Lung cancer is the leading cause of cancer death in the U.S with 142,670 deaths expected in 2019, 90% caused by cigarette smoking. Worldwide, there are 1 billion smokers and 1.6 million lung cancer deaths per year. Smokeless tobacco, a cause of oral cavity, esophagus, and pancreas cancer, is another deadly tobacco product. The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) are among the most important carcinogens in tobacco products, considered by the International Agency for Research on Cancer to be “carcinogenic to humans.” Here, we extend our research on NNN and NNK by developing new urinary biomarkers from interactions of their metabolites with amino acids and DNA. We also propose unique new methodology which can determine which oral cell DNA adducts – in addition to those derived from tobacco-specific nitrosamines – result from exposure to compounds in cigarette smoke. This will be accomplished using high resolution DNA adductomics techniques to identify DNA adducts in oral cells of people who have smoked cigarettes containing [13C]-labelled tobacco. We will use our biomarker and DNA adduct data to analyze oral cell DNA, lung DNA, and urine samples in our Lung Cancer and Pulmonary Nodule Biorepository, with the goal of using readily obtainable samples (e.g. oral cells or urine) to predict DNA adduct levels in smokers' lungs. The specific aims are: 1. Develop human urinary biomarkers of NNN exposure based on interactions of its metabolites with amino acids. These will be the first metabolite biomarkers specific to exposure to this powerful oral cavity carcinogen. 2. Develop an analytical method for quantitation of 7-[4-(3-pyridyl)-4-hydroxybutyl]guanine (7-PHB-G) as a human urinary DNA/RNA adduct biomarker of tobacco-specific nitrosamine exposure plus metabolic activation. 7-PHB-G will be the first urinary biomarker of specific DNA adduct formation by NNN and NNK. 3. Prepare cigarettes from specially grown fully [13C]-labelled tobacco and use high resolution mass spectrometry-based DNA adductomics techniques to specifically identify which oral cell DNA adducts originate from tobacco in people who smoke these cigarettes. This aim promises to resolve longstanding uncertainty about which DNA adducts result from cigarette smoke exposure. 4. Compare tobacco-specific and tobacco-related DNA adduct formation in lung tissue and oral cells from cigarette smokers, and relate to urinary biomarkers of tobacco exposure. These specific aims can result in new biomarkers of tobacco-related exposures and cancer risk and ultimately test our overall hypothesis that biomarkers, combined with certain genetic information, can identify those tobacco users at highest risk for cancer, so that effective preventive measures can be initiated. The biomarkers developed in this program also have potential application in tobacco product evaluation and regulation.