Summary
Eight compounds (chloral hydrate, colchicine, hydroquinone, DL menthol, mitomycin C, sodium iodoacetate, thimerosal, and valinomycin) were chosen as compounds to establish the Comet assay (single cell test) as an in vitro genotoxicity screen in Genetic Toxicology of Syngenta AG (former Novartis Crop Protection AG).
Furthermore, these compounds were used to study the ability of the Comet assay to discriminate between genotoxins and cytotoxins.
All compounds were tested in the Standard Comet assay as well as in the whole cell Standard Comet assay specially designed for adherent growing cells. Selected compounds were also tested in the modified Comet assay using lysed cells (chloral hydrate, hydroquinone, sodium iodoacetate, mitomycin C, and thimerosal).
Compounds (chloral hydrate, hydroquinone, mitomycin C, and thimerosal) which are known or suspected to form DNA-DNA cross-links or DNA-protein cross-links were also checked for their ability to reduce ethyl methanesulfonate induced DNA damages. The results have been discussed and are summarized in the following table.
Evaluating all results, mitomycin C and sodium iodoacetate were finally assessed as positive and thimerosal äs inconclusive in the Comet assay. All other compounds were found to be negative in the Comet assay.
Further general conclusions could be drawn out regarding the Comet assay procedure:
- Six, instead of the four or five categories usually used in the common literature, should be taken for the comet scoring. These categories depend on DNA migration level: intact cells (IC), slightly damaged cells (SDC), damaged cells (DC), highly damaged cells (HDC), extremely damaged cells with small head (EDC I), and extremely damaged cells without head (EDC II). The characterization of cells with small but still existing heads and intensive tailing äs apoptotic cells seemed to be not completely accurate because of the fmdings with valinomycin and ethyl methanesulfonate (EMS).
- The statistical evaluation of test data on the basis of the Student's f-test is not appropriate since the categories of damage do not follow a normal distribution.
Therefore we decided, in contrast to the common literature, to use the Dunn test, a nonparametric analysis rank test based on Kruskal-Wallis rank sums.
The advantage of this test is to avoid false positive results.
- The Standard Comet assay is not able to detect genotoxins with the potential to produce DNA-DNA or DNA-protein cross-links (mitomycin C as a cross-linker gave a negative result). For the identification of compounds with the suspected ability to cross-link, a special protocol has to be performed in the presence of EMS.
- The most important thing is to set up test conditions which really give the possibility to discriminate between genotoxins and cytotoxins. The whole cell Standard Comet assay is clearly able to differentiate because of the discussed criteria. The most important criteria depend on the toxicity of the assessed concentrations. In order to discriminate a secondary cytotoxic effect from a primary genotoxic effect, the absolute toxicity of cells investigated should not exceed 10 % to avoid positive results potentially associated with cytotoxicity- induced DNA degradation. All cells (at least 25) on a slide should be included in the evaluation.
- Depending on the conditions of the modified Comet assay with lysed cells, control cells show extremely high tau moments and might therefore give false positive or false negative results. However, the modified Comet assay (no cellular metabolism and membrane barrier) is helpful in analyzing chemical and/or biological characteristics of genotoxic agents when performed in combination with the Standard Comet assay or the whole cell Standard Comet assay.
- Further work is required to evaluate the Comet assay against Standard regulatory Genetic Toxicology assays in respect to standardization and validation.
In summary, the Comet assay is an easy-to-learn, easy-to-perform, rapid, and inexpensive in vitro (and in vivo) screen for Genetic Toxicology. However, the evaluation and Interpretation of data from the Comet assay must be done very carefully and need a lot of experience (i.e., should not be used äs a time-to-time assay).
