Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/%28SICI%291098-2280%282000%2935%3A3%3C206%3A%3AAID-EM8%3E3.0.CO%3B2-J

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals.

Single cell gel electrophoresis assay: methodology and applications.

This review considers the potential for DNA strand breaks, particularly as measured by the comet assay, to act as a biomarker of genetic toxicity in fish and other aquatic species. The background need for such biomarkers is introduced in relation to carcinogenicity, reproductive effects and other adverse effects of pollution. Sensitive measurements of DNA strand breakage can be achieved, e.g., by alkaline elution, alkaline unwinding or by single cell gel electrophoresis (comet) techniques. The DNA damage can be a reflection, not only of direct strand breakage, but also of alkali-labile sites and of repair enzyme-mediated breakage (i.e., is non-specific). A range of genotoxic chemicals (both with and without the requirement for metabolic activation) give positive effects in various cell types of vertebrate and invertebrate aquatic species, following in vitro and in vivo exposures under laboratory conditions. A limited number of analyses of organisms exposed to polluted waters or sediments in the field have implicated DNA strand breakage as a relatively sensitive, rapid and broad specificity indicator of genotoxic pollutant exposure. The comet assay deserves further exploitation to assess inter-individual and inter-cell variability in response to pollutants and naturally occurring genotoxic stimuli, and to assess the persistence of these effects.

DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring

A review of the literature on the mutagenicity/genotoxicity of surface waters is presented in this article. Subheadings of this article include a description of sample concentration methods, mutagenic/genotoxic bioassay data, and suspected or identified mutagens in surface waters published in the literature since 1990. Much of the published surface water mutagenicity/genotoxicity studies employed the Salmonella/mutagenicity test with strains TA98 and/or TA100 with and/or without metabolic activation. Among all data analyzed, the percentage of positive samples toward TA98 was approximately 15%, both in the absence and the presence of S9 mix. Those positive toward TA100 were 7%, both with and without S9 mix. The percentage classified as highly mutagenic (2500-5000 revertants per liter) or extremely mutagenic (more than 5000 revertants per liter) was approximately 3-5% both towards TA98 and TA100, regardless of the absence or the presence of S9 mix. This analysis demonstrates that some rivers in the world, especially in Europe, Asia and South America, are contaminated with potent direct-acting and indirect-acting frameshift-type and base substitution-type mutagens. These rivers are reported to be contaminated by either partially treated or untreated discharges from chemical industries, petrochemical industries, oil refineries, oil spills, rolling steel mills, untreated domestic sludges and pesticides runoff. Aquatic organisms such as teleosts and bivalves have also been used as sentinels to monitor contamination of surface water with genotoxic chemicals. DNA modifications were analyzed for this purpose. Many studies indicate that the 32P-postlabeling assay, the single cell gel electrophoresis (comet) assay and the micronucleus test are sensitive enough to monitor genotoxic responses of indigenous aquatic organisms to environmental pollution. In order to efficiently assess the presence of mutagens in the water, in addition to the chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs. This is because according to this review they proved to be sensitive and reliable tools in the detection of mutagenic activity in aquatic environment. Many attempts to identify the chemicals responsible for the mutagenicity/genotoxicity of surface waters have been reported. Among these reports, researchers identified heavy metals, PAHs, heterocyclic amines, pesticides and so on. By combining the blue cotton hanging method as an adsorbent and the O-acetyltransferase-overproducing strain as a sensitive strain for aminoarenes, Japanese researchers identified two new type of potent frameshift-type mutagens, formed unintentionally, in several surface waters. One group has a 2-phenylbenzotriazole (PBTA) structure, and seven analogues, PBTA-type mutagens, were identified in surface waters collected at sites below textile dyeing factories and municipal wastewater treatment plants treating domestic wastes and effluents. The other one has a polychlorinated biphenyl (PCB) skelton with nitro and amino substitution group and it was revealed to be 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl derived from chemical plants treating polymers and dye intermediates. However, the identification of major putative mutagenic/genotoxic compounds in most surface waters with high mutagenic/genotoxic activity in the world have not been performed. Further efforts on chemical isolation and identification by bioassay-directed chemical analysis should be performed.

Mutagens in surface waters: a review

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a "comet with tail" formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp.

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) (“comet”) assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a “comet-with-tail” formation. Exposure of R. clamitans tadpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans tadpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment. © 1996 Wiley-Liss, Inc.

Alkaline single‐cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles

The alkaline single cell gel (SCG) or 'comet' and peripheral blood micronucleus (pbMN) assay have been used to compare the effects of the direct acting mutagens, methyl methanesulfonate (MMS) and N-nitroso-N-methylurea (NMU), and the indirect acting mutagens, benzo[a]pyrene (BAP), cyclophosphamide (CP) 9, 10-dimethyl-1,2-benzanthracene (DMBA), and mitomycin C (MMC) in an inbred house mouse (Mus domesticus) strain. The alkaline SCG assay was able to detect DNA damage from direct acting mutagens. However, it appears that, even at the highest concentrations tested, the SCG assay was not able to detect DNA damage caused by 3 of 4 indirect acting mutagens tested. The exception was BAP. The pbMN assay was sensitive to DNA damage caused by both groups of mutagens. Multiple injections did not increase the sensitivity of the SCG assay to the indirect acting mutagen CP. Further, simultaneous injections of CP and MMS, in one experiment, resulted in significantly lower (p < 0.05) average DNA ratios and micronucleated polychromatic erythrocyte counts than those obtained after treatment with MMS alone. Although the SCG assay has been shown to be sufficiently sensitive to detect DNA damage caused by both direct and indirect acting mutagens in deermice (Peromyscus maniculatus) and bullheads (Ameiurus nebulosus), similar results are not seen in the inbred house mouse strain tested.

Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens

The alkaline single-cell gel (SCG) or `comet' assay has been applied to the detection of DNA damage from a number of chemical and biological factors in vivo and in vitro. In the past, a number of cell types has been used with peripheral blood lymphocytes being the most readily accessible. This study was designed to determine whether lymphocytes sequestered in the spleen might prove more sensitive to DNA damage than those in the peripheral circulation. This would result in a more effective SCG assay. Baseline DNA length to width ratios for peripheral blood and splenic lymphocytes did not differ significantly from each other (1.27 and 1.21, respectively). Neither did ratios of lymphocytes from the two sources, sampled 20 and 48 h after injection with 100 mg/kg methyl methanesulfonate (MMS) (3.81 and 3.62 at 20 h, respectively; and 1.96 and 2.21 at 48 h, respectively). Recovery from MMS damage at 168 h postinjection was also not different in the two groups of cells (1.13 and 1.16, respectively). However, an examination of cell profiles of DNA damage showed that splenic lymphocytes had a significantly higher percentage of damaged cells (63.33%) than did peripheral blood lymphocytes (40.67%) 48 h postinjection. Of the hypotheses proposed for this difference, the most likely seems to involve the different proportions of B- and T-lymphocytes present in the peripheral blood and the spleen. Since the difference between peripheral blood and splenic lymphocytes was seen only at 48 h postinjection, the use of splenic lymphocytes in the SCG assay is not advantageous under most circumstances.

Comparison of DNA damage in peripheral blood and spleen lymphocytes using the single-cell gel assay.

The alkaline single-cell gel (SCG) assay, which determines DNA damage in mice treated with 100 mg/kg methyl methanesulfonate (MMS), was run by using three power supplies from Bio-Rad (models 3000Xi, 200/2.0, and Power Pac 300). Comparisons of the results obtained from the use of these power supplies showed differences in mean DNA tail length to width ratios and profiles of these ratios in individual cells. Model 200/2.0 power supply appeared to enhance significantly the sensitivity of the assay. In purchasing power supplies, there is a need to evaluate their effect on the sensitivity of the test.

Maria Vrzoc

Papers

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp.

Alkaline single‐cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles

Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens

Comparison of DNA damage in peripheral blood and spleen lymphocytes using the single-cell gel assay.

Comparison of three power supplies used for the single-cell gel assay.