Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay.
The single cell gel electrophoresis (SCGE) or comet assay, which measures DNA strand breaks in individual cells, was used to analyse DNA damage and repair induced by the SN1-type alkylating carcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea in CHO cells. The comet assay was comparable in sensitivity to the alkaline elution assay. The alkyl-adducts detected as DNA single-strand breaks (ssb) by this technique were completely repaired within 24 h after treatment. These data indicate that long-lived lesions, such as alkylphosphotriesters, are not converted into 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 excision repair process of N-alkylpurines. When SCGE was performed at pH 12.5 instead of pH 13.5 a lower level of ssb was detected and these breaks were completely resealed within 2 h after treatment. These data suggest that different subsets of lesions are detected under different pH conditions. The SCGE combined with inclusion within the cells of endonuclease III revealed that a high portion of AP sites induced by alkylation damage were not converted into ssb by alkali. The level of endonuclease III-sensitive sites decreased as a function of the repair time and by 24 h after treatment no sites were left on the DNA. The use of this modified SCGE assay allows the estimation of the total amount of unrepaired AP sites present on DNA. Alkylation-induced ssb as detected by the comet assay should be regarded as an indicator of repair rate and balance more than a measure of actual DNA damage.
Fortini
P
P
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Roma, Italy.
Raspaglio
G
G
Falchi
M
M
Dogliotti
E
E
D000478:Alkylation; D000818:Animals; D002273:Carcinogens / Q000633:toxicity; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005240:Feasibility Studies; D005260:Female; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence
D002273:Carcinogens; D004247:DNA