Abstract
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 non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or 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 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 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.
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Introduction
Ostling and Johanson (1984) were the first to quantify DNA damage in cells using a microgel electrophoresis technique known as “single cell gel electrophoresis or Comet assay”. However, the neutral conditions they used, allowed the detection of only DNA double-strand breaks. Later, the assay was adapted under alkaline conditions by Singh et al. (1988), which led to a sensitive version of the assay that could assess both double- and single-strand DNA breaks as well as the 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 assessing various kinds of damage in different cells (Collins 2004; Speit and Hartmann 2005). 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 as qualitatively in individual cell populations (Olive and Banath 2006). Some other lesions of DNA damage such as DNA cross-links (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 (Speit and Hartmann 2005), genotoxicity testing (Moller 2005), and human biomonitoring (Kassie et al. 2000; Moller 2006a).
Relative to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution, and micronucleus assay, the advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage (one break per 1010 Da of DNA; Gedik et al. 1992), 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 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 (COM 2000), 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 (Fig. 1).
Schematic diagram of the use of Comet assay in assessing DNA damage in different models from bacteria to humans
Bacteria
The first study to assess the genetic damage in bacteria treated with 12.5–100 rad of X-rays using Comet assay was conducted by Singh et al. (1999). In the study, neutral Comet assay was used for direct (visual) determination of DNA double-strand breaks in the single electrostretched DNA molecule of Escherichia coli JM101. Significant increase in the 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 strands of the electrostretched human DNA in the Comet assay represented individual chromosomes.
Plant models
Plant bioassays help detect genotoxic contamination in the environment (Maluszynska and Juchimiuk 2005). 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 (Grant 1999) and micronucleus (Ma et al. 1995). During the last decade, the Comet assay has been extensively applied to plants (leaves, shoot, and roots) to detect DNA damage arising due to chemicals and heavy metals in polluted soil (Table 1).
Comet assay in lower plants
Fungi
Schizosaccharomyces pombe has been used as a model organism to investigate DNA damage due to chlorinated disinfectant, alum, and polymeric coagulant mixture in drinking water samples (Banerjee et al. 2008). 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 (1999) used mycelia of Sordaria macrospora grown and treated with a variety of DNA-damaging agents directly on agarose minigels for 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.
Algae
Aquatic unicellular plants like algae provide information of 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 utilizing 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 than dark conditions (Erbes et al. 1997). 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 (Aoyama et al. 2003). 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 (Sastre et al. 2001).
Comet assay in higher plants
V. faba has been widely used for the assessment of DNA damage using 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 (Angelis et al. 2000; Menke et al. 2000). 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. (2000) also suggested that the Comet assay was able to detect a phenomenon resembling clastogenic adaptation at molecular level. Gichner and Plewa (1998) 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 could be incorporated for in situ plant environmental monitoring.
The Comet assay has also been used to study the effect of age of plant on DNA integrity (Koppen et al. 1999) as well as kinetics of DNA repair (Gichner et al. 2000) 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 metal (Gichner et al. 2006). 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 (Vandenhove et al. 2006). The ornamental plant Impatiens balsamina was used as a model to understand the genotoxic effect of Cr+6 and airborne particulate matter (Poli et al. 1999), 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 non-availability of the toxicant due to its soil binding affinity. Therefore, Vajpayee et al. (2006) 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. monneiri 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 environment, as 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 runoffs.
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. Comet assay has globally been used for assessment of DNA damage in various animal models (Table 1).
Lower animals
Tetrahymena thermophila is a unicellular protozoan widely used for genetic studies due to its well-characterized 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 standardized with known mutagens such as phenol, hydrogen peroxide, and formaldehyde (Lah et al. 2004). The method was then used for the assessment of genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant (Lah et al. 2004). 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.
Invertebrates
Studies have been carried out on various aquatic (marine and freshwater) and terrestrial invertebrates (Table 1). The genotoxicity assessment in marine and freshwater invertebrates using the assay has been reviewed (Cotelle and Ferard 1999; Lee and Steinert 2003; Mitchelmore and Chipman 1998a). Cells from hemolymph, embryos, gills, digestive glands, and coelomocytes from mussels (Mytilus edulis; Rank et al. 2005), 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 (Salagovic et al. 1996; Rajaguru et al. 2003) and fruit fly, Drosophila, (Bilbao et al. 2002; Mukhopadhyay et al. 2004). 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 widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage (Mitchelmore and Hyatt 2004).
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 pollution in the water bodies and thus provide the potential for environmental biomonitoring. The Comet assay in mussels can be used to detect a reduction in water quality caused by chemical pollution (Frenzilli et al. 2001; Jha et al. 2005; Rank et al. 2005; Steinert et al. 1998). 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; Large et al. 2002) and oil spills with petroleum hydrocarbons (Hamouten et al. 2002). 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 (Large et al. 2002). The green-lipped mussels (Perna viridis) also showed a similar result to B[a]P in water (Siu et al. 2004).
Significant levels of interindividual variability, including seasonal variations in DNA damage, have been reported from some studies in both laboratory and field (Wilson et al. 1998; Shaw et al. 2000, 2004; Frenzilli et al. 2001). Baseline monitoring has thus to be carried out over long time intervals. Temperature-dependent DNA damage was observed in hemocytes of freshwater mussel Dreissena polymorpha (Buschini et al. 2003), 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 (Wilson et al. 1998).
Villela et al. (2006) used the golden mussel (Limnoperna fortunei) as a potential indicator organism for freshwater ecosystems due to its sensitivity to water contaminants. Comet assay in haemocytes of freshwater Zebra mussel, D. polymorpha Pallas, was used as a tool in determining the potential genotoxicity of water pollutants (Bolognesi et al. 2004; Klobucar et al. 2003; Pavlica et al. 2001; Riva et al. 2007). Klobucar et al. (2003) suggested the use of Comet assay in haemocytes from caged, non-indigenous 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 naturally contaminated deep-sea environment (Dixon et al. 2004; Pruski and Dixon 2003). 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 cells (Mitchelmore et al. 1998b), hemocytes (Rank and Jensen 2003), and gill cells (Wilson et al. 1998, Rank and Jensen 2003) 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 (Labieniec and Gabryelak 2004, 2006). Wilson et al. (1998) demonstrated potential application of the Comet assay to gill cells of M. edulis as a potential in vitro screen for agents destined for release or disposal into the marine environment.
Comet assay in other bivalves
Coughlan et al. (2002) 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 (Coughlan et al. 2002; Hartl et al. 2004). 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 (Gwo et al. 2003). Gielazyn et al. (2003) 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 hemocytes from oyster (Crassostrea virginica) and clam (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 adapability of the animals to high concentrations of contaminants (e.g., B[a]P), which may pose a major problem (Large et al. 2002). Also, seasonal variation and temperature altered both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants under in vitro conditions (Buschini et al. 2003; Hartl et al. 2004). 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 (Machella et al. 2006).
Comet assay in earthworm
The Comet assay applied to earthworms is a valuable tool for monitoring and detection of genotoxic compounds in terrestrial ecosystems (Salagovic et al. 1996; Zang et al. 2000; Table 1). As 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. (1993) demonstrated a dose–response with the extent of DNA damage in coelomic leucocytes (coelomocytes) of earthworms (Eisenia foetida) from soil treated with different chemicals as an indication of soil pollution.
Coelomocytes from E. foetida demonstrated increased DNA damage when worms were exposed to soil samples from polluted coke oven sites (Salagovic et al. 1996) or industrialized contaminated areas (Xiao et al. 2006) and even sediment samples from polluted river system (Rajaguru et al. 2003). An insecticide, parathion, produced DNA strand breaks at all time points and doses in the sperm cells of E. foetida (Bustos-Obregon and Goicochea 2002), while dose–effect relationships were displayed by two pesticides, Imidacloprid and RH-5849, in the same species (Zang et al. 2000), showing that pesticides could also have adverse effects on non-target 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 (Reinecke and Reinecke 2004). The eleocytes, a subset of coelomocytes, exhibited increased DNA strand breaks under both in vitro and in vivo conditions and could be used as a sensitive biomarker for genotoxicity in earthworms (Di Marzio et al. 2005). Another earthworm, Aporrectodea longa (Ude), when exposed to soil samples spiked with B[a]P and/or lindane, demonstrated intestinal cells to be more sensitive to the effect of the genotoxicants than the crop/gizzard cells (Martin et al. 2005).
Fourie et al. (2007) used five earthworm species (Amynthas diffringens, Aporrectodea caliginosa, Dendrodrilus rubidus, Eisenia foetida, and Microchaetus benhami) to study genotoxicity of sublethal concentrations of cadmium sulphate, with significant DNA damage being detected in E. foetida followed by D. rubidus and A. caliginosa. The study showed a 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.
Comet assay in Drosophila
The simple genetics and developmental biology of Drosophila melanogaster has made it the most widely used insect model and has been recommended as an alternate animal model by the European Centre for the Validation of Alternative Methods (ECVAM; Benford et al. 2000). Recently, Drosophila has evolved as a model organism in toxicological studies (Mukhopadhyay et al. 2003; Nazir et al. 2003). D. melanogaster has also been used as an in vivo model for assessment of genotoxicity using Comet assay (Bilbao et al. 2002; Mukhopadhyay et al. 2004; Siddique et al. 2005a, b; Table 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 (Bilbao et al. 2002).
Third instar larvae of D. melanogaster (Oregon R+) were validated for genotoxicity assessment using a modified Comet assay (Siddique et al. 2005a, b). As 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 dimethyl sulfoxide (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), which were mixed in standard Drosophila diet and produced a significant dose-dependent response (Siddique et al. 2005a, b). Cypermethrin, a synthetic pyrethroid, even at low concentrations (at 0.002 ppm), and leachates of industrial waste produced significant dose-dependent increase in DNA damage in the brain ganglia and anterior midgut of D. melanogaster (Mukhopadhyay et al. 2004; Siddique et al. 2005b). Results from Comet assay have also shown a direct correlation between the concentrations of cisplatin adducts and DNA damage in somatic cells of D. melanogaster (García Sar et al. 2008).
In vitro studies using Drosophila S2 cells demonstrated that the ectopically expressed DNA glycosylases (dOgg1 and RpS3) reduced the oxidized guanosine (8-OxoG) but contributed to increased DNA degradation due to one of the constituents of the DNA repair system (Radyuk et al. 2006).
The studies in Drosophila have shown it to be a good alternate to animal model for the assessment of in vivo genotoxicity of chemicals using the Comet assay.
Comet assay in other invertebrates
Nereis virensa, a polychaete, plays an important role in the distribution of pollutants in sediments due to their unique property of bioturbation. These worms are similar to earthworms in soil and can be used for genotoxicity assessment of sediments. Intracoelomic injection of B[a]P was given to the worms, and Comet assay was conducted on coelomocytes (De Boeck and Kirsch-Volders 1997). 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 (De Boeck and Kirsch-Volders 1997).
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 insects due to industrial pollutants (Augustyniak et al. 2006).
The estuarine grass shrimp, Palaemonetes pugio, exposed to coal combustion residues from coal-fired electrical generation, were studied for DNA damage using Comet assay. Chronic exposure caused DNA damage in hepatopancreas cells of adult shrimps as compared to reference shrimp (Kuzmick et al. 2007). Comet assay in planarians is an important test for environmental monitoring studies, as these are simple organism with high sensitivity, low cost, and high proliferative rate (Prá et al. 2005). The genotoxic potential of water from Diluvio’s Basin was evaluated in planarians where increase in pollutants towards the basin led to an increase in the DNA damage in these species (Prá et al. 2005). Significant increase of primary DNA damage was observed in planarian cells due to Norflurazon, a bleaching herbicide (Horvat et al. 2005), and copper sulfate (Guecheva et al. 2001) when compared to the control animals.
These studies have also shown the use of Comet assay in biomonitoring diverse environmental conditions utilizing sentinel species.
Vertebrates
Studies of vertebrate species where the Comet assay is used include fishes, amphibians, birds, and mammals. Cells (blood, gills, kidneys, and livers) of different fishes, tadpoles and adult frogs, as well as rodents have been used for assessing in vivo and in vitro genotoxicity of chemicals, and human biomonitoring has also been carried out employing the Comet assay (Table 1).
Comet assay in fishes
Various fishes (freshwater and marine) have been used for environmental biomonitoring, as they are endemic organisms, which serve as sentinel species for a particular aquatic region, to the adverse effects of chemicals and environmental conditions. The Comet assay has found wide application as a simple and sensitive method for evaluating in vivo as well as in vitro DNA damage in different tissues (gills, liver, blood) of fishes exposed to various xenobiotics in the aquatic environment (Table 1).
Environmental biomonitoring to assess the genotoxic potential of river waters has been carried out in hepatocytes of chub (Leuciscus cephalus; Winter et al. 2004), erythrocytes of mullet (Mugil sp.), sea catfish (Netuma sp.; de Andrade et al. 2004a, b), bullheads (Ameiurus nebulosus), and carps (Cyprinus carpio; Pandrangi et al. 1995; Buschini et al. 2004). Basal level of DNA damage has been shown to be influenced by various factors, such as temperature of water in erythrocytes of mullet and sea catfish (de Andrade et al. 2004a, b), age and gender in dab (Limanda limanda; Akcha et al. 2003), and exhaustive exercise in chub (Aniagu et al. 2006). Therefore, these factors should be accounted for during environmental biomonitoring studies for genotoxicity. The sensitivity of the assay may be affected by high intra-individual variability (Akcha et al. 2003). The protocol and experimental conditions used for the Comet assay for monitoring marine ecosystems may lead to differences in the results obtained (Belpaeme et al. 1998). The use of chemical and mechanical procedures to obtain cell suspension may also lead to DNA damage (Kosmehl et al. 2006). 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 (de Miranda Cabral Gontijo et al. 2003). Hence, keeping in mind animal welfare, multi-sampling in the same fish can be conducted.
In vitro studies on fish hepatocytes (Risso-de Faverney et al. 2001), primary hepatocytes and gill cells (Schnurstein and Braunbeck 2001), as well as established cell lines (with metabolic competence; Nehls and Segner 2001, 2005) 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 (Villarini et al. 1998), tannins (Fedeli et al. 2004), and low concentrations (<10 μM) of diaryl tellurides and ebselen, an organoselenium compound (Tiano et al. 2000), in oxidative DNA damage has been studied in nucleated trout (Oncorhynchus mykiss) erythrocytes for use of these compounds in biological systems. Kammann et al. (2000) 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 the use of DNA repair inhibitor, 1-beta-d-arabinofuranosylcytosine (ara C). 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 (Cabrita et al. 2005; Labbe et al. 2001), sea bass (Dicentrarchus labrax; Zilli et al. 2003), and gilthead sea bream (Sparus aurata; Cabrita et al. 2005). The DNA damage was prevented by the addition of cryopreservants such as bovine serum albumin and dimethyl sulfoxide (Zilli et al. 2003). These studies have demonstrated the sperm Comet assay as a useful model in determining the DNA integrity in frozen samples for commercially cultured species.
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.
Comet assay in amphibians
Comet assay in amphibians has been carried out at adult and larval stages for eco-genotoxicity of aquatic environments, and studies until 1999 have been well reviewed by Cotelle and Ferard (1999). The animals chosen for the Comet assay act as sensitive bioindicators of aquatic and agricultural ecosystems (Table 1). The animals were either collected from the site (in situ) or exposed to chemicals under laboratory/natural conditions.
Erythrocytes from tadpole of two sentinal species Rana clamitans and Rana pipiens have been used for in situ genotoxicity monitoring of water bodies (Ralph and Petras 1997). 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 the 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 time (Ralph and Petras 1997) and during various metamorphosis states (Ralph and Petras 1998a) 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 indigenous population is not present due to ecological imbalance from pollution. R. clamitans and American toad (Bufo americanus) tadpoles were caged at polluted reference site and demonstarted significant (P < 0.05) increases in DNA damage, relative to control tadpoles in the laboratory (Ralph and Petras 1998b). 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. (2007) 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. Effect of polyploidy on bleomycin-induced DNA damage and repair in Xenopus laevis (pseudotetraploid) and Xenopus tropicalis (diploid) was studied using Comet assay (Banner et al. 2007). The X. tropicalis was more sensitive with 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.
Comet assay in birds
There are few studies involving Comet assay in birds (Table 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 (Baos et al. 2006; Pastor et al. 2001a, b, 2004); however, species-specific and intraspecies differences were observed. Faullimel et al. (2005) 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.(2006) reported that T-2 toxin and deoxynivalenol (DON) induced DNA fragmentation in chicken spleen leukocytes which was abrograted by dietary nucleotides. Kotłowska et al. (2007) have demonstrated increased DNA fragmentation in turkey sperm after 48 h of liquid storage and might be helpful in evaluating the DNA integrity for artificial insemination.
Comet assay in rodents
Mice and rats have been widely used as animal models for the assessment of in vivo genotoxicity of chemicals using the Comet assay (Table 1). The in vivo Comet assay has been accepted by the UK Committee on Mutagenicity testing of chemicals in food, consumer products, and environment (COM 2000) 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, Comet assay finds place as a supplemental in vivo test which has been accepted by international guidelines (Brendler Schwaab et al. 2005). There are specific guidelines for the performance of Comet assay in vivo for reliable results (Tice et al. 2000; Hartmann et al. 2003; Burlinson et al. 2007).
Multiple organs of mouse/rat including brain, blood, kidney, lungs liver, and bone marrow have been utilized for the comprehensive understanding of the systemic genotoxicity of chemicals (Meng et al. 2004; Patel et al. 2006; Sasaki et al. 2000; Sekihashi et al. 2002). 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 non-target organs can also be seen (Sasaki et al. 2000). A 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 were compiled by Sasaki et al. (2000, 2002). 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 (Sasaki et al. 2002). The Comet assay can be used as an in vivo test apart from the cytogenetic assays in hematopoietic cells and also for those compounds which have poor systemic bioavailability.
Different routes of exposure in rodents have been used, e.g., intraperitoneal (Ansari et al. 2004; Patel et al. 2006), oral (Risom et al. 2007; Wang et al. 2006), and inhalation (Meng et al. 2005; Valverde et al. 2002), 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 (Sekihashi et al. 2002). The in vivo comet assay helps in hazard identification and assessment of dose–response relationship as well as mechanistic understanding of a substance’s mode of action. Besides being used for testing the genotoxicity of chemicals in laboratory-reared animals, Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation (Mateos et al. 2008).
In vivo Comet assay in rodents is an important test model for genotoxicity studies, as many rodent carcinogens are also human carcinogens, and hence, this model not only provides an insight into the genotoxicity of human carcinogens but also is suited for studying their underlying mechanisms.
Comet assay in humans
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 characterization (Albertini et al. 2000; Dusinska and Collins 2008; Moller 2005, 2006a; Table 1). DNA damage assessed by the Comet assay gives an indication of recent exposure and at an early stage where it could also undergo repair (Maluf and Erdtmann 2001), and thus, it provides an opportunity for intervention strategies to be implemented timely. 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 sample (~2–3 ml) as well as proliferating cell population (or cell culture). Human biomonitoring using the Comet assay is advantageous, as it is rapid, cost-effective, easy compilation of data and concordance with cytogenetic assays (Faust et al. 2004a).
The assay has been widely used in studying DNA damage and repair in healthy individuals (Bajpayee et al. 2002, 2005; Betti et al. 1995; Collins 2004) in clinical studies (Corrie et al. 2005; Wynne et al. 2007; McKenna et al. 2008) as well as in dietary intervention studies (Glei et al. 2005; Moller et al. 2004; Moller and Loft 2002; Porrini et al. 2005; Wilms et al. 2005) and in monitoring the risk of DNA damage resulting from occupational (Güerci et al. 2006; Garaj-Vrhovac and Zeljezic 2002; Piperakis et al. 2006; Srám and Binková 2000), environmental (Gutiérrez-Castillo et al. 2006; Pandey et al. 2005), oxidative DNA damage (Cavallo et al. 2006b; Palus et al. 2005), exposures or lifestyle (Avogbe et al. 2005; Dhawan et al. 2001). White blood cells or lymphocytes are the most frequently used cell type for Comet assay in human biomonitoring studies (reviewed by Angerer et al. 2007; Faust et al. 2004a, b; Moller 2006b); however, other cells have also been used, e.g., buccal cells (Szeto et al. 2005), nasal (Mussali- Galante et al. 2005), sperm (Delbes et al. 2007; Fraser 2004; Schmid et al. 2007; Singh et al. 2003), epithelial (Graham-Evans et al. 2004; Emri et al. 2004; Rojas et al. 2000), and placental cells (Augustowska et al. 2007).
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 (Djuzenova et al. 1999; Lin et al. 2007; Schabath et al. 2003; Shao et al. 2005). The in vitro Comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates (Witte et al. 2007) 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 (Anderson 2001; Moller et al. 2000).
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.
Specificity, sensitivity, and limitations of the Comet assay
The Comet assay has found worldwide acceptance for detecting DNA damage and repair in prokaryotic and eukaryotic cells. However, there are issues relating to the specificity, sensitivity, and limitations of the assay which need to be addressed by the 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 (Moller 2006a). Cell to cell, gel to gel, culture to culture, animal to animal variability as well as use of various image analysis systems or visual scoring (Forchhammer et al. 2008) 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 and DNA adduct formation cannot be measured. The specific and sensitive detection of these lesions requires the use of lesion-specific enzymes (Collins 2004). These enzymes are bacterial glycosylase/endonuclease enzymes, which recognize a particular 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 amount of DNA in the tail (Moller 2006a). Oxidized pyrimidines are detected with use of endonuclease III, while oxidized purines with formamidopyrimidine DNA glycosylase (FPG). Modifications have been made in the protocol to specifically detect double-strand breaks (neutral Comet assay; Singh 2000), single-strand breaks (at pH 12.1; Miyamae et al. 1997), DNA cross-links (decrease in DNA migration due to cross-links, Singh 2000), and apoptosis (Singh 2000). 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 (Yasuhara et al. 2003). An adaptation of the Comet assay was also developed which enables the discrimination of viable, apoptotic, and necrotic single cells (Morley et al. 2006). Use of proteinase-K specifically removes DNA–protein cross-links, leading to increased migration but would not affect the DNA–DNA cross-links, thereby indicating a specific type of lesion (Singh 2000).
Tail (%) DNA and Olive tail moment give a good correlation in genotoxicity studies (Kumaravel and Jha 2006), and as 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 considered a better parameter (Kumaravel and Jha 2006).
It is therefore required that the in vitro and in vivo testing be conducted according to the Comet assay guidelines and appropriately designed multi-laboratory international validation studies be carried out.
Guidelines for the in vitro as well as in vivo Comet assay have been formulated (Hartmann et al. 2003; Tice et al. 2000). Recently, issues relating to study design and data analysis in Comet assay were discussed by the International Workgroup on Genotoxicity Testing where particular attention was given to the alkaline version (pH > 13) of the in vivo Comet assay and recommendations were made for a standardized protocol, which would be acceptable to international agencies (Burlinson et al. 2007). 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 inter-laboratories studies.
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 (COM 2000), and international validation studies are underway supported by 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 (Burlinson et al. 2007).
There has been only one multi-laboratory 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 Comet assay in human lymphocytes (Gedik and Collins 2005). 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 optimize their research work (Moller 2006a). Moller (2006b) 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 (Moller 2006a).
Conclusion
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 proven it to be a Rossetta Stone in the garden of Genetic Toxicology.
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Dhawan, A., Bajpayee, M. & Parmar, D. Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biol Toxicol 25, 5–32 (2009). https://doi.org/10.1007/s10565-008-9072-z
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DOI: https://doi.org/10.1007/s10565-008-9072-z