The Comet Assay in Toxicology is the first book of its kind to be devoted exclusively to the Comet assay and its applications as a very important tool in toxicology. This multi-author book will serve as both a reference and a guide to investigators in the biomedical, biochemical and pharmaceutical sciences. It is written by authors from the fields of genetic toxicology and human epidemiology, with first-hand knowledge of their chosen sub-specialities, and who actively contribute to the peer-reviewed literature. The book is divided into four sections, reflecting the range of interest in the exploitation of this assay. The book begins with an introductory section reviewing the genesis of the assay for those new to the technique and the current knowledge of the various fields in which it finds wide acceptance. This section sets the scene by explaining why the assay has become the most sensitive and sought after assay in modern toxicology. The next section considers various procedures being followed to assess different types of DNA damage in various cell types. The third section brings together the specific applications of the assay in diverse fields ranging from clinical applications, human monitoring and environmental toxicology through to genetic toxicity testing. The fourth section considers guidelines for the conduct of the assay in in vitro and in vivo systems, based on the recommendations of the International Workgroups on genotoxicity test procedures. Finally, the book draws to a close with an assessment of imaging and image analysis and statistics used for understanding data generated by the assay. This is a unique reference book as it provides the scientific community with the latest advances of the Comet assay as well as its applications. It is aimed at students and scientists in the fields of molecular epidemiology and genetic toxicology.

Professor Alok Dhawan currently holds the position of Senior Assistant Director at the Indian Institute of Toxicology Research, Lucknow, India. He was awarded the Young Scientist Medal in Biomedical Sciences by the Indian National Science Academy in 1993. He was also awarded with the coveted CSIR Young Scientist Award in Biological Sciences in 1999 by the Council of Scientific & Industrial Research, India. The Indian Council of Medical Research also conferred its Shakuntala Amir Chand prize on Professor Dhawan for his contributions in the area of neuro- and geno- toxicology. He was elected as Vice President – Environmental Mutagen Society of India in 2006. He is also an elected fellow of the Society of Toxicology (India), and a member of various scientific societies in India as well as a member of the Asian Association of Environmental Mutagen Societies, Japan, and the United Kingdom Environmental Mutagen Society. He has published 67 papers in peer reviewed journals and has also has been the Associate Guest Editor of a special issue of Teratogenesis, Carcinogenesis and Mutagenesis journal published by Wiley-Liss, Inc, U.K in 2003 as well as Guest Editor of a special issue of the Nanotoxicology journal in 2008. He is actively involved in developing and establishing alternate animal models and substitute technologies for toxicity evaluation as well as in identifying peripheral biomarkers of exposure, effect and susceptibility to xenobiotics. Professor Dhawan is currently working in the area of nanomaterial and in silico toxicology. Professor Diana Anderson currently holds the Established Chair at the Department of Biomedical Sciences at the University of Bradford, UK and is Research Co-ordinator for the Department. In 1974, Professor Anderson was appointed as Head of Mutagenesis Studies at ICI’s Central Toxicology Laboratory and she joined BIBRA International in 1981 as Head of Genetic and Reproductive Toxicology and became Assistant Director and Group Forum Co-ordinator in 1987. In 1992, she became Senior Associate and Co-ordinator of External Affairs at BIBRA. She has served on the editorial board of 8 international journals, has over 300 publications, has edited/authored 6 books and guest-edited 9 special issues of 4 international journals. She has been/is Series Editor of books in Current Toxicology for John Wiley and Sons and Issues in Toxicology for the Royal Society of Chemistry. As an active Committee member, she has been Vice-President of the Institute of Biology and is Chair of the Scientific Committee of the International Association of Environmental Mutagen Societies. New research laboratories in India and Korea have been established with her help under the auspices of the British Council and UNIDO. She has organised both national and international meetings and is/was a member of various national (e.g. MRC Advisory Board and Veterinary Products Committee) and international committees, including the European Union Scientific Committee for Animal Nutrition (SCAN) and is a consultant for many international organisations, such as the WHO, NATO, TWAS, UNIDO and the OECD.

The Comet Assay in Toxicology

By Alok Dhawan Diana Anderson

The Royal Society of Chemistry

Copyright © 2009 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-199-2

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, 3,
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,
Chapter 3 Microplate-Based Comet Assay Elizabeth D. Wagner and Michael J. Plewa, 79,
Chapter 4 The Use of Higher Plants in the Comet Assay Tomas Gichner, Irena Znidar, Elizabeth D. Wagner and Michael J. Plewa, 98,
Chapter 5 Methods for Freezing Blood Samples at -80 °C for DNA Damage Analysis in Human Leukocytes Narendra P. Singh and Henry C. Lai, 120,
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, 129,
Chapter 7 Detection of DNA Damage in Drosophila and Mouse Alok Dhawan, Mahima Bajpayee and Devendra Parmar, 151,
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, 173,
Chapter 9 Applications of the Comet Assay in Human Biomonitoring Andrew R. Collins and Maria Dusinska, 201,
Chapter 10 The Comet Assay in Human Biomonitoring Mahara Valverde and Emilio Rojas, 227,
Chapter 11 Comet Assays in Dietary Intervention Trials Armen Nersesyan, Christine Hoelzl, Franziska Ferk, Miroslav Misik and Siegfried Knasmueller, 267,
Chapter 12 The Comet Assay for the Evaluation of Genotoxic Exposure in Aquatic Species G. Frenzilli and B. P. Lyons, 297,
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, 310,
Chapter 14 The Comet Assay in Sperm – Assessing Genotoxins in Male Germ Cells Adolf Baumgartner, Eduardo Cemeli, Julian Laubenthal and Diana Anderson, 331,
Section IV: Regulatory, Imaging and Statistical Considerations,
Chapter 15 Comet Assay – Protocols and Testing Strategies Andreas Hartmann and Günter Speit, 373,
Chapter 16 Imaging and Image Analysis in the Comet Assay Mark Browne, 390,
Chapter 17 Statistical Analysis of Comet Assay Data David P. Lovell, 424,
Subject Index, 451,

CHAPTER 1

The Comet Assay: A Versatile Tool for Assessing DNA Damage

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 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 Johanson 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., 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. 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. Some other lesions of DNA damage such as DNA cross-linking (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, genotoxicity testing and human biomonitoring.

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 DNA), requirement for small number of cells (~10 000) 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, 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 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. 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. 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 aberrations and micronuclei. 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 The Comet Assay in Lower Plants

1.3.1.1 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. The authors observed a significantly higher (P<0.001) DNA damage in chlorinated water (i.e. tap water) when compared to untreated (negative control) or distilled water (laboratory control). Hahn and Hock 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.

1.3.1.2 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. 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. 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.

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. 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. also suggested that the Comet assay was able to detect a phenomenon resembling clastogenic adaptation at the molecular level. Gichner and Plewa 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.

The Comet assay has also been used to study the effect of age of plant on DNA integrity as well as the kinetics of DNA repair 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. 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. The ornamental plant Impatiens balsamina was used as a model to understand the genotoxic effect of Cr6+ and airborne particulate matter, 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.

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. 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 µM) for 2, 4, and 18 h resulted in dose- and 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 µM cadmium resulted in significant (P<0.05) 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. The method was then used for the assessment of genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant. The method provided an excel- lent, 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. Cells from haemolymph, embryos, gills, digestive glands and coelomocytes from mussels (Mytilus edulis), zebra mussel (Dreissena polymorpha), clams (Mya arenaria), and polychaetes (Nereis virens), have been used for ecogenotoxicity studies using the Comet assay. DNA damage has also been assessed in earthworms and fruit flies, Drosophila. 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.

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 pollution-indicator 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. 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), and oil spills with petroleum hydrocarbons. 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. The green lipped mussels (Perna viridis) also showed a similar result on exposure to B[a]P in water.

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