posted on 2018-08-07, 00:00authored byRomel Dator, Linda B. von Weymarn, Peter W. Villalta, Cory J. Hooyman, Laura A. Maertens, Pramod Upadhyaya, Sharon E. Murphy, Silvia Balbo
The
tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), is a potent lung carcinogen that exerts its carcinogenic effects
upon metabolic activation. The identification and quantitation of
NNK metabolites could identify potential biomarkers of bioactivation
and detoxification of this potent carcinogen and may be used to predict
lung cancer susceptibility among smokers. Here, we 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 a custom script for automated d0–d4-m/z-pair-peak detection, enabled unbiased
identification of numerous NNK metabolites. The structures of the
metabolites were confirmed using targeted LC-MS2 with retention-time
(tR) and MS2-fragmentation
comparisons to those of standards when possible. Eleven known metabolites
and unchanged NNK were identified simultaneously. More importantly,
our workflow revealed novel NNK metabolites, including 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 distributions
of a panel of nitroso-containing NNK-specific metabolites in rats
before and after phenobarbital (PB) treatment, and this served as
a demonstration of a general strategy for the detection of metabolic
differences in animal and cell systems. Lastly, we generated a d4-labeled NNK-metabolite mixture to be used
as internal standards (d4-rat urine) for
the 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.