Ethyl alcohol (henceforth ethanol) is a human carcinogen.1,2 Its consumption has been associated with cancers at various sites, including the oral cavity. Despite strong epidemiological evidence, the mechanisms of ethanol carcinogenicity remain unclear, hampering the ability to develop efficacious preventive strategies, identify individual susceptibility, and effectively face the challenges deriving from the projected increase in consumption. Ethanol major metabolite, acetaldehyde (AA), is suggested to play a crucial role in head and neck cancers by reacting with DNA. These reactions generate chemical modifications (DNA adducts) that, if not repaired, may result in mutations and ultimately lead to cancer. Individuals with genetic deficiencies in ALDH2, the enzyme responsible for AA detoxification, were shown to have a 15% increased risk of developing oral cancer when drinking.5 Additionally, Fanconi Anemia (FA) patients, who have impaired mechanisms to repair AA-related DNA damage, have an average 500-fold higher chance of developing oral cancer.6 Previous studies have shown a direct and dose-dependent connection between ethanol consumed and AA-derived DNA damage, in the oral cavity of healthy volunteers.7 This effect was not as evident in blood DNA from the same individuals, indicating a distinct contribution of AA exposure coming from oral ethanol metabolism by the mucosa and oral microbiome. Our hypothesis is that AA resulting from oral metabolism of ethanol is playing a crucial role in oral cancer through the formation of DNA adducts, and that levels and persistence of driver adducts will increase in individuals with increasing oral cancer risk. Using cutting-edge analytical approaches, our objective is to characterize ethanol's oral metabolism and its corresponding DNA damage and mutational profiles, to develop a systematic assembly of biomarkers for identifying oral cancer risk and for developing strategies for early detection and prevention. This will be done by completing 3 aims. The first one will characterize DNA damage profiles in oral cells, collected after exposure to a controlled alcohol dose from participants from 3 groups at increasing risk of AA-related oral cancer (active ALDH2*1/1* homozygotes, inactive ALDH2*1/2* heterozygotes and FA patients). Driver adducts will be identified as those increasing in the groups following the increased cancer risk and persisting over time in FA patients. The second aim will focus on investigating the role of the oral microbiome in ethanol metabolism, by characterizing the oral microflora and measuring the aldehyde profile resulting from the ethanol dose in the saliva of study participants. Additionally, the oral microbiome will be characterized in samples from non-drinkers, included to investigate if ethanol consumption results in a specific profile. Finally, the third aim will analyze genome-scale mutational signatures in oral cell lines exposed to AA, for which DNA adducts will be profiled, a...