FORMATION AND PERSISTENCE OF DNA-ADDUCTS IN POUCH SKIN FIBROBLASTS AND LIVER-TISSUE OF RATS EXPOSED IN-VIVO TO THE MONOFUNCTIONAL ALKYLATING-AGENTS N-METHYL-N-NITROSOUREA OR N-ETHYL-N-NITROSOUREA

Base substitutions and frameshifts induced by genotoxic agents are considered to result mainly from incomplete repair and incorrect replication of modified nucleotides in DNA. In this study, induction and persistence of O-6-alkyl- and 7-alkylguanine adducts were determined by reverse phase HPLC and electrochemical detection in DNA of pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU). Although an exposure dependent increase in the level of adducts was found for both chemicals, a much lower frequency of both O-6-alkylguanine and 7-alkylguanine was detected after ENU treatment than after MNU treatment, indicating that MNU is much more reactive with DNA than ENU. The persistence of O-6-alkyl- and 7-alkylguanine was studied for up to 48 h at exposure levels of 60 mg/kg for MNU and 100 mg/kg for ENU. A time-dependent decline in the levels of both adducts was observed, but O-6-alkylguanine was more rapidly lost than 7-alkylguanine in both pouch skin fibroblasts and liver. Furthermore, DNA adducts were faster lost from liver than from pouch skin fibroblasts. The loss of O-6-alkylguanine adducts is probably mediated by the action of O-6-alkylguanine-DNA alkyltransferase (AGT) in the target tissues since AGT activity was detectable in protein extracts of pouch skin fibroblasts and liver from unexposed rats and from exposed rats, 48 h but not 1 h after MNU and ENU treatment. AGT activity recovered faster in liver tissue than in pouch skin fibroblasts, and after ENU exposure an induction of AGT activity was observed in the liver but not in pouch skin fibroblasts. The difference in the level of O-6-alkylguanine in DNA of pouch skin fibroblasts introduced upon exposure to MNU and ENU may explain the molecular nature of most base pair changes observed previously in spectra of hprt mutants induced in these cells in vivo. The frequency of O-6-methylguanine upon MNU exposure remains relatively high with time and these adducts most likely cause GC to AT transitions. In the case of ENU, O-6-ethylguanine was detected at very low frequencies resulting in a low contribution of GC to AT transitions. Rather, the ENU spectrum is dominated by base pair changes at AT base pairs.