The ability of the Comet assay to discriminate between genotoxins and cytotoxins

Abstract

The Comet assay has been used widely in genetic toxicology, radiation biology and medical and environmental research. This assay detects single-strand breaks and alkali-labile sites in DNA and DNA degradation due to necrosis or apoptosis. It may also be modified to detect DNA cross-linking. Although a considerable number of chemicals have been tested in the assay there are many aspects of validation to be considered before the method could be considered to provide definitive evidence of genotoxic potential. For example, very few non-genotoxins have been tested to assess specificity of the Comet assay and there has been only one reported study which investigated whether the in vitro Comet assay is prone to false positive responses due to cytotoxicity. We have investigated the response of the alkaline Comet assay in TK6 human lymphoblastoid cells to cytotoxic damage and genotoxic damage. Several compounds which are toxic by different mechanisms were tested in the assay. Cycloheximide and trypsin gave a negative comet response at a highest dose of 5 mg/ml and no toxicity was observed. Sodium lauryl sulphate and potassium cyanide produced a significant increase in DNA migration at cell survival levels of < or = 75%. The distribution 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 glyoxal gave a positive comet response. Mitomycin C was negative at survival levels of approximately 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 mitomycin C was not detected if a cut-off point of 75% viability is used as the criterion of a positive response.

In vitro and in vivo toxicity assessment of nanoparticles

“…DNA-specific fluorescent dye is used to stain the samples. The gel is then analysed for the amount of fluorescence in the head and tail and the tail length [62][63][64]. Comet assay was used to assess the toxicity of zinc oxide nanoparticles at 25 mg Zn/L on D. tertiolecta which resulted in 55% nuclei damage [65].…”

&lt;i&gt;In Vitro&lt;/i&gt; Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients

“…The viability of the treated cells was measured after the initial one-hour incubation with oxaliplatin or satraplatin by using Trypan blue exclusion according to Henderson et al [33]. Treated cells were mixed 50:50 with 0.4% Trypan blue, and then 100 cells were scored for viability using a phase-contrast microscope.…”

The genotoxicological evaluation of several local raw foods extracts on Chang liver cells by Single Cell Electrophoresis Assay

“…In this study, preliminary cytotoxicity measurement with the tetrazolium reduction assay (MTT) was performed. It is recommended in the Comet Assay Guidelines to avoid the testing of doses that decrease the viability by more than 30 (Anderson et al, 1998;Henderson et al, 1998;Tice et al, 2000). In order to fulfill these requirements and to be able to discern between cytotoxicity and genotoxicity, a short incubation time of 24 h at 37 had been applied.…”