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 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 (∼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,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 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).

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.

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).

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 (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 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 

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 

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 Cr⁶⁺ 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.

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 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.

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).

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.

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 (Mya arenaria), and polychaetes (Nereis virens), have been used for ecogenotoxicity studies using the Comet assay. DNA damage has also been assessed in earthworms^61,63  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 

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.^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.

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 cells^208,212  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.

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 

The Comet assay applied to earthworms is a valuable tool for monitoring and detection of genotoxic compounds in terrestrial ecosystems^61,66  (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 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.

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 assay^70–72,206  (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 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.

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.

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).

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 carpio^90,226 ). 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 (<10 μM) 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 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.

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 (P<0.001) DNA damage than tadpoles collected from sites of little or no agriculture. Similarly R. pipiens tadpoles collected from industrial sites showed significantly higher (P<0.001) 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 (P<0.05) 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 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.

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.

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 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  oral^241,242  and inhalation^130,243  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.

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 characterisation^6,8,245,246  (Table 1.1). The DNA-damage 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 (∼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.²⁴⁸ 

The assay has been widely used in studying DNA damage and repair in healthy individuals,^{3,194,249,250}  in clinical studies^31,251,252  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}  epithelial^269–271  and placental cells.²⁷² 

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.

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 (pH>13) 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 assay²⁸⁰ ), single-strand breaks (at pH 12.1,²⁸¹ ), 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 (pH>13) 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 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 

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.

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).