Assessing the Physiological Relevance of the Bioactivation of Tobacco Specific Carcinogen NNK and its Metabolite NNAL: Quantitative Analyses of Hydrolysis Products from Their Protein Adducts under Different Carcinogen Exposure Conditions

4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are tobacco-specific lung carcinogens. NNK is widely used in preclinical models to induce lung tumorigenesis for elucidating carcinogenesis mechanisms and developing preventive agents, because of the high tumor burden in a cost-effective manner. Typical NNK doses employed in these models substantially exceed levels in humans and the routes of administration (e.g., intraperitoneal injection, oral gavage, or delivery via drinking water) differ fundamentally from inhalational exposure through cigarette smoke. At the molecular level, NNK and NNAL undergo metabolic bioactivation by cytochrome P450 enzymes, generating reactive intermediates capable of covalently modifying DNA and proteins, thereby initiating carcinogenesis. NNK and NNAL are also subject to competing metabolism. Their portion undergoing carcinogenic bioactivation is expected to vary as a function of dose and route of exposure, raising concerns of physiological relevance of these preclinical models. Systematic comparison of NNK and NNAL bioactivation profiles in humans with these preclinical models can help assess their physiological relevance and inform translational application. Using LC-MS/MS methods, this study quantified 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, an NNK-derived bioactivation hydrolysis product) and 1-(3-pyridyl)-1,4-butanediol (PBD, an NNAL-specific bioactivation hydrolysis product) in lung, liver and serum samples from two mouse models and in human lung and plasma samples. Marketed differences were observed between NNK intraperitoneal preclinical models and human samples; in contrast, the tobacco smoke-based preclinical model better recapitulates human NNK and NNAL bioactivation. These results provide guidance for the future use of these models in investigating lung carcinogenesis and developing preventive agents.