Human cytochrome P450-mediated metabolism of nicotine and tobacco-specific carcinogens: N-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Smoking is the principal risk factor for developing lung cancer, the leading cause of cancer-related deaths in the United States. Two potent tobacco-specific carcinogens are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N '-nitrosonornicotine (NNN). Metabolic activation of NNK and NNN is required in order to exert their carcinogenic potential. Cytochrome P450 enzymes catalyze the activation of NNK and NNN via hydroxylation of carbons positioned alpha to the N-nitroso group of the nitrosamine.;In this thesis, the role of human cytochrome P450 enzymes in the metabolism of NNK. NNN, and nicotine was investigated. Specifically, the contribution by CYP2A6 and CYP2B6 to the metabolism of NNK and nicotine in human liver microsomes was studied. CYP2B6 was determined to be an efficient catalyst of NNK metabolism but not nicotine metabolism in human liver microsomes. Although CYP2B6 is not as abundant as CYP2A6 in the liver; it expressed in the lung. Therefore, its ability to catalyze NNK alpha-hydroxylation efficiently may have important implications in the lung.;Another P450 expressed in the human lung is CYP2A13, an excellent catalyst of NNK alpha-hydroxylation. Recently several genetic polymorphisms have been identified for CYP2A13 including polymorphisms that result in single amino acid changes. Functional differences in CYP2A13-catalyzed NNK and NNN metabolism as a result of these genetic polymorphisms was investigated. No significant differences were observed for NNN metabolism. However, modest but significant decreases in NNK metabolism were determined for three variants, R257C, D158E, and V323L. This work should provide better guidelines for design and interpretation of epidemiology studies.;Lastly, a novel key active site residue of CYP2A13 was identified for NNK metabolism. Site-directed mutagenesis was used to generate a single mutation in the active site of CYP2A13. The characterization of NNK metabolism by mutant, Asn297Ala CYP2A13 confirmed the importance of this residue to the catalytic efficiency of NNK alpha-hydroxylation by CYP2A13. Computational docking of NNK into the active site of CYP2A13 indicated that residue Asn297 is also important for substrate orientation. This thesis research has contributed to the understanding of carcinogenesis by tobacco-specific carcinogens by further elucidating the cytochrome P450-catalyzed metabolic activation pathways of NNN and NNK.