Effect of in vitro exposure time on comet assay results

Some mutagens are inactivated rapidly by components included in culture media, especially by serum. Long incubation periods may not be appropriate for the comet assay because DNA lesions may be repaired during the time that mutagens are inactivated, leading to false negative results. We questioned how the exposure period of Chinese hamster ovary cells to 8 unstable mutagens affected outcome of the assay.Although the longest biological half-life of the test mutagens was 1.98 h, four were positive following 0.5—24 h incubations while other four were positive only when the incubation period was ≤ 4 h, suggesting that the DNA damage was repaired and the mutagens were inactivated. The rapid inactivation of mutagens in the medium did not affect whether the outcome of the comet assay was positive or negative when cells were exposed for 1—4 h. Based on these results, we concluded that long exposure should not be employed for the compounds that are unstable in culture media, and appropriate incubation time should be determined for them individually.