LAUSR

As an abundantly present tobacco component, carcinogen 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) has also been detected in atmospheric particulate matter, suggesting the ineluctable exposure risk of this contaminant. NNK metabolic activation by cytochrome P450 enzymes (CYPs) is a prerequisite to exert its genotoxicity, but the metabolic regioselectivity and mechanism are still unknown. Here the binding feature and regioselectivity of CYP 1A1, 1A2, 2A6, 2A13, 2B6, 3A4 toward NNK are unraveled through molecular docking and molecular dynamics (MD) simulations. Binding mode analyses reveal that 1A2 and 2B6 have definite preference for NNK α-methyl hydroxylation, while the other four CYPs preferentially catalyze α-methylene hydroxylation. The binding affinities between NNK and CYPs evaluated by the binding free energies follow the order of 2A13 > 2B6 > 1A2 > 2A6 > 1A1 > 3A4. The density functional theory (DFT) calculations are further performed to characterize the mechanism of NNK biotransformation. Results show that the α-hydroxyNNK generated from α-hydroxylation may undergo nonenzymatic decomposition to form genotoxic diazohydroxide and aldehyde, and further oxidation by P450 to yield nitrosamide, which mainly contributes to NNK toxification capacity. Meanwhile the pyridine N-oxidation and denitrosation of Cα-radical intermediate play an important role in detoxifying NNK. Overall, the present study provides the molecular basis for CYP-catalyzed regioselectivity and mechanism of NNK biotransformation, which can enable the identification of metabolites for assessing the health risk of individual NNK exposure.