DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay

Abstract

Using the alkaline comet assay (single cell gel electrophoresis), we studied the induction and persistence of DNA damage induced by methyl methanesulfonate (MMS) and neocarzinostatin (NCS) in the repair-deficient Chinese hamster cell lines V-E5 and XR-V15B. Effects in the comet assay were analyzed directly after treatment as well as after a postincubation period in mutagen-free medium to gain insight into the DNA repair capacities of the mutant cell lines in relation to different primary DNA lesions. Both mutagens caused a concentration-related increase in DNA strand breakage in both mutant cell lines and in the normal parental cell lines. Repair of MMS-induced DNA damage during postincubation was similar in normal and mutant cell lines, while diminished repair was seen after NCS treatment in XR-V15B cells. Our data show that XR-V15B cells only repaired about 30% of NCS-induced DNA damage within 1 h, while the parental V79 cell line repaired about 70%. Since this cell line is defective in the repair of DNA double-strand breaks (DSB), the results indicate that NCS-induced DSB significantly contribute to the genotoxic effects seen in the comet assay. However, compared to previously studied induction of gene mutations and chromosome aberrations, detection of NCS-induced DNA effects with the comet assay was less sensitive and increased DNA migration only occurred under strong cytotoxic conditions.