Biotransformation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in peripheral human lung microsomes.

The contributions of different enzymes to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) biotransformation were assessed in human lung microsomes prepared from peripheral lung specimens obtained from seven subjects. Metabolite formation was expressed as a percentage of total recovered radioactivity from [5-3H]NNK and its metabolites per milligram of protein per minute. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol was the major metabolite formed in the presence of an NADPH-generating system, with production ranging from 0.5186 to 1.268%/mg of protein/min, and total NNK bioactivation (represented by the sum of the four alpha-carbon hydroxylation endpoint metabolites) ranged from 0.002100 to 0.005685% alpha-hydroxylation/mg of protein/min. Overall, production of bioactivation metabolites was greater than that of detoxication (i.e., N-oxidation) products. Based on total bioactivation, subjects could be classified as high or low NNK bioactivators. In the presence of an NADPH-generating system, microsomal formation of the endpoint metabolite 1-(3-pyridyl)-1-butanone-4-carboxylic acid (keto acid) was consistently higher than that of all other alpha-carbon hydroxylation endpoint metabolites. Contributions of cytochrome p450 (p450) enzymes to NNK oxidation were demonstrated by NADPH dependence, inhibition by carbon monoxide, and inhibition by the nonselective p450 inhibitors proadifen hydrochloride (SKF-525A) and 1-aminobenzotriazole (ABT), particularly in lung microsomes from high bioactivators. At 5.0 mM, ABT inhibited total NNK bioactivation by 54 to 100%, demonstrating the importance of ABT-sensitive enzyme(s) in human pulmonary NNK bioactivation. Contributions of CYP2A6 and/or CYP2A13, as well as CYP2B6, to NNK bioactivation were also suggested by selective chemical and antibody inhibition in lung microsomes from some subjects. It is likely that multiple p450 enzymes contribute to human pulmonary microsomal NNK bioactivation, and that these contributions vary between individuals.

Investigation of the role of lipoxygenase in bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung.

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen believed to play a role in human lung cancer. Bioactivation of NNK involves alpha-carbon hydroxylation that could be catalyzed by cytochrome P450, hemoglobin, and lipoxygenases (LOX). In the present study, the role of LOX in NNK bioactivation was investigated. Formation of keto acid, the endpoint metabolite of alpha-methylene NNK hydroxylation, was observed in human lung cytosols incubated with 4.2 microM [5-(3)H]NNK (N = 6). Following concanavalin A affinity chromatography to enrich human lung lipoxygenase (HLLO), the fraction containing cytosolic components less LOX (fraction 1) retained the ability to bioactivate NNK. Although enriched HLLO exhibited the characteristic dioxygenase and hydroperoxidase activities, it did not bioactivate NNK. The LOX inhibitor nordihydroguaiaretic acid inhibited dioxygenase activity of HLLO by 83 +/- 19% (P < 0.05, N = 6), but did not inhibit keto acid formation in the crude cytosols (N = 6, P > 0.05). Failure of soybean LOX to catalyze NNK bioactivation supported the results observed in human lung cytosols, and failure of chemically generated alkylperoxyl radicals to bioactivate NNK further suggested that the dioxygenase activity of LOX is not likely to be involved in NNK bioactivation. Horseradish peroxidase and myeloperoxidase catalyzed NNK bioactivation were also nondetectable. Our results demonstrate that, although human lung cytosols can bioactivate NNK to form keto acid, LOX is not involved. We have attributed the ability of crude human lung cytosols to bioactivate NNK to hemoglobin. The inhibitory effect of 1-aminobenzotriazole and arachidonic acid on keto acid formation in the crude cytosols and in fraction 1, respectively (P < 0.05, N = 6), is consistent with hemoglobin-catalyzed NNK bioactivation.