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 journalArticle

2 Citations (Scopus)

Abstract

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
Volume90
Issue number20
DOIs
StatePublished - Oct 16 2018

Fingerprint

Tobacco
Metabolites
Isotopes
Carcinogens
Labeling
Mass spectrometry
Rats
Oxides
Nitrosamines
Detoxification
Acetylcysteine
Biomarkers
Phenobarbital
Animals
Demonstrations
Chemical activation

PubMed: MeSH publication types

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

Cite this

In Vivo Stable Isotope Labeling and Mass Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen in Rats. / Dator, Romel; von Weymarn, Linda B.; Villalta, Peter W.; Hooyman, Cory J.; Maertens, Laura A.; Upadhyaya, Pramod; Murphy, Sharon E.; Balbo, Silvia.

In: Analytical Chemistry, Vol. 90, No. 20, 16.10.2018, p. 11863-11872.

Research output: Contribution to journalArticle

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abstract = "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.",
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AU - Hooyman, Cory J.

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