Pyridyloxobutyl DNA Adducts Inhibit the Repair of O6-Methylguanine

The carcinogenic tobacco-specific nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), both methylates and pyridyloxobutylates DNA. 06-Methylguanine (06-mG) persistence has been correlated to NNK-induced lung tumor formation in A/J mice. The pyridyloxobutyla-tion pathway enhances the tumorigenicity of the methylation pathway. In this paper we test the hypothesis that DNA pyridyloxobutylation increases O6-mG persistence by inhibiting the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Pyridyloxobutylated DNA was generated by reacting calf thymus DNA with the model pyridyloxobutylating agent 4-(ac-etoxymethylnitrosamino)-l-(3-pyridyl)-l-butanone (NNKOAc) in the presence of esterase. The alkylated DNA inhibited the ability of partially purified rat liver AGT to repair 06-mG when it was incubated with AGT prior to the addition of 3H-methylated DNA. The extent of inhibition was dependent on the amount of NNKOAc reacted with DNA. The ability of NNKOAc-treated DNA to inhibit AGT was destroyed when the DNA was subjected to neutral thermal hydrolysis. Approximately 1 pmol of AGT was inhibited for every 25 to 50 pmol of 4-hydroxy-l-(3-pyridyl)-l-bu-tanone-releasing adducts present in NNKOAc-treated DNA. The inhibitory activity of this alkylated DNA was relatively stable under physiological conditions (pH 7.4,37C). Only 13% of the AGT reactive activity was lost after 7 days. When pyridyloxobutylated DNA was incubated simultaneously with 3H-methylated DNA and AGT, a significant reduction in [3H]methyl transfer to AGT was observed. The levels of reduction were similar to those observed when unlabeled methylated DNA containing comparable levels of O6-mG was substituted for NNKOAc-treated DNA. Based on these results, a cocarcinogenic role for pyridyloxobutylation in NNK-induced lung tumorigenesis is proposed in which pyridyloxobutyl DNA adduct(s) compete with 06-mG for reaction with AGT resulting in sustained levels of O6-mG. These enhanced levels then increase the probability of tumor induction by NNK.