PROJECT SUMMARY The tobacco alkaloid nornicotine is a precursor to N'-nitrosonornicotine (NNN), a potent tobacco-specific carcinogen. NNN is believed to play an important role in the induction of esophageal and oral cavity cancers in tobacco users. Nornicotine contributes to NNN exposure in tobacco users in two ways. In addition to being a precursor to NNN formation in tobacco, it may also be nitrosated endogenously. Endogenous nitrosation of nornicotine that is present in tobacco products can occur in the oral cavity, in the stomach, or elsewhere in the body of tobacco users. This is particularly relevant to the use smokeless tobacco products - a form of tobacco that is kept in the oral cavity for prolonged periods of time, creating favorable conditions for nornicotine nitrosation. Given the high carcinogenic potency of NNN and the association of smokeless tobacco use with oral and esophageal cancers, regulation of nornicotine content in smokeless tobacco products is urgently needed. The Food and Drug Administration (FDA) has authority to regulate harmful and potentially harmful tobacco constituents under the Family Smoking Prevention and Tobacco Control Act. The information on the extent of endogenous nitrosation of nornicotine as a function of its content in smokeless products is crucial for the adequate regulation of this tobacco constituent by the FDA. Aims of this proposal are: (1) To determine the variation of nornicotine content in smokeless products currently marketed in the U.S. There is no information on nornicotine content in individual smokeless brands, and the extent of variation among the currently marketed U.S. products is unknown. Aim 1 will address this important gap and will also provide critical information for Aim 2 of this proposal. (2) To investigate the endogenous formation of NNN in smokeless tobacco users. Smokeless tobacco users will be randomized to a tobacco-free herbal snuff containing various amounts of stable isotope-labeled nornicotine added to it. Isotope-labeled urinary total NNN - a biomarker of exposure to NNN - will be measured in these subjects by using liquid chromatography-tandem mass- spectrometry. This methodology will allow for specific identification of urinary total NNN from endogenous nitrosation of nornicotine, versus that resulting from exposure to NNN in tobacco products. The effect of nornicotine reduction on the extent of endogenous NNN formation will be studied by gradually decreasing the amount of isotope-labeled nornicotine added to study snuff. The results of this study will provide extremely important new information on the endogenous formation of the carcinogenic nitrosamine NNN upon exposure to nornicotine from tobacco use. An understanding of this process is absolutely necessary for the adequate regulation of nornicotine in tobacco products with the purpose of minimizing public harm.