| Objective This study analyzed the involvement of the methylation level and rs2941740 polymorphism in the promoter region of the ERα gene with bone changes in adults exposed to fluoride from the perspective of epigenetics and genetics,and provided a basis for exploring the relationship among fluoride exposure,ERα gene and bone metabolism at the molecular level.Materials and methods1 Study population The cross sectional study was conducted in endemic fluorosis villages in Kaifeng City of Henan Province from 2011 to 2012.A total of 1131 local residents were recruited via cluster sampling,who were aged from 18 to 55 years old and lived in the villages for more than 5 years.Then questionnaire collection and physical examination were conducted.Based on the cross sectional study,residents with bone tissue abnormalities in X-ray radiographs were selected as the case group.Matched by age(±5 years old)and gender,residents with bone tissue normalities in X-ray radiographs were taken as the control group according to the proportion of about 1:1,and then a case-control study was conducted.A total of 252 subjects were recruited including 144 subjects in bone change cases and 108 subjects in controls.Biological samples including fasting blood and morning urine were collected and biomarkers were analyzed.2 Detection of biomarker X-ray diagnosis was performed to detect bone changes by a medical professional in a certified hospital.Urinary fluoride content was detected by fluoride ion selective electrode method.The levels of serum calcium and magnesium were determined by atomic absorption spectrophotometry.Chemiluminescence immuno-assay was used to detect the level of serum estradiol.The level of methylation in promoter region of ERα gene was detected by quantitative methylation specific polymerase chain reaction.Genotypes were identified by Taqman probe and Real-time PCR.3 Statistical analysis The data were entered into the database using Epidata 3.0 software.Statistical analysis was performed using the SPSS statistical software package,version 21.0.The main statistical methods were Chi-square test,t test for independent samples,rank sum test,multiple linear regression and binary logistic regression.It was considered statistically significant when P≤ 0.05.Results1 Analysis of biomarkers in bone change cases and controls(1)In bone change cases,there were 67.9%(72/106)subjects with urinary fluoride level exceeding 1.6 mg/L,and 32.7%(34/106)subjects in controls.The urinary fluoride level in cases was higher than that in the controls,and the difference was statistically significant(P<0.05).The urinary fluoride level in cases was higher than that in the controls,and the difference was statistically significant(P<0.05).(2)In the female subjects,the serum magnesium level in cases was higher than that in controls,and the difference was statistically significant(P<0.05).2 Relationship between ERα gene rs2941740 polymorphism and bone changes in adults exposed to fluoride(1)The distribution of ERα rs2941740 genotype was similar between the bone change cases and controls,and the difference was not statistically significant(P>0.05).(2)After adjusted for age,gender and urinary fluoride,compared to the CC genotype,subjects carrying CT genotype(OR=1.059,95% CI: 0.545-2.056)and TT genotype(OR=3.144,95% CI: 0.577-17.140)did not increase the risk of bone changes.(3)After adjusted for age,gender and urinary fluoride,carrying the C allele as reference and subjects carrying the T allele did not increase the risk of bone changes(OR=1.333,95% CI: 0.770-2.307).(4)After adjusted for age and gender,the level of fluoride exposure was the influencing factor of bone changes(β=0.470,P<0.05).There was no interaction between fluoride exposure and ERα gene rs2941740 polymorphism on bone changes(P>0.05).(1)There were significant differences in serum calcium and serum magnesium levels among subjects with CC genotype in the different fluoride exposure groups(P<0.05).Serum calcium and serum magnesium levels were higher in the high fluoride exposure group than that in the low fluoride exposure group.However,no significant differences were observed in serum calcium,serum magnesium and estrogen levels in subjects with CT and TT genotypes in different fluoride exposure groups(P>0.05).(2)The serum calcium,serum magnesium and estrogen levels were no significant differences in the same fluoride exposure group when subjects carrying different genotypes of ERα gene rs2941740(P>0.05).4 Relationship between the ERα gene methylation level in promoter region and bone changes in adults exposed to fluoride(1)In the male subjects,the methylation level in the promoter region of ERα gene in bone change cases was lower than that in controls,and the difference was statistically significant(P<0.05).(2)After adjusted for age and gender,the levels of serum calcium(β=-0.634),fluoride exposure(β=0.552)and methylation(β=-4.986)had significant effects on bone changes(P<0.05,respectively).(3)The methylation level in male subjects was negatively correlated with the urinary fluoride(r=-0.221,P=0.027).There were no significant correlation between methyla-tion and the levels of serum calcium,serum magnesium,and estrogen in both male and female subjects(P>0.05,respectively).(4)For subjects who carrying the different genotypes of ERα gene rs2941740 locus,there was no significant difference of the promoter methylation level in the same fluoride exposure group(P>0.05).No significant difference was found in the ERαgene promoter methylation level between the high fluoride exposure group and low fluoride exposure group who carrying the same genotype of ERα rs2941740(P>0.05).(5)After adjusted for age and gender,no interaction was observed between fluoride exposure level and ERα rs2941740 genotypes on the methylation level in the ERαgene promoter region(P>0.05).3 Relationship between ERα rs2941740 polymorphism and serum biomar-kers in adults exposed to fluorideConclusion 1 In the male subjects,the ERα gene methylation level in promoter region may be an important link in the impact of environmental fluoride exposure on bone changes.2 Increased levels of serum calcium and ERα gene methylation in promoter region were protective factors for bone changes in adults exposed to fluoride.3 No interaction was found between ERα gene rs2941740 polymorphism and fluoride exposure on the methylation level in ERα gene promoter region and bone changes. |
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