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Objective   This prospective clinical study comparatively investigated the effects of tobacco smoking on global methylation and hydroxymethylation in oral epithelial cells.Methods   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 unpaired two-tailed t-tests or the Mann–Whitney U test; P &lt; 0.05 was considered statistically significant.Results   There was no statistically significant difference in the average number of cigarettes between smoker and former smoker groups. Although methylation levels were lower for smokers (3.1%) and former smokers (2.16%), compared with never smokers (4.16%), these differences were not statistically significant. There was a two-fold increase in hydroxymethylation level in never smokers, compared with smokers.Conclusions   Our findings suggest that smoking leads to global reductions in both methylation and hydroxymethylation levels in oral epithelial cells in a manner influenced by the intensity and length of exposure to tobacco smoke.

Changes in global DNA methylation and hydroxymethylation in oral mucosa according to tobacco smoke exposure