Squamous cell carcinoma of the head and neck (HNSCC) is a devastating, frequently disfiguring, and often fatal disease, expected to affect more than 53,000 people in the U.S. in 2020 and kill more than 10,000. Prevention of this terrible disease is critical. Cigarette smoking, alcohol consumption, and human papilloma virus (HPV) are well established major causes of HNSCC; only smoking and alcohol consumption are considered here. Cigarette smoking and alcoholic drinks are sources of multiple DNA adducts that are critical in the carcinogenic process. This proposal will establish a liquid chromatography-nanoelectrospray ionizationhigh resolution tandem mass spectrometry (LC-NSI-HRMS/MS) profile analysis of 12 oral cell DNA adducts that are likely causes of HNSCC. This was inspired by our recent analysis of DNA adducts in oral cells, in which we found levels more than 20 times higher in cigarette smokers than in non-smokers. These exciting results encouraged us to propose a profile analysis of important carcinogen-derived DNA adducts in oral cells according to the following specific aims: 1. Develop an LC-NSI-HRMS/MS profile analysis method for quantitation of 12 important and representative carcinogen and toxicant – DNA adducts in human oral cells and tissue. The adducts are derived from various carcinogens and DNA reactive compounds in cigarette smoke and alcoholic beverages. 2. Apply the profile analysis to oral cells from currently healthy individuals: a) 100 non-smokers who are non drinkers or light drinkers; b) 100 cigarette smokers who are non-drinkers or light drinkers; and c) 100 cigarette smokers who are moderate or heavy drinkers. Comparisons of adduct levels in groups a and b will identify adducts enhanced by cigarette smoking while comparisons of groups b and c will identify adducts that are enhanced by the combination of smoking and moderate or heavy drinking. 3. Test the longitudinal stability of the oral cell DNA adduct profile analysis over a 6 month period in 50 smokers who are non-drinkers or light drinkers. 4. A) Determine the DNA adduct profile in oral cells collected from 75 smokers with HNSCC and compare to that in 200 smokers without HNSCC recruited in Specific Aim 2 with the goal of identifying an adduct profile that is characteristic of HNSCC incidence. B) Compare the oral cell DNA adduct profile from part A of this aim to that in tissue, both normal and tumor, in a subset of 60 patients from part A who undergo surgery, to determine whether oral cell DNA adduct patterns are consistent with those in tissue. Our results will potentially identify individuals who are susceptible to HNSCC but are unable to quit smoking. Once identified, aggressive lifestyle and monitoring interventions in these subjects such as oral examinations 2-4 times per year can be initiated for prevention or early detection of this disfiguring and often fatal cancer.