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

Romel Dator, Linda B. von Weymarn, Peter W. Villalta, Cory J. Hooyman, Laura A. Maertens, Pramod Upadhyaya, Sharon E. Murphy, Silvia Balbo

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)11863-11872
Number of pages10
JournalAnalytical Chemistry
Issue number20
StatePublished - Oct 16 2018

Bibliographical note

Funding Information:
This work was supported by grant CA-138338 from the National Cancer Institute. R.D. was partially supported by the 2017 American Society for Mass Spectrometry Postdoctoral Career Development Award. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant CA-077598. Salary support for P.W.V. was provided by the U.S. National Institutes of Health and the National Cancer Institute (Grant R50-CA211256). We thank Dr. Stephen Hecht (Univ. of Minnesota); Dhimant Desai, Ph.D.and Shantu Amin, Ph.D. (Penn State Milton S. Hershey Medical Center, Hershey, PA), for providing the synthesized β-OH NNK. We also thank Drs. Lisa Peterson and Irina Stepanov (Univ. of Minnesota) for their helpful comments on this work and Robert Carlson for editorial assistance.

Publisher Copyright:
Copyright © 2018 American Chemical Society.

Copyright 2018 Elsevier B.V., All rights reserved.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

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