The Effects Of Triclosan And BDE-209 And Their Metabolites On DNA Methylation In Human Target Cells And Possible Mechanism

Triclosan(TCS), widely used in pharmaceuticals and personal care products as an antimicrobial agent, has been reported to be an liver tumor promoter in mice. Our previous work showed that exposure of TCS at high-doses(1.25-10 μM) for 24 h induced global DNA hypomethylation in human liver hepatocellular Hep G2 cells, while the low-doses and long-term exposure is the characteristic of environmental pollution to human. The concentration of TCS in sea water, river water, wastewater and sediments ranges from 0.035 to 7.945 n M. However, whether such low-doses long-term TCS exposure could regulate tumor suppressor gene expression by DNA methylation, and the underlying mechanisms, if so, is not clear. Besides, Decabromodiphenyl ether(BDE-209), a widely used flame retardant in furniture, textiles and electronic products, has been found in human blood, milk and liver tissues, and serum concentration in electronic waste dismantling workers even reaches as high as 3.2 μM. Therefore,(BDE-209) and its environment debromination product Monocholoro-nonabromo diphenyl ethers(Cl-BDE-208) are also explored their DNA methylation toxicity in human breast cancer MCF-7 cells, for they have similar structure with TCS. In this study, we investigated whether TCS and BDE-209 and their metabolites could cause DNA methylation changes of several DNA methylation biomarkers in their target human cells, including global DNA methylation(GDM), repetitive elements and tumor suppressor genes, and the possible underlying mechanisms of DNA methylation aberrant was also studied. The results might provide scientific basis for health risk assessment of TCS and BDE-209 and their metabolites..The main contents as the following:1.According to the concentration levels of TCS in environmental samples, 0.625-5 n M were chosen as exposure concentration to investigate the effects of low-doses and long-term exposure(two weeks) of TCS on DNA methylation of global DNA(GDM), repetitive element genes and liver tumor suppressor gene(p16) in Hep G2 cells using HPLC-MS/MS, Methylight, Q-MSP, Pyrosequencing, and Massarray methods. Furthermore, the changes of DNMTs(DNA methyltransferases) activity, the gene and protein expression levels of DNMT1(DNA methyltransferase 1) and Me CP2(Methylated DNA binding domain), and levels of 8-hydroxy-2-deoxyguanosine(8-OHd G) were also detected to explore the potential mechanisms of abnormal DNA methylation.2. Based on the concentration levels of BDE-209 in human tissue samples, 0.375-3 μM were chosen as exposed concentrations to investigate the effects of Cl-BDE-208 and BDE-209 exposure on DNA methylation of repetitive elements LINE-1 gene, global DNA(GDM), and estrogen receptor gene(ESR1 and ESR2) in MCF-7 cells. Moreover, the changes of DNA oxidative stress index 8-OHd G levels, 5hmd C levels, activity of DNMTs, gene expression levels of DNMTs(DNMT1、DNMT3a and DNMT3b)and ESR1 were detected to explore the possible mechanism of gene methylation after Cl-BDE-208 and BDE-209 exposure in MCF-7 cells.Through researches above, the followings are the main conclusions:1. The results of low-dose TCS long-term exposure showed that 0.625-5 n M TCS down-regulated repetitive elements LINE-1 methylation levels, but not global DNA methylation, through down-regulating DNMT1(DNA methyltransferase 1) and Me CP2(methylated DNA binding domain) expression, and accumulating 8-hydroxy-2-deoxyguanosine(8-OHd G) levels. More intertesting, low-dose TCS increased p16 gene methylation levels and decreased p16 gene expression, rather than high-dose(10 μM) TCS. Further, the results of MTCS(methyl-triclosan, the environmental metabolite of TCS) contrast to TCS showed that, hydroxyl group may contribute to TCS-mediated DNA methylation changes.2. The results of environmental relative concentrations of Cl-BDE-208 and BDE-209 short-term exposure showed that Cl-BDE-208 and BDE-209 decreased LINE-1 and global DNA methylation through down-regulating DNMTs activity, and 8-OHd G level increase may be involved in BDE-209-induced hypomethylation. Interestingly, Cl-BDE-208 and BDE-209 elevated ESR1 gene methylation and inhibited ESR1 expression, while both compounds have no effects on ESR2 gene.3. TCS and BDE-209 decreased repetitive elements LINE-1 methylation levels through down-regulating DNMTs activity. Meanwhile, 8-OHd G levels increase may be involved in TCS and BDE-209.