The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress.Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl-N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48 h after treatment with the monofunctional alkylating agents.

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The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips 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 assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. 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.

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The genotoxic effect of the monofunctional alkylating agent N:-methyl-N:-nitrosourea (MNU) on root-tip nuclei of the field bean, Vicia faba, has been tested by comparative application of three protocols of the comet assay. While the alkaline denaturation/alkaline electrophoresis (A/A) procedure proved to be most sensitive at low doses, the alkaline denaturation/neutral electrophoresis (A/N) procedure yielded an optimal dose-response curve within a wider dose range. With the neutral electrophoresis without alkaline denaturation (N/N) procedure only minimal response was found. MNU-mediated single-strand breaks occurred in nuclei of all interphase stages. Detection of tandemly repeated FOK:I elements on comets by fluorescence in situ hybridization showed an average involvement of these heterochromatin-specific sequences in MNU-mediated single-strand breaks. This, together with previous results, suggests that the pronounced clustering of chromosomal aberrations in heterochromatic regions after treatment with S phase-dependent mutagens is mainly due to an error-prone interference of recombinative repair and replication in damaged basic repeats of large tandem repeat arrays.