Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O⁶ position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O⁶-methyldeoxyguanosine (O⁶-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O⁶-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O⁶-methylguanine (O⁶-meG). Exposure of cells to 0.6 mM O⁶-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O⁶-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O⁶-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced Osu6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O⁶-medGuo, it is concluded that this lesion is highly pro-mutagenic.