The Comet Assay

Purpose. The purpose of the comet assay is to evaluate the potential of the test substance to induce primary DNA damage—that is, DNA strand-breaks in treated animals, usually rodents (Ostling and Johanson 1984 Singh et al. 1988 Ohve et al. 1990a,b 1991 Ohve 2002 Collins 2002). It is applicable to any tissue from which a sufficient amount of ceUs or nuclei can be isolated without damaging DNA or triggering DNA repair processes. 

Regulatory Acceptance. The comet assay is widely accepted by regulatory agencies, even though it was only relatively recently developed and is still being validated. No OECD guidelines are yet available, but several publications provide internationally agreed protocol recommendations (Tice et al. 2000 Hartmann et al. 2003 Burhnson et al. 2007). 

Study Design. The compound is administered by the most appropriate route to at least four to five animals per group and at a minimum of two doses (MTD and 25-50% MTD). After a single administration, the tissues are sampled respectively 

2-6 hours (preferably 3) and 16-26 hours (preferably 21) after treatment, in order 

An increase in DNA migration parameters indicates that the test substance has induced DNA strand-breaks, while a decrease suggests DNA-DNA and DNA-protein crosslinks. 

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Sasaki, Y.F. et al., The comet assay with 8 mouse organs results with 39 currently used food additives, Mutation Res., 519, 103, 2002. 

Collins, A. R. (2004). The COMET assay for DNA damage and repair. Molecular... 

Betti, C. and M. Nigro. 1996. The Comet assay for the evaluation of the genetic hazard of pollutants in cetaceans preliminary results on the genotoxic effects of methyl-mercury on the bottle-nosed dolphin (Tursiops truncatus) lymphocytes in vitro. Mar. Pollut. Bull. 32 545-548. 

Olive, P.L., Banath, J.P., and Durand, R.E., Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay, Radiat. Res., 122, 86, 1990. 

Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., and Tsuda, S., The comet assay with 8 mouse organs results with 39 currently used food additive, Mutat. Res., 519,103, 2002. 

Sekihashi, K., Yamamoto, A., Matsummura, Y., Ueno, S., Watanabe-Akanuma, M., Kassie, F., Knasmuller, S., Tsuda, S., and Sasaki, Y.F., Comparative investigation of multiple organs of mice and rats in the comet assay, Mutat. Res., 517, 53, 2002. 

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

Kleinsasser NH, Harreus UA, Kastenbauer ER, Wallner BC, Sassen AW, Staudenmaier R, Rettenmeier AW (2004) Mono(2-ethylhexyl) phthalate exhibits genotoxic effects in human lymphocytes and mucosal cells of the upper aerodigestive tract in the comet assay. Toxicol Lett 148 83-90... 

Analysis of Deoxyribonucleic Acid Damage Using the Comet Assay Two... 

DAB was genotoxic in the comet assay inducing DNA damage in the stomach, colon liver, bladder, lung, and bone marrow. It is also mutagenic to Salmonella in the Ames test. Because of its demonstrated carcinogenicity in animals, human exposure to DAB by any route should be avoided. In recent years, this compound has been used only in laboratories as a model of tumorigenic activity in animals. It is not produced commercially in the United States and is of little occupational health importance. 

Tsuda S, Matsusaka N, Madarame H, et al The comet assay in eight mouse organs results with 24 azo compounds. MutatRes 465 11-26, 2000... 

Jaloszynski P, Kujawski M, Wasowicz M, et al Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay. Mutat Res-. 439(2) 199-206, 1999... 

Witte, I., Plappert, U de Wall, H. and Hartmann, A. (2007) Genetic toxicity assessment Employing the best science for human safety evaluation part III The comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection. Toxicological Sciences, 97, 21-26. 

Figure 19.5 Visualization of DNA damage induction in cultured human keratinocytes by photo-activated lomefloxacin using the comet assay. The presence of DNA breaks (induced either by ROS or by excision of DNA lesions) leads to fragmentation and electrophoretic migration to produce the comet tails, whereas bulky genomic DNA remains in the comets heads.

Figure 19.6 Cell type-dependent induction of DNA damage as revealed by the comet assay performed on melanocytes, fibroblasts and keratinocytes (see [43]).

Reconstructed skin models may also be used to study photogenotoxicity. In fact, the comet assay was recently adapted to such models, using a specific technique, that is, dissociation and separation ofkeratinocytes after UV exposure of the reconstructed epidermis. Using a mixture of specific enzymes cocktail, it was possible to obtain suspension of cells without damaging them. For instance, the photocomet assay could be successfully performed for lomefioxacin after UVA exposure of reconstmcted epidermis [76], as shown in Figure 19.7. 

B2. Benjamin, E. N., Measuring apoptosis in individual cells with the comet assay. Promega Notes 64, 13 (1997). 

F6. Florent, M., Godard, T., Ballet, J. J., Gauduchon, R, and Sola, B., Detection by the comet assay of apoptosis induced in lymphoid cell hnes after growth factor deprivation. Cell Biol. Toxicol. 15,185-192 (1999). 

A study using the Comet assay in mucosal cells isolated from human biopsies of the upper aerodigestive tract indicated that 7V-nitrosodiethanolamine caused significant DNA damage in oral cavity epithelia and also in the mucosa of the phaiynx and larynx. 

Anderson. D.. Yu. T.-W. Schmezer, P. (1995) An investigation of the DNA-damaging ability of benzene and its metabolites in human lymphocytes, using the comet assay. Environ, mol. Mutag.. 26. 305-314... 

Klaude, M., Eriksson, S., Nygren, J. Ahnstrdm, G. (1996) The comet assay mechanisms and technical considerations. Mutat. Res., 363, 89-96... 

Duthie, S.J. Collins, A.R. (1997) The influence of cell growth, detoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the Comet assay) in human cells. Free Rad. Biol. Med., 22, 717-724... 

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