Effects Of Fluoride On DNA Methylation In Mouse Early Embryo

Fluorosis caused by non-pHrenology tissue damage has attracted extensive attention. The reproductive toxicity of fluorine and the impact on the offspring’s health has become a hot topic, but so far its molecular mechanism is not fully understood. Drinking water fluorosis is a systemic disease caused by a large number of long-term high fluoride content in drinking water, which is a type of chronic endemic fluorosis. This study is a fluorine-containing drinking water (100mg/L) in mice as the animal model. We examine the effects of fluoride on mouse liver, sperm and early embryo of genomic DNA methylation and imprinting gene DNA methylation. The epigenetics is introduced to study the reproductive toxicity of fluoride.1. Effects of fluoride on DNA methylation in mouse liverTo understand the effects of fluoride on mouse liver genomic DNA methylation and retrotransposon LINE1gene DNA methylation, we used bisulfite sequencing (BSP), and enzyme-linked immunosorbent assay (ELISA) to examine the effects of male mice were fed with fluorine-containing water for35days, the genomic DNA methylation and LINE1gene DNA methylation in the liver. The results showed that the genomic DNA methylation level in the treatment group and the control group was1.20%±0.11%and1.20%±0.14%, the difference was not significant; DNA methylation level of LINE1in the treatment group and control group was85%±3.15%and81.87%±5.39%, the difference was not significant. The results showed that male mice, were fed with fluorine-containing water for35days, did not directly affect the level of DNA methylation in mouse liver.2. Effects of female mice fluoride intake on DNA methylation in mouse early embryoTo understand the effects of female mice fluoride intake on DNA methylation in mouse early embryo. We used BSP and COBRA to examine the effect of female mice were fed with fluorine-containing water for2days, DNA methylation of the imprinted genes and retrotransposon LINE1gene DNA methylation in the embryo. The results showed that DNA methylation level of H19decreased from the control group49.33%±7.60%for the9.12%±5.28%, significant difference; DNA methylation level of Peg3in the treatment group and the control group was4.14%±0.32%and4.66%±0.45%, the difference was not significant; DNA methylation level of LINE1in the treatment group and the control group was42.33%±7.99%and41.67%±7.42%, the difference was not significant, the results of COBRA to further validate the sequencing results. This suggests that female mice, during pregnancy, fluoride intake, DNA methylation of the paternal imprinted genes, H19, was anomalous, but DNA methylation of the maternal imprinted genes, Peg3, and retrotransposon LINE1gene DNA methylation in the embryo did not change significantly.3. Effects of male mice fluoride intake on DNA methylation in mouse early embryoTo understand the influences of male mice, before fertilization, fluoride intake DNA methylation of in mouse early embryo. We used BSP and COBRA to examine the effect of the normal female mated with male mice, which were fed with fluorine-containing water, on DNA methylation of the imprinted genes and retro transpo son LINE1gene DNA methylation in the embryo. The results showed that DNA methylation level of H19decreased from the original49.33%±7.60%to27%±8.54%, significant difference; DNA methylation level of Peg3elevated from the control group,4.66%±0.45%to the treatment group96.55%±0%, the difference was significant; DNA methylation level of LINE1in the treatment group and the control group was45.50%±7.66%and41.67%±7.42%, the difference was not significant, the results of COBRA to further validate the sequencing results. This suggests that the normal female mated with male mice, which were fed with fluorine-containing water. The analysis results in the early embryo showed that DNA methylation of the imprinted genes, H19, Peg3, had increased significantly, whereas the LINE1gene methylation levels in normal.4. Effects of fluoride on DNA methylation in mouse spermTo understand the effects of fluoride on DNA methylation in mouse sperm, we used BSP and ELISA to examine the effect of male mice were fed with fluorine-containing water for35days, the genomic DNA methylation and H19, Rasgrfl, Peg3, LINE1genes DNA methylation in the sperm. The results showed that DNA methylation level of H19in the treatment group and control group was88.63%±5.14%and91.11%±0.95%; DNA methylation level of Rasgrfl in the treatment group and the control group was97.50%±1.12%and97.78%±1.21%; DNA methylation level of Peg3in the treatment group and the control group was0.71%±0.014%and0%±0%; DNA methylation level of LINE1in the treatment group and the control group was 85.96%±3.59%and90.06%±2.39%; the genomic DNA methylation level in the treatment group and the control group was0.44%±0.13%and0.41%±0.10%, the difference were not significant. The results showed that male mice, were fed with fluorine-containing water for35days, did not directly affect the level of DNA methylation in mouse sperm.