The Role Of Synaptotagmin 10 Gene Promoter Methylation In Fluoride-induced Neurotoxicity

Part 1.Relationship between fluoride exposure,synaptotagmin 10 gene promoter methylation and intelligence in childrenObjective: Excessive fluoride exposure could induce neurotoxicity,but the mechanism has not been fully elucidated.This study conducted cross-sectional survey among 668 children to investigate the relationship between fluoride exposure,DNA methylation and intelligence in children.Methods: According to the historical water fluoride monitoring data and the results of fluoride concentration analyses,we randomly selected 325 and 343 children aged 7 to 13 years in the fluorosis areas(water fluoride concentration > 1.0 mg/L)and nonfluorosis areas(water fluoride concentration ? 1.0 mg/L)of Baodi District in Tianjin,respectively.And then we collected first-spot morning urine samples and fasting peripheral blood samples from each recruited child.The children's intelligence quotient(IQ)was evaluated using the combined Raven test.The basic information of each child was collected using a self-made questionnaire.And then we performed physical examinations on the children and collected medical examination data.We detected fluoride concentration in urinary using fluoride ion selective electrode method,and then extracted DNA samples from peripheral blood using DNA extraction kit.We selected DNA samples from 10 pairs of children living in endemic fluorosis areas and nonfluorosis areas to screen the methylation differential genes using Human Methyl-Seq.And then we verify the methylation level of the target gene in 668 DNA samples using quantitative methylation specific PCR.The difference of continuous variable between two groups were compared by t-test.And the difference of categorical variables was compared using ?2 tests.The relationship of fluoride exposure,the methylation level of synaptotagmin 10(Syt10)gene,and the IQ scores in children were analyzed by general linear regression.Subsequently,we analyzed the relationship between fluoride exposure and intelligence levels of children(as same as the relationship between the methylation level of Syt10 gene and the intelligence levels of children)were analyzed by logistic regression model.Finally,we used mediating effects analysis to analyze the mediating effects of Syt10 gene methylation changes in fluoride-induced intelligence loss.Result: The water fluoride and urinary fluoride concentrations of children in the endemic fluorosis group were significantly higher than those of children in the nonfluorosis group(P < 0.001,respectively).The IQ scores of children in the endemic fluorosis group(105.78 ± 11.65)were significantly lower than those of children in the non-fluorosis group(108.37 ± 12.55)(P = 0.006).There was no significant gender difference in children's IQ scores in the endemic fluorosis group,non-fluorosis group or total population(P > 0.05).After grouping the population according to IQ scores,we found that compared with the non-fluorosis group,the children in the endemic fluorosis group have lower proportion of superior and above intelligence and higher proportion of dull normal and below intelligence(P < 0.001).In the discovery study,after screening DMRs using human methyl-Seq,we revealed 7 DMRs(each DMR was associated with a gene)associated with fluoride exposure,and one of the DMRs overlaps with the promoter region of Syt10 gene,while the function of Syt10 gene is related to neurodevelopment.In the replication study,we found that the methylation level of the Syt10 gene promoter region in children with fluorosis(3.32% ± 1.27%)was significantly higher than that of the non-fluorosis group(3.11% ± 1.16%).We observed an elevation of 0.12%(95% CI: 0.02%,0.22%)of methylation level of the promoter region in Syt10 gene,a reduction of 1.00 units(95% CI:-1.85,-0.15)of children's IQ scores and a reduction of 5%(OR = 0.95,95% C: 0.92,0.97)in the odds of superior and above intelligence with each 1 mg/L increase in the concentration of water fluoride adjusting for potential confounding factors.Similarly,we observed an elevation of 0.08%(95% CI: 0.01%,0.16%)of methylation rate of the promoter region in Syt10 gene,a reduction of 1.01 units(95% CI:-1.69,-0.33)of children's IQ scores and a reduction of 4%(OR = 0.96,95% CI: 0.93,0.98)in the odds of superior and above intelligence with each 1 mg/L increase in the concentration of urinary fluoride.Additionally,we also found a reduction of 2.56 units(95% CI:-3.39,-1.74)of children's IQ scores,a reduction of 5% in the odds of excellent and above intelligence,a reduction of 5% in the odds of upper intelligence,and an elevation of 4% in the odds of dull normal and below intelligence with each 1% increase in the methylation rate of promoter region of Syt10 gene.The mediation analyses showed that 29.3%(95% CI:7.5%,68.1%)of the relationship between water fluoride and the IQ scores,and 20.8%(95% CI:6.2%,51.2%)of the relationship between urinary fluoride and the IQ scores were mediated by the alteration of the methylation rate of Syt10 gene.Additionally,16.3%(95% CI: 5.4%,39.8%)of the relationship between water fluoride and the odds of superior and above intelligence,and 14.8%(95% CI: 4.9%,37.0%)of the relationship between urinary fluoride and the odds of superior and above intelligence in children were mediated by the alteration of the methylation rate of Syt10 gene.Conclusions: Excessive fluoride exposure is associated with children's intelligence loss in,and this relationship is mediated by the methylation level of promoter region in the Syt10 gene.Part 2.Relationship between Syt10 promoter methylation,gene expression level and fluoride-induced neurotoxicityObjective: A Na F-treated rat model and a Na F-treated cell model were established to analyze the relationships of fluoride-induced neurotoxicity and the alterations of methylation and gene expression levels of Syt10 gene.Methods: Sixty adult SD rats(8 weeks old,180-220 g,female/male = 2:1)were purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention.After one-week acclimation,rats within each sex were randomly divided into 4 groups,including a control group(tap water,F-concentration < 0.5 mg/L)and three Na F(10,50 and 100 mg/L Na F)treated groups(Na F was administered orally via drinking water).The rats were then treated with Na F.After 2 months of Na F treatment,female and male rats(2:1)in each group were randomly mated overnight.The pregnant rats were housed in separate cages with the same environmental and exposure conditions until the weaning of pups.The male pups were given the same concentration of Na F as their parents did until 2 months after birth.Morris water maze test was used to observe whether fluoride exposure affected rats' learning and memory ability,and the effect of Na F on rat hippocampal structure was observed by Nissl staining.And we observed the number of synaptic vesicles using transmission electron microscopy.Methyl TargetTM-Seq was used to detect the methylation level of Cp G island in the promoter region of Syt10 gene.The expression level of Syt10 m RNA was detected by real-time RT-PCR.The expression level of Syt10 protein was detected by western blot and immunohistochemical.Furthermore,we have established a Na F-treated rat adrenal pheochromocytoma(PC12)cell model.The CCK8 kit was used to detect the cell viabilities in PC12 cells treated by Na F.The levels of gene methylation and gene expression were detected by the same methods used in in vivo models.Besides,we also used laser confocal microscopy to detect the expression and localization of Syt10 protein in PC12 cells.Results: The results of the Morris water maze showed that in the place navigation test,the average escape latency on the first day were not significantly different across groups demonstrating similar initial performance,however,the average escape latency of rats exposed to 50 and 100 mg/L Na F were significantly longer than those in control group on the 2nd day,3rd day and the 4th day(P < 0.05).In the spatial probe test,Na F-infected rats.The number of crossings of the rats exposed to 50 mg/L and 100 mg/L Na F was significantly lower than that of the rats in control group(P < 0.05).Nissl staining showed that when compared with the control rats,the number of Nissl bodies in hippocampal neurons in the Na F-treated rats was decreased,and the Nissl bodies is lighter in color and lower in density.The transmission electron microscopy revealed that the number of synaptic vesicles near the presynaptic membrane in the Na F-treated rats was decreased when compared with the control.The results of Methyl TargetTM sequencing showed that the methylation level of the Syt10 gene promoter region in rat hippocampi and PC12 cells increased with increasing fluoride exposure.Real-time RT-PCR results showed that the level of Syt10 m RNA in hippocampi and PC12 cells decreased with increasing fluoride exposure.In addition,the results from western blot,immunohistochemistry and confocal showed that the level of Syt10 protein in rat hippocampi and PC12 cells decreased with increasing fluoride exposure.Conclusion: Fluoride exposure can elevate the methylation levels of Syt10 gene promoter region and decrease the expression of Syt10 m RNA,resulting in decreased expression of Syt10 protein,which in turn decrease the number of synaptic vesicles,inducing neurotoxicity.