Study On The Effects Of DNA Methyltransferase In Primary Cultured Human Skin Fibroblasts Induced By Inorganic Arsenics And Its Methylated Metabolites

Objective:To observe different effects of iAsⅢ,iAsⅤ,DMAⅤ,MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts and possible molecular mechanism in the skin lesions of arsenic. Methods:1.The different effects of iAsⅢ,DMAⅤ,MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts was tested by MTT reduction assay.2-The cell cycle distribution was detected by flow cytometry when cells were treated with or without different concentrations of iAsⅢ,iAsⅤ,DMAⅤ,MMAⅢin human skin fibroblasts.3.Cell structure examined by electron microscopy.4. Semi-quantitative RT-PCR method was applied to detect the mRNA expression level of DNMT1,3A and 3B,As3MT. Results: 1. The results of MTT assay showed that with different concentration of iAsⅢ,DMAⅤ,MMAⅢ(0.5-5μmol/L), the skin fibroblasts were markedly stimulated, and a dose-effect relationship was observed; F13 skin fibroblasts grew slowly, the fast growing phase of skin fibroblasts was at the 96 h.2. The different doses of sodium arsenic can injury the normal microstructure of skin fibroblasts. After be treated with 0.5μmol/l sodium arsenite,Cell morphology appeared several changes:cell surface microvillus change more,thin and long, cell nucleus is big, core is irregular, nucleoli is obviously, There are small number of organelle in the cytoplasm, there are vacuoles in the part of mitochondria. After be treated with 10μmol/l sodium arsenite, Cell morphology appeared several changes:cell surface microvillus showed significantly reduced than 5μmol/l sodium arsenite. Lissome have significantly increased than 5μmol/l sodium arsenite. There are a lot of vacuoles in the mitochondria. Nucleolus has disappeared.3.High or middle doses of iAsⅢ,iAⅤ,DMAⅤ,MMAⅢcould increase cell percentage in G1 phase, That was showed G1 phase of cell cycle was being stilled.4. The result of RT-PCR showed that the gene expression of DNA methyltransferase were not in the same levels among different groups on the skin fibroblasts. Conclusion:The proliferation of the skin fibroblasts is regulated in a double-phase manner by inorganic arsenic and its metabolites. Low concentration induces stimulation effect, but high concentration inhibits the cell proliferation. High or middle doses of iAsⅢ,iAsⅤ,DMAⅤ,MMAⅢincreased cell percentage in G1 phase, blocked G1 phase. The different doses of sodium arsenic can injury the normal structure of skin fibroblasts. It plays a vital role in the skin lesions of arsenic.