

ii) N¼15 Mild Cognitive Impariment (MCI), 6F; 9M age range¼55–76 years and iii) N¼15 controls, 9F; 6M.

Visual grading of comets (0, without detectable tail; 4, highest damage/ lymphocytes)

pH413, 20 min, 25 V, 300A

N¼14 Fancony Anemia patients; 8M; 6F; age range¼3–16 years, N¼30 Down Syndrome patients; 20M; 10F; age range¼0–10 years and N¼30 controls; 18M; 12F age range¼0–17 years Mild cognitive impairment and Alzheimer’s Disease

Down syndrome and Fancony anemia patients

Oxidative stress

Sensitivity to DNA damage

Results give an indication that oxidative stress, at least at the DNA level, is an early event in the pathogenesis of AD.

DNA damage was significantly higher in cells for DS patients that in controls. The FA group presented higher damage when compared to controls. 10

9

Clinical Applications of the Comet Assay 177

pH not given, 20 min; E¼25V, 300 mA, 20 min


(continued ).

N¼12, 5 F, 7 M; 6 malnourished severely infected children, age range¼6–29 months; 6 well-nourished severely infected children, age range¼7–36 months

Study subject

Table 8.1

TL/lymphocytes

Parameter of DNA damage/indicator cells Malnourished children

Clinical case Treatment with antibiotics/ malnutrition. Sulfatrimetroprim, salbutamol, ambroxol, amikacin, cefotaxime, dicloxacillin, metronidazole, penicillin, ampicillin, cefalexin; malnutrition

Genotoxic factor

Ref. 11

Results and remarks Increase in DNA damage in malnourished children compared to well-nourished children; significantly increased comet tail length in both wellnourished and malnourished children after treatment with antibiotics; the effect in malnourished children was twice as much as that in wellnourished children. In both malnourished and well-nourished children, there was a cell population resistant to druginduced DNA damage; however, the proportion of resistant cells was higher in the latter group.

178 Chapter 8

VS/L

Mean tail length

pH¼13, 20 min: E¼25 V and 300 mA, 20 min

Alkalne, 25 V, 300 mA, 20 min

N¼31 couples; 18 couples patients, 13 couples controls; age¼ns

i) N¼10 well-nourished children: without infection and treatment (WN); 3F;7M. ii) N¼4F mildly infected children: well nourished, without treatment (MI). iii) N¼ 8, 6F; 2M severely infected children: well nourished, without treatment (SEI). iv) N¼5, 2F; 3M severely infected children: well nourished, with treatment (SEI-T). v) N¼4, 3F; 1M malnourished children with infection, without treatment (MN). vi) N¼5, 2F; 3M malnourished children with infection, with treatment (MN-T).

Bronhopneumonia, pharyngitis, rhonopharyngitis, sepsis, tuberculosis meningea, gastroenteritis typhoid,

Couples with a history of more than fatal loss

Great increase in DNA damage in patients compared to controls; frequency of cells with limited and extensive migration varied within the same group. Great increase in damaged cells in smokers of both control and patient groups. Severe infection is associated with a significant increase in DNA damage.

Uknown

Uknown

13

12


Visual grading of DNA damage/ lymphocytes

Tailed cell percentage; length/ width ratios of DNA mass/lymphocytes

pH¼13, 40 min; E¼19 V, 300 mA, 40 min

pH 4 13, 30 min; E¼25V (0.8 V/cm), 300 mA, 30 min

N¼13, 9 F, 4 M; age range¼21–29 years

N¼100, 26 F, 74 M; 50 leprosy patients undergoing therapy, average age¼35 years; 50 healthy controls, average age¼31 years


Study subject

(continued ).


Table 8.1

Increase in the number of damaged buccal cells and peripheral blood lymphocytes of antiplaque users (0.12% CHX solution for 18 days) as compared to controls; mean grade of damage in buccal cells was significantly higher than that in lymphocytes. No information on the effect of confounding factors was given although questionnaires were filled out.

Use of antiplaque agent (chlorhexidine digluconate)

Leprosy status/the drugs dapsone, rifampicin, clofazimine, ofloxacin

Buccal cells and peripheral blood lymphocytes of antiplaque users

Leprosy/multidrug treatment against leprosy

Increased DNA damage in treated leprosy patients as compared to controls; also, disease factor alone significantly influenced DNA damage.

Results and remarks

Genotoxic factor

Clinical case

15

14

Ref.

180 Chapter 8

pH¼13, 24 V, 300 mA, 20 min

pH¼13, 40 min; E¼19 V (1.6 V/cm), 300 mA, 40 min

N¼36 uremic patients, 12 F; 24 M, age range¼ 21–75 years, undergoing maintenance hemodialysis and N¼36 controls, 12F; 24M, age range¼20–75 years

N¼36, 7 M, 29 F; 24 exposed, age range¼20–66 years; 12 unexposed, mean age 43 years Visual grading of damage (no tail, short tail, long tail) in lymphocytes

Visual grading of damage

Medical use of anaesthetics in patients

Peripheral blood lymphocytes from 36 dialysis patients before and after Vitamin E

Sevoflurane, isoflurane

Antioxidant supplementation of Vitamin E

Increase of DNA damage at 1 and 2 h after anaesthesia as compared to controls; DNA repair was observed 3 days after anaesthesia and was completed on the 5th day. Elevated comet responses after 120 min of exposure and began to decrease on the first day after operation; mean comet responses observed on the third day and fifth day after operation were not statistically different compared to control group and before operation.

The DNA breakage observed in the lymphocytes of patients before Vitamin E supplementation was significantly higher than in the controls but a clear protective effect of Vitamin E supplementation was observed after 14 weeks of therapy. 17

16


18

19 Increased scores of comet parameters in oral contraceptive users (150 g desogestrel and 20 or 30 g ethinyl estradiol for 24 months); s. higher comet scores were observed for pregnant women compared to controls. No statistically s. differences between oral contraceptive users and pregnant women in regard to the level of DNA damage; smoking did not affect the level of DNA damage in oral contraceptive users.

Pregnancy hormones (estrone and estradiol); oral contraceptives (ethinyl estradiol)

Use of oral contraceptives/ pregnancy

Visual grading of damage (no tail, short tail, long tail)/ lymphocytes

pH¼13, 20 min; E¼1.6 V/cm, 300 mA, 20 min

i) N¼36 F; 18 exposed to oral contraceptives, mean age 24 years; 18 controls. ii) 17 women in the last trimester of pregnancy, mean age 28 years, 17 controls

Ref.

Increase in the proportion of moderately and severely damaged cells on the first day after being exposed to the anaesthetic agent. Damage decreased 3 days after exposure and was almost identical to control values 5 days later.

Results and remarks

Isoflurane (exposure for 60 and 120 min)

Genotoxic factor

Patients anesthesized with isofluran

Clinical case

VS/L


pH¼13, 40 min; E¼19 V and 300 mA, 20 min


(continued ).

N¼24; 4 M, 20 F; 12 patients and 12 controls; age range¼22–66 years

Study subject

Table 8.1

182 Chapter 8


Median tail moment and percent tail DNA

pH¼13, 20 min; E¼1V/ cm, 20 min

pH¼13, 0.66 V/cm, 20 min

N¼82, 51M, 31F, 44 healthy controls age range¼44–78 years and 38 patients with squamous cell carcinoma of the head and neck, age range¼13–78 years

N¼62 patients; 34 with no history of UCC; 28 with a history of Urothelial Cell Carcinoma (UCC).

Urothelial Cell Carcinoma patients

Squamous cell carcinoma of the head and neck

Smoking

Cancer related pathological changes

The background DNA damage was significantly increased in UCC patients as compared to controls.

Increased background DNA damage in the tumour patient group; following irradiation, lymphocytes of tumour patients showed higher DNA damage, slower repair and higher residual unrepaired damage than those of healthy subjects. 21

20


TM/lymphocytes

Percentage and Tail Moment

pH¼13, 0,78 V/cm, 20 min

N¼40 breast cancer patients (clinical stage III) and N¼60 controls. All non smokers


pH alkali, 1 h; E¼B0.8 V/cm, 20 min


(continued ).

N¼140 F; 70 breast cancer patients, mean age 53 years; 70 controls, mean age 53 years

Study subject

Table 8.1

Breast cancer patients and controls

Breast cancer

Clinical case

Oxidative DNA damage

Cancer related pathological changes and in vitro exposure to ionising radiation

Genotoxic factor

23

22

Increased background DNA damage in the breast cancer patient group; DNA damage in lymphocytes after in vitro exposure to ionising radiation was significantly higher in patients; significantly lower repair capacity after treatment with radiation in patients. Level of DNA damage was not influenced by age; level of DNA damage increased in patients with increasing BMI; level of DNA damage decreased in controls with increasing BMI. PBL of cancer patients show a greater number (percentage) and a higher degree TM of DNA strand breaks in PBL and double strand breaks are distinctively higher in breast cancer patients than in controls

Ref.

Results and remarks

184 Chapter 8

Total image length/L

TM

TM/lymphocytes

pH ns, 20 min; E¼25 V and 300 mA, 20 min

pH¼13,5; 20 min; 25 V (0,83Vcm1)

pH¼13, 20 min; E¼20V (0.8 V/cm), 300 mA, 25 min

N¼11; F; age range¼29–55 years

i) N¼50 unselected BC patients ii) cancer patients with an adverse early skin reaction to RT, and included seven patients with BC, one with tongue carcinoma and one with plasmacytoma iii) 16 controls.

N¼397 F; 188 first-degree female relatives; 88 newly diagnosed untreated patients, 121 controls; age range¼18–70 years; non-smokers

Breast cancer

Breast cancer patients and controls

Breast cancer patients treated with high doses of CP

Cancer related pathological changes and in vitro exposure to N-methyl-N-nitro-Nnitrosoguanidine (MNNG)

Radiosensitivity

CP (l875 mg/ m2 /day) and cisplatin (165 mg/ m2/day)

25

26 Great increase in the frequency of basal TL level of firstdegree female relatives as compared to controls and in patients as compared to their first-degree female relatives; similar pattern of effect was observed upon exposing lymphocytes of the three groups to MNNG in vitro or in the study of the repair capacity of the cells.

24

The Comet assay did not reveal any differences among the three groups in terms of their initial and residual levels of DNA damage.

Great increase but variable increase in DNA damage; level of damage did not correlate with serum levels of chemotherapeutic agents or with lymphocyte toxicity. Mean level of DNA migration was not affected by cryopreservation.


pH¼13.5, 30 min; E¼22 V, 30 min

pH¼13, 20 min; E¼25 V, 300 mA, 20 min

Study subject

N¼8 F; age range¼25–50 years

N¼10, 8 F, 2 M, all nonsmokers; age range¼37–58 years

(continued ).


Table 8.1

TL/TM, leukocytes

TM/lymphocytes, tumour cells from breast


5-Fluorouracil, adriamycin, cyclophosphamide, metho-trexate, leucovorin calcium, cisplatin

Ifosfamide, doxorubicin

Treatment for breast cancer

Polychemotherapy of various kinds of solid tumours

Genotoxic factor

Clinical case 27

28 Increased comet parameters in cells of all cancer patients after administration of various antineoplastic drugs (amount of administered drugs specified). s. pretreatment and posttreatment interindividual variations in the level of DNA damage were found.

Ref.

Increase of DNA damage in lymphocytes 1 day after treatment and the effect lasted or even increased at 48 h; DNA damage fell back to pretreatment values 3 weeks after treatment; in tumour cells, a shift towards increased DNA damage was seen 3 weeks after treatment. Amount of DNA damage in either tissue did not significantly correlate with clinical response or toxicity.

Results and remarks

186 Chapter 8

TM/lymphocytes

Visual grading of DNA damage/ lymphocytes

pH¼13, 20 min; E¼25 V, 300 mA, 20 min

pH¼13, 20 min; E¼24 V, 300 mA, 20 min

N¼113 breast cancer patients; age range¼36–80 years

N¼72, 24 F, 48 M,36 uremic patients undergoing haemodialysis, mean age 49 years; 36 healthy controls, mean age 49 years

Haemodialysis due to chronic renal failure/uraemia

Radiation treated breast cancer patients

Oxidative stress

Total dose of 50 Gy to the whole breast

Great increase DNA damage of lymphocytes from patients undergoing dialysis as compared to controls. Patients treated with Vitamin E (600 mg/day for 14 weeks) had a significant decrease in DNA strand breakage; no significant association between level of DNA damage and duration of dialysis.

DNA repair capacity showed large differences among patients; 11 patients showed considerably enhanced induction of DNA damage and 7 patients had severely impaired DNA repair capacity. No apparent correlation between acute skin reactions upon radiotherapy and in vitro radiation effects. 29

29


DNA content and mean tail moment

TL, % F/WBC

TM/WBC

alkaline, 0.6 V/cm, 25 min

pH¼13, 20 min; E¼25 V and 300 mA, 20 min

pH¼13, 60 min; E s 25 V (0.8 V/cm) and 300 mA, 30 min

N¼22 patients undergoing palliative radiotherapy for treatment of accessible metastatic tumours were exposed to two doses of radiation. On the second day of radiation, 13 patients were given 80 mg/kg nicotinamide postoperatively on an empty stomach 2 h before treatment; the remaining nine patients acted as controls.

N¼28; 8 M, 20 F; age range¼39–45 years

Study I, N¼46; 24 M, 22 F; 11 thyroid carcinoma patients, 35 controls; age range¼25–80 years

Study subject


(continued ).


Table 8.1

Patients subjected to radiotherapy and chronically exposed to irradiation from Chernobyl area

Thyroid cancer patients treated with 131I sodium iodide

Metastatic tumours

Clinical case

Study I¼131I sodium iodide (5,55 GBq)

131 I sodium iodide (3700-5550 MBg)

Radiation, nicotinamide

Genotoxic factor

Both groups showed decrease, in repair capacity; in the group residing in Chernobyl area, TM after in vitro irradiation was decreased compared to the control group. No adaptive response was seen since the pre-exposure dose was high and equal doses were used every time.

Small increase in TL but not significant.

Decrease in hypoxic fraction

Results and remarks

32

31

30

Ref.

188 Chapter 8

MTL and MTM

TL and TM

pH413, 20 min, 25 V, 300 A

pH413, 20 min, 25 V, 300 A

pH¼13, 5; 20 min; 25 V (0, 83 V/cm)

pH413, 20 min, 25 V, 300 A

N¼24; 8M; 16F; age range¼35–79 years and N¼23 controls; 7M; 16F; age range¼27–85 years

N¼33; 14M; 19F; age range¼35–79 years and N¼33 controls 14M; 19F; age range¼32–85 years

N¼3 invasive transitional cell bladder carcinoma (HT1376, UMUC-3, RT112)

N¼13; N¼12 controls age range¼29–74 years

% TDNA

TM

TM/tumor cells

PH ns, 60 min; E¼0.67 V/cm and 300 mA, 25 min

N¼6; all F; age range¼44–81 years

see above

see above

Study II, N¼ 48; 11 exposed children and 2 exposed adults; Sex and age¼ns 35 controls (20 M and 15 F)

Mitochondrial disease’s patients

Invasive transitional cell bladder carcinoma

Different kinds of cancer (9 mastocarcinoma patients, 3 lung cancer patients, 2 esophagus cancer patients, 3 nasopharyngeal carcinoma)

Various types of cancer

Breast cancer patients treated with radiotherapy

see above

Ubidecarenone

Not any significant difference in primary DNA damage between patients and control.

The UMUC-3 shows the greatest DNA damage and HT1376 displays the least damage at each dose examined.

Lower DNA repair capacity.

UVC, bleomycin

Radiosensitivity

No significant difference between untreated cancer patients and controls. The MTLs and MTMs at 10, 30 and 50 Gy were significantly higher than the MTL and MTM at 0 Gy for each patient.

Radiobiologically hypoxic cells showed a decrease in TM.

see above

Radiation 10, 30 and 50 Gy

Radiation (5-10 Gy)

Study II¼environmental radiation from Chernobyl area (120–8, 170 Bq)

37

36

35

34

33

32


pH¼13, 40 min; E¼25 V (0.86 V/cm) and 300 mA, 20 min

pH alkaline; 10 min, 25 V (0,714 Vcm1; 300 mA


Study subject

N¼32; 15 M, 17 F; 16 patients, 16 controls; age range¼22–60 years

N¼33 with cancer; all M; N¼14; all M with fertility

N¼24; 17 M, 7 F; 9 patients and 15 controls; age range¼57–71 years

(continued ).


Table 8.1

TM, %F/UT

Percentage head DNA

Total image length/ WBC


Patients with transitional cell carcinoma (TCC)

Cancer (testicular cancer, lymphoma and leukaemia) patients

Vasculitis/ collagen disease patients

Clinical case

DNA baseline damage

DNA integrity

CP (a total dose of 50–200 mg daily)

Genotoxic factor

Increase in damage in samples from TCC patients compared to controls. No significant difference between TCC grades.

DNA integrity was reduced by cancer.

Increase in DNA damage in CPtreated patients compared to controls or patients without chemotherapy; increased in DNA damage was variable and not clearly related to CP dose; CP-induced DNA effects persisted in vivo for a period of several days, but for less than 2 weeks.

Results and remarks

40

39

38

Ref.

190 Chapter 8


pH¼12.5, 2 V (0.714 V/ cm); 300 mA, 10 min

pH 13, 40 min; E¼25 V (0.8 V/cm) and 300 mA, 30 min

N¼41; M; 13 fertile and 28 infertile; age¼ns

N¼50 patients

N¼20; M; 10 patients and 10 controls; age range¼33–59 years %F/L

Percentage head DNA

% DNA head area

Insulin-dependent diabetes patients

Fertile and infertile males

Fertile and infertile males

Reactive oxygen species

X-ray irradiation

DNA baseline damage

Mean values of strand breaks and oxidized pyrimidines were significantly increased in diabetics; strong correlation between altered purine sites and serum glucose concentration. Significant correlation between body mass index and strand breaks only in diabetics.

DNA damage increases with X-rays dose.

Baseline levels and damage in sperm samples from fertile and infertile groups were found to be similar. Background DNA damage in sperm cell was increased compared to somatic cells.

42

41

40


Tail length

Visual grading of comets (0, without detectable tail; 4, highest damage/ lymphocytes) Visual grading of comets (0, without detectable tail; 4, highest damage/ lymphocytes)

24 min at 20 V

pH¼13, 25 min; 25 V/ 300 mA

pH413, 25 V (1 V/cm, 300 mA), 30 min

N¼17

N¼25 Rhematoid Arthritis patients; 17F; 8M, N¼26 control; 16F; 10M

N¼20; all M (9 of whom have heritable predisposition to schizophrenia); age range¼37–41 years. N¼20 controls

% DNA in tail


pH alkaline; 20 min; 0,8 V/cm; 300 mA


(continued ).

N¼14 type 2 diabetic patients (9M; 5F) and N¼14 controls (7M; 7F)

Study subject

Table 8.1

Oxydative stress

Oxidative stress

Rheumatoid arthritis patiens

Schizophrenic populations

Not increased DNA damage from controls.

Lymphocyte DNA damage level increases in patients with RA.

Prolonged exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks.

Normal healthy subjects exhibited lower levels of both DNA breaks and FPG-sensitive oxidative DNA damage than diabetics, the difference was more relevant and statistically significant for oxidative damage.

Oxidative DNA damage

Cytokines

Results and remarks

Genotoxic factor

Islets human heart beating donors

Peripheral blood cells from patients with Type 2 diabetes mellitus

Clinical case

46

45

44

43

Ref.

192 Chapter 8

pH413, 25 V (1 V/cm, 300 mA), 30 min

N¼35; age range¼50–70 years, obstructive sleep apnea population; N¼35 controls Visual grading of comets (0, without detectable tail; 4, highest damage/ lymphocytes)


Oxidative stress

Oxidative stress

Chronic psychogenic stressed population

Obstructive sleep apnea

Obstructive Sleep Apnea patients had higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than lymphocytes from controls. OSA patients had a reduced capacity to repair the DNA damage induced by the three agents.

Cells from the stressed population were more sensitive to the induction of DNA damage and had higher level of residual damage. Stress conditions may cause the affected individuals to be susceptible to environmental mutagenic agents. 48

47

Abbreviations used: CP: cyclophosphamide, E: electrophoresis, %F: percentage fluorescence in the tail, F: female, L: lymphocytes, M: male, N¼number, ns: not specified, s.: significant, SCGE: single cell gel electrophoresis, TL: tail length, TM: tail moment, WBC: white blood cells, UT: urothelial cells, VS: visual scoring

pH413, 25 V (1 V/cm, 300 mA), 30 min

N¼30; 15M; 15F age range¼35–41years, stressed exposed population. N¼30 controls 15M; 15F


194

Chapter 8

the background DNA damage was significantly increased in patients as compared to controls. In studies of newly diagnosed untreated breast cancer patients and patients free of cancer/treatment for at least 6 months significantly higher DNA damage was found in lymphocytes if compared to controls.22,23 A higher susceptibility to in vitro treatment of lymphocytes with N-methyl-N-nitro-N-nitrosoguanidine or ionising radiation and a decrease in the DNA-repair capacity was also demonstrated. Sanchez et al.24 using both alkaline and neutral Comet assays found that blood lymphocytes from breast cancer patients exhibited higher single- and double-strand breaks by comparison with healthy individuals. High doses of cyclophosphamide and cisplatin administered to breast cancer patients resulted in a significant increase in DNA damage of lymphocytes from these patients.25 Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer was examined using the single-cell gel electrophoresis.26 No differences in the background or radiation-induced DNA damage were observed when compared with controls. Rajeswari et al.27 showed that lymphocytes from first-degree female relatives of breast cancer patients showed an increased DNA damage upon exposure to mutagens in vitro. The repair capacity of first-degree relatives was also decreased. Three studies28–30 have assessed the risk of DNA damage in lymphocytes of cancer patients who received treatment against breast cancer or various kinds of solid tumours. In all three studies, the level of genetic damage in lymphocytes was significantly increased through treatment. Twenty-two patients undergoing palliative radiotherapy for treatment of accessible metastatic tumours were given nicotinamide. Both nicotinamidetreated tumours and controls demonstrated a significant increase in the percentage of cells containing heavily damaged DNA.31 Nicotinamide, however, was found to reduce hypoxia in the tumour cells. Monitoring of genetic damage induced by therapeutic exposure to 131I was undertaken in a group of thyroid cancer patients.32,33 The single cell gel electrophoresis was also employed to assess the effects of radio- and chemotherapy.34 Jianlin et al.35 in their study of cancer patients during radiotherapy found no significant differences in the amount of singlestrand DNA damage between untreated cancer patients and controls. However, a dose–response relationship between radiation dose and genetic damage in patients during radiotherapy was found, as well as interindividual variance in response to radiation.35 Wei et al.36 investigated the repair capacity of lymphocytes from various types of cancer patients after being exposed to bleomycin and UVC irradiation. The results indicate a reduced DNA-repair capacity for the lymphocytes of all the examined cancer patients when compared to healthy controls. Approximately 50% of patients with invasive transitional cell bladder carcinoma fail to respond to radiotherapy. These patients are disadvantaged by the absence of predictive information regarding their radiosensitivity, thus allowing the tumour to gain additional time for metastatic spread before


195

cystectomy is performed. In this case, the Comet assay can be used in order to investigate the response of this malignancy to radiotherapy.37 The Comet assay was used to quantify primary and oxidative DNA damage in leukocytes of mitochondrial disease (MD) patients. The assay indicated a slightly higher level of primary DNA damage in patients compared with controls. A difference in oxidative DNA damage was also observed, this, however, was not statistically significant.38 Treatment of patients with vasculitis/collagen disease with cyclophosphamide resulted in a significant increase in DNA damage compared to controls.39 O’Donovan40 evaluated the DNA integrity in spermatozoa of men with different types of cancer before and after therapy. His results indicated a reduced DNA integrity if compared to controls. No significant difference in DNA damage between the different cancer groups was observed. A marginally significant difference in comet values in spermatozoa of cancer patients before and after therapy was, however, found. McKelvey-Martin et al.41 compared the baseline DNA damage in sperm cells of fertile and infertile males and did not find a significant difference between the two groups. Hughes et al.42 compared the sensitivity of the Comet assay technique and enzyme-linked immunosorbent assay (ELISA) for the assessment of human sperm integrity. Diabetic patients43 were found to have increased DNA damage when the Comet assay was employed in order to measure the amount of DNA breaks. Evaluation of DNA base oxidation measured as formamidopyrimidine DNA glycosylase (FPG) sensitive sites in peripheral blood cells from type 2 diabetes patients revealed an oxidative DNA damage increase in diabetic compared to normal subjects. Delaney et al.44 reported that prolonged exposure of human pancreatic islets to a mixture of cytokines induces DNA-strand breaks. Rheumatoid arthritis patients were found to have increased DNA-damage levels when compared to controls.45 In addition, the DNA damage was found to be related to the severity of the disease in the patients. The Comet assay was also used to study DNA damage and repair efficiency in schizophrenic populations46 as well as on populations exposed to chronic psychogenic stress.47 The results revealed that lymphocytes from schizophrenic populations showed response similar to the controls. In the case of the stressed population, however, the results indicated that this population was more sensitive to the induction of DNA damage and had a higher level of residual DNA damage than the controls. It was found that lymphocytes from patients with obstructive sleep apnoea syndrome had higher basal levels of DNA damage and were more sensitive to the effects of external DNA-damaging agents than the controls.48

8.4 Discussion and Conclusions The Comet assay has already demonstrated its sensitivity as a technique for the evaluation of DNA damage among a variety of cell types.

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The great usefulness of the Comet assay and its rapid spread among researches are due to several advantages in comparison with other techniques, such as: 1) It has been applied in a wide variety of eukaryotic cells in any organ, with successful results. 2) Sample size is very small (from 10 000 to 50 000 cells). 3) It detects damage at the single-cell level. 4) Cell lines are suitable. 5) Highly sensitive (50–15 000 breaks/cell). 6) Results are obtained on the same day. 7) Damage can be detected in cycling as well as in noncycling cells. 8) Noninvasive technique. 9) Fresh or frozen samples are suitable. 10) It is fast, simple and inexpensive. The technique is now being widely used in many studies for measuring DNAstrand breaks in single cells. However, the studies listed in this review had some flaws due to inconsistent Comet protocols and study design problems. The most common shortcomings found in the design of the studies were: i. Use of small study groups weaken the statistical power. ii. Gender distribution between the groups was usually unequal. iii. Data on external or internal exposure to agents were not always provided. iv. Size of the study group and the control usually was unequal, making the comparison of the two groups rather difficult. v. No data were always provided on profession, disease status or ethnicity. All these factors should be seriously taken into account in forthcoming clinical studies in order to make possible interlaboratory comparisons of the findings.

References 1. O. Ostling and K. Johanson, Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun., 1984, 123, 291. 2. P. Cook, I. Brazell and E. Jost, Characterization of nuclear structures containing superhelical DNA, J. Cell Sci., 1976, 22, 303. 3. B. Rydberg and K. J. Johanson, in DNA-repair mechanisms, eds. P. C. Hanawalt, E. C. Friedberg, C. F. Fox, Academic Press, New York, NY, 1978, 465. 4. N. Singh, M. McCoy, R. Tice and E. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 1988, 175, 184.


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5. P. Olive, J. Banath and R. Durand, Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the ‘‘comet’’ assay, Radiat. Res., 1990, 122, 86. 6. C. S. Djuzenova, D. Schindler, H. Stopper, H. Hoehn, M. Flentje and U. Oppitz, Identification of ataxia telangiectasia heterozygotes, a cancerprone population, using the single cell gel electrophoresis (Comet) assay, Lab Invest., 1999, 79, 699. 7. M. H. L. Green, J. E. Lowe, S. A. Harcourt, P. Akinluyi, T. Rowe, J. Cole, A. V. Anstey and C. F. Arlett, UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the Comet assay: A potential diagnostic technique, Mutat. Res., DNA Repair, 1992, 273, 137. 8. S. Bu¨rger, D. Schindler, M. Fehn, B. Mu¨hl, H. Mahrhofer, M. Flentje, H. Hoehn, E. Seemanova´ and C. S. Djuzenova, Radiation-induced DNA damage and repair in peripheral blood mononuclear cells from nijmegen breakage syndrome patients and carriers assessed by the comet assay, Environ. Mol. Mutagen., 2006, 47, 260. 9. S. W. Maluf and B. Erdtmann, Genomic instability in Down’s Syndrome and Fanconi anemia assessed by micronucleus analysis and single-cell gel electrophoresis, Cancer Genet. Cytogenet., 2001, 124, 71. 10. L. Migliore, I. Fontana, F. Trippi, R. Colognato, F. Coppede`, G. Tognoni, B. Nucciarone and G. Siciliano, Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients, Neurobiol. Aging, 2005, 26, 567. 11. C. Gonzalez, O. Najera, E. Cortes, G. Toledo, L. Lopez, M. Betancourt and R. Ortiz, Susceptibility to DNA damage induced by antibiotics in lymphocytes from malnourished children, Teratogen. Carcinogen. Mutagenesis, 2002, 22, 147. 12. V. Baltaci, N. Aygu¨n, D. Akyol, A. E. Karakaya and S. Sardas, Chromosomal aberrations and alkaline Comet assay in families with habitual abortion, Mutat. Res., 1998, 417, 47. 13. M. Betancourt, R. Oritz, C. Gonzalez, P. Perez, L. Cortes, L. Rodriguez and L. Villasenor, Assessment of DNA damage in leukocytes from infected and malnourished children by single-cell gel electrophoresis/Comet assay, Mutat. Res., 1995, 331, 65. 14. K. Eren, N. O¨smeric and S. Sardas, Monitoring of buccal epithelial cells by alkaline Comet assay (single-cell gel electrophoresis technique) in cytogenetic evaluation of chlorhexidine, Clin. Oral Investig., 2002, 6, 150. 15. K. Kalaiselvi, P. Rajaguru, M. Palanivel, M. V. Usharani and G. Raumu, Chromosomal aberration, micronucleus and Comet assays on peripheral blood lymphocytes of leprosy patients undergoing multidrug treatment, Mutagenesis, 2002, 17, 309. 16. E. Kan, U¨. U¨ndeger, M. Bali and N. Basaran, Assessment of DNA-strand breakage by the alkaline COMET assay in dialysis patients and the role of vitamin E supplementation, Mutat. Res., 2002, 520, 151.

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17. L. Karabiyik, S. Sardas, U. Polat, N. A. Kocabas and A. E. Karakaya, Comparison of genotoxicity of sevoflurane and isoflurane in human lymphocytes studied in vivo using the Comet assay, Mutat. Res., 2001, 492, 99. 18. S. Sardas, N. Aygun, M. Gamili, Y. Unal, N. Unal, N. Berk and A. Karakaya, DNA damage evaluated by the alkaline Comet assay in lymphocytes of humans anaesthesized with isofluorane, Mutat. Res., 1998, 418, 93. 19. A. Biri, E. Civelek, B. Karahalil and S. Sardas, Assessment of DNA damage in women using oral contraceptives, Mutat. Res., 2002, 521, 113. 20. O. Palyvoda, J. Polanska, A. Wygoda and J. Rzeszowska-Wolny, DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the Comet assay and micronucleus tests, Acta Biochim. Pol., 2003, 50, 181. 21. A. M. de Miranda Cabral Gontijo, J. P. de Castro Marcondes, F. N. Elias, M. L. C. S. de Oliveira, R. O. A. de Lima, D. M. F. Salvadori and J. L. V. de Camargo, DNA damage in cytologically normal urothelial cells of patients with a history of urothelial cell carcinoma, Environ. Mol. Mutagenesis, 2002, 40, 190. 22. T. R. Smith, M. S. Miller, K. K. Lohman, L. D. Case and J. J. Hu, DNA damage and breast cancer risk, Carcinogenesis, 2003, 24, 883. 23. P. Sańchez, R. Penãrroja, F. Gallegos, J. L. Bravo, E. Rojas and L. Benı´ tez-Bribiesca, DNA damage in peripheral lymphocytes of untreated breast cancer patients, Arch. Med. Res., 2004, 35, 480. 24. R. R. Tice, G. H. S. Strauss and W. P. Peters, High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single-cell gel electrophoresis assay, Mutat. Res., 1992, 271, 101. 25. C. S. Djuzenova, B. Mu¨hl, M. Fehn, U. Oppitz, B. Mu¨ller and M. Flentje, Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays, J. Cancer, 2006, 94, 1194. 26. N. Rajeswari, Y. R. Ahuja, U. Malini, S. Chandashekar, N. Balakrishna, K. V. M. Rao and A. Khar, Risk assessment in first degree female relatives of breast cancer patients using the alkaline Comet assay, Carcinogenesis, 2000, 21, 557. 27. E. C. Johnstone, M. J. Lind, M. J. Griffin and A. V. Boddy, Ifosfamide metabolism and DNA damage in tumour and peripheral blood lymphocytes of breast cancer patients, Cancer Chemother. Pharmacol., 2000, 46, 433. 28. N. Kopjar, V. Garaj-Vrhovac and I. Malis, Assessment of chemotherapyinduced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline Comet assay, Teratogen. Carcinogen. Mutagen., 2002, 22, 13. 29. O. Popanda, R. Ebbeler, D. Twardella, I. Helmbold, F. Gotzes, P. Schmezer, H. W. Thielmann, D. von Fournier, W. Haase, M. L. Sautter-Bihl, F. Wenz,


30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

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H. Bartsch and J. Chang-Claude, Radiation-induced DNA damage and repair in lymphocytes from breast cancer patients and their correlation with acute skin reactions to radiotherapy, Int. J. Radiat. Oncol. Biol. Phys., 2003, 55, 1216. D. B. McLaren, T. Pickles, T. Thomson and P. L. Olive, Impact of nicotinamide on human tumour hypoxic fraction measured using the Comet assay, Radiother. Oncol., 1997, 45, 175. S. Gutie´rrez, E. Carbonell, P. Galofre´, A. Creus and R. Marcos, Application of the single-cell gel electrophoresis (SCGE) assay to the detection of DNA damage induced by 131I treatment in hyperthyroidism patients, Mutat. Res., 1998, 13, 95. U. G. Plappert, B. Stocker, H. Fender and T. M. Fliedner, Changes in the repair capacity of blood cells as a biomarker for chronic low-dose exposure to ionizing radiation, Environ. Mol. Mutagen., 1994, 30, 153. P. L. Olive, R. E. Durand, J. L. Riche, I. A. Olivotto and S. M. Jackson, Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers, Cancer Res., 1993, 53, 733. L. Jianlin, H. Jiliang, J. Lifen, Z. Wei, W. Baohong and Deng, Measuring the genetic damage in cancer patients during radiotherapy with three genetic endpoints, Mutagenesis, 2004, 19, 457. Z. Wei, J. Lifen, H. Jiliang, L. Jianlin, W. Baohong and Deng, Detecting DNA-repair capacity of peripheral lymphocytes from cancer patients with UVC challenge test and bleomycin challenge test, Mutagenesis, 2005, 20, 271. V. J. McKelvey-Martin, T. S. Ho Edwin, S. R. McKeown, S. R. Johnston, P. J. McCarthy, N. Fadilah Rajab and C. S. Downes, Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells, Mutagenesis, 1998, 13, 1. L. Migliore, S. Molinu, A. Naccarati, M. Mancuso, A. Rocchi and G. Sicilian, Evaluation of cytogenetic and DNA damage in mitochondrial disease patients: effects of coenzyme Q10 therapy, Mutagenesis, 2004, 19, 43. A. Hartmann, K. Herkommer, M. Gluck and G. Speit, DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single cell gel test Comet assay, Environ. Mol. Mutagen., 1995, 25, 180. M. M. O’Donovan, An evaluation of chromatin condensation and DNA integrity in the spermatozoa of men with cancer before and after therapy, Andrologia, 2005, 37, 83. V. J. McKelvey-Martin, N. Melia, I. K. Walsh, S. R. Johnston, C. M. Hughes, S. E. M. Lewis and W. Thompson, Two potential clinical applications of the alkaline single cell gel electrophoresis assay: 1. human bladder washings and transitional cell carcinoma of the bladder; and 2. human sperm and male infertility, Mutat. Res., 1997, 375, 93.

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41. C. M. Hughes, V. J. McKelvey-Martin and S. E. M. Lewis, Human sperm DNA integrity assessed by the Comet and ELISA assays, Mutagenesis, 1999, 14, 71. 42. A. R. Collins, K. Rasˇ lova´, M. Somorovska´, H. Petrovska´, A. Ondrusˇ ova´, B. Vohnout, R. Fa´bry and M. Dusˇ inska´, DNA damage in diabetes: Corellation with a clinical marker, Free Radic. Biol. Med., 1998, 25, 373. 43. V. Pitozzi, L. Giovannelli, G. Bardini, C. M. Rotella and P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes, Mutat. Res., 2003, 529, 129. 44. C. A. Delaney, D. Pavlovic, A. Hoorens, D. G. Pipeleers and D. L. Eizirik, Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells, Endocrinology, 1997, 138, 2610. 45. O. Altindag, M. Karakoc, A. Kocyigit, H. Celik and N. Soran, Increased DNA damage and oxidative stress in patients with rheumatoid arthritis, Clinic. Biochem., 2007, 40, 167. 46. D. Psimada, N. Messini-Nikolaki, M. Zafiropoulou, A. Fortos, S. Tsilimigaki and S. M. Piperakis, DNA damage and repair efficiency in lymphocytes from schizophrenic patients, Cancer Lett., 2004, 204, 33. 47. E. Dimitroglou, M. Zafiropoulou, N. Messini-Nikolaki, S. Doudounakis, S. Tsilimigaki and S. M. Piperakis, DNA damage in a human population affected by chronic psychogenic stress, Int. J. Hyg. Environ. Health, 2003, 206, 39. 48. K. Kontogianni, N. Messini-Nikolaki, K. Christou, G. Gourgoulianis, S. Tsilimigaki and S. M. Piperakis, DNA damage and repair capacity in lymphocytes from obstructive sleep apnea patients, Environ. Mol. Mutagen., 2007, 48, 722.

CHAPTER 9

Applications of the Comet Assay in Human Biomonitoring ANDREW R. COLLINSa,* AND MARIA DUSINSKAb, c a

Department of Nutrition, Faculty of Medicine, University of Oslo, Norway; Norwegian Institute of Air Research (NILU), Kjeller, Norway; c Research Base of Slovak Medical University, Bratislava, Slovakia

b

9.1 Biomonitoring and Biomarkers – An Introduction Human biomonitoring depends on the use of biomarkers, defined as quantitative indicators of molecular and cellular events in biological systems, relevant to human health, development, aging, etc. Biomarkers are measured in biological material (generally blood or urine) collected from patients or volunteer subjects in observational or intervention studies. The molecular epidemiological approach, measuring molecular or cellular biomarkers as indicators of disease risk or of exposure to causative or preventive factors, has applications in studies of environmental and occupational exposure, disease aetiology, nutrition, lifestyle, etc. It is a valuable adjunct to conventional epidemiology, and has the advantage that it requires far fewer subjects and much less time (and is therefore more economical) than the conventional approach. In addition, the biomarkers, if carefully chosen, can give useful information about molecular mechanisms involved in disease aetiology, for example if they reflect an early stage in the progression of the disease. This review will focus on biomarkers of genotoxic exposure and cancer risk, and in particular on the measurement of DNA damage and DNA repair using the Comet assay. Molecular biomarkers can be applied in the context of case-control, cohort or intervention studies. As in conventional epidemiology, * Corresponding author Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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study design and statistical considerations (especially power calculations) are critically important – as is the use of validated, reliable biomarker assays with sufficient sensitivity to detect individual differences with accuracy and precision. This seems an obvious point, but many commonly used biomarker assays have not yet been properly validated.1,2 In this respect, considerable effort has gone into validating the Comet assay for use in monitoring DNA damage.3–9

9.2 The (Modified) Comet Assay The Comet assay is essentially a simple, rapid and sensitive method for measuring DNA breaks in small numbers of cells (typically lymphocytes, in human biomonitoring studies). Cells embedded in agarose on a glass slide or plastic film are lysed with 2.5 M NaCl and Triton X-100 to remove membranes and soluble cell constituents, as well as histones, leaving the DNA, still supercoiled and attached to the nuclear matrix, as a nucleoid. Electrophoresis (usually but not necessarily carried out in an alkaline solution) causes DNA loops containing breaks to move towards the anode, forming ‘‘comets’’ when stained and visualised by fluorescence microscopy. The relative content of DNA in the tail indicates the frequency of breaks. Strictly speaking, ‘‘breaks’’ in the context of the alkaline Comet assay include apurinic sites, which are alkali-labile. The basic Comet assay was modified to detect specific lesions, by digesting the nucleoids with lesion-specific enzymes: formamidopyrimidine DNA glycosylase (FPG), which recognises the oxidised purine 8-oxoGua, endonuclease III to detect oxidised pyrimidines, T4 endonuclease V to detect UV-induced pyrimidine dimers, AlkA (3-methyladenine DNA glycosylase) for alkylated bases, or uracil DNA glycosylase, which removes misincorporated uracil from DNA.10 This modification greatly increases the scope of the assay, since specific kinds of DNA damage are related to different environmental exposures and can be related to different physiological factors. Careful use of the enzymes can give useful information on the likely cause of damage. However, it is important to remember the following: Digestion conditions (especially concentration of enzyme and time of incubation) should be optimised for the quantitative detection of the appropriate lesions. Ideally, a titration should be carried out with a substrate of cells containing a known amount of a specific lesion. Enzymes have varying specificities. Thus, FPG recognises not just 8oxoGua but also the formamidopyrimidine breakdown products of oxidised purines, as well as some alkylation products;11,12 while AlkA has a tendency to attack even undamaged DNA.13 Damage may be underestimated if lesions are inaccessible in the DNA, or occur in clusters in such proximity that they register as a single break.

9.3 Guidelines for Biomonitoring Studies Several general articles on biomonitoring1–2,14–16 are recommended for help in the design of biomonitoring studies using the Comet assay. Certain basic

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principles of study design should be followed in order to avoid obtaining falsepositive and -negative results. Exposed groups should be matched with control (referent) groups with respect to sex, age, smoking habit, alcohol consumption, nutrition and lifestyle. Investigated groups should be large enough to give valid and meaningful results, and so should be estimated in advance by means of a power calculation. For this, an assumption of the minimum difference or change expected to have a biologically meaningful effect is needed, together with information on the precision of the assay method, and the coefficient of variation (or standard deviation) of the biomarker. As an example, the coefficient of variation for separate analyses of 8-oxoGua in identical cell samples using the Comet assay is typically 10–20%.17 As a rule of thumb, it is usually advisable to have at least 50 subjects in each category within a study population. Appropriate inclusion and exclusion criteria have to be clearly defined in advance, and applied when recruiting subjects. Confounding factors (such as age, sex, smoking), which influence the background level of DNA damage and may bias the study, should be taken into consideration. Environmental and occupational monitoring relies on data from exposure measurement and personal monitoring, and information on dose–response relationships, if available, is valuable. Time of sampling, seasonal and geographical details, and operational aspects (e.g. retrieval conditions, transport and storage conditions) should be recorded as all these might increase variability. There follows a check list to help in planning a biomonitoring study, for example monitoring populations for effects of exposure to a genotoxic agent, or effects of differences in diet or lifestyle or age, or carrying out intervention studies. We assume that the samples consist of lymphocytes (the usual biomonitoring material for the Comet assay). Carry out a power calculation to ensure that there are sufficient subjects in the study to obtain statistically meaningful results.18 Apply clear inclusion and exclusion criteria for selection of subjects for the study. Include a matched control group of subjects, i.e. unexposed, or untreated, or taking a placebo (according to the type of study). Carry out a pilot study for every critical aspect of the study – from sample collection to data analysis – to check for unforeseen problems, and to assess (and if possible control for) experimental variation. Store aliquots of lymphocytes at 80 1C or in liquid nitrogen. Freeze them slowly in freezing medium. (See protocol below, Section 9.7.1.2.) When using frozen stored samples, consider whether to select them completely randomly for analysis, or in batches (e.g. all samples from one subject together in the same experiment). Code samples, to ensure that analysis of samples is done ‘‘blind’’. While carrying out a particular study, always use the same protocols, and chemicals from the same company, and avoid making any change in procedure, however slight it may seem.

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As far as possible follow the principles of good laboratory practice. Always obtain ethical approval before starting a study. Points of specific relevance to the Comet assay are given in the following section.

9.4 Biomonitoring with the Comet Assay: Special Considerations While the Comet assay is eminently suitable for use in biomonitoring, consideration should be given to certain practical and theoretical questions. Every step from initial sampling to final evaluation of data plays an important role and can influence the reliability of results. Attempts have been made to introduce standard protocols for use in different laboratories,6,10,19–21 but they are not universally adopted, and minor variations inevitably arise. Several individual and interlaboratory investigations are in progress to establish which variables are important in the sense of having a real effect on experimental results. Meanwhile, it is obviously advisable for each laboratory to set up and implement its own standard protocols for all experimental procedures, manipulations of samples and analyses. Here, we have collected together scattered observations and recommendations to improve the reliability and robustness of the assay. Consider using lesion-specific endonucleases to increase the sensitivity and selectivity of the Comet assay. Use enzyme from the same batch, and identical digestion conditions, throughout the study. Make at least two parallels from each sample (i.e. two gels, or slides, for each endpoint – strand breaks, FPG-sensitive sites, etc.) When analysing results in terms of the overall effect of exposure or treatment, it is the overall comet score for each subject/sample that counts – not the values for all the comets scored (which would give a misleading view of variation). Archive slides (leave on the bench for a day or two to dry and then store in boxes at room temperature). Read the article by Lovell and Omori18 for help with statistical aspects of planning and executing the study.

9.4.1

Surrogate and Target Cells; The Use of White Blood Cells

In most biomonitoring studies there is little choice but to use white blood cells. White blood cells are not representative of all cells in the body, and in particular they are not target cells for cancer. However, because they circulate in the body, their cellular, nuclear and metabolic state (including DNA) reflects overall body exposure. More appropriate cells from various tissues and organs that have occasionally been used include exfoliated bladder cells, nasal and buccal epithelial

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Applications of the Comet Assay in Human Biomonitoring 22–26

27

28

cells, tear duct epithelial cells, and cells from biopsies; but the comets tend to have high levels of damage compared with lymphocytes, and probably this relates to the necessary physical or enzymic disaggregation of tissue, or to the presence of dead or senescent cells. Sperm have been used in several studies27–31 but the DNA in sperm is packaged very differently from DNA in somatic cells, and this affects comet production; methods have not yet been fully optimised. Usually, lymphocytes are isolated from whole blood and used in the Comet assay either at once or after cryopreservation (see protocols below, Section 9.7.1). Though differences in the level of DNA damage between fresh and frozen samples of cells may be observed, they are generally slight and consistent, and the use of frozen white blood cells has logistic advantages when numerous samples are collected in a short time, as in many biomonitoring studies. Hininger et al.32 developed a protocol for the evaluation of DNA damage in frozen whole blood: there were no differences in levels of DNAstrand breaks between fresh and frozen blood.

9.4.2

Sampling Time and Transport

Betti et al.33 found that the time of year when sampling takes place plays a greater role in the Comet assay than in other cytogenetic assays, namely chromosome aberrations and micronuclei. This variation with time is loosely referred to as ‘‘seasonal variation’’. Avoid collecting samples from all exposed subjects and then from all controls (or vice versa), over different time frames. If possible, sample both exposed (treated) and control (reference) individuals on the same day to reduce the likelihood of day-to-day experimental variation influencing results. If a study extends over more than one year, ideally samples should be taken in the same season in each year. The quality of biological material used for measurements of markers is dependent on sampling conditions. This is particularly important for the Comet assay, as damage to DNA can arise if environmental conditions are suboptimal. In the course of a biomonitoring study in Slovakia, blood samples were collected from three different towns always at the same time in the morning; the variability of estimates of DNA-strand breaks and FPG-sensitive sites was not affected by time of transport/storage up to 4 h.34 It is recommended to collect samples of early morning fasted blood, and to transport them in cool conditions (but not directly in contact with ice). A joint study in two independent laboratories showed that storage of samples for up to 4 days at 4 1C or at room temperature had no effect on the level of DNA breaks.35 However, nothing is known of the stability of oxidised bases under these conditions.

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Reference Standards

The use of a standard protocol with quality assurance throughout a study should minimise variation within one laboratory. Reference standards (positive/negative controls with known levels of damage) should ideally be included in each electrophoresis run, or batch of samples, or at least tested every week. Freshly isolated or cryopreserved human lymphocytes or mammalian cell lines have been used as a negative control, and cells exposed to genotoxic agents as a positive control (sometimes incorrectly called an internal standard). The effort to develop a true internal standard by using molecular probes or specific cells that can be clearly distinguished from the cells under investigation in the same gel is still continuing. Include a negative control (e.g untreated lymphocytes) with real samples for Comet assay analysis. Preferably, these cells should be frozen aliquots from a single collection of lymphocytes. Include a positive control (e.g. lymphocytes treated with H2O2 of a concentration that gives a substantial but not saturating level of damage) with real samples for Comet assay analysis. Preferably, these cells should be from a single collection of lymphocytes, treated with the damaging agent and then frozen. Results from the standard lymphocytes should not show variation from week to week; if they do, there is a problem with the procedure that should be sorted out. If the variation is large, then the samples may need to be reanalysed. If the reference standards are exchanged between laboratories, results from those laboratories can be directly compared. Otherwise, calibration against Xor g-irradiated cells (with defined amounts of damage) (see Section 9.9.3 below on calibration) can control for interlaboratory variation. If specific DNA lesions are measured by the Comet assay using repair enzymes, digestion conditions should be optimised (see below, Section 9.8.10).

9.4.4

What Affects the Background Level of DNA Damage?

The basal level of DNA damage in leukocytes is influenced by various lifestyle factors and environmental exposures, including exercise, air pollution, sunlight, and diet, as reviewed by Møller.21 To establish the baseline or background level of DNA damage against which individual levels (or population means) should be compared, Møller pooled results from 125 studies. (Where studies reported data as a visual score, rather than computer-generated parameters, the score was adjusted to a scale of 0–100 to allow direct comparison with % tail DNA.) Median values of % tail DNA/visual score (with range, 25% from median, in brackets) were: DNA-strand breaks 8.6 (4.4–14.5) Endonuclease III-sensitive sites 11.0 (4.2–19.5) FPG-sensitive sites 7.6 (3.2–14.2)


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Apparent seasonal and geographical differences in comet scores have been detected in several studies.6,21,36–39 Fluctuations in strand breaks through the year in a group of subjects who were repeatedly sampled were attributed to differences in sun exposure.21,38,39 A significant (but weak) negative correlation between latitude and basal damage has been identified, with relatively low levels of damage in northern Europe.21

9.5 DNA Damage as a Marker of Environmental Exposure and Risk To be of practical use in risk assessment, a biomarker assay should be fully validated. There are few assays that meet the requirements for accuracy, reproducibility, specificity, sensitivity and reliability. The Comet assay is partly validated, thanks to efforts such as the EC-funded concerted action ESCODD and the current network of excellence ECNIS. There are by now hundreds of environmental and occupational biomonitoring studies making use of the alkaline Comet assay to measure, in most cases, strand breaks and AP sites, and several review articles have been published.4–6,21,40 DNA damage as measured by the Comet assay (whether strand breaks, or specific base damage) is regarded as a biomarker of exposure to genotoxic agents, and as an index of biologically effective dose. It is worth noting that some of the strand breaks detected will be intermediates in the excision of damaged bases (rather than frank breaks) and so are an indirect indicator of DNA damage. DNA damage is also commonly regarded as a marker of cancer risk (since there is an obvious involvement of DNA damage in mutagenesis), but – in contrast to chromosome aberrations and micronuclei, which have been shown to have cancer-predictive value41,42 – there is as yet no evidence that a high level of DNA damage measured in white blood cells reflects an increased risk of cancer. Since most DNA damage is quickly repaired, the lesions are transient and so must be regarded as a marker of exposure rather than effect. While clearly most relevant to cancer, DNA lesions indicating exposure to oxidative damage can be an appropriate biomarker of risk of other diseases related to oxidative stress, such as cardiovascular or inflammatory diseases.43,44 Protection against DNA oxidation is afforded by endogenous and possibly also dietary antioxidants, and so antioxidant status has come to be regarded as a relevant phenotypic feature. The Comet assay can be used to assess antioxidant status by challenging subjects’ lymphocytes with H2O2 and measuring the breaks produced.45

9.6 DNA Repair as a Biomarker of Individual Susceptibility DNA repair removes potentially mutagenic changes in DNA and is therefore a crucial element in protection against cancer. Individual differences in capacity

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for repair will influence susceptibility to cancer. It is likely that genetic, environmental as well as lifestyle and nutritional factors influence individual repair capacities. DNA repair is therefore increasingly recognised as an important marker in biomonitoring studies, and methods based on the Comet assay have been developed. The simplest approach to measuring DNA repair in human cells is to monitor the time course of rejoining of DNA breaks or removal of enzymesensitive sites during incubation of cells after treatment with a specific damageinducing agent. The dose of damage inflicted on the cells should be low (as close as possible to the levels expected under ‘‘physiological’’ conditions), and in this respect the Comet assay is particularly useful because of its high sensitivity. This approach has been used to investigate individuals exposed to traffic pollution,46,47 workers in a rubber tyre factory,48 styrene-exposed workers,49 and nuclear power plant workers.50 An alternative in vitro approach has been developed, in which a cell extract is incubated with a damage-containing DNA substrate. The substrate consists of agarose-embedded nucleoids derived from cells treated with a specific DNAdamaging agent; the repair activity assayed in the extract is defined by the kind of damage present in the substrate. Initially, the assay was designed to measure 8-oxoguanine DNA glycosylase (OGG) activity, by treating the cells with photosensitiser Ro 19-8022 plus light to induce 8-oxoGua.51 Langie et al.52 modified the assay to measure nucleotide excision repair (NER), on a substrate containing bulky adducts induced by treatment with benzo(a)pyrene diolepoxide (BPDE). Gaivaõ et al.53 applied in vitro assays for both NER (with UVdamaged substrate) and base excision repair (BER, using Ro 19-8022+light) and estimated inter- and intraindividual variability in a group of 430 healthy subjects. Individual repair rates were consistent across time. Between individuals, variation was high; the range was about 4-fold for BER and 10-fold for NER, implying considerable scope for investigation of environmental and genetic influences. This in vitro approach was applied to measure repair in samples from individuals exposed to asbestos and other mineral fibres.54–56

9.7 Protocols 9.7.1

Protocol for Blood Sample Collection and Long-Term Storage of Lymphocytes for the Measurement of DNA Damage and Repair

This protocol is appropriate for collecting samples of lymphocytes in a population study or intervention trial. Lymphocytes are stored frozen, in aliquots appropriate for (1) measuring DNA damage with the normal Comet assay plus lesion-specific endonucleases, and (2) assessing DNA repair activity using in vitro assays for BER and NER. It is important to carry out a trial run of the entire procedure, well in advance of the start of the actual study, to ensure that all steps are satisfactory and that meaningful results will be obtained.


9.7.1.1

209

Materials and Reagents Required

Blood; 7–8 ml from each subject in Becton–Dickinson cell-preparation tubes (CPT) containing sodium citrate as anticoagulant. Samples in CPT should be kept at room temperature before centrifugation for a maximum of 2 h (but the less time the better). Lymphoprep (Nycomed, Oslo) – used as alternative to CPT PBS (cold) Ice Cryovials (1 ml) and microcentrifuge tubes (1.5 ml), labelled with coded sample ID Freezing medium; PBS or medium (MEM, or RPMI) with 20% calf serum and 10% DMSO (cold) Liquid nitrogen

9.7.1.2

Procedure

Mix blood by gently inverting CPT a few times. Centrifuge for 30 min at 1500 g, room temperature, no brake on centrifuge. If required (for other assays), remove plasma and freeze in aliquots. Remove band containing lymphocytes, just above the gel. Use a plastic Pasteur pipette and remove about 2 ml. (Red blood cells can also be recovered from the bottom of the CPT if required.) Add cells to 10 ml of PBS in 15 ml centrifuge tube, on ice. Centrifuge at 4 1C for 10 min at 700 g (the brake can be used). Discard supernatant. Disperse cells in pellet by tapping tube hard. Add 10 ml of cold PBS and mix by pipetting a few times (not too vigorously). Keep on ice. Take a sample (50 ml) for cell count. Calculate and record on spreadsheet (Figure 9.1) the total number of cells. Invert tube to mix, and divide contents into 2 portions by transferring 7 ml to a 15 ml centrifuge tube (labelled ‘‘II’’) for the repair assays. The remaining 3 ml of cell suspension, labelled ‘‘I’’, is for assaying DNA damage: Tube I: Centrifuge for 10 min at 700 g, 4 1C. Discard supernatant. Tap the tube to disperse the pellet. Add 1 ml of freezing medium for every 3 106 cells and gently mix by pipetting a few times. Divide into equal aliquots of approximately 0.3 ml in cryovials. The volume and hence the number of cells here is not critical. Prepare at least 4, and no more than 6 aliquots from each sample. Place tubes in a thick-walled box of expanded polystyrene in a 80 1C freezer and leave overnight. Slow freezing is essential to preserve DNA without shearing.

1 2 3 4 5 6 7

2.7.2008 2.7.2008 2.7.2008 3.7.2008 3.7.2008 3.7.2008 3.7.2008

9.7 6.5 11.1 4.5 8.2 15.3 10

C Total cells x10*6

D Cells for comets (I) = C2*0.3 2.91 1.95 3.33 1.35 2.46 4.59 3 In: (ml) =D2/3 0.97 0.65 1.11 0.45 0.82 1.53 1.00

E

4 4 4 4 4 5 4

Aliquots

F

H Cells/ aliquot =D2/F2 0.73 0.49 0.83 0.34 0.62 0.92 0.75

G Volume/ aliquot =E2/F2 0.24 0.16 0.28 0.11 0.21 0.31 0.25

I J K Cells for repair Cells\ (II) Aliquots aliquot =C2*0.7 =I2/J2 6.79 2 3.40 4.55 2 2.28 7.77 2 3.89 3.15 2 1.58 5.74 2 2.87 10.71 3 3.57 7 2 3.50

small blood sample volume

AC

L M N Notes e.g.who did the isolation; problems

O

Excel spreadsheet devised to help with allocating lymphocytes isolated from a blood sample for DNA-damage measurement (standard Comet assay) and for making extracts for DNA repair assays. Simple macros are entered in the first data row (as in row 2 in this example) and transferred to each cell in the column by dragging down. For each sample, the total number of cells is entered in column C; the macro divides this into 30% for comets for DNA damage, and 70% for repair extract pellets, and calculates the number of cells for each (columns D and I, respectively). The number of aliquots required is entered in column F (DNA damage) and J (repair), and the macros calculate the number of cells per aliquot and in column G the volume to be dispensed per aliquot for the DNA damage assay. (For the repair aliquots, the cells are taken up in 1 ml – see protocol – and a macro is not required for the calculation)

Subject

B

Date

Figure 9.1

1 2 3 4 5 6 7 8 9 10

A

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Transfer to labelled boxes and store at 80 1C. (The cryovials can be transferred to a liquid-nitrogen storage tank. Samples stored for a period of 5 months either in LN2 or at 80 1C show no difference in damage levels. Disadvantage of LN2: If you are unlucky, a cryovial may become filled with liquid nitrogen; on warming up, the sudden vaporisation of the liquid can cause the tube to shatter. Always wear eye protection when working with LN2.) Tube II (for assay of DNA repair): 7 ml of cell suspension. Centrifuge for 10 min at 700 g, 4 1C. Discard supernatant. Suspend pellet in 1 ml of PBS and divide equally into 0.5 ml microcentrifuge tubes so that each tube contains 3–5 106 cells (recommended minimum 2 tubes, maximum 4). NB: It is crucially important that these aliquots are equal, and that all the suspension is used, so that all samples have identical cell concentrations; this is because the repair rate will depend on the concentration of cells in the extract. Spin at B14 000 g (i.e. top speed on a microcentrifuge), 3 min, at 4 1C. Discard supernatant, removing as much as possible (tap upside down on tissue and then use pipettor to remove last drop). NB: It is important not to lose cells at this stage, so take care not to disturb the pellet. Close tubes. Flash-freeze by dropping tubes into liquid N2. Store tubes at 80 1C. As an alternative to CPT, ordinary vacutainers can be used: Collect 10 ml of blood in vacutainers containing citrate or EDTA. Mix blood with equal volume of PBS. Gently and slowly layer onto 20 ml of Lymphoprep in a 50 ml conical plastic centrifuge tube. Centrifuge for 30 min at 700 g, room temperature, no brake on centrifuge. Remove band containing lymphocytes, just above Lymphoprep, using 1 ml Pasteur pipette, or pipettor; collect about 5 ml and transfer to a 15 ml plastic centrifuge tube. Add 10 ml of PBS; mix. Spin for 20 min at 700 g, room temperature. Decant supernatant, add PBS to volume of 10 ml, resuspend cells, count sample. Split into two samples, 3 ml (tube I) and 7 ml (tube II), and proceed as above.

9.7.2

Comet Assay – Determination of DNA Damage (Strand Breaks and Oxidised Bases)

This protocol gives data on detection of strand breaks (including alkali-labile sites) and oxidised bases. It can be adapted for the determination of antioxidant resistance by treating lymphocytes with H2O2 (50 mM for 5 min on ice) and measuring the damage induced; and measurement of lesions other than oxidised bases simply requires substitution of the appropriate endonuclease.

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Slide Preparation: Precoating Slides with Agarose

(Early versions of the Comet assay used fully frosted slides, which give good anchorage for agarose, but can only be used a few times before agarose layers begin to detach. Agarose-precoated slides are cheaper, and give better visualisation, especially after drying the slides. When frosted slides are dried, the frosted surface is too close to the comets, and there is great interference from light reflected from the frosting. Drying of slides is advantageous for archiving, and also for viewing of comets, since the comets are all in virtually the same plane, obviating the need for constant refocusing.) The slides for precoating should be grease-free; clean if necessary by soaking in alcohol and then wiping dry with a clean tissue. Dip slides in a (vertical) staining jar of melted 1% standard agarose in H2O. Drain off excess agarose, wipe the back clean, and dry by leaving on a clean bench overnight.

9.7.2.2

Preparation of Cells

Place frozen aliquot of lymphocytes in a 37 1C water bath and as soon as the ice has melted, tip into a 15 ml centrifuge tube containing 10 ml of PBS on ice. (Do not allow cells to stand in warm freezing medium, as the DMSO tends to cause DNA damage.) Centrifuge 10 min at 700 g, 4 1C. Resuspend pellet in PBS at 106 cells per ml (based on the original cell count, retrieved from the spreadsheet). Keep on ice.

9.7.2.3

Embedding Cells in Agarose

Two gels are made on each slide. Work quickly as the agarose sets quickly at room temperature! Pipette 40 mL of cell suspension into a microcentrifuge tube and add 140 mL of 1% low melting point (LMP) agarose in PBS at 37 1C. Mix by tapping tube and then sucking agarose up and down (once) with pipette. Take 2 70 mL aliquots and place as two well-separated drops on a precoated (and appropriately labelled) slide. Cover each gel with an 18 18 mm glass coverslip. Leave slides at 4 1C for at least 5 min to set.

9.7.2.4

Lysis

Add 1 ml Triton X-100 to 100 ml of lysis solution (4 1C) and mix thoroughly. Remove coverslips from gels (by sliding off between finger and thumb) and place in this solution in a staining (Coplin) jar. Leave at 4 1C for 1 h or longer (up to 24 h is generally OK, but there is a tendency for gels to detach from slides with prolonged lysis).


9.7.2.5

213

Enzyme Treatment (Endonuclease III, FPG)

Prepare 300 ml of enzyme reaction buffer. Put aside 1 ml for enzyme dilutions. Wash slides in 3 changes of this buffer (4 1C) in a staining jar, for 5 min each. Meanwhile, prepare dilutions of enzyme. The final dilution of the working solution will vary from batch to batch. Remove slides from last wash, and dab off excess liquid with tissue. Place 30–40 mL of enzyme solution (or buffer alone, as control) onto gel, and cover with a 20 20 mm coverslip, or a square cut from Parafilm (approx. 2.5 cm square). Put slides into moist box (to prevent desiccation) and incubate at 37 1C for 30 min.

9.7.2.6

Alkaline Treatment

The electrophoresis solution should be cooled before use, e.g. by pouring into the electrophoresis tank in the cold room an hour or so before it is needed. The solution can be reused (at least twice) but should not be stored for a long period as it will absorb CO2 from the air and the pH will change. Gently place slides (minus coverslips) on platform in tank, immersed in solution, forming complete rows (gaps filled with blank slides). Make sure that tank is level and gels are just covered. Leave for 40 min.

9.7.2.7

Electrophoresis

For most tanks (i.e. of standard size), run at 20–25 V (constant voltage setting) for 20 min. If there is too much electrolyte covering the slides, the current may be so high that it exceeds the maximum, so set this at a higher level than you expect to need. If necessary, i.e. if the set voltage is not reached, remove some solution. Normally the current is around 300 mA but this is not crucial. (The voltage depends on tank dimensions. 0.8 V/cm between the electrodes is often recommended, but the voltage drop is considerably greater across the platform where the conducting layer is least deep and the resistance highest. The changes in voltage–current–resistance across the tank from electrode to electrode, and the conditions within the gel, provide an interesting exercise in simple theoretical physics.)

9.7.2.8

Neutralisation

Wash for 10 min with neutralising buffer in a staining jar at 4 1C, followed by 10 min in water. Proceed to staining while gels are wet: OR (Optional: fix with ethanol.)

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Dry (room temperature or a warm oven) for storage. (Drying has the advantage that in the thin agarose layer the depth of comets varies much less than in wet gel, so less refocusing is needed during microscopy.) Store slides at room temperature.

9.7.2.9

Staining

Alternative stains: 4 0 6-diamidine-2-phenylindol dihydrochloride (DAPI) (1 mg/ml); propidium iodide (2.5 mg/ml); Hoechst 33258 (0.5 mg/ml); SYBRGold (0.1 ml/ml); ethidium bromide (20 mg/ml). Make up stain in distilled H2O or according to manufacturer’s instructions, and store 1 ml aliquots in the dark at 20 1C. Place 20 mL of a stain solution onto each slide and cover with a 22 22 mm coverslip. Keep slides in a dark, moist chamber until they are viewed. They may be left overnight before viewing, either stained or unstained (however, if stained, some fluorescence is lost). If gels are dried before viewing, simply add stain to the slide on the position of the dried gel.

9.7.2.10

Storage and Re-Examination

After staining, wash slides briefly in water and drain. Leave slides at room temperature or place in a warm oven until the gel has dried. Store slides at room temperature. For re-examination, stain as above.

9.7.3 In Vitro Assays for DNA Repair These in vitro assays provide a measure of the excision repair activity of an extract of cells (such as lymphocytes) by providing the extract with a DNA substrate containing specific damage. In principle, any mammalian cells can be used to produce the substrate DNA. It is convenient to use HeLa cells, or similar, growing as a monolayer. Cells should be near but not at confluence, and should not be under stress (as this can lead to increased background breaks). It is good practice to change the medium the previous day. To assess BER of oxidised Gua, the substrate cells are treated with a photosensitiser plus visible light. To assess NER, the substrate cells are UVCirradiated. The treated cells are embedded in agarose on a microscope slide, and lysed, to form nucleoids. This substrate is then incubated with the extract. Incision at damage sites is detected using the alkaline Comet assay. The protocol here is a modification of the original published version.51


9.7.3.1

215

Preparation of Substrate Cells for BER

Remove culture medium and wash cells on a dish with PBS. Add 5 ml of cold PBS containing Ro 19-8022 (1 mM). NB Avoid excessive light during this stage. Place on ice 33 cm from a 500 W halogen lamp and irradiate for 5 min. Remove Ro 19-8022 solution, wash monolayer with PBS, and trypsinise (do not leave in trypsin for longer than necessary to detach cells). Add a few ml of medium with serum (to inactivate trypsin). Take a sample to carry out a cell count. Centrifuge cells at 400 g, 5 min, 4 1C. Suspend the pellet in freezing medium (culture medium with double the normal concentration of fetal calf serum and 10% DMSO) at 3 106 cells per ml. Split into 0.4 ml aliquots (enough to make 28 slides, 2 gels per slide) in microcentrifuge tubes, and freeze slowly (in an expanded polystyrene box) to 80 1C. Store at this temperature.

9.7.3.2

Preparation of Substrate Cells for NER

Remove medium from cells and wash with PBS. Irradiate the cells, after removing the lid of the dish, with UVC light to give a dose of 1 J m2. (To measure the dose, a radiometer is needed. Simple filters to reduce the dose rate to a manageable level can be devised from certain plastics, or metal gauze.) Remove cells from the dish by gentle trypsinisation. When they are detached, add medium with serum to stop the action of the trypsin. Subsequent steps are the same as for BER (Section 9.7.3.1).

9.7.3.3

Preparation of Cell Extract

On the day of the experiment, thaw the aliquot and resuspend pellet in 65 mL of buffer A to which Triton X-100 is added to 0.25% just before use. Vortex-mix for 5 s at top speed. Incubate for 5 min on ice. Spin at B14 000 g (or top speed on microcentrifuge), 4 1C, 5 min. Remove 55 mL of supernatant and combine with 220 mL of cold buffer F (for BER) or buffer F+Mg (for NER).

9.7.3.4

The Reaction: BER or NER

Thaw the frozen photosensitiser and light-treated substrate cells (for BER), or UV-irradiated cells (for NER); wash twice in cold PBS, centrifuging for 5 min at 800 g, 4 1C. Suspend in 100 mL of PBS, and add 4 ml of 1% LMP agarose at 37 1C. Mix thoroughly.

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Prepare Comet slides in the usual way with 2 gels each of 70 mL of the cell suspension in agarose (approx. 2 104 cells per gel). Place coverslip over each gel. Place slides in refrigerator for 5 min for agarose to set. Remove coverslips and place the slides in cold lysis solution (with Triton X100) for one hour (minimum). Wash the slides 3 times (5 min each) with cold buffer F (for BER) or F+Mg (for NER). After last wash, dab off excess buffer with paper tissue. Place 30 mL of extract on each gel and cover with a glass coverslip or with a square of Parafilm. It is recommended to include incubations with FPG (a positive control) and with buffer F (+ Mg) alone as a negative control. Place slides in a moist box (previously kept at 37 1C). The box should contain suitable racks sitting in water to ensure humidity without the slides getting wet. Incubate at 37 1C for 10 min and 20 min. Include a 0 min incubation for the buffer F control. After incubation, remove coverslip, transfer immediately to alkaline electrophoresis solution in tank and continue the Comet assay as normal. A wash in a solution of 1 mM EDTA is recommended after the NER assay before placing in alkali, to remove Mg. Note: To arrange for all gels to have the same alkaline incubation period, start the reaction at different times for the 20, 10 and 0 min incubations, so that they all end at the same time. Alternatively, start all at the same time and at the end place slides on an ice-cold metal plate until all are ready for transfer to alkaline solution together. Generally, one incubation period – either 10 or 20 min – is sufficient to estimate the repair capacity of normal extracts. The optimal incubation period should be established by experimentation before analysis of real samples. The incision rate should be expressed relative to the number of cells in the extract.

9.8 Solutions, etc. Prepare solutions from appropriate stocks, such as 0.5 M Na2EDTA, 1 M Tris, 1 M KCl etc. Keep solutions at 4 1C.

9.8.1.

Lysis Solution

2.5 M NaCl 0.1 M EDTA 10 mM Tris Prepare 1 litre. Set pH to 10 with either solid NaOH, or preferably concentrated (10 M) NaOH solution. (Add 35 ml of NaOH straight away to


217

ensure that EDTA dissolves, and then add dropwise to pH 10.) Add 1 ml Triton X-100 per 100 ml immediately before use. (Many people include Sarkosyl and DMSO in the lysis solution. Others have found that they serve no purpose.)

9.8.2.

Buffer F (Enzyme Reaction Buffer for FPG, Endonuclease III, and In Vitro BER Assay)

40 mM HEPES 0.1 M KCl 0.5 mM EDTA 0.2 mg/ml BSA pH 8.0 with KOH (can be made as 10 stock, adjusted to pH 8.0 and frozen at 20 1C)

9.8.3

Buffer F+Mg (Used for In Vitro NER Assay)

As buffer F but includes 1 mM MgCl2.

9.8.4

Buffer A (Used in In Vitro Repair Assays)

45 mM HEPES 0.4 M KCl 1 mM EDTA 0.1 mM dithiothreitol 10% glycerol Adjust pH to 7.8 with KOH

9.8.5

Triton Solution

1% Triton X-100 in buffer A (can be kept for 2 days at 4 1C – perhaps longer).

9.8.6

Ro 19-8022 (Photosensitiser)

Obtained from Hoffmann La Roche. Dissolve in 70% ethanol at 1 mM and store in small aliquots in microtubes at 20 1C. Avoid excessive light during preparation and wrap tubes in aluminium foil. Working solution: 1 mM in PBS.

9.8.7

Electrophoresis Solution

0.3 M NaOH 1 mM EDTA

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Neutralising Buffer

PBS (PBS is just as good a neutralising solution as the originally recommended Tris buffer, and is much cheaper.)

9.8.9

Agarose

Electrophoresis grade, e.g. Gibco BRL 5510UA LMP (low melting point), e.g. Gibco BRL 5517US

9.8.10

Enzymes

Endonuclease III (endo III) and formamidopyrimidine DNA glycosylase (FPG) are isolated from bacteria containing overproducing plasmids. Because such a high proportion of protein is the enzyme, a crude extract is perfectly satisfactory; in our experience there is no nonspecific nuclease activity at the concentrations employed. The enzyme extracts are best obtained from a research laboratory producing them, although some enzymes are commercially available. On receipt, the enzyme (which should have been frozen in transit) should be dispensed into small aliquots (say, 5 mL) and stored at 80 1C. This minimises repeated freezing and thawing. The final dilution of the working solution will vary from batch to batch. The following assumes a dilution of 1/3000. FPG: Dispense the stock solution into 5 mL aliquots and refreeze at 80 1C. Take one of these aliquots and dilute to 0.5 ml using the regular FPG/ endoIII reaction buffer – with the addition of 10% glycerol. Dispense this into 10 mL aliquots (label as ‘‘100 diluted’’) and freeze at 80 1C. For use, dilute one of these 10 mL aliquots to 300 mL with buffer (no glycerol) and keep on ice until adding it to the gels: do not refreeze this working solution. Endonuclease III is more stable: Dispense the stock solution into 5 mL aliquots and refreeze at 80 1C. Take one of these aliquots and dilute to 0.5 ml using the regular FPG/ endoIII reaction buffer (no need to add glycerol). Dispense this into 10 mL aliquots (label as ‘‘100 diluted’’) and freeze at 80 1C. For use, dilute one of these 10 mL aliquots to 300 mL with buffer (no glycerol) and keep on ice until it is added to the gels. The buffer in which enzyme is stored may contain b-mercaptoethanol or similar to preserve the enzyme. However, inclusion of sulfhydryl reagents in the reaction buffer would significantly increase background DNA breakage.

9.9 Analysis and Interpretation of Results 9.9.1

Quantitation

Computer-assisted image analysis gives results that are easily compared between labs. With commercially available software linked to a charge-coupled


219

device camera mounted on the microscope, individual comet images can be analysed with a range of parameters including tail length; % of total fluorescence in head and tail; and ‘‘tail moment’’. We recommend % DNA in tail as the most informative parameter. Score 50 comets per gel (fewer if most of the comets show very similar levels of damage, e.g. from control, undamaged cells) and calculate the mean % tail DNA. It is possible to analyse comets quantitatively without image-analysis software. The human eye readily discriminates comets representing different levels of damage, and a widely used scheme for visual scoring is based on 5 recognisable classes of comet, from class 0 (undamaged, no discernible tail) to class 4 (almost all DNA in tail, insignificant head). Select 100 comets at random from each slide, avoiding the edges of the gel, where anomalously high levels of damage are often seen. Each comet is given a value according to the class it is in, so that an overall score can be derived for each gel, ranging from 0 to 400 arbitrary units. When slides are analysed in parallel by visual scoring and by computer image analysis, the match between results is excellent.57 With practice, visual scoring is very quick.

9.9.2

Calculation of Net Enzyme-Sensitive Sites

The control gels (no enzyme treatment) provide an estimate of the background of DNA-strand breaks (SB). The enzyme-treated gels reveal strand breaks and lesions recognised and cleaved by the enzymic treatment (SB+OX). Assuming a linear dose response, whether working in % DNA in tail or in arbitrary units (from visual scoring), subtraction of (SB) from (SB+OX) gives a measure of ‘‘net’’ oxidised pyrimidines/altered purines.

9.9.3

Calibration

Comet assay results, whether obtained as % tail DNA or as arbitrary units, can be expressed in ‘‘real’’ units, such as DNA breaks per 109 Daltons, if the assay is calibrated. Calibration is indirect, is based on a dose–response curve using ionising radiation to induce DNA breaks, and relies on the equivalence of 0.31 breaks per 109 Daltons per Gy that was established many years ago using alkaline sucrose sedimentation techniques.58 See ref. 59 for examples of calibration curves.

9.9.4

How to Deal with Comet Assay Data Statistically

In most biomarker studies, when samples of comets are assessed using computer-based image analysis, the important parameter is the mean comet score for each sample; the distribution of comets within a gel is irrelevant (indeed, if individual comet scores are used for statistical analysis of population groups, a misleadingly low standard error will be obtained). At least two replicate gels are normally scored to obtain the mean value for each sample. These mean values are taken forward into the statistical analysis. Using appropriate statistical tests

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is crucial for evaluating results and identifying differences between exposed and control (or treated and untreated) groups.18 The choice of test depends on whether or not results are normally distributed. The correct evaluation of results and consequently their interpretation are dependent on appropriate statistical analysis. It should be emphasised that false-negative or -positive results can result from a poor design (selection criteria, poor matching of groups, inappropriate biomarkers, too few subjects, etc.). If – as is customary – a P value of 0.05 is set to define statistical significance, then by definition one in 20 apparently significant results will have arisen by chance – an important consideration when numerous biomarkers are measured and many comparisons and correlations are studied. While individual studies may be relatively small in size, meta-analysis (i.e. pooling the results of different studies, assigning weight to the results according to aspects of the study design) can lead to statistically much stronger and more credible conclusions.

9.10 Conclusions The Comet assay, in its various modified forms, plays an important role in human biomonitoring studies. It serves as a valuable general method for detection of genotoxic exposure in humans,4–7,40,60–62 even though not all types of carcinogenic exposures are expected to induce direct DNA damage in white blood cells. In addition to providing data on the effects of genotoxic exposure in human populations, the Comet assay has yielded a great deal of fundamental information on mechanisms of genotoxicity, and cellular responses to DNA damage. While DNA damage has not been correlated with cancer risk in individuals, it is generally assumed that elevated levels of DNA damage will have implications for health. The use of lesion-specific endonucleases allows the measurement of different kinds of DNA base damage, which can give important clues as to the nature of the environmental agent causing the damage. Oxidative DNA damage, measured in lymphocytes with the Comet assay, may have relevance not only for cancer risk but also for other diseases associated with oxidative stress, such as cardiovascular disease. Individual susceptibility to carcinogen-exposure is assumed to depend in part on the individual’s ability to repair damage before it can be fixed as mutations. Modifications of the Comet assay allow measurement of cellular repair (monitoring the removal of lesions from the DNA), and of the in vitro repair capacity of cell extracts on a DNA substrate containing specific lesions. Phenotypic markers of DNA damage and repair are affected by environmental but also by genetic factors. Great emphasis is currently given to the analysis of genotype. The combination of phenotypic data with polymorphism analysis has already given valuable information on gene–environmental interactions and on the coordinated regulation of the various repair pathways and other defence mechanisms.34 However, genotyping requires very large


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numbers of subjects, especially when examining rare SNPs or interactions between SNPs. To match this demand, high throughput versions of the Comet assay (and other phenotype assays) will be needed. The EC-funded COMICS project (Comet assay and cell array for fast and efficient genotoxicity testing) aims to increase by orders of magnitude the numbers of samples that can be analysed in a given time, by using multiwell formats and ‘‘cell arrays’’ to increase the number of samples per experiment, and developing an alternative, automated method of scoring based on differential fluorescence.

Acknowledgements We acknowledge the support of EC-funded projects, in particular the Centre of Excellence in Environmental Health (HEAR NAS, QLK6-2002-90445), NewGeneris (FOOD-2005-016320) and COMICS (LSHB-CT-2006-037575).

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10. A. R. Collins, The Comet assay for DNA damage and repair: principles, applications, and limitations, Mol Biotechnol., 2004, 26, 249–261. 11. G. Speit, P. Schu¨tz, I. Bonzheim, K. Trenz and H. Hoffmann, Sensitivity of the FPG protein towards alkylation damage in the Comet assay, Toxicol. Lett., 2004, 15, 151–158. 12. C. C. Smith, M. R. O’Donovan and E. A. Martin, hOGG1 recognizes oxidative damage using the Comet assay with greater specificity than FPG or ENDOIII, Mutagenesis, 2006, 21, 185–190. 13. K. G. Berdal, R. F. Johansen and E. Seeberg, Release of normal bases from intact DNA by a native DNA repair enzyme, EMBO. J., 1998, 17, 363–367. 14. R. J. Albertini, D. Anderson, G. R. Douglas, L. Hagmar, K. Hemminki, F. Merlo, A. T. Natarajan, H. Norppa, D. E. G. Shuker, R. Tice, M. D. Waters and A. Aitio, IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans, Mutat. Res., 2000, 463, 111–172. 15. M. N. Bates, J. W. Hamilton, J. S. LaKind, P. Langenberg, M. O’Malley and W. Snodgrass, Workgroup report: biomonitoring study design, interpretation, and communication – lessons learned and path forward, Environ. Health Perspect, 2005, 113, 1615–1621. 16. W. W. Au, Usefulness of biomarkers in population studies: from exposure to susceptibility and to prediction of cancer, Int. J. Hyg. Environ. Health, 2007, 210, 239–246. 17. ESCODD, Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus, Carcinogenesis, 2002, 23, 2129–2133.. 18. D. P. Lovell and T. Omori, Statistical issues in the use of the comet Assay, Mutagenesis, 2008, 23, 171–182. 19. A. R. Collins, Oxidative DNA damage biomakers: A need for quality control, In: Critical Reviews of oxidative stress and ageing, eds. R. G. Cutler and H. Rodriguez, World Scientific, New Jersey, 2003, p. 469. 20. G. Speit and A. Hartmann, The Comet assay a sensitive genotoxicity test for the detection of DNA damage and repair, Methods Mol. Biol., 2006, 2314, 275–286. 21. P. Møller, Assessment of reference values for DNA damage detected by the Comet assay in human blood cell DNA, Mutat. Res., 2006, 612, 84–104. 22. E. Rojas, M. Valverde, M. Sordo and P. Ostrosky-Wegman, DNA damage in exfoliated buccal cells of smokers assessed by the single-cell gel electrophoresis assay, Mutat. Res., 1996, 370, 115–20. 23. D. Pinhal, A. M. Gontijo, V. A. Reyes and D. M. Salvadori, Viable human buccal mucosa cells do not yield typical nucleoids: impacts on the singlecell gel electrophoresis/Comet assay, Environ. Molec. Mutagen., 2006, 47, 117–126. 24. K. Eren, N. Ozmeriç and S. SardaS, Monitoring of buccal epithelial cells by alkaline Comet assay (single-cell gel electrophoresis technique) in cytogenetic evaluation of chlorhexidine, Clin. Oral Investig., 2002, 6, 150–154.


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25. T. I. Fortoul, M. Valverde, M. C. Lo´pez, M. R. Avila-Costa, M. C. AvilaCasado, P. Mussali-Galante, A. Gonzalez-Villalva, E. Rojas and P. OstroskyShejet, Genotoxic differences by sex in nasal epithelium and blood leukocytes in subjects residing in a highly polluted area, Environ. Res., 2004, 94 243–248. 26. Y. T. Szeto, I. F. Benzie, A. R. Collins, S. W. Choi, C. Y. Cheng, C. M. Yow and M. M. Tse, A buccal cell model Comet assay: development and evaluation for human biomonitoring and nutritional studies, Mutat. Res., 2005, 578, 371–81. 27. E. Rojas, M. Valverde, M. C. Lopez, I. Naufal, I. Sanchez, P. Bizarro, I. Lopez, T. I. Fortoul and P. Ostrosky-Wegman, Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single-cell gel electrophoresis assay, Mutat. Res., 2000, 468, 11–17. 28. B. L. Pool-Zobel, I. Dornacher, R. Lambertz, M. Knoll and H. K. Seitz, Genetic damage and repair in human rectal cells for biomonitoring: sex differences, effects of alcohol exposure, and susceptibilities in comparison to peripheral blood lymphocytes, Mutat. Res., 2004, 551, 127–34. 29. C. M. Hughes, S. E. Lewis, V. J. McKelvey-Martin and W. Thompson, A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified Comet assay, Molec. Hum. Reprod., 1996, 2, 613–619. 30. M. Sergerie, G. Bleau, R. Teule´, M. Daudin and L. Bujan, Sperm DNA integrity as diagnosis and prognosis element of male fertility, Gynecol. Obstet. Fertil., 2005, 33, 89–101. 31. L. Migliore, R. Colognato, A. Naccarati and E. Bergamaschi, Relationship between genotoxicity biomarkers in somatic and germ cells: findings from a biomonitoring study, Mutagenesis, 2006, 21, 149–52. 32. I. Hininger, A. Chollat-Namy, S. Sauvaigo, M. Osman, H. Faure, J. Cadet, A. Favier and A. M. Roussel, Assessment of DNA damage by Comet assay on frozen total blood: method and evaluation in smokers and non-smokers, Mutat. Res., 2004, 558, 75–80. 33. C. Betti, T. Davini, L. Giannessi, N. Loprieno and R. Barale, Comparative studies by Comet test and SCE analysis in human lymphocytes from 200 healthy subjects, Mutat. Res., 1995, 343, 201–207. 34. M. Dusinska and A. R. Collins, The Comet assay in human biomonitoring: gene-environment interactions, Mutagenesis, 2008, 23, 191–205. 35. D. Anderson, T. W. Yu, M. M. Dobrzyn´ska, G. Ribas and R. Marcos, Effects in the Comet assay of storage conditions on human blood, Teratog. Carcinog. Mutagen., 1997, 17, 115–125. 36. M. Dusˇ inska´, B. Vallova´, M. Ursı´ nyova´, V. Hladı´ kova´, B. Smolkova´, L. Wso´lova´, K. Rasˇ lova´ and A. R. Collins, DNA damage and antioxidants: fluctuation through the year in a central European population group, Food Chem. Toxicol., 2002, 40, 1119–1123. 37. B. Smolkova´, M. Dusˇ inska´, K. Rasˇ lova´, G. McNeill, V. Spustova´, P. Blazı´ cˇek, A. Horska´ and A. Collins, Effects of seasonal variation in diet

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40. 41.

42.

43. 44.

45.

46.

47.

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on markers of oxidative damage to lipids and DNA in defined population groups in Slovakia, Mutat. Res., 2004, 551, 135–144. P. Møller, H. Wallin, E. Holst and L. E. Knudsen, Sunlight induced DNA damage in human mononuclear cells, FASEB J., 2002, 16, 45–53. L. Giovannelli, C. Saieva, G. Masala, G. Testa, S. Salvini, V. Pitozzi, E. Riboli, P. Dolara and D. Palli, Nutritional and lifestyle determinants of DNA oxidative damage: a study in a Mediterranean population, Carcinogenesis, 2002, 23, 1483–1489. P. Møller, Genotoxicity of environmental agents assessed by the alkaline Comet assay, Basic Clin. Pharmacol. Toxicol., 2005, 96(Suppl. 1), 1–42. L. Hagmar, S. Bonassi, U. Stro¨mberg, A. Brøgger, L. E. Knudsen, H. Norppa and C. Reuterwall and the European Study Group on Cytogenetic Biomarkers and Health, Chromosome aberrations in lymphocytes predict human cancer: A report from the European study group on cytogenetic biomarkers and health (ESCH), Cancer Res., 1998, 58, 4117–4121. S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W. P. Chang and N. Holland, et al., An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogenesis, 2007, 28, 625–631. L. Rison, P. Møller and S. Loft, Oxidative stress-induced DNA damage by particulate air pollution, Mutat. Res., 2005, 592, 119–137. M. Dusˇ inska´, K. Rasˇ lova´, J. Lietava, M. Somorovska´, H. Petrovska´, P. Dobrı´ kova´ and A. R. Collins, Elevated DNA damage in lymphocytes from ankylosing spondylitis, diabetes and hyperlipidemic patients, In Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease, ed. J. T. Kumpulainen and J. T. Salonen, Royal Society of Chemistry, Cambridge, 1999, 433–436. M. Panayiotidis and A. R. Collins, Ex vivo assessment of lymphocyte antioxidant status using the Comet assay, Free Rad. Res., 1997, 27, 533–537. A. Cebulska-Wasilewska, A. Wiechec´, A. Panek, B. Binkova´, R. J. Sra´m and P. B. Farmer, Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity, Mutat. Res., 2005, 30, 73–81. A. Cebulska-Wasilewska, I. Pawzyk, A. Panek, A. Wiechec´, I. Kalina, T. Popov, T. Georgieva and P. B. Farmer, Exposure to environmental polycyclic aromatic hydrocarbons: influences on cellular susceptibility to DNA damage (sampling Kosice and Sofia), Mutat. Res., 2007, 620, 145–154. P. Vodicka, R. Kumar, R. Stetina, L. Musak, P. Soucek, V. Haufroid, M. Sasiadek, L. Vodickova, A. Naccarati, J. Sedikova, S. Sanyal, M. Kuricova, V. Brsiak, H. Norppa, J. Buchancova and K. Hemminki, Markers of individual susceptibility and DNA repair rate in workers exposed to xenobiotics in a tyre plant, Environ. Mol. Mutagen., 2004, 44, 283–292.


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49. P. Vodicka, J. Tuimala, R. Stetina, R. Kumar, P. Manini, A. Naccarati, L. Maestri, L. Vodickova, M. Kuricova, H. Jarventaus, Z. Majvaldova, A. Hirvonen, M. Imbriani, A. Mutti, L. Migliore, H. Norppa and K. Hemminki, Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers, Environ. Health Persp., 2004, 112, 867–871. 50. P. Aka, R. Mateuca, J.-P. Buchet, H. Thierens and M. Kirsch-Volders, Are genetic polymorphisms in Ogg1, XRCC1 and XRCC3 genes predictive for the DNA-strand break repair phenotype and genotoxicity in workers exposed to low dose ionising radiation? Mutat. Res., 2004, 556, 169–181. 51. A. R. Collins, M. Dusˇ inska´, E. Horva´thova´, E. Munro, M. Savio and R. Sˇtetina, Inter-individual differences in repair of DNA base oxidation, measured in vitro with the Comet assay, Mutagenesis, 2001, 16, 297–301. 52. S. A. S. Langie, A. M. Knaapen, K. J. J. Brauers, D. van Berlo, F.-J. van Schooten and R. W. L. Godschalk, Development and validation of a modified Comet assay to phenotypically assess nucleotide excision repair, Mutagenesis, 2006, 21, 153–158. 53. I. Gaivao, A. Piasek, A. Brevik, S. Shaposhnikov and A. R. Collins, Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation, Cell Biol. Toxicol., 2009, 25, 45–52. 54. M. Dusˇ inska´, A. R. Collins, A. Kazimı´ rova´, M. Barancˇokova´, V. Harrington, K. Volkovova´, M. Staruchova´, A. Horska´, L. Wso´lova´, A. Kocˇan, J. Petrı´ k, M. Machata, B. Ratcliffe and S. Kyrtopoulos, Genotoxic effects of asbestos in humans, Mutat. Res., 2004, 553, 91–102. 55. M. Dusˇ inska´, A. Kazimı´ rova´, M. Barancˇokova´, A. Horska´, K. Burghardtova´, K. Volkovova´, M. Staruchova´, L. Wso´lova´ and A. R. Collins, Does occupational exposure to mineral fibres cause DNA or chromosome damage? Mutat. Res., 2004, 553, 103–110. 56. M. Dusˇ inska´, Z. Dzupinkova´, L. Wso´lova´, V. Harrington and A. R. Collins, Possible involvement of XPA in repair of oxidative DNA damage, deduced from analysis of damage, repair and genotype in a human population study, Mutagenesis, 2006, 21, 205–211. 57. A. R. Collins, V. L. Dobson, M. Dusˇ inska´, G. Kennedy and R. Sˇteˇtina, The Comet assay: what can it really tell us? Mutat. Res., 1997, 375, 183–193. 58. G. Ahnstro¨m and K. Erixon, Measurement of strand breaks by alkaline denaturation and hydroxyapatite chromatography, in DNA Repair. A Laboratory Manual of Research Procedures, eds. E.C. Friedberg and P. C. Hanawalt, Marcel Dekker, New York, 1981, 403–18. 59. A. R. Collins, A. A. Oscoz, G. Brunborg, I. Gaivaõ, L. Giovannelli, M. Kruszewski, C. C. Smith and Rudolf Stetina, The Comet assay: topical issues, Mutagenesis, 23, 143–151. 60. F. Kassie, W. Parzefall and S. Knasmu¨ller, Single-cell gel electrophoresis assay: a new technique for human biomonitoring studies, Mutat. Res., 2000, 463, 13–31.

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61. F. Faust, F. Kassie, S. Knasmu¨ller, S. Kevekordes and V. Mersch-Sundermann, Use of primary blood cells for the assessment of exposure to occupational genotoxicants in human biomonitoring studies, Toxicology, 2004, 198, 341–50. 62. U. Plappert, K. Raddatz, S. Roth and T. M. Fliedner, DNA-damage detection in man after radiation exposure – the Comet assay – its possible application for human biomonitoring, Stem Cells., 1995, 13(Suppl 1), 215–22.

CHAPTER 10

The Comet Assay in Human Biomonitoring MAHARA VALVERDE AND EMILIO ROJAS* Depto. Medicina Geno´mica y Toxicologı´ a Ambiental, Instituto de Investigaciones Biome´dicas, Universidad Nacional Autońoma de Me´xico, C.U. CP 04510, Me´xico D.F., Me´xico

10.1 Introduction Several versions of the Comet assay are currently in use; the main version also known as the Single-cell gel electrophoresis (SCGE) assay was introduced by Singh et al.1 in 1988. This alkaline version (pH413), which is capable of detecting DNA damage involving DNA single-strand breaks, alkali-labile sites, repair sites and crosslinks in individual cells, is actually the most widely employed. Subsequently, Olive and coworkers2 developed versions of the neutral technique of Ostling and Johanson,3 which involved lysis in alkali treatment followed by electrophoresis at either neutral or mild alkaline4 (pH 12.3) conditions to detect DNA double- and single-strand breaks. The aim of the present chapter is to summarise data published to mid-2007 that addresses the use of the Comet assay in evaluating the possible exposure hazard of humans to genotoxic agents. An increasing number of manuscripts in this area are being published, particularly with respect to risk evaluations of occupational exposure to a broad range of toxicants; including data from the Comet assay as well as other endpoints such as sister chromatid exchanges (SCEs), micronuclei (MN), chromosomal aberrations (CAs), and some polymorphisms. *



227

228

Figure 10.1

Chapter 10

Percentages of human monitoring publications employing the Comet assay since 1993.

At the time that this work was written, 166 studies were located through the National Center for Biotechnology Information (NCBI) that address human biomonitoring using the alkaline Comet assay (pH413); studies performed with other pH conditions were not included. We also exclude studies of human biomonitoring with any clinical application, although we recognise that this was the first application of the Comet assay in terms of biomonitoring and exposed populations. We consider that the pre-existing disease complicated their analysis in terms of biomonitoring and needs attention in an individual review. Also, studies with an in vitro or ex vivo assessment were excluded. The studies included, can be distributed into four categories: environmental exposure, lifestyle conditions, occupational exposure, and reviews of the biomonitoring literature. It is important to note that between 1988 and 1993 there was only one publication addressing human monitoring and it was a review by McKelvey-Martin and colleagues.5 Subsequently, periodical publications about lifestyle and studies of human exposure have increased greatly since 1999; showing in conjunction with occupational exposure publications a linear trend line to increase year by year (Figure 10.1). Although most of the publications (59%) have analysed occupational exposure, articles addressing environmental and lifestyle exposure risks constituted 17%, and reviews represented 7% of the publications (Figure 10.2).

10.2 Human Monitoring In the last couple of years, there has been an increase in the number of studies monitoring the genotoxic effects of several xenobiotics in humans with the aim

The Comet Assay in Human Biomonitoring

Figure 10.2

229

Percentages of human monitoring publications by categories; environmental exposure, lifestyle, occupational exposure and reviews.

of identifying hazards for risk-assessment purposes. Several markers are now available to monitor exposure of humans to mutagens and carcinogens. The advantages of human monitoring for the individuals being studied include: identification of exposure, identification of environmental mutagens/carcinogens, and determination of the possible range of susceptibility of humans to specific mutagens and carcinogens. Given that most human carcinogens are genotoxic, but not all genotoxic agents have been shown to be carcinogenic in humans, it is important to recognise that human monitoring of genotoxicity is independent of cancer as an endpoint. Genotoxicity endpoints classically evaluated CAs, MN, and DNA damage (e.g. adducts, strand breaks, crosslinking, alkali-labile sites). Assays for these endpoints involve biochemical or electrophoretic assays, such as the Comet assay, sister chromatid exchanges (SCEs), and assays for protein or DNA adducts, and hypoxanthine-guanine phosphorybosyltransferase (HPRT) mutations.6 All of these endpoints can be considered biomarkers of exposure, its effects, and in some cases susceptibility, which are the hallmarks of molecular epidemiology. Exposure biomarkers include determination of xenobiotic agents or associated metabolites in biological fluids that can reflect the extent of internal exposure (e.g. DNA-adducts, etc.). The effects on the body that can serve as biomarkers include alterations of physiology, biochemistry, cell structure or function directly attributable to exposure to a xenobiotic substance (e.g. CAs, MN, DNA damage). Closely related to these biomarkers are biomarkers of susceptibility, which indicate increased vulnerability of individuals to diseases such as cancer (e.g. GSTM1 polymorphisms). Data are accumulating that support the hypothesis that genotoxicity endpoints are predictors of human cancer risk. Most carcinogens are genotoxic and have been associated with various types of DNA damage. However, the

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relationships between DNA damage, persistence and repair, and mutagenic endpoints are complex. The alkaline Comet assay has been shown to detect DNA damage in eukaryotic cells treated in vitro or in vivo with genotoxic agents. With its ease of application, the Comet assay has been increasingly used in human monitoring.6 The Comet assay is useful for evaluating xenobiotic impacts based on its use of small cell samples, and its ability to evaluate DNA damage in nonproliferating cells such as lymphocytes. In addition, the ability to obtain sufficient numbers of cells for analysis from different tissues, e.g. lymphocytes and buccal cells, provides a relatively noninvasive procedure for analysis. Most of the publications that describe the use of the Comet assay in human monitoring employed it as either a biomarker of exposure or effect, or in parallel with other early effect biomarkers (e.g. CA and MN, etc.). In addition to the versatility of the assay in detecting different kinds of DNA damage, including those present in the earliest steps of carcinogenesis, the ability to correlate results of the assay with detection of other biomarkers, supports the continued use of the Comet assay for risk assessment. The studies reviewed here reflect the increasing role the Comet assay has had in the field of human monitoring in the last decade. Expanded knowledge of the International Programme on Chemical Safety (IPCS) guidelines for standard parameters,6 as well as reproducibility of results for the same genotoxic agent assessed by different laboratories around the world, has further contributed to the successful application of the Comet assay.

10.3 Environmental Exposure The environmental impact of natural events, as well as man-made interventions, will always be present; and developing the capacity to recognise and minimise these impacts and their harmful consequences on human health is a daunting task. There have been 19 studies to evaluate the effects of air pollution on humans using biomarkers. Most of the studies of air-pollution exposure sampled blood cells and analysed seasonal variations or sunlight exposure,7–9 the effects of polycyclic aromatic hydrocarbons (PAHs) with heavy metals,10 or without heavy metals,11,12 the consequences of living in close proximity to a mining site and being exposed to arsenic and lead13 or the results of exposure to PM 2.5 and ultrafine particles.14–16 However, some studies evaluated the effects of air pollution on nasal epithelial cells,17,18 on exfoliated tear duct epithelial cells,19 or on a combination of leukocytes, nasal and buccal epithelial cells20,21 (Table 10.1). Environmental exposure to radiation has also been evaluated. The only negative correlation between radiation and DNA damage was obtained in a study performed by Garcia and Mandina22 in which the effects of consuming 137 Cs-contaminated food were evaluated. Other studies assessing the effects of the Chernobyl radiation accident found increased levels of DNA damage23–26 (Table 10.1). Environmental pesticide exposure evaluated by Comet assays of peripheral blood specimens yielded positive results and showed a correlation

Leukocytes

Leukocytes

Lymphocytes

Leukocytes

Leukocytes

Nasal epithelial cells

Nasal mucosa cells

Benzene exposure

PM 2.5

Ultrafine particles

Ultrafine particles

Air pollution

Air pollution (cigarette smoke)

Lymphocytes (children)

Leukocytes

Environmental pollutants Environment, seasonal variations

PAH, benzene metabolites and heavy metals Air pollution (PAH)

Cell type

Induction of DNA damage and FPG sites. Induction of DNA damage and FPG sites after outdoor exposure. Induction of DNA damage associated with changes in nasal cytology. Induction of DNA damage associated with changes in nasal cytology.

Induction of DNA damage in summertime and higher levels of 1-hydroxypyrene. Induction of DNA damage in children close to a coke oven plant. Induction of DNA damage and correlation with PAH-adducts. GSTM1 genotype effect on Comet parameters. Induction of DNA damage and decreased DNA-repair capacity in gasoline service attendants, petrochemical factory workers and Bangkok school children. Induction of DNA damage.

Comet assay result

GSTM1, GSTT1, GSTP1, NADPH, quinine reductase genotypes had no effect on DNA–PHA adducts and oxidative damage. NQO1 and GST genes modulate the effect.

Induction of PAH adducts, and GSTM polymorphisms

Other biomarkers

Studies employing the Comet assay for biomonitoring of environmental exposures.

Exposure

Table 10.1

18

17

16

15

14

12

11

10

7

Ref.

The Comet Assay in Human Biomonitoring 231

Induction of DNA damage in environmental tobacco smoke of more than 20 cigarettes/day.

Venous and cord blood

Leukocytes Leukocytes

Lymphocytes

Air pollution

Air pollution and smoking Organic concentrates in the drinking water Environmental tobacco smoke (passive smoking)

Radiation Sunlight Sunlight

Leukocytes Lymphocytes

Induction of DNA damage. Induce DNA damage.

Venous and cord blood

Air pollution

Air pollution, fluorine and arsenic in drinking water, gasoline fumes and pesticides (DDT, DDE)

Induction of DNA damage. Higher DNA damage in summer time and older individuals (40–55 years).

Negative

Induce DNA damage and correlation with ozone exposure. Induction in DNA damage in leukocytes and nasal epithelial cells. Induction of DNA damage in peripheral blood by air pollution, fluorine and arsenic drinking water, and gasoline fumes. Negative for pesticides. Air pollution induces damage in nasal cells and negative effects for buccal epithelial cells. Negative

Exfoliated tear duct epithelial cells Leukocytes, nasal and buccal epithelial cells Leukocytes, nasal and buccal epithelial cells

Air pollution (hydrocarbons and ozone) Air pollution

Comet assay result

Cell type

(continued ).

Exposure

Table 10.1

Increase of MDA levels, and decrease of GPX and tocopherol.

GTM1 polymorphisms (negative) Negative CA. Induction of DNA adducts and aneuploidies, polymorphisms of GSTM1 and NAT2.

Other biomarkers

8 9

119

118 29

117

116

21

20

19

Ref.

232 Chapter 10

Negative Induction of DNA damage and alterations in DNA-repair capacity. Induction of DNA damage and alterations in DNA-repair capacity. Induction of DNA damage. Induction of DNA damage. Induction of DNA damage and its correlation with DDE, DDD, and DDT levels. Induce DNA damage and correlation with DDE and DDT levels. Negative

Lymphocytes

Leukocytes

Leukocytes

Lymphocytes

Leukocytes (Children)

Leukocytes (Women)

Leukocytes

Leukocytes

Cs internal contamination caused by food consumption Chronically irradiated volunteers from the Chernobyl region Chernobyl accident liquidators Radon indoor air Metals Arsenic and lead (mining site) Pesticides DDE, DDD and DDT

DDE and DDT

Deltamethrin

137

Positive results for MN in exfoliated cells, and CA

30

28

27

13

26

25

23

22


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between DNA damage and dichlorodiphenyl dichloroethylene (DDE), dichlorodiphenyl dichloroethane (DDD), and dichlorodiphenyl trichloroethane (DDT) levels,27,28 as well as in the presence of organic concentrates in drinking water.29 However, exposure to deltamethrin did not induce DNA damage.30 DNA damage was induced in human peripheral blood lymphocytes by organic concentrates in drinking water.29 We analysed 28 studies of environmental exposure in humans; 86% of them contained data with positive Comet assay results, while 25% of the studies employed other biomarkers such as adducts, gene polymorphisms, CAs, MN, malondialdehyde (MDA), glutathion peroxidase (GPX) and tocopherol levels. Overall, most of these studies (71%) revealed a positive correlation between the presence of DNA damage evaluated by the Comet assay and the results obtained using other biomarkers.

10.4 Lifestyle Exposure Lifestyle is a convenient portmanteau term, which in relation to the causes of cancer has come to mean all aspects of the way people behave, whether determined voluntarily or as imposed by economic, cultural, or geographic circumstances, including reproduction.31 Lifestyle conditions such as smoking, recreational sun tanning, or insufficient intake of cancer-preventive compounds (e.g. fruits and vegetables), may render individuals susceptible to cancer. In this respect, the Comet assay has been used to explore the influence of smoking habits and vitamin C consumption on DNA damage. Diet, exercise, smoking habits, buccal health, and gender are variables that can contribute to DNA damage and thereby induce genotoxicity. Detection of DNA damage in blood, buccal, and urinary bladder cells32–39 using the Comet assay produced the results shown in Table 10.2. Studies without damage induction were also found in reports by Mohankumar et al.40 in which the nucleotide excision repair (NER) capacity was not altered in a smoking population. Other studies have determined that a smoking habit was not a confounding factor for occupational exposure to ionising radiation.41 Other negative reports that examined blood and buccal cells have been published.42,43 Eight studies have evaluated the antioxidant capacity of vitamin C and vegetable consumption in terms of extent of DNA damage as detected by Comet assay analysis. Studies of vitamin C consumption after breakfast44 as well the consumption of tomatoes, carrots, spinach,45,46 green tea, or berries for 28 days,47 all showed decreased levels of DNA damage. However, similar studies using the Comet assay and parallel biomarker analysis, including plasma cholesterol levels, and ras and p21 levels, have reported negative findings,48,49 Other experiments were conducted in a Gambian population to determine whether aflatoxin consumption induces DNA damage. The results from the Comet assay and other biomarkers (e.g. CAs, MN, SCEs and GSTM1 polymorphisms) came back negative for DNA damage.50 On the other hand, Betancourt and collaborators51 determined that DNA damage was increased in

Lymphocytes Leukocytes Frozen and fresh leukocytes Lymphocytes Lymphocytes

Lymphocytes

Smoking Smoking Smoking

Smoking

Smoking Smoking

Lymphocytes Leukocytes

Leukocytes

Leukocytes

Gender Indian population Malnourished children

Rectal cells Buccal cells, leukocytes and lymphocytes Mucosa of cheeks

Alcohol abuse Buccal health chlorexidine

Leukocytes

Lymphocytes

Aflatoxin

Buccal health, use of orthodontic apparatus Exercise, short distance triathlon Exhaustive exercise and Vitamin E supplementation Ex-smokers

Cell type

Decrease in DNA damage after 1 year of cessation. Increased DNA damage in women. Increase in DNA damage of malnourished children, as well as children under treatment for infection. Negative Negative Increase in DNA damage and no effects on frozen cells. Increased DNA damage Increased DNA damage, more damage in men than women. Increased DNA damage.

Increased DNA damage and correlation with cobalt and nickel levels. Increased DNA damage and decrease after 5 days postexercise. Decreased damage after 14 days of vitamin E consumption.

Increased DNA damage. Increased DNA damage.

Negative but females presented higher values than males.

Comet assay result

Human biomonitoring of lifestyle variable risks by using the Comet assay.

Exposure

Table 10.2

SCE less sensitive

SCE negative

NER capacity not altered

MDA levels did not change

MN negative

CA negative, MN negative, SCE negative GSTM1 genotype negative

Other biomarkers

33

174 32

40 42 37

120 51

36

53

54

56

52 55

50

Ref.


Urinary bladder cells Buccal mucosa Lymphocytes

Leukocytes

Smoking Smoking Smoking and antioxidant supplementation

Smoking as confounding factor Smoking habit in mothers

Negative Increased DNA damage. Negative

Lymphocytes

Buccal cells

Leukocytes

Lymphocytes


Lymphocytes

Vegetable consumption (green tea and berry)

Vitamin C after breakfast

Vitamin C supplementation

Vitamin C supplementation Women cooking with biomass fuels Women menstrual cycle

Increase in DNA damage for those who smoked more than 10 cigarettes/day. Increased DNA damage. Negative Decrease in damage after 20 weeks of vitamin C, vitamin E and carotene supplements. Negative in a population exposed to low dose of ionising radiation. Increased DNA damage in mothers with long smoking periods. Decreased DNA damage by supplementation with tomatoes, carrots or spinach. Decreased DNA damage may be due to enhancement of cytosolic GSTP1 and DNA-repair proteins by tomato and carrot juices. Decrease in DNA damage after 28 days of a carotenoid-rich berry and green tea supplements. Decrease in DNA damage 4 h after consumption. Negative

Comet assay result

Vegetable consumption

Vegetable consumption

Exfoliated buccal cells

Smoking

Lymphocytes of newborns and mothers Lymphocytes

Cell type

(Continued ).

Exposure

Table 10.2

Cholesterol levels, ras and p21 levels were not affected

GSTM1, GSTP1 and GSTT1 polymorphisms

Other biomarkers

122

49 121

48

44

47

46

45

34

41

39 43 35

38

Ref.

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237

malnourished children, and in children receiving treatment for infection. In a study designed to determine if alcohol abuse could be considered a lifestyle genotoxicant, the Comet assay was tested as a biomonitoring tool to detect genotoxicity in human rectal cells.52 It is important to take into account the physical activity of the subjects in human monitoring studies employing the Comet assay as a genotoxicity tool. For example, the German group of Speit, reported that physical exertion above the aerobic–anaerobic threshold can produce DNA damage in blood leukocytes.53,54 There have also been two interesting studies of buccal health. One study reported DNA damage induced by chlorexidine in several cell types (buccal cells, peripheral blood, and lymphocytes); while the other determined that an orthodontic apparatus could induce DNA damage in cells of the cheek mucosa as a result of cobalt and nickel exposure.55,56 In summary, 71% of publications that used biomonitoring to evaluate lifestyle variables reported positive Comet assay results, while 29% employed other biomarkers such as CAs, MN, gene polymorphisms, cholesterol levels, ras and p21 protein levels, NER capacity, and MDA levels. This is in contrast to biomonitoring studies of environmental factors where all biomarkers showed negative results. Three studies employing both the Comet assay and other endpoint analyses reported negative findings.40,48,50 Interestingly, studies that evaluated lifestyle-associated DNA damage had a similar percentage of positive reports (67%).

10.5 Occupational Exposure Occupational biomonitoring examines the exposure of workers to a variety of chemical, biological, or physical (e.g., noise, heat, and radiation) agents to determine if the exposures may result in an increased risk of adverse health outcomes. Alternatively, occupational biomonitoring studies may also evaluate a population of workers with a common adverse health situation in order to determine if workers’ disease states can be attributed to an agent or a set of agents. In the last decade, the Comet assay has been used extensively as a hazard-evaluation tool in these cases. Table 10.3 presents data from 98 studies which we classified into four exposure categories: 1) air pollutants, including volatile organic compounds (VOCs), PAHs, and asbestos, which constitutes 54% of studies in this category; 2) medical personnel exposed to antineoplastic drugs, anaesthetics or radiation, which constitutes 16% of occupational studies; 3) metal exposure (13% of the reports); and 4) pesticide exposure (16% of works). For these four categories of occupational hazards, 81% of them showed DNA damage detected by Comet assay analysis and 53% of the studies showed DNA damage using other endpoints. Many studies have looked at the exposure of workers at rubber factories, lamination plants, footwear factories, within the plastics industry, cigarette factories, and pharmaceutical production plants for exposure to different kinds of hydrocarbons and VOCs. Most of the studies found an induction of DNA

Benzene or benzene metabolites Benzo[a]pyrene, benzofluoranthene, naphthalene, acetonaphthene, alkenes and 1,3-butadiene (rubber tyre factory) Bromopropane

Lymphocytes

Benzene (elevator manufacturing workers) Benzene (printing company)

Increased DNA damage that correlated with MN test.

Increased DNA damage by gender, facility, and GSTM1 polymosphism had an effect on the damage.

Leukocytes

Increased DNA damage.

Increased DNA damage influenced by smoking habits. Increased DNA damage.

MN (positive), Positive results in CA only chromatid and chromosome breaks

63

62

61

60

59

83 58

57

Increased DNA damage in outdoor workers of Mexico city. DNA damage was positively correlated with PM 2.5 and ozone exposure. Negative Increased DNA damage in asbestosexposed workers, and delayed DNA-repair capacity detected.

Ref. 81

Negative SCE DNA damage was higher in asbestos-exposed workers with Gln/Gln than in those with Arg/Arg XRCC1 codon 399 polymorphism

Other biomarkers

Negative

Comet assay result

Lymphocytes

Granulocytes, T and B lymphocytes Lymphocytes


Air pollutants, traffic fumes Asbestos

Workers exposed to pollutants in work places Lymphocytes 4,4 0 -methylenediphenyldiisocyanate, 2,4-toluenediisocyanate and 2,6toluenediisocyanate Air pollutants (VOCs, PM Leukocytes 2.5, ozone)

Cell type

Studies of occupational hazards identified by human biomonitoring using the Comet assay

Exposure/work place

Table 10.3

238 Chapter 10

Frozen leukocytes

Leukocytes

Diesel exhaust (shale-oil mine workers)

Environmental pollutants (waste disposal) Flight personnel (cosmic radiation, airborne pollution, ozone and electromagnetic fields Formaldehyde (plywood factory)

Hydrocarbons and jet fuel derivatives (airport personnel) Methotrexate plant production workers Oxidation hair dyes

Leukocytes

Diesel exhaust

Increased damage. Increased DNA damage, smoking workers had more damage. Increased DNA damage in underground workers (drivers and dieselpowered excavators). Increased DNA damage in underground workers vs. surface workers. Smoking workers had more damage. Negative

Increased DNA damage. Increased DNA damage. Negative

Lymphocytes

Leukocytes

Lymphocytes


Buccal cells Lymphocytes

Carbon disulfide Cigarette factory

Negative

Lymphocytes

Leukocytes

Butadiene

Negative

Lymphocytes

Butadiene

Increased DNA damage and was affected by smoking. Negative

Lymphocytes

Lymphocytes

Bus manufacturing workers

Induction of MN, HPRT, and TCR Induction of SCE in smokers

No association between XRCC1 polymorphisms and DNA damage SCE, MN and p21 plasma levels were negative

Genotype of GSTM1, GSTP1 and GSTT1 had no effect Positive results for CA. Negative SCE

Increase O(6)-alkylguanine DNA adducts

Positive results for CA, and SCE, no effect of GSTM1 or GSTT1 genotypes Negative HPRT, CA and MN

78

134

70

69

77

72

68

67

65 66

80

79

64


Lymphocytes

Lymphocytes

Lymphocytes T and B-lymphocytes, granulocytes Lymphocytes and buccal cells Leukocytes

Lymphocytes

Lymphocytes

PAH coke oven workers

PAH coke-oven and graphite electrode production plant

PAH in aluminium industry PAHs (emission inspection and incineration workers) PAHs (airport personnel)

PAHs (coke oven workers)



Cell type

(continued ).

Exposure/work place

Table 10.3

Increased DNA damage and correlation with 1-OHP. More damage in workers with GA genotype of G27466A polymorphism of XRCC1 than those with GG genotype. Increased DNA damage. Higher damage in Ahr Lys (554) variant genotype.

Negative Increased DNA damage in all cell types monitored. Increased DNA damage and FPG Comet assay. Negative


Negative and no influence of GSTM1 and GSTT1 polymorphisms.

Comet assay result

Induction of SCE, CA and negative MN CA negative and induction of SCE, MN. Association between SCE and 1-hydroxyptrene

Increase 1-OH pyr levels, -SCE, -DNA adducts and negative MN induction (urothelial cells) GSTT1, GSTM1 and CYP1A1 polymorphisms have no effect, increase of 8-oxodGuo levels Negative MN

Other biomarkers

140

139

75

138

136 137

135

74

Ref.

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Lymphocytes

Lymphocytes


Rubber processing

Rubber tyre factory workers

Sewage workers Silica (foundry and pottery workers) Solvents in a complex mixture (footwear workers)

Leukocytes

Negative

Lymphocytes

Increased DNA damage, associated with smoking and alcohol drinking. Increased DNA damage, but Comet plus FPG and Endo III was negative. Negative Increased DNA damage, smoking was related to damage. Increased DNA damage, influence of GSTP1 polymorphisms.

Negative

Lymphocytes

Lymphocytes

Lymphocytes

PAHs (policemen)


Lymphocytes

PAHs (policeman)

Increased DNA damage and FPGComet. Increased DNA damage and reduced DNA-repair capacity. Increased DNA damage and decrease in repair efficiency. Increased DNA damage.

Leukocytes

Leukocytes

PAHs (paving workers)

Increased DNA damage. Factors having influence on damage were 1-OHP, XRCC1-exon 9 variant genotype, ERCC2-exon 10 variant genotype and XRRC1-exon 6 variant genotype.

PAHs (fireproof-material producing plant) Petroleum derivates, hydrocarbons, benzene, toluene and xylene (BTX) Radio-frequency radiation (RFR) Rubber factory workers

Lymphocytes


Induction of MN, decrease of 6-beta-hydrocortisol and increase of 17-hydrocorticoteroid

Negative CA

BPDE adducts negative, and increase of 8-oxodGuo Induction of CA and MN correlate with age

MN frequencies were influenced by coke oven exposure, 1-OHP, age, mEH3 variant genotype, ERCC2exon 10 variant genotype and XRCC1-exon 6 variant genotype Increased levels of urine OH-pyrene

164, 165

71 163

162

161

76

82

160

145

144

143

142

141


Leukocytes

Sperm and lymphocytes

Leukocytes

Lymphocytes

Styrene, plastic lamination plant

Styrene

Toluene and organic solvents (shoe workers) Vinyl chloride monomerPVC

White blood cells

Lymphocytes

Increased DNA damage in workers exposed to vinyl chloride monomer values greater than 5 ppm. Dose– response relationship of urinary thiodiglycolic acid and DNA breaks. Increased DNA damage, correlates with time of exposure. Increased DNA damage, smoking enhanced the effect.

Lymphocytes

Styrene, hand lamination

Vinyl chloride monomerPVC (plastic industry) Wooden furniture plant workers

Negative

Lymphocytes

Styrene (lamination plant)

Increased DNA damage in both cell types.

Increased DNA damage, correlation with years of employment and DNA adducts. Increased DNA damage, correlates with years of exposure.

Increased DNA damage with respect to exposure time.

Increased DNA damage, correlates with DNA-adducts.

Lymphocytes

Styrene (lamination plant)

Comet assay result

Cell type

(continued ).

Exposure/work place

Table 10.3

Induction of DNA adducts, and increase of HPRT mutation frequency Haemoglobin DNA-adduct, CA, HPRT correlate with the exposure time Induction of O6-guanine adducts, increase of HPRT mutant frequency Induction of CA and increased expression of adhesion molecules CD62L, CD18, CD11a, CD11b, CD49a and CD54 MN frequency correlates with Comet assay in sperm and lymphocytes GSTT1 and GSTM1 polymorphisms have no effect

Other biomarkers

173

172

171

71

170

169

168

167

166

Ref.

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Lymphocytes

Lymphocytes

Leukocytes

Lymphocytes

Lymphocytes

Lymphocytes

Lymphocytes

Radiation to low dose



Waste anesthetics gases and supplementation of vitamin E and C X-rays

X-rays

Anesthetic gases (operating room personnel) Antineoplastic drugs

Antineoplastic drugs (nurses)


Radiation to low dose Radiation to low dose

Leukocytes

Lymphocytes


Ultrasound Radiation to chronic low dose

Medical personnel exposed to drugs and radiation Drug mixtures Buccal cells and lymphocytes

Increased DNA damage that correlates with CA only if including gaps. Negative with correlation with comet and micronucleus test results. Increased DNA damage that was reduced after vitamin supplementation. Increased DNA damage, comet more sensitive than MN. Increased DNA damage, correlated with age. Increased DNA damage, smokers have more damage. Increased DNA damage in workers that used latex gloves only as safety protection. Increased DNA damage. XRCC1, XRCC3 and APE1 polymorphisms may influence the chronic exposure.

Increased DNA damage only in nurses handling the highest amount of drugs. In buccal cells, was negative. Increased DNA damage. Increased DNA damage. Smoking only increased damage in control subjects. Increase of DNA damage. Increase of DNA damage, but comet plus FPG and Endo III was negative. Increased DNA damage.

Negative MN

Induction of cytokinesisblocked MN Induction of MN and dicentic bridges

Negative MN

Induction of MN and correlate with age CA (positive results)

CA (positive results)

Higher environmental levels of cyclophosphamide, 5fluorouracil and ifosfamide

96

95

94

93

92

91

99

90

89

87 88

85 86

84


Lymphocytes

Antineoplastic drugs (nurses) Antineoplastic drugs (nurses)

Leukocytes

Lymphocytes

Chromium and nickel (welders occupational exposure) Cobalt or hard metal dust


Increased DNA damage and was influenced by XRCC3 and hOGG1 polymorphisms. Negative Increased DNA damage as well as FPG-comet.

Lymphocytes

Chromium (chrome-plating workers)

Cobalt-containing dust Copper smelter exposed to inorganic arsenic

Increased DNA damage, correlated with chromium lymphocyte concentrations. Increased DNA damage.

Leukocytes

Mercury vapours

Increased DNA damage and correlation with Mn exposure. Increased DNA damage, correlation with duration of working in factory, age and smoking. Negative, but DNA-repair capacity altered.

Leukocytes

Lymphocytes

Increased DNA damage in nurses without safety protection. Increased DNA damage even when work schedule was on limit.

Comet assay result

Workers exposed to metals Al, Cd, Co, Cr, Ni, Pb (welding fumes) Arsenic (glass workers)

Lymphocytes

Cell type

(continued ).

Exposure/work place

Table 10.3

Induction of MN

Increase MN in buccal epithelial cells

Induction of MN in buccal cells and positive correlation with Comet assay Negative SCE, increase of CA frequency. Association between DNA-repair alterations and duration of occupational exposure.

Increased MN when working out of schedule limit, and negative working on limit

Other biomarkers

101 128

127

126

125

100

124

123

98

97

Ref.

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Leukocytes

Lymphocytes

Lymphocytes

Leukocytes

Lead

Lead

Lead and cadmium

Lead smelters and battery industry

Lymphocytes

Leukocytes

Lymphocytes

Lymphocytes

Leukocytes

Pesticide mixture

Pesticide production

Pesticides

Pesticides

Pesticides (sprayers from Cayambe, Ecuador)

Workers exposed to pesticides Pesticide (fruit growers) Leukocytes

Lymphocytes

Lead


Increased DNA damage correlates with age, pesticide exposure level, CYP3A5 and GSTP1 genotype. Increased DNA damage. Workers using safety protection present less damage. Increased DNA damage, smoking workers had more damage. Increased DNA damage, after 6 months exposure decreased but still high vs. control. Increased DNA damage, after 8 months of exposure saw decrease, but remained high vs. control.


Increased DNA damage and smoking habit have influence. Increased DNA damage, highest values in workers exposed to 4500 mg/L. Increased DNA damage.


Increased frequency of CA and after 8 months of exposure decrease, but still high. Induction of CA. No association between polymorphisms and DNA damage

CYP3A5 and GSTP1 genotype

Induction of SCE, FISH, and MN

Decrease of GSH levels and down-regulation of PKC

151

150

149

148

147

146

133

132

131

130

129



Increased DNA damage. Increased damage 1 day after spraying (herbicide, isoproturon and fungicide, chlorothalonil plus insecticide mixture) and correlation with spraying tanks used. Increase in DNA damage, smoking influenced damage.

Leukocytes

Spermatozoa

Leukocytes

Pesticides, fungicide, insecticide mixture

Increased DNA damage, after 8 months of exposure saw decrease but still high vs. control.

Leukocytes

Leukocytes

Lymphocytes

Negative Increased DNA damage after 5 years of work. Negative


Pesticides Pesticides (banana-packing workers, women) Pesticides (Greek greenhouses workers) Pesticides in a complex mixture (atracine, alachlor, cyanazine, 2,4-dichlorophenoxyacetic acid, and malathion) Pesticides in a complex mixture (atracine, alachlor, cyanazine, 2,4-dichlorophenoxyacetic acid, and malathion) Pesticides, Fenvalerate (pesticide factory workers) Pesticides, fungicide, herbicide and insecticide mixture (farmers)

Comet assay result

Cell type

(continued ).

Exposure/work place

Table 10.3

Low DNA integrity

Induction of CA and MN

Increased CA, SCE and MN and 8 months after exposure decrease but still high vs. control

Other biomarkers

157

156

155

154

153

102

103 152

Ref.

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Pesticides: organophosphates, carbamates, pyrethroids (pesticide-manufacturing workers) Pesticides: organophos phorus insecticide formulators Increased DNA damage, smokers had more damage. Increased DNA damage.

Leukocytes

Leukocytes

Increased catalase, superoxide dismutase and glutathione peroxidase activity in workers exposed more than 6 months and lipid peroxidation was negative

159

158


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damage due to the occupational exposure (60%), and some of these found corroborating data using multiple endpoints.57–70 However, some Comet assay studies did not reveal DNA damage associated with environmental exposure in the work place. For example, biomonitoring studies of a variety of workers, such as sewage,71 waste disposal,72 shoe factory,73 coke oven,74,75 rubber factory,76 flight personnel exposed to cosmic radiation and environmental pollutants,77 workers exposed to hair dyes,78 butadiene,79,80 4.4 0 -methylenediphenyldiisocyanate, 2,4-toluenediisocyanate and 2,6-toluenediisocyanate,81 radiofrequency radiation82 and air pollutants of traffic fumes,83 did not show increased levels of DNA damage. Most studies of medical personnel exposed to antineoplastic drugs, anaesthetics, or radiation, found an induction of DNA damage using the Comet assay biomonitoring;84–98 however, one report did not show occupational DNA damage.99 In exposure studies concerning metals and pesticides, only two studies did not find induction of DNA damage. In a study of mercury vapour exposure, Cebulska and colleagues100 reported that although exposure did not induce single-stranded DNA breaks, alterations in the DNA-repair capacity were detected. Furthermore, a report by De Boeck101 evaluating the genotoxic capacity of cobalt-containing dust did not detect any DNA damage, and studies examining pesticide exposure in two different populations did not find DNA damage.102,103

10.6 Reviews The Comet assay was originally presented as a rapid, simple, visual, and sensitive technique for detecting breaks in DNA, alkali-labile sites, crosslinks and delayed repair sites.5,104 These properties of the assay also suggested its potential for use in human biomonitoring and other related fields such as clinical research, environmental monitoring, genotoxicity studies, and DNArepair studies. A subsequent review published by Kassie and colleagues105 addressed the application of the Comet assay in identifying dietary protective factors in clinical studies, as well as its application in monitoring the risk of DNA damage resulting from occupational, environmental or lifestyle factors compared with conventional cytogenetic tests. In the same year, Panavello and Clonfero106 reviewed the international scientific literature of studies examining the influence of metabolic genotypes on biological indicators of genotoxic risk in environmental or occupational exposure. In 2004, five reviews of human biomonitoring were published; one of them included 30 studies of occupational exposure,107 and another included 45 studies monitoring human exposure to genotoxic agents as a result of occupation, drug treatment, disease, or environmental pollution.108 These reviews of published studies not only highlighted the consistent observation that results obtained using the Comet assay were consistent with concurrently performed cytogenetic assays, but also demonstrated that the Comet assay is sensitive enough to detect low levels of DNA damage in human lymphocytes and provides acceptable specificity and


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reliability in providing negative results in the absence of exposure to occupational agents that are suspected as genotoxicants. In 2004, Maluf et al.109 discussed the use of peripheral blood lymphocytes to evaluate the health risk of a population exposed to ionising radiation. This study also considered that individual factors can interfere with the frequency of DNA mutations and could thereby impact the selection of control individuals and the interpretation of the results. Maluf et al.109 concluded that the combination of the alkali Comet assay and a cytokinesis-block micronucleus assay was accurate in monitoring the risk of populations chronically exposed to ionising radiation. Collins et al.110 reviewed applications of the Comet assay in the testing of novel chemicals for possible genotoxicity, monitoring environmental contamination by genotoxins, for human biomonitoring and molecular epidemiology, and in fundamental research of DNA damage and repair. It is important to note that this review emphasises that the sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognise specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. Another interesting review published in 2004 considered the use of the tail moment of lymphocytes to evaluate DNA damage in human biomonitoring studies.111 The authors evaluated four tail parameter cells containing high vs. low levels of DNA damage using epidemiological assays. Their conclusions were that both the tail moment and the tail DNA (%) were suitable parameters for human biomonitoring. Alternatively, Møller et al.112 evaluated the Comet assay as a test of genotoxicity of environmental agents in both experimental animal models and biomonitoring studies. The conclusion from the aggregated data of the publications reviewed indicated that the Comet assay is a reliable method for the detection of DNA damage. However, not all types of genotoxic exposure should be expected to produce DNA damage in mononuclear blood cells. The following year the same author published a review evaluating the strength of the Comet assay in biomonitoring studies, concluding that the Comet assay was a valuable method for detecting genotoxic exposure in humans, although the predictive value of the assay was unknown because it had not been investigated in prospective cohort studies.113 The last published review about human monitoring by Grandi et al.114 discussed the use of the Comet assay in occupational medicine and industrial toxicology. The limitations and critical features associated with the Comet assay were a lack of sensitivity to aneugens and a possible underestimation of genotoxic potency of agents with multiple mechanisms of action.

10.7 Usefulness of the Comet Assay in Human Monitoring The field of biomonitoring has gained interest with scientists and organisations around the world. Biological monitoring has been an important tool for the

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surveillance of medical health programmes in European countries. In the United States it is employed in the monitoring of occupational hazards. Human biomonitoring is the only way to identify and quantify human exposure and risk, elucidate the mechanisms of toxic effects and to ultimately decide if measures have to be taken to reduce exposure. Human biomonitoring studies with exposure to genotoxic chemicals, especially the measurement of DNA-strand breaks in lymphocytes and 8-hydroxy2 0 -deoxyguanosine (8-OHdG) in white blood cells, have been popular; however, there is still a lack of well-established dose–response relations between occupational and environmental exposures and the induction of 8-OHdG or formation of strand breaks, which limits the applicability of these markers. Comet assay data have been very informative in these biomonitoring applications. Additional applications of biomonitoring are in the development of health-based biomarkers that are sensitive to dose–effect and dose–response relationships.115 The Comet assay has the potential to be applied to all of the previously described categories of human biomonitoring it is worth noting that almost half of the studies were performed in lymphocytes, meanwhile leukocytes represent 38%, nasal cells only 2.5% and the rest of the cells used (buccal, rectal cells, etc.) represent 10% of the studies. In general, approximately 80% of the Comet assay studies report a positive result, however, studies using lymphocytes have bigger rates of negative results (26% versus 17% in leukocytes, probably because lymphocytes with more damage could be lost during isolation procedures. Studies performed with other cells such as nasal, buccal or rectal, have a low rate of negative results, probably because these cells are primary targets of the genotoxic agents studied. The congruence of results between the Comet assay and other endpoints such as MN or SCEs, has been one of the principal reasons to increase the use of the Comet assay as a biomarker for hazard assessment. The usefulness of this assay in human monitoring studies certainly depends on the xenobiotics involved, their molecular mechanisms of action, as well as the experimental design (e.g. timing of sample collection). According to IPCS guidelines,6 the optimal sample collection timing for any cell population is during long-term chronic exposure when the induction and repair of DNA damage is presumed to be maintained at a steady-state equilibrium. Such timing maximises the likelihood of an agent being identified as having DNA-damaging potential. For the sampling of cells after an acute exposure, or after termination of chronic exposure to a genotoxic agent, the optimal collection time for detecting induced DNA damage is most likely within a few hours of exposure termination. This window of sampling can affirm that the extent of DNA damage in a population of cells decreases as the amount of time between exposure termination and sampling increases. In addition, the repair of DNA damage through DNA-repair processes, and the loss of heavily damaged cells through apoptosis, necrosis, or cell turnover, also depends on the agent of exposure. An additional advantage of the Comet assay for human biomonitoring is the feasibility of its application to a broad spectrum of cells, including both proliferating and nonproliferating cells, and cells of tissues that are the first sites of


251

contact with xenobiotics. With the application of the Comet assay to these various cell types, a better estimation of hazard exposure can be made.

10.8 Conclusions Over the past twenty years, the DNA damage in the Comet assay has been employed in molecular epidemiology as a robust biomarker of hazardous effects on human populations, particularly in monitoring the effects of occupational hazards. The versatility of the Comet assay combined with the use of specific enzymes, has allowed the identification of specific regions of DNA damage in human populations. The ability to monitor larger populations of people is an important advantage of the assay, and represents the potential of the assay to facilitate advances in mechanistic studies rather than observational studies. Although the Comet assay is able to evaluate DNA breaks that are present, additional studies are necessary to identify the molecular mechanisms underlying this DNA damage. Therefore, it is important to consider DNA damage evaluation by the Comet assay as a biomarker in studies where the exposure results in damage such as DNA-strand breaks, alkali-labile sites, repair sites and DNA crosslinks. Due to the high concordance of Comet assay results with results of cytogenetic assays conducted in parallel, the negative results obtained in the absence of genotoxins, and the sensitivity of the Comet assay enabling it to detect low levels of DNA damage in different cell types (blood cells, nasal, buccal, tear duct epithelial cells, rectal cells), the Comet assay represents a powerful tool with which to identify DNA damage specificity. Negative or inconclusive results obtained using the Comet assay in studies where smoking habits are involved could be supported by cotinine detection. These negative data could be diminished by use of Comet assay protocols outlined by IPCS guidelines.3 The most important parameters for using the assay include the identification of differences between exposed and control groups with respect to lifestyle variables (diet, smoking habits, medical treatments, history of chronic diseases, physical activity, etc.) and the timing of sample collection. Additionally, our review of the literature revealed the importance of establishing standard methodological conditions that affect unwinding and electrophoresis times (that is why it is important to use in every step of the technique an internal control, cells with a known level of DNA damage, that will help us to analyse the data between different electrophoresis sessions), or tail values (tail length, tail DNA (%), tail moment), with the intention of being able to compare data collected from numerous studies conducted in different laboratories around the world. We found this to be a very important point since the assay is sometimes used without discussion of the type of information provided; the fact that it is such a successful assay for demonstrating DNA damage is often used as sufficient proof to justify its use. However, the Comet assay is susceptible to subtle manipulations depending on the type and timing of sampling performed. Therefore in the reporting of DNA damage detected by

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the Comet assay, the context of how the DNA damage was created also needs to be considered in the interpretation of the results. The success of the Comet assay is reflected by its use over the past 20 years in the field of genetic biomonitoring, and by the increasing number of studies that continue to use it. As its shortcomings may eventually be overcome, the data from the Comet assay will become more robust and further validated for use as a suitable biomarker for human monitoring.

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138. D. Cavallo, C. L. Ursini, G. Carelli, I. Iavicoli, A. Ciervo, B. Perniconi, B. Rondinone, M. Gismondi and S. Iavicoli, Occupational exposure in airport personnel: characterization and evaluation of genotoxic and oxidative effects, Toxicology, 2006, 223, 26–35. 139. S. Leng, J. Cheng, Z. Pan, C. Huang, Y. Niu, Y. Dai, B. Li, F. He and Y. Zheng, Association between XRCC1 and ERCC2 polymorphisms and DNA damage in peripheral blood lymphocytes among coke oven workers, Biomarkers, 2004, 9, 395–406. 140. Y. Chen, Y. Bye, J. Yuang, W. Chen, J. Sun, H. Wang, H. Liang, L. Guo, X. Yang, H. Tan, Y. Su, Q. Wei and T. Wu, Association of polymorphisms in AhR, CYP1A1, GSTM1, and GSTT1 genes with levels of DNA damage in peripheral blood lymphocytes among coke-oven workers, Cancer Epidemiol. Biomarkers Prev., 2006, 15, 1703–1707. 141. L. Qiu, S. Leng, Z. Wang, Y. Dai, Y. Zheng and Z. Wang, Path analysis of biomarkers of exposure and early biological effects among coke-oven workers exposed to polycyclic aromatic hydrocarbons, Cancer Epidemiol. Biomarkers Prev., 2007, 16, 1193–1199. 142. D. Cavallo, C. L. Ursini, P. Bavazzano, C. Cassinelli, A. Frattini, B. Perniconi, A. Di Francesco, A. Ciervo, B. Rondinone and S. Iavicoli, Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs, Ann. Occup. Hyg., 2006, 50, 211–218. 143. A. Cebulska-Wasilewska, A. Wiechec´, A. Panek, B. Binkova´, R. J. Sra´m and P. B. Farmer, Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity, Mutat. Res., 2005, 588, 73–81. 144. A. Cebulska-Wasilweska, I. Pawlyk, A. Panek, A. Wiechec, I. Kalina, T. Popov, T. Georgieva and P. B. Farmer, Exposure to environmental polycyclic aromatic hydrocarbons: influences on cellular susceptibility to DNA damage (sampling Kosice and Sofia), Mutat. Res., 2007, 620, 145–154. 145. B. Marczynski, R. Preuss, T. Mensing, J. Angerer, A. Seidel, A. El Mourabit, M. Wilhelm and T. Braning, Genotoxic risk assessment in white blood cells of occupationally exposed workers before and after alteration of the polycyclic aromatic hydrocarbon (PAH) profile in the production material: comparison with PAH air and urinary metabolite levels, Int. Arch. Occup. Environ. Health, 2005, 78, 97–108. 146. Y. J. Liu, P. L. Huang, Y. F. Chang, Y. H. Chen, Z. L. Xiu and R. H. Wong, GSTP1 genetic polymorphism is associated with a higher risk of DNA damage in pesticides-exposed fruit growers, Cancer Epidemiol. Biomarkers Prev., 2006, 15, 659–666. 147. U. Undeg˘er and N. Basaran, Assessment of DNA damage in workers occupationally exposed to pesticide mixtures by the alkaline Comet assay, Arch. Toxicol., 2002, 76, 430–436. 148. P. Grover, K. Danadevi, M. Mahboob, R. Rozati, B. S. Banu and M. F. Rahman, Evaluation of genetic damage in workers employed in

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150.

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152.

153.

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155.

156.

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CHAPTER 11

Comet Assays in Dietary Intervention Trials ARMEN NERSESYAN, CHRISTINE HOELZL, FRANZISKA FERK, MIROSLAV MISˇI´K AND SIEGFRIED KNASMUELLER* Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria

11.1 Introduction A large number of investigations indicate that dietary factors have an impact on DNA stability in humans.1 On the one hand, consumption of food is one of the main factors in oxidative stress,2 which causes damage to the genetic material, also the lack of micronutrients such as Fe, Se, Zn and folic acid3,4 as well as genotoxic carcinogens contained in the human diet such as nitrosamines, polycyclic aromatic hydrocarbons, heterocyclic aromatic amines5,6 that cause damage and instability of genetic material that leads to adverse health effects including various forms of cancer,7,8 neurodegenerative disorders,9 cardiovascular diseases,10 infertility,11 allergy12 and aging.13 It has been estimated by Doll and Peto14,15 that approximately 35% of all cancer deaths in western countries are due to nutrition and would be avoided by the development of dietary strategies. On the other hand, a large number of bioactive compounds have been identified in human foods that protect DNA via different modes of action including direct scavenging of reactive molecules such as ROS,

*



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induction of detoxifying and inhibition of activating enzymes, as well as induction of alterations of DNA-repair processes to name but a few.16–18 We have critically discussed the methods that are currently used for the identification of DNA-protective effects in the human diet (for reviews see refs. 19 and 20). In particular, in vitro approaches that are widely used, do not yield results that can be extrapolated to humans and approaches that provide information about protective effects in men are needed in order to draw firm conclusions and justify health claims.21–23 The different models that have been developed for human studies include micronucleus assays with peripheral blood cells,24 chemical quantification of oxidised DNA-bases in blood cells,2 DNAadduct measurements, chromosomal analyses of metaphase cells as well as the single-cell gel electrophoresis assays (SCGE) or Comet assays, for reviews see refs. 18 and 25). Since its first use in a human intervention trial by the group of Collins26 in 1996 the latter method is increasingly used. This chapter gives a brief overview on the present state of knowledge and also on recent use of the SCGE assay in human dietary intervention studies for prevention of DNA damage.

11.2 Experimental Design of Human Studies The experimental design of SCGE assays in human trials has been critically discussed in the articles by Moller and Loft27–29 and the authors attempted to define criteria for the quality of such studies. In principle, the trials can be conducted with sequential or crossover design. It has been emphasised that the inclusion of placebo groups will increase the scientific quality of trials.27,29 Furthermore, questionnaire-based trials also have been carried out. Most of the studies used a sequential design (about 50%) followed by placebo controlled (32%) and crossover trials (18%). Questionnaire-based studies are rarely conducted (only one among 81 trials) and results obtained were found to be contradictory to the animal experiments. The study of Giovannelli et al.,30 for example, reported increased formation of oxidised bases after consumption of coffee and lycopene while in other experiments including animal investigations protective effects were found.31,32 In a number of experiments (see for example, refs. 25 and 33–35), run-in phases were included in which the participants consumed a controlled diet and did not consume the items under study. We strongly recommend the use of such phases in which the participants control the consumption of dietary factors that are known to cause protective effects and also avoid physical exercises that may lead to comet formation.36,37 Also wash-out periods will increase the quality of intervention studies but the definition of their duration is problematic; for example, Bub et al.38 showed that the reversal of the protective effects of a polyphenolics enriched diet takes several weeks. Also, the duration of the intervention period itself is problematic. It can be expected that direct-acting scavengers cause effects already after a few hours, while compounds that act via induction of protective enzymes will require longer periods to elicit protective effects. In the case of

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phenolics, it was shown that the reduction of oxidative DNA damage occurs with some delay.39 Multiple sampling, which was used in some of the studies,29,35,40 may help to solve this problem. Moller and Loft29 stressed that crossover trials and placebo-controlled studies are preferable to sequential design studies as the impact of the intervention on dietary habits as well as seasonal effects can be avoided. This may be relevant for long-term investigations but in most studies, the intervention periods were below three weeks and no substantial changes of the aforementioned factors can be expected.27,29,41 Also, the inclusion of different dose groups in intervention trials should be desirable, but usually financial limitations as well as the capacity of laboratories are limiting factors. The number of participants which should be included is a further crucial question. In the 1990s, only a few individuals were enrolled in most of the trials but the criticism raised by Moller and Loft27 has led to an increase of the number of participants. An adequate strategy to determine the ideal number of participants is the calculation of the statistical power. Data for different studies can be found in the review of Moller and Loft,27 where the number of participants were calculated to detect a 50% effect. To receive such an effect, the number of participants of the different studies vary substantially. The quality of intervention trials can be increased when factors are taken into consideration that affect their outcome; these include the age,42 gender,42,43 body weight,44 seasonal effects12,45 and life style of the participants including nutritional habits, alcohol and tobacco consumption30,46–48 as well as physical activity.49 Ethnicity may also affect the outcome of SCGE trials.50 Data concerning the influence of age and gender on DNA damage are, however, controversial.43,48,51,52 Most dietary studies (7% out of 81) were carried out with healthy individuals. However, some of the newer investigations were conducted with participants who suffered from diseases related to oxidative stress (such as, e.g., diabetes, renal failure), or had increased oxidative DNA damage due to physical exercises.53–57 The results of some of these studies indicate that the protective effects of specific dietary factors are more pronounced in individuals under oxidative stress, for example, Glei et al.58 found a protective effect with supplemented bread in smokers but not in nonsmokers.

11.3 Indicator Cells and Media The majority of investigations (92%) were carried out with peripheral lymphocytes while only in a few studies (6 out of 81 studies) leucocytes were used. When cells are treated with ROS-generating chemicals or radiation they are in most cases kept in artificial media. However, it is also possible to conduct experiments in which the cells are maintained in the plasma of the donors. The latter protocol enables the detection of extracellular ROS scavenging effects caused by dietary factors in plasma itself, while the former reflects changes in the intercellular ROS defense system.

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In a few newer intervention trials, buccal cells have been used that can be easily obtained from the oral cavity. Szeto et al.59 described the development of an improved protocol in which agarose embedded cells of epithelial origin from the mouth were lysed with trypsin and proteinase K treatment and showed a reduction of migration in a small intervention trial with carotene-rich berry juice. According to the authors it is not possible to use the earlier protocol that was developed by Rojas et al.60 as it leads to extremely high background levels. Another approach was developed recently by Osswald et al.61 who isolated lymphocytes from mouthwashes and developed a technique for SCGE analysis that was used successfully in an intervention trial with supplemented bread carried out by Glei and coworkers.58 Kleinsasser et al.62,63 have shown that it is possible to establish miniorgan cultures with oral mucosal cells and use them to study the DNA-damaging effects of dietary genotoxins in SCGE trials. This approach may also be used to monitor the protective effects of dietary compounds in ex-vivo studies in humans. Buccal cells could be ideal for intervention trials, as they can be collected with a noninvasive method. The drawback is the high background values. Since more than 90% of human cancers arise from epithelial cells it has been postulated that the experiments with these cells may have relevance for the detection of cancer-preventive effects.64 In this context it is notable that micronucleus assays with exfoliated oral cells have been successfully used to predict health risks due to occupational exposure, life style factors and also to identify protective factors in the human diet.65–67

11.4 Conventional SCGE Trials with Complex Foods and Individual Components – The Current State of Knowledge The results of studies that were conducted between 1997 and December of 2005 have been reviewed in the papers of Moller and Loft;27,29,41 findings of newer trials are listed in Table 11.1. In total, 17 investigations were carried out after 2005 under standard conditions, with restriction (lesion-specific) enzymes ENDOIII and FPG, and/or after challenge with chemicals or gradiation. Figure 11.1 depicts results obtained with the different protocols in all studies published in peer-reviewed articles so far. It can be seen that in about 50% of intervention trials, protection towards FPG-sensitive sites, and exogenous DNA damage by ROS were found, while protection against oxidised pyrimidines (ENDOIII-sensitive sites) and endogenous formation of single- and double-strand breaks apurinic sites was seen less frequently (i.e. in 40% and 32% of the studies). It is notable that in one trial with H2O2, there was evidence for a gender-specific effect, i.e. the effect was observed only in males.73

blinded, placebo controlled parallel study n ¼ 37 ~, postmenopausal, 57 d 5 groups (different carotenoid intervention/placebo) sampling: 0 d, 15 d, 29 d, 43 d,57 d

sequential n¼8 #+~, 5 d run in, 5 d intervention sampling: 0 d, 5 d



coffee 600 mL/p/d (150 mL metal filtered+450 mL paper filtered)

coffee 1 L/p/d metal filtered

Brussels sprouts, 300 g/p/d

Design of the study2

LY/TL k ENDOIII k FPG k PhIPk 2 Trp-P-2 k H2O2

LY/TL 2 SC k BPDE

LY/TL/TM 2 SC k ENDOIII k FPG kTrp-P-2 m PhIP k H2O2

LY/VIS k SC (in all carotenoid treatment groups) after 57 d, with b- carotene and mixed carotin formulation at day 15 2 H2 O 2

SCGE results3

68

69

70

2 plasma and saliva overall GST m plasma GST-p, 2 plasma GST-a 2 creatinine, cholesterol, alanine and aspartate aminotransferase, alkaline phosphatase, cholesterol k SULT (which activates PhIP), 2 SOD 2GPx m plasma vitamin C

40

plasma: m carotinoid

LY: m SOD 2GPx

Ref.

Remarks

Examples of dietary intervention studies (2005 and newer) based on the Comet assay.

1

carotenoids lutein lycopene b-carotene 12 mg/p/d each or 4 mg each in a mix/p/d

Dietary factor

Table 11.1

Comet Assays in Dietary Intervention Trials 271

(continued ).

placebo-controlled parallel design n¼27 # (treatment:18, placebo: 9) 3 w run in 4 w intervention or placebo 3 w wash out sampling: 0 d, after each week, from 1 to 9

blinded, placebo-controlled crossover study n¼13 #+13 ~, sampling: 26 d per phase (placebo wash out intervention)

sequential design 5 d wash-out 4 w intervention n¼168 (54 #+114 ~) sampling: befor and after intervention

red mixed berry juice (rich in polyphenols TEAC 19:1) 700 mL/p/d

tomato-drink (rich in caroteniods) 250 mL/p/d

blueberry/apple juice (1 L/d) – 97 mg quercetin+16 mg ascorbic acid/ d


randomised crossover design (high phenol oil vs. low phenol oil) n¼10 ~, postmenopausal, 8 w sampling: 2 w (5 times first period, 5 times second period)

1

high phenol extra virgin olive oil 50 g/p/d (equals 30 mg phenols/p/d)

Dietary factor

Table 11.1

LY/TM k H2O2 #: stronger effect than ~

LY/%DNA 2 SC

whole blood /%DNA k SC k FPG k H2O2 during the intervention 2 after the intervention

LY/%DNA high phenol oil: k SC k FPG 2 H2 O2

SCGE results3

73

plasma: m quercetin m vitamin C m TEAC

35

plasma: 2 malondialedehyde blood: m total glutathione, 2 oxidised glutathione

72

71

high phenol oil: m plasma antioxidant capacity m olive oil phenolics in human urine (hydroxytyrosol, homovanillyl alcohol)

production of INF-g by stimulating blood cells mafter placebo intake, 2 after tomato drink, but m TNF-a

Ref.

Remarks

272 Chapter 11

single-blind, randomised, crossover study 8 w intervention 7 w wash-out n¼30 #+30 ~ sampling: weeks 0, 8, 15 and 23

double-blind randomised-control trial 4 w intervention n¼80 (40 #+40 ~) sampling: weeks 0 and 4

randomised, crossover clinical trial 4 w intervention 4 w wash-out period n¼60 # smokers, control n¼30 # nonsmokers sampling: weeks 0 and 4

placebo-controlled, randomised 3d n¼16 (8 sumach, 8 placebo) sampling: 0 d, 3 d

placebo-controlled, randomised 3d n¼16 (8 GA, 8 placebo) sampling: 0 d, 3 d

raw watercress, 85 g/d

(Vitasay) complex of 10 vitamin+10 minerals

almond consumption 84 g/day

sumach (Rhus coriaria L) 3 g/d

gallic acid (GA) 0.2 mg/kg bw/d

LY/TL k SC k ENDO III k FPG k H2O2

LY/TL k SC k ENDO III k FPG k H2O2

LY/%DNA k SC

LY/%DNA k SC k H2O2

LY/%DNA k SC k FPG 2 H2 O2

plasma: m SOD m GPX 2GST m GST-p

78

77

76

serum: m a-tocopherol m SOD mGPX only in smokers

plasma: m SOD k GPX m GST m GST-p

75

74

no effect of age and gender

beneficial changes were significantly higher in smokers than in nonsmokers; plasma: m lutein m b-caretene


(continued ).


double-blind, randomised, crossover design n¼6 #, consumption of apples, wash-out one w, again consumption sampling: 0 h, 1–6, 9, 12 and 24 h

coffee 600 mL/p/d (200 ml metal filtered+400 mL paper filtered)

apples 1000 g/d 1 either organically or conventionally produced

LY/%DNA 2 SC k ENDOIII 2 FPG k FeCl3 2 H2O2

LY/TL/TM 2 SC k ENDOIII k FPG kTrp-P-2 k H2O2

LC/%DNA 2 SC k H2O2 after consumption of orange juice

LY/TL 2 SC k FPG after the intervention, 2 after wash-out phase

SCGE results3

33

80

plasma: 2 antioxidant capacity of low-density lipoproteins

79

plasma: m vitamin C after orange and supplemented drink till 8 h after consumption

LY: m SOD 2 GPx

34

Ref.

in parallel CBMN assay in LY was carried out: k apoptosis 2 MN 2 NPB 2 Nbud 2 necrosis, 2 NDI 2 NDCI

Remarks

Abbreviations : BPDE – benzo(a)pyrene-7,8-diol-9,10-epoxide, d – days, ENDOIII – endonuclease III, FPG – formamidopyrimidine glycosylase, GPx – glutatione peroxidase, GST – glutathione-S-transferase, INF-g – interferon-g, L – liter, LC – leucocytes, LY – lymphocytes, m – months, Nbud – nuclear buds, NDI – nuclear division index, NDCI – nuclear cytotoxicity division index, NPB – nucleoplasmatic bridges, p – person, PhIP – 2-amino-1-methyl-6-phenylimidiazo[ 4,5-b]pyridine,SC – standard conditions of the Comet assay (single-strand breaks, double-strand breaks, alkali-labile sites), SOD – superoxide dismutase, SULT – sulfotransferase, TEAC – trolox equivalent antioxidative capacity, TL – tail length, TM – tail moment, TNF-a – tumor necrosis factor a, Trp-P-2 – 3-amino-1methyl-5H-pyrido[ 4,3-b]indole, w – weeks k decrease, m increase, 2 no alteration

repeated-measure design, seven ~ received each drink on three different occasion, 2 weeks apart, sampling: every h till 8 h, and 24 h after the intake of each drink

blood orange juice (300 mL 1), drink supplemented with the same amount of vitamin C (150 mg), and sugar drink (control)


sequential n¼13 #+~, 5 d run in, 5 d intervention, 10 d wash out sampling: 0 d, 5 d, 10 d

1

wheat sprouts 70 g/d

Dietary factor

Table 11.1 274 Chapter 11


Figure 11.1

275

Evaluation of results obtained in SCGE experiments in human dietary intervention trails. SC – standard conditions, ROS – damage induced by reactive oxygen species (H2O2 treatment or radiation); white bars: positive results, grey bars: negative results.

11.5 Use of SCGE Trials to Detect Protection Against DNA-Reactive Carcinogens Humans are exposed to a broad variety of genotoxic carcinogens either via contaminated environment (water, air) or by consumption of foods that contain various groups of DNA-reactive carcinogens. Over recent years, we have attempted to develop protocols for SCGE experiments with peripheral lymphocytes that can be used in human intervention trials. Table 11.2 lists representatives of several groups of DNA-reactive chemicals that can be used in such studies. It can be seen that it is possible to conduct experiments with a number of genotoxic human carcinogens. However, the activities of phase I enzymes that are required for the activation of procarcinogens are quite low in lymphocytes,81 therefore negative results are obtained with some compounds such as benzo(a)pyrene, a representative of the PAHs and also with the mycotoxin aflatoxin B1 (AFB1). In order to include these agents in SCGE trials, it is possible to use DNA-reactive metabolites, for example benzo(a)pyrene-7,8diol-9,10-epoxide (BPDE), which is the most reactive metabolite of benzo(a)pyrene.82 In the case of aflatoxin B1, the reactive metabolite (the exo isomer of AFB1 8,9-epoxide) is highly unstable,83 but it is possible to activate the mycotoxin by preincubation with exogenous liver homogenate (S9 mix) that

most abundant HA in fried beef and chicken; act.: CYP1A1/1A2, SULT; detox.: UGT, microsomal NADH-dependent reductase IC: 2B second most abundant HA in meat; act.: CYP1A1/1A2, SULT; detox.: UGT, microsomal NADH-dependent reductase IC: 2B found in charred fraction of cooked fish, carcinogenic in rodents (liver); act.: CYP1A1/1A2; detox.: GST IC: 2B found in cooked meat and fish; act.: CYP1A1/1A2, NAT; detox.: UGT, (GST); IC: 2A

model compound of polycyclic aromatic hydrocarbons, found in foods and in environmental compartments (e.g., water sediments, air); act.: CYP1A1/ EH; detox.: GST; IC: 1

reactive metabolite of B(a)P

Benzo(a)pyrene (B(a)P)

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

Polycyclic aromatic hydrocarbons (PAHs)

2-amino-3-methylimidazo[ 4,5-f]quinoline (IQ)

3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)

2-amino-9H-pyrido[2,3-b]-indole (AaC)

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (PhIP)

Heterocyclic aromatic amines

Occurrence/metabolism

0.025 (0.01–0.253) 0.01 (with FPG treatment)

negative 0.05 (with S9) (0.02–0.5)

0.6 (0.5–1.0)

0.2 (0.15–0.45)

1.0 (0.1–1.5)

0.7 (0.13–0.7)

LOEL and dose range (mM)

Compounds tested with the SCGE assay in peripheral human lymphocytes.

Compound group

Table 11.2

93

92

90 91

87,89

70,86

88

70,86

Ref.

276 Chapter 11

Cadmium chloride (CdCl2)

Vanadium pentoxide (V2O5)

Metals

Cyclophosphamide (CP)

Miscellaneous compounds

Furan

Glycidamide (GA)

Acrylamide (AA)

Thermal degradation products

industrial chemical, used for production of sulfuric acid and steel alloys; genotoxic in vivo and in vitro, inhalative exposure to V2O5 causes lung cancer in rodents; IC: 2B used in a variety alloys; exposure occurs mainly via airborne dust and fume, genotoxic in vivo and in vitro, induces lung cancer in humans and lung cancer leukaemia in rodents; IC: 1

cytostatic, the active metabolite is 4-OH CP; act.: CYP-izozymes; detox.: GST IC: 1

found in different food items: cooked, fried and smoked meat, coffee, cocoa, bread, roasted hazelnuts IC: 2B

found in fried or baked foods (e.g., potato chips), neurotoxic, genotoxic, induces lung tumors in mice and in various organs of rats; act.: CYP2E1; detox.: GST; IC: 2A main metabolite of AA, formed by epoxidation; evidence that it acts as the ultimate mutagenic metabolite of AA

0.1 (0.5–5.0)

0.0003 (0.0003–0.006)

0.1 (with S9) (0.10–0.75) negative

0.3 (0.5–6.0) 0.01 (with FPG treatment) (0.01–2.0) 10 (0.01–10)

negative (0.5–6.0)

99,100

96–98

90,95

Hoelzl, unpublished data

92

93

93,94


0.25 (0.125–0.25)

0.025 (0.001-0.025)

negative (0.16–0.64)

0.05 (0.01–0.5)

LOEL and dose range (mM)



101–105

Ref.

Abbreviations: act. – activation; CYP – cytochrome P450; detox. – detoxification; EH – epoxide hydrolase; GST – glutathione-S-transferase; IC – classification according to IARC; LOEL – lowest observed effect level; NAT – N-acetyltransferase; SULT – sulfotransferase; UGT – UDP-glucuronosyltransferase.

Ochratoxin A

Aflatoxin B1

produced by Aspergillus flavus, found in maize and groundnuts especially in areas with humid, hot climate, potent liver carcinogen; act.: CYP1A1, detox.: GST ‘‘activated’’ Aflatoxin B1 (incubation with S9 mix); IC: 1 produced by Aspergillus ochraceus and Penicillium verrucosum, found in coffee beans, grain and pork products, nephrotoxic, induces renal tumors; act.: CYP-system; detox.: not known; IC: 2B

used in a variety of alloys; has been in widespread commercial use for more than 100 years; potent genotoxin, induces lung cancer in humans; IC: 1

Chromium (VI)

Mycotoxins

Occurrence/metabolism

(continued ).

Compound group

Table 11.2

278 Chapter 11

279


has been developed for routine testing of chemicals by Malling. Also, heterocyclic aromatic amines (HAs) can be preactivated with this procedure,85 but we, as well as others86,87 showed that positive effects can be obtained in lymphocytes also with the parent compounds, albeit relatively high concentrations are required.70,86,87 Intervention studies, which we conducted over recent years with selected genotoxic carcinogens in our laboratory are included in Table 11.2. It can be seen that in some cases protective effects were observed that could be partly explained by alterations of the activities of activating and/or detoxifying enzymes. For example, the inhibition of DNA damage by PhIP after consumption of Brussels sprouts could be attributed to inhibition of sulfotransferase (SULT1A1) that is required for the activation of this heterocyclic aromatic amine.70 Interestingly, no protective effect was observed in the same study towards Trp-P-2, which is structurally related but does not require SULT-mediated activation.70 In a study on coffee consumption (800 ml/d for 5 days), a significant increase of PhIP-induced DNA migration (Figure 11.2) was observed. The reason for this adverse effect may be the induction of enzymes such as CYP1A1 and CYP1A2 by coffee as demonstrated in rats by Huber et al.106 In the same intervention study, changes of the sensitivity of the cells towards the alkylating agent methyl methanesulfonate (MMS) were also found, whereas no clear effect was seen with dimethyl nitrosamine.106 It was shown recently in animal experiments that the coffee diterpenoids cahweol and kafestol induce the activity of the enzyme O6-methylguanine-DNA methyltransferase.106 This removes methyl groups from guanosine and the induction of this repair enzyme may account for the protective effect towards MMS, which we also observed in humans.68 One of the most important detoxification pathways of xenobiotics is their conjugation with glutathione.107,108 It is known that many food compounds, for example, glucosides in Brassicas as well as sulfo-group-containing amino acids in Allium vegetables109,110 cause induction of gluthathione transferases (GSTs), which catalyse these reactions. We also demonstrated in an investigation that consumption of coffee over a period of 5 days leads to substantial protection against BPDE-induced DNA migration, which could be explained by induction of this enzyme.69 Also, studies show that the spice sumach (Rhus coriaria L.) has demonstrated a DNA-protective effect against BPDE which was paralleled by an increase of the GST activity in lymphocytes.78

11.6 Use of SCGE Experiments to Monitor Alterations of the DNA-Repair Capacity In 2001, Collins et al.111 published a protocol for modified SCGE experiments that enables monitoring of the DNA-repair capacity in human lymphocytes. The technique is based on the use of cell-free extracts of white blood cells that are incubated with nucleoids of cells having increased levels of 8-oxoG caused by treatment with photosensitisers. Accumulation of breaks due to incision of

280

Figure 11.2

Chapter 11

Results of a preliminary intervention study in which the impact of coffee consumption on DNA migration induced by heterocyclic aromatic amines (3-amino-1-methyl-5H-pyrido[ 4,3-b]indole acetate, Trp-P-2 and 2-amino-1-methyl-6-phenylimidazo[ 4,5-b]pyridine, PhIP) and by alkylating agents (N-methyl-N-nitrosourea, MNU and N-nitroso-dimethylamine, DMNA) was monitored in lymphocytes. The participants (n ¼ 8) consumed 800 ml of metal-filtered (French Press) coffee over a period of 5 days. Subsequently, the blood cells were treated with the methylating agents and the HAs for 30 min at 37 1C. For each experimental point, three cultures were evaluated in parallel and from each, 50 cells were analysed for DNA migration. Bars show mean valuesSD, stars indicate significance (Wilcoxon signed rank test, **Po0.01, ***Po0.001).


281

oxidised bases by the repair enzyme 8-oxoguanine DNA glycosylase (OGG1) leads to increased comet formation. In two human intervention studies, one with coenzyme Q10, and one with kiwi food extract, evidence for an increase of DNA-repair capacity was found,112,113 but it is notable in this context that several nutritional studies failed to find an increase of OGG1 at the transcriptional level.28,114–116 Another approach that has been developed by Evans et al.117 is based on the use of bleomycin. Comparison of the time kinetics of disappearance of the comets induced by the cytostatic drug serves as an indication of the repair capacity.118 This protocol has been used to assess the differences in repair capacity of DNA of cancer patients and healthy subjects119–122 and also to investigate the molecular mechanisms of repair inhibition.98,123 This test was applied successfully for evaluation of the protective role of various food components in vitro,124,125 but to our knowledge no results from dietary intervention studies are available. Overall, the investigation of the modulation of DNA repair by dietary factors is currently a ‘‘hot topic’’ and SCGE-based techniques may contribute substantially to the elucidation of the impact of dietary factors on DNA-repair processes.

11.7 What Have We Learned from Intervention Studies so Far? The overall evaluation of the currently available studies shows that single treatment protocols were successful in demonstrating DNA protection in 8 studies out of 12 (67% positive results) and in 45 out of 69 (65% positive results) in trials with longer intervention and repeated consumption. Furthermore, it is interesting that individual compounds were in general less effective than complex foods or juices. Also in a recent study by Astley et al.126 in which a mixed carotene capsule, a daily portion of cooked minced carrots, a portion of mandarin oranges and vitamin C tablets were tested after different time intervals, negative results were obtained with the chemicals. These findings indicate that complex foods may contain active, yet unidentified components. In this context it is notable that the strong effects observed with kiwi fruit juice and Brussels sprouts could not be attributed to their vitamin C contents.70,112 Interestingly, two studies indicated that the consumption of increased levels of fruits and vegetables (600g/d) is not as beneficial as expected, because no protective effect was detected.127 Also, in an earlier study of van den Berg et al.,128 no protective effects were observed after consumption of 330 ml of a fruit and vegetable juice. On the contrary, Bichler et al.33 reported a significant reduction of DNA migration caused by H2O2 as well as decrease of FPG- and ENDOIII-sensitive sites after coffee consumption (600 ml/person/day) indicating that coffee may be more effective with regard to ROS protection than fruit- and vegetable-enriched diets. In this context, it is notable that several

282

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recent reports have indicated that the contribution of coffee consumption to the overall antioxidant status calculated on the basis of TROLOX (in rats129) as well as TRAP130 and TAC131 equivalents (in humans) is higher than that of fruits and vegetables. It is likely that the diet contains specific compounds, which are highly active antioxidants in vivo as they are well absorbed and act partly via interaction with signaling pathways, which leads to the induction of antioxidant enzymes. One typical example is the phenolic compound gallic acid (GA) that was identified as the active principle of the spice sumach (Rhus coriaria L.) and is also present in specific fruits such as mangoes and rhubarb and also in red wine. Ferk and coworkers78 showed in a placebo-controlled study that GA which is a component of sumach is 30–40 times more active than the vitamins C and E in SCGE trials with humans.

11.8 Future Perspectives The currently available data indicate that SCGE trials are a fast and costeffective approach, which enables the study of the DNA-protective effects of dietary factors in humans. At present, the majority of available data concerns the prevention of oxidative DNA damage, while only a few investigations have been conducted in which modulation of repair processes and alterations of the sensitivity towards genotoxic carcinogens were monitored. Recently, we evaluated the reliability and predictive value of different methods that are used to identify dietary constituents with antioxidant properties and concluded that most biochemical approaches are not reliable as they are either conducted under nonphysiological conditions and/or detect only a limited spectrum of antioxidant mechanisms.2 Also, in vitro experiments with cell lines lacking the representation of signaling pathways and/or transcription factors, which induce antioxidant enzymes are unlikely to yield reliable data. Therefore, one of the most promising approaches for the justification of health claims of antioxidant properties of foods requested increasingly by regulatory authorities may be human intervention trials in which effects are monitored in SCGE experiments.21–23 As pointed out by Moller and Loft,27,29 one of the crucial problems of these trials concerns their adequate design. It could be solved by formation of an international working group that evaluates critically the different parameters that affect the outcome of the studies and aims to establish standardised guidelines. Recent standardisation efforts132,133 for SCGE experiments address the problems that are relevant for human studies, only partly, and the existing data are insufficient to draw firm conclusions as they are not based on coordinated efforts. One important issue that has not been addressed by the critical discussions of Moller and Loft27,29,41 concerns mechanistic explanations of the results obtained in the Comet assays. Such explanations can strongly improve the scientific quality of the results obtained in human trials. In other words, small


283

studies without placebo or crossover design can also provide valuable information when the results can be interpreted on the basis of mechanistic experiments. Typical examples are explanations of the protective effects of coffee towards BPDE by induction of GST69 and the demonstration of reduction of PhIP-induced DNA damage by consumption of Brussels sprouts due to inhibition of SULT activity.70 The development of targeted microarrays for human studies, which enable the monitoring of alterations of genes encoding for signalling pathways,

Figure 11.3

Schematic representation of results of human and animal studies with gallic acid (GA). The compound was tested in a placebo-controlled human intervention trial, and the extent of DNA migration before and after consumption of 0.2 mg/kg/day for 3 days was monitored in lymphocytes under standard conditions, with lesion specific enzymes (FPG, ENDO III) and after H2O2 treatment. (for protocols see Collins et al.146). In subsequent animal studies, rats (HimOFA, male 200–220 g, n ¼ 3/ group) received GA in drinking water (identical amount as in the human trial, 0.2 mg /kg b. w.) and DNA damage was monitored in inner organs in nonirradiated and gamma-irradiated animals (the experiments were carried out as described by Sasaki et al.147). In further experiments, rats (n ¼ 8/group) received GA (0.2 mg/kg/d) in drinking water for 8 days before gamma-irradiation (3.0 Gy/min weekly for 4 weeks, in total 12 Gy). After 21 weeks, the frequencies of preneoplastic lesions (GSTp+) were determined in the livers (Grasl-Kraupp et al.148). Numbers in (a) and (b) indicate in % the reduction of DNA migration (tail length) in different treatment groups.

284

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antioxidant defense and metabolism of xenobiotics as well as proteome analyses, will strongly contribute to the explanation of results obtained in SCGE trials.2 For example, Hoelzl et al.134 found in a proteomic study with lymphocytes that consumption of Brussels sprouts leads to a pronounced increase of SOD protein that explains the strong antioxidant effects seen in a previous study. The most critical issue of human studies using the Comet assay with dietary components concerns their interpretation with regard to beneficial health effects. It has been shown in recent meta-analyses that genetic alterations, i.e. micronuclei and chromosomal aberrations (which have been used in contrast to SCGE assays for several decades) are reliable biomarkers for human cancer risks.135–137 However, it is not well known if DNA migration monitored in SCGE is associated with increased cancer rates and diseases such as coronary heart diseases, diabetes, hepatitis B and C, renal failure and HIV.138–140 It is also unclear if oxidant effects play a causal role in the etiology of these diseases, although in the case of cancer and coronary heart diseases mechanistic studies strongly suggest such associations.70,141,142 It is notable that some of the compounds detected as protective in Comet assay trials, for example vitamins and lycopene have been shown to reduce DNA damage in inner organs and protect against chemically induced cancer.143–145 In the case of gallic acid,78 we demonstrated that it is highly protective against g-radiationinduced DNA damage in various organs of rats and also against radiationinduced formation of preneoplastic lesions. Figure 11.3. depicts the results of these experiments that strongly support the assumption that protective properties of gallic acid against ROS-induced cancers can be expected in humans. Similar strategies could be used in studies concerning prevention of other forms of cancer and other diseases for which adequate animal models exists (e.g. for liver cirrhosis, diabetes and neurodegenerative disorders). This is provided that promising results can be obtained from long-term supplementation trials as evidence for beneficial effects in humans.

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135. S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W. P. Chang, N. Holland, M. Kirsch-Volders, E. Zeiger, S. Ban, R. Barale, M. P. Bigatti, C. Bolognesi, A. Cebulska-Wasilewska, E. Fabianova, A. Fucic, L. Hagmar, G. Joksic, A. Martelli, L. Migliore, E. Mirkova, M. R. Scarfi, A. Zijno, H. Norppa and M. Fenech, An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogenesis, 2007, 28, 625–631. 136. S. Bonassi, L. Hagmar, U. Stromberg, A. H. Montagud, H. Tinnerberg, A. Forni, P. Heikkila, S. Wanders, P. Wilhardt, I. L. Hansteen, L. E. Knudsen and H. Norppa, Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. European Study Group on Cytogenetic Biomarkers and Health, Cancer. Res., 2000, 60, 1619–1625. 137. H. Norppa, S. Bonassi, I. L. Hansteen, L. Hagmar, U. Stromberg, P. Rossner, P. Boffetta, C. Lindholm, S. Gundy, J. Lazutka, A. CebulskaWasilewska, E. Fabianova, R. J. Sram, L. E. Knudsen, R. Barale and A. Fucic, Chromosomal aberrations and SCEs as biomarkers of cancer risk, Mutat. Res., 2006, 600, 37–45. 138. H. Z. Pan, D. Chang, L. G. Feng, F. J. Xu, H. Y. Kuang and M. J. Lu, Oxidative damage to DNA and its relationship with diabetic complications, Biomed. Environ. Sci., 2007, 20, 160–163. 139. F. A. Domenici, M. T. Vannucchi, A. A. Jordao Jr., M. S. Meirelles and H. Vannucchi, DNA oxidative damage in patients with dialysis treatment, Ren. Fail., 2005, 27, 689–694. 140. C. Bolukbas, F. F. Bolukbas, A. Kocyigit, M. Aslan, S. Selek, M. Bitiren and M. Ulukanligil, Relationship between levels of DNA damage in lymphocytes and histopathological severity of chronic hepatitis C and various clinical forms of hepatitis B, J. Gastroenterol. Hepatol., 2006, 21, 610–616. 141. S. P. Hussain, L. J. Hofseth and C. C. Harris, Radical causes of cancer, Nat. Rev. Cancer, 2003, 3, 276–285. 142. M. M. Elahi and B. M. Matata, Free radicals in blood: evolving concepts in the mechanism of ischemic heart disease, Arch. Biochem. Biophys., 2006, 450, 78–88. 143. L. R. Ferguson and M. Philpott, Cancer prevention by dietary bioactive components that target the immune response, Curr. Cancer. Drug Targets, 2007, 7, 459–464. 144. P. Talalay and J. W. Fahey, Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism, J. Nutr., 2001, 131, 3027S–3033S. 145. A. Sengupta and S. Das, The anti-carcinogenic role of lycopene, abundantly present in tomato, Eur. J. Cancer Prev., 1999, 8, 325–330. 146. A. R. Collins, M. Dusinska, C. M. Gedik and R. Stetina, Oxidative damage to DNA: do we have a reliable biomarker?, Environ. Health Perspect., 1996, 104(Suppl 3), 465–469. 147. Y. F. Sasaki, S. Kawaguchi, A. Kamaya, M. Ohshita, K. Kabasawa, K. Iwama, K. Taniguchi and S. Tsuda, The Comet assay with 8 mouse

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organs: results with 39 currently used food additives, Mutat. Res., 2002, 519, 103–119. 148. B. Grasl-Kraupp, B. Ruttkay-Nedecky, L. Mullauer, H. Taper, W. Huber, W. Bursch and R. Schulte-Hermann, Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression, Hepatology, 1997, 25, 906–912.

CHAPTER 12

The Comet Assay for the Evaluation of Genotoxic Exposure in Aquatic Species G. FRENZILLIa AND B. P. LYONSb,* a

Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy; b Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK

12.1 Introduction Lakes, rivers and marine coastal areas are the receptacles for huge amounts of waste, derived either directly from industrial and municipal sources or indirectly from the atmospheric deposition of airborne emissions. This leads to the aquatic environment becoming a sink for complex mixtures of both well-known and emerging toxicants.1 A number of these contaminants are highly persistent and possess mutagenic and/or clastogenic properties.2,3 In the late 1970s the importance of detecting the mutagenic/genotoxic risks associated with water pollution was perceived and the Salmonella bioassay4 or sentinel species, such as mussels5 and fish6,7 were used to monitor the aquatic environment. Over the following decades a suite of tests have been developed for evaluating DNA alterations in aquatic animals, these are based on potentially premutagenic lesions, such as DNA adducts, base modifications, DNA–DNA and DNA– proteins crosslinking and DNA-strand breaks.8 The analysis of modified or damaged DNA has been shown to be a highly suitable method for assessing exposure to genotoxic contaminants in aquatic *



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environments. In general, the methods developed are sensitive to a range of contaminant concentrations, applicable to a wide range of species and have the advantage of detecting and quantifying exposure to genotoxins without a detailed knowledge of the contaminants present. Tests directly assessing DNAstrand breaks, or downstream alterations following DNA-strand damage, are commonly used to assess genotoxic exposure in aquatic animals. Initially, the procedures for measuring DNA-strand breaks were based on the separation of double-stranded DNA, which was then assessed by centrifugation or filtration; or on the denaturation rate under alkaline conditions and determined by the incorporation of a fluorescent dye by the double-stranded DNA. The development and application of alternative procedures based on cytogenetic investigations, such as the sister chromatid exchange (SCE) assay or the micronucleus (MN) test have also been widely used. However, while the MN test has been used extensively in fish and aquatic invertebrates, use of metaphase-based assays (e.g. SCE) has been hindered by the fact that many aquatic species have karyotypes consisting of numerous, small chromosomes. The single-cell gel electrophoresis (SCGE) or Comet assay was first applied to ecotoxicology over 15 years ago, and has since become one of the most widely used tests for detecting DNA-strand breaks in aquatic animals.9–13 The Comet assay has many advantages over other methods commonly used to assess genotoxic exposure, including (1) genotoxic damage can be detected in most eukaryotic cell types at the single-cell level; (2) only a small number of cells are required; (3) it is a rapid and sensitive technique; (3) Due to the nature of DNA-strand-break formation it provides an early-warning response to genotoxic exposure. As a consequence of the advantages listed above the Comet assay has been used widely in both laboratory- and field-based studies to assess genotoxic exposure in many freshwater and marine organisms. However, unlike mammalian genotoxicology, where the focus is limited to a small number of model species, efforts in the aquatic field have generally lacked coordination and have used an extensive range of sentinel species.9,11,13 While guidelines relating to the use of the Comet assay have been published for mammalian genotoxicology,14,15 no standard protocols currently exist for environmental studies. Consequently, the variations in protocols can lead to major differences in results and an inability to directly compare studies. Despite these obvious limitations the Comet assay provides a well-researched tool for addressing various aspects relating to genotoxicity in aquatic studies. This review will focus on recently published examples of the assay and its application to assessing genotoxic exposure in both marine and freshwater organisms.

12.2 Protocols, Cell Types and Target Organs The majority of aquatic studies published to date have used circulating blood cells (either haemocytes or erythrocytes), as target cells for Comet assay analysis. This is likely to be due to the practical advantage of processing tissues

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from a ready-made supply of nucleated cells in suspension. Solid tissues such as gill or fish hepatocytes require dissociation prior to analysis, with the potential of introducing damage through enzymatic or mechanical processes. However, when comparing cell types it is usually reported that circulating cells are less sensitive than hepatocytes or gill cells.16–21 Hepatocytes and mussel digestive cells are known to both bioaccumulate and biotransform pollutants, two processes that can lead to the activation of genotoxins or promote the activities of reactive oxygen species and other radicals.17 Blood, and to a lesser extent the haemolymph, of bivalve molluscs (e.g. mussels) are ‘‘buffered’’ tissues, in which contaminants arrive having crossed numerous biological barriers. Furthermore, mature erythrocytes have a relatively low metabolising capacity, which acts to minimise the production of potentially DNA-damaging metabolites.17 Studies have also demonstrated that different cell types responded with different sensitivities to contaminant exposure.20 Gill cells appeared to be the most sensitive following MNNG exposure, while liver and digestive gland were more sensitive to B(a)P, suggesting that uptake routes and bioaccumulation mechanisms need to be taken into account when designing experiment systems. Previous mammalian studies have demonstrated that certain tissue types may have higher background levels of DNA damage due to the presence of alkalisensitive sites in cells with highly condensed chromatin.22 Similar studies comparing basal levels of DNA migration in mussel gill cells, haemocytes and fish erythrocytes under both mild alkaline (pH 12.1) and alkaline versions (pH413) of the Comet assay have supported this assumption.23,24 Indicating that the mild alkaline version of the assay should be employed when dealing with certain cell types (e.g. fish erythrocytes), in order to prevent higher background levels of DNA-strand breaks inhibiting data interpretation. Indeed, this problem has been highlighted in other studies using fish species where excessive DNA tail migration has inhibited the interpretation of results.25 In addition to the variation in response depending on cell type, it is also apparent that a range of Comet assay protocols (differing in terms of agarose concentrations, lysing and electrophoresis parameters) have been used in studies with aquatic organisms.9–13 Therefore, effort is required to establish standardised protocols for the main species and cell type commonly used in environmental studies. The production of standard protocols, or the initiation of interlaboratory ring testing workshops focusing on aquatic species are essential if the Comet assay is to develop further as an environmental monitoring tool.

12.3 Application of the Comet Assay to Invertebrate Species 12.3.1

Freshwater Invertebrates

Most studies published to date using freshwater invertebrates have focused on filter-feeding organisms, as the test species of choice in field- and laboratory-based experiments. The genotoxic potential of common environmental contaminants,

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including tannins and polyphenols have been investigated using the freshwater mussel Unio tumidus.26 The same species has also been used to investigate the potential for B[a]P and ferric iron, to induce DNA damage.27 Studies conducted in parallel demonstrated a correlation between B[a]P exposure, 8-oxodGua levels and strand breaks, suggesting that oxidative stress was the mediator of genotoxicity. The zebra mussel (Dreissena polymorpha) has been used to evaluate the effect of standard (sodium hypochlorite and chlorine dioxide) and emerging (peracetic acid, PAA) disinfectant products on the formation of mutagens in surface waters.28 Mussels maintained in PAA-treated water failed to show a difference when compared with controls animals, whereas those treated with the two chlorinated disinfectants actually displayed a reduction in DNA damage, which was thought to be associated with the induction of detoxification processes or crosslinks inhibiting DNA migration. The golden mussel (Limnoperna fortunei) has been used to assess the genotoxic potential of Brazilian lake waters using a combination of the MN test and the Comet assay.29 Mussels were exposed for 7 days to either water or sediment samples from various contaminated locations and it was observed that micronuclei formation was responsible for almost 60% of the positive comet results observed. The golden mussel has also been used to investigate genotoxic effects of the pesticide copper sulfate (CuSO4) and biocide pentachlorophenol (PCP).30 Dose–response relationships were observed for both chemicals and the mussels, demonstrated a significant capacity for DNA repair within 2 h of the exposure ending. Moreover, the exposure to an environmental sample over 7 days confirmed the species sensitivity to waterborne contaminants and indicated its potential use as a biomonitoring organism. The Comet assay has also been applied to the study of naturally occurring toxins, including biotoxin (microcystin) producing strains of cyanobacteria.31 Zebra mussels were used to detect strain-specific DNA damage profiles, which persisted over the 3-week study period, confirming the sublethal genotoxicity of these toxins.

12.3.2

Marine Invertebrates

Marine invertebrates have been widely used as sentinel species in environmental monitoring programs. This is mainly due to their sessile nature, ability to bioaccumulate contaminants and general ease of capture.32–34 The majority of work has focused on coastal and estuarine environments. For example, Hartl et al. used the clam (Tapes semidecussatus) as an indicator species for the presence of potentially genotoxic substances in estuarine environments, demonstrating an increase in DNA damage in haemocytes, gill and digestive gland cells of animals exposed to contaminated sediments.16 The study also highlighted the differences in sensitivity between cell types, with gill and digestive gland cells appearing to be the most sensitive target tissues for detecting genotoxic exposure. The Mediterranean mussel (Mytilus galloprovincialis) has also been extensively deployed as a sentinel organism to assess the genotoxic effects of crude-oil spills.35–37 Studies have demonstrated the sensitivity of mussels to oil exposure


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and laboratory studies have clearly linked the total polycyclic aromatic hydrocarbon (TPAHs) content of oils with the level of DNA damage observed.35 In Northern European studies the Blue mussels (M. edulis) have also been used to differentiate sites receiving waste treatment effluent, with positive correlations detected between the presence of selected contaminants and the level of DNA damage.38 Again, the study highlighted the differences observed between sensitivity to contaminant exposure and cell type. Mussels have also been used extensively in the field as part of transplantation studies.39–41 The use of indigenous organisms is often hampered by the absence of a suitable sentinel species, or if present, the genotoxic responses obtained may be influenced by local physiological adaptations. Furthermore, the use of transplanted organisms also offers advantages over indigenous species, such as ensuring genetic homogeneity, developmental/reproductive status and controlling the precise exposure window. Validation studies have been undertaken with the Comet assay to assess the time-course variations in DNA damage following field transplantation experiments.39–40 It was observed that within the first 7 days following transplantation the level of DNA damage can fluctuate, which is likely to be caused by manipulation disturbance, then after 2 weeks the level reaches a plateau. Such data suggests that transplantation experiments lasting less that 2 weeks may give spurious results, with the levels of DNA damage detected attributable to artifacts associated with the sampling procedure rather than genotoxic exposure. Studies conducted in a coastal area of Denmark, impacted by a disused chemical site, have also highlighted that the levels of DNA damage in mussels can be affected by seasonal variations in baseline levels.39 Such results are likely to be influenced by the seasonal variations, which are known to exist for a range of physiological and reproductive processes in mussels.42,43 The sampling location has also been shown to influence the results of fieldbased surveys. For example, mussels (M. edulis) sampled from the intertidal zone in Reykjavik harbour had higher levels of DNA damage when compared with mussels collected from the subtidal zone at the same site.44 While the study supports the use of DNA-strand breaks as a measure of environmental pollution it also highlights the high levels of intrasite variability in DNA damage that can occur. As such the study further serves to underline the importance of validating experimental protocols and sampling procedures to ensure that noncontaminant related factors (e.g. physiological and biochemical responses to variations in oxygen availability and temperature stress) do not adversely affect biomarkers data.

12.4 Application of the Comet Assay to Vertebrate Species 12.4.1

Freshwater Vertebrates

The Comet assay has been applied to a range of freshwater vertebrate species to help inform chemical risk assessment or as part of site-specific investigative

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monitoring. Due to their small size and well-documented husbandry protocols freshwater fish are often seen as ideal candidates for laboratory studies. Fish such as goldfish (Carassius auratus), carp (C. carpio) and rainbow trout (Oncorhyncus mykiss) have been used extensively to study the genotoxic effects of model mutagenic compounds including, agent N-methyl-N-nitro-Nnitrosoguanidine (MNNG) and benzo[a]pyrene B(a)P.45,46 The Comet assay has also been applied to chemical risk assessment studies to screen a range of herbicides and pesticides for genotoxic potential.47–49 A range of environmental samples have also been screened for genotoxicity in the laboratory and field using the Comet assay in conjunction with a number of small freshwater fish species. Embryos of zebrafish (Danio rerio) have been used to assess freeze-dried sediment and sediment extracts, rich in perylene and copper, from Laguna Lake in the Philippines.50 Goldfish have also been screened using the Comet assay and MN test to study the effects of leachates from landfill sites.51 Laboratory experiments have detected elevated levels of DNA damage in C. carpio erythrocytes exposed in vivo for 20 days to lake waters treated with disinfectants (sodium hypochlorite, peracetic acid, chlorine dioxide).52 The genotoxic effects of agriculture chemical runoff have been studied in a combined laboratory and field caging experiment. The Comet assay detected an elevation in DNA damage in the Sacramento sucker (Catastomus occidentalis), which was linked to runoff events and supported by Ames assay test data.53 Chub (Leuciscus cephalus) have also proved to be an ideal sentinel species for investigating the genotoxicity of UK rivers, with the level of DNA damage detected correlated to a decrease in chemical water quality at the sites investigated (contaminated with organochlorine pesticides (OCPs), heavy metals, PAHs and PCBs).54 Increasingly, the Comet assay has been applied to amphibian species due to their perceived sensitivity to environmental conditions and importance as biodiversity indicators. Tadpoles (Rana hexadactyla) have been used to screen sulfur dyes used in the tannery industry, with dose-dependent levels of DNA damage observed along with efficient DNA-repair mechanisms following the cessation of exposure.55 Xenopus laevis larvae have been used in genotoxic risk evaluation studies of extracted soil leachates and of bottom ash resulting from municipal solid waste incineration processes.56 Species of toad (Bufo raddei) have been used to assess petrochemical contamination in the Lanzhou region of China.57

12.4.2

Marine Vertebrates

There are a limited number of Comet assay studies utilising marine fish species in comparison to those using freshwater species (for detailed review see refs. 9,11,13). This is mainly due to the logistical problems associated with collecting fish at sea (e.g. the need for research vessels) and technical problems inherent within the assay, such as the difficulty of performing electrophoresis reproducibly at sea (e.g. dealing with adverse weather conditions). To date, those


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studies undertaken have mainly focused on flatfish and bottom-feeding species, which due to their close association with sediment-bound contaminants are widely used in marine monitoring programmes.58,59 In vivo studies have been undertaken to investigate oxidative stress in the European eel (Anguilla anguilla).60 The Comet assay has also proven to be a useful tool for studying the genotoxic effects of nonbioaccumulating contaminants in the marine environment. For example, the environmental effects of the known mutagen and potential carcinogen, styrene, have been studied in the mussel (M. edulis) and fish (Symphodus mellops).61 Styrene has not previously been considered to be harmful to marine fauna due to its high volatility and low capacity to bioaccumulate. However, it was shown to cause a statistically significant increase in DNA damage in blood cells, probably due to the formation of a radical styrene metabolite, which is thought to have potent oxidative capacity. Hatcheryreared turbot (Scophthalmus maximus L.) have been used successfully to investigate the genotoxic potential of PAH and heavy metal contaminated sediment from sites in Cork Harbour (Ireland).62 Eelpout (Zoarces viviparus) have been used in site-specific investigative monitoring following a bunker oil spill in Go¨teborg harbour, Sweden. The Comet assay was deployed alongside a battery of other bioassays and elevated levels of DNA damage were correlated to the presence of PAH metabolites in the bile of fish.63 The marine flatfish dab (Limanda limanda) is a commonly used flatfish species in offshore monitoring programmes and it has been used in a number of studies investigating the impacts of genotoxic contaminants in coastal and estuarine waters.64–66 Studies have shown that both sex and age of the fish have a significant effect on the presence of DNA-strand breaks, which again highlights the influence of other factors (i.e. reproductive status) may have on the extent of DNA damage.64,65

12.5 Conclusions In conclusion, the Comet assay has broad applicability when applied to aquatic organisms, providing researchers and environmental managers with a sensitive and rapid tool for assessing environmental exposure to genotoxins. However, a wide variety of personalised Comet assay protocols are evident in the literature. Standardisation and interlaboratory calibration of the Comet assay, as applied to aquatic species, will be required if the technique is to be used routinely by national bodies charged with monitoring water quality.

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30. I. V. Villela, I. M. de Oliveira, J. da Silva and J. A. P. Henriques, DNA damage and repair in haemolymph cells of golden mussel (Limnoperna fortunei) exposed to environmental contaminants, Mutat. Res., 2006, 605, 78–86. 31. G. Juhel, J. O’Halloran, S. C. Culloty, R. M. O’Riordan, J. Davenport, N. M. O’Brien, K. F. James, A. Furey and O. Allis, In vivo exposure to microcystins induces DNA damage in the haemocytes of the zebra mussel, Dreissena polymorpha, as measured with the Comet assay, Environ. Mol. Mutagen., 2007, 48, 22–29. 32. B. L. Bayne, Watch on mussels, Mar. Pollut. Bull., 1976, 7(12), 217–128. 33. R. Seed, Ecology, In: B. L. Bayne, Marine Mussels, their ecology and physiology, International Biological Programme, Cambridge University Press, Cambridge, 1976, 13–65. 34. M.H. Salazar and S. M. Salazar, In situ bioassays using transplanted mussels: I. Estimating chemical exposure and bioeffects with bioaccumulation and growth, in J. S. Hughes, G. R. Biddinger, E. Mones, Eds. Environmental Toxicology and Risk Assessment – Third Volume. Philadelphia: American Society for Testing and Materials STP, 1995, 2118, 216–241. 35. B. Perez-Cadahia, B. Laffon, E. Pasaro and J. Mendez, Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil, Comp. Biochem. Physiol. C: Toxicol. Pharmacol., 2004, 138, 453–460. 36. B. Laffon, T. Rabade, E. Pasaro and J. Mendez, Monitoring of the impact of Prestige oil spill on Mytilus galloprovincialis from Galician coast, Environ. Int., 2006, 32, 342–348. 37. I. C. Taban, R. K. Bechmann, S. Torgrimsen, T. Baussant and S. Sanni, Detection of DNA damage in mussels and sea urchins exposed to crude oil using Comet assay, Mar. Environ. Res., 2004, 58, 701–705. 38. J. Rank, K. Jensen and P. H. Jespersen, Monitoring DNA damage in indigenous blue mussels (Mytilus edulis) sampled from coastal sites in Denmark, Mutat. Res./Genet. Toxicol. Environ. Mutagen., 2005, 585, 33–42. 39. J. Rank, K. K. Lehtonen, J. Strand and M. Laursen, Aquatic toxicology, DNA damage, acetylcholinesterase activity and lysosomal stability in native and transplanted mussels (Mytilus edulis) in areas close to coastal chemical dumping sites in Denmark, Aquat. Toxicol., 2007, 84, 50–61. 40. F. Regoli, G. Frenzilli, R. Bocchetti, F. Annarumma, V. Scarcelli, D. Fattorini and M. Nigro, Time-course variations of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment, Aquat. Toxicol., 2004, 68, 167–178. 41. M. Nigro, A. Falleni, I. Del Barga, V. Scarcelli, P. Lucchesi, F. Regoli and G. Frenzilli, Cellular biomarkers for monitoring estuarine environments: transplanted versus native mussels, Aquat. Toxicol., 2006, 77, 339–347. 42. A. Hines, G. S. Oladirana, J. P. Bignell, G. S. Stentiford and M. R. Viant, Direct sampling of organisms from the field and knowledge of their


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phenotype: key recommendations for environmental metabolomics, Environ. Sci. Tech., 2007, 41, 3375–3381. J. P. Bignell, M. J. Dodge, S. W. Feist, B. P. Lyons, P. D. Martin, N. G. H. Taylor, D. Stone, L. Travalent, G. D. Stentiford and G. D. Mussel, histopathology: effects of season, disease and species, Aquatic Biology, 2008, 2, 1–15. H. P. Halldorsson, G. Ericson and J. Svavarsson, DNA-strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavik harbour, Mar. Environ. Res., 2004, 58, 763–767. S. Masuda, Y. Deguchi, Y. Masuda, T. Watanabe, H. Nukaya, Y. Terao, T. Takamura, K. Wakabayashi and N. Kinae, Genotoxicity of 2-2-(acetylamino)-4-bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3 0 -dichloro-5,4 0 -dinitrobiphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the Comet assay, Mutat. Res., 2004, 560, 33–40. T. Kosmehl, A. V. Hallare, G. Reifferscheid, W. Manz, T. Braunbeck and H. A. Hollert, A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos, Environ. Toxicol. Chem,., 2006, 25(8), 2097–2106. B. Ateeq, M. Abul Farah and W. Ahmad, Detection of DNA damage by alkaline single-cell gel electrophoresis in 2,4-dichlorophenoxyacetic-acidand butachlor-exposed erythrocytes of Clarias batrachus, Ecotoxicol. Environ. Saf., 2005, 62, 348–354. S. Pandey, N. S. Nagpure, R. Kumar, S. Sharma, S. K. Srivastava and M. S. Verma, Genotoxicity evaluation of acute doses of endosulfan to freshwater teleost Channa punctatus (Bloch) by alkaline single-cell gel electrophoresis, Ecotoxicol. Environ. Saf., 2006, 65, 56–61. T. Cavas and S. Ko¨nen, Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the Comet assay, Mutagenesis, 2007, 22, 263–268. T. Kosmehl, A. V. Hallare, T. Braunbeck and H. Hollert, DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the Comet assay, Mutat. Res., 2008, 650, 1–14. Y. Deguchi, T. Toyoizumi, S. Masuda, A. Yasuhara, S. Mohri, M. Yamada, Y. Inoue and N. Kinae, Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and Comet assay using goldfish, Mutat. Res., 2007, 627, 178–185. A. Buschini, A. Martino, B. Gustavino, M. Monfrinotti, P. Poli, C. Rossi, M. Santoro, A. J. Dorr and M. Rizzoni, Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization, Mutat. Res., 2004, 557, 119–129.

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53. K. M. Whitehead, J. L. Kuivila, S. Orlando, S. L. Kotelevtsev and Anderson, Genotoxicity in native fish associated with agricultural runoff events, Environ. Toxicol. Chem., 23, 2868–2877. 54. M. J. Winter, N. Day, R. A. Hayes, E. W. Taylor, P. J. Butler and J. K. Chipman, DNA-strand breaks and adducts determined in feral and caged chub (Leuciscus cephalus) exposed to rivers exhibiting variable water quality around Birmingham, UK, Mutat. Res., 2004, 552, 163–175. 55. P. Rajaguru, R. Kalpana, A. Hema, S. Suba, B. Baskarasethupathi, P. A. Kumar and K. Kalaiselvi, Genotoxicity of some sulfur dyes on tadpoles (Rana hexadactyla) measured using the Comet assay, Environ. Mol. Mutagen., 2001, 38, 316–322. 56. F. Mouchet, L. Gauthier, C. Mailhes, M. J. Jourdain, V. Ferrier, G. Triffault and A. Devaux, Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests), Sci. Total Environ., 2006, 355, 232–246. 57. D. Huang, Y. Zhang, Y. Wang, Z. Xie and W. Ji, Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China, Mutat. Res., 2007, 629, 81–88. 58. S. W. Feist, T. Lang, G. D. Stentiford and A. Ko¨hler, Biological effects of contaminants: use of liver pathology of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring, ICES Tech. Mar. Environ. Sci., 2004, 38, 42. 59. JAMP guidelines for general biological effects monitoring. Joint Assessment and Monitoring Programme. Oslo and Paris Commissions, 1998, 38 pp. 60. F. Regoli, G. W. Winston, S. Gorbi, G. Frenzilli, M. Nigro, I. Corsi and S. Focardi, Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla: toward development of a more holistic biomarker assessment, Environ. Toxicol. Chem., 2003, 22, 2120–2129. 61. E. Mamaca, R. K. Bechmann, S. Torgrimsen, E. Aas, A. Bjornstad, T. Baussant and S. L. Floch, The neutral red lysosomal retention assay and Comet assay on haemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops) exposed to styrene, Aquat. Toxicol., 2005, 75, 191–201. 62. M. G. J. Hartl, M. Kilemade, D. Sheehan, C. Mothersill, J. O’Halloran, N. M. O’Brien and F. N. A. M. van Pelt, Hepatic biomarkers of sedimentassociated pollution in juvenile turbot, Scophthalmus maximus L, Mar. Environ. Res., 2007, 64, 191–208. 63. G. Frenzilli, V. Scarcelli, I. Del Barga, M. Nigro, L. Forlin, C. Bolognesi and J. Sturve, DNA damage in eelpout (Zoarces viviparus) from Goteborg harbour, Mutat. Res., 2004, 552, 187–195. 64. F. Akcha, G. Leday and A. Pfohl-Leszkowicz, Potential value of the Comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds, Mutat. Res., 2003, 534, 21–32.


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65. F. Akcha, F. Vincent Hubert and A. Pfohl-Leszkowicz, Measurement of DNA adducts and strand breaks in dab (Limanda limanda) collected in the field: effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA damage, Mutat. Res., 2004, 552, 197–207. 66. B. P. Lyons, G. D. Stentiford, J. Bignell, F. Goodsir, D. B. Sivyer, M. J. Devlin, D. Lowe, A. Beesley, C. K. Pascoe, M. N. Moore and E. Garnacho, A biological effects monitoring survey of Cardigan Bay using flatfish histopathology, cellular biomarkers and sediment bioassays: findings of the Prince Madog Prize 2003, Mar. Environ. Res., 2006, 62, S342–S346.

CHAPTER 13

The Alkaline Comet Assay in Prognostic Tests for Male Infertility and Assisted Reproductive Technology Outcomes SHEENA E. M. LEWIS AND ISHOLA M. AGBAJE Reproductive Medicine, Queen’s University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UK

13.1 Introduction Infertility affects one in six couples in Europe during their reproductive years with dysfunctional sperm being one of the most common causes. Conventional semen analysis has proven variable and lacking in prognostic value so, over the past decade, more useful molecular fertility biomarkers have been explored. Amongst the tests showing most promise are those measuring sperm DNA quality. Sperm DNA damage has been closely associated with numerous indicators of reproductive health including fertilisation, embryo quality, implantation, spontaneous abortion and childhood diseases.1 It therefore has great potential as a prognostic test for assisted reproductive treatment (ART), when couples are presenting with male infertility. Unlike somatic cells, sperm have a unique tightly compacted chromatin structure. Our group has modified the alkaline Comet assay for use with sperm. Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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Sperm DNA also differs from somatic cells in its high susceptibility to oxidative damage; this is largely due to the presence of abundant polyunsaturated fatty acids acting as substrates for reactive oxygen species (ROS) and its lack of repair mechanisms. Consequently, the effects of ROS and antioxidant protection on sperm DNA fragmentation have been widely investigated. In this review the relationship between actual sperm DNA damage as determined by the alkaline Comet assay and potential DNA damage as measured by DNA adduct testing will also be examined and the potential of routine clinical practices such as cryopreservation and prolonged incubation to induce further DNA damage investigated. Finally, the usefulness of sperm DNA tests as prognostic markers and in particular, the opportunities and challenges provided by DNA testing in male-fertility determination will be discussed.

13.2 Sites of DNA Damage in Sperm Sperm chromatin differs from somatic cells in both constituents and arrangement. During spermiogenesis, protamines, which are half the size of histones,2 replace the majority of histones and the chromatin is wound into unique supercoiled doughnut structures named toroids.3,4 As the sperm pass through the epididymis, the protamines are crosslinked by disulfide bonds reducing the chromatin to one-sixth the volume taken up in somatic cell nuclei.2 This dense compaction gives protection against exogenous assault to the sperm DNA as reflected by the high levels of irradiation required to damage sperm DNA, compared with somatic cells5 and also by the relative resistance of sperm nuclear and mitochondrial genomes to damage when treated with hydrogen peroxide.6,7 Despite this protection, basal levels of sperm DNA damage are very high in infertile8,9 and even fertile men.5 In addition to exhibiting higher basal levels of DNA damage, sperm from infertile men are more susceptible to damage from hydrogen peroxide, X-ray irradiation5,10 and cryoinjury.11 Damaged DNA has been observed in testicular, epididymal and ejaculated sperm. Sperm DNA first becomes susceptible to damage if chromatin packing is not completed during spermatogenesis.12,13 Some strand breaks may be necessary to reduce the torsional stress experienced by DNA during the rearrangement of its tertiary structure, a time when protamine replacement is occurring in elongating spermatids.14–16 However, these are temporary nicks and if they are not repaired17,18 increased DNA fragmentation in mature, ejaculated sperm may result. Damage can also occur as a result of suboptimal compaction19 due to incomplete disulfide crosslinking during epididymal transit. Although DNA repair does occur in developing sperm20 it is terminated as transcription and translation cease postspermiogenesis.21,22 As a result, sperm have no mechanism to repair DNA damage incurred during their transit and storage in the epididymis or postejaculation. In addition, the cellular machinery that allows these cells to complete apoptosis is discarded. As a result, advanced stages of germ cell differentiation into spermatocytes, spermatids and

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subsequently spermatozoa cannot be deleted, even though they may be defective or even partially apoptosed as evidenced by Fas expression or endonuclease activation.23,24 This may explain the large numbers of defective sperm present even in fertile mens’ semen.25 Further DNA damage can occur as sperm pass through the epididymis. This damage can be either induced in mature sperm by adjacent ROS-producing immature sperm in the epididymis, by ROS-producing epithelial epididymal cells or through toxic factors present as sperm undergo epididymal maturation. The latter hypothesis is supported by work that shows lower levels of DNA damage in testicular sperm that increases in caudal epididymal and ejaculated sperm.26–28 This has recently been confirmed in a study by Greco et al.,29 where sperm DNA damage was markedly lower in testicular than ejaculated sperm. Suganuma’s group is also in agreement showing that defective sperm experienced an increase in DNA damage during passage through the epididymis.30 It is acknowledged that even the proximal epididymis has substantial proportions of senescent sperm28 releasing ROS as they age and die31 and damaging those adjacent to them. This may at least in part explain the higher levels of sperm DNA damage in the epidydimis.

13.2.1

Oxidative Stress, a Major Cause of DNA Damage

As with other indications of sperm dysfunction, the importance of reactive oxygen species (ROS), caused by either increased ROS generation or impaired antioxidant defence, as a primary instigator of sperm DNA damage is well established.32–34 Sperm are particularly vulnerable to damage from ROS because of their high polyunsaturated fatty acid content and limited ability to repair damage. Sperm from infertile men are often associated with high levels of ROS caused by either increased generation or impaired antioxidant defence.35,36 Associations between oxidative stress and sperm DNA damage have been reported in numerous studies.9,31,37–39 + The oocyte can provide limited repair to damaged sperm DNA postfertilisation.40,41 However, if inadequately repaired, such damage can predispose to mutations in the developing embryo with the potential to induce disease in the offspring.34 It is acknowledged that a greater proportion of inherited diseases have their origin in the paternal germ line.42 Furthermore, cancers arising from germ cell mutations show a much greater paternal than maternal contribution.43 This fact is further illustrated by the finding of higher rates of haematological cancers (leukaemias and lymphomas) in offspring of men who smoke44 with the suspected causal link being the increased level of oxidative sperm DNA damage.45

13.2.2

Oxidative Stress, Antioxidant Therapies

Although there is now consensus as to oxidative stress as a major source of sperm DNA damage, less progress has been made in developing useful


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antioxidant therapies. The production of oxidative stress from hydroxyl, superoxide and hydrogen peroxide radicals can be kept in check by chainbreaking antioxidants such as vitamins C and E. Vitamin C concentrations are ten times higher in seminal plasma than blood plasma36 emphasising their physiological importance. Further, our group has also reported reduced levels of chain-breaking antioxidants; vitamins C and E in both sperm and seminal plasma of infertile men compared with fertile males36,46 and protective benefits when added during sperm preparation for ART.47 Metal chelators can also be useful in reducing ROS generation and preventing lipid peroxidation of sperm membranes, thereby protecting sperm DNA (reviewed by ref. 48). Paradoxically, the addition of combinations of antioxidants such as vitamins C and E can have damaging effects to DNA in vitro37 and in vivo where it causes an increase in DNA decondensation49 or they can be ineffective.50 Indeed, Vitamin E alone has been described as ‘‘a double-edged sword’’, its effects being strictly dependent on dosage51 and ineffective if given to males whose infertility aetiology is not oxidative stress. This is reflected in a study of couples who had had failed ART treatments,29 where the male partners had high levels of sperm DNA damage. Antioxidant treatment of vitamin C (1 g) plus vitamin E (1 g) daily for two months was administered before a further ICSI attempt. Following treatment, significantly lower levels of sperm DNA damage concomitant with higher success rates from ICSI (48% vs. 7% pregnancy rates) were observed. However, some patients did not respond to antioxidant therapy despite evidence of sperm DNA damage, perhaps suggesting either the damage was occurring through an nonoxidative pathway or other complicating factors, such as, Vitamin E’s nonantioxidant functions,52 preventing its efficacy. Further research is urgently needed to find the most effective antioxidant therapy for sperm DNA protection in the greatest range of patients.

13.2.3

Sperm DNA Damage Tests

Traditionally, male infertility diagnoses have depended on microscopic analyses and biochemical assays to determine human semen quality. The commonly measured parameters are sperm concentration, motility and morphology in the ejaculate. Most laboratories also include ‘‘sperm suitability’’ tests where the subpopulations of fastest swimming sperm are separated by density centrifugation. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, their clinical value in predicting fertility is questionable. Over the past decade, a number of laboratory tests have been developed to determine specific aspects of sperm function. These include quantitative sperm motion parameters, capacitation, basal and induced acrosome reactions and sperm-zona pellucida interactions. However, few have been proven to have strong prognostic value and thus have not become routine clinical tests. There is general acceptance that sperm nuclear DNA tests show the most promise in the diagnosis and treatment of male infertility. This has led to the development of numerous techniques to

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assess sperm DNA integrity. Of these, the alkaline Comet, terminal deoxynucleotidyl transferase (TdT, TUNEL) and sperm chromatin structure (SCSA) assays (reviewed in refs. 26,48,53) have been shown to be most robust. Each of these tests determines different aspects of DNA damage. The alkaline Comet assay (originally known as the single-cell gel electrophoresis assay) assesses actual DNA-strand breaks and alkali-labile sites when used under alkaline conditions. It has been tested in vitro and in vivo in a wide variety of mammalian cells54–56 employing a number of different genotoxic stimuli including UV radiation, carcinogens, radiotherapy and chemotherapy.57 It has been proven to be rapid,57,58 reproducible10 and have higher sensitivity than alkaline elution or nick translation (NT) assays. Even with prior chromatin decondensation,9,59 this assay can detect damage equivalent to as few as 50 single-strand breaks per cell. One of its unique and powerful features is the ability to characterise the responses of a heterogeneous population of cells by measuring DNA damage within individual cells as opposed to just one overall measure of damaged cells versus undamaged cells as in the TUNEL. This is important, since DNA damage may be the pivotal factor in determining the sperm’s capacity to achieve a pregnancy. Semen is one of the most heterogeneous biological fluids in humans. ART outcomes are improved by isolating the best subpopulations for clinical use. By using the alkaline Comet assay, the actual damage load of small cohorts of sperm may be measured. As the alkaline Comet assay only requires 100 cells for analysis it has also been particularly useful for studies involving DNA of testicular sperm and for men with low sperm concentrations where sperm numbers are limited.60

13.2.4

Modifications to the Alkaline Comet Assay for Use with Sperm

The alkaline Comet assay has been extensively used to study DNA fragmentation in a number of cell types. The study of sperm DNA fragmentation using this technique requires the use of a modified protocol primarily because of the differences in DNA packaging between sperm and somatic cells as described earlier. The formation of disulfide bonds between protamines and DNA is the key in facilitating the high level of DNA compaction in sperm. In addition, it is acknowledged that following ejaculation additional bonds are formed, further enhancing chromatin stability.61 However, the extent of this DNA compaction prevents DNA strands migrating during electrophoresis in conventional alkaline Comet assay protocols. To overcome this difficulty, our group has performed a number of studies modifying alkaline Comet protocols for use with sperm.10,37 In early studies, the use of conventional alkaline Comet protocols designed for somatic cells, failed to facilitate lysis indicated by the sperm head and tails remaining intact. In subsequent studies, the protease enzyme Proteinase K was added to remove protamines, thereby allowing sperm DNA to decondense and migrate. However, this was only effective with relatively high concentrations of Proteinase K (100 mg/mL) and after prolonged (overnight)


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incubation. This had the concomitant adverse effect of inducing sperm DNA damage. Baseline damage for tail DNA in sperm was variable, often reaching 25%.10,47,62 In our current protocol, lysis and decondensation steps involve immersion (within agarose gels) in freshly prepared cold lysis solution (2.5 M NaCl, 100 mM Ethylenediamine tetraacetic [EDTA] and 10 mM Tris (pH10), with 1% Triton X-100 added just prior to use), for 1 h at 4 1C. This is followed by incubation with dithiothreitol (DTT, 10 mM) for 30 min at 4 1C followed by lithium diiodosalicyclate (LIS, 4 mM) for 90 min at 20 1C to reduce the disulfide bonds. The use of this modified protocol has been shown to be a reliable and reproducible method of assessing DNA damage in sperm with baseline damage stabilising at B10–15% in sperm with normzoospermic profiles.37 One criticism of the alkaline version of the Comet assay for sperm DNA is that it measures alkali-labile sites that are not specific for infertility.63 In addition, it has limited ability to distinguish between endogenous and induced strand breaks or between single- and double-strand breaks.53,64 It has been suggested65 that markers of double-strand breaks may be more important in relation to + fertility because although sperm DNA damage can be repaired by oocytes between sperm entry and initiation of the next S-phase, this DNA-repair capacity is limited and DSB are more difficult to repair than SSB.40,65,66 In contrast, others (reviewed by ref. 67) suggest that total DNA damage is a more valuable indicator. The alkaline Comet assay, under alkaline conditions, measures single- and double-strand DNA breaks and those alkali-labile sites that, at high pH, are susceptible to breakage and conversion into single-strand breaks. Since these are all included in the analysis, proponents of the alkaline Comet assay suggest it is the optimal assay for the assessment of overall DNA damage. The most commonly used alkaline Comet measures are tail DNA (percentage of DNA in the tail compared to the percentage in the ‘‘head’’ or unfragmented DNA), tail length (the length of the tail measured from the leading edge of the head), or Olive tail moment (OTM) (percentage of DNA in the tail [tail DNA] times the distance between the means of the tail and head fluorescence measures). The OTM is expressed in arbitrary units. Each of these parameters describes endogenous DNA damage corresponding to DNA-strand breakage and/or alkali-labile sites. In the optimisation of the alkaline Comet assay for use with sperm we found tail DNA to be the most reproducible parameter,10 therefore, sperm DNA damage has been expressed as tail DNA throughout our studies. Clinical thresholds for ART success have not yet been established for the alkaline Comet assay. However, its clinical value has been shown in diagnosis of suboptimal semen profiles and associations with classic parameters of semen analyses such as sperm concentration,9 morphology,11 mitochondrial + penetration.69 The subpopulations of sperm isolated function68 and oocyte from semen by density centrifugation for ART, have also been shown to have less DNA fragmentation.70 Furthermore, the predictive value of DNA damage in embryo quality71 and pregnancy with ejaculated and testicular sperm60 have also been reported. A second commonly used test for sperm DNA damage is the SCSA. It is also believed to measure both single- and double-strand breaks,72 although

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primarily single-stranded DNA using a DNA fragmentation index (now called DFI, formerly COMPa1). It can also be used to measure what the authors call ‘‘immature sperm populations’’ that have higher than normal stainability (high-density stainability; HDS). It can be used in conjunction with light microscopy or flow cytometry, enabling very large numbers of sperm to be assayed rapidly. In addition, it benefits from individual laboratory training prior to use by its initiator Don Evenson and a standard protocol closely adhered to by all users minimising interlaboratory variation. The SCSA is less specific than alkaline Comet or TUNEL in determining DNA fragmentation in that it detects changes in protamine content and disulfide crosslinkage as well as DNA-strand breaks. However its advantages are that it is highly repeatable and clinical thresholds have been established showing that there is a greater chance of pregnancy after intrauterine insemination, in vitro fertilisation and intracytoplasmic sperm injection and if the semen has o27–30% DFI, respectively.72 In the TUNEL assay, TdT preferentially labels the blunt 3 0 -OH ends of double-stranded DNA breaks, but also measures single-strand breaks.73 It has the advantage of being relatively quick and easy to perform. However, one major limitation of this assay in sperm results from the high levels of sperm DNA compaction combined with the absence of a lysis step. Whilst the protocol may not induce further damage, it may limit accessibility of the TdT enzyme to all 3 0 -OH ends. This may account for the range of suggested clinical thresholds as reviewed by Tesarik et al.,74 showing a number with considerably lower values (12, 15, 18%,75–78) than that proposed by Evenson (30%). There are just a few studies comparing alternative assays within the same study (SCSA and alkaline Comet,79 alkaline Comet and TUNEL,68 and SCSA and TUNEL,80–82 which give surprisingly close correlations between assays despite the differences in protocol and in the parameters measured by each assay.

13.2.5

Sperm DNA Adducts and their Relationship with DNA Fragmentation

In addition to discrete strand breaks, ROS can induce other types of DNA damage, including base loss or modification/adduct formation, the most common of which is 7,8-dihydro-8-oxo-2 0 -deoxoguanosine (8-OHdG), an oxidative adduct of the purine guanosine.83 If sperm with DNA adducts are successful in achieving a pregnancy, paternally originating errors in DNA replication, transcription and translation can occur, potentially predisposing the offspring to a number of cancers and other degenerative disorders.84,85 Furthermore, given time, such base modifications may also lead to discrete DNA-strand breaks.86 In a study from our group,87 sperm from type-1 diabetic men had significantly higher 8-OHdG 10–5dG as well as DNA fragmentation (as assessed by the alkaline Comet) than those of nondiabetic men. A significant association (rs ¼ 0.7) between DNA fragmentation assessed by the alkaline Comet assay


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and levels of 8-OHdG 10 dG was also reported. Therefore, the measurement of DNA adducts combined with fragmentation assays gives an insight to potential as well as actual DNA damage.88 The direct effects of oxidative sperm DNA adducts on pregnancy have been reported in one study where the likelihood of pregnancy occurring in a single, menstrual cycle was inversely associated with 8-oxodG,89 further emphasising its prognostic value.

13.3 Can Sperm DNA Integrity Predict Success? Relationships with Assisted Conception Outcomes Aitken et al.,33 have reviewed the relationships between sperm DNA damage and fertilisation. This group has shown that at low levels of oxidative stress, DNA damage is induced yet the fertilising potential of the sperm is actually enhanced, reflecting the importance of cellular redox status in driving tyrosine phosphorylation events associated with functions such as sperm capacitation.33,90 These results are clinically significant since they support the studies of Ahmadi et al.66,91 showing that sperm with damaged DNA can still achieve + fertilisation. Furthermore, the oocyte controls both fertilisation and early cleavage stages of embryonic development92 suggesting that sperm with damaged DNA can retain their fertilising potential resulting in damaged DNA becoming part of the next generation’s genome. In clinical studies, although fertilisation in vitro (by IVF) has been shown to be negatively correlated with DNA damage,93 this is not the case with the recent modification of IVF; namely intracytoplasmic sperm injection (ICSI) that now accounts for approximately 50% of ART cycles. Intracytoplasmic sperm injection is an invaluable innovation in the treatment of infertile males with poor quality sperm.94 However, ICSI has removed many of the cellular checkpoints that prevent poor quality or immature sperm from successfully + fertilising oocytes. In addition, it has de-emphasised the importance of sperm selection and allowed the arbitrary choice of sperm for injection. For the first time in history, we have perfected a technique that circumvents all natural barriers to fertilisation thus facilitating the union of potentially defective gametes. While fertilisation may be independent of sperm DNA integrity, the postfertilisation development of the embryo can be seriously disrupted by such damage. After the third stage of cleavage, the paternal genome exerts a major influence92,95 and evidence of DNA damage is reflected in impaired embryonic development. Thus, in assisted-conception cycles, preimplantation development is negatively correlated with DNA-damage assessed by a variety of methods including nick translation,96 the alkaline Comet assay,71 TUNEL91,97 and SCSA.98,99 Pregnancy rates have a negative correlation with high levels of sperm DNA fragmentation in artificial insemination cycles.77 An inverse relationship has also been reported between pregnancy rates with ICSI and the level of DNA fragmentation in immature60 and mature sperm.60,75,98,100,101 In addition,

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even fertile couples took longer to conceive naturally, with the time to pregnancy increasing as a function of the proportion of sperm with abnormal chromatin.102 Evenson8 also demonstrated that the miscarriage rate was higher in couples who conceived naturally but where the partner’s sperm had poor chromatin. Higher rates of pregnancy loss have also been documented in ICSI than in IVF cycles.103 As these pregnancies were almost inevitably achieved with sperm of + poor quality that would have been incapable of fertilising an oocyte naturally, the link may well be sperm DNA damage. Poor subpopulations of sperm (discarded after density centrifugation but equivalent to those used in ISCI70 also have markedly higher levels of DNA fragmentation. However, because of the success of ICSI in bypassing rather than addressing the problem of poor sperm quality, these sperm with potentially damaged DNA continue to be used indiscriminately in ART. While sperm with damaged DNA may show a reduced capacity, fertilisation and implantation do occur with unknown consequences on the health of the next generation. There are a growing number of studies associating high mutational risk paternal occupations (such as exposure to metals, solvents and pesticides) and an increase in birth defects and childhood diseases.104 Animal studies have also demonstrated very clearly that sperm damaged by paternal exposure to cancer therapeutic agents can have adverse effects on the offspring.105–107 Tobacco, another source of mutagenic xenobiotics108 that can induce sperm DNA damage has been associated with a higher incidence of childhood cancer in the next generation.44,109 Indeed up to 14% of all such cancers have been linked to paternal smoking.109 New reports also show increases in schizophrenia, achondroplasia and Apert’s syndrome in children of older men with high levels of sperm DNA damage.67 This further suggests that sperm DNA damage can impact negatively upon the health of offspring.

13.4 Clinically Induced DNA Damage The advent of ISCI in 1995 has facilitated the use of immature testicular sperm + that would be incapable of fertilising an oocyte in vivo. The DNA of these sperm is even more vulnerable to damage than that of ejaculated sperm29,60,86 perhaps because they have not completed the process of disulfide crosslinking. The selection of sperm for ICSI usually involves an evaluation of motility, as this gives an indication of the viability of the sperm.110 This is problematical in testicular sperm due to lack of inherent motility. The use of a nonviable testicular sperm may lead to lower fertilisation rates than obtained with an ejaculated sperm.111 The technique of culturing testicular sperm in vitro prior to ICSI has been recommended by a number of groups112–114 in order to promote an increase in sperm motility. A period of 24 h has been suggested as optimal for the development of motility in a sufficient number of testicular sperm to give choice in the treatment cycle.114 However, incubation of fresh testicular sperm for ICSI appears to be beneficial only in terms of the development of motility


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and morphology. Worryingly, this procedure also damages sperm DNA.115 In this study from our lab we provided evidence that both fresh and frozenthawed testicular sperm from men with obstructive azoospermia display substantial DNA fragmentation following routine incubation in vitro prior to ICSI injection.115 To avoid this, we recommended that all testicular sperm should be injected without delay in order to protect the genetic health of the resulting child.

13.4.1

Cryopreservation

Cryopreservation is a core technique in the preservation of male fertility before cytotoxic chemotherapy or radiotherapy and during ART. The use of frozen semen is also mandatory in donor insemination programmes where samples are stored until donors are screened for infections such as HIV and hepatitis B. However, despite many refinements in methodology, the quality of post-thaw samples remains suboptimal and ART success rates with frozen sperm are lower than with fresh samples.116 Sperm lose most of their cytoplasm during maturation and therefore lose the enzymatic defences present in somatic cells, including chain-breaking antioxidants. This leaves them at a considerable disadvantage. However, sperm are protected during ejaculation, by the high levels of antioxidants in seminal plasma. For example, seminal ascorbate is present at ten times the concentration of that in blood plasma.36 By returning prepared sperm to seminal plasma to prepared sperm before freezing the DNA of those subpopulations of sperm with greatest fertility potential can be protected from cryoinjury.11 This is supported by a previous study in which similar protection of sperm DNA was observed if sperm were prepared for ART in the presence of antioxidants.47 Cryopreservation of testicular sperm is also very important; ensuring the availability of sperm for subsequent treatment cycles without the need to perform additional invasive biopsies.117 However, cryoinjury to DNA is common in testicular sperm from fertile and infertile men. This may be due to the fact that all testicular sperm are more vulnerable to oxidative damage than ejaculated sperm since they have not undergone epididymal transit and maturation, where their DNA will be crosslinked conveying protection.

13.4.2

Vasectomy

Traditionally, vasectomy has been considered an irreversible form of contraception. Today, however, many postvasectomised men wish to have a second family with a new partner. Many vasectomy reversals have been replaced by testicular biopsy performed at an outpatient clinic and subsequently used in ICSI.118 It has always been accepted that previously fertile men would suffer no impairment to sperm since the vasectomy was simply a forced blockage as opposed to defective spermatogenesis. However, it has recently been reported that the postvasectomised men have markedly reduced sperm yields.119

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In addition, significant increases in DNA damage have been observed in testicular sperm postvasectomy compared to those from fertile men with a positive correlation between DNA fragmentation and time following vasectomy. The impairment of sperm quantity and quality is most significantly reflected in a reduction in pregnancy rates after ART.120

13.5 A Major Barrier to Progress The advent of ICSI has provided a means of treatment for infertile couples with severe male factor infertility previously considered untreatable by conventional ART. However, its success has also impeded progress in the development of prognostic tests for sperm health because this therapeutic technique allows us to bypass the natural hurdles to fertilisation so that even unfit sperm succeed. The consequence of this has been a further reduction in the diagnostic significance of parameters such as sperm concentration, motility and morphology but without the inclusion of more appropriate selection tests. Since short-term (i.e. pregnancy) success rates with ICSI are as good as those of IVF, where dysfunctional sperm are not the primary problem, there has been little incentive for the development of sperm-selection tests for ICSI. This approach has been described as a human experiment, since ICSI is in its ‘‘infancy’’, with comparatively little long-term data on the health and well being of subsequent generations. Given the plethora of studies associating damaged sperm DNA with impaired fertility at every milestone, a more cautious approach would be to select sperm with genomic integrity.

13.6 Opportunities and Challenges – The Establishment of Clinical Thresholds and the Integration of DNA Testing into Clinical Practice Since ART, in particular ICSI, facilitates reproduction using sperm that would not achieve a pregnancy spontaneously, it is important to assess the quality of paternal genetic material and to establish criteria by which to choose appropriate cohorts of sperm. Assisted reproduction is expensive financially and emotionally, highly invasive and the long-term consequences remain unknown. Therefore, couples justifiably want to know the likelihood of success before embarking on a treatment cycle. These patients have a right to the fullest information that we can provide and scientists need to provide reliable tests for the clinician to give couples quantitative estimates of their chances of a pregnancy from their treatment. Two challenges face the scientists working in the field of ART. The first is to establish robust DNA tests with high prognostic strength and the second is to persuade the clinicians and managers of fertility centres that the inclusion of such testing in male infertility will be beneficial to patient and centre alike. At present there is no consensus as to the best test to use whether it be the alkaline


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Comet, SCSA or TUNEL. This will require international collaboration to standardise protocols, patient groups, tests and scientific and clinical outcome parameters. Ideally this should be facilitated by the formation of a subcommittee within a recognised professional society such as European Society of Human Reproduction and Embryology (ESHRE) to oversee the validation studies. This precedence was set by the establishment of a Special Interest group in 1996121 whose remit was to establish guidance on sperm-function tests prior to the discovery of sperm DNA testing. The development of a similar group to assess sperm DNA tests was proposed by the working group at the MaleMediated Developmental Toxicity conference in 2003;64 to our knowledge, it has not yet been implemented. The formation of such groups and collaborations should be considered a high priority.

Acknowledgements The authors would like to thank Mrs Margaret Kennedy, Biomedical Scientist, Andrology Laboratory, Royal Jubilee Maternity Service, Belfast and the staff of the Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, for their contributions to this work. The authors thank their sponsors The Wellcome Trust, The Fertility Research Trust, and Northern Ireland Research and Development Office without whose support these studies would not have been possible.

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91. A. Ahmadi and S. C. Ng, Developmental capacity of damaged spermatozoa, Hum. Reprod., 1999, 14(9), 2279–85. 92. P. Braude, V. Bolton and S. Moore, Human gene expression first occurs between the four- and eight-cell stages of preimplantation development, Nature, 1988, 332(6163), 459–61. 93. R. J. Aitken, Founders’ Lecture. Human spermatozoa: fruits of creation, seeds of doubt, Reprod. Fertil. Dev., 2004, 16(7), 655–64. 94. P. Devroey, J. Liu, Z. Nagy, A. Goossens, H. Tournaye, M. Camus, A. Van Steirteghem and S. Silber, Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia, Hum. Reprod., 1995, 10(6), 1457–60. 95. J. Tesarik, E. Greco and C. Mendoza, Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation, Hum. Reprod., 2004, 19(3), 611–5. 96. D. Sakkas, F. Urner, D. Bizzaro, G. Manicardi, P. G. Bianchi, Y. Shoukir and A. Campana, Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development, Hum. Reprod., 1998, 13(Suppl 4), 11–9. 97. J. G. Sun, A. Jurisicova and R. F. Casper, Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro, Biol. Reprod., 1997, 56, 602–607. 98. J. Erenpreiss, M. Bungum, M. Spano, S. Elzanaty, J. Orbidans and A. Giwercman, Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications, Hum. Reprod., 2006, 21(8), 2061–4. 99. R. A. Saleh, A. Agarwal, E. A. Nada, M. H. El-Tonsy, R. K. Sharma, A. Meyer, D. R. Nelson and A. J. Thomas, Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility, Fertil. Steril., 2003, 79(Suppl 3), 1597–605. 100. M. Bungum, P. Humaidan, M. Spano, K. Jepson, L. Bungum and A. Giwercman, The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI, Hum. Reprod., 2004, 19(6), 1401–8. 101. M. R. Virro, K. L. Larson-Cook and D. P. Evenson, Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development and ongoing pregnancy in In-Vitro fertilization and intracytoplasmic sperm injection cycles, Fertil. Steril., 2004, 81(5), 1289–95. 102. M. Spano, J. P. Bonde, H. I. Hjollund, H. A. Kolstad, E. Cordelli and G. Leter, Sperm chromatin damage impairs human fertility, The Danish First Pregnancy Planner Study Team, Fertil. Steril., 2000, 73(1), 43–50. 103. I. Bar-Hava, J. Ashkenazi, M. Shelef, A. Schwartz, M. Brengauz, D. Feldberg, R. Orvieto and Z. Ben-Rafael, Morphology and clinical outcomes of embryos after in vitro fertilization are superior to those after intracytoplasmic sperm injection, Fertil. Steril., 1997, 68(4), 653–7. 104. A. Olshan and D. Mattison, Male Mediated Developmental Toxicity, Plenum Press, New York, 1994.


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105. B. Robaire and B. F. Hales, The male germ cell as a target for drug and toxicant action, In: The male gamete: From basic science to clinical applications, ed. C. Gagnon. Cache River Press, Vienna, IL, USA, 1999, 469–474. 106. J. M. Trasler and T. Doerksen, Teratogen update: paternal exposuresreproductive risks, Teratology, 1999, 60(3), 161–72. 107. J. M. Trasler, B. F. Hales and B. Robaire, Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny, Biol. Reprod., 1986, 34(2), 275–83. 108. C. J. Smith, T. A. Perfetti, M. A. Mullens, A. Rodgman and D. J. Doolittle, ‘‘IARC group 2B Carcinogens’’ reported in cigarette mainstream smoke, Food Chem. Toxicol., 2000, 38(9), 825–48. 109. T. Sorahan, P. Prior, R. J. Lancashire, S. P. Faux, M. A. Hulten, I. M. Peck and A. M. Stewart, Childhood cancer and parental use of tobacco: deaths from 1971 to 1976, Br. J. Cancer, 1997, 76(11), 1525–31. 110. M. Nijs and W. Ombelet, Intracytoplasmic sperm injection in assisted reproductive technology: an evaluation, Hum. Fertil. (Camb.), 2000, 3(3), 221–225. 111. W. R. Edirisinghe, S. M. Junk, P. L. Matson and J. L. Yovich, Changes in motility patterns during in vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection, Hum. Reprod., 1996, 11(11), 2474–6. 112. B. Balaban, B. Urman, A. Sertac, C. Alatas, S. Aksoy, R. Mercan and A. Nuhoglu, In Vitro culture of spermatozoa induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection, Hum. Reprod., 1999, 14(11), 2808–11. 113. S. Emiliani, M. Van den Bergh, A. S. Vannin, J. Biramane, M. Verdoodt and Y. Englert, Increased sperm motility after in vitro culture of testicular biopsies from obstructive azoospermic patients results in better post-thaw recovery rate, Hum. Reprod., 2000, 15(11), 2371–4. 114. Y. Hu, W. S. Maxson, D. I. Hoffman, S. J. Ory, M. R. Licht and S. Eager, Clinical application of intracytoplasmic sperm injection using in vitro cultured testicular spermatozoa obtained the day before egg retrieval, Fertil Steril, 1999, 72(4), 666–9. 115. L. H. Dalzell, C. M. McVicar, N. McClure, D. Lutton and S. E. Lewis, Effects of short and long incubations on DNA fragmentation of testicular sperm, Fertil. Steril., 2004, 82(5), 1443–5. 116. W. V. Holt, Basic aspects of frozen storage of semen, Anim. Reprod. Sci., 2000, 62(1–3), 3–22. 117. S. J. Silber, A. C. Van Steirteghem, J. Liu, Z. Nagy, H. Tournaye and P. Devroey, High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy, Hum. Reprod., 1995, 10(1), 148–52.

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118. V. Abdelmassih, J. P. Balmaceda, J. Tesarik, R. Abdelmassih and Z. P. Nagy, Relationship between time period after vasectomy and the reproductive capacity of sperm obtained by epidydimal aspiration, Hum. Reprod., 2002, 17(3), 736–40. 119. C. M. McVicar, D. A. O’Neill, N. McClure, B. Clements, S. McCullough and S. E. Lewis, Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI, Hum. Reprod., 2005, 20(10), 2795–800. 120. D. A. O’Neill, C. M. McVicar, N. McClure, P. Maxwell, I. Cooke, K. M. Pogue and S. E. Lewis, Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis, Fertil. Steril., 2007, 87(4), 834–41. 121. ESHRE Andrology Special Interest Group andrology: Consensus workshop on advanced diagnostic andrology techniques. Hum. Reprod., 1996. 11(7), 1463–1479.

CHAPTER 14

The Comet Assay in Sperm – Assessing Genotoxins in Male Germ Cells ADOLF BAUMGARTNER, EDUARDO CEMELI, JULIAN LAUBENTHAL AND DIANA ANDERSON* University of Bradford, Division of Biomedical Sciences, Richmond Road, Bradford, BD7 1DP, United Kingdom

14.1 Introduction We are constantly confronted with an increasing number of potentially harmful toxic/genotoxic chemicals. Confounding factors such as our lifestyle, the environment in which we live, medical treatments and our innate susceptibility due to our genetic make-up has also to be taken into account. Some of these chemical assaults and stress factors can be detrimental to cells, in particular to our genome, leading to an increase in mutations. Sometimes, such mutations could even give rise to cancer. Hence, there is concern not only about damage to our somatic cells but also damage to our germ cells, i.e. reproductive cells, as they pass genetic material on to our progeny in the next and successive generations. Therefore, the DNA integrity of germ cells is of crucial importance. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is the Comet or single-cell gel electrophoresis (SCGE) assay. The following chapter will provide an overview of the use of the Comet assay utilising sperm or testicular cells in reproductive toxicology. This includes *



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considerations of damage assessed by protocol modification, cryopreservation versus the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro Comet assay studies employing sperm as well as a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, the examination of sperm as mature male germ cells in humans seems to be the easier and logical choice for research and for testing possible detrimental effects serving reproductive toxicological purposes. Consequently, there is a growing interest in the evaluation of effects of genotoxins on male germ cells and the sperm Comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins, presumed or proven to damage the DNA, to be reliably determined.

14.2 Single-Cell Gel Electrophoresis In 1984, Ostling and Johanson published a method using microgel electrophoresis of immobilised cells lysed at high salt concentrations.1 When they applied an electrophoretic field with pH conditions less than pH 10, ‘‘tails’’ were observed where some DNA from the nucleus migrated faster than the rest of the nuclear DNA. The basic principles of this so-called Comet assay were derived from previous results, which characterised the nuclear structure of lysed cells as containing looped superhelical DNA being attached to a nuclear scaffold comprised of proteins and RNA.2,3 Alkaline denaturation at a higher pH and DNA unwinding were incorporated later and seemed to be an important step in detecting DNA damage.4 This allowed, at a pH of Z 13, the detection of doublestrand breaks (DSB), single-strand breaks (SSB) and alkali-labile sites (ALS). The induced damage forming the tail of the comet consisted mainly of singlestranded DNA,5 most likely originating from predominant relaxation of supercoiled loops, rather than alkaline unwinding. Nevertheless, separation of both DNA strands, i.e. unwinding, occurs under alkaline conditions. By choosing different pH conditions for electrophoresis and the preceding incubation, different damage types and different levels of sensitivity can be assessed. Under neutral conditions (pH 7–9) almost exclusively DSB can be detected by merely subjecting lysed cell nuclei to an electrophoretic field at neutral pH.6,7 Although, some SSB due to the relaxation of supercoiled loops containing the breaks might also contribute to the observed comet.8 Alternatively, under alkaline conditions DSB and SSB (at around pH 12.3 within a range of pH 12.0 to 12.5) and additionally ALS (above pH 12.6 up to pH Z 13) can be visualised resulting in increased DNA migration in the electrophoretic field.9,10 The extensive applications of the Comet assay with its various modifications led to the establishment of guidelines for its use,10 which have been very valuable as a basis to standardise protocols when carrying out the Comet assay with a variety of cell types and tissues either in vivo or in vitro. However, these guidelines are not entirely applicable when investigating reproductive cells like sperm in the Comet assay, unless several adjustments are made – particularly to relax the highly compacted sperm chromatin structure.

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14.3 The Use of Sperm with the Comet Assay For the compilation of toxicological data on genotoxins that target and affect the reproductive system, it is essential to assess germ cells for DNA damage and DNA integrity. It is not yet known if lymphocytes11 can be used as surrogates instead of mature germ cells for the evaluation of reproductive genotoxins. Thus, especially sperm as mature male germ cells have several major advantages when compared to other reproductive cells: provided ethical approval has been obtained, sampling is noninvasive and fairly easy, and quite a large number of cells can be collected at one sampling time. Samples from a wide age range are possible. However, not only male germ cells have been used, the Comet assay has also been carried out with mammalian oöcytes12 and embryos.13

14.3.1

Human Sperm

During mammalian spermiogenesis, the last part of spermatogenesis,14 the postmeiotic haploid spermatocytes undergo major morphological changes when the genome is repacked and compacted to form spermatids and finally spermatozoa. Besides the loss of most of the cytosol and the formation of a tail, the spermatids’ nuclear chromatin condenses to a very tightly packed, crystalline-like structure initiated by an exchange of the histones by protamines.15,16 These protamines covalently stabilise the DNA through the formation of intraand intermolecular disulfide bonds.17,18 During the chromatin condensation and compaction process, about 85% of the histone-bound DNA in human sperm is transformed into compact nucleoprotamine chromatin.19 Mammalian sperm chromatin is approximately six times more compacted than metaphase chromosomes, even though it seems to be organised very specifically.20

14.3.2

Modifying Existing Comet Protocols for the Use of Sperm

Based on the protocol of Singh et al.4,21 various groups have adjusted the basic method of single-cell gel electrophoresis for the use of human or animal sperm. Essential to the recent protocols used22–27 is that sperm are immobilised within a layer of low melting point agarose (0.5–1%) and spread out onto dry agarosecoated slides. An optional cover layer of agarose may serve as protection of the cell-containing layer. Subsequently, in the lysis step, cell membrane, cytosol and nuclear membrane are removed via incubation in lysing buffer (100 mM EDTA, 10 mM Tris, pH 10) containing a high concentration of salt (2.5 M NaCl) and a nonionic detergent (1% Triton X-100).4 The use of 10% DMSO in the lysing solution varies; however, it can be added as a protectant against free radicals within the lysing solution. Because of the highly compacted chromatin human sperm, nuclei have to be decondensed by incubation in 4–10 mM dithiothreitol (DTT)21,28 and/or 0.05–0.1 mg/ml proteinase K (PK)29 or 10 mg/ml RNase.24 The decondensation procedure of the sperm chromatin is known to vary with temperature, length and strength of incubation depending on the species,

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e.g. when compared to mice. After lysis and equilibration in electrophoresis buffer, the agarose-embedded nuclei are subject to electrophoresis, which can be performed with different pH conditions achieving detection of different sensitivities of DNA damage.31 The layer of buffer above the slides should be around 1–2 mm, resulting in a current of approximately 300 mA (for alkaline buffer, pH Z 3) at 0.7–0.9 V/cm. Depending on the pH of the electrophoresis buffer, a neutralisation step might be necessary. In order to stain the nuclei for microscopic examination various fluorescent dyes can be used, such as ethidium bromide, YOYO-1, SYBR Green or SYBR Gold. The Comet assay has also been successfully applied to previously dried and methanol-fixed sperm before applying the agarose.32,33 This brief itemised protocol for the alkaline Comet assay on sperm is an example of a current version of this assay: - Resuspend treated or untreated sperm in low melting point agarose (in PBS) to reach a final concentration of 1%. - Spread 100 ml of this agarose-cell suspension onto a dry agarose-coated (1% in water) glass slide and let it set (apply a coverslip, preferably done on a cold surface). - Cover the above layer with 100 ml low melting point agarose and let it set (apply a coverslip, preferably carried out on a cold surface, for approx. 5 min). - Incubate the slides for 60 min at 4 1C in cold lysing solution containing dithiothreitol (DTT) [2.5 M NaCl,100 mM EDTA,10 mM Tris, pH 10, and 10 mM DTT]. - Transfer the slides into the second cold lysing solution containing proteinase K (PK) and incubate for 60 min at 4 1C [2.5 M NaCl,100 mM EDTA, 10 mM Tris, pH 10, and 0.05 mg/ml PK]. - Place the slides onto a tray from an electrophoresis unit and preincubate for 20 min at 4 1C (unwinding) in cold alkaline electrophoresis buffer [1 mM EDTA, 300 mM NaOH, pH 13.5]. - Run the electrophoresis for 20 min at 4 1C at a constant voltage of 25 V/ cm. Adjust the current to 290–300 mA at the start of electrophoresis by adding or removing electrophoresis buffer in the tank. - Drain and neutralise the slides three times for about 5 min by covering them with neutralising buffer [400 mM Tris, pH 7.5]. - Code the slides and stain them with 60 ml of 20 mg/ml ethidium bromide. - After applying coverslips, assess the slides under a microscope with a fluorescent attachment.

14.3.3

Sperm DNA and the Comet Assay

For the sperm Comet assay, decondensation of the highly condensed sperm DNA is required prior to unwinding and electrophoresis.21 Using alkaline electrophoresis conditions at pH Z 13, human and mouse sperm yield surprisingly high amounts of SSB (106–107 per genome), mostly due to ALS.


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Neither human lymphocytes under alkaline conditions, nor sperm under neutral conditions show these DNA breaks, which may represent functional characteristics but not pre-existing SSB.21 In addition to intracellular processes like DNA replication and DNA repair, which utilise DNA-nicking enzymes34,35 during parts of spermatogenesis, the reason for this elevated number of ALS seems to be the high degree of chromatin condensation.21,36 Under alkaline conditions, nicks in the DNA seem to provide a starting point for DNA unwinding by transforming the breaks into single strands.37,38 Published data suggest that sperm nuclei compared to leucocytes contain more than double the amount of SSB.39 More importantly, sperm chromatin is nine-fold more enriched in single-stranded segments – potentially prone to becoming ALS. Due to the higher degree of compaction, those partially denatured sections may be the result of elevated torsional stress of DNA loops.39 The susceptibility of human sperm to alkaline DNA denaturation seems to be strongly correlated with DNA-strand breaks indicating an important physiological relevance in terms of sperm quality and fertility.40 Fertile sperm tend to be more resistant to chemically induced DNA breakage than sperm from infertile men,41 which makes carrying out semen analysis according to WHO criteria42 as well as completing a reproductive questionnaire for the donors, an absolute prerequisite. As mature sperm lack DNA-repair capacity43 three potential mechanisms, which may be independently or codependently accountable for basic sperm damage, have been identified.44 These involve (a) defective chromatin condensation during spermiogenesis,35,45,46 (b) apoptotic events during spermatogenesis, epididymal maturation or within the ejaculate47,48 as well as (c) oxidative stress from reactive oxygen species.26,49 During the first stages of spermatogenesis, many toxicants do not primarily cause strand breaks, but damage the DNA by crosslinking50 or introducing AP (apurinic or apyrimidinic) sites,51 which are alkali labile and develop into SSB under alkaline conditions. Intermediates as well as base-free positions in the DNA introduced by glycosylases through base excision repair52 are just two examples of detectable ALS. A high innate activity of enzymes involved in rapid excision repair, however, can also create high levels of incision-related breaks in the Comet assay and therefore lead to a larger comet tail.53 When performing the Comet assay it has to be taken into account that crosslinked DNA, in contrast to other DNA damage, inhibits DNA migration in the electrophoretic field by stabilising the DNA.54,55

14.3.4

The Sperm Comet Assay and the Use of Repair Enzymes

When assessing DNA breakage with the Comet assay the apparent damage above the negative control originates from the genotoxic compound of interest and/or yet unrepaired DNA breaks generated by DNA repair enzymes as a consequence of the chemical insult. DNA-repair mechanisms,56 namely nucleotide excision repair (NER) and base excision repair (BER), involve the enzymatic generation of DNA nicks to replace misleading events such as DNA

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adducts or oxidised nucleotides. This concept of repair does not apply to repair-deficient late spermatids and sperm. However, nicks from DNA repair events of earlier repair-proficient stages of spermatogenesis could still remain. Two repair enzymes, formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (EndoIII), which recognise and remove oxidised pyrimidines and purines and subsequently create nicks in the DNA, have been commonly introduced as an addition to the general Comet assay for the detection of this kind of DNA damage.58–60 Despite the fact that such a modified Comet assay is currently used as a biomarker for oxidative DNA damage,11,60,61 scant literature data are available on the detection of bulky DNA adducts and oxidised bases in male germ cells. Cellular repair studies showed that human testicular cells have limited capacity to repair oxidative lesions, which were recognised by Fpg or Nth, an homologue of EndoIII, using the alkaline Comet assay.62 Additionally, Cemeli and colleagues63 have found that the repair enzymes Fpg and EndoIII are not a valid option for detecting oxidised bases after chemical treatment in sperm in vitro, which might be due to the occurrence of DNA breaks generated by the chemical itself and by the enzymatic action, thus confounding the results. However, these enzymes were able to clearly detect oxidised bases in negative controls, namely untreated sperm, implying new possibilities for human male biomonitoring63 since this has not been carried out before. Repair of bulky adducts and oxidised bases does occur in somatic cells as well as in germ cells other than late spermatids and mature spermatozoa. For this reason, the sperm Comet assay per se is not sensitive enough to detect such DNA lesions in sperm,64 unlike chemically induced structural DNA breaks via radical species, which are readily detected with the sperm Comet assay.22,65 To overcome this lack of sensitivity, Cordelli and colleagues64 developed a modified sperm Comet assay based on the addition of a protein extract from HeLa cells, containing various repair enzymes, to agarose-embedded bull sperm. In the presence of the HeLa cell extract a clear-cut dose response was observed when treating with alkylating chemicals like methanesulfonate and melphalan, which was not seen in the absence of the extract.

14.3.5

Assessing the Sperm Comet

The Comet assay with human sperm is able to identify low levels of DNA damage7,26,66–68 even if the scored comet images are more heterogeneous than those seen with lymphocytes. The preferred comet parameter to describe the observed DNA damage is the tail moment or Olive tail moment, which provides the most stable estimate for DNA damage because it has a larger degree of uniformity in quantile dispersions.69 However, for sperm, additionally the % head DNA is used due to high levels of basic/baseline DNA damage. Untreated sperm only show about 80% head DNA.41,70,71 Using image analysis in combination with the Comet assay, apoptotic or necrotic cells can also be identified due to their small or nonexistent head and large diffuse comet tails. These


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comet images are termed ghosts or ‘‘hedgehogs’’ when looking at lymphocytes with excessive damage.72 For sperm, such highly damaged cells showing extensive DNA fragmentation cannot be excluded.73 There is still an ongoing debate about these ghost cells arising from apoptotic events and showing extensive damage. They do not seem to originate from early stages of apoptotic events but rather from dead cells. The extensive fragmentation into low molecular weight DNA fragments seem to be associated with late apoptotic events.74 Thus, it has been suggested that these ghost cells cannot be used for the interpretation of induced genotoxic DNA damage when a risk from apoptosis is present.75 But other findings suggest that results obtained with the Comet assay are not confounded by concomitant processes leading to apoptosis.76 The mutagen-induced DNA-damage measured with the Comet assay appears to be the principal cause for the induced damage77 making the Comet assay a valuable tool for evaluating agent-induced DNA damage. However, when late apoptotic events are intentionally targeted with a highly sensitive method like the Comet assay, it has been suggested to omit the electrophoresis step for quantifying the late apoptotic fraction.78

14.3.6

Comet-FISH on Sperm

Comet-FISH, the combination of the Comet assay and fluorescence in situ hybridisation (FISH)79 comparatively assesses the overall DNA damage and genetic instability (Comet assay) along with chromosomal abnormalities at specific gene loci (FISH). Since its development80 around ten laboratories have published research data using the Comet-FISH technique on somatic cells focusing on chromosome specific targets (e.g. telomeres, centromeres or whole chromosomes),80,81 strand breaks within the p53 gene,82 SSB induced by UV-A light,83 region-specific repair activities,84,85 transcription-coupled DNA repair,86 fragility of telomeres (using peptide nucleic acid probes)81 and also on genetic instability in breast cancer or oropharnyx cancer cells.87,88 In comparison, the hybridisation procedure differs in four major points within the ten laboratories using Comet-FISH: (a) thermal denaturation at 70–74 1C vs. alkali denaturation with 0.3–0.5 M NaOH, (b) hybridisation signal detection with enzyme-coupled antibodies, e.g. 2-hydroxy-3-naphthoic acid-2 0 -phenyl-anilide phosphate (HNPP) fluorescence-enhancing system, vs. direct fluorochromelabelled DNA probes vs. signal amplification via antibody sandwiches, (c) posthybridisation washing with 2 SSC at 70–72 1C vs. 50% formamide/ 2 SSC not exceeding 37 1C and (d) dehydration in 100% ethanol for three days up to several weeks vs. dehydration for 30–60 min. So far, no data have been published utilising Comet-FISH on sperm. However, the first steps have been done to perform Comet-FISH on sperm (J. Laubenthal, personal communication) mainly focusing on the detection of site-specific DNA breaks at certain loci in the germ-line, which may contribute as initial events for the onset of hereditary disease in somatic cells of the next generation. In brief, the sperm Comet assay22 has been slightly modified to

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adjust for included FISH steps, thus, not using the optional cover layer of agarose and using normal melting point agarose instead of low melting point agarose to allow thermal denaturation of the comet head and tail DNA. Comet-FISH on sperm can be a promising technique for environmental and reproductive sciences focusing on DNA damage as well as chromosomal loci within the haploid genome with a high spatial resolution. Using DNAstretching techniques for the nuclear sperm chromatin with subsequent fibreFISH89 might prove helpful for the sperm Comet-FISH and is currently being investigated (J. Laubenthal, personal communication).

14.3.7

Cryopreserved versus Fresh Sperm

For larger studies, it is more practical to evaluate the DNA integrity of sperm in cryopreserved semen rather than fresh sperm;90 however, freezing living cells can cause unfavourable and damaging effects due to ice crystal formation and/ or severe osmotic changes. There are reports that freezing might affect chromatin structure and sperm morphology.91 Also, DNA damage from cryopreservation in semen from infertile men has been detected using the alkaline Comet assay.92 Moreover, the sperm chromatin structure assay (SCSA) revealed that the sperm quality might deteriorate upon cryopreservation93 and cryopreservation of testicular spermatozoa by itself may reduce pregnancy rates.94 The freezing-thawing process affects the DNA integrity of boar spermatozoa when assessing the post-thaw quality of boar semen using the neutral Comet assay95 as well as SCSA.96 Conventional cryopreservation and storage in liquid nitrogen caused DNA damage in thawed macaque sperm but with the exception of the motile sperm fraction.97 Freezing sperm in seminal plasma improved post-thaw motility and DNA integrity.98 However, a vital role for the integrity of the cell membrane is the way of freezing sperm in terms of speed, stepwise changes in temperature and the cryopreservative used.99,100 In another study, no differences were found with the Comet assay when fresh and frozen human sperm were compared.101 For the use of sperm with the Comet assay in reproductive toxicology, flash freezing of aliquoted semen samples in liquid nitrogen seems to be the method of choice, which most closely resembles reproducibly results obtained with fresh sperm.90

14.3.8

Viability Considerations

For the Comet assay, it is imperative to test cells for viability after treatment to exclude cytotoxic effects. A viability of greater than 75% should be produced for the maximum concentration of a tested compound to avoid a false-positive response due to cytotoxicity.50 It was suggested that when only 50 cells are being scored, cell viability should be above 95%.102 It should also be noted that it is not feasible to measure cell viability on cells from solid tissues due to the disruption of the cell membranes when separating the cells.103 A variety of viability tests for germ cells exist and are also used for sperm, e.g. the Trypan


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22,104,105

blue exclusion test. As this exclusion test only indicates an intact membrane and not necessarily a viable cell, genuine viability tests like the dual fluorescence method with SYBR-14/Propidium iodide staining are more suitable.106,107 Especially for sperm, viability can vary and is considered normal under WHO criteria (Appendices IA and IV) when above 50% for Trypan Blue exclusion and 460% for the hyperosmotic swelling test.42 It has also been shown that freezing sperm increases the rigidity of the membrane and decreases the viability to values of 45% using an eosin-nigrosin viability test.108 Thus, for the in vitro sperm Comet assay, it is perhaps necessary to determine the cytotoxicity of chemicals in parallel via the viability of lymphocytes according to published guidelines8 to ensure against possible artefacts. In general, untreated cells show background levels of DNA damage of around 0–10% DNA in the tail depending on the cell type8 and approximately 20% DNA in the tail for sperm.41

14.3.9

Statistical Analysis

The number of sperm cells evaluated with the Comet assay must represent a balance between accuracy and precision. It has been suggested in the Comet guidelines to blindly score at least 50 cells per culture or individual on independently coded slides with 25 cells scored per duplicate.10 However, published studies display a large variability of approaches to the number of cells scored. For instance, 100 cells were scored in three independent repeats,109 100 cells per sample (based on 2 duplicates)110 or 150 cells per sample (50 cells per slide).111 It is crucial to understand the hierarchy that accounts for many experimental designs where a number of cells are scored on a number of slides for each sample and that the sample rather than the cell is the unit for statistical analysis.112 Otherwise, the degrees of freedom will vary greatly and result in a type-2 statistical error. It also has to be taken into consideration that in vivo experiments can result in lower sensitivity than expected due to the internal variability within the groups. Despite the higher sensitivity observed in vitro, variability might be overlooked if no repeats or duplicates have been included in the experimental design. It is important to identify an appropriate number of individuals for in vivo studies or repeat experiments in vitro to perform a suitable statistical analysis. Finally, it is worth mentioning that in addition to the statistical significance of a finding, it is necessary to understand and interpret the biological relevance of the data obtained.

14.4 Utilising Male Germ Cells with the Comet Assay The next two sections summarise the in vivo and in vitro usage of the Comet assay with male germ cells under alkaline (pH 10 up to pH Z 13) and neutral (pH 7 up to pH 9) conditions in genotoxicology and biomonitoring. A more detailed list of studies using the Comet assay on human testicular cells, particularly on sperm, as well as on male germ cells from at least 13 animal species can be found in Tables 14.1 and 14.2, respectively.

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N A1 A2 A3 A A1 A3 N N2 ¼ A1 N1 N1 N1 A1 A2 N1 A1 ¼ N3 N1 N1 A2 A2 N2 A1 A1 A1 A1 A1

Comet assay In In In In In In In In In In In In In In In In In In In In In In In In In In In In In In

vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo

Treatment Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

Species Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm

Targeted cells

In vivo and in vitro Comet assay responses in human sperm or testicular cells.

Abu-Hassan et al., 2006 Agbaje et al., 2007 71 Ahmad et al., 2007 40 Aravindan et al., 1997 150 Belcheva et al., 2004 151 Bertolla et al., 2006 110 Bian et al., 2004 152 Caglar et al., 2007 153 Chatterjee et al., 2000 154 Ding et al., 2003 155 Donnelly et al., 2000 24 Duty et al., 2003 156 Hauser et al., 2003 157 Hauser et al., 2007 67 Hughes et al., 1997 26 Irvine et al., 2000 158 Larson et al., 2001 159 Lewis et al., 2004 160 Lu et al., 2002 161 McVicar et al., 2004 127 Meeker et al., 2004 162 Meeker et al., 2008 163 Migliore et al., 2002 164 Migliore et al., 2006 129 Morris et al., 2002 165 Esfahani et al., 2005 166 O’Connell et al., 2002 167 O’Connell et al., 2002 168 O’Connell et al., 2003 169 O’Donovan, 2005

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Publication

Table 14.1

Untreated Untreated Untreated Untreated Cigarette smoke Untreated Fenvalerate Untreated Fludarabine Hypothermia Untreated Phthalates PCBs, HCB, DDT, DDE Phthalate Untreated Untreated Untreated Untreated Untreated Untreated Chlorpyrifos, carbaryl Untreated Styrene Styrene Untreated Untreated Untreated Untreated Untreated Chemotherapy

Toxicant

340 Chapter 14

Anderson et al., 1997

23

Arabi, 2004 Baumgartner et al., 2004 182 Bennetts et al., 2008 132 Cemeli et al., 2004

181

22

180

Anderson et al., 1997 Anderson et al., 1998 Anderson et al., 2003

131

65

171

O’Neill et al., 2007 Sakkas et al., 2002 172 Schmid et al., 2003 128 Schmid et al., 2007 173 Shen and Ong, 2000 174 Singh et al., 2003 175 Song et al., 2005 176 Steele et al., 1999 27 Tomsu et al., 2002 44 Trisini et al., 2004 130 Verit et al., 2006 126 Xu et al., 2003 177 Xu et al., 2007 178 Aitken et al., 1998 111 Ambrosini et al., 2006 179 Anderson et al., 1997

170

A2 A1 A1 A1

A1 A1 A1

A1

A1 A3, N3 A1 A1, N2 A3 N1 ¼ A1 A1 N1 A2 A3 ¼ A2 A1 A1 vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vivo vitro vitro vitro

In In In In

vitro vitro vitro vitro

In vitro In vitro In vitro

In vitro

In In In In In In In In In In In In In In In In

Human Human Human Human

Human Human Human

Human

Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

Sperm Sperm Sperm Sperm

Sperm Sperm Sperm

Sperm

Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm

Untreated Untreated Untreated Untreated Untreated Untreated Benzene Untreated Untreated Untreated Untreated Acrylonitrile Untreated Hydrogen peroxide Oleoylethanolamide Oestrogens, dibromochloropropane, butadiene metabolites Oestrogens, lead, dibromochloropropane, ethylene glycol monoethyl ether, butadiene metabolites Trp, IQ, antioxidants Trp, IQ, PhiP, flavonoids Hydrogen peroxide, SOD, catalase, vitamin C Nicotine Doxorubicin Oestrogenic compounds Hydrogen peroxide, oestrogen-like compounds, flavonoids

The Comet Assay in Sperm – Assessing Genotoxins in Male Germ Cells 341

186

185

184

Donnelly et al., 2000

Donnelly et al., 1999

Ding et al., 2004 Dobrzynska et al., 2004

192

Singh and Stephens, 1998 Song et al., 2002 101 Steele et al., 2000 94 Thompson-Cree et al., 2003 193 Van Kooij et al., 2004 194 Xu et al., 2000

7

Hughes et al., 1999 Li et al., 2006 Li et al., 2007

McKelvey-Martin et al., 1997a Sierens et al., 2002

191

41

190

189

188

98

Donnelly et al., 2001 Donnelly et al., 2001 90 Duty et al., 2002 70 Hughes et al., 1996 187 Hughes et al., 1998

92

(Continued ).

Chan et al., 2001 Connelly et al., 2001

183

32

33

Publication

Table 14.1

N1 ¼ A1 A1 N A3

A1 A2

A1 N2 ¼

A1 A1 N1 A1 A1

A1

A1

¼ A1

N3 N3

Comet assay

vitro vitro vitro vitro vitro

In In In In In In

vitro vitro vitro vitro vitro vitro

In vitro In vitro

In vitro In vitro In vitro

In In In In In

In vitro

In vitro

In vitro In vitro

In vitro In vitro

Treatment

Human Human Human Human Human Human

Human Human

Human Human Human

Human Human Human Human Human

Human

Human

Human Human

Human Human

Species

Sperm Sperm Sperm Sperm Sperm Sperm

Sperm Sperm

Sperm Sperm Sperm

Sperm Sperm Sperm Sperm Sperm

Sperm

Sperm

Sperm Sperm

Sperm Sperm

Targeted cells

Hydrogen peroxide E6-E7 HPV DNA fragments Microwave radar Triiodothreonine, L-thyroxine, noradrenaline Vitamins C & E, hydrogen peroxide Hydrogen peroxide, glutathione, hypotaurine Freezing Freezing Freezing Hydrogen peroxide, X-rays Vitamins C & E, urate, acetyl cystein, X-rays Vitamins C & E, X-rays Hydrogen peroxide Freezing, cryoprotectants, antioxidants X-rays, hydrogen peroxide Hydrogen peroxide, isoflavones, vitamins C & E X-rays Freezing Freezing Freezing X-rays Hydrogen peroxide

Toxicant

342 Chapter 14

Olsen et al., 2003 Bjorge et al., 1996

Olsen et al., 2001

A1

A1 A1

¼ N1 A, N1

In vitro

In vivo In vitro

In vitro In vitro In vivo

Human, rat

Human Human Human, mouse Human, rat Human, rat Testicular cells

Testicular cells Testicular cells

Sperm Sperm Sperm Untreated 1,2-Dibromo-3-chloropropane, 4-nitroquinoline N-oxide, X-rays Methylmethane sulfonate

Sodium nitroprusside, zinc Holding at RT Untreated

Footnote: A list of publications that use the Comet assay on human sperm or testicular cells In vivo and/or In vitro. Electrophoresis preincubation (unwinding) and electrophoresis conditions: alkaline A (pH 10–13) [when detailed information was published: A1 (pH Z 13), A2 (pH 12–12.5), A3 (pH 10–11.9)]; neutral N (pH 7– 9) [detailed: N1 (pH 9), N2 (pH 8–8.5), N3 (pH 7–7.5)]; ‘‘¼‘‘indicates that the information is not accessible because the publication is printed in Chinese or Russian. a These publications use both in vivo and in vitro treatment.

52

197

62

21

196

Yang et al., 2004 Young et al., 2003 Singh et al., 1989

195


Dietrich et al., 2005

217

212

Baumber et al., 2005

Zhou et al., 2006b Zilli et al., 2003 213 Morse-Gaudio and Risley, 1994 214 Chen et al., 2002 215 Chen et al., 2003 216 Baumber et al., 2003

211

210

205

Boe-Hansen et al., 2005 Gloor et al., 2006 206 Bustos-Obregon and Goicochea, 2002 207 Labbe et al., 2001 208 Cabrita et al., 2005 209 Ciereszko et al., 2005

204

A1

A1 A2 A1 A1, N1 A1 A1

A1 vitro vitro vitro vitro vitro vitro

In vitro

In In In In In In

In vitro

In vivo In vitro In vitro

A1 A2 A1, N2

vitro vitro vitro vitro vitro vitro vitro vitro

In vitro In vitro In vivo

In In In In In In In In

Treatment

N2 N1 A1

¼ N2 N1 N1 N1 A3a A1 A2

199

Arabi and Heydarnejad, 2007 Fraser and Strzezek, 2004 95 Fraser and Strzezek, 2005 200 Fraser and Strzezek, 2007 201 Fraser and Strzezek, 2007 202 Jiang et al., 2007 203 Arabi, 2005 64 Cordelli et al., 2007

Comet assay

198

Horse

Fish Fish Frog Hamster Hamster Horse

Fish

Fish Fish Fish

Bull Cat Earthworm

Boar Boar Boar Boar Boar Boar Bull Bull

Species

In vivo and in vitro Comet assay responses in male animal germ cells.

Publication

Table 14.2

Sperm

Sperm Sperm Testicular cells Sperm Sperm Sperm

Sperm

Sperm Sperm Sperm

Sperm Sperm Male germ cells

Sperm Sperm Sperm Sperm Sperm Sperm Sperm Sperm

Targeted cells

Untreated Freezing Hydrogen peroxide, UV, bisazir Hydrogen peroxide, UV Duroquinone Freezing Teniposide (VM-26) NADPH NADPH, SOD Xanthine, xanthine oxidase, catalase, SOD, glutathione Vitamins C&E, catalase, SOD, glutathione

Cadmium Freezing Freezing Freezing Freezing Freezing Mercury chloride Methanesulfonate, melphalan Mechanical stress X-ray, freezing Parathion

Toxicant

344 Chapter 14

6

Villani et al., 2007 Zhang et al., 2001 Zhang et al., 2006

Haines et al., 1998

232

231

230

Laberge and Boissonneault, 2005 119 Leopardi et al., 2005 227 Marty et al., 1999 228 Osipov et al., 2002 229 Samanta et al., 2004

226

123

Garagna et al., 2005 Haines et al., 2001 25 Haines et al., 2002 225 Hong et al., 2005

120

222

Baulch et al., 2007 Brinkworth et al., 1998 223 Cho et al., 2003 121 Cordelli et al., 2003 224 Dobrzynska et al., 2005 122 Dobrzynska, 2005

30

97

Linfor and Meyers, 2002 Li et al., 2007 219 Legue et al., 2001 118 Altamirano-Lozano et al., 1996 220 Banks et al., 2005 221 Barber et al., 2006

218

A, N2

A1 ¼ ¼ In vitro

In vivo In vivo In vivo

vivo vivo vivo vivo

In In In In

A2 N1 ¼ A1

vivo vivo vivo vivo

vivo vivo vivo vivo vivo vivo

vitro vitro vivo vivo vivo vivo

In vivo

In In In In

In In In In In In

In In In In In In

A2, N2

A1 N2 N2 ¼

N2 A1 A1 A1 A1 A1

A1 A1 A1 A1 A2 A1

Mouse

Mouse Mouse Mouse

Mouse Mouse Mouse Mouse

Mouse

Mouse Mouse Mouse Mouse

Mouse Mouse Mouse Mouse Mouse Mouse

Horse Monkey Mouse Mouse Mouse Mouse

Sperm

Testicular cells Testicular cells Testicular cells

Sperm Sperm Sperm Testicular cells

Spermatids

Sperm Sperm Sperm Testicular cells

Sperm Sperm Sperm Testicular cells Sperm Sperm

Sperm Sperm Testicular cells Testicular cells Sperm Sperm

Sodium ortho-vanadate Untreated g-rays X-rays, RP-1 herb extract (Podophyllum hexandrum) Vanadyl sulfate Smoking Lead acetate, vitamin C, thiamin g-rays

Freezing Freezing X-rays, interleukins Vanadium pentoxide Heat Untreated F1 generation g-rays 1,3-butadiene Untreated X-rays X-rays, vincristine X-rays, cyclophosphamide, mitomycin C Bentazon X-rays, Indium-114m X-rays 50 Hz electromagnetic fields Untreated


Labaj et al., 2004b

Lazarova et al., 2004b

Lazarova et al., 2006b

242

243

Bjorge et al., 1995

Li et al., 2006 Atorino et al., 2001

Codrington et al., 2004) Delbes et al., 2007

241

134

240

239

125

124

235

Tang and Xuan, 2003 Zheng and Olive, 1997b 236 Garagna et al., 2001 237 Anderson et al., 1997 238 Gwo et al., 2003 66 Anderson et al., 1996

234

A1

A1

A1

A1

¼ A3

A2/N3 A2/N3

¼ A A1 A1 A3 A1

¼

Huang et al., 2003

Comet assay

233

(Continued ).

Publication

Table 14.2

vitro vitro vitro vitro vitro vivo

In vitro

In vitro

In vitro

In vitro

In vivo In vitro

In vivo In vivo

In In In In In In

In vitro

Treatment

Rat

Rat

Rat

Rat

Rat Rat

Rat Rat

Mouse Mouse Mouse, rabbit Mouse, rat Oyster Rat

Mouse

Species

Testicular cells

Testicular cells

Testicular cells

Testicular cells

Sperm Testicular cells

Sperm Sperm

Sperm Testicular cells Sperm Testicular cells Sperm Testicular cells

Testicular cells

Targeted cells

Hydrogen peroxide, lycium barbarum polysaccharides Carbon disulfide X-rays TCDD Butadiene metabolites Freezing Cyclophosphamide, ethyl methanesulfonate, bleomycin, ethylene glycol monoethyl ether Cyclophosphamide Bleomycin, etoposide, cis-platinum Cumene hydroperoxide Hydrogen peroxide, g-rays 1,2-Dibromo-3chloropropane Lignin, hydrogen peroxide, N-methyl-N 0 nitrosoguanine Carboxymethyl chitinglucan, hydrogen peroxide Carboxymethyl chitinglucan, methylene blue

Toxicant

346 Chapter 14

Kotlowska et al., 2007

Perrin et al., 2007 Wellejus et al., 2004

Lazarova et al., 2006

N2

A1 A1

A1

In vivo

In vitro In vitro

In vitro

Turkey

Rat Rat

Rat

Sperm

Spermato-cytes Testicular cells

Testicular cells

Hydrogen peroxide, Nnitrosomorpholine, methylene blue, benzo[a]pyrene g-rays 17alphaethinylestradiol Untreated

A list of publications that use the Comet assay on male animal germ cells In vivo and/or In vitro. Electrophoresis preincubation (unwinding) and electrophoresis conditions: alkaline A (pH 10–13) [when detailed information was published: A1 (pH Z 13), A2 (pH 12–12.5), A3 (pH 10–11.9)]; neutral N (pH 7–9) [detailed: N1 (pH 9), N2 (pH 8–8.5), N3 (pH 7–7.5)]; a separation by ‘‘/’’ indicates that two solutions with different pH have been used for preincubation and the electrophoresis; ‘‘¼’’indicates that the information is not accessible because the publication is printed in Chinese or Russian. a Within this publication the use of the neutral Comet assay has been described; however, in this table the assay has been grouped into the alkaline Comet assay because of the pH 10 buffer used for preincubation and electrophoresis. b These publications use both in vivo and in vitro treatment.

246

245

133

244


348

14.4.1

Chapter 14

In Vivo Comet Assay

The Comet assay is a well-established biomarker system for in vivo biomonitoring of occupational exposures. It is able to rapidly and sensitively test DNAdamaging genotoxins and confounding factors influencing responses.113 The alkaline version of the Comet assay with a pH Z 13 has become a reliable and an accepted assay for in vivo genotoxicological evaluations and has been approved by the UK Committee on Mutagenicity of Chemicals in Food, Consumer Products and Environment and US Food and Drug Administration,114 due to the development of standardised protocols.8,10,115,116 In the regulatory setting, the Comet assay is primarily employed as a very useful follow-up or supplementary in vivo test for mutagenic compounds, which have been shown to produce a positive response in in vitro mutagenicity tests and a negative response in the bone marrow assay, as it demonstrates several advantages over other in vivo indicator tests that are generally accepted. The Comet assay can be applied to virtually any organ and differentiated cell type (local genotoxicity), provided an acceptable and suitable cell preparation method exists, and it covers a broader spectrum of primary DNA lesions by evaluating single cells.117 It has been recommended116 that 100–150 cells per individual animal have to be evaluated in the in vivo Comet assay applications, depending on the number of animals per group. At least two dose levels are required to be tested: a high dose, which produces signs of toxicity, and a low dose (25–50% of the high dose). Due to undergoing rapid DNA repair in cells other than mature sperm, e.g. spermatocytes, SSB of primary lesions may only be short lived. Therefore, this kind of DNA damage could be missed by inadequate sampling times.117 Various studies facilitating the in vivo Comet assay on sperm or testicular cells have been done to toxicologically evaluate reprotoxins and genotoxins. A variety of toxicants has been investigated in vivo in mice including vanadium,118,119 herbicides like bentazon120 and X-rays.25,121–123 In rats, chemotherapeutic drugs like cyclophosphamide66,124 and bleomycin either on its own66 or in combination with etoposide and cis-platin125 have been tested with the Comet assay on sperm and testicular cells. Also, chemicals like ethyl methanesulfonate and the testicular toxin ethylene glycol monomethyl ether66 have been examined. The in vivo Comet assay has also been used with human sperm for evaluating sperm DNA damage of occupational exposure for toxicants like acrylonitrile,126 phthalates24 and pesticides such as fenvalerate.110 Also, monitoring populations for environmental exposure to carbaryl and chlorpyrifos, both pesticides, which appeared to be associated with increased DNA damage in human sperm, has been carried out.127 When monitoring populations it became evident that a positive correlation between age as well as caffeine intake and DNA damage could be observed in sperm.128 Regression analysis showed that DNA damage was positively associated with age (29–44 years), abnormal sperm and motility, and negatively associated with sperm concentration.129 It has been long known that the baseline DNA damage in human and mouse sperm in the Comet assay is high when compared to somatic cells due


349

21

to the presence of alkali-labile sites. Also, ejaculated sperm DNA is significantly more damaged than testicular sperm DNA.101 Studies comparing baseline DNA damage in sperm from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile men did not show a significant difference between the three groups. However, after a challenge with X-rays and hydrogen peroxide it was concluded that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.41,70 Irvine and coworkers26 stated that a significant proportion of infertile men had elevated levels of DNA damage in their ejaculated spermatozoa. Highly significant negative correlations were observed between DNA fragmentation and semen quality, particularly for sperm count. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage.26 Verit and coworkers130 did not find any relationship between sperm DNA damage and oxidative stress in normozoospermic infertile men and considered that the pathophysiology of idiopathic infertility cannot be explained by sperm DNA damage or seminal oxidative stress. An attempt was made to find associations between semen parameters and sperm DNA damage with the neutral Comet assay. Although associations between semen and Comet assay parameters were found, their magnitudes were weak, suggesting that the Comet assay provides additional independent information on sperm function.44

14.4.2

In Vitro Comet Assay

Studies with the in vitro Comet assay on sperm have mainly focused on the investigation of the potential genotoxic damage of compounds such as flavonoids (silymarin, myricetin, quercetin, kaempferol, rutin, and kaempferol-3rutinoside) and food mutagens (3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp) and 2-amino-3-methylimidazo-4,5-f)quinoline (IQ)) either on their own or in combination.131 Further research has been carried out on oestrogens (diethylstilbestrol, beta-estradiol, daidzein, genestein, equol and nonylphenyl) either on their own,65 combined with antioxidants (catalase, vitamin C, SOD)22 or combined with flavonoids (quercetin, kaempferol).132 Other toxicants investigated included X-rays,7 gamma-radiation,6 doxorubicin,23 lead sulfate, nitrate and acetate, dibromochloropropane, ethylene glycol monoethyl ether, 1,2-epoxybutene, and 1,2,3,4-diepoxybutane.65 All compounds produced positive responses in general, but ethylene glycol monoethyl ether produced a positive response only in sperm and not in peripheral lymphocytes. Similarly, the phytoestrogens, genistein and daidzein, were less responsive in the peripheral lymphocytes in the male than in the sperm. This may be due to greater sensitivity of mature spermatozoa because of their lack of repair.65 However, since damage was generally seen over a similar dose range, a one-to-one or a

350

Chapter 14

one-to-two ratio of somatic and germ cell damage was observed and this has implications for man for risk-assessment purposes.65,131 It was later concluded that human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.62 By contrast, the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells in conjunction with the Comet assay has also been reported.133 This revealed the presence of DNA-strand breaks in nontreated cells, whose numbers decreased with the duration of the culture, suggesting the involvement of DNA-repair mechanisms related to meiotic recombination. Besides repair capacity, it should also be taken into account that when using cells from testes for in vitro studies, various testicular cell types show differences in metabolic activation of chemical compounds.134 Anderson and colleagues131 believe that there are low levels of metabolic activity even in sperm because the heterocyclic amines normally requiring metabolic activation have shown positive responses.

14.5 The Sperm Comet Assay versus Other Assays Used in Reproductive Toxicology As sperm DNA integrity is essential for successful fertilisation and the subsequent embryo development135 several assays on spermatozoa have been developed in the last few years to evaluate DNA integrity and to determine DNA fragmentation.136 These include the sperm chromatin dispersion (SCD) and the DNA-breakage detection fluorescence in situ hybridisation (DBDFISH) assays, utilising like the Comet assay agarose-embedded cells but without applying an electrophoretic field.36,137 Other approaches like in situ nick translation (ISNT)26 and terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL)48 take advantage of enzymes, which are able to incorporate in situ marker-molecule-labelled deoxynucleotides into the DNA to detect DNA damage very accurately. Another well-known, highly efficient assay, the sperm chromatin structure assay (SCSA), avails the metachromatic dye acridine orange and flow cytometry to assess the ratio of single-stranded to double-stranded DNA in a large number of individual sperm.138,139 Despite the advantage of SCSA being a rapid, precise and objective measure of sperm DNA fragmentation,140 the Comet assay seems to be more selective as it can detect various types of DNA damage, like DSB, SSB, ALS as well as crosslinks, and advantageously only a few cells are needed for an exact evaluation.141 Nevertheless, the sperm Comet assay and SCSA measure DNA damage by different principles, but the conclusions arising from the data are similar.142 Focusing on DNA integrity of male germ cells in general but on different endpoints like detection of nicks in situ (e.g. TUNEL), chromatin dispersion pattern (e.g. SCD) or the difference between single-stranded and doublestranded DNA in sperm chromatin (e.g. SCSA) all these tests can perfectly corroborate findings when done in combination with the Comet assay, even if it seems to be the most sensitive assay.


351

14.6 Conclusions In humans, more than 80% of all structural aberrations occur de novo and are of paternal origin.143 Additionally to basic sperm damage,44 the DNA from reproductive cells may sustain even further damage from genotoxins due to lifestyle, environmental and medical exposure. Knowing this fact, it is of great importance to protect the integrity of our genome as effectively as possible. However, by only detecting major numerical and structural abnormalities, minor but potentially global chromosomal damage, which might play an important role in paternal genome abnormalities in miscarriage, is often underestimated.144 It is known that sperm DNA damage higher than 8% cannot be completely repaired in the zygote and might lead to impaired embryo development and early pregnancy loss.145 It is therefore very important to have a standardised assay in reproductive toxicology at hand, which can effectively target male germ cells. To assess DNA damage per se in reproductive cells, the single-cell gel electrophoresis (SCGE) or Comet assay has proven to be a reliable and rapid method,9 hence being the most sensitive way to detect DNA damage.146 The in vivo Comet assay on somatic cells is already in use for regulatory purposes, e.g. UK Guidance on a strategy for testing of chemicals for mutagenicity, and it is also considered for obtaining data on germ cell DNA damage.147 The importance of the sperm Comet assay as a relatively new technique providing a sensitive assessment of genetic damage seems to have become widely recognised. This can be seen in the various publications available on baseline and toxicology studies utilising male germ cells. Also, changes and sophisticated modifications during the last few years have improved the potential of the Comet assay even further, for example by introducing treatment with repair enzymes.60 Even though guidelines are available for the assessment of DNA damage in the Comet assay in somatic cells, unfortunately, no standardised Comet protocol for sperm is available so far. This also accounts for a wide range of related procedures like sperm storage, which may lead to different baseline damage. It is therefore crucial for future reproductive toxicology studies on male germ cells to improve existing sperm DNA-damage assessments and to develop more accurate diagnostic tests. At present, the Comet assay with sperm, used in reproductive toxicology studies, seems to provide the necessary sensitivity, accuracy and flexibility for becoming a reliable test system for biomonitoring of genotoxins and reprotoxins.

Acknowledgements The authors wish to thank the EU for funding (New Generis Grant: Food-CT2005-016320) and UKIERI (UK-India Education and Research Initiative Grant: SA07-067).

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34. 35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

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198. M. Arabi and M. S. Heydarnejad, Mechanism of the dysfunction of the bull spermatozoa treated with cadmium, Zhonghua. Nan. Ke. Xue., 2007, 13, 291–296. 199. L. Fraser and J. Strzezek, The use of Comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C, Folia. Histochem. Cytobiol., 2004, 42, 49–55. 200. L. Fraser and J. Strzezek, Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing, Anim. Reprod. Sci., 2007, 99, 317–329. 201. L. Fraser and J. Strzezek, Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezingthawing?, Theriogenology, 2007, 68, 248–257. 202. Z. L. Jiang, Q. W. Li, W. Y. Li, J. H. Hu, H. W. Zhao and S. S. Zhang, Effect of low density lipoprotein on DNA integrity of freezing-thawing boar sperm by neutral Comet assay, Anim. Reprod. Sci., 2007, 99, 401–407. 203. M. Arabi, Bull spermatozoa under mercury stress, Reprod. Domest. Anim., 2005, 40, 454–459. 204. G. B. Boe-Hansen, I. D. Morris, A. K. Ersboll, T. Greve and P. Christensen, DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay, Theriogenology, 2005, 63, 1789–1802. 205. K. T. Gloor, D. Winget and W. F. Swanson, Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa, J. Zoo. Wildl. Med., 2006, 37, 327–335. 206. E. Bustos-Obregon and R. I. Goicochea, Pesticide soil contamination mainly affects earthworm male reproductive parameters, Asian J. Androl., 2002, 4, 195–199. 207. C. Labbe, A. Martoriati, A. Devaux and G. Maisse, Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout, Mol. Reprod. Dev., 2001, 60, 397–404. 208. E. Cabrita, V. Robles, L. Rebordinos, C. Sarasquete and M. P. Herraez, Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm, Cryobiology, 2005, 50, 144–153. 209. A. Ciereszko, T. D. Wolfe and K. Dabrowski, Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir, Aquat. Toxicol., 2005, 73, 128–138. 210. G. J. Dietrich, A. Szpyrka, M. Wojtczak, S. Dobosz, K. Goryczko, L. Zakowski and A. Ciereszko, Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa, Theriogenology, 2005, 64, 1809–1822.


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SECTION IV: REGULATORY, IMAGING AND STATISTICAL CONSIDERATIONS

CHAPTER 15

Comet Assay – Protocols and Testing Strategies ANDREAS HARTMANNa,* AND GU¨NTER SPEITb a

Novartis Pharma AG, Preclinical Safety, WKL105.4. 09, CH-4002 Basel, Switzerland; b Universita¨t Ulm, Institut fu¨r Humangenetik, D-89069 Ulm, Germany

15.1 Introduction The assessment of a genotoxic hazard of chemicals and pharmaceuticals is an important component of the preclinical safety assessment. Experience with genetic toxicology testing over the past several decades has demonstrated that no single assay is capable of detecting all genotoxic effects. Therefore, the potential for a compound to cause genotoxicity is typically determined through a battery of in vitro and in vivo genotoxicity tests. In the case of pharmaceuticals, these assays are typically conducted at an early time point in the development of a new drug as they are relatively short in duration, inexpensive, and provide an early means to identify potential genotoxic carcinogens, which otherwise would not be detected until the completion of carcinogenicity assays. Internationally harmonised genotoxicity testing guidance ICHS2A and S2B that have been in operation since 1995 and 19971,2 are under revision as genetic toxicology testing has evolved since. Recently, a number of changes has been proposed by an ICH expert working group that has issued a new guidance draft, termed ‘‘S2R: Guidance on Genotoxicity Testing and Data Interpretation for * Corresponding author Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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Pharmaceuticals Intended for Human Use’’. One major concern driving the revision of the guidance takes into account accumulating evidence demonstrating that in vitro cytogenetic assays are oversensitive towards positive results associated with cytotoxicity.3–5 As a consequence, the limitation of test concentrations and cytotoxicity levels were proposed for the mammalian cell tests to address concerns over growing numbers of nonrelevant positive findings. The basis for this proposal is built by an extensive review of results obtained with in vitro hazard identification testing and in vivo risk assessment testing in the pharmaceutical industry as well as regulatory review. In the context of the revision of the ICH guidance, supplementary in vivo assays such as the Comet assay are becoming more important in the assessment of genotoxicity. The options for a standard battery of genotoxicity tests will be expanded by the possibility to choose to conduct an in vivo test with investigation of genotoxic damage in two tissues instead of conducting an in vitro test with mammalian cells followed by an in vivo test. As pharmaceuticals are generally tested for toxicity in rodent repeat-dose toxicity tests and as there is no requirement for an acute high dose rodent toxicity test any longer, the assessment of genotoxicity (e.g., bone marrow micronucleus test or other tissue/ endpoint) is proposed to be integrated into the rodent repeat-dose toxicity study to optimise animal usage. Finally, in the context of the Food and Drug Administration (FDA) Critical Path Initiative and the European Medicines Agency Road Map to 2010, opportunities for more efficient drug development are sought that include abbreviated genotoxicity testing. One of the initiatives that has emerged in this context is the elaboration through guidance of exploratory investigational new drugs (INDs)/clinical trial applications (CTAs).6,7 With regards to genetic toxicology testing it is acceptable to conduct a standard bacterial mutation assay as well as a test for chromosomal aberrations either in vitro or in vivo. Furthermore, according to the new guidances it is acceptable to perform the in vivo cytogenetics assessment in conjunction with the repeat-dose toxicity study in the rodent.6,7 This approach reflects a proposal in which repeated daily treatments with subchronic duration of exposures were shown to produce similar results (magnitude of response) in comparison to acute treatment.8

15.2 Applications of the In Vivo Comet Assay for Regulatory Purposes The use of the in vivo Comet assay for regulatory purposes mainly focused on applications as a supplementary test to follow up on in vitro positives or to investigate potential target organ genotoxicity.9,10 As such, the Comet assay has potential advantages over other in vivo genotoxicity test methods, which are reliably applicable to rapidly proliferating cells only or have been validated preferentially in a single tissue only. The Comet assay may detect a broader spectrum of primary DNA lesions, including single-strand breaks and oxidative base damage, which may not be detected in the UDS test because they are not

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repaired by nucleotide excision repair. The advantages of the Comet assay over the alkaline elution test include the detection of DNA damage at the single-cell level and the requirement for only small numbers of cells per sample. In contrast, when using the alkaline elution assay, large quantities of cells are necessary for the determination of genotoxic effects and, therefore, only a limited number of organs/tissues can be evaluated using this technique. In particular, this seems important for the investigation of suspected tissue specific genotoxic activity, which includes ‘‘site-of-contact’’ genotoxicity (cases of high local versus low systemic exposure). The main focus of this chapter is the regulatory use of the in vivo Comet assay for genotoxicity testing of pharmaceuticals with special emphasis on recommendations on test performance that have been issued by international expert panels. As part of the International Workshop on Genotoxicity Testing (IWGT), expert working groups on the Comet assay were convened to review and discuss the procedures and methods and to issue recommendations for the Comet assay in vitro and in vivo in 199912 and more focused on refinement of recommendations for the in vivo assay at the IWGT in 2005.13 In the latter meeting, protocol areas that were unclear or that required more detail in order to produce a standardised protocol with maximum acceptability by international regulatory agencies were discussed with regards to guidance for conducting the in vivo Comet assay that had been issued following exert panel discussions at the 4th International Comet Assay Workshop in 2001.11

15.3 Recommendations for Test Performance For the application of the in vivo Comet assay in genetic toxicity it is important to understand under which circumstances data from this test system can contribute to hazard identification and risk assessment. A review on the use and status of the Comet assay in current strategies for genotoxicity testing summarised the state-of-the-art and is recommended for further reading. The review lists specific examples for practical applications of the in vivo Comet assay and potential consequences of positive and negative test results are provided.9

15.3.1

Genetic Endpoint of the Comet Assay

For regulatory use of the Comet assay it is important to understand that this assay is an indicator test that detects primary DNA lesions and, therefore, can not be used as a primary in vivo genotoxicity test such as the micronucleus test. Indicator tests (or supplemental tests) do not directly measure mutations but detect effects related to the process of mutagenesis, such as DNA damage, recombination and repair. These assays differ with respect to the endpoints assessed. Induction of primary DNA lesions, that is, measurement of exposure, uptake and reactivity to DNA can be measured by the Comet assay, the 32Ppostlabeling assay, the alkaline elution or unwinding assays.14,15 For the determination of the repair of DNA lesions the unscheduled DNA synthesis

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(UDS) test is being used. Finally, measurement of induction of genetic changes using transgenic animal assays for point mutations is utilised. Results of supplemental tests can provide additional useful information in the context of extended genotoxicity testing. It has to be emphasised that primary DNA lesions may be repaired error-free and do not necessarily result in formation of mutations. Neither the magnitude of DNA migration in the Comet assay nor the shape of the comet can reveal the types of DNA damage causing the effect or their biological significance, that is, their mutagenic potential. Therefore, conclusions regarding the mutagenicity of a test compound cannot be made solely on the basis of Comet assay effects. However, a negative Comet result can be considered as a strong indicator for the absence of a mutagenic potential. Among the various versions of the Comet assay, the alkaline (pH of the unwinding and electrophoresis buffer Z 13) method enables detection of the broadest spectrum of DNA damage16 and is, therefore, recommended (in the first instance) for regulatory purposes.9,11,12 The alkaline version detects DNA damage such as strand breaks, alkali-labile sites (ALS), and single-strand breaks associated with incomplete excision repair. Under certain conditions, the assay can also detect DNA–DNA and DNA–protein crosslinking, which (in the absence of other kinds of DNA lesions) appears as a relative decrease in DNA migration compared to concurrent controls. In contrast to other DNA alterations, crosslinks may stabilise chromosomal DNA and inhibit DNA migration.17 Thus, reduced DNA migration in comparison to the negative control (which should show some degree of DNA migration) may indicate the induction of crosslinks, which are relevant lesions with regard to mutagenesis and should be further investigated. Increased DNA migration indicates the induction of DNA-strand breaks and/or ALS. Furthermore, enhanced activity of excision repair may result in increased DNA migration, which can influence Comet assay effects in a complex way. While DNA repair generally reduces DNA migration by eliminating DNA lesions, ongoing excision repair may increase DNA migration due to incision-related DNA-strand breaks. Thus, the contribution of excision repair to the DNA effects seen in the Comet assay depends on the types of induced primary DNA damage and the time point of analysis.18

15.3.2

Basic Considerations for Test Protocol

The recommendations issued by Tice et al.12 and Hartmann et al.11 were refined by Burlinson et al.13 and describe aspects of the test procedure regarding test animals, test substance, use of concurrent negative and positive control animals as well as dose selection for the design of a cytogenetic assay in much detail. In brief, either a single treatment or repeated treatments (generally at 24-h intervals) are equally acceptable. In both experimental designs, the study is acceptable as long as a positive effect has been demonstrated or, for a negative result, as long as an appropriate level of animal or tissue toxicity has been demonstrated or the limit dose with appropriate tissue exposure has been used.


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For repeated treatment schedules, dosing must be continued until the day of sampling. On a daily basis, test substances may be administered as a split dose (i.e. two treatments separated by no more than a few hours), to facilitate administering a large volume of material. The test may be performed in two ways. If animals are treated with the test substance once, then tissue/organ samples are obtained at 2–6 h and 16–26 h after dosing. The shorter sampling time is considered sufficient to detect rapidly absorbed as well as unstable or direct acting compounds. In contrast, the late sampling time is intended to detect compounds that are more slowly absorbed, distributed and metabolised. When a positive response is identified at one sampling time, data from the other sample time need not be collected although it might be useful for the interpretation of the test result. Alternatively, if multiple treatments at 24-h intervals are used, tissue/organ samples need be collected only once. The sampling time should be 2–6 h after the last administration of the test substance. Alternative sampling times may be used when justified on the basis of toxicokinetic data. One important aspect for the validity of a study is the inclusion of positive and negative controls. A positive control substance needs to demonstrate that the conditions of the study, in particular, the electrophoresis, were appropriate to demonstrate the induction of DNA damage. In addition, the stability of the negative/positive control data over time and criteria for determining the acceptability of new studies, based on historical control data, should be established for each tissue. Finally, minimal reporting standards should follow current OECD standards for the in vivo genotoxicity test and should ensure that all studies can be independently evaluated. Previous recommendations have covered some aspects of reporting standards.11,12

15.3.3

Selection of Tissues and Cell Preparation

In principle, any tissue of the experimental animal, provided that a high-quality single-cell/nucleus suspension can be obtained can be used for a Comet assay. Selection of the tissue(s) to be evaluated should be based, wherever possible, on data from absorption, distribution, metabolism, excretion studies, and/or other toxicological information. A tissue should not be evaluated unless there is evidence of, or support for, exposure of the tissue to the test substance and/or its metabolite(s). In the absence of such information and, unless scientifically justified, two tissues should be examined. Recommended tissues are liver, which is the major organ for the metabolism of absorbed compounds, and a site of first contact tissue, e.g. gastrointestinal for orally administered substances, respiratory tract for substances administered via inhalation, or skin for dermally applied substances. Which tissue is evaluated first is at the discretion of the investigator and both tissues need not be evaluated if a positive response is obtained in the first tissue evaluated. Single-cell suspensions can be obtained from solid tissue by mincing briefly with a pair of fine scissors,19 incubation with digestive enzymes such as

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collagenase or trypsin, or by pushing the tissue sample through a mesh membrane. In addition, cell nuclei can be obtained by homogenisation.21 During mincing or homogenisation, EDTA can be added to the processing solution to chelate calcium/magnesium and prevent endonuclease activation. In addition, radical scavengers (e.g., DMSO) can be added to prevent oxidantinduced DNA damage. Any cell-dissociation method is acceptable as long as it can be demonstrated that the process is not associated with inappropriate background levels of DNA damage.

15.3.4

Image Analysis

DNA migration in individual cells can be assessed by using image analysis or by manual scoring. While the use of image analysis enables various parameters to be analysed, manual scoring usually determines the length of DNA migration, the percentage of cells with and without migration, or the proportion of comets that can be ‘‘binned’’ into various migration categories.22 However, a limitation of manual scoring may be a potential inability to take into account the density or shape of tails that can include short but dense tails and long but sparse tails depending on the effects of compounds tested. With image-analysis systems, the most common parameters analysed are the tail intensity, i.e. the percentage DNA in the tail (% tail DNA), tail moment, and tail length and/or image length (referring to nucleus plus migrated DNA). Some parameters (e.g., tail moment) may be calculated differently among image-analysis systems that can lead to quantitative differences which can be problematic when comparing interlaboratory data. The percentage DNA in the tail is considered the parameter that can best be compared between laboratories. The consensus of the IWGT was that image analysis is preferred but not required and that the parameter % tail DNA appeared to be the most linearly related to dose and the easiest to intuitively understand.14 There was no consensus that % tail DNA must be the only parameter used. However, if some measure of tail moment is used, than % tail DNA and tail length data must be provided as well. Heavily damaged cells exhibiting a specific microscopic image (commonly referred to as hedgehogs) consisting of small or nonexistent head and large, diffuse tails23 potentially represent dead or dying cells and may be excluded from data collection. However, determining their frequency may be useful for data interpretation. Data on the distribution of migration among cells should also be presented. This is accomplished by sorting cells within ‘‘bins’’ based on the metric used to evaluate DNA migration and presenting the data as the percentage of cells within each bin.

15.3.5

Assessment of Cytotoxicity – A Potential Confounding Factor

A general issue with DNA-strand break assays such as the Comet assay is that indirect mechanisms related to cytotoxicity may lead to enhanced strand-break formation. However, since DNA damage in the Comet assay is assessed on the


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level of individual cells, dead or dying cells may be identified on microscopic slides by their specific image. Necrotic or apoptotic cells can result in comets with small or nonexistent head and large, diffuse tails24 as observed in vitro upon treatment with cytotoxic, nongenotoxic articles.25–27 However, such microscopic images are not uniquely diagnostic for apoptosis or necrosis since they may also be detected after treatment with high doses of radiation or high concentrations of strong mutagens.28 For the in vivo Comet assay, only limited data are available to establish whether cytotoxicity results in increased DNA migration in tissues of experimental animals. In a comprehensive investigation with genotoxic and nongenotoxic kidney carcinogens, the ability of the Comet assay to distinguish genotoxicity versus cytotoxicity was assessed by investigating five known genotoxic renal carcinogens acting through diverse mechanisms of action and two rodent renal epigenetic carcinogens. The authors concluded that the Comet assay using kidney cells of rats is not prone to falsepositive results due to cytotoxicity.29 Other investigations showed that despite necrosis or apoptosis in target organs of rodents such as kidneys,30 stomach,31 liver or duodenum,10 no elevated DNA migration was observed. However, enhanced DNA migration was seen in homogenised liver tissue of mice dosed with carbon tetrachloride32 when histopathological examination showed evidence of necrosis in the liver. Therefore, to avoid potential false-positive effects resulting from cytotoxicity, recommendations regarding a concurrent assessment of target organ toxicity have been made, including dye viability assays, histopathology and a neutral diffusion assay.11,12

15.3.6

Ongoing Validation Exercises

The Japanese Center for the Validation of Alternative Methods (JaCVAM) is organising an international validation study of the in vivo Comet assay, in cooperation with the US National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Mammalian Mutagenicity Study Group (MMSG)/Japanese Environmental Mutagen Society (JEMS). The purpose of this validation study is to evaluate the ability of the in vivo Comet assay to identify genotoxic chemicals as a potential predictor of rodent carcinogenicity. A more validation-type study investigated several aspects of an experimental design such as positives controls, tissue toxicity and sources of experimental variability.31 To examine cytotoxicity the neutral diffusion assay and histopathological/haematological analysis were used. Based on analyses of pooled data from several studies tissue preparations were identified as a source of high variability. The authors’ conclusion was that a higher number of samples/slides may be required to achieve sufficient power to detect a positive effect in certain tissues.31 Clearly, more such validation exercises are required to better define optimised protocols.

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15.4 Applications of the In Vivo Comet Assay for Regulatory Purposes A comprehensive review on the use and status of the in vivo Comet assay has been issued recently.9 For regulatory purposes, the in vivo Comet assay is currently being used (1), to follow up on positive findings in one or more tests of the standard genotoxicity battery; (2) to elucidate a potential contribution of genotoxicity to the induction of preneoplastic and/or neoplastic changes detected in long-term tests in rodents; (3) to investigate local genotoxicity. Additional areas that have been proposed are testing of industrial chemicals33 or assessment of photochemical genotoxicity.34,35 In addition, the Comet assay can be applied as a mechanistic tool to distinguish clastogenic from aneugenic effects. Since aneugenicity is well accepted to result from mechanisms of action for which thresholds exist, demonstration that micronucleus formation is a result of chromosome loss should allow an acceptable level of human exposure to be defined.36 No matter the trigger for conducting supplemental in vivo genotoxicity testing, it is critical that the approach utilised, for example the endpoint and target tissue assessed, is scientifically valuable, such that the results will aid in interpreting the relevance of the initial finding of concern. Ultimately, the goal of supplemental genotoxicity testing is to determine if a mutagenic risk is posed to humans under the intended use of a compound.

15.4.1

Follow-Up Testing of Positive In Vitro Cytogenetics Assays

An analysis of positive and negative in vitro chromosomal aberration results in various cell types amongst data that had been submitted to the German Federal Institute for Drugs and Medical Devices (BfArM) between 1995 and 2005 showed that approximately 30% of the compounds had positive in vitro genotoxicity data.37 The dataset consisted of 804 chromosomal aberration studies on nearly 600 pharmaceuticals and showed that the frequency of positive results in four different cell types studied for chromosomal aberrations and in the mouse lymphoma assay (detecting gene mutations as well as chromosomal damage) was very similar. It is noteworthy that such a high percentage of positive mammalian cell results is seen in submission dossiers assuming companies have already screened out compounds that are not considered suitable for development. In contrast to the high percentage of positive in vitro studies, results from bone marrow cytogenetic assays are frequently negative. This discrepancy may result from a number of major differences that exist when testing in cultured cells versus the intact animals. One important difference is the external metabolic system that is used in in vitro systems while in the intact organism, intact metabolic pathways exist. Metabolic inactivation can occur in the intact animal, the parent compound or active metabolite may not reach the target cell in vivo, rapid detoxification and elimination may occur, or plasma levels in vivo may not be comparable to


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concentrations that generate positive responses in the in vitro assays that are often accompanied by high levels of cytotoxicity. It is also worth noting that positive results generated in vitro may be secondary to effects, such as cytotoxicity, which may never be achieved under in vivo exposure conditions. At present, data from in vivo experiments are therefore essential before definitive conclusions are drawn regarding the potential mutagenic hazard to humans from chemicals that produce positive results in one or more in vitro tests. There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus test is negative. A recent analysis compared the usefulness of the in vivo Comet assay as a second in vivo test in comparison with the UDS test and the transgenic mutation (TG) assay.38 While the UDS test gave only positive results witho20% of carcinogens, the TG assay gave positive results with 450% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus negative or equivocal carcinogens. Although more data are needed before a general recommendation can be made, this study clearly indicated that the Comet assay should play a more prominent role in regulatory testing strategies in the future.

15.4.2

Follow-Up Testing of Tumourigenic Compounds

Carcinogenicity testing of compounds such as pharmaceuticals negative in the standard in vitro and in vivo genotoxicity assays may yield evidence of a tumourigenic response in rodents. The ICH guidance S2B2 stipulates that such compounds shall be investigated further in supplemental genotoxicity tests, if rodent tumourigenicity is not clearly based on a nongenotoxic mechanism. Typically, supplemental in vivo genotoxicity tests are performed with cells of the respective tumour target organ to distinguish between genotoxic and nongenotoxic mechanisms of tumour induction. For such purposes it is important that the method applied has a high specificity to distinguish genotoxic from nongenotoxic modes of action. In a study investigating genotoxic and nongenotoxic kidney carcinogens in rats, the in vivo Comet assay demonstrated the induction of DNA damage in the target organ by genotoxic carcinogens but not by kidney carcinogens that act through nongenotoxic modes of action.29 In a comprehensive summary of investigations of more than 200 compounds, a high specificity for Comet assay data was demonstrated by a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens.39 Finally, a recent review summarised published data and concluded that one of the major advantages of the in vivo Comet assay was the ability to evaluate virtually any tissue of experimental animals. It was concluded that a negative result from such a study would provide strong evidence that tumour induction may rather result from an epigenetic mechanism than from organ-specific genotoxicity.9

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15.4.3

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Assessment of Local Genotoxicity

The Comet assay is considered a very useful tool to investigate genotoxicity at the first site of contact, such as oral or stomach mucosa cells or the nasal cavity of rodents. This approach is of interest for investigations of compounds with a low systemic bioavailability or for very short-lived compounds or their metabolites.11,12 Furthermore, the evaluation of genotoxic effects in directly exposed organs may address certain human exposure scenarios. Target organs in this respect include nasal or oral cavity, lung, oesophagus, stomach mucosa, duodenum or skin. A comprehensive review of applications and available data has been published.9

15.4.4

Assessment of Germ Cell Genotoxicity

A recent review by Speit et al. assessed the use of the Comet assay for investigating germ cell genotoxicity.40 While current test strategies focus on somatic cells from different organs to detect the genotoxic activity, the Comet assay also has the potential to be a useful tool for investigating germ cell genotoxicity. The ‘‘Globally Harmonised System of Classification and Labeling of Chemicals (GHS)’’ has published classification criteria for germ cell mutagens, i.e. chemicals that may cause mutations in the germ cells of humans that can be transmitted to the progeny.41 Category 1 B defines ‘‘chemicals which should be regarded as if they induce heritable mutations in the germ cells of humans’’. Among the criteria that are given for this category, one requires ‘‘positive results(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has the potential to cause mutations in germ cells. This supporting evidence may, for example, be derived from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells’’. Although GHS does not explicitly mention the Comet assay but only lists the Sister Chromatid Exchange (SCE) analysis in spermatogonia and the Unscheduled DNA Synthesis test (UDS) in testicular cells as examples for genotoxicity tests in germ cells, the Comet assay might play an important role in this context in the future. GHS requires that classification for heritable effects in human germ cells has to be made on the basis of well-conducted, sufficiently validated tests, preferably as described in OECD test guidelines. The standard alkaline in vivo Comet assay can easily be adapted to investigations with cells from the gonads (testis and ovary) for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, standardisation and validation studies are necessary before the Comet assay can be usefully applied in risk assessment of germ cell mutagens.

15.4.5

Assessment of Photogenotoxicity

The assessment of photochemical genotoxicity (‘‘photogenotoxicity’’) is an important part of photosafety testing that has become a regulatory requirement


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for medicinal products, dermatological and sunscreen products. Photochemical genotoxicity of drugs was described to be involved in the generation of skin tumours42 and can lead to injuries to the eye.43 In the European Union (EU), pharmaceutical products are regulated under the European Agency for the Evaluation of Medicinal products (EMEA), whilst dermatological and sunscreen products are regulated under the EU Cosmetics Directive. The conditions for the photosafety evaluation of pharmaceuticals and cosmetics are laid out by The EMEA Committee for Proprietary Medicinal Products ‘‘Notes for Guidance on Photosafety Testing’’44 and the FDA/CDER Guidance on industry photosafety testing.45 The main objective of photogenotoxicity testing is to make an assessment of the potential of a compound to turn into a photochemical carcinogen upon activation with UV or visible light. Several standard genotoxicity assays such as the photo-Ames, photochromosome aberration (CA) and photo-Comet assays have been described in the literature and are based on standard ‘‘dark’’ versions of regulatory assays used for genotoxicity assessment. The tests have been adapted towards use in photogenotoxicity testing, such as the photoclastogenicity test in CHO cells and tentative guidelines have been issued.46 Considerable concern regarding the biological plausibility of the response of certain chemicals in the in vitro photoclastogenicity assay has been raised, suggesting that this assay is oversensitive and lacks specificity.47 Specifically, given that more than 55% of all substances tested yielded positive results in these tests, the definition of in vitro photogenotoxicity for substances that are clastogenic in the dark requires reconsideration, especially taking into account the absence of validated in vivo tests that could distinguish genuine from pseudophotoclastogens.47 In addition, several compounds that did not absorb UV light were shown to elicit a photoclastogenic response in the photochromosome aberration assay using a CHO cell line.47,48 Therefore, the biological significance of in vitro photoclastogenicity data for hazard identification and risk assessment remains questionable and alternative methods need to be considered. In vivo methods may, therefore, be considered an alternative. Compared to in vitro tests on isolated cells, additional parameters may influence the photogenotoxicity in vivo such as the metabolism of a compound, systemic distributed or disposition into skin. In addition, the skin and the eye are composed of different layers, which can function as protective barriers and have an impact on penetration and absorption of wavelengths from sunlight. Furthermore, binding and retention time of the compound in the different layers of the skin or eye as well as their DNA-repair mechanisms may have an impact on the photochemical toxicity. Most of the photochemical reactions involve the generation of free-radical oxygen species. Oxygen content and the antioxidant status may have an important impact on the photogenotoxicity outcome.46 Due to higher oxygen pressure in vitro, higher amounts of radical oxygen species may be generated, with the consequence that a photogenotoxic effect might be overestimated under in vitro conditions. Therefore, more reliable test systems should enable a more thorough assessment of a photogenotoxic hazard. An in vivo Comet assay

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has been established to assess the photogenotoxic response of fluoroquinolones in the skin of mice.34 In addition to the analysis of skin keratinocytes, the use of cells of the retina and cornea of rats treated with model compounds sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen has been established.35 These investigations demonstrate that the photo-Comet assay in rodents is a reliable method to elucidate drug-induced photogenotoxicity under conditions that are relevant to the human situation.

15.4.6

Genotoxicity Testing of Chemicals

The genotoxicity testing requirements for chemicals differ from regulations for pharmaceuticals. The identification of possible genotoxic effects has long been fundamental for toxicity testing of chemicals. Different strategies for genotoxicity testing are applied depending on the regulated ‘‘substance class’’ (use of substances, type and degree of exposure, risk-benefit considerations, etc.). While genotoxicity testing originally focused on the detection of germ cell mutagens, in current regulatory practice the emphasis is put on screening for possible carcinogenic substances. In addition, genotoxicity testing is increasingly being used to clarify the contribution of genotoxicity to findings in carcinogenicity studies. A working group sponsored by the German-speaking section of the European Environmental Mutagen Society (GUM) proposed a simplified approach to genotoxicity testing of chemicals.33 The proposed strategy consists of basic testing using a bacterial gene mutation test plus an in vitro micronucleus test (stage I) and follow-up testing in vivo (stage II) in the case of relevant positive results observed in stage I. For the follow-up testing a single study combining the analysis of micronuclei in bone marrow with the Comet assay in appropriate tissues was suggested. Negative results for both endpoints in relevant tissues would generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds recognised as in vivo somatic cell mutagens/genotoxicants in this hazard identification step would need further assessment or would be considered as potential genotoxic carcinogens and potential germ cell mutagens in the absence of additional data.33

15.5 Conclusions The in vivo Comet assay is increasingly used to contribute to hazard identification (i.e. how likely an agent is to be genotoxic/mutagenic to humans) and to dose–response assessment (i.e. the relationship between the dose of a substance and the probability of induction of an adverse effect). Data from the in vivo Comet assay data are increasingly considered by regulatory agencies in the process of risk assessment and may be requested under specific circumstances. The Comet assay was shown to be a reliable test system with high sensitivity that enables detection of DNA damage in organs that cannot be investigated in the classical assays such as the micronucleus test or the unscheduled DNA


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synthesis (UDS) test. One important area in which it is relevant to assess for DNA damage in specific target organs such as stomach, kidney, bladder, the Comet assay at present is the most feasible method. The quality of the study performance is critical and recommendations have been issued by international expert panels.11–13 Systematic investigations aiming at more optimised study designs have been issued more recently.31 A negative result in the Comet assay is considered as supportive evidence of a lack of genotoxic activity of a test compound in the tissues tested. When a compound induced genotoxic effects in vitro, a negative in vivo Comet assay – generally in combination with other negative in vivo genotoxicity tests – provides supporting evidence that genotoxic effects detected in vitro have no relevance for the in vivo situation. According to published experience with agrochemicals, pharmaceuticals and hair dyes, a negative in vivo Comet assay would allow further development of a compound to proceed. However, to fulfil regulatory requirements, additional testing may be considered. A positive result indicates a genotoxic effect of the test compound in the respective tissues of the species tested and, therefore, an indication for a mutagenic potential of the test compound. If positive in vitro data were obtained for the compound, a positive in vivo Comet assay signal should be considered evidence that the in vitro signal is of biological significance in vivo. For substances in developmental stages, a positive in vivo Comet assay generally represents a major hurdle and will frequently result in discontinuation of further development. If further testing is considered necessary, the testing strategy needs to be determined on a case-by-case basis that much depends on the mode of action of the compound and the already existing data. Finally, the quality of the test performance and the plausibility of the result should be critically evaluated in the context of existing genotoxicity data for this compound as well as available data on absorption and disposition of the compound investigated. Isolated positive in vivo Comet assay results in the context of otherwise negative datasets have been reported.9 Such cases should initiate a critical reevaluation of the existing genotoxicity data and the need for additional testing should be defined. Further testing should be performed to enable a careful risk assessment of the compound by means of the ‘‘weight of evidence’’ approach.

References 1. ICH S2A (1995). Specific aspects of regulatory genotoxicity tests. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 1995, http://www.ich.org/. 2. ICH S2B (1997). A standard battery for genotoxicity testing of pharmaceuticals. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 1997, http://www.ich.org/. 3. D. J. Kirkland and L. Mu¨ller, Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds, Mutat. Res., 2000, 464, 137.

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4. D. Kirkland, M. Aardema, L. Henderson and L. Muller, Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res., 2005, 584, 1. 5. D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H. Glatt, P. Hastwell, M. Hayashi, P. Kasper, S. Kirchner, A. Lynch, D. Marzin, D. Maurici, J. R. Meunier, L. Mu¨ller, G. Nohynek, J. Parry, E. Parry, V. Thybaud, R. R. Tice, J. van Benthem, P. Vanparys and P. White, How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop, Mutat. Res., 2007, 628, 31. 6. Guidance for Industry, Investigators, and Reviewers. Exploratory IND Studies. US of Health and Human Services, FDA, CDER, 2006. 7. Guidance to the Conduct of Exploratory Trials in Belgium. Federal Agency for Medicines and Health Products in Belgium (FAMHP), 2007. 8. G. Krishna, G. Urda and J. Theiss, Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: A pharmaceutical industry perspective, Environ. Molec. Mutagen., 1998, 32, 115. 9. S. Brendler-Schwaab, A. Hartmann, S. Pfuhler and G. Speit, The in vivo Comet assay: use and status in genotoxicity testing, Mutagenesis, 2005, 20, 245. 10. A. Hartmann, M. Schumacher, U. Plappert-Helbig, P. Lowe, W. Suter and L. Mueller, Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations, Mutagenesis, 2004, 19, 51. 11. A. Hartmann, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. R. Collins, A. Smith, G. Speit, V. Thybaud and R. R. Tice, Recommendations for conducting the in vivo alkaline Comet assay, Mutagenesis, 2003, 18, 45. 12. R. R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.-C. Ryu and Y. F. Sasaki, The single cell gel/Comet assay: Guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen., 2000, 35, 206. 13. B. Burlinson, R. R. Tice, G. Speit, E. Agurell, S. Y. Brendler-Schwaab, A. R. Collins, P. Escobar, P. Honma, T. S. Kumaravel, M. Nakajima, Y. F. Sasaki, V. Thybaud, Y. Uno, M. Vasquez and A. Hartmann, Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup, Mutat. Res., 2007, 627, 31. 14. G. Ahnstrom, Techniques to measure DNA single-strand breaks in cells: a review, Int. J. Radiat. Biol, 1988, 54, 695. 15. M. C. Elia, R. D. Storer, T. W. McKelvey, A. R. Kraynak, J. E. Barnum, L. S. Harmon, J. G. DeLuca and W. W. Nichols, Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays, Environ. Mol. Mutagen., 1994, 24, 181. 16. R. R. Tice, The single cell gel/Comet assay: a microgel electrophoretic technique for the detection of DNA damage and repair in individual cells,


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in Environmental Mutagenesis, ed. D.H. Phillips, S. Venitt, Bios Scientific Publishers, Oxford, 1995, p. 315. O. Merk and G. Speit, Detection of crosslinks with the Comet assay in relationship to genotoxicity and cytotoxicity, Environ. Mol. Mutagen., 1999, 33, 167. G. Speit and A. Hartmann, The contribution of excision repair to the DNA-effects seen in the alkaline single cell gel test (Comet assay), Mutagenesis, 1995, 10, 555–559. R. R. Tice, P. W. Andrews, O. Hirai and N. P. Singh, The single cell gel (SCG) assay: an electrophoretic technique for the detection of DNA damage in individual cells, in Biological Reactive Intermediates. IV. Molecular and Cellular Effects and Their Impact on Human Health, eds. C.R. Witmer, R.R. Snyder, D.J. Jollow, G.F. Kalf, J.J. Kocsis, I.G. Sipes, Plenum Press, New York, 1991, p. 157. S. Y. Brendler-Schwaab, P. Schmezer, U. Liegibel, S. Weber, K. Michalek, A. Tompa and B. L. Pool-Zobel, Cells of different tissues for in vitro and in vivo studies in toxicology: Compilation of isolation methods, Toxicol. in vitro, 1994, 8, 1285. Y. Miyamae, M. Yamamoto, Y. F. Sasaki, H. Kobayashi, M. Igarashi Soga, K. Shimol and M. Hayashi, Evaluation of a tissue homogenization technique that isolates nuclei for the in vivo single-cell gel electrophoresis (comet) assay: a collaborative study by five laboratories, Mutat. Res., 1998, 418, 131. A. R. Collins, A. Ai-guo and S. J. Duthie, The kinetics of repair of oxidative DNAdamage (strand breaks and oxidised pyrimidines) in human cells, Mutat. Res., 1995, 336, 69. P. L. Olive, J. P. Banath and R. E. Durand, Heterogeneity in radiation induced DNA damage and repair in tumor and normal cells measured using the ‘‘Comet’’ assay, Radiat. Res., 1990, 122, 86. P. L. Olive, G. Frazer and J. P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the Comet assay, Radiat. Res., 1993, 136, 130. L. Henderson, A. Wolfreys, J. Fedyk, C. Bourner and S. Windebank, The ability of the Comet assay to discriminate between genotoxins and cytotoxins, Mutagenesis, 1998, 13, 89. A. Hartmann, E. Kiskinis, A. Fjaellman and W. Suter, Influence of cytotoxicity and compound precipitation on test results in the alkaline Comet assay, Mutat. Res., 2001, 497, 199. M. Kiffe, P. Christen and P. Arni, Characterization of cytotoxic and genotoxic effects of different compounds in CHO K5 cells with the Comet assay (single-cell gel electrophoresis assay), Mutat. Res., 2003, 537, 151. M. S. Rundell, E. D. Wagner and M. J. Plewa, The Comet assay: genotoxic damage or nuclear fragmentation? Environ. Molec. Mutagen., 2003, 42, 61. F. Nesslany, N. Zennouche, S. Simar-Meintie`res, I. Talahari, E. N. NkiliMboui and D. Marzin, In vivo Comet assay on isolated kidney cells to

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distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds, Mutat. Res., 2007, 630, 28. T. Mensing, P. Welge, B. Voss, L. M. Fels, H. H. Fricke, T. Bruning and M. Wilhelm, Renal toxicity after chronic inhalation exposure of rats to trichloroethylene, Toxicol. Lett., 2002, 128, 243. C. C. Smith, D. J. Adkins, E. A. Martin and M. R. O’Donovan MR, Recommendations for design of the rat Comet assay, Mutagenesis, 2008, 23, 233. Y. F. Sasaki, A. Saga, M. Akasaka, S. Ishibashi, K. Yoshida, Y. Q. Su, N. Matsusaka and S. Tsuda, Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline singlecell gel electrophoresis (comet) assay in multiple mouse organs, Mutat. Res., 1998, 419, 13. S. Pfuhler, S. Albertini, R. Fautz, B. Herbold, S. Madle, D. Utesch and A. Poth, Genetic toxicity assessment: employing the best science for human safety evaluation part IV: Recommendation of a working group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a simple and straightforward approach to genotoxicity testing, Toxicol. Sci., 2007, 97, 237. U. Wirnitzer, N. Gross-Tholl, B. Herbold and E. von Keutz, Photochemically induced DNA effects in the Comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA, Mutat. Res., 2006, 609, 1. M. Struwe, K. O. Greulich, U. Junker, C. Jean, D. Zimmer, W. Suter and U. Plappert-Helbig, Detection of photogenotoxicity in skin and eye in rat with the photo-Comet assay, Photochem. Photobiol. Sci., 2008, 7, 240. K. S. Bentley, D. Kirkland, M. Murphy and R. Marshall, Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization, Mutat. Res., 2000, 464, 41. D. Kirkland, M. Aardema, L. Muller and H. Makoto, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles, Mutat. Res., 2006, 608, 29. D. Kirkland and G. Speit, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and noncarcinogens. III. Appropriate follow-up testing in vivo, Mutat. Res., 2008, 654, 114. Y. F. Sasaki, K. Sekihashi, F. Izumiyama, E. Nishidate, A. Saga, K. Ishida and S. Tsuda, The Comet assay with multiple mouse organs: comparison of Comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and US NTP Carcinogenicity Database, Crit. Rev. Toxicol., 2000, 30, 629. G. Speit, M. Vasquez, and A. Hartmann, The Comet assay as an indicator test for germ cell genotoxicity, Mutat. Res., 2009, 681, 3. GHS, Globally Harmonized System of Classification and Labelling of Chemicals (GHS): First Revised Edition, United Nations, Geneva, 2005.


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42. K. S. Loveday, Interrelationship of photocarcinogenicity, photomutagenicity and phototoxicity, Photochem. Photobiol., 1996, 63, 369. 43. R. D. Glickman, Phototoxicity to the retina: Mechanisms of damage, Int. J. Toxicol., 2002, 21, 473. 44. CPMP/SWP/398/01: The Committee for Proprietary Medicinal Products (CPMP) ‘‘Notes for Guidance on Photosafety Testing. http://www. emea.europa.eu/pdfs/human/swp/039801en.pdf. 45. CDER/FDA Guidance for industry photosafety testing, 2003, http:// www.fda.gov/cder/guidance/index.htm. 46. S. Brendler-Schwaab, A. Czich, B. Epe, E. Gocke, B. Kaina, L. Mu¨ller, D. Pollet and D. Utesch, Photochemical genotoxicity: principles and test methods. Report of a GUM task force, Mutat. Res., 2004, 566, 65. 47. A. M. Lynch, S. A. Robinson, P. Wilcox, M. D. Smith, M. Kleinman, K. Jiang and R. W. Rees, Cycloheximide and disulfoton are positive in the photoclastogencity assay but do not absorb UV irradiation: another example of pseudophotoclastogenicity? Mutagenesis, 2008, 23, 111. 48. E. K. Dufour, T. Kumaravel, G. J. Nohynek, D. Kirkland and H. Toutain, Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in preirradiated or simultaneously irradiated Chinese hamster ovary cells, Mutat. Res., 2006, 607, 215.

CHAPTER 16

Imaging and Image Analysis in the Comet Assay MARK BROWNE Andor Technology, Microscopy Systems Division, Morrisville, NC 27560, USA

16.1 Introduction This chapter addresses aspects of digital imaging and analysis of specimens prepared by the Comet assay. Comet assay protocols and study designs vary widely depending on the scientific questions being asked of the assay, but they always require repeatable characterisation of the DNA distribution of individual treated cell nuclei (comets) in specimen slides. Characterisation of the slides is normally performed by computer image analysis and the output data are subject to statistical analyses, which are varied according to the nature of the scientific experiment, but principles of data preparation and summarisation are consistently applied. Hence, we will discuss principles and practice for comet visualisation and imaging, analysis, data collection and summarisation as the key steps between the ‘‘wet assay’’ and statistical analyses prior to drawing reliable scientific conclusions from Comet assay studies.

16.1.1

Experimental Design and Applications

As described elsewhere in this book, the Comet assay involves a well-characterised series of steps to harvest a subsample of cells (the specimen) from one or more individuals or samples in one or more exposure groups. In a Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org

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well-designed study there will be at least ‘‘negative controls’’ or an unexposed group as well as a number of ‘‘test’’ specimens at one or more exposure levels. The negative control serves as a ‘‘baseline’’, while the positive control group (if includeda) provides the assurance that the experimental procedure is adequate to detect a statistically significant effect. The use of internal controls is essential in comet studies because the flexibility of the assay is enormous and so good experimental design is critical to the final statistical power of the conclusions. Because the Comet assay can be used to quantify DNA integrity, structure, damage and repair, depending on the design of the specific assay, its uses are extensive. Moreover, it can be used with cells from virtually any organism: studies include, but are not limited to mammals, fish, invertebrates, plants and cell cultures of all these. The Comet assay has been used for applications as diverse as safety testing of pharmaceuticals and chemical products, fertility research, dietary impact and protection, tumour radio- and chemosensitivity, environmental impact studies of pollutants from power station plumes to chemical run-off into lakes and rivers and occupational or habitual exposure to potential carcinogens. In other chapters of this book many of these topics are explored in depth, but suffice to say the flexibility demands that good study design is observed and developed for specific cases.

16.2 Comet Sample Preparation The harvested specimens are treated to create a suspension of individual cells in low melting point agarose gel, further treatment (e.g. DNA unwinding and lysis of cellular membranes) allows their DNA to be revealed and subject to electrophoresis.2,3 During electrophoresis the electric field applied results in migration of DNA towards the anode (positive voltage terminal) and the resulting distribution can be compared to gel electrophoresis of bulk DNA, except it is at the single-cell level. Smaller fragments of DNA will move faster as they are subject to less ‘‘drag’’ from the gel.1 Thus, the analysis of the distribution of DNA in the individual cells provides a means of evaluating the migration pattern under the specific electrophoresis conditions. The ensemble of measured distributions is used as an indicator of the impact of the preharvest exposure of the organism or culture to DNA-damaging agents and is subject to statistical analyses as mentioned above to establish a confidence level on the observations.31 Following electrophoresis in a high-pH buffer (typically 412.5 depending on the protocol and study in hand), the Comet specimens are neutralised (pH 7) and optionally air dried for storage until ‘‘scoring’’ or analysis is performed. To speed drying, the slides are commonly immersed in (70%) ethanol, which is hydrophilic and therefore helps to remove moisture form the agarose gel. Ethanol dehydration of the specimens can be useful for long-term storage (months) because a dry gel will maintain the DNA distributions created by the a

Note: In biomonitoring or environmental exposure studies there are only exposed and unexposed groups.

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comet treatment. Storage of comet slides should be in a cool, low relative humidity (RH) environment. High RH (above 60%) for even a few days will lead to partial rehydration of the agarose gel, DNA diffusion and consequent ‘‘blurring’’ of the comet DNA distributions. Since most DNA stains are pH sensitive, neutralisation provides the dual purpose of limiting further damage to the DNA and preparing the specimen for stainingb. Dried specimens are rehydrated and stained by addition of the fluorophore in solution. A coverslip is placed over the specimen, which is left to hydrate in darkness to avoid potential fluorophore bleaching. Adequate safety precautions should be taken when handling DNA dyes since many are genotoxic, especially those that bind to DNA by intercalation.4

16.3 Comet Fluorescence Staining and Visualisation DNA staining or labelling is performed by applying a solution of a chosen fluorophore (probe) that binds selectively to the DNA. The specimen can then be viewed with a microscope under epifluorescence illumination. A CCD camera is used to capture the resultant image and its digital output is transferred to a computer for quantitative analysis. Details of the microscopy, CCD cameras and analysis are discussed in subsequent sections. The goal of labeling is to produce an image suitable for detection by a CCD camera (or eye) and of sufficient quality for reliable analysis. Analysis requires both high (or adequate) signal-to-noise ratio (SNR) and high signal-to-background ratio (SBR). High SNR ensures repeatable (low variance) analysis, while SBR ensures adequate dynamic range to differentiate similar but different DNA distributions. Since damaged DNA is commonly observed as small fragments in the ‘‘tail’’ of the comet, which are of relatively low intensity, the noise determines the smallest fragment that can be detected. Dynamic range, DR is defined as DR ¼ ðsaturation value backgroundÞ=detection limit SNR depends primarily on the probe brightness and camera sensitivity and because both are functions of wavelength it is best to try to match fluorophore spectral emission to the camera’s spectral response. We will explore this further in the section on CCD cameras. For practical reasons, the detection limit of the camera is commonly considered to be three times the ‘‘noise floor’’ of the detector. In a CCD camera the noise floor is set by the read noise, which establishes a limit to the system sensitivity and dynamic range. Other factors in the imaging process will contribute to noise and reduce SNR and DR further. Our goal is to limit these other factors as far as possible and to understand their sources to allow suitable selection among the many options available. b

Note: Ethanol must be completely evaporated before staining as it impedes the labelling of the DNA by some fluorophores.


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SBR depends on a number of factors including the fluorescence enhancement of the probe when bound to DNA, compared to its free form,7 autofluorescence in the sample and instrumentation, the fluorescence microscope filter (blocking) performance and on the quality of slide preparation. Autofluorescence can be reduced by avoiding UV excitable DNA probes – see later. Some key properties of widely used DNA fluorophores or ‘‘optical probes’’ are listed in Table 16.1. A useful measure of relative intensity for a given fluorophore can be computed from the product of extinction coefficient, e and quantum yield, QY,5 and we can define the brightness index, B as B ¼ e QY=1000 The extinction coefficient, sometimes called the molar absorptivity is usually measured in solution with a fluorescence or absorption spectrometer and quantifies how strongly a species absorbs light (photons) at a given wavelength. Its unit of measurement is cm1 M1, i.e. it depends on the optical path length and concentration6 and is the species constant, e in the Beer–Lambert law described by the well-known formula Ir ¼ Io 10ect where Ir is the intensity after absorption, Io is the initial intensity; c is the concentration and t the path length in cm. Quantum yield is the ratio of the number of photons emitted to the number of photons absorbed, with a limiting value of one. QY is a wavelengthdependent property. Note that other factors, including bleach rate can have an impact on a given probe’s performance in imaging. Fluorophore spectral absorption, emission and QY are sensitive to local molecular environment, such as pH, electrical potential, etc. In fact, some probes are selected or designed to exploit specific sensitivities that enable the study of microenvironment dynamics within living cells and organisms. In comet samples, however, our goal is to label the DNA with the probe to prepare for imaging and quantification, so the brightness index provides a useful tool for comparison. Figure 16.1 shows the absorption and emission spectra of the fluorophores in Table 16.1. Of particular note are the spectra of ethidium bromide (EB) and propidium iodide (PI) that have been normalised to their UV absorption peaks. On the other hand, ethidium homodimer (EH) has been normalised to its visible absorption maximum, but had it been shown with the UV maximum it would exhibit a similar curve to PI and EB. The point here is that the visible excitation of PI, EB and EH shows much lower brightness than UV excitation, but for reasons of safety, background autofluorescence and low transmission in optical components, UV excitation is not recommended. Consequently, probes with high extinction coefficients in the visible wavelengths are preferred for comet studies and of this group SYBR Green delivers the brightest results.

502 502 360 528 521 305(UV) 538 498

Acridence Orange DAPI in DMSO Ethidium Homodimer Ethidium Bromide Propidium lodide UV Propidium lodide SYBR Green

526 640** 455 617 603 632 632 522

Emission peak, nm 0.43 0.34 0.08 0.14 0.10 0.10 0.7

24000 20000* 20000 36000 5900 50000

QY(DNA)

53000

Extinction Coefficient, cm1 M1

14.85 1.6 2.8 3.6 0.59 35

21.4

Brightness index

Notes: QY (DNA) indicates the quantum yield when bound to DNA. * Some manufacturers’ do not publish all the relevant data on probes for proprietary reasons, but we have made estimates in those cases marked with an asterisk based on observed brightness in imaging. ** indicates RNA fluorescence since Acridine orange labels DNA and RNA differentially.

Excitation peak, nm

Commonly used nucleic acid fluorescent probes with comet samples.

Fluorophore

Table 16.1

394 Chapter 16


Figure 16.1

395

Normalised excitation and emission spectra for the nucleic acid dyes bound to DNA and listed in Table 16.1. Spectral data provided courtesy of Iain Johnson, Invitrogen, Inc. See the Fluorescence Spectral Viewer (http://www.invitrogen.com).

16.4 Fluorescence Microscopy for Comet Imaging Many aspects of the instrumentation can affect overall imaging performance. The principles of fluorescence microscopy and some practical issues around the selection and operation of the instrumentation components will be considered here. Comet slides are viewed on a fluorescence microscope, because use of fluorescence for staining DNA is so convenient and widespread. Figure 16.2 shows the principal components of the epifluorescence microscope that is in universal use for life science imaging today. Correct configuration and adjustment of the microscope is of central importance in obtaining goodquality results from the Comet assay. The following sections discuss the

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Figure 16.2

Microscope schematic showing key components of the epifluorescence light path.

Table 16.2

Some important properties of light sources used for fluorescence microscopy. Brightness Ranking

Coupling

Alignment

Mercury arc Hg 100

1

Direct

Xenon arc Xe

100

2

Direct

Metal halide LED

120 10

1 3

LG LG

Manual 300 center Manual 300 center Pre-aligned 2000 Pre-aligned 10,000

Light Source

Power W

Approx Lifetime hrs

properties of the microscope components and are intended to provide practical advice on the configuration and use of the instrument.

16.4.1

Light Sources

Until recently, the primary means of delivering excitation light to a fluorescence microscope was the high-pressure mercury (Hg) or Xenon (Xe) arc lamp. Hg lamps still predominate in labs with basic microscope equipment for historical reasons, but new devices have come on to the market in recent years that make the old standards obsolete. For a summary of light source properties see Table 16.2.


Figure 16.3

397

Emission spectrum of a high-pressure mercury (Hg) arc lamp (HBO100).

Hg lamps are hazardous to use if not handled with care since they contain pressurised vapour, operate at high temperature, emit UV light and require careful alignment for high performance. A typical lamp will be rated at 100 W and because these lamps are usually mounted in a lamp house that couples directly to the microscope epi-illumination optics they irradiate heat and UV light to the microscope, the specimen and the user. The output spectrum of the Hg light source is shown in Figure 16.3 with characteristic UV emission at 365 nm and strong peaks at 405, 440, 550 and 580 nm. Disposal of mercury lamps requires appropriate hazardous waste management and their typical lifetime is restricted to 200 or 300 h. As mercury lamps age they lose intensity quite rapidly, output dropping by about 50% over their lifetime. Further, as the electrodes erode in the hostile conditions of the arc, arc position can wander leading to significant fluctuations in intensity in the few Hz to 100 Hz frequency range, which can seriously affect imaging and effectively adds noise to the image-derived data. Arc-lamp alignment and focus strongly affect illumination uniformity, which if not corrected can lead to further spatially dependent variations (noise) in imaging. Of course, a well-aligned, wellmaintained mercury lamp can deliver excellent results, but in terms of reducing potential sources of error in fluorescence microscopy, the light source is recommended as the first candidate for updating. A newer generation of arc lamps is based on MH sources, which have lifetimes in the order of 2000 h, are supplied in prealigned cartridges and typically housed in a remote air-cooled enclosure. Light is coupled to the epifluorescence optics via a liquid light guide (LLG). These factors provide a major performance improvement over traditional mercury lamps. The extended lifetime and reduced fall-off (B30% over lifetime) results in less maintenance and higher-quality data. The LLG and remote housing results in lower hazard for the user and the

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microscope is not subject to the mechanical stresses of heating and cooling induced by the lamp housing. These lamps offer many advantages and are strongly recommended, but they also require managed disposal since they contain mercury (Figure 16.3). Xenon (Xe) arc lamps were traditionally lamp-house mounted like the Hg arc lamp, but in more recent developments they have been housed remotely and coupled with a light guide in much the same way as the metal halide source described above. New-generation Xe lamps have lifetimes of 2000 h and instrumentation companies have developed sophisticated tunable sources based on the Xe lamp because it has a broad, relatively flat emission spectrum, as shown in Figure 16.4. This is desirable when optical output power should remain fairly constant over the full spectrum and finds application in fields such as spectral and ratio imaging.8 Most recently, sources based on solid-state light-emitting diodes or LEDs have become commercially available.9 LED array modules (LAMs) are many more times efficient than arc lamps (B50:1) and have lifetimes of at least 10 000 h, i.e. in the order of 500 times that of a conventional Hg arc lamp and 5–6 times that of a MH source. The primary drawback of current LED technology is the relative lack of brightness, but this has been addressed with good optical design and the latest-generation units are LLG coupled and provide very compelling benefits. In time, it is likely that LED technology will become the standard in light sources for fluorescence microscopy, but the initial cost of purchase is still rather more than MH sources. However, they are exceptionally stable and introduce very little noise to the measurement process and because their spectra are usually restricted to about 20–40 nm about their peak emission wavelength they tend to result in very low background images.

16.4.2

Epifluorescence Light Path

The light from the source is gathered with collection optics and a near-collimated beam is used to fill the ‘‘field aperture’’. This aperture is essentially a user-controlled iris, which can be adjusted to restrict the illuminated field of view if required. In a well-configured instrument, this aperture will be clearly visible with the in-focus specimen. It is the equivalent of Kohler illumination in transmitted light microscopy and the instrument optics are designed to provide minimum aberrations in this configuration. It is for this reason that a badly aligned lamp can be such a problem, if the light arriving at the field aperture is not uniform and collimated then it will not uniformly illuminate the specimen and poor imaging and analysis results will follow. The epifluorescence light path also includes the fluorescence (interference) filter set as shown in Figure 16.2. The light from the source is spectrally bandlimited by the excitation filter, whose job is to define the excitation wavelengths delivered to the fluorescent specimen. The excitation light is reflected onto the sample by a dichroic mirror, whose function is to separate excited and emitted light, as described in the next section.


Figure 16.4

16.4.3

399

(a) Emission spectrum of a high pressure xenon arc lamp (XBO100) (b) Emission spectra of a number of LED arrays used for fluorescence excitation.

Fluorescence Filter Sets

The fluorescence filter set performs a central role in ensuring an image is detected with high intensity and low background, especially in broadband light sources such as Hg, Xe and MH arc lamps. The filter set must be matched to the

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fluorescent probe spectral characteristics. This means making sure of the following points:

16.4.3.1

Excitation Filter

The central transmission wavelength should closely match the peak excitation wavelength of the fluorescence label and bandwidth should be sufficient to deliver enough light to the specimen. The filter should reject other wavelengths as strongly as possible to help ensure low background. Referring to the spectral emission curve of the light source, Ex(l) and the transmission curve of the excitation filter, X(l), we can estimate the relative excitation intensity, IE reaching the specimen for a given source and filter as a sum of products: IE ¼

X

Ex ðli Þ:Xðli Þ

i

where li represents the ith wavelength.

16.4.3.2

Dichroic Filter

The transmission cut-on edge wavelength should be close to the minimum overlap between the excitation and emission spectra of the fluorescence label. The dichroic mirror must strongly reflect the excitation light and show minimal transmission in the excitation spectral range. Typically, the transmission might be as low as 106 to ensure a low background image. The transmission side of the dichroic spectrum should match the fluorophore emission with high efficiency, typically 80–95%.

16.4.3.3

Emission Filter

The transmission spectrum should be close to the peak emission of the fluorescent label and emission bandwidth defines the wavelength range over which fluorescence light will be gathered. In order to ensure a resultant image with low background and good dynamic range, the emission filter should also exhibit strong rejection of the excitation light. Once again, we can estimate the relative emission intensity, IM reaching the detector for given a fluorophore and filter set represented by their normalised spectra, Em(l) and M(l), respectively: IM ¼

X

Em ðli Þ:Mðli Þ

i

There are many useful resources to visualise and explore the spectra of fluorescent probes and filters available on the World Wide Web e.g. http:// www.mcb.arizona.edu/ipc/fret/default.htm Investment in a tailored filter set for the chosen fluorophore is a worthwhile investment. Several companies manufacture high-quality filters, including


Figure 16.5

401

Spectral transmission of a matched filter set overlaid with SYBR Green normalised excitation and emission fluorescence spectra.

Chroma, Newport, Omega and Semrock. The transmission spectra of excitation, dichroic and emission filters of a set designed for fluorophores similar to FITC is shown in Figure 16.5. The filter spectra are overlaid with the excitation and emission spectra of SYBR Green one of our favoured nucleic acid dyes for Comet assay. Interference filters may be fabricated by evaporation of very thin layers of dielectric materials onto glass or silica substrates and consequently should be handled with gloves. Scratches lead to reduce contrast, and deposits of oil or other surface contamination will also adversely affect the performance of the filters. More recent technologies introduced by Semrock10 modify the substrate surface by ‘‘ion beam sputtering’’ in which ions are embedded into the substrate to modify its optical properties and provide interference filter performance. The resultant filters are hard, resistant to heat damage and scratching and can deliver exceptional spectral performance, as illustrated in Figure 16.5. Some older microscopes were designed primarily for observation by eye and may use a long-pass emission filter, which transmits infrared (IR) light. Some CCD cameras exhibit substantial sensitivity in the IR region that may result in high background or low contrast images. The solution is to obtain a bandpass emission filter, or, where suitable, fit an IR cut filter to the CCD camera. The latter will reduce sensitivity, so the former is preferred when performance is limited by light throughput.

16.4.4

Microscope Objectives

Since both the excitation and emission light pass through the microscope objective, it is important to make sure that the lens is capable of a high level of transmission at both wavelengths. Objectives designed for fluorescence imaging

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should always be used for best performance, these are usually marked as ‘‘Fluo’’ somewhere in their description. Objectives are also characterised by other imaging properties. For example, a plan objective is ‘‘flat field’’ corrected, meaning that it provides an image completely in focus across the full field of view. Achromats and apochromat objectives are corrected for chromatic aberrations at 2 and 3 wavelengths, respectively. Chromatic correction is important for fluorescence because it ensures that the excitation and emission focal planes are coincident. The most common lens for comet imaging is the 20 objective, which will typically have a numerical aperture (NA) of 0.5. NA is a measure of an objective lens’ ability to collect light, since the NA defines the collection half angle, y/2 as follows: NA ¼ n: sinðy=2Þ and n is the coupling medium refractive index For ‘‘air’’ or ‘‘dry’’ lenses, n ¼ 1.0, while for water n ¼ 1.33 and oil n ¼ 1.515. It is most common to use a dry lens for comet imaging as the resolution requirements are not extreme and it is important to ensure imaging of the entire comet distribution, which this usually accommodates. Note that use of a higher NA lens may produce a brighter image, but it will also reduce the depth of field as defined in the equation below. Depth of field, DF can be understood as the axial range over which the image remains in focus. DF ¼ 2 l:n=ðNAÞ2 where l is wavelength of operation Depth of field can be significant when it is wished to avoid too much refocusing during viewing of comet specimens, which tend to be distributed at different heights or focal planes in the LM agarose gel used in their preparation. A typical lens used for comet observation would offer 20 magnification with NA ¼ 0.5 (y/2 ¼ 30 degrees) and hence DF B 4 mm. It is clear then that focus of the specimen is important even though image analysis can overcome a small amount of defocus without a serious impact on results. Obvious, though frequently overlooked, is the fact that lenses must be kept scrupulously clean. Use only proper lens tissues and 70% ethanol for cleaning. Dry lenses must be kept free of immersion oil and oil lenses must be cleaned with alcohol after use. Dirty lenses can result in image distortion, low light throughput and generally poor imaging performance.

16.4.5

Beam-Splitter and C-Mount Adapter

Although the excitation light is delivered via the objective lens, fluorescence emission from the specimen occurs in all directions with equal probability, so only a fraction of the emission is collected by the objective lens and contributes to image formation in the microscope. This light passes back through the dichroic filter and is further selected by the emission filter, to produce an image of the specimen. The beam-splitter/prism directs the image of the specimen to


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the eyepiece or to the CCD camera, and in some cases, to both. The best condition for electronic imaging results when 100% of the light from the specimen is directed to the CCD camera. Such a prism is commonly referred to as a 100/100 port switch. In some older microscopes stray light from eyepieces and elsewhere in the instrument can find its way to the camera, resulting in poor contrast. For this reason, fluorescence microscopy is often carried out in a darkened room, with eyepieces covered. The fluorescence image, which is finally projected onto the CCD camera, must arrive in the correct optical plane for a sharp image. This plane should be ‘‘parfocal’’ with the eyepiece image to avoid refocusing between the eyepiece and camera. To achieve this and mechanical compatibility with most CCD cameras, a C-mount adapter is used. The C-mount is the almost universal CCTV connection and has been adopted by the majority of digital CCD camera manufacturers. The standard C-mount has a one-inch (25 mm) aperture and a thread of 32 turns per inch with the image plane located 17.5 mm from the shoulder of the adapter. In many cases the camera will have a CCD chip that is much less than one inch (commonly two-thirds-, half- or third-inch CCD chip) and so the field of view it sees will be a fraction of that projected into the eyepieces and the camera image plane. Hence, some C-mounts have de-magnifying optics built in to increase the camera field of view. Demagnification has the effect of concentrating light onto a smaller area and so can be used to increase effective intensity at the cost of resolution (distance per pixel). Common C-mount magnifications are 0.63, to match two-thirds-inch CCDs, and 0.5, to match half-inch CCDs. A 1 C-mount will not adversely affect imaging, however, provided the image is bright enough.

16.5 Image Detection – CCD, EMCCD and CMOS Cameras Imaging Comet assay specimens involves detection of the fluorescence emission image and conversion to a quantifiable electrical signal. The emission light is many orders of magnitude less intense than that delivered in the excitation beam and depends on source intensity, optical transmission of microscope components, and brightness index of the fluorophore as well as camera sensitivity. In a well-configured, modern microscope comet specimens are relatively bright and a moderately sensitive camera is usually adequate. At the time of writing there are essentially two forms of electronic sensor in widespread use. These are CCD (charge coupled device) and CMOS (complementary metal oxide semiconductor device). Both rely on planar silicon processing that is used in the production of integrated circuits. In this technology, electronic structures are created on crystalline silicon wafers using photolithography, epitaxial growth and surface modification by vapour deposition and/or ion implantation. Photolithography masks are designed to

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define the geometry of the ‘‘chip’’ from millions of individual circuit elements. After exposure to UV light the masks develop areas of ‘‘photoresist’’ where no modification is required and in a multistep process complex 3-dimensional structures can be created by modifying the electrical properties of the silicon crystal close to its surface.11 Silicon is not only a semiconductor of great value in manufacturing planar transistor circuitry, but also has excellent photoelectric conversion properties for visible and near-infrared (NIR) light. A combination of these properties is exploited to create sensors that convert twodimensional photon density distributions (images) into a matrix of electrical charge packets. The relationship of charge to intensity in CCD sensors is extremely linear over the dynamic range of the sensor. The matrix of charge packets is converted to a serial analogue voltage signal by sequential access and is converted to a digital image for computer analysis. The vast majority of scientific cameras in use today utilise the CCD image detector and the operating principles remain are outlined in an excellent review by McKay.12 As an example, a schematic of a 3-phase CCD crosssectional structure is shown in Figure 16.6(a). Each of the ‘‘pixels’’ or picture elements is defined by a set of three transparent, low-resistivity polysilicon electrodes that are isolated from the substrate and from each other by an insulating SiO2 layer. The pixel has a structure similar to a MOSFET transistor with an n-type implant in a p-type substrate. As voltage patterns are applied to the electrodes they control the electrical properties of the silicon substrate below. Incident photons in the visible wavelength range have a high probability of passing through the polysilicon gates, resulting in photoconversion to electron– hole pairs in the substrate. During the exposure or integration period the central electrode is held at a relatively high positive voltage, producing a depletion region or ‘‘storage well’’ below the electrode. Electrons resulting from photoconversion are attracted to the depletion region, while holes diffuse away into the p-type substrate. This structure, sometimes called a photogate, stores the resulting ‘‘charge packet’’ which is proportional to the local photon flux. Depletion regions have a finite capacity known as the ‘‘well-depth’’ that represents saturation of the detector and limits the exposure or peak signal intensity that can be measured. The photogate has three important strengths. First, it is simple to manufacture, second it has a high fill factor, i.e. much of the pixel area can be photosensitive, and third it can have a high well capacity. However, the polysilicon gates tend to reduce sensitivity, especially in the blue region of the spectrum, so to achieve sensitivity in the UV coatings are applied or other methods of illumination are employed, such as back thinning.12 CCDs and CMOS sensors differ primarily in their read-out methods. CCDs are constructed in one of three ways: frame transfer, full frame and interline devices are all widely used. In essence, they all rely on a series of parallel charge shift operations to a serial readout register. The charge packets in this register are transferred pixel by pixel to a single output stage, where charge is converted to voltage. The parallel shift is implemented differently in the three patterns, but the mechanism of charge transfer is similar. In commercial and scientific CCDs charge transfer is extremely efficient (typically more than 99.999%) and


Figure 16.6

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Schematics of the structure of silicon charge coupled device (CCD) imager. (a) Cross section of the planar device illustrating the charge integration (exposure) phase. (b) Front view of an ‘‘interline’’ CCD illustrating the parallel shift of pixel charge packets into vertical storage registers. The vertical registers are shifted incrementally into the horizontal readout register and then to the charge-conversion stage resulting in a voltage sequence representing the image data.

because there is a single output stage the detectors show very low PRNU (photoresponse nonuniformity). The read-out process, especially the charge conversion stage, generates ‘‘read noise’’, which ultimately limits the sensitivity of the detector. The lower the read noise, the lower the signal that can be detected with confidence. In modern high-performance CCD cameras, read noise as low as 6–10 electrons rms can be achieved, while in a mid-range camera this is likely to be in the range 20–100 electrons rms.

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In CMOS sensors the readout method is by sequential access to each photosensor by row and column decoders much like memory chips, but the readout is analogue so that the signal is proportional to the charge stored in the photosensor. The charge-conversion stages are switched for each line or even each pixel and therefore show much higher variations than CCDs, but access can be much faster. Because CMOS access circuitry requires metallised electrodes for row and column access in addition to the photogate, the sensitivity of frontilluminated CMOS sensors is typically rather low. Consequently, CMOS detectors have found most application in high-speed, high-light applications and are much less common in scientific imaging, where sensitivity and uniformity of response are more desirable. An important source of noise in all silicon detectors is ‘‘dark current’’, which results form the spontaneous creation of charge pairs in the semiconductor even when not exposed to light. Dark current increases rapidly with substrate temperature and so scientific devices are often thermoelectrically cooled. Moderate cooling, e.g. 20–40 1C below ambient results in very low dark current and is sufficient for cameras used in most fluorescence imaging applications. Silicon has a bandgap of about 1.124 eV (the energy that has to be injected to release an electron following the absorption of a photon) which makes it sensitive to light in the visible-NIR part of the spectrum from 400–1100 nm.13 This matches well the fluorophores developed for labelling cells for visual inspection. Figure 16.7 shows the spectral response of a typical mid-range CCD detector. The average number of photoelectrons collected from a given number of incident photons defines the quantum efficiency (QE) of the detector – usually quoted as a percentage. The QE is equivalent to the quantum yield defined in the section on DNA labelling. This figure shows that CCD and EMCCD

Figure 16.7

Spectral response of a typical mid-range CCD detector.


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cameras show good spectral response in the visible range, especially the green (480–540 nm) and the detector is well matched to a number of the fluorophores listed in Table 16.1. The EMCCD detector is very similar in structure to the CCD, but in the read-out phase it includes a ‘‘gain register’’ that can multiply the photoelectron signal charge. The operation of the gain register relies on ‘‘impact ionisation’’, a stochastic process that can occur when charge transfer is subject to an increased electric field. In the readout sequence, one electrode is driven at an elevated voltage resulting in a high electric field and electrons are rapidly accelerated towards the next storage area. Energetic electrons may impact other electrons in the silicon lattice and transfer sufficient energy to free them from their bound states (B1.124 eV). Electrons freed in this way will be added to the charge packet and transferred to the output resulting in signal gain. The probability of impact ionisation in a high-performance EMCCD sensor is only 1.5%, but after 500 transfer stages the net gain can be many thousands.14 EM amplification is quieter than external electronic amplification because it occurs before the injection of readout noise. EMCCD-based cameras are primarily used for very low light imaging and not generally used for comet studies, where signals are considered to be of a moderate light level. However, we have used EMCCD cameras for comet imaging with considerable success and recently introduced a comet analysis system including an EMCCD camera as standard.

16.5.1

Practical Matters

Many laboratories use inexpensive 8-bit video CCD cameras to deliver highquality signals for analysis. However, if any part of the imaging system is compromised, an 8-bit camera risks inadequate dynamic range. If the signal is weak or has low dynamic range subsequent analysis may be unable to detect the less bright fragments in the comet tail, resulting in underestimation of damage. On the other hand, cooled digital CCD or EMCCD cameras with grey-scale resolution of 12–16 bits are more expensive, but deliver a higher dynamic range and ease the task of detecting the Comet tail without saturating the head region in the detector. EMCCDs have the advantage that in the case of weak signals they can amplify the signal on the sensor and effectively improve the final signal to noise ratio, but this is only of value when the background is low. In practice, the real choice comes down to flexibility and the ability of the imaging system to measure biologically significant changes in the samples. There are many factors that can be adjusted to adapt the specimen processing to provide that sensitivity apart from the camera, and as stated previously an 8bit camera is adequate when the remainder of the system is well configured and maintained. Some examples of images from a well-configured instrument operating with an 8-bit CCD camera are shown in Figures 16.8 and 16.9. It will be apparent that the images show all the necessary properties of high contrast, low

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Figure 16.8

(a) Frozen duodenum cells from untreated rat (b) Frozen urinary bladder cells from same untreated rat.

Figure 16.9

Frozen duodenum cells from rat exposed to 300 mg/kg EMS for 4 h.

background and wide dynamic range. Images of this quality are more than adequate for the digital image analysis that follows.

16.6 Image Processing and Comet Scoring Over the last twenty years, a wide range of imaging and analysis systems and software have been developed for the Comet assay.15 Both interactive and


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automated imaging systems are used for analysis, but in automatic systems the absence of a supervisory operator can result in a number of pitfalls and the potential for erroneous analysis. Publications16,17 have shown that automated systems can be used to establish concordance with interactive systems. But issues remain with automated systems such as scoring of artifacts or potential bias in machine selection rules choosing features for analysis within a narrow morphology or intensity range. Whether manual or automatic analysis is involved, the image capture and visualisation processes are similar. Therefore, we will address the principle tools used for comet imaging and analysis rather than specific implementations. Scoring specimen slides is typically done in a blind manner – the scorer has no prior knowledge of which slide is being evaluated. The intention is to avoid any scorer bias. Whether the scoring system is manual or automated, the following sequence will normally be followed. Select and mount specimen slide and capture/enter slide ID. Scan the slide by manual or motorised stage and select a field with candidate comets for analysis. Focus slide prior to analysis. Capture digital image. Execute image segmentation and analysis sequence. Present results to scorer or machine and save data and images for review. Review may be immediately after capture in an interactive system or in a subsequent step after storage to ensure data are robust. Repeat for required number of cells per specimen slide. Choose next slide and repeat from step 1 until scoring complete or suspended.

16.6.1

Image Analysis

Image analysis requires a series of operations, which we will consider in broad outline here. Specifics will vary among the various private, commercial and public-domain programs available, but will be broadly similar. Note that the following assumes that the imaging system, including microscope and camera are set up to ensure relatively uniform illumination, high contrast fluorescence images that lie within the dynamic range of the camera as defined in the previous sections.

Segment the image into regions of background and comet and ‘‘debris’’. Select contiguous comet region for analysis. Correct for background fluctuations. Identify comet components – head and tail. Perform distribution intensity analysis. Report and record results.

We will discuss these steps in a general way to avoid too many specific details.

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Segmentation

Segmentation involves separation of features of interest from background and requires a second-level algorithm to distinguish valid features from debris. Interactive systems rely on a user to highlight the feature of interest with a cursor of some kind and then the analysis can proceed to identification of comet, head and tail. Automatic systems commonly use intensity differences between features of interest and background. The features of interest are separated or ‘‘masked’’ from the rest of the image and analysed further. The simplest automated technique relevant to fluorescence-labeled comet specimens is to use a so-called threshold, where pixels in the image are classified to one of two states – ‘‘1’’ or ‘‘0’’. Classification is by comparison of each pixel in the image, P(x, y) to the threshold value T and the results in M(x, y) a binary mask image – a template for feature selection. IF Pðx; yÞ > T then Mðx; yÞ ¼ 1 ELSE Mðx; yÞ ¼ 0 In this technique selection of T becomes a key step in the analysis. One popular approach to threshold selection is based on analysis of the image histogram, which describes the distribution of pixel grey-level values within the image. An example of a histogram from an image region of a comet slide and is shown in Figure 16.11, where two distributions have been overlaid to illustrate the next point. Algorithms for the selection of T are usually designed to maximise the probability of selecting the feature versus the background and, provided the image background is uniform, a global threshold can be estimated. A popular approach is to assume that the image is made up of two classes – features and background as shown in Figure 16.11 – and to estimate the grey level that minimises the classification error. The simplest method and the one used here is not rigourous in that it does not actually minimise error, but is a good

Figure 16.10

(a) untreated CHO-K1 cells (b) CHO-K1s treated with 10 mM EMS for 3 h.


Figure 16.11

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Comet image histogram with background and comet distributions – scaling of frequency by log10 allows easier visualisation of the real data. The simple smoothed method (SSM) of estimating threshold value selects the grey level corresponding to the first local minimum after the background peak frequency.

approximation and relies on identifying the first local minimum of the smoothed histogram to the right of the grey-level mode (background). The histogram is smoothed to reduce the influence of noise and we refer to this method as SSM (simple smoothed minimum). A more rigourous approach was first described almost 30 years ago and is known as Otsu’s method.18 This calculates the threshold separating two classes to minimise their combined variance in a simplified iterative algorithm that incrementally cycles through each grey level of the image and chooses the global minimum. There are many other algorithms aimed at achieving global error minimisation. On the other hand, algorithms have been developed to deal with images that show uneven illumination (shading) resulting in varying background and/or feature intensities. These algorithms operate on smaller regions of the image, where local statistics are considered more uniform and are referred to as ‘‘adaptive’’ threshold methods.19,20 In the examples we present here, we assume that the images have been acquired on a well-configured system and that the illumination is uniform. Hence, we apply SSM segmentation. Figure 16.12(a)) shows an original digital image captured from a comet slide and Figure 16.12(b)) shows the mask image M(x, y) resulting from the SSM threshold. Further morphological processing of the mask image M(x, y) is required to remove isolated pixels resulting from noise in the image, to join groups of pixels to create contiguous regions and finally to apply region size ‘‘filters’’ to define Comets. Figure 16.12(b)) shows M(x, y) with threshold noise (isolated pixels

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Figure 16.12

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(a) Captured image of a field from a comet slide. (b) Mask image after automatic threshold with background noise speckles. (c) Mask image after median and closing processing operations. (d) Selected comets after masking and prior to analysis – white rectangles show the maximum extent of the comets.

throughout the image) that is easily removed by single-pass median and closing operations.21 The result is shown in Figure 16.12(c)), where some debris remains on the left-hand side, but this is easily removed by size filtering. Filters for comet analysis are often based on properties such as area, A (number of connected pixels of value 1), perimeter, P (e.g. number of edge pixels), which may be combined into shape descriptors of the region, e.g. circularity, C: C ¼ 4pA=ðP2 Þ note that for a circular feature C ¼ 1.


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Figure 16.12(d) shows the extracted comets from the raw image using the mask regions. Comet intensity distributions are analysed further to provide quantitative measures of migration.

16.6.3

Further Segmentation – Identifying Head and Tail of the Comet

The idea of head and tail was developed to distinguish between the intact DNA in the comet (head) and the damaged DNA, separated by electrophoresis into the tail. It should be noted however, that although this idea is an accepted convention it is not a necessary concept and direct analysis of the comet intensity distributions can deliver meaningful (possibly more meaningful!) results. We will discuss this briefly in the comet parameter section. The separation of head and tail regions from the comet intensity distribution requires that a model be fitted to the data. The model most commonly applied assumes the head is a circular feature or at least a symmetrical profile – similar to the distribution seen in ‘‘undamaged’’ (or endogenously damaged) negative control cells – see Figure 16.10). A common approach to fitting either a circular region to the grey-level segmented image or a symmetrical intensity distribution utilises the leading edge of the comet as the template for the region and defines the circle diameter and front edge. A typical fitted circular feature is shown in Figure 16.13. Pixels falling on or within the circumference of the circle are considered comet head pixels, while those falling within the comet mask, yet outside the circumference and to the right of the centre of the circle are tail pixels. The final step in preparing the image data for intensity distribution analysis is to subtract the mean grey level of the image background in each column to correct for offsets resulting from specimen, fluorophore and camera background levels. The mathematical result of the overall segmentation and correction exercise is a two-dimensional distribution for each feature as follows: C(x, y) – the comet x, y distribution; H(x, y) – the comet head x, y distribution; T(x, y) – the comet tail x, y distribution; and C(x, y) ¼ H(x, y)+T(x, y).

16.6.4

Analysis of the Comet, Head and Tail Distributions

Once the comet has been segmented and background corrected, the detailed analysis of the intensity distributions is performed. Note that all of these data need to be considered as relative, i.e. no absolute analysis is possible because there are so many variables in the system. But in the context of a well-designed experiment with positive and negative controls the comparisons provide extremely valuable and biologically relevant information. For this discussion we will consider that the comet, head and tail distributions are converted to 1D intensity profiles for final-stage analysis because they

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Figure 16.13

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The image shows segmented comets with head region identified by the white circles – intensities outside of this region and to the right of the centre of the circle are considered comet tail. Comet length is defined as the length of the white rectangle. Head diameter is the diameter of the white circle. Tail length is defined as the distance from the right-hand edge of the circle to the right-hand edge of the white rectangle.

have been subject to a uniform 1D electric field aligned with the X-axis to induce the migration. This is achieved by integration in the Y-axis and results in profiles shown in Figure 16.14 and defined in the equations below, where start to end refers to the top and bottom of the rectangular regions of interest (ROI) shown in Figures 16.12(d) and 16.13. P C(x)¼ P C(x, y) for y from top to bottom ROI H(x)¼P H(x, y) for y from top to bottom ROI T(x)¼ T(x, y) for y from top to bottom ROI The resulting profiles are considered as distributions and subject to distribution analysis. Parameters extracted from the comet, head and tail intensity distributions were defined in the early days of comet development by imageanalysis developers15 and researchers interested in establishing key descriptors of biological significance. Some relevant publications include22–27 the last being a review of distribution parameters.

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Imaging and Image Analysis in the Comet Assay Integrated Intensity

Cmax

Ht k

2cmd-k

a

cmd

Figure 16.14

Example of a comet intensity distribution after integration in the vertical axis (perpendicular to the electrophoresis axis) and background subtraction. Cmax is the peak intensity of distribution. Ht is the head threshold setting. Cmd corresponds to the centre of the white circle in Figure 16.13, k is the front edge of the white circle and (2Cmd–k) is the right hand edge of the white circle. a is the right hand edge of the white rectangle.

Prior to distribution analysis descriptors included comet length as measured by image analysis16 and visual scoring using a classification scheme (5 classes, 0–4 with 0 ¼ no damage and 4 ¼ very heavy damage) as described by Collins.32 In 1990, the first measurement of tail moment was proposed,17 which was later named ‘‘Olive tail moment’’ (OTM) to distinguish it from a similar, but different measure called the tail moment (TM) that was proposed by Browne15 and published in Ashby.22 The OTM is the product of the mean migration distance and % tail DNA, while the TM is the product of tail length and % tail DNA. OTM is a more robust parameter than TM because it is intensity weighted and therefore relatively insensitive to the threshold setting which defines the tail length. Table 16.3 and Figures 16.13 and 16.14 explain the calculation of the basic distribution parameters. The idea of a moment is borrowed from physics, e.g. moment of inertia, and also from mathematics. The nth moment of a real-valued function f(x) of a real variable about a value c is defined as follows: m 0n

¼

ZN N

ðx cÞn f ðxÞdx

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Table 16.3

Calculation of the key comet distribution parameters, see Figure 16.14.

Measure

Units

Integrated Intensity %HDNA %TDNA Comet Length Head diameter Tail Length

arb None None mm mm mm

Comet

Head

Tail

P C(xi)

P PH(xi) P H(xi)*100/ C(xi)

P T(xi) P P T(xi)*100/ C(xi)

See figure 16.13 See figure 16.13 See figure 16.13

In discrete functions, like the comet intensity distributions, the nth moments Mn can be calculated as: Mn ¼

X

ðxi cÞn Cðxi Þ with xi ¼ xs . . . xe

The first moment about zero is the mean, m or the centre of gravity of the distribution, so OTM can be further defined as the first moment about zero of T(x) multiplied by % tail DNA. Higher-order moments are more recognisable if c in the equations above is set to m, when they are called the central moments. Then, the second central moment is the variance, the third central moment is the skewness and the fourth central moment is the kurtosis. These measures may all be extracted from the comet, head and tail distributions, but their biological significance in characterising DNA damage in the comet assay is not at all obvious. Nonetheless, the correlation between these parameters and known levels of DNA damage has been studied,21,22 and concludes that only OTM and % tail DNA are strongly correlated with known levels of damage induced by gamma radiation and therefore suggests that we dispense with other measures. Note: The primary comet measures accepted for publication and recommended by the scientific community are % tail DNA, tail length and Olive tail moment. This was agreed by the working groups of the ‘‘International Comet Workshop’’ held at the University of Ulm in Germany during July 2001 and is still accepted today.

16.6.5

Comet Analysis – Other Approaches

As mentioned previously, although comet head and tail became the standard way of considering the DNA distribution analysis, it is clear that analysis of the entire comet distribution has some advantages. Not least is the fact that that the separation into head and tail regions, a potential source of error and an arbitrary step is avoided.

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The normalised comet moment analysis is a measure of the whole comet distribution that is defined as follows: CM ¼

X

CðiÞ:xðiÞ=

X

CðiÞ

This measurement weights each column integrated intensity by its distance from the start of the comet distribution as defined in the segmented comet image. So, like the Olive tail moment, it takes into account not only the migration distance, but also the quantity of DNA that has migrated at each distance. The result is a parameter that has what is sometimes called a ‘‘fat zero’’, i.e. the negative control value has a significant baseline. But this does not alter the capacity to detect statistically significant variations in its value. Correlation of comet moment parameters with dose of known DNA-damaging agents, e.g. X-radiation shows it is as powerful as the Olive tail moment, without the need for head–tail segmentation. In 1995 Hellman24 presented the comet moment and comet inertia, first and second moments of the comet distribution and found them to be sensitive measures of damage. In the same year, Kent published a paper25 evaluating the use of the comet ‘‘moment of inertia’’ in which the segmented 2D Comet distribution, e.g. Figure 16.12(d)) was treated as a rigid body and this approach also showed good sensitivity and correlation with DNA damage. In 2004, Dehon26 published yet another approach based on global curve fitting to comet distributions. It seems this topic will be revisited in the literature as researchers continue to explore new ways of presenting and evaluating the DNA distributions resulting from the Comet assay.

16.7 How Many Cells, How Many Replicates? A number of studies have been published showing that the accumulation of data from multiple single-cell analyses rapidly converge to stable mean values.30 Even in comet specimens where heterogeneity can be high, the mean settles down to a stable estimate of any of the comet parameters after about 50–75 cells in a single replicate. Further, from the same and other published studies, it is recommended that two or three replicate specimens should be scored per exposure unit or individual.29 One common failure in study design is the failure to perform a prestudy statistical power calculation, resulting in an inadequate sample size.28 Based on the goal of 80% power to detect a 20% effect, Smith29 shows that the optimal group size in pharmacological exposures is at least 4 individuals per group and recommends 6 individuals per group to cope with unforeseen losses. In environmental or biomonitoring studies where exposure levels are unknown or a weak effect is under examination, groups will be much larger, even in the hundreds, to improve statistical power. More individuals can help to compensate for high variability in biological response.

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16.7.1

Chapter 16

Data Presentation and Preparation for Analysis

Data are normally stored at a single-cell level in a comet database or in textbased numerical files. Examples of data presentation are shown in Figure 16.15 where we can see three-dimensional histograms comparing exposure responses. This may be presented by exposure unit or by group as desired. This is a useful visual presentation and helps us to appreciate the shape of the distributions of the data, but statistical analysis is required to establish the scientific meaning of the results. For comparative analysis the data need to be aggregated in some way. This is important for statistical analysis, which is the ultimate tool used to establish relationships between exposure and biological effects. An important part of this process is to relate the data gathered during the ‘‘blind scoring’’ process (see Section 16.6) back to the exposure information of the original specimens. This is done by decoding the preassigned exposure unit ID back to the real exposure information. Decoding is carried out by a supervisor or study director, who will be responsible for ensuring the scientific integrity of the work. Once the data are reassociated with the exposure information, data summarisation is performed. We have developed a software tool for this task that is

Figure 16.15

Data presentation in the 3D histogram from a database viewer, which allows graphical comparison of the comet data from multiple exposures, either between individual exposure units or exposure groups.


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summarised here. The summary approach follows the recommendations of Hartmann.25 A table is prepared that presents exposure group statistics on the outer level with exposure units statistics on the inner level. Individual exposure units are presented under their exposure group heading. For each individual the mean or median value of the % tail DNA, OTM and perhaps tail length is presented, along with standard error (SEM) for each parameter and Nc the number of comets scored. Then, for each group the exposure level is presented along with the mean of the means or mean of medians of the individual exposure units, the standard deviation (SD) for the group and Ni, the number of individuals in the group. These data are listed on a group by group basis. The groups will consist of at least a negative control (unexposed) and one or more exposed groups and where possible a positive control as indicated previously. The data summary is now complete and ready for use with the statistical package for analysis. There are many packages available, but they all provide the tools outlined in the next section.

16.7.2

Statistical Analyses

Statistical tests are a subject of debate and many errors, but the following is proposed based on the recommendations of De Muth.31 First, test for the normality of the data in the individual exposure units, i.e. are the data normally distributed? If the answer is yes, parametric tests such as the t-tests can be used to compare exposure groups. Prior to use of parametric analysis it is necessary to test for, and correct if necessary, the equality of variances between the groups. In the case of positively skewed data log transformation of the data may be sufficient. When parametric tests are used it is important to apply the tests as paired or unpaired. Unpaired means that the exposure groups under study are independent, i.e. come from separate individuals, while paired means that the groups come from the same individuals and would include the ‘‘before and after’’ exposure regimes. Paired tests tend to be more powerful because individuals serve as their own control. Data that are not normally distributed should be subject to nonparametric statistical analysis. These tests include Mann–Whitney. If an exposure related response is under examination, as in pharmaceuticals for example, then regression testing may also be applied. These techniques should also be selected based on the normality of the data, i.e. selection of parametric vs. nonparametric statistical tools. One final point relates to use of the confidence value (1p) and the null hypothesis. Note that p indicates the predicted error rate and so (1p) is the probability that the conclusion is correct, i.e. if p ¼ 0.05, then 95% of the time the conclusion is correct. (1p) is also called the confidence level and it is strongly recommended that 95% confidence is chosen before rejecting the null hypothesis. The null hypothesis is the outcome if the statistical test does not

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result in po0.05 (95% confidence). As De Muth hypothesis is never proven, we only fail to reject it’’.

16.7.3

31

points out, ‘‘the null

Data Storage and Management

The Comet assay is becoming increasingly important not only as a research tool, but also in safety assessment for human exposure to chemicals, pharmaceuticals and medical devices. The flexibility of the assay allows its application in a multiplicity of exposure regimes from acute testing to chronic environmental effects. When handled with appropriate attention to detail, it provides a most sensitive tool to detect biological events that may be precursors to disease. Since the inception of good laboratory practice (GLP) in the 1970s, the rigour of the analytical process and the management of the resultant data have been of major concern to regulatory bodies, whose job it is to protect the general population from exposure to hazardous or potentially hazardous materials. Therefore, comet data should be stored in a manner that allows their review and further evaluation for the future. As more information about test substances or environments becomes available, the information may have more to reveal. For this reason, we recommend storing comet data in an accessible database format with appropriate detailed descriptions of format for future readability. In addition, maintaining images of the comet cells along with the

Figure 16.16

Example of a database gallery from a comet study (courtesy of Helix3 Inc.). Data stored in this way is amenable to long-term archival, training, review and further statistical analysis.


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extracted data and circumstances of quantification (audit trail) provides both visual and analytical information for future review. An example of such a storage system is presented in Figure 16.16 that shows an example of a ‘‘gallery’’ of some comet images in a particular treatment group from the comet database of an acute exposure study (courtesy of Helix3 Inc.).

16.8 Conclusions The Comet assay has grown over the last twenty years from an interesting research tool to the most sensitive and flexible assay available for safety testing and exposure risk analysis. Although not validated in the generally accepted sense, Comet data are now in demand by regulatory agencies the world over. The scientific insight they can provide with appropriately designed studies is substantial. As comet data become increasingly widely used it is important to consider the life cycle of the data and its protection for the future. Rigour in analysis applies not only to the exposure, sampling and preparation of specimens, but also to their subsequent quantification, data extraction, organisation and storage. For these data to maintain their value we must manage them and ensure their availability to future generations of scientists.

References 1. T. A. Brown, in Essential Molecular Biology, Ed. T.A. Brown, Oxford University Press, Oxford, 2000. 2. N. Singh, M. McCoy, R. Tice and E. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 1988, 175, 184–191. 3. P. Olive, Banath and R. Durand, Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using a ‘‘Comet’’ assay, Radiat. Res., 1990, 122, 86–94. 4. J.-M. Exbrayat, in Visualization of Nucleic Acids, Ed. G. Morel, CRC Press, 1995, p. 17. 5. G. McNamara, M. J. Difilippantonio and T. Ried, in Current Protocols in Human Genetics, John Wiley & Sons, Inc., 2005, 4.4.1–4.4.34. 6. J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, Springer Science+Business Media, LLC, 3rd edn., 2006, pp. 59. 7. I. Gryczynsk, J. Malicka, Z. Gryczynsk, J. Lakowicz and C. Geddes, Dramatic Increases in Resonance Energy Transfer Have Been Observed Between Fluorophores Bound to DNA Above Metallic Silver Islands: Opportunities for Long-Range Immunoassays and New DNA Arrays, J. Fluoresc., 2002, 12(2). 8. S. Inoue and K. Spring, in Video Microscopy: The Fundamentals, Springer, 1997. 9. J. Beacher, LEDs for Fluorescence Microscopy, Biophotonics, 2008, 2.

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10. T. Erdogan, New Optical Filters Improve High Speed Multicolor Fluorescence Imaging, Biophotonics, 2006, 3. 11. B. Jahne, in Practical Handbook on Image Processing for Scientific and Technical Applications, CRC Press, 2nd edn, 2004. 12. C. Mackay, Charge Coupled Devices in Astronomy, Astron. Astrophys., 1986, 24, 255–283. 13. D. P. Murphy, in Fundamentals of Light Microscopy and Electronic Imaging, Wiley Liss, 2001. 14. A. Basden, C. Haniff and C. Mackay, Photon counting strategies with low light level CCDs, Mon. Not. R. Astron. Soc., 2003, 345(3), 985–991. 15. M. Browne, ‘‘Fenestra Comet 1.0-the first commercial system developed specifically for Comet Image Analysis’’, Kinetic Imaging, Liverpool, UK, 1991. 16. G. Dehon, L. Catoire, P. Duez, P. Bogaerts and J. Dubois, Validation of an automatic Comet assay analysis system integrating the curve fitting of combined Comet intensity profiles, Mutat. Res., 2008, 650, 87–95. 17. W. Frieauff, A. Hartmann and W. Suter, Automatic analysis of slides process in the Comet assay, Mutagenesis, 2001, 16, 133–137. 18. N. Otsu, A threshold selection method from gray-level histograms, IEEE Trans. Magn., 1979, 9, 62–66. 19. J. Kittler, J. Illingworth and J. Fo¨glein, Threshold selection based on a simple image statistic, Comp. Vision Graph. Image Proc., 1985, 30, 125–147. 20. M. Wilkinson, Optimizing edge detectors for robust automatic threshold selection: coping with edge curvature and noise, Graph. Mod. Image Proc., 1998, 60, 385–401. 21. J.-L. Starck, F. Murtagh and A. Bijaoui, in Image Processing and Data Analysis: The Multiscale Approach, Cambridge University Press, Cambridge, 1998. 22. J. Ashby, H. Tinwell, P. Lefevre and M. Browne, The single cell gel electrophoresis assay for induced DNA damage (Comet assay): measurement of tail length and moment, Mutagenesis, 1995, 10, 85–90. 23. T. Kumaravel and N. Awadesh, Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals, Mutat. Res., 2006, 605(1-2), 7–16. 24. B. Hellman, H. Vaghef and B. Bostro¨m, The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay, Mutat. Res., 1995, 336(2), 123–31. 25. C. R. Kent, J. J. Eady, G. M. Ross and G. G. Steel, The comet moment as a measure of DNA damage in the Comet assay, Int. J. Radiat. Biol., 1995, 67(6), 655–660. 26. G. Dehon, P. Bogaerts, P. Duez, L. Catoire and J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the Comet assay, Chemometrics and Intelligent Laboratory Systems, 2004, 73(2), 235–243. 27. T. Kumaravel, B. Vilhar, S. Faux and N. Awadesh, Comet Assay Measurements: A Perspective, Cell Biology and Toxicology, 2009, 25(1), 5–32.


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28. P. Duez, G. Dehon, A. Kumps and J. Dubois, Statistics of the Comet assay: a key to discriminate between genotoxic effects, Mutagenesis, 2003, 18, 159–166. 29. C. Smith, D. Adkins, E. Martin and M. O’Donovan, Recommendations for design of the rat Comet assay, Mutagenesis, 2008, 23, 233–240. 30. A. Hartmann, M. Schumacher, U. Plappert-Helbig, P. Lowe, W. Suter and L. Mueller, Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations, Mutagenesis, 2004, 19, 51–59. 31. J. De Muth, in Basic Statistics and Pharmaceutical Statistical Applications, CRC Press, 2006. 32. A. Collins, M. Dusinska, M. Franklin, M. Somorovska, H. Petrovska, S. Duthie, L. Fillion, M. Panayiotidis, K. Raslova and N. Vaughan, Comet assay in human biomonitoring studies: reliability, validation and applications, Environ. Mol. Mutagen., 1997, 30, 139–146.

CHAPTER 17

Statistical Analysis of Comet Assay Data DAVID P. LOVELL Department of Biostatistics, Postgraduate Medical School, University of Surrey, Daphne Jackson Road, Manor Park, Guildford, Surrey, GU2 7WG, UK

17.1 Introduction The single-cell gel electrophoresis (SCGE) or Comet assay is a quick, relatively simple and economic method for the investigation of single- and double-strand breaks in DNA. The assay has been used in in vivo and in vitro experimental approaches across a range of species as well as in human studies and other biomonitoring investigations. It is now used to assess the genotoxicity of chemical and physical agents. The method is increasingly accepted by regulatory authorities in their assessment of the genotoxicity of chemicals1 and initiatives are underway to develop OECD guidelines for an in vivo version, while in vitro methods are being investigated with the objective of future validation.2 Special issues of Mutagenesis (2008, 23, 143–240), Mutation Research (2009, 681, 1–109), and Cell Biology and Toxicology (2009, 25, 1–98) provide overviews of the fields of research where the Comet assay is now used. The standard alkaline Comet assay detects strand breaks and acid-labile sites3 but since its first description in the 1980s the fields of research the assay has been applied to have grown and it now exists in a number of forms with new applications of the methods continuing to be developed, such as the development of assays including lesion-specific enzymes.4 This has resulted in a series of protocols that have undergone various modifications depending upon


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the proposed use. However, general recommendations and guidelines for carrying out the Comet assay have been produced.5–7 The purpose of this chapter is to discuss some of the experimental design and statistical analysis issues associated with the use of the Comet assay. The objective is to discuss some of the statistical concepts underlying the design of comet experiments with emphasis on aspects of experimental design as opposed to a detailed mathematical treatment of different statistical methods. In this context, the link between the experimental unit (a term that has a very precise meaning in the context of statistical methodology) and the statistical analysis is critical. More detailed discussion of statistical methods can be found in Lovell et al.8 and Lovell and Omori9 that include sets of recommendations.

17.2 Experimental Design and Statistical Analysis Although researchers often concentrate much of their attention on the specific methods used for carrying out statistical tests it is important to appreciate that this is only part of the statistical input into the design of Comet assay studies. Experimental design can be viewed as strategic, while the statistical analysis of the data obtained is more tactical. The analysis applied may, thus, be somewhat secondary or consequential to the work that had gone before into the design of a successful study. It is crucial, therefore, to involve statistical expertise at the design stage. It is frequently stated, but unfortunately sometimes ignored, that a statistician should be consulted before starting a study. This continues to be extremely relevant. Failure to seek or act on statistical advice can lead to a poor design with the consequence that subsequent statistical analysis is either suboptimal or impossible. Such an event, particularly where it involves human subjects or experimental animals, is both ethically and economically unsatisfactory. No amount of statistical ‘‘wizardry’’ or virtuosity can rescue a badly designed experiment. If this were the only point the reader takes away from this chapter then a major objective would have been achieved. The advice is, perhaps, even more relevant than in the past. Statistical software has become increasingly easy to use, some comes with the sales ‘‘pitch’’ that statistical analyses can be carried out without needing the help of a statistician, instrumentation systems and apparatus often includes statistical analysis options embedded in the equipment. This increased convenience comes with the risk, unless the researcher is careful, of introducing serious errors into the analysis and interpretation of studies.

17.3 Study Design Many of the experimental design and statistical issues related to the use of the Comet assay are also relevant to other mutagenicity tests as well as, in general, to other biological systems. Many of the points made in this chapter can, therefore, be applied more generally.

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Statistical methods used in mutagenicity and other toxicology studies have traditionally been based upon approaches using hypothesis testing with the reporting of the probability associated with the testing of a null hypothesis. The findings are often reported as statistically significant when the probability or P values are below certain critical values. Formerly, the researcher had to compare the test statistic obtained from the experiment with critical values published in special sets of statistical tables.10 Now, the test statistics and their associated P values are provided as part of the output of a software package. One main criticism of the hypothesis-testing approach is that the fact that a result is statistically significant does not mean that it is a large or a biologically important result. This is a symptom of the much greater problem of equating significance testing and P values with decision making. Many statisticians have argued for a move away from formal hypothesis testing to one based more upon the estimation of the size of effects detected in a study together with some measure of the uncertainty associated with the estimate (such as a confidence interval).11,12 A number of journals have followed the British Medical Journal’s approach13 in their guidelines for publications, stressing estimation over P values. A clear objective, with a realistic chance of achieving this objective, is a crucial aspect of any study design. An example is whether a study is a dose– response investigation and whether the objective is to identify an effect of a given size. The objective ties in with the concept of the power of a study where power is the probability of detecting an effect of a given size if it is really present (or the probability of rejecting the null hypothesis where it is false). Comet assays can be divided into three main areas of investigations: human, animal (in vivo) and in vitro studies. These studies may be inferential or descriptive and may also be observational or experimental. Inferential studies require a comparator (or control) group and the objective is to identify differences between the groups. Descriptive studies do not, generally, involve hypothesis testing, instead they focus on providing an accurate description of the variables under some specified conditions. They can often be considered as hypothesis-generating studies. Experimental and observational studies differ in the degree of intervention and the relationship with causality. Most in vivo and in vitro studies are experimental studies. In the case of human studies the potential is for a ‘‘gold standard’’ randomised control trial (RCT) or a less well controlled (quasiexperimental) comparative study. In both cases, the treatment is administered and a cause–effect relationship is sought. The statistical methods for the analysis of experimental design and observation studies are similar but differ in some major respects. In the experimental study an intervention is applied to one group (such as the treated group in the classic RCT) and the effects ‘‘caused’’ by this intervention assessed. In observational studies an attempt is made to find an association (using, for instance, the Bradford Hill causality criteria14). In the observational study membership of the groups is a consequence of how the groups are defined, while in the experimental case it is (or should be) by randomisation. It is important to remember that the statistical tests (such as the


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t-tests or analysis of variance) applied to both observational and experimental studies will produce numerical results even though the assumption is that the data are from a designed experiment. However, not all the assumptions underlying the statistical tests will be met in the observational study and this can result in biases being introduced. Copas and Li wrote: ‘‘observational studies are often analysed as if they had resulted from a controlled study, and yet the tacit assumption of randomness can be crucial for the validity of inference’’.15 The key concepts of experimental design are: independence of experimental units, randomisation, replication and local control. These concepts were first proposed by R.A. Fisher in the 1920–30s and have undergone much subsequent development.16–18 Fisher’s work on the factorial and similar designs provides powerful methods for the investigation of planned factors while controlling inaccuracy and estimating precision. Fisher’s work laid the basis for the important and increasingly influential field of design of experiment (DOE) methodology.18 Factorial designs are particularly powerful as they provide an efficient way of exploring both the main and interaction effects of experimental factors using relatively small numbers of experimental units (which, of course, has implications for the 3Rs and animal usage). The use of DOE approaches instead of the traditional OFAT (one factor at a time) approach is an important methodological development. It is also an entry into more complex designs suitable for the investigations of mixtures and interactions. Factors that could be examined without further use of resources include the effect of sex, different treatments and diets. An example might be the investigation of factors relevant to optimising electrophoretic conditions in the Comet assay.

17.4 Endpoints The Comet assay is a quantitative or semiquantitative method. The identification of the endpoint to be measured is an important aspect of the study. The measurements taken need to be consistent and repeatable so that valid comparisons can be made between sets of samples from different treatments. The assay has the advantage of being relatively straightforward to carry out and does not need particularly sophisticated equipment. However, the analysis of the images of comets is not simple. Comets can have complex shapes and quantifying these shapes in terms of simple measures is challenging. Increasingly computer-based image analysis is preferred to a visual classification of comets based on the morphology and degree of damage,7 although both methods are still acceptable.4 Computer-based methods can increase the precision and reduce the subjectivity of the measurement process. Image-analysis programmes are capable of collection of large quantities of data but one challenge is to reduce these data, which Collins et al.4 call a ‘‘surfeit of information’’, into a smaller number of informative values that summarises and describes the comet from that particular cell.

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Table 17.1

Chapter 17

Various measures obtainable from image-analysis programmes.

Head DNA Tail DNA % head DNA % tail DNA Head radius Tail length Comet length Head CoG Tail CoG Tail moment Olive tail moment

Amount of DNA in the comet head Amount of DNA in the comet tail Percent of DNA in the comet head Percent of DNA in the comet tail Radius of the comet head Length of the comet tail measured from right border of head area to end of tail Length of the entire comet from left border of head area to end of tail ‘‘Centre of gravity’’ of DNA in the head ‘‘Centre of gravity of DNA’’ in the tail % tail DNA tail length % tail DNA (tail CoG–head CoG)

A number of measures, some derived from the use of automated image analysis, can be taken to describe and quantify the comet (Table 17.1). Examples include measures of the absolute amount of DNA as measured by the sum of the intensities of the pixels in the head or tail, the values of the relative amount of DNA in the head or tail, measures of absolute length, the tail length and the head radius of the comet. Measures representative of the ‘‘centre of gravity’’ in the head and tail as well as ‘‘moment’’ measures (composites measures taking into account both comet length and the intensity of staining) can be derived. The Olive tail moment, for instance, attempts to combine two aspects of the comet shape: the length of the comet and the intensity of the comet by calculating the product of the percentage DNA in the tail and the difference between the head and tail centres of gravity. Three measures – % tail DNA, tail length and tail moment – are now commonly used as measures of DNA migration with an increasing tendency for the endpoint, % tail DNA, to be the preferred measure for assessment.2,7 Collins et al.4 discuss scoring methods and point to the limitation of some of the quantitative methods such as a lack of a standardised measurement for comparisons across studies. Some measurements, such as tail length, are made in pixels and are, in effect, ‘‘arbitrary values’’. As damage increases the intensity of the staining of the DNA in the tail increases rather than the tail length increasing. However, the tail length may be the most sensitive endpoint at very low levels of damage. Measures may be difficult to generalise across studies, limiting, for instance, their use in quantitative comparisons across studies or for deriving power calculations. Standardisation of measurements between laboratories can be problematic as, for instance, the choice of where to begin and end tail length measures can vary between laboratories.3 Collins et al.4 also suggest there is an indication of nonlinearity at low doses in calibration curves using the tail moment. Collins et al.4 state that % tail DNA is ‘‘strongly recommended as the parameter of choice’’. The % tail DNA value has the advantage of being expressed on a scale from 0 to 100% making comparisons across studies easier


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and being linearly related to dose in calibration studies. Electrophoretic conditions should be adjusted to allow cells from negative control samples to show some migration of DNA, such as 5–10% tail DNA, which gives a measure of variability for the statistical analysis. Collins3 suggests that untreated control cells should, in general, have a low level of damage probably less than 10% (but more than 0). Suggestions have been made that the % tail DNA values of negative control cells should be between 10–20% (or 5–15%) to allow for the detection of both an increase and a decrease in migration to be detected. A decrease may represent a reduction in apparent damage as a consequence of crosslinking agents. A statistical point is that a formal hypothesis test would be one-sided if any change would only be expected in one direction, while a two-sided test would be appropriate if the results could go in either direction. A two-sided test is slightly less powerful than a one-sided test. Semiquantitative approaches have also been used to score comets on a scale from 0 to 4. Collins et al.4 show a photograph of different grades of damage. (A grade 4 comet is equivalent to the ‘‘hedgehog’’ cell where all the DNA is in the tail.) These grading scores correlate well with quantitative values of % tail DNA with the difference between each grade being equivalent to an extra 20% tail DNA.3,4 The values (0–4) given to the comets can be summed to provide a quantitative measure for 100 cells on a scale from 0 to 400. Collins et al.19 showed a close agreement in the relationship between visual- and image-analysis-based methods. Similarly, Pitarque et al.20 calculated a genetic damage index (GDI) based upon differential weightings given to the different grades of damage for five ‘‘arbitrary’’ categories from Type 0 (undamaged) to Type IV (highly damaged)) and used this categorisation to obtain a quantitative measure for the slide based upon a weighting applied to the number of cells with the different grades of damage where the GDI ¼ (Type I+2 Type II+3 Type III+4 Type IV)/ (Type 0+I+II+III+IV). Cells can also be assessed using a binary endpoint as either a ‘‘responder’’ or ‘‘nonresponder’’ based on an assessment of the degree of migration. The percentage of ‘‘responder’’ cells per slide is then recorded. One approach to the analysis of the data given the number of alternative endpoints is to analyse a number of them. Analyses should give similar results because studies have shown appreciable correlations between the measures of different endpoints. However, if the conclusions drawn by using different endpoints differ this would indicate that the data should be examined closely to identify the reasons for the divergent results. Extra data on toxicity may also be collected. Collins et al.4 discuss the use and limitations of the trypan blue exclusion test for viability but point out that viability should not be a problem in the in vivo assay but may be for in vitro studies. Counts of the numbers of ‘‘hedgehog’’ cells and ‘‘ghosts’’ comets (consistent with the complete migration of DNA) can also be made. Such data are usually not included in the formal statistical analysis of the comet measures but provide an important aid in the assessment of the quality of a study.

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It is good practice to sample cells from different parts of a slide to reduce introducing biases because the comets are not homogeneous across the slide, for instance, because of, ‘‘edge effects’’, where comets at the edge of the slide have different measures to those nearer the centre of a slide. Sampling to reduce such potential biases could consist of ensuring that if 50 cells are measured per slide, then no more that 5 cells are taken from each of 10 of more randomly chosen areas on the slide. Bowden et al.21 suggested that the shape of the comet may be informative and may be preferred to measures of comet mass or length. The multiple measurements possible on the comet image would allow such a suggestion to be investigated using multivariate methods to try to distinguish whether there are particular comet shapes indicative of particular types of damage. Collins et al.4 note that the Comet assay is very sensitive and capable of detecting between 100 to several thousand breaks per human cell. They stress the use of rigorously controlled calibration studies using ionising radiation. These show near-linear slopes from 0 to 10 Gy, suggesting that it is possible to express data as Gy equivalents that can then be converted into lesion frequencies per 106 Da (Daltons). Forchhammer et al.22 have suggested that the most informative way to present Comet assay results is as lesions per unaltered nucleotides or diploid cells. Collins et al.4 point out that there is considerable interlaboratory variability in the steepness of the calibration curves, probably reflective of protocol differences. Interlaboratory comparisons are in progress to try to reduce the discrepancies through, presumably, subtle differences in protocols such as variability in electrophoretic conditions. There is appreciable potential for DOE methodology to identify the important factors involved. Statistical analysis can be carried out using the values for the individual cells but it is often carried out at the level of the experimental unit (the animal or the culture/subculture that the treatment is applied to) or of the individual slide. Identifying a representative value for the comet measures for each experimental unit is not straightforward. Complications arise because the distribution of the individual cell endpoints is unlikely to match any of the common statistical distributions such as the normal distribution. The distributions observed, especially after treatment, are complex, and even if a function could be fitted to the distribution, it would require a number of parameters. Simple transformation such as the logarithm of the measures may not produce a normal distribution. A number of summary statistics of the cell measures have been suggested including the geometric mean (equivalent to the antilog of the mean of the log10 transformed data), the median (the 50th percentile) and various other percentiles: 75th, 90th and 95th. The untransformed mean is usually not recommended because the distribution, particularly of treated cells, is often skewed.23 However, the central limit theorem implies that the means of samples are normally distributed even when the distribution of the population they were derived from is distinctly non-normal, provided the sample sizes are above about 30, so that concern about analysis using untransformed means may not be crucial.


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17.5 The Experimental Unit and Experimental Design A central concept of experimental design, critical to a successful statistical analysis, is the identification of the experimental unit. The US NIST defines it as ‘‘the entity to which a specific treatment combination is applied’’ (NIST http://www.itl.nist.gov/div898/handbook/pri/section7/pri7.htmref) and is the unit to which treatments are randomised. In the case of in vivo studies this is the animal,6 while for in vitro studies it will be an independent culture or subculture.7 The linking of the experimental unit to the level at which randomisation occurs is related to the concept of independence of the measures; this is an important assumption underlying many statistical tests. An incorrect specification of the experimental unit in the statistical analysis can lead to a serious misinterpretation of the results of the statistical analysis. Replication is an important aspect of experimental design as it provides an estimate of the ‘‘error’’ variability used in the statistical tests. Replication can be either by biological or technical replicates. The former are taken from separate experimental units; the latter are repeat samples from within the same unit. In general, it is better, if the opportunity arises, to increase the number of biological as opposed to technical replicates. Pooling of samples from different experimental units before measuring should, in general, be avoided. Repeated sampling from this pooled sample will then result in a set of technical replicates but would provide no estimate of the variability between biological replicates. In general, the Comet assay is a hierarchical or ‘‘nested’’ design (Figure 17.1). In a hierarchical design the experimental unit (the animals in the in vivo design and the cultures in the in vitro design) are ‘‘nested’’ or replicated within doses, while a number of slides or gels from each animal or culture are prepared and a number of cells from each slide or gel are ‘‘scored’’. For instance, in a study of goldfish exposed to a glyphosate formulation five fish per dose per duration

Figure 17.1

Hierarchical or nested design. Example of a hierarchical in vitro design based upon 4 dose levels including a negative control, 5 cultures/subcultures at each does level, 3 slides/gels per subculture, 50 cells per slide/ gel. (From Lovell and Omori, 2008).

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were studied, five slides prepared per fish and 200 cells were scored from each slide.24 Such designs thus have a number of levels of variability – experimental groups, animals, slides, cells – and the statistical analysis involves developing methods that model these different levels of variability. Ideally a statistical analysis should ‘‘account’’ for the different levels of variability in the design and avoid the error of not taking into account ‘‘hidden layers’’ of variability in the design. A serious error is the use of the individual cell as the experimental unit in the statistical analysis as this may overestimate the statistical significance of a comparison. Wrongly treating repeated measures on the same individual as independent can result in a superficially more powerful test and an overestimation of statistical significance. This problem of what is termed ‘‘pseudoreplication’’ has been recognised for some time in ecological studies and its implications and effects on experimental design and analysis are well documented and widely, if not completely, appreciated.25,26 Similar points have been made in the neurosciences.27 Failing to take the experimental unit into account in the statistical analysis is a serious error. In the case of in vitro designs it is important to clarify the relationship between the cultures and subcultures with respect to the experimental units and to ensure adequate replication. Lovell and Omori9 illustrate the different types of in vitro designs and the distinction between repeat experiments using different cultures and the use of different cultures within the same experiment. In the absence of appropriate replication there is a danger that any variability in subculture is confounded with treatment effects leading to potential artifactual results. Studies, for instance, which fail to take into account this hidden variability can result in apparent significant differences between treatments. The more cells scored per subculture in these designs the more likely a significant result will occur. Wiklund and Agurell23 have provided specific recommendations for Comet assay designs. Based upon simulation studies, they recommended a design with 50 cells from 3 slides per experimental unit and 4 to 5 animals per group for an in vivo study and 2 or 3 cultures for an in vitro study. Recently, Smith et al.28 have provided recommendations for the design of the rat Comet assay designs. They suggest that a design with 6 animals per group, 3 gels per animal and 50 cells per gel would have 80% power to detect a 2-fold difference for studies using liver, bone marrow and stomach and a 3-fold increase in studies using blood. They recommend that investigators using the rodent Comet assay should carry out a similar analysis to determine the optimal experimental design for their own laboratory.

17.6 Statistical Methods There is no consensus on a single statistical method for the analysis of Comet assay data.6 This is not surprising as there is probably no statistical method that can adequately handle data from the individual cell values given the complexity

Statistical Analysis of Comet Assay Data 9

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of the distribution of the values. However, concentrating on a single representative value for each experimental unit (animal or cultures) will probably result in data that can, with care, be analysed by a number of standard statistical methods. Some care may, however, be needed in the interpretation. Duez et al.,29 for instance, suggested using either the median or 75% percentile of the sample. They concluded that a trend analysis on medians of the samples was satisfactory. In practice, though, any statistical analysis is a trade-off between the sophistication of the model being fitted and the practicalities of the conduct and reporting of the data. In broad terms there are three types of statistical comparisons that may be made. Firstly, a comparison between the negative and positive control groups (see below); secondly, a test for differences between a number of groups and for a dose–response relationship and thirdly, pairwise comparisons between the individual treated groups and the negative control group. Statistical tests usually involve a test of a null hypothesis. In a simple case this means that there is no difference between a treated and control group. The alternative hypothesis is that there is a difference. The statistical test applied can be either one- or two-sided. One-sided means the experimental effect will either have no effect or have an effect in one predefined direction, two-sided meaning the effect, if any, could go in either direction. It is argued that if, when using a one-sided test, an effect in the wrong (unpredicted) direction is found that this should be ignored no matter how significant it might be because if there is any interest at all in a result in the opposite direction then a two-sided test should be used. The outcome of a statistical test of a null hypothesis can be illustrated by a 2 2 table (Figure 17.2). This table shows there are two types of correct results and two types of incorrect results: the Type 1 error (or a) (falsely rejecting the null hypothesis) which is related to the significance level of the test and the Type II error (or b) (wrongly accepting the null hypothesis) related to the power of the test. The power of the test is (1 – b). A range of statistical methods (both parametric and nonparametric) are available, and have been used, for the analysis of comet data.8,9 Each test makes some assumptions about how the study was carried out and the nature of the data. In the case of parametric statistical tests (those based upon an underlying parameterised distribution such as the normal) these are: independence, normal distribution of the residual errors and equal variability within the groups. There is also the assumption (which is often violated in observational studies) that the experimental units were randomly assigned to the treatments. If the analysis is based upon a hypothesis-testing approach there are a range of parametric test that include the t-tests for two-group and the analysis of variance methods for multiple-group comparisons and specific tests of dose–response relationships. These tests are special cases of the wider general linear model (GLM) methodology. For most of the simpler parametric tests there is a nonparametric test equivalent: the Mann–Whitney for two groups, the Kruskal–Wallis for multiple-group comparisons and the Jonkheere–Terpstra trend test for specific tests

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Null Hypothesis

False

True False Rejection

Decision

Reject

Correct Result

Type I error Significance level (α)

False Acceptance Accept

Type II error

Correct Result

Power (1-β)

Figure 17.2

Hypothesis testing. 2 2 table showing possible results of a test of a null hypothesis illustrating the occurrence of Type 1 (or a) error that is related to the significance level of the test and Type 2 (or b) error that is related to the power of the test.

of dose–response relationships. Table 17.2 lists parametric tests and their nonparametric equivalent. In general terms, the parametric test will be more powerful if the assumptions underlying it are met. The nonparametric tests are slightly less powerful under these circumstances but may give more accurate Type 1 errors (Figure 17.2) when the assumptions are violated. However, nonparametric tests are not assumption free and violations affecting the distributions may also affect the probability values associated with nonparametric tests. Small sample sizes or number of experimental units (such as n ¼ 4 or 5) will also reduce the power of the nonparametric tests. A test for a dose-related effect will have greater statistical power than pairwise comparisons. The dose–response test can be thought of as a more ‘‘sophisticated’’ hypothesis with the potential to define a set of orthogonal (statistically independent) components, testing in a four-group design, linear, quadratic and cubic contrasts. A curvilinear response may have two or more of the components statistically significant. The greater power of these tests of specific hypotheses may mean that a shallow but real, dose–response relationship can be detected by the specific linear trend test even though the overall test of the equality of the four means in the ANOVA can be nonsignificant. Some decision trees/flow charts used for choosing statistical tests suggest no further testing if the overall or omnibus ANOVA test of the equality of all the group means is not significant. This is clearly inappropriate if a more specific hypothesis is implicit in the experimental design. A more general approach taking account of the hierarchical design is possible using the general linear model (GLM). The GLM is a specific case of an even more general approach, confusingly, called generalised linear modelling

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Table 17.2

Parametric tests and their nonparametric equivalents.

Objective

Parametric

Nonparametric

Description of a group

Mean and standard deviation (SD) One-sample t-test

Median and interquartile range (IQR) Wilcoxon rank sum test

Unpaired t-testa

Mann–Whitney

Paired t-test

Wilcoxon rank sum test

One-way ANOVA Repeated-measures ANOVA Linear component in ANOVA Pearson’s product moment correlation Linear regression

Kruskal–Wallis Friedman test

One group with standard value Compare two groups (unpaired) Compare two groups (paired) Compare Z 2 groups Compare Z 2 matched groups Test for linear trend Association (2 variables) Predict dependent variable from independent

Jonkheere–Terpstra trend test Spearman correlation Nonparametric regression

a

Note: there are a number of versions depending upon whether the within group variability is assumed to be the same and pooled for the analysis.

(GZM). GZM provides a modelling approach that can be used with a variety of theoretical data distributions. A range of more sophisticated methods can be used to model the hierarchical nature of the designs. These include random effects modelling (REM), generalised estimating equations (GEEs) and hierarchical linear models (HLMs). Some of these have been applied to Comet assay data.8 The computing facilities for these methods are becoming more widely available and the use of such methods by statisticians is likely to increase in the future. A number of statistical software packages such as SAS (through its GLM and MIXED procedures), SPSS, Genstat and as well as R (a publicdomain open-source statistical analysis software language) can be used to carry out analyses using some of these models. Lovell and Omori8 provide more details of the range of methods available. It is not clear yet whether these methods will provide appreciably more information than the less sophisticated methods currently in use. Decision trees for statistical tests often include tests for whether data fit a particular distribution (Kolmogorov–Smirov and Shapiro–Wilks tests) or have equal variability (homogeneity of variances) between groups (Levene and Bartlett’s tests). Tests for normality are likely to have high power to detect deviations because of the large datasets produced by the Comet assay. The tests are, consequently, capable of detecting relatively minor deviations from distributions such as the normal. This means that small datasets that are nonnormally distributed may show a nonsignificant departure for a goodness of fit test while a large set showing a slight departure will show significance. This again illustrates some of the potential pitfalls of selecting statistical tests on the basis of the results of other tests.

436

Chapter 17

Care should be taken in choosing statistical tests if the cells have been classified as either responders or nonresponders or some such similar binary response. Both the chi-square and Fisher exact tests of 2 2 tables assume independence of the data but if individual cells are used in the analysis rather than the correct experimental unit such as the culture or the animal then the tests are vulnerable to producing highly significant but incorrect results. Data in this form can be expressed as the percentage of responder cells and analysed using methods developed for handling proportional data such as appropriate logistic regression models or by analysis of variance after an arcsin, angular or logit transformation. Escobar et al.,30 for instance, used ordered logistic regression to investigate the use of the Comet assay combined with fluorescence in situ hybridisation (Comet–FISH) to detect DNA breakage in the specific chromosomal regions in in vitro TK6 lymphoblastoid cells. In conclusion, there is no general consensus as to which method should be the standard.8 In practice, because of the different statistical philosophies underlying statistical analysis this is likely to continue especially as approaches based upon estimation rather than hypothesis testing, Bayesian methods and modelling become more widely used. A range of different methods can, therefore, be used. It may be useful to see if different methods give broadly similar results. If not, and the conclusion differs, it would be sensible to explore the data to try to identify what causes the difference in interpretation.

17.7 Use of Control Groups Many experimental studies will have two types of concurrent controls included in the design: a negative (vehicle) control and a positive control using a compound known to produce comets. Further control groups may be included if comparisons between, for instance, untreated and vehicle-treated groups were considered relevant. The negative vehicle control is a comparator for the various treated groups and is involved in the formal statistical comparison between groups. The role for the positive control is different. It may be included to characterise the sensitivity of the test method and to provide a check or an evaluation of the testing techniques of the laboratory. Statistical tests between the negative and positive control groups can misleadingly producing nonsignificant results because of the small sample sizes together with the high variability sometimes found in the positive control group resulting from variability in response such as can arise from a mixture of responders and of nonresponders. This would create problems if there was a decision rule that an experiment was rejected as unsatisfactory if a significant difference was not found between the two groups. It is, therefore, not necessary to make formal statistical tests between the two control groups. Rather, as the purpose of the positive control is to check the technique, consideration should be given to methods that minimise the number of animals needed to provide this reassurance. An approach that made more


437

use of historical control data or used a few or even one concurrent positive control animal to demonstrate technical capability would reduce animal usage. In this context, although methods could be developed that took into account the difference between the two control groups, it does not seem sensible to include the positive control data in any formal testing method for the test material. The development of sets of both negative and positive historical control data by a laboratory is a resource that could be used in conjunction with quality control (QC) methods31,32 to assess the quality of the concurrent experimental work and to identify and, if necessary, correct any long-term drift in the performance of the assay by the laboratory. Such an assessment could be part of the evaluation of whether the experimental study is satisfactory.

17.8 Assessment of Results Controversies over the use of the statistics to assess the results of studies often relate to the use of probability values to draw a conclusion. The equating of a significant effect with a positive result and a nonsignificant effect with a negative result is a serious problem. This is a symptom of the greater problem of equating significance testing and P values with decision making. Increasingly statistical opinion is moving from the concept of hypothesis testing to the idea of estimation and model testing.12 Much more emphasis, therefore, should be given to the estimation of the size of an effect and the confidence interval associated with it than the specific statistical significance level. One example of this philosophy is the increased emphasis on the iterative and data-driven aspect of model building that contrasts with the development of codified statistical analysis plans (SAPs) increasingly required in regulatory science.33 Longford and Nelder, in particular, criticise what they call the ‘‘cult of the single study’’, the use of P values, multiple comparison and of nonparametric tests in the provision of evidence to regulatory authorities. Nester34 discusses some of the philosophical underpinning of statistical analyses and has produced a set of quotes criticising the use of significance testing. (This is reproduced at http://welcome.warnercnr.colostate.edu/~anderson/nester.html.) The comet experiment is a test of whether a compound is biologically active. The statistical tests of whether a test such as the Comet assay is a good predictor of say, genotoxicity, is different from whether it is detected as positive in an experiment. The finding of a significant effect of a treatment in a comet experiment does not mean that this compound is predicted to be, say, a carcinogen. Dichotomisation of results into genotoxic or nongenotoxic classification based upon a decision rule may be a convenient management/regulatory endpoint. However, dichotomisation leads to a loss of information.33,35 A consequence is that some weak mutagens will be ‘‘called’’ negative and disagreements will occur when different criteria are used by different laboratories. Longford and Nelder point to the potential of modelling approaches to handle the dichotomisation

438

Chapter 17 Disease or carcinogenic status

Diagnostic or STT result

Present / Carcinogenic D

Absent / noncarcinogenic

a

b

c

d

Positive

+ Negative

−

Sensitivity Specificity Positive Predictive Value (PPV) Negative Predictive Value (NPV) Prevalence Where N = a+b+c+d

Figure 17.3

= a/(a+c) = d/(b+d) = a/ (a+b) = d/ (c+d) = (a+c)/N

Diagnostic test statistics. 2 2 table showing statistics derived from the use of a diagnostic test or a short term-test (STT) to predict disease or carcinogenic status.

problem. It is also important to note that the two statistical procedures: assessing the results of a study and for measuring the methods predictive ability are different processes and that equating a negative result as a consequence of a dichotomisation into a mechanistic threshold is a serious error. Comparisons against some Gold Standard provide a test of whether the assay is a good discriminator of carcinogens and noncarcinogens.36 A 2 2 table for the properties of a diagnostic test are shown in Figure 17.3. Estimates of statistics such as sensitivity and specificity can be derived. Although the 2 2 table is superficially similar to that in Figure 17.2 the false-positive and falsenegative errors are different from the Type 1 and Type 2 errors associated with hypothesis testing.

17.9 Multiple Comparison Issues Many studies involve multiple comparisons such as between each dose level and the concurrent negative control or between sets of subgroups or correlations. This can raise the concern that when a number of hypothesis tests are carried out some results will be significant by chance alone. Figure 17.4 shows that if 20 independent comparisons are made at the significance level P ¼ 0.05 then there is a 64% chance that one or more of these comparisons will be significant by chance alone even though none of the groups are, in fact, different from one another. Using a significance level P ¼ 0.01 the corresponding percentage is 18%.

439


This is of particular concern when a series of post hoc comparisons are carried out after the study has been completed and there is a danger of ‘‘data dredging’’. A number of multiple comparison methods have been proposed to try to address this problem.37 Multiple comparison methods aim to control the Type 1 error (false rejection rate) by managing the experiment- or family wise error rate (EER or FWER) or the individual or comparisonwise error rates (CWER). The consequence of their use is to lower the power of the study, in effect, ‘‘damping’’ down the significance of the results. Two widely used multiple comparison methods in toxicology are Bonferroni’s correction and Dunnett’s test. The Bonferroni correction adjusts the significance level that a hypothesis test is carried out by taking the number of comparisons (n) being made into account. A simple approximation is to use a/n as the significance level for rejecting the null hypothesis or by multiplying the actual P value obtained by n and comparing this with the significance level of, say, 0.05. This is a highly conservative method. Other multiple comparisons methods are somewhat less conservative. Dunnett’s test was originally designed to test multiple treatments against a common control. It was designed to maintain the EER at 0.05 meaning that experiments where one or more of the comparisons with the negative control were falsely declared as significant would occur on average only one in every 20 similar experiments. It is arguable whether its use is appropriate for studies

Probability of a significant result with increasing numbers of tests 1.0

P(one or more tests sig.)

P

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The Comet Assay in Toxicology (Issues in Toxicology)

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In Vitro Methods in Toxicology

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