Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity

The alkaline comet assay is a sensitive test for the detection of a variety of DNA lesions. However, crosslinks are not readily detected under standar d test conditions. Recently, modifications have been introduced measuring c rosslinks by determining the reduction of induced DNA migration. We used th e comet assay to comparatively investigate in V79 cells the effect of three different crosslinkers: formaldehyde (FA), which predominantly induces DNA -protein crosslinks, cisplatin (DDP), which mainly produces DNA-DNA-intrast rand crosslinks, and mitomycin C (MMC), which mainly leads to DNA-DNA-inter strand crosslinks. In the standard alkaline comet essay, only MMC induced a slight increase in DNA migration at high toxic concentrations. FA and DDP did not induce any DNA migration under the test conditions used. In the mod ified comet assay, all three crosslinkers led to a clear reduction of gamma -ray-induced DNA migration. This reduction was seen in the case of FA paral lel to the induction of cytotoxicity and SCE, while for MMC and DDP inducti on of cytotoxicity, SCE and HPRT gene mutations occurred at much lower conc entrations than the effects in the comet assay. The DNA-DNA crosslinkers ca used a reduction of induced DNA migration only at cytotoxic concentrations. Our results indicate that the modified comet essay protocol is a sensitive test for the detection of DNA-protein crosslinks. However, the results For MMC and DDP suggest that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. Furthermore, the relationship between c rosslinking and genotoxicity seems to be very different for the th ree diff erent types of crosslinking substances. (C) 1999 Wiley-Liss, Inc.