Effect Of Cadmium On DNA Methylation And Autophagy In Rat Hepatocytes

Cadmium(Cd), an mainly industrial and environmental pollutant, has been classified as a carcinogen by the International Agency for Research on Cancer (I ARC). Liver is one of the most important targeted organs for Cd accumulation and damage. However, it is still not clear about the exact molecular mechanism of Cd induced liver injury. Epigenetic modification including DNA methylation is an important molecular target of bodies or cells damaged by the environmental harmful substances. In the present research, a model of SD rat primary hepatocytes with Cd treatment in vitro was established to investigate the effect of Cd on hepatocytes DNA methylation and autophagy.1Establishment of the Cd induced hepatocytes modelIn this study, the rat primary hepatocytes culture system in vitro was used to study the changes of cell viability in different concentrations of Cd. In primary hepatocytes cultured at different time points in vitro (4、7、10and24h), which were incubated with cadmium acetate of different concentrations (0、0.5、1、2、4μmol/L) for3h. The results of MTT assay showed that Cd reduced the cell viability of rat primary hepatocytes at a dose-depentant manner. After exposed to4μmol/L Cd, the cell viability decreased to78.35%, showing a significantly difference from the control group (P<0.01). Hepatocytes were cultured for4、7and24h then treated with4μmol/L Cd for3h in vitro, cell viability was significantly decreased (P<0.01). The results suggested that Cd induced cell viability was at a concentration-dependent manner.2Effect of Cd on DNA methylation in rat primary hepatocytesThe SD rat primary hepatocytes were treated with4μmol/L Cd for3h. Genome DNA methylation were determined by5-MC methyl immunofluorescence analysis method. Bisulfite sequencing (BSP) and combined bisulfite restrition analysis (COBRA) were used to examine the DNA methylation status of P53, MT and Linel. qRT-PCR method was applied to detect the effects of Cd on DNMTs、P53、PARP、PNK and OGG1mRNA expression level. The results showed that the level of genome DNA methylation slowly decreased as culture time elapsed, and the trend was enhanced after Cd treatment, consistent with DNMTs activity decreased. P53, MT and Linel methylation level showed no significant difference. P53, PNK and PARP mRNA level increased after Cd treatment. In conclusion, Cd might injure hepatocytes by destroying the stability of the genomic DNA methylation. Although the DNA methylation of MT, a key gene of heavy metal damage, was at a lower expression level before the treatment of Cd, the protective mechanisms of MT and DNA repair may too weak to save the liver cells from Cd injury.3Effect of Cd on autophagy in rat primary hepatocytesAutophagy is a cellular defense process to degradate the damaged cytoplasm and organelles. The rat primary hepatocytes with Cd treatment were used to investigate the effect of Cd on autophagy. LC3protein levels were examined by western blot and immunofluorescence assay. After the rate of autophagy increased by the inducement of rapamycin, the effect on cell viability was tested by MTT method. The results showed that Cd reduced the level of hepatocyte autophagy and cell viability. Rapamycin induced autophagy could hardly attenuate the damage of Cd. In conclusion, Cd inhibited the protection of autophagy in hepatocytes.