DNA methylation and fluoride exposure in school-age children: Epigenome-wide screening and population-based validation.

Excessive fluoride exposure and epigenetic change can induce numerous adverse health outcomes, but the role of epigenetics underneath the harmful health effects induced by fluoride exposure is unclear. In such gap, we evaluated the associations between fluoride exposure and genome-wide DNA methylation, and identified that novel candidate genes associated with fluoride exposure. A total of 931 school-age children (8-12 years) in Tongxu County of Henan Province (China) were recruited in 2017. Urinary fluoride (UF) concentrations were measured using the national standardized ion selective electrode method. Participants were divided into a high fluoride-exposure group (HFG) and control group (CG) according to the UF concentrations. Candidate differentially methylated regions (DMRs) were screened by Infinium-Methylation EPIC BeadChip of DNA samples collected from 16 participants (eight each from each group). Differentially methylated genes (DMGs) containing DMRs associated with skeletal and neuronal development influenced by fluoride exposure were confirmed using MethylTarget™ technology from 100 participants (fifty each from each group). DMGs were verified by quantitative methylation specific PCR from 815 participants. Serum levels of hormones were measured by auto biochemical analyzer. The mediation analysis of methylation in the effect of fluoride exposure on hormone levels was also performed. A total of 237 differentially methylated sites (DMSs) and 212 DMRs were found in different fluoride-exposure groups in the epigenome-wide phase. Methylation of the target sequences of neuronatin (NNAT), calcitonin-related polypeptide alpha (CALCA) and methylenetetrahydrofolate dehydrogenase 1 showed significant difference between the HFG and CG. Each 0.06% (95% CI: -0.11%, -0.01%) decreased in NNAT methylation status correlated with each increase of 1.0 mg/L in UF concentration in 815 school-age children using QMSP. Also, each 1.88% (95% CI: 0.04%, 3.72%) increase in CALCA methylation status correlated with each increase of 1.0 mg/L in UF concentration. The mediating effect of NNAT methylation was found in alterations of ACTH levels influenced by fluoride exposure, with a ß value of 11.7% (95% CI: 3.4%, 33.4%). In conclusion, long-term fluoride exposure affected the methylation pattern of genomic DNA. NNAT and CALCA as DMGs might be susceptible to fluoride exposure in school-age children.

Fluoride exposure and intelligence in school-age children: evidence from different windows of exposure susceptibility.

BACKGROUND: The intellectual loss induced by fluoride exposure has been extensively studied, but the association between fluoride exposure in different susceptibility windows and children's intelligence is rarely reported. Hence, we conducted a cross-sectional study to explore the association between fluoride exposure in prenatal and childhood periods and intelligence quotient (IQ). METHODS: We recruited 633 local children aged 7-13 years old randomly from four primary schools in Kaifeng, China in 2017. The children were divided into four groups, of which included: control group (CG, n = 228), only prenatal excessive fluoride exposure group (PFG, n = 107), only childhood excessive fluoride exposure group (CFG, n = 157), both prenatal and childhood excessive fluoride exposure group (BFG, n = 141). The concentrations of urinary fluoride (UF) and urinary creatinine (UCr) were determined by fluoride ion-selective electrode assay and a creatinine assay kit (picric acid method), respectively. The concentration of UCr-adjusted urinary fluoride (CUF) was calculated. IQ score was assessed using the second revision of the Combined Raven's Test-The Rural in China (CRT-RC2). Threshold and saturation effects analysis, multiple linear regression analysis and logistic regression analysis were conducted to analyze the association between fluoride exposure and IQ. RESULTS: The mean IQ score in PFG was respectively lower than those in CG, CFG and BFG (P < 0.05). The odds of developing excellent intelligence among children in PFG decreased by 51.1% compared with children in CG (OR = 0.489, 95% CI: 0.279, 0.858). For all the children, CUF concentration of ≥1.7 mg/L was negatively associated with IQ scores (ß = - 4.965, 95% CI: - 9.198, - 0.732, P = 0.022). In children without prenatal fluoride exposure, every 1.0 mg/L increment in the CUF concentration of ≥2.1 mg/L was related to a reduction of 11.4 points in children's IQ scores (95% CI: - 19.2, - 3.5, P = 0.005). CONCLUSIONS: Prenatal and childhood excessive fluoride exposures may impair the intelligence development of school children. Furthermore, children with prenatal fluoride exposure had lower IQ scores than children who were not prenatally exposed; therefore the reduction of IQ scores at higher levels of fluoride exposure in childhood does not become that evident.

Association between low-to-moderate fluoride exposure and bone mineral density in Chinese adults: Non-negligible role of RUNX2 promoter methylation.

Bone mineral density (BMD) changes were reported to be associated with excessive fluoride exposure and abnormal expression of RUNX2. However, whether the alteration of methylation status, a most commonly used marker for the alteration of gene expression in epidemiological investigation, of RUNX2 is associated with low-to-moderate fluoride exposure and BMD changes has not been reported. Our study aims to explore the role of RUNX2 promoter methylation in BMD changes induced by low-to-moderate fluoride exposure. A total of 1124 adults (413 men and 711 women) were recruited from Kaifeng City in 2017. We measured BMD using ultrasound bone densitometer. Concentrations of urinary fluoride (UF) were measured using ion-selective electrode, and the participants were grouped into control group (CG) and excessive fluoride group (EFG) according to the concentration of UF. We extracted DNA from fasting peripheral blood samples and then detected the promoter methylation levels of RUNX2 using quantitative methylation-specific PCR. Relationships between UF concentration, RUNX2 promoter methylation and BMD changes were analyzed using generalized linear model and logistic regression. Results showed in EFG (UF concentration > 1.6 mg/L), BMD was negatively correlated with UF concentration (ß: -0.14; 95%CI: -0.26, -0.01) and RUNX2 promoter methylation (ß: -0.13; 95%CI: -0.22, -0.03) in women. The methylation rate of RUNX2 promoter increased by 2.16% for each 1 mg/L increment in UF concentration of women in EFG (95%CI: 0.37, 3.96). No any significant associations between UF concentration, RUNX2 promoter methylation, and BMD were observed in the individuals in CG. Mediation analysis showed that RUNX2 promoter methylation mediated 18.2% (95% CI: 4.2%, 53.2%) of the association between UF concentration and BMD of women in EFG. In conclusion, excessive fluoride exposure (>1.6 mg/L) is associated with changes of BMD in women, and this association is mediated by RUNX2 promoter methylation.

The role of maternal methylation in the association between prenatal meteorological conditions and neonatal H19/H19-DMR methylation.

Meteorological conditions during pregnancy can affect birth outcome, which has been linked to the H19/H19-differentially methylated region (DMR). However, the detailed mechanisms underlying this association are unclear. This was investigated in the present study to provide epidemiological evidence for elucidating the pathogenesis of adverse birth outcomes. A total of 550 mother-newborn pairs were recruited in Zhengzhou, China from January 2010 to January 2012. Meteorological data including temperature (T), relative humidity (RH), and sunshine duration (SSD) were obtained from the China Meteorological Data Sharing Service System. Bisulfite sequencing PCR was performed to determine the methylation levels of H19/H19-DMR using genomic DNA extracted from maternal peripheral and umbilical cord blood. The results showed that H19-DMR methylation status in cord blood was positively associated with that in maternal blood. Neonatal H19-DMR methylation was negatively associated with T and RH during the first trimester and positively associated with these variables during the third trimester. There was a positive correlation between neonatal H19-DMR methylation and SSD during the second trimester and a negative correlation during the third trimester. Similar associations were observed between maternal H19-DMR methylation and prenatal meteorological conditions. We also observed significant interaction effects of maternal H19/H19-DMR methylation and most prenatal meteorological factors on neonatal methylation, and found that changes in the methylation status of maternal H19-DMR were responsible for the effects of prenatal meteorological conditions on neonatal methylation. In summary, neonatal H19-DMR methylation was significantly associated with prenatal meteorological conditions, which was modified and mediated by maternal H19-DMR methylation changes. These findings provide insights into the relationship between meteorological factors during pregnancy and adverse birth outcomes or disease susceptibility in offspring, and can serve as a reference for environmental policy-making.

Prenatal ambient air pollution exposure and SOD2 promoter methylation in maternal and cord blood.

The evidence is increasing that prenatal air pollutant exposure contributes to elevated oxidative stress in children, but the underlying mechanism is unclear. A pilot study was conducted in China to explore the associations between prenatal ambient air pollution exposure and superoxide dismutase 2 (SOD2) promoter methylation in maternal and cord blood. After detection and analyses, SOD2 promoter methylation levels in umbilical cord blood were elevated as maternal SOD2 promoter methylation levels increased. In addition, the SOD2 promoter methylation levels in umbilical cord blood were positively associated with the particulate matter 10 (PM10) exposure concentrations during the entire pregnancy and the second trimester. In maternal peripheral blood, the SOD2 promoter methylation levels were positively associated with the exposure concentrations of PM10 (during the entire pregnancy and the second trimester) and nitrogen dioxide (NO2) (during the first trimester of pregnancy), whereas the levels were negatively associated with the exposure concentrations of NO2 during the third trimester of pregnancy. Additionally, interaction analyses revealed that the maternal SOD2 promoter methylation level and sulfur dioxide (SO2) exposure (during the entire pregnancy and the third trimester), as well as NO2 exposure (during the third trimester of pregnancy), had an interaction effect on the SOD2 promoter methylation level in umbilical cord blood. Furthermore, mediation analysis revealed that the associations between SOD2 promoter methylation in umbilical cord blood and PM10 exposure during the entire pregnancy and the second trimester were partly mediated by maternal SOD2 promoter methylation. In conclusion, prenatal exposure to air pollutants was significantly associated with SOD2 promoter methylation levels in umbilical cord blood, and this association may be affected by SOD2 promoter methylation levels in maternal peripheral blood. These associations may be one of the mechanisms by which prenatal air pollutant exposure leads to oxidative stress in newborns.

Ambient air pollution, H19/DMR methylation in cord blood and newborn size: A pilot study in Zhengzhou City, China.

Prenatal exposure to air pollutants is believed to be associated with adverse birth outcomes. However, the potential mechanisms, especially the epigenetic modified effects, still remain unclear. This study was designed to explore the association of air pollution, H19/DMR methylation levels, and birth weight and length. A total of 527 mother-infant pairs were recruited from Houzhai Center Hospital, Zhengzhou. Air pollution data during the study period was collected. The methylation at H19 promoter region and H19 DMR in maternal and cord bloods were determined using real-time PCR analysis. Ridge regression was used to analyze the association of air pollutants exposure during gestation with H19/DMR methylation and birth weight and length respectively. Results showed that prenatal exposure to NO2 was associated with higher H19 methylation in cord blood. Whereas SO2 and PM10 exposure were associated with lower H19 and H19 DMR methylation respectively. After stratification by pregnancy trimesters, the association of H19 methylation in cord blood with PM10 exposure also was found. Furthermore, prenatal exposures to air pollutants also were associated with birth weight and length. Specifically, with the increase of maternal SO2 exposure during the entire pregnancy, birth weight and length significantly decreased. While birth weight and birth length were significantly increased with NO2 exposure. The stratified analysis also found the associations between PM10 exposure and birth sizes in different trimesters. In conclusion, the gene methylation level in cord blood might be associated with prenatal environmental exposures. Birth weight and length were associated with both prenatal environmental exposures and genetic factors.

Do Environmental Fluoride Exposure and ESRα Genetic Variation Modulate Methylation Modification on Bone Changes in Chinese Farmers?

Although increasing evidence suggests that estrogen receptor α (ESRα) genetic variation could modify bone damage caused by environmental fluoride exposure, little is known about epigenetic mechanisms in relation to bone changes. A case-control study was conducted among farmers aged 18-55 years in Henan Province, China. X-ray was used to detect bone changes. Methylation status was determined by methylation-specific PCR. Genotypes were identified by Taqman probe and real-time PCR. In this study, we found that methylation status in the promoter region of the ESRα gene was lower in bone change cases than that in controls, which was only observed in male farmers after stratification by gender. Furthermore, methylation level was negatively associated with the urinary fluoride concentration in male farmers. No significant association was found between the distribution of ESRα rs2941740 genotypes and the risk of bone changes. Multivariate logistic regression analysis showed that after adjusting for age and gender, increased serum calcium and methylation status were protective factors for bone changes. No interaction effect was observed between fluoride exposure and ESRα rs2941740 polymorphism on bone changes. In conclusion, the current work suggests that bone changes are associated with methylation status, which might be modulated by fluoride exposure in male farmers. Methylation status and bone changes were not modified by ESRα gene rs2941740 polymorphism in the promoter region.