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A polymorphism of the methionine synthase reductase gene increases chromosomal damage in peripheral lymphocytes in smokers.The cytogenetic effects of cigarette smoke has been evaluated as one of many potential confounders in a large number of biomonitoring studies of occupationally or environmentally exposed populations and control subjects. Despite the well-known presence of carcinogens in the cigarette smoke, the results in the scientific literature linking smoking habits to micronuclei (MN) frequency, one of the cytogenetic markers, are rather controversial. Here, we investigated the relationships among MN frequency, smoking habits and five folate metabolic enzyme gene polymorphisms (MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TYMS 3'UTR) in 132 healthy Japanese men who were non-habitual drinkers. In never- and former-smokers, no statistically significant differences in the mean MN frequencies were observed according to the five folate metabolic enzyme gene polymorphisms. In current-smokers, however, subjects with the AA genotype for MTRR had a significantly higher mean MN frequency than the AG genotypes for MTRR (p<0.05). Furthermore, among subjects with the AA genotype for MTRR, current-smokers were found to have a significantly higher mean MN frequency than never- and former-smokers (p<0.05). To further characterize this association, we stratified the smoking status into five groups: non-smokers (never-smokers and former-smokers), 1-10 cigarettes/day, 11-20 cigarettes/day, 21-30 cigarettes/day and >or=31 cigarettes/day. There was an overall trend for the mean MN frequency in subjects with the MTRR AA genotype to increase as the number of cigarettes smoked per day increased (p<0.01, Jonckheere-Terpstra test). The results of our preliminary study suggest that the MTRR AA genotype acts to increase the MN frequency resulting from cigarette smoking. Therefore, studies on human genotoxicity based on cytogenetic markers of MN should take into account both the MTRR polymorphism and the potential confounding effect of smoking, although these preliminary findings need to be validated in larger populations because of the relatively small sample size.