The Relationship Between Arsenic Exposure And DNA Methylation In ABCA1 Gene Promoter Region

Objective:To explore the effect of arsenic on ABCA1 gene promoter region methylation level,gene expression,cholesterol outflow and apoptosis of THP-1 macrophage.Methods:THP-1 macrophage was exposed to 0μM,4μM,8μM,12 μM sodium arsenate for 48 h,pretreatment with n-acetyl cysteine(NAC)for 1 h and then co-exposed to12μM sodium arsenate for 48 h.We detected cell survival rate by CCK-8,intracellular reactive oxygen species generation by flow cytometry DCFH-DA staining method,the p47 phox localization in macrophage by immunofluorescence,the DNA methylation of ABCA1 gene promoter region by pyrophosphate sequencing,ABCA1 expression by q RT-PCR and Western blotting.We then added 50 mg/m L ox-LDL to induce macrophage,and detect the lipid enrichment in the cells by oil red staining,the concentration and outflow of cholesterol by the total cholesterol and free cholesterol detection kit.We finally detect apoptosis by Hoechst staining and flow cytometry Annexin V-FITC/PI double dying.Results:Significant negative correlation was found between the survival rate of THP-1 macrophage and the concentration of arsenic exposure.The cellular survival rate was 66.9% and 20.7% after exposure to 12 and16 μM sodium arsenate,so we choose 0,4,8,12 μM as the concentration of arsenic exposure in subsequent experiments.The survival rate of macrophages had significantly recovered after pretreatment with 3000 μM NAC.Significant positive correlation was found between the production of ROS in THP-1 macrophage and the concentration of arsenic exposure,the production of ROS in 4,8,12 μM sodium arsenate group increased by 0.31,0.5,0.78 times compared with control,respectively.After pretreatment with 3000 μM NAC,the production of ROS in macrophages decreased significantly.The percentage of the cell with p47 phox transferring to the cell membrane was 0.5%,26.5%,20%,26.5% in 0,4,8,12 μM sodium arsenate group,respectively.The percentage of the cell with p47 phox transferring to the cell membrane was 3.5% after pretreatment of 3000 μM NAC.Arsenic exposure could increase ABCA1 gene methylation in macrophages.The methylation level of Cp G3 was increased by 17.9% in 4 μM sodium arsenate group;In 8 μM sodium arsenate group,the methylation level of Cp G1,Cp G2,Cp G3,Cp G4,Cp G6 increased by 48%,9.7%,7.6%,18.3%,15.0%,respectively;In 12 μM sodium arsenate group,the methylation level of Cp G1,Cp G2,Cp G3,Cp G6 increased by 20.5%,9.7%,28.1%,16.3%,respectively.Pretreatment with 3000 μM NAC could inhibit the effect of the hypermethylation induced by arsenic.Significant negative correlation was found between the expression of ABCA1 gene and protein and the concentration of arsenic exposure,which was 30.2% and 22.5% of control in 12μM sodium arsenate,respectively and had a significant increase in macrophages after pretreatment with 3000 μM NAC.The lipid accumulation of foam cells had a significantly increase with arsenic exposure concentration increasing by Oil red staining,total cholesterol levels increased significantly and the rate of cholesterol outflow reduced significantly.The lipid accumulation decreased significantly,the total cholesterol decreased significantly and the rate of cholesterol outflow increased significantly after pretreatment of 3000 μM NAC in macrophages.Arsenic exposure could increase the number of shrinked cell nucleus and cell apoptosis rate,which could be inhibited after pretreatment of 3000 μM NAC.Conclusion: Arsenic could promotthe assembly of NADPH oxidase in THP-1 macrophages,enhance the production of ROS,induce oxidative stress,then induce ABCA1 gene promoter region DNA hypermethylation,and reduce ABCA1 expression in the macrophages,causing lipid accumulation,cholesterol outflow reduction and apoptosis.