Objective To investigate the effects of fluoride exposure dose and exposure time on the expression of 5-methylcytosine (5-mC) in blood,liver,kidney and brain of rats;and to understand whether there is a difference in the effects of fluoride on DNA methylation levels in different tissues.Methods Eighty three-week old SPF male Wistar rats were randomly divided into four groups according to body weight [(82.34 ± 10.60) g],with 20 rats in each group.The rats of control group drank distilled water and the fluoride group's drank distilled water containing 25,50 and 100 mg/L of F ion,respectively.Rats were sacrificed after fed for 1 month and 3 months (n =10),and peripheral blood and tissue samples were collected.The incidence of dental fluorosis was observed in rats.Bone and urine fluoride content was detected by ion selective electrode method.The content of 5-mC in blood,liver,kidney and brain was detected by enzyme-linked immunosorbent assay (ELISA).The independent and interactive effects of fluoride exposure dose and exposure time on 5-mC in rat peripheral blood and different tissues were analyzed by factorial design anova.Results After feeding for 1 month and 3 months,all rats in the fluoride group had dental fluorosis with different severities,while none dental fluorosis was found in the control groups.Fluoride exposure dose and exposure time had a main effect on bone fluoride contents [1 month:(324.985 + 127.094),(846.148 ± 331.861),(1 886.601 + 250.140),(2 420.971 + 135.883) mg/kg;3 months:(417.591 ± 88.324),(1 582.243 ± 347.975),(2 163.519 ± 614.932),(2 755.434 ± 265.370)mg/kg;F =96.692,13.077,P < 0.01],respectively,but there was no interaction effect (F =2.013,P > 0.05);fluoride exposure dose had a main effect on urinary fluoride contents (F =62.358,P < 0.01),the exposure time had no effect on it (F =0.862,P > 0.05),and there was no interaction effect (F =0.081,P > 0.05).Fluoride exposure dose had a main effect on the 5-mC content in the blood (F =8.446,P < 0.01),the exposure time had no effect on it (F =0.095,P >0.05),and there had an interaction effect (F =4.676,P < 0.01).Fluoride exposure dose and exposure time had a main effect on the 5-mC content in the liver,respectively (F =4.737,7.064,P < 0.01 or < 0.05),and an interaction effect was exist (F =8.302,P < 0.01).Fluoride exposure time had a main effect on the 5-mC content in the kidney (F =6.340,P < 0.05),the exposure dose had no effect on it (F =0.140,P > 0.05),and there was no interaction effect (F =1.269,P > 0.05).Fluoride exposure dose and exposure time had no effect on 5-mC content in the brain (F =0.633,2.065,P > 0.05).Conclusion Fluoride exposure dose and exposure time have the different effect on the levels of 5-mC in blood,liver,kidney and brain,suggesting that there may be differences in the effects of fluoride on DNA methylation levels in different tissues.