The Function Of Methylation Of CYP1A1 Promoter Region In Anti-tuberculosis Drug-induced Hepatic Injury

Objectives In present study,we investigated the association between CpG island methylation status of CYP1A1 gene promoter region and anti-tuberculosis drug-induced liver injury(ADIH)and the effect of 5-aza-2'-deoxycytidine(5-Aza-Cd R)on ADIH.Methods HL-7702 cells were divided into control group,isoniazid group(INH),ri fampicin group(RFP),pyrazinamide group(PZA),two-drug group(INH+RFP=50?g/ml+100?g/ml),three-drug group(INH+RFP+PZA=85.5?g/ml+427.5?g/ml+171?g/ml).Firstly,the cells were cultured in different anti-tuberculosis drugs for 24 h,and C CK8 assay was performed to test the cell viability of damaged hepatocytes in ord er to ensure the concentrations of different anti-tuberculosis drugs.Secondly,all of the hepatocytes were cultured in 6h,12 h,24h on the basis of various concentrati ons identified above,and Lai method were used to check the changes of LDH,S OD,MDA at different time points of different drugs.The changes of CYP1A1 m RNA expression were tested through q RT-PCR.The relationship between CYP1A1 m RNA and LDH,SOD,MDA were determined through correlation analysis,and we selected the time point on which CYP1A1 m RNA had the biggest change an d used sequencing method to test the CpG island methylation status of CYP1A1 gene promoter region after treated by different anti-tuberculosis drugs.Finally,hep atocytes were treated with 5-Aza-Cd R,and q RT-PCR was used to detect the m RN A of CYP1A1,DNMT1,DNMT3 a and DNMT3 b.We used MSP method for dete cting the CpG island methylation status of CYP1A1 gene promoter region in diffe rent anti-tuberculosis drugs,ELISA method for CYP1A1 protein,DNMT1,DNMT3 a,DNMT3b protein and enzyme activity,and Lai method for the changes of LD H,SOD,MDA.Results According to survival rate,the drug concentrations were determined to be800?g/ml INH,300?g/ml RFP,500?g/ml PZA,150?g/ml(INH+RFP),and 684?g/ml(INH+RFP+PZA).After cultured for 6h,12 h,24h in the concentrations of these drugs above,we found LDH,SOD,MDA which were the indicators of liver injury were all changed abnormally.The expression of LDH and MDA of each drug group were higher than the control group,while the level of SOD was lower than the control group.With the extension of time,LDH and MDA increased more obviously,and reached the maximum level at 24h(P<0.01),and the level of LDH and MDA showed the largest increasing in the three-drug group at 24 h.While the SOD of each drug group also decreased to the lowest value at 24 h,and the three-drug group had the largest decreasing(P<0.01),suggesting that different types of anti-tuberculosis drugs can lead to liver injury,and the combination of the three drugs is more serious.After treated with INH,PZA,two drugs,three drugs,the expression of CYP1A1 m RNA was decreased firstly and then recovered,and all of them were decreased to the lowest value(P<0.05)at 12 h,and the three drugs has the largest decreasing.While RFP group was increased first and then decreased,and reached the lowest value at 24h;except for the RFP group,CYP1A1 m RNA was negatively correlated with LDH and MDA in other drug groups,while positively correlated with SOD.These results suggest that CYP1A1 has a low expression,and reached the lowest level at 12 h during the process of liver injury induced by INH,PZA,two-drug group,three-drug group.The low expression of CYP1A1 may be regulated by promoter region methylation.We used sequencing to test the CpG island methylation status of CYP1A1 gene promoter region,the result showed that the CYP1A1 promoter region methylation rate was increased(P<0.05)in INH,PZA,two-drug group and threedrug group.The methylation rate of three-drug group was higher than the two-drug group.The methylation rate of RFP group was increased,but there was no statistical significance(P>0.05).These results suggest that low expression of CYP1A1 may be affected by its promoter region methylation in INH,PZA,two-drug group and three-drug group.To further ensure the relationship between methylation and liver injury,we cultivated HL-7702 with 5-Aza-Cd R.According to the protein expression and enzyme activity of DNMT1,DNMT3 a,DNMT3b,10umol/L 5-Aza-Cd R was selected.The result of MSP showed that,in comparison with the control group,CYP1A1 promoter region methylation rate of the group treated with 5-Aza-Cd R was decreased in INH,PZA,twodrug group and three-drug group,and thethree-drug group possessed the most decreasing.At the same time,compared to the group treated with different kinds of anti-tuberculosis drugs but without inhibitor,the expression of CYP1A1 protein and transcription of m RNA were increased in the group added with inhibitor,and the protein expression level is significantly higher than the transcription level of m RNA.The protein and m RNA of CYP1A1 changed most significantly in the three-drug group added with inhibitor(P<0.01).These results suggest that 5-Aza-Cd R inhibits the CpG island methylation of CYP1A1 gene promoter region and promotes its expression.In addition,we found that in the two drugs combined with 5-Aza-Cd R group and three drugs combined with 5-Aza-Cd R group,SOD activity increased and the protein content of MDA decreased compared to that without 5-Aza-Cd R.These all had statistical significance(P<0.05),and the levels of LDH decreased only in the three drugs combined with 5-Aza-CdR group(P<0.01).Conclusions The CpG island high methylation status of CYP1A1 gene promoter region regulates the low expression of CYP1A1,and is relative to liver injury induced by INH,PZA,INH+RFP and INH+RFP+PZA.5-Aza-Cd R can increase the expression of CYP1A1 by demethylation,and alleviate the damage of liver injury caused by the combination of two drugs and combination of three drugs.