4. Discussion

Water-borne endemic fluorosis has been widely prevalent in China, especially in the central, western and northern parts of China. In order to explore the possible epigenetic changes caused by fluoride exposure, we conducted a screening and validation study in step by step. We undertook, for the first time, microarray analyses of DNA methylation on samples from school-age children who had been exposed to/not exposed to excessive fluoride in drinking water. Gradually, we conducted population-based verification of DMRs. In total, 237 DMSs and 212 DMRs were found in different fluoride exposure groups in the epigenome-wide phase. In addition, we found that the methylation of the target sequences of NNAT, CALCA and MTHFD1 was significantly different between the HFG and CG according to MethylTarget™ method. Only the methylation status of NNAT and CALCA were susceptible to fluoride exposure in the population-based validation phase. Notably, NNAT methylation could modify the influence of fluoride exposure on the ACTH level in children.

According to the study design, we first screened the DMSs and DMRs sensitive to fluoride exposure in the epigenome-wide phase using the 850 K BeadChips. Notably, DNA methylation profile in girls is relatively inconstant from the beginning of puberty (Thompson et al., 2018). Hence, we selected sixteen boys to screen the DMRs susceptible to fluoride in the BeadChip analyses in the discovery phase. We found 70 significantly enriched biological process entries related to DMSs using the GO database. These biological-process entries were mainly involved in the positive regulation of transcription of Notch receptor targets, morphogenesis of a branching structure and processing of peptide hormone, which participate in multiple functions in the body. Studies have shown that fluoride exposure can affect regulation of apoptotic process, receptor activity and nervous system development (Bartos et al., 2018, Wei et al., 2018). Analyses of pathway enrichment using the KEGG database provided a pathway map of gene functions, including dopaminergic synapse, thyroid hormone synthesis and the NF-kappa B signaling pathway. These results are similar to previous studies (Kupnicka et al., 2020, Wang et al., 2020, Zhang et al., 2008). Interestingly, we found some fluoride-related pathways that have not been discovered (e.g., renin secretion, toll-like receptor signaling pathway, and cholinergic synapse). In light of the development of targeted interventions for gene methylation (Papikian et al., 2019, Tian et al., 2019), these findings provide novel clues for the basic research and clinical treatment of fluoride-related diseases.

In the subsequent validation phase, we first recruited 100 children including fifty boys and fifty girls to validate the identified target DMRs. Nine DMRs were validated by MethylTarget™ method. Methylation status of the target sequences of CALCA, NNAT and MTHFD1 was significantly altered. In the population validation phase, only methylation status of CALCA and NNAT were affected by fluoride exposure.

Calcitonin, encoded by CALCA gene, plays a crucial role in bone metabolism by restraining bone resorption (Meleleo and Picciarelli, 2016). It also prevents osteoporosis and maintains bone mass (Naot et al., 2019). We observed that CALCA methylation status positively correlated with the UF concentration, which suggested that fluoride exposure may have affected CALCA methylation, and verified the result in our published study of adult samples (Sun et al., 2020), which showed that CALCA methylation in children and adults were easily affected by fluoride exposure. An increase in CALCA methylation was caused by increased exposure to fluoride, which indicated that the expression of CALCA might be affected.

An epigenetic study using high-throughput technique on fluoride exposure was reported by Daiwile and colleagues (Daiwile et al., 2019). They observed that the promoter regions of genes of BMP1, METAP2, MMP11 and BACH1 were hypermethylated in human osteosarcoma cells exposed to fluoride. However, as compared with in vitro experiments, the internal environment of the body is sophisticated. In addition, unlike blood cells in the peripheral circulation, bone cells are the target, which may be more sensitive to fluoride exposure (Jiang et al., 2020).

Apart from CALCA methylation, NNAT methylation related to neurodevelopment negatively correlated with the UF concentration. It has been reported that NNAT gene is involved in the regulation of ion channels in brain (Pitale et al., 2017), chemical reprogramming of astrocytes into neurons, and assists regeneration of new neurons for brain repair (Ma et al., 2019). The location of NNAT in rat brain cells reflects various functions, including differentiation and maintenance of cells (Kanno et al., 2019). The influence of fluoride on development of the nervous system has attracted increasing attention (Dec et al., 2017). Our study indicated that fluoride exposure may alter NNAT methylation, which may further explain the neurotoxicity of fluoride at the molecular level. Accordingly, changes in NNAT methylation may also impact the development of intelligence. This finding provided a clinical reference that alterations in methylation induced by exposure to excessive amounts of fluoride might be involved in a change in neural development.

ACTH is a very important hormone secreted by the pituitary–adrenaline axis. One animal study demonstrated that the ACTH level positively correlated with fluoride exposure (Kinawy and Al-Eidan, 2018). It has been reported that NNAT could participate in maintaining the overall structure of the nervous system in the brain (Wijnholds et al., 1995), and the NNAT gene is also expressed in the adrenal glands. NNAT is also thought to be involved in development and maturation of the pituitary glands (Aikawa et al., 2003). We found that the UF concentration negatively correlated with NNAT methylation, and that the ACTH level positively correlated with the level of fluoride exposure. Therefore, we hypothesized that NNAT methylation might play an important role in the changes of the serum ACTH level caused by fluoride exposure. Accordingly, we investigated the modification effect of methylation using mediation analyses, and we found that NNAT methylation mediated 11.7% of the alteration in the ACTH level influenced by fluoride exposure. Hence, NNAT methylation might be an important biomarker in the effect of fluoride on ACTH. However, the specific harmful mechanism needs further study.

One of the strengths of our study was that we used high-throughput sequencing to screen DMRs that are sensitive to fluoride exposure, and then coherently validated the DMRs in population-based samples to ensure a scientific and rigorous experiment. In addition, one-carbon metabolism is a major metabolic network through which nutrients can regulate DNA methylation (Steluti et al., 2019). Different dietary patterns can lead to changes in gene methylation profile and subsequently affect hormone secretion. Study participants were students living in boarding school, and they had consistent diets and living habits, which eliminated the interference of confounding factors in our study.

Several limitations of our study should be stated. First, due to the restrictions of a cross-sectional design, we could not determine a causal relationship between fluoride exposure and DNA methylation. Second, our study was based on sequencing using the 850?K BeadChip, which is a popular method for epigenome-wide association studies, but it is not specifically a genome-wide sequencing tool. Third, the sample size in the discovery phase was relatively small. Nevertheless, at the subsequent validation phase, we expanded the sample size and adjusted multiple confounding factors to make our results more statistically robust. Further research should be analyzed with all methylation markers related to fluoride exposure, and comprehensively validate the corresponding disease outcomes in large cohort studies to better determine the use of such markers in research and clinical practice.

5. Conclusions

For the first time, we used microarray technique to analyze the DNA methylation status of school-age children with different exposure levels at the epigenome level, and the 237 DMSs and 212 DMRs associated with fluoride exposure were screened. We identified that NNAT and CALCA as DMGs were susceptible to fluoride exposure in school-age children using high-throughput sequencing and subsequent population-based validation. Finally, we observed that NNAT methylation could modify the influence of fluoride exposure on the ACTH level in children. These findings provide useful predictors and therapeutic targets of adverse health outcomes induced by fluoride exposure.