N'-Nitrosonornicotine (NNN) is a critical component of smokeless tobacco due to its relatively high abundance and ability to cause tumors in the esophagus and oral cavity of laboratory animals. For these reasons, NNN is considered a likely cause of tobacco-related cancers. To exhibit these effects, NNN must be bioactivated by cytochrome P450 oxidation, implying that individuals who extensively activate NNN may have a higher risk for cancer. P450 oxidation generates highly reactive α-hydroxyNNN species that can bind to DNA. These species also hydrolyze and are further oxidized to 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) or 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), which are rapidly excreted in urine and have potential as human biomarkers for NNN activation; however, these products are also minor metabolites of nicotine. Because nicotine is &gt;10,000 fold higher than NNN in tobacco, it is unclear what amount of keto acid and hydroxy acid result from NNN bioactivation. To overcome this issue, we will measure NNN activation in humans who use smokeless tobacco containing [pyridine-D4]NNN (0.2 µg/g tobacco). After a week of acclimation, 24-hour urine will be collected for three days and analyzed by an established assay for the sum of both metabolites. Because [pyridine-D4]NNN is 4 amu heavier, NNN-derived metabolites will be completely distinct from nicotine-derived metabolites. In pursuit of this goal, our lab has developed a spray-based method for enriching tobacco with [pyridine-D4]NNN, gained full FDA and IRB approval, optimized the GMP process for manufacturing [pyridine-D4]NNN-enriched smokeless tobacco, and tested our assay on [pyridine-D4]NNN-treated rats. Ongoing work includes recruiting subjects and analyzing urine samples as they become available. This study will provide the first data on NNN bioactivation in humans and identify inter-individual differences in NNN bioactivation.

Development of a novel approach for measuring N'-nitrosnornicotine bioactivation in humans by using deuterium-labeled analogs

256th National Meeting (2018)

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