Kinetic of induction and disappearance of DNA-strandbreaks and mutations of different alkylating agents: 3-nitrosooxazolidine-2-one (NOZ-2), 3-nitrosooxazolidin-5-one (NOZ-5) and glycidamide using comet-assay and hPRT-gene-mutation-assay in V79-cells.

A71

Genotoxic N-Nitrosooxazolidinones have been reported to be generated from nitrosated amino acids in the presence of dietary aldehydes. These compounds can rapidly hydrolyse, generating highly reactive diazonium-ions supposed to covalently interact with nucleophilic centers in DNA. The induction and disappearance kinetics of DNA-strandbreaks was studied with the putative hydroxyethylating NOZ-2 and its carboxymethylating/methylating analogue NOZ-5. Glycidamide (GA), a preferentially N7-guanine-alkylating agent, was tested in comparison. Mutagenicity was determined by the hPRT-gene-mutation-assay. NOZ-2 strongly induced DNA-strandbreaks (SSB) already after 15' incubation (≥3µM). Additional treatment with the DNA-repair enzyme formamido-pyrimidine-DNA-glycosylase (FPG) did not substantially increase DNA-strandbreaks. In contrast, GA and NOZ-5 were only marginally active without FPG-treatment (≥300µM), but FPG-treatment elevated the response significantly (≥10µM). Moreover, whilst NOZ's induced DNA-strandbreaks very fast (15' incubation), GA reacted much slower (≥ 1h). Within 8h, GA- and NOZ-5-induced DNA-strandbreaks disappeared nearly completely. In contrast, within the same time-interval the NOZ-2-induced DNA-lesions were persistent. Both NOZ's were strongly mutagenic, inducing significant hPRT-mutations already at ≥10µM. GA became significantly mutagenic only at about 80-fold higher concentrations. In terms of DNA-lesions, NOZ-2 is expected to hydroxyethylate, NOZ-5 to carboxymethylate/methylate DNA at various nucleophilic centers, including O6 and N7 of guanine. Predominantly however, alkylation at the phosphodiester groups of the DNA-backbone is to be expected. Since V79 cells are repair-deficient concerning O6-alkyltransferase, the strong hPRT-mutagenicity of NOZ's might be ascribed to proportionate O6-alkylation and to further more persistent lesions. Given, that N7-adducts are subject to FPG-repair, the strong comet-signal-enhancement by FPG for NOZ-5 finds an explanation. In contrast, the FPG-effect on the NOZ-2-induced comet-signal is small, most probably because phosphate group hydroxyethylation triggers spontaneous SSB-induction. At variance to NOZ-2 however, phosphotriesters generated by NOZ-5 are not supposed to give rise to spontaneous SSB, thus allowing the FPG-effect to become detectable. In contrast to NOZ-induced SSB, GA-induced SSB are effectively removed within 8h in V79 cells which might explain in part the rather weak hPRT-mutagenicity.