Nornicotine Nitrosation in Saliva and Its Relation to Endogenous Synthesis of N′-Nitrosonornicotine in Humans

Introduction: We recently reported that certain amounts of the carcinogen N'-nitrosonornicotine (NNN) can be formed endogenously from nicotine and/or nornicotine in some users of oral nicotine replacement therapy products. Although the acidic environment of the stomach creates the most favorable conditions for nitrosation, this reaction could also occur in the oral cavity in the presence of bacteria that catalyze nitrosation at neutral pH. Methods: To test the hypothesis that endogenous formation of NNN could occur in the oral cavity, we investigated nitrosation of nicotine and nornicotine in human saliva. To specifically identify NNN as derived from precursors added to saliva, we incubated saliva samples with [pyridine-D-4] nicotine and [pyridine-D-4] nornicotine, with and without the addition of nitrite, and subsequently analyzed [pyridine-D-4] NNN by liquid chromatography-tandem mass spectrometry. Results: Consistent with kinetic studies on nicotine and nornicotine nitrosation, incubation of saliva with [pyridine-D-4] nornicotine alone produced detectable amounts of [pyridine-D-4] NNN, whereas only traces of [pyridine-D-4] NNN were found in samples incubated with [pyridine-D-4] nicotine and sodium nitrite. Incubation of saliva samples from 10 nonsmoking volunteers with [pyridine-D-4] nornicotine resulted in the formation of [pyridine-D-4] NNN in 8 samples, with yields ranging from 0.003% to 0.051% of the added alkaloid. Conclusion: Our results demonstrate that NNN can be formed from nornicotine in human saliva without deliberate addition of any other substance. Therefore, nornicotine, as present in tobacco or in nicotine replacement products, is a carcinogen precursor.