Effects of phenobarbital and 3-methylcholanthrene induction on the formation of three glucuronide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco specific carcinogen believed to be a causative agent for human lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated, by a-hydroxylation, at either the methyl or methylene carbons adjacent to the N-nitroso group. We recently reported the presence of a glucuronide conjugate of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (α-hydroxymethylNNK-Gluc) in the urine of Phenobarbital (PB) treated rats, and in the media of PB induced hepatocytes incubated with NNK. PB induces the α-hydroxylation of NNK, which generates the aglycon, as well as several UDP-glucuronosyl transferases. In the study presented here, we compared the metabolism of NNK to α-hydroxymethylNNK-Gluc by PB induced, 3-methylcholanthrene (3-MC) induced and control rat hepatocytes. Media was analyzed for the products of α-hydroxylation, N-oxidation and glucuronidation by radioflow HPLC. PB induced both N-oxidation and α-hydroxylation of NNK. 3-MC did not induce N-oxidation but induced α-hydroxylation more than 10-fold. α-HydroxymethylNNK-Gluc was not detected (< 0.05% total metabolites) when control hepatocytes were incubated with 1 to 100 μM NNK. When 3-MC and PB induced hepatocytes were incubated with 1-100 μM NNK α-hydroxymethylNNK-Gluc, expressed as the average percent of metabolites, accounted for 0.725 ± 0.27 and 1.35 ± 0.24% (± S.D.) of the NNK metabolites, respectively. The percent of NNK metabolized to α-hydroxymethylNNK-Gluc is small. But this glucuronide is potentially important in NNK carcinogenesis, since its formation results in the direct conjugation of an active metabolite responsible for DNA adduct formation. When PB induced rats were injected with NNK the level of NNK hemoglobin adducts, which can serve as surrogates for DNA adducts, decreased 50% compared to control rats administered NNK. Hepatic microsomal metabolism increased 2-fold and urinary α-hydroxymethylNNK-Gluc increased more than 10-fold in PB treated rats. One explanation for the decrease in NNK hemoglobin adducts may be a PB induced increase in the glucuronidation of α-hydroxymethylNNK, the metabolite responsible for adduct formation.