Abstract 4650: Nornicotine nitrosation in human saliva and its relation to endogenous formation of N′-nitrosonornicotine in oral nicotine replacement therapy users
Authors: Stepanov Irina , Knezevich Aleksandar , Muzic John , Hatsukami Dorothy K. , Hecht Stephen S. 
Published: June 2012

We recently reported occasional significant increases in urinary biomarkers of the potent human carcinogen N′-nitrosonornicotine (NNN) in some users of oral nicotine replacement therapy (NRT) products, such as nicotine gum or lozenge, compared with baseline smoking levels in the same subjects. We hypothesized that this is due to endogenous synthesis of NNN through the reaction of nicotine and/or nornicotine that is metabolically formed from nicotine or originally present in NRT products, with nitrosating agents supplied by diet. While the acidic environment of the stomach creates the most favorable conditions for nitrosation, this reaction can also occur in the oral cavity in the presence of bacteria that catalyzes nitrosation at neutral pH. To test the hypothesis that endogenous formation of NNN can occur in the oral cavity of NRT users, we investigated nitrosation of nicotine and nornicotine in human saliva. To specifically identify NNN as derived from the precursors added to saliva, we used [pyridine-D4]nicotine and [pyridine-D4]nornicotine and subsequently analyzed [pyridine-D4]NNN by liquid chromatography-tandem mass spectrometry. Aliquots of pooled saliva from nonsmoking volunteers were incubated for 30 min at 37 °C with either [pyridine-D4]nicotine or [pyridine-D4]nornicotine, in the presence of various concentrations of sodium nitrite. [Pyridine-D4]NNN was not detected in the blank saliva aliquot that was added as a negative control. Incubation of saliva with 50 ng of [pyridine-D4]nornicotine alone produced 38 pg [pyridine-D4]NNN, or 0.08% yield. The aliquot incubated with 200 ng of [pyridine-D4]nornicotine and 7 mg of sodium nitrite formed 5,503 pg of [pyridine-D4]NNN (nearly 150-fold increase). Only traces of [pyridine-D4]NNN were detected in samples incubated with [pyridine-D4]nicotine. These results are in agreement with kinetic studies on nicotine and nornicotine nitrosation. To investigate the effect of natural inter-individual variations in salivary nitrite and/or oral microflora on the amount of orally synthesized NNN, saliva samples from 10 nonsmoking volunteers were incubated with deuterium-labelled nicotine and nornicotine. Formation of [pyridine-D4]NNN was observed in 6 samples treated with [pyridine-D4]nornicotine, with yields ranging from 0.001 to 0.037% of the added alkaloid. Trace amounts of [pyridine-D4]NNN were detected in some samples incubated with [pyridine-D4]nicotine. In summary, our results demonstrate that NNN can be formed in human saliva in the presence of nornicotine, supporting the hypothesis that NNN can be synthesized endogenously in oral NRT users. Removal of nornicotine from NRT products could improve the safety of these products. Additional studies are needed to evaluate the contribution of metabolically formed nornicotine to the endogenous formation of NNN in oral NRT users.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4650. doi:10.1158/1538-7445.AM2011-4650