Adaptation to alkylation damage in DNA measured by the comet assay

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methonesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root rips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used For detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet essay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease ill. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation. (C) 2000 Wiley-Liss, Inc.