Stereo- and regioselectivity in the cytochrome P450-mediated metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed from nicotine during the curing and pyrolysis of tobacco, is found in both mainstream and sidestream cigarette smoke, and is a lung carcinogen in all animal models in which it has been tested; it is thus a putative human lung carcinogen. Elucidation of the mechanisms by which NNK and other environmental carcinogens cause cancer is necessary to expedite the search for cancer therapeutic and preventive agents. In an attempt to facilitate the excretion of NNK by increasing its hydrophilicity, cytochrome P450 (P450) monooxygenases paradoxically convert NNK into DNA-reactive electrophiles. Subsequent covalent modification of DNA can lead to DNA mutations and ultimately neoplasia—if these mutations persist in genes critical for control of cell proliferation. DNA damage does not occur in the absence of P450-mediated bioactivation, making it important to investigate the molecular details of this deleterious event.; In this thesis, NNK bioactivation was investigated by studying in vitro metabolism, by determining tumorigenicity and levels of covalently modified DNA in a mouse model of NNK-induced lung cancer, and by constructing molecular models of P450 enzymes with NNK docked in the active site. Importantly, it R hydrogen of NNK. Stereospecific deuterium substitution at this position attenuates tumorigenicity, DNA adduct formation, and metabolism of NNK. These results provide important insight into enzyme-substrate interactions leading to NNK bioactivation. In additional studies, striking differences in the regioselectivity of NNK metabolism were observed when comparing several closely related P450 enzymes. The experimentally garnered biological and biochemical data was rationalized with the aid of molecular models of P450 enzymes constructed using the crystal structure of a related mammalian enzyme as a template. These models should aid in our understanding of the mechanisms of chemical carcinogenesis and serve as guides for the rational design of enzyme inhibitors that may hold promise as chemopreventive agents.