The Pyridyloxobutyl DNA Adduct, O6-[4-Oxo-4-(3-pyridyl)butyl]guanine, Is Detected in Tissues from 4-(Methylnitrosamino)- 1-(3-pyridyl)-1-butanone-treated A/J Mice

Abstract

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent pulmonary carcinogen. This unsymmetric nitrosamine can be metabolically activated to lung DNA methylating and pyridyloxobutylating intermediates. The methyl DNA adducts are well characterized. The pyridyloxobutyl adducts are unstable under DNA hydrolysis conditions and decompose to release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). One of the HPB-releasing adducts,O⁶-[4-oxo-4-(3-pyridyl)butyl]guanine (O⁶-pobG), has been detected in DNA reacted in vitro with the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). To determine whether this adduct was formed in vivo, A/J mice were treated with 10 μmol of [5-³H]NNK and sacrificed 24 h postinjection. The mutagenic O⁶-pobG was detected in liver but not lung DNA from these animals. Since 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of NNK, it is also possible that these animals are activating NNAL to a pyridylhydroxybutylating agent. Therefore, we also measured the levels of O⁶-[4-hydroxy-4-(3-pyridyl)butyl]guanine (O⁶-phbG) in these DNA samples. While radioactivity did coelute with synthetic standard for this potential NNAL adduct in one lung DNA sample, significant levels of O⁶-phbG were not detected in any other lung or liver DNA samples. The pyridyloxobutyl adduct, O⁶-pobG, was also observed in lung and liver DNA from mice treated with 4.2 μmol of [5-³H]NNKOAc in the presence but not absence of 2.5 μmol of O⁶-benzylguanine, a known depletor of the repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT). These data indicate that this adduct is formed in vivo but is repaired in part by AGT. Cell-free extracts from A/J mouse lung and liver were used to determine the relative rate of O⁶-alkylguanine repair. O⁶-mG and O⁶-pobG were removed from DNA to the same extent in a competitive assay, suggesting that low levels of O⁶-pobG in lungs of NNK-treated mice did not result from preferential repair of O⁶-pobG by AGT. It is more likely that initial levels of O⁶-pobG are much lower than initial levels of O⁶-mG in lung DNA from NNK-treated A/J mice. These data are consistent with previous studies, which indicate that DNA methylation is the critical pathway for NNK-induced lung carcinogenesis in A/J mice.