THE ABILITY OF THE COMET ASSAY TO DISCRIMINATE BETWEEN GENOTOXINS ANDCYTOTOXINS

The Comet assay has been used widely in genetic toxicology, radiation biology and medical and environmental research. This assay detects sin gle-strand breaks and alkali-labile sites in DNA and DNA degradation d ue to necrosis or apoptosis. It may also be modified to detect DNA cro sslinking. Although a considerable number of chemicals have been teste d in the assay there are many aspects of validation to be considered b efore the method could be considered to provide definitive evidence of genotoxic potential, For example, very few non-genotoxins have been t ested to assess specificity of the Comet assay and there has been only one reported study which investigated whether the in vitro Comet assa y is prone to false positive responses due to cytotoxicity. We have in vestigated the response of the alkaline Comet assay in TK6 human lymph oblastoid cells to cytotoxic damage and genotoxic damage, Several comp ounds which are toxic by different mechanisms were tested in the assay . Cycloheximide and trypsin gave a negative comet response at a highes t dose of 5 mg/ml and no toxicity was observed, Sodium lauryl sulphate and potassium cyanide produced a significant increase in DNA migratio n at cell survival levels of less than or equal to 75%. The distributi on of damaged cells indicated that cells at various stages of necrotic cell death were present. Hydrogen peroxide, 4-nitroquinoline oxide, 9 -aminoacridine, ethyl methanesulphonate, N-nitroso-N-ethylurea and gly oxal gave a positive comet response, Mitomycin C was negative at survi val levels of similar to 70%. These results indicate that the maximum concentration of test substance tested should produce viabilities > 75 % in order to avoid false positive responses due to cytotoxicity, The assay was able to detect DNA damage induced by an alkylating agent, an intercalating agent and oxidative damage. The cross-linking agent mit omycin C was not detected if a cut-off point of 75% viability is used as the criterion of a positive response.