Role Of DNA Methylation In Genetic Damage Of Hepatocytes Induced By VC In Rats

Objectives:To investigat DNA damage induced by vinyl chloride in rats and to explore the dose-response and time-response relationship.Detected the status of genome-wide methylation,aberrant methylation in the promoter and mRNA expression of KRAS,CDKN2 A,RASSF1A,MGMT,SYK in rat liver to discuss the relationship between genetic and epigenetic mechanism of vinyl chloride.Methods:Ninety-six healthy SD male rats were randomly divided into a negative control group(0 mg/kg)and three VC exposed groups(5mg/kg?25mg/kg?125mg/kg).There are 24 rats in each group.Different dose of VC was given to SD rats by intraperitoneal injection every other day,totally three times a week.Eight rats in each group were killed at the end of 6,8 and 12 weeks respectively.The liver was sacrificed and the DNA damage level of hepatocytes was evaluated by comet assay.We used Methyl Flash Methylated DNA Quantification Kit to detect the status of genome-wide methylation.The methylation status of the 5' region of KRAS,CDKN2 A,RASSF1A,MGMT,SYK were detected by Quantitative Real-time methylation specific PCR(QMSP).The mRNA expression of these genes was examined by Real-time PCR(QPCR).SPSS22.0 was used to analyze the experimental data.The experimental data were in accordance with the normal or approximate distribution,ANOVA was used to compare the data.We chose the correlation coefficient of Pearson for analysis of bivariate correlation,the significance level of 0.05.Result:1.Vinyl chloride can induce liver tissue necrosis,hepatic steatosis,cirrhosis and other histopathological changes.The level of DNA damage in rat hepatocytes increased with the increase of exposure dose and prolonged exposure time.Genome-wide methylation level was significantly higher in the exposed group than in the control group(P <0.05).2.Changes of DNA methylation in five promoter regions of rats:2.1In different groups: The methylation of MGMT promoter in hepatocytes of rats in exposed groups was higher than the control group(P <0.05),the mRNA expression in rat increased with the increasing dose at 6 weeks.At 8 weeks,the methylation of KRAS and SYK in the exposed groups decreased with the increasing dose,the mRNA of SYK increased with the increasing dose.At 12 weeks,the methylation level of RASSF1 A and MGMT increased significantly with the increase of the dose,and the expression of RASSF1 A mRNA decreased with the dose increasing.2.2 At different times of exposure: There was no significant difference in the methylation and mRNA expression of KRAS,CDKN2 A,RASSF1A,MGMT and SYK in the control group(P> 0.05).In the group of 5mg/kg,the methylation of MGMT and SYK decreased with the prolongation of exposure time and the mRNA of MGMT increased with the prolongation of time.The methylation of KRAS,MGMT and SYK in 25mg/kg was lower at 6 weeks than at 8 weeks and 12 weeks(P <0.05)and the mRNA increased with the prolongation of exposure time.At 6 weeks and 8 weeks,the methylation of CDKN2 A and RASSF1 A were less than at 12 weeks(P <0.05),the mRNA of RASSF1 A was significantly higher than at 6 weeks and 8 weeks(P <0.05).3.The damage of DNA was positively correlated with the methylation level of RASSF1 A and MGMT promoter(P <0.05).Conclusions:1.Vinyl chloride can lead to the damage of DNA,elevate the level of genome-wide methylation.2.Vinyl chloride can decrease the methylation of KRAS,SYK.It can also increase the methylation of CDKN2 A,MGMT.The methylation of RASSF1 A decreases and mRNA expression increases in the short-term and low-dose.But the methylation of RASS1 A will decline when the exposure is at longer-term and higher-dose.3.The increase methylation of RASSF1 A,MGMT may affect the repair of DNA damage of rat liver cell induced by vinyl chloride and increase the DNA damage of hepatocytes.