Effects Of Chronic Hepatitis B Virus Infection On The Expression Of Cytochrome P450 2C9

Hepatitis B virus(HBV) infection is a serious global health problem,with 2 billion people infected worldwide,350 million suffering from chronic HBV infection,and estimated 112 million chronic carriers in China.For this particular population,there are needs to take drugs for treating other diseases,which poses a challenge to the doctor in selecting safe regimens for these patients.A family of enzymes known as cytochrome P450,which are the most important metabolizing enzymes,located in the endoplasmic reticulum of liver cells,and are responsible for the metabolization of about 90%of drugs intaken.Epidemiological studies indicated that drug induced liver injury is closely related to P450.A lot of factors including genetic diversity and illness can influence the activities of P450 enzyme, For example,individual variability occurs in the metabolism of CYP2C9 substrates in humans and a principal factor is the presence of genetic polymorphisms in humans.Most notably,the CYP2C9^*2 and CYP2C9^*3 alleles have significantly lower intrinsic clearances of CYP2C9 substrates both in vivo and in vitro.As to patients with chronic HBV infection,it has been documented that HBV infection could affect the expression of cytochrome P450,but the exact relationship between HBV infection and biochemical activities of major CYP subfamilies remains unknown.CYP2C9 is one of the most important P450s for drug metabolisation in human liver,it is rate-limiting enzyme in the metabolic clearance of a large scale of clinically used drugs,such as the hypoglycemic agents tolbutamide and glipizide,the anticonvulsant phenytoin,the S-enantiomer of the anticoagulant warfarin,and numerous nonsteroidal anti-inflammatory agents including flurbiprofen,diclofenac,torsemide and ibuprofen.Approximately 16%of clinically used drugs are metabolized by CYP2C9.Because CYP2C9 enzyme play a prominent role in the metabolism of many pharmaceutical agents and activation or inactivation of potential carcinogens,it would be necessasy to know whether CYP2C9 activities are likely to be altered by HBV infection.To address this issue, we investigated the effect of HBV infection on activity of CYP2C9.Potential mechanisms under our experiments' results were further probed at cell and molecular levels.Genetic polymorphisms of CYP450 2C9 in Chinese populationObjective:The purpose of this part is to identify CYP2C9 allele frequency and the prevalence of CYP2C9^*2 and CYP2C9^*3 allele.Methods:Peripheral blood samples were obtained from 20 patients received liver resection under informed consents with(n=10) or without(n=10) chronic HBV infection.Genomic DNA were isolated from whole blood with Blood DNA Isolation Kit.CYP2C9 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method.Sequence of some samples were confirmed by TA cloning and direct sequencing.Results:OD260/OD280 ratio of the genomic DNA was between 1.8 and 1.9,the yield of genomic DNA was approximately 90ng/μl～230ng/μl.The results of agarose gel electrophoresis analysis showed that the DNA extracted was integrity.PCR-RFLP results showed all 20 samples were CYP2C9 wild-type allele CYP2C9^*1^*1,CYP2C9^*2 and CYP2C9^*3 were not detected.Conclusions:All the samples showed CYP2C9 wild-type allele,no CYP2C9^*2 and CYP2C9^*3 allele variation was found,the low frequency of CYP2C9^*2 and CYP2C9^*3 allele variation in Chinese may partly be explained by these results.However,the results obtained in this part may provide basic research for future investigations.Effects of chronic HBV infection on human hepatic CYP2C9Objective:To investigate the effect of chronic HBV infection on human hepatic CYP2C9 in patients with chronic HBV infection.Methods:Liver tissue samples were obtained from 20 patients received liver resection under informed consents with(n=10) or without(n=10) chronic HBV infection.Liver S9 fractions were prepared utilizing differential centrifugation at 1,0000×g from the liver homogenate.The activity of CYP2C9 was detected by high performance liquid chromatography(HPLC).The expression of CYP2C9 mRNA and protein were determined by RT-PCR and western blotting.Results:The Vmax of CYP2C9 enzyme in patients with chronic HBV infection was(40.4±10.4) pmol.mg^-1.min^-1,significantly reduced(P=0.0367) comparing to the non-infected control group:(52.6±13.4) pmol.mg^-1.min^-1.RT-PCR showed the expression of CYP2C9 mRNA in patients with chronic HBV infection was(0.39±0.28) which was significantly lower than that of non-infected control(0.65±0.13,P=0.0171), Similarly,western-blotting showed the protein expression in patients with chronic HBV infection was lower than non-infected contro(10.26±0.13 vs 0.60±0.19, P=0.0002).However there was no significant difference for Km values for tobutamide 4-hydroxylation between HBV infected(263.5±66.4μmol/L) and non-infected controls(284.6±85.9μmol/L)(P=0.5471).Conclusions:Chronic hepatitis B virus infection may down-regulate activity of human hepatic CYP2C9 both in mRNA and protein level,but does not affect its structure.Clinical significance is addressed that chronic HBV infected patients might be subjected to drug to drug interaction by CYP2C9. Effects of HBx on the induction of CYP2C9 in HepG2 cellObjective:To invetigate the effect of HBx protein on enzymatic activity of CYP2C9 in HepG2 cell line in vitro,and to explore the regulatory mechanism of CYP2C9.Methods:Rifampicin was selected as inducer to enhance CYP2C9 expression in HepG2 cell line cultured in DMEM medium,For testing cytotoxicity of rifampicin(5μmol/L～50μmol/L),the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay were used.Eukaryotic expression vector for hepatitis B virus X(HBV X) gene with enhanced green fluorescence protein(EGFP) was constructed,and pEGFP-N1(encoding green fluorescent protein) and pEGFP-X(encoding HBx) was transfected to HepG2 cell line in vitro treated with 50μmol/L rifampicin.After transient transfection,EGFP gene were detected by fluorescence microscope in different time,cell received no treatment used as normal control group.HepG2 cells were harvested after 72h,and the expression of CYP2C9 protein and mRNA in cells were determined by western blotting and RT-PCR,respectively.Results:MTT testing showed that various concentrations of rifampicin had no obvious toxicity to HepG2 cell.Compared with normal control group,the expression of CYP2C9 mRNA and protein increased significantly in group transfected with pEGFP-N1 and rifampicin,by 224%and 300%,respectively.Similarly,group transfected with pEGFP-X and rifampicin demonstrated significantly increasement in mRNA and protein,by 163 %and 245%,respectively.There was significant difference between two groups in mRNA and protein level(P＜0.05).Conclusions:The induction of CYP2C9 by rifampicin in HepG2 Cell line can interfered by HBx,which suggested that HBx has suppressive effect on the expression of CYP2C9.