Effect Of C-fos Demethylation On Fluoride-induced Apoptosis In L-02 Cells

Endemic fluorosis is prevalent in many parts of the world. Fluorosis can cause damage not only to skeletal tissue and teeth, but also to soft tissues. The liver is the primary organ for detoxification in human body, so the fluoride-induced liver toxicity has aroused wide public concern. Previous animal experiments have demonstrated that fluoride has toxic effects on the liver and cause lipid peroxidation. In addition, fluoride can lead to cell deformation, cords disorders, cytoplasmic, nuclear pyknosis, and nucleolar margination in hepatic cells. Apoptosis has been demonstrated to play an important role in toxicity of excessive fluoride in liver. Furthermore, some in vitro experimental studies shown that the toxicity of fluoride can lead to hepatocyte apoptosis and reduce antioxidation, and oxidative stress can induce apoptosis to some extent.The proto-oncogene c-fos encodes a transcription factor that plays a pivotal role in cell proliferation, differentiation, and apoptosis. A lot of research showed that fluoride could induce fibroblast, osteoblast apoptosis and c-fos mRNA expression. Further study suggests the main reason of c-fos expression disorder is abnormal DNA methylation. Tumor research reported that the methylation of c-fos is the pivotal reason which inhibits tumor cells apoptosis. Ischemical reperfusion injury and induced injury in rat liver also came to similar conclusions.DNA methylation is the major modification of eukaryotic genomes and is known to have a profound effect on gene expression. It is one of the regulatory processes that are referred to as epigenetic. It is a process that DNA methyltransferase (Dnmts) catalyst to S-adenosylmethionine (SAM) as a methyl donor to methyl added to cytosine dinucleotide CpG carbon atoms of the fifth to make it into a 5-methyl cytosine. Currently, three DNA methyltransferase members have been identified in humans. The most well-characterized DNA methyltransferase is Dnmt1. This enzyme is required for proper embryonic development in mammals, and is involved in copying the methylation pattern from an existing DNA strand to the newly synthesized DNA strand following DNA replication. For this reason, Dnmt1 is called the maintenance DNA methyltransferase. In contrast, Dnmt3a and Dnmt3b are believed to be de novo methyltransferases that can add a methyl group to a cytosine at a new location in the DNA strand, instead of just copying one that already exists.So far, whether excessive floride can induce c-fos methylation and the relationship between hepatotoxicity and the c-fos methylation are not clear. In order to study the effect of c-fos demethylation on fluoride-induced apoptosis and provide some useful clues for further study of fluoride-induced hepatotoxicity. The expression levels of c-fos, Dnmt1, Dnmt3a and Dnmt3b, and the level of c-fos demethylation in human embryo hepatocyte L-02 cells were measured after in vitro cultured L-02 was exposed to sodium fluoride (NaF) at different doses. This paper consists of three parts:Part I Effects of fluoride on apoptosis, mRNA and protein expression levels of c-fos in L-02 cells in VitroObjective To investigate the effects of NaF on apoptosis, c-fos mRNA and protein expression levels in L-02 cells in Vitro. Methods L-02 cells were exposed to different concentrations of NaF (0, 20, 40, 80μg/ml) for 24 h, and then the cell survival rates, percentage of apoptosis, mRNA and protein expression levels of c-fos were measured. Results Compared with the control group, the cell survival rates in 20, 40 and 80μg/ml NaF-treated groups were significantly lower (P<0.05). The percentage of apoptosis in 40 and 80μg/ml NaF-treated groups were significantly higher than that of the control group (P<0.05), and increased as the dose of NaF increased concentrations; The mRNA and protein expression levels of c-fos in 40 and 80μg/ml NaF-treated groups were significantly higher than those of the control group (P<0.05). Conclusion NaF exposure can increase the mRNA and protein expression levels of c-fos and cause apoptosis in L-02 cells in vitro.Part II Effects of fluoride on mRNA expression levels of Dnmt1, Dnmt3a, and Dnmt3b in L-02 cells in VitroObjective To investigate the effects of NaF on Dnmt1, Dnmt3a, Dnmt3b mRNA expression levels in L-02 cells in Vitro. Methods L-02 cells were exposed to different concentrations of NaF (0, 20, 40, 80μg/ml) for 24 h, and then the mRNA expression levels of Dnmt1, Dnmt3a, and Dnmt3b were measured respectively. Results The mRNA expression levels of Dnmt1 in 20, 40 and 80μg/ml NaF-treated groups were significantly lower than those of the control group (P<0.05); The mRNA expression levels of Dnmt3a in 80μg/ml NaF-treated groups was significantly lower than that of the control group (P<0.05); The mRNA expression levels of Dnmt3b in 40 and 80μg/ml NaF-treated groups were significantly lower than those of the control group (P<0.05). Conclusion NaF exposure can decrease the mRNA expression levels of Dnmtl, Dnmt3a, Dnmt3b in L-02 cells in vitro. Part III Effects of floride on c-fos demethylation measured by bisulfite sequencingObjective To analysis the methylation status of CpG islands in c-fos promoter in L-02 cells by using bisulfite sequencing. Methods The genomic DNA were extracted from L-02 cells which were exposed to different concentrations of NaF (0, 20, 40, 80μg/ml) for 24 h, and then the cells were treated by bisulfite sodium, the methylation status of c-fos amplified by polymerse chain reaction, then the amplified DNA were sequencing. Results c-fos sequencing showed that there were 144 bases to be sequenced, in which 10 CG have not changed in control group, 1 CG has not changed in 20, 40, 80μg/ml NaF-treated groups respectively. Conclusion The methylation levels of c-fos is decrease during NaF exposure. Demethylation of c-fos possible involved in the process of c-fos activation which induced by NaF in L-02 cells.