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This prospective clinical study comparatively investigated the effects of tobacco smoking on global methylation and hydroxymethylation in oral epithelial cells. Buccal cells from the inside of the cheeks were collected from 47 individuals, including smokers, former smokers, and never smokers. DNA was extracted using dedicated kits. Methylated and hydroxymethylated DNA fractions were measured using assays similar to enzyme-linked immunosorbent assays. The levels of methylation and hydroxymethylation were compared among groups using unpaire