Chapter 1: The Comet Assay: A Versatile Tool for Assessing DNA Damage

Toxic substances and newer chemicals being added each year into the environment have led to increasing pollution of ecosystems as well as deterioration of air, water and soil quality. Excessive agricultural and industrial activities also 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 cause deleterious effects in somatic or germ cells of the sentinel species as well as non-target species. Hazard prediction and risk assessment of chemicals, therefore, becomes imperative for assessing the genotoxic potential of chemicals before their release into the environment or for commercial use as well as to evaluate DNA damage in flora and fauna affected by contaminated or 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 examine the use of the Comet assay in diverse cell types from bacteria to humans, to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species or bioindicator organisms in a particular ecosystem are the first to be affected by adverse changes in their environment. Determination of DNA damage in these organisms provides 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  (in 1984) were the first to quantify DNA double stranded breaks in cells using a microgel electrophoresis technique, known as the single cell gel electrophoresis (SCGE) or Comet assay. Later, the assay was adapted by Singh et al.,²  using alkaline conditions, which 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, the assay has been modified at various steps (cell isolation, lysis, electrophoresis, staining) to make it suitable for detecting 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,³  genotoxicity testing⁶  and human biomonitoring.^7,8  The field of ecotoxicology also provides a potential for use of Comet assay in natural ecosystems and has recently been reviewed to include the common experimental models used for studies, developments and/or modifications in protocols and improvements for future tests.⁹ 

Relative to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution and the micronucleus assay, 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 non-proliferating 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 allows for robust types of statistical analysis.

A limitation of the Comet assay is that aneugenic effects,¹⁰  and epigenetic mechanisms of indirect 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 humans, used in the Comet assay for assessing DNA damage (Figure 1.1).

Singh et al.¹¹  first used the Comet assay to assess the genetic damage in bacteria treated with 12.5–100 rad of X-rays. In the study, DNA double-strand breaks in the single electrostretched DNA molecule of Escherichia coli JM101 were determined using the neutral Comet assay. A significant increase in DNA breaks was induced by a dose as low as 25 rad, which was directly correlated to X-ray dosage (Table 1.1). The study supported the hypothesis that the strands of the electrostretched human DNA in the Comet assay represented individual chromosomes.

A modified version of the above Comet assay was used to assess the genotoxicity of antibacterial clay mineral mixture (CB) in Escherichia coli. CB leachate caused a significant increase in the double strand breaks in the bacterial cells, showing antimicrobial-mediated genotoxicity and suggesting the use of CB as an alternative bactericidal therapeutic.¹² 

Plant bioassays are important tests which 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. Genotoxicity assessment in roots of plants like Vicia faba, Nicotiana and Allium cepa, have been widely conducted.^14,15  However, during the last decade, the plant Comet assay has been extensively applied to plants (leaves, shoots and roots) to detect DNA damage arising due to chemicals, radiation and heavy metals in polluted soil and comprehensively reviewed¹⁶  (Table 1.1).

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.³⁹  The authors observed significantly higher (P<0.001) DNA damage in chlorinated water (i.e. tap water) when compared with untreated (negative control) or distilled water (laboratory control). Hahn and Hock⁴⁰  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. This model allowed for the rapid and sensitive detection of DNA damage by a number of chemicals simultaneously. Few studies of the Comet assay in Saccharomyces cerevisiae have been reported, possibly due to the presence of the cell wall and the small amount of cellular DNA, however, it has been optimized as a model system to study oxidative DNA damage and repair,^41,42  as well as genotoxicity of chemicals^13,17,18  and food additives.¹⁹ 

Algae are aquatic unicellular plants, which provide information regarding the potential genotoxicity of the water in which they grow. Being single-celled organisms, they can be used as a model for risk assessment monitoring of environmental pollution of aquatic environments using the Comet assay. The freshwater green algae species, Pseudokirchneriella subcapitata and Nannocloris oculata revealed DNA damage by the insecticide Chlorpyriphos and fungicide Tebuconazole at low concentrations.⁴³  The unicellular green alga Chlamydomonas reinhardtii has shown DNA damage due to known genotoxic chemicals^21,44  and the herbicide paraquat²²  and also demonstrated that oxidative stress was better managed by the algal cells under light than dark conditions.⁴⁴  The Comet assay successfully evaluated chemically-induced DNA damage and repair in Euglena gracilis and the responses were found to be more sensitive than those of human lymphocytes under the same treatment conditions.⁴⁵  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 and it can be used as a part of bioassay for ecotoxicology studies. E. gracilis demonstrated increased genotoxicity in Comet assay parameters due to organic extracts from Taihu Lake (China), and has thus been selected as a bioindicator organism to provide early warning of organic pollutants.²⁰  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.²³ 

Recently there has been an increase in the use of the Comet assay in higher plants to study DNA damage and repair, to understand the effects of genotoxicity of pollutants and the environment. The effect of various stressors on DNA damage in plants, the correlation of the DNA damage with cellular responses¹⁶  and DNA repair^46,47  have been reviewed and recommendations regarding the method have also been made for increasing the reliability and throughput of the Comet assay in plants.⁴⁸ 

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.^49,50  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.⁴⁹  also suggested that the Comet assay was able to detect a phenomenon resembling clastogenic adaptation at molecular level. Vicia faba used as a bioindicator plant has shown increased DNA damage due to inorganic arsenic in water (correlated with abnormal molecular changes at 20 and 30 mg l⁻¹ concentration),²⁴  lead (due to oxidative stress at 10 µM concentration),²⁵  and persistent organic pollutant-containing agricultural soils from Tlaxcala, Mexico.²⁶ 

Gichner and Plewa⁵¹  developed a sensitive method for isolation of nuclei from leaf tissue of Nicotiana tabacum, which, due to its high resolution and constant low tail moment values for negative controls, could be incorporated in in situ plant environmental monitoring.⁵¹  The Comet assay has been used to study the effect of alkylating agents in tobacco seedlings.⁵²  A small but significant increase in DNA damage compared with controls was noted in heterozygous tobacco and potato plants grown on soil contaminated with heavy metals.²⁹  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. Detection of concentration-dependent genotoxicity of urban air pollutants in leaf nuclei³¹  and titanium dioxide (TiO₂) nanoparticles,³²  in Nicotiana using the Comet assay has shown it to be useful for environmental monitoring.

No DNA damage was observed in the root or shoot cells of Phaeseolus vulgaris treated with different concentrations of uranium.³⁵  Cr(vi) showed concentration-dependent increases in DNA damage as detected by Comet assay and complemented by flow cytometry in leaves and roots of Pisum sativum, revealing clastogenic action of chromium.³⁶  The alkaline Comet assay was used to measure DNA damage and repair in the model plant Arabidopsis and rye grass exposed to X-rays.⁴⁷  Rapid and slow phases of repair were observed for acute exposures of 5 and 15 Gy, and a possible explanation of homologous repair (HR) of double-strand breaks during the slow phase was proposed.⁴⁷  For the first time Comet–fluorescence in situ hybridization (FISH) was conducted in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH), demonstrating 5S rDNA in the tail of the Comets, and suggesting Comet–FISH as a tool for environmental monitoring.⁵³ 

The major drawback with plant models was the fact that exposure needs to be given through the soil and it is difficult to say whether the result demonstrates synergies with other chemicals in the soil or non-availability of the toxicant due to its soil binding affinity. To circumvent this disadvantage, Vajpayee et al.,³⁷  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. Another bioindicator plant duckweed (Lemna) was used to study effects of industrial wastewater samples from environmental monitoring sites along the river Sava (Croatia) and showed a marked increase in DNA damage.³⁸ 

Reviews of the use of Comet assay in higher plants have been recently published which discuss protocols and its use in environmental genotoxicity research,⁵⁴  as well as applications in DNA repair studies and mutation breeding.⁵⁵  These studies revealed that DNA damage measured in plants using the Comet assay is a good model for in situ monitoring and screening of genotoxicity of polluted environments. Higher plants can also be used as an alternative first-tier assay system for the detection of possible genetic damage resulting from polluted waters or effluents due to industrial activity or agricultural run offs.

Animal models have long been used to assess the safety or toxicity of chemicals and finished products. With the advancements in technology, use of knockouts and transgenic models has become common for mimicking the effects in humans. The Comet assay has globally been used for assessment of DNA damage in various animal models.

The Comet assay has been used in a unicellular protozoan and invertebrates for establishing the safety of the environment in which these species are found (Table 1.2)

Tetrahymena thermophila is a unique unicellular protozoan, with both somatic and germ nucleus present in the same cell, and is widely used for genetic studies due to its well characterized genome. Therefore it was validated as a model organism for assessing DNA damage using a modified Comet assay protocol standardized with known mutagens such as phenol, hydrogen peroxide and formaldehyde.⁵⁶  The method was then used for the assessment of genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant.⁵⁶  The method provided an excellent, low level detection of genotoxicants and proved to be a cost-effective and reliable tool for genotoxicity screening of waste water. Ecological risk assessment of the organic pollutant dechlorane plus (DP) was conducted in Tetrahymena using the Comet assay, which showed its potential genotoxicity at high levels.⁵⁷  Melamine was found to be highly toxic to the Tetrahymena genome which also caused apoptosis.⁵⁸  An acellular Comet assay in Tetrahymena has also been used to study the genotoxicity of TiO₂ nanoparticles.⁵⁹ 

Various aquatic (marine and freshwater) and terrestrial invertebrates have been used for genotoxicity studies employing the Comet assay (Table 1.2) which have also been reviewed.^9,93,125,126  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 fly (Drosophila). The Comet assay has been employed to assess the extent of DNA damage at polluted sites in comparison to reference sites in the environment and, in the laboratory, it has been used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage.⁷⁸ 

Adverse effects of contaminants in the aquatic environment have been studied in freshwater and marine mussels as they are important pollution indicator organisms. These sentinel species provide the potential for environmental biomonitoring of aquatic environments which they inhabit. The Comet assay in mussels can be used to detect a reduction in water quality caused by chemical pollution.^75,127  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),⁷⁰  and oil spills with petroleum hydrocarbons.⁹²  However, the damage returned to normal levels, after continued exposure to 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.⁷⁰  Repairable DNA damage with B[a]P was also observed with Mytilus galloprovincialis and the green lipped mussels (Perna viridis).⁸⁵  Effects of ionizing radiation, due to anthropogenic addition of radionuclides in aquatic environment, have been found to alter DNA damage and RAD 1 genes in Mytilus tissues.⁷³  Since the biomonitoring of the indicator organisms in situ may cause time constraints and not all samples may be processed at the same time, the cryopreservation of samples for later analysis in laboratory would be beneficial. Kwok et al.¹²⁸  used different media for this study and found that preserved haemocytes samples of Mytilus may be stored at cryogenic temperatures for a month without change in DNA damage for analysis in Comet assay.¹²⁸ 

Inter-individual variability, including seasonal variations in DNA damage have been reported from some studies, both in laboratory and field,^71,130,131  hence baseline monitoring has to be carried out over long time intervals. Haemocytes of freshwater Zebra mussel Dreissena polymorpha have shown temperature-dependent DNA damage showing that the mussels are sensitive to changes in water temperatures,⁶⁴  and monitoring ecogenotoxicity with these species should account for variations in temperatures. The Comet assay in haemocytes of D. polymorpha was used as a tool in determining the potential genotoxicity of water pollutants,^60–63  and Klobucar et al.⁶⁵  suggested that haemocytes from caged, non-indigenous mussels could be used for Comet assay for monitoring genotoxicity of freshwater. The hOGG1 enzyme was used in the Comet assay to evaluate 8-oxo-2′-deoxyguanosine (8-oxo-dG) as a marker of oxidative DNA damage in D. polymorpha.¹²⁹ 

DNA damage and repair studies in vent mussels, Bathymodiolus azoricus, have been carried out to study the genotoxicity of naturally contaminated deep-sea environment.^83,84  The vent mussels demonstrated similar sensitivity to environmental mutagens to that of coastal mussels and thus could be used for ecogenotoxicity studies of deep sea waters using the Comet assay. Villela et al.¹³²  used the golden mussel (Limnoperna fortunei) as a potential indicator organism for freshwater ecosystems due to its sensitivity to water contaminants.

In vitro Comet assay has also been used in cells of mussels, which can be used to screen genotoxic agents destined for release or disposal into the marine environment. Dose-responsive increases in DNA strand breakages were recorded in digestive gland cells¹³³  haemocytes¹³⁴  and gill cells¹³⁴  of M. edulis exposed to both direct-acting (hydrogen peroxide and 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone) and indirect-acting (B[a]P, 1-nitropyrene, nitrofurantoin and N-nitrosodimethylamine) genotoxicants. Digestive gland cells^78,135  and haemocytes⁸⁰  of Unio tumidus were also used for in vitro studies of DNA damage and repair by different compounds.

Coughlan et al.⁹⁰  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.^90,91  Comet assay was used for the assessment of sperm DNA quality of cryopreserved semen in Pacific oyster (Crassostrea gigas) as it is widely used for artificial fertilization.⁸⁸  The Comet–FISH assay, conducted in haemocytes of C. gigas, was shown to have potential for detecting DNA damage of target genes, induced by toxicant exposure and to allow better understanding of the impact of genotoxicity on animal physiology and fitness.¹³⁶  Gielazyn et al.¹³⁷  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 oyster (Crassostrea virginica) and clam (Mercenaria mercenaria). The herbicide diuron induced significant DNA damage in oyster spermatozoa at 0.05 μg l⁻¹ upwards while its environmental concentrations significantly affected embryo–larval development, showing deleterious effects of herbicide in non-target organisms.⁸⁹ 

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.¹³⁸ 

The Comet assay applied to earthworms is a valuable tool for monitoring and detection of genotoxic compounds in terrestrial ecosystems^94–105  (Table 1.2). 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.

Coelomocytes from Eisenia foetida have been used for biomonitoring purposes, to assess DNA damage in worms exposed to soil samples from industrialized contaminated areas⁹⁴  and sediment samples from polluted river systems.⁹⁵  Ecogenotoxicity studies have shown dose dependent DNA strand breaks caused by insecticide⁹⁷  and pesticides⁹⁸  in E. foetida as well as Pheretima species¹³⁹  demonstrating that pesticides could also have adverse effects on non-target species. Ionizing radiation affects the soil ecology, as it induced oxidative damage in spermatogenic cells of E. foetida and also reduced reproduction at dose rates at or >4 mGy h⁻¹.¹⁰²  Radiation with exposure to mercury produced synergistic effects and increased damage to DNA.¹⁰³  Humic acid was found to alleviate nickel- and deltamethrin-induced toxicity in earthworms, and could be used to reduce oxidative damage to DNA, lipids and proteins.¹⁰⁴  Medicinal therapy using peloids (natural mud), despite usually being beneficial, may also pose a risk of toxic effects as was seen in a study with E. foetida exposed to peloids.¹⁴⁰ 

In vitro exposure of primary cultures of coelomocytes to nickel chloride as well as exposure of whole animals either in spiked artificial soil water or in spiked cattle manure substrates exhibited increased DNA strand breaks due to the heavy metal.¹⁰⁰  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.⁹⁹  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 or gizzard cells.¹⁰⁷ 

Fourie et al.¹⁴¹  used five earthworm species (Amynthas diffringens, Aporrectodea caliginosa, Dendrodrilus rubidus, Eisenia foetida and Microchaetus benhami) to study genotoxicity of cadmium sulphate, 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.

The simple genetics and developmental biology of Drosophila melanogaster has made it the most widely used insect model. It has been recommended as an alternate animal model by the European Centre for the Validation of Alternative Methods¹⁴²  and evolved into a model organism for toxicological studies.^143,144  D. melanogaster has been used as an in vivo model (Table 1.2) for assessment of genotoxicity^108–115  and oxidative DNA damage¹⁴⁵  as well as for in vitro studies¹⁴⁶  using the Comet assay. Cisplatin induced adducts in D. melanogaster are influenced by conditions of nucleotide excision repair, and this correlates well with DNA damage as seen in Comet assay.¹⁴⁷  Recently, the Comet assay in Drosophila as an in vivo model has been used to assess the genotoxicity of zinc, copper and cadmium nanomaterials, which have demonstrated oxidative DNA damage.^113–115 

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.

Nereis virensa, a polychaete, plays an important role in the distribution of pollutants in sediments due to its unique property of bioturbation. These worms are similar to earthworms in soil and can be used for genotoxicity assessment of sediments. They have been used to study sediment-associated toxicity of silver nanoparticles, and bioaccumulation in the body was also shown.¹²⁴  Genotoxicity of intracoelomically injected B[a]P was assessed in worm coelomocytes using Comet assay, however, Nereis species was not found to be suitable for assessing PAH genotoxicity due to their lack of metabolic capability to convert B[a]P to its toxic metabolite.¹⁴⁸ 

DNA damage was assessed in neuroblast cells of brains of first instars of grasshoppers (Chorthippus brunneus) exposed to various doses of zinc from a polluted site, to understand the mechanism of toxicity in the insects due to industrial pollutants.¹⁴⁹  Comet assay parameters in brain cells of larvae originating from eggs of grasshoppers from different polluted sites have shown an association between increased DNA damage and heavy environmental pollution.¹¹⁶  Paraquat caused increased DNA damage in brain cells in both in vitro and in vivo administrations.¹¹⁷ 

Chronic exposure to coal combustion residues from coal-fired electrical generation in estuarine grass shrimp, Palaemonetes pugio, caused DNA damage in hepatopancreatic cells of adult shrimps as compared with the reference shrimp as seen in the Comet assay.¹²¹  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.¹⁵⁰  The genotoxic potential of polluted waters from Diluvio's Basin, Norflurazon, a bleaching herbicide¹⁵¹  and copper sulfate¹⁵²  was evaluated in planarians, where, significant increases in primary DNA damage were observed in these species. These studies have also demonstrated the use of the Comet assay in biomonitoring diverse environmental conditions utilizing sentinel species.

Studies of vertebrate species where the Comet assay is used have included 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 1.3).

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 and blood) of fishes exposed to various xenobiotics in the aquatic environment (Table 1.3).

The basal level of DNA damage detected in the Comet assay has been shown to be influenced by various factors, such as the temperature of water in erythrocytes of mullet and sea catfish,^156,157  age and gender in dab (Limanda limanda¹⁷⁹ ), exhaustive exercise¹⁵⁴  and seasonal changes¹⁵⁵  in chub. Therefore, these factors should be accounted for during environmental biomonitoring studies. The high intra-individual variability may also affect the sensitivity of the assay.¹⁷⁹  The protocol and experimental conditions used for the Comet assay for monitoring marine ecosystems may lead to differences in the results obtained. Also, chemical and mechanical procedures to obtain cell suspensions may lead to additional DNA damage.³¹⁸  Anaesthesia did not contribute towards DNA damage in vivo in methyl-methanesulfonate (MMS)-treated fishes and the anaesthetic benzocaine did not alter the DNA damage in erythrocytes after in vitro exposure to MMS or H₂O₂.³¹⁹  Hence keeping in mind animal welfare, multi-sampling of the same fish can be conducted. Recently, nanomaterials toxicity has gained importance in aquatic toxicology as nanomaterials synthesis and use has increased. Its impact on the aquatic environment and on fishes needs to be elucidated and this calls for development and implementation of protocols for nanomaterial genotoxicity in ecotoxicology.^320–322 

In vitro studies on fish hepatocytes,^182,185  primary hepatocytes and gill cells¹⁸⁶  as well as established cell lines (with metabolic competence^189–191 ) using the Comet assay have also been conducted to assess the genotoxicity of chemicals in water samples. The effect of tannins¹⁸³  and low concentrations (<10 µM) of diaryl tellurides and ebselen—an organoselenium compound¹⁸⁴  in oxidative DNA damage has been studied in nucleated trout (Oncorhynchus mykiss) erythrocytes for use of these compounds in biological systems. Kammann et al.¹⁸¹  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 method with use of the DNA repair inhibitor, 1-beta-d-arabinofuranosylcytosine (ara C). The base excision repair Comet assay has been used to examine DNA repair capacity after exposure to coal tar runoff on fish hepatocytes, to examine the clearance of DNA damage caused.¹⁹¹  The Comet assay with fish cell lines may be a suitable tool for in vitro screening of environmental genotoxicity, however, the metabolizing capabilities of the cell line need to be taken into account.

Cryopreservation has been shown to induce DNA strand breaks in spermatozoa of trout,^174,323  gilthead sea bream (Sparus aurata³²³ ) and sea bass (Dicentrarchus labrax³²⁴ ). The DNA damage was prevented by the addition of cryopreservants such as BSA and dimethyl sulfoxide.³²⁴  These studies have demonstrated the sperm Comet assay to be a useful model for determining the DNA integrity in frozen samples for commercially cultured species. The DNA damage due to xenobiotics as observed in Comet assay is repairable and this DNA repair can also be measured by Comet assay. However, the more permanent alterations caused by genotoxic compounds are not evaluated through the Comet assay. In such cases, amplified fragment length polymorphism (AFLP) has been found to reveal alterations in DNA even after repair was complete, suggesting supplementation of Comet assay with additional methods to get a holistic picture.³²⁵ 

These studies have demonstrated the usefulness of the Comet assay in fishes as a model for monitoring genotoxicity of aquatic habitats using these indicator animals.

The Comet assay in amphibians has been carried out at adult and larval stages for ecogenotoxicity of aquatic environments and studies have been reviewed by de Lapuente et al.⁹  The animals chosen for the Comet assay, act as sensitive bio-indicators of aquatic and agricultural ecosystems and are either collected from the site (in situ) or exposed to chemicals under laboratory or natural conditions.

Erythrocytes from tadpoles of Rana species have been used for the assessment of genotoxicity of water bodies as in situ sentinel organisms for environmental biomonitoring.^202–204  R. pipiens tadpoles collected from industrial sites showed significantly higher (P<0.001) DNA strand breaks than samples of R. clamitans tadpoles from agricultural areas while those collected from agricultural regions, showed significantly higher (P<0.001) DNA damage than tadpoles collected from sites of little or no agriculture. The higher levels of DNA damage may be attributed to the pesticides used in the agricultural region. Variation in DNA damage due to sampling time²⁰²  and during various metamorphosis states³²⁶  was also observed in the Comet assay. Hence, for biomonitoring environmental genotoxicity using the Comet assay, pooling of early tadpole phases could be helpful. Studies have been conducted on caged tadpoles in areas where the indigenous population is not present, due to ecological imbalance from pollution e.g. large lakes and aquatic areas near high industrial activity. R. clamitans and the American toad (Bufo americanus) tadpoles were caged at the polluted reference site and demonstrated significant (P<0.05) increases in DNA damage.³²⁷  The effects of ionizing radiation,¹⁹⁷  heavy metal pollution²⁰⁶  and agrochemicals²⁰⁷  on DNA damage in blood cells of tadpoles as well as adults of toads or frogs have shown that these animals can provide information about the environment that these species inhabit.

Huang et al.¹⁹⁶  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 which require metabolic activation. The effect of polyploidy on bleomycin-induced DNA damage and repair in Xenopus laevis (pseudotetraploid) and Xenopus tropicalis (diploid) was studied using the Comet assay.¹⁹⁸  The X. tropicalis was more sensitive with a lower capacity for repair than X. laevis, showing that polyploidy protects against DNA damage and allows rapid repair, and hence these species may be used as a good model for DNA damage and repair studies.

There are few studies involving the Comet assay in birds (Table 1.3). 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,^210–212  however, species-specific and intra-species differences were observed. Frankic et al.²¹⁷  reported that T-2 toxin and deoxynivalenol (DON) induced DNA fragmentation in chicken spleen leukocytes, which was abrogated by dietary nucleotides. The DON induced DNA damage was also shown to reduce with supplementation of Mycofix select²¹⁴  in broiler chicken. Sperm cryopreservation is an important genetic resource in the poultry industry for artificial insemination and the Comet assay is helpful in evaluating the DNA integrity of preserved sperms. Kotłowska et al.²¹³  have demonstrated increased DNA fragmentation in turkey sperm after 48 hours of liquid storage, and Gliozzi et al.³²⁸  have shown increased DNA fragmentation and decreased motility in chicken spermatozoa after cryopreservation and storage at −196 °C. Faullimel et al.²¹⁸  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.

Mice and rats have been widely used as animal models for the assessment of in vivo genotoxicity of chemicals using the Comet assay (Table 1.3). The in vivo Comet assay has been recently included in the ICH SR1 guidelines³²⁹  for regulatory genotoxicity testing and is accepted by the UK Committee on Mutagenicity testing of chemicals in food, consumer products and environment¹⁰  as a test for assessing DNA damage. Within a battery of tests, the Comet assay in liver cells can be used as an in vivo test along with mammalian bone marrow micronucleus test and AMES test, which has been accepted by international guidelines.³²⁹  A positive result in the in vivo Comet assay assumes significance if mutagenic potential of a chemical has already been demonstrated in vitro. There are specific guidelines for the performance of the Comet assay in vivo for reliable results.^330,331  Recently, the Japanese Center for the Validation of Alternative Methods (JaCVAM), organized an international validation study to evaluate the reliability and relevance of the in vivo rat alkaline Comet assay for identifying genotoxic carcinogens, using liver and stomach as target organs. Pre-validation studies were carried out to optimize the test protocol to be used and chemicals to be tested were decided, which would be used in five laboratories for the validation studies.^332,333  The comprehensive data obtained has been published in Mutation Research, Genetic Toxicology and Environmental Mutagenesis (2015, Volumes 786–788, Mutation Research).

Multiple organs of mouse or rat including brain, blood, kidney, lungs, liver and bone marrow have been utilized for the comprehensive understanding of the systemic genotoxicity of chemicals.^{231,232,334,335}  The most important advantages of the use of Comet assay is that DNA damage in any organ can be evaluated without the need for mitotic activity and that DNA damage in target as well as non-target organs can also be seen.³³⁵  The mouse or rat organs exhibiting 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.³³⁵ 

Different routes of exposure in rodents have been used e.g. intraperitoneal,^229,231  oral^336,337  and inhalation^221,338  to study the genotoxicity of different chemicals, as the route of exposure is an important determinant of the genotoxicity of a chemical due to its mode of action. 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.³³⁹ 

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.

The Comet assay is a valuable method for biomonitoring occupational and environmental exposures to genotoxicants in humans and can be used as a tool in risk assessment for hazard characterization^6,8  (Table 1.3). The DNA damage assessed by the Comet assay gives an indication of recent exposure and at an early stage where it could also undergo repair³⁴⁰  and thus it provides an opportunity for intervention strategies to be implemented in a timely manner. Follow-up studies conducted in the same population after removal of genotoxicant or dietary intervention can detect the extent of reduction in DNA damage.³⁴¹  It is a non-invasive technique compared with other techniques (e.g. chromosomal aberrations, micronucleus) which require larger samples (∼2–3 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.^6–8 

The assay has been widely used in studying DNA damage and repair in healthy individuals^{3,250,342,343}  in clinical studies^{246–249,344,345}  as well as in dietary intervention studies^260–267  and in monitoring the risk of DNA damage resulting from occupational exposures,^{268–293,346,347}  environmental,²⁵⁰  oxidative DNA damage^345,348  or lifestyle.^294–301  The wide applications of the assay and factors (e.g. age, gender, lifestyle) which can affect the result, have been discussed recently in the ComNet project to establish baseline data on DNA damage for all laboratories.⁶  Though white blood cells or lymphocytes are the most frequently used cell type for the Comet assay in human biomonitoring studies,³⁴⁹  other cells have also been used for the Comet assay e.g. epithelial,³⁵⁰  (including buccal and nasal cells),²  sperm,^{266–268,282,351,352}  urothelial cells²⁷³  and placental cells.³⁵³ 

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.^354–356  The in vitro Comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity or photogenotoxicity screening of drug candidates^357,358  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 of human genotoxicity.^3,8 

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.

The Comet assay has found worldwide acceptance for detecting DNA damage and repair in prokaryotic and eukaryotic cells.³⁵⁹  However, issues relating to the specificity, sensitivity and limitations of the assay need to be addressed before it gets accepted in the regulatory framework, including inter-laboratory validation of in vitro and in vivo Comet assay. Though the in vivo assay has recently been implemented in regulatory toxicity testing, the in vitro assay is not included.³⁶⁰ 

The variability in the results of the Comet assay is largely due to its sensitivity and minor differences in the experimental conditions used by various laboratories as well as the effect of confounding factors in human studies (lifestyle, age, diet, inter-individual and seasonal variation). Cell to cell,³⁶¹  gel to gel, culture to culture and animal to animal variability as well as use of various image analysis systems or visual scoring,³⁶²  number of cells scored³⁶³  and use of different Comet parameters,³⁶⁴  e.g. Olive tail moment and tail (%) DNA, are the other factors contributing to inter-laboratory differences in the results, which can be controlled.^365,366  A multi-laboratory DNA base-excision repair study, in three cell lines using the modified Comet assay also showed large inter-laboratory variation attributed to the cell extract and substrate cells incubation step.³⁶⁷ 

The limitation of the Comet assay is that it only detects DNA damage in the form of strand breaks. The alkaline (pH >13) version of the assay assesses direct DNA damage or alkali-labile sites; base oxidation and DNA adduct formation can measured with the use of lesion-specific enzymes.³  These enzymes are bacterial glycosylase or endonuclease enzymes, which recognize 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 increased amounts of DNA in the tail. Oxidized pyrimidines are detected with use of endonuclease III, while oxidized purines are detected with formamidopyrimidine DNA glycosylase (FPG). Modifications have been made in the protocol^3,331  to specifically detect double-strand breaks (neutral Comet assay), single-strand breaks (at pH 12.1), DNA crosslinking (decrease in DNA migration due to crosslinks) and apoptosis. 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.³⁶⁸  An adaptation of the Comet assay was also developed which enables the discrimination of viable, apoptotic and necrotic single cells.³⁶⁹  DNA repair can also be measured using the Comet assay and has been reviewed.³⁷⁰  With integration of biological and engineering principles, a Comet chip has been devised, which potentiates robust and sensitive measurements of DNA damage in human cells and can be utilized for various applications of the Comet assay.³⁷¹  The Comet–FISH assay was successful in detecting damage and repair in different genes regions in a cell and could be used for clinical purposes.³⁷² 

Tail (%) DNA and Olive tail moment (OTM) give a good correlation in genotoxicity studies⁴¹  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.³⁶⁴ 

It is therefore required that the in vitro and in vivo testing be conducted according to the Comet assay guidelines and that appropriately designed multi-laboratory international validation studies should be carried out. Guidelines for the in vitro as well as in vivo Comet assay have been formulated.^373,374  Study design and data analysis in the Comet assay have been discussed by the International Workgroup on Genotoxicity Testing (IWGT), where recommendations were made for a standardized protocol, which would be acceptable to international agencies.³⁷⁵  Critical parameters of the protocol, sensitivity of the protocol used, combination and integration with other in vivo studies, use of different tissues, freezing of samples and choice of appropriate measures of cytotoxicity were some of the areas covered in the recommendations.³⁷⁵ 

The in vivo Comet assay was the first-tier screening assay for assessment of DNA damage in rodents by the Committee on Mutagenicity, UK.¹⁰  International validation studies with genotoxic chemicals were carried out by the Japanese Centre for Validation of Alternative Methods (JaCVAM),^332,376  supported by the U.S. NTP 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 Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS).

Multi-laboratory validation studies in the European countries have been conducted to study the FPG-sensitive sites and background level of base oxidation in DNA using the Comet assay, in human lymphocytes.^367,377  It was found that half of the laboratories demonstrated a dose–response effect.³⁷⁷  However, many laboratories have carried out their own validation studies of DNA damage to optimize their research work. The large number of biomonitoring studies have 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.

The Comet assay is now well established and its versatility has imparted a sensitive tool to toxicologists for assessing DNA damage and repair. This has been demonstrated by its wide applications in assessing genotoxicity in plant and animal models, both aquatic and 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 Genetic Toxicology.