In Vivo Stable-Isotope Labeling and Mass-Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen in Rats

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen and exerts its carcinogen effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bio-activation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we have used in vivo isotope labeling and high-resolution mass spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample enrichment, LC/MS, and data analysis workflow, including the custom script for automated d(0)-d(4) m/z pair peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS(2) with retention time (t(R)) and MS(2) fragmentation comparison to standards when possible. Eleven known metabolites and the unchanged NNK were identified simultaneously. More importantly, our workflow revealed new and novel NNK metabolites including: the 1,3-Diol (13); α-OH-methyl-NNAL-Gluc (14); nitro-NK-N-oxide (15); nitro-NAL-N-oxide (16); γ-OH NNAL (17); and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distribution of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this serves as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we have generated a d(4)-labeled NNK metabolite mixture to be used as internal standards (d(4)-rat urine) for relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung cancer risk and susceptibility in smokers.