This proposal's specific objective is to evaluate the validity of using gene expression in buccal cells to accurately reflect gene expression in human lung when both are tobacco smoke-exposed, as they are in smokers. The induction of genes encoding carcinogen metabolizing enzymes simultaneously in an easily-accessible surrogate tissue as well as the lung suggests the possibility of expression phenotyping smokers for bioactivating gene inducibility. If this carcinogen metabolism pathway is indeed relevant to lung cancer susceptibility, then a high phase I and low phase II enzyme inducibility phenotype might be hypothesized to confer risk for lung cancer. The power of studies on carcinogen-metabolizing gene induction in lung cancer to date have been limited by the number of subjects enrolled, as quality human lung tissues suitable for expression or activity assays are in short supply. Additionally, studies of coding sequence polymorphisms in these genes have presented very mixed results as to conferred risk for lung cancer. This study proposes to take advantage of newly developed real-time methods for quantifying gene expression in an appropriate, smoke-exposed epithelium, and comparing that expression to that in precisely-defined microdissected smoke-exposed human lung epithelia. Therefore, the specific aims are: 1) Develop quantitative real-time RT-PCR assays for the evaluation of gene expression of a panel of carcinogen metabolizing enzymes in buccal mucosa, including Ahr, ER, CYP1B1, CYP1A1, GSTM1, GSTM3, GSTP1, GSTT1, NQO1. 2) Compare the gene expression in buccal mucosa to simultaneously sampled plasma assayed for nicotine and cotinine by state-of-the-art LC-MS-MS techniques. 3) Quantitatively compare the expression of carcinogen metabolizing enzyme genes from in situ tobacco-exposed human buccal mucosal cells with that from microdissected human lung bronchial and alveolar epithelial cells also exposed in situ. 4) Measure the variability of this buccal cell carcinogen-metabolizing gene expression across individuals, accounting for expression confounders such as tobacco smoke exposure and dietary constituents, and thereby identify phase I and phase II high or low expressors. The long-term goal is to develop a panel of biomarkers in an easily-accessible, smoke-exposed epithelium that identifies smokers at high-risk for lung cancer, and thereby allows smoking-cessation, chemoprevention and early-disease detection strategies to be focussed on those most at risk.
