Berg, Jeannette Zinggeler2010-02-102010-02-102009-11https://hdl.handle.net/11299/57438University of Minnesota Ph.D. dissertation. November 2009. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Sharon E. Murphy, PhD. 1 computer file (PDF); xvi, 191 pages. Ill. (some col.)Nicotine is the addictive agent in tobacco and differences in nicotine metabolism may affect tobacco use, and consequently exposure to tobacco carcinogens. A lung procarcinogen in tobacco is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carcinogenic effect is dependent on metabolic activation and is counter-balanced by metabolic detoxification. Nicotine and NNK are structurally related and both are metabolized by cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and flavin-monoxygenase (FMO) enzymes. The goal of this thesis research was to explore variation in nicotine metabolism in vivo and to probe specific enzyme-catalyzed reactions of NNK in vitro. Low nicotine and cotinine glucuronidation were observed among African Americans compared to Europeans, and among individuals with a variant UGT2B10 genotype. In a controlled dose study of ethnic differences in nicotine metabolism (n= 93 smokers), African Americans excreted 30-40 % less nicotine and cotinine as their glucuronide conjugates than European Americans. This difference in glucuronidation explained the higher free cotinine concentrations observed in African Americans compared to European Americans. The most efficient in vitro catalyst of nicotine and cotinine glucuronidation is UGT2B10. We demonstrated that UGT2B10 contributes to in vivo nicotine metabolism in a genotype-phenotype analysis of 325 smokers. Individuals who were heterozygous for the UGT2B10 Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type; the ratio of cotinine glucuronide:cotinine was decreased by 60 %, while increases in urinary and plasma cotinine and trans-3'-hydroxycotinine were observed. Strikingly, a robust biomarker of nicotine intake, nicotine equivalents, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95 % CI, 48.9 - 68.2) versus 69.2 nmol/ml (95 % CI, 64.3 - 74.5). Individuals with low activity UGT2B10 may smoke less intensely, as reported for individuals with CYP2A6 polymorphisms that cause decreased nicotine C-oxidation. In contrast to nicotine, NNK is a carcinogen. It is metabolized to reactive intermediates that can form DNA and protein adducts, or it is detoxified by glucuronidation. P450 2A13 is the most efficient catalyst of NNK oxidation. We explored the effect of an active site mutant, Asn297Ala, on enzyme function and found that loss of hydrogen bonding to substrate in the active site affected substrate orientation and product formation. The orphan P450 2A7 was considered as a potential catalyst for NNK oxidation, but expression of wild-type or two naturally-occurring variants failed to yield protein with a P450 spectra and no appreciable activity towards P450 2A substrates was observed. Preliminary experiments were conducted to search for the glucuronide conjugate formed from the unstable oxidation product alpha-hydroxymethyl NNK, which has not been identified in any human system. The extent to which variation in metabolism mediates smoking behavior and cancer risk warrants consideration. The enzymes involved are potential drug targets for smoking cessation pharmacotherapy and cancer chemoprevention.en-USCytochrome P450GlucuronidationNicotineNNKTobaccoUGT2B10Biochemistry, Molecular Bio, and BiophysicsThesis or Dissertation