Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay

The single cell gel electrophoresis (SCGE) or comet assay, whichmeasures DNA strand breaks in individual cells, was used toanalyse DNA damage and repair induced by the SN₁-type alkylatingcarcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosoureain CHO cells. The comet assay was comparable in sensitivityto the alkaline elution assay. The alkyl-adducts detected asDNA single-strand breaks (ssb) by this technique were completelyrepaired within 24 h after treatment. These data indicate thatlong-lived lesions, such as alkylphosphotriesters, are not convertedinto ssb under the standard SCGE alkaline conditions (pH 13.5).The lesions revealed by the comet assay are mainly apurinic/apyrimidinic(AP) sites and breaks formed as intermediates in the base excisionrepair process of N-alkylpurines. When SCGE was performed atpH 12.5 instead of pH 13.5 a lower level of ssb was detectedand these breaks were completely resealed within 2 h after treatmentThese data suggest that different subsets of lesions are detectedunder different pH conditions. The SCGE combined with inclusionwithin the cells of endo-nuclease III revealed that a high portionof AP sites induced by alkylation damage were not convertedinto ssb by alkali. The level of endonuclease III-sensitivesites decreased as a function of the repair time and by 24 hafter treatment no sites were left on the DNA. The use of thismodified SCGE assay allows the estimation of the total amountof unrepaired AP sites present on DNA. Alkylation-induced ssbas detected by the comet assay should be regarded as an indicatorof repair rate and balance more than a measure of actual DNAdamage. ³To whom correspondence should be addressed