Computational Insight into the Activation Mechanism of Carcinogenic N'-Nitrosonornicotine (NNN) Catalyzed by Cytochrome P450.

Tobacco-specific N'-nitrosonornicotine (NNN), a genotoxic nitrosamine classified as Group 1 carcinogen, is also present in atmospheric particulate matter and has even been detected as a new disinfection byproduct in wastewaters. NNN generally requires metabolic activation by cytochrome P450 enzymes to exert its genotoxicity, but the respective biotransformation pathways have not been described in detail. In this work, we performed density functional theory (DFT) calculations to unravel possible NNN activation pathways including α-hydroxylation, β-hydroxylation, pyridine N-oxidation, and norcotinine formation. The results reveal an initial rate-determining Hα-atom abstraction step for α-hydroxylation, followed by an unexpected kinetic competition between denitrosation and OH rebound, leading to ( iso-)myosmine as a detoxified product and α-hydroxyNNNs as the precursor of carcinogenic diazohydroxides, respectively. Further detoxification routes are given by β-hydroxylation with relative high reaction barrier and N-oxidation with comparable barrier to the toxifying α-hydroxylation. Moreover, we show for the first time how norcotinine can be generated as a minor NNN metabolite that is formed from iso-myosmine through a unique porphyrin-assisted H atom 1,2-transfer mechanism. These results demonstrate that the carcinogenic potential of NNN is subject to a kinetic competition between activating and deactivating metabolic routes, and identify respective biomarkers to inform about the individual risk associated with NNN exposure.