乙型肝炎病毒慢性感染对细胞色素P450 2C9表达的影响
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摘要
乙型肝炎病毒感染是全球主要的公共安全问题之一,国内有近10%的人口感染乙型肝炎病毒,感染者已超过1.2亿。药物在这类人群中的代谢规律是否跟正常人一致呢?是否会由于HBV感染影响药物的代谢,从而导致药物不良反应(药物性肝损害)的概率增加呢?这个问题一直是临床关注的热点,也是临床医学的一个盲区。
肝脏是药物代谢的主要场所,肝脏进行药物代谢依赖于微粒体中的多种酶系,其中最重要的是细胞色素P450酶系。越来越多的证据表明,药物代谢过程所致肝损害与P450酶系密切相关,任何导致P450酶活性变化的因素都可以影响药物在体内的代谢,其中,遗传基因多态性和疾病的状况是公认的非常重要的因素。基因突变可以用来解释人们在服用药物时出现的药效学和药代学的个体差异,例如细胞色素P450亚家族CYP2C9酶,CYP2C9~*2和CYP2C9~*3突变基因能显著降低酶活性。另一个影响酶活性的因素是疾病的状况,对于慢性乙型肝炎患者而言,既往研究表明HBV可以对P450酶表达产生影响,但是HBV对P450酶各主要亚家族酶活力的影响是不一致的,而且先前的工作大部分都是在动物和细胞水平进行的,没有HBV感染影响人肝细胞色素P450表达的研究。
由于CYP2C9在人类肝脏中含量丰富,占总量的20%,仅次于CYP3A,是肝内含量第二的CYP亚家族酶,能代谢大约16%不同性质的临床药物,并在前致癌物、前毒物和致突变剂的活化中起一定作用。既然CYP2C9酶在临床药物代谢中有如此重要的作用,那么慢性HBV感染时,CYP2C9酶的活性是如何变化以及影响酶变化的机制是什么呢?这是我们关注的重点。本实验选取CYP2C9酶作为研究对象,收集临床肝脏标本,检测慢性HBV感染肝组织和无HBV感染的肝组织中CYP2C9酶的活性,并检测该酶在mRNA水平和蛋白水平变化情况,同时在细胞水平对临床研究结果进行机制的探讨,为慢性HBV感染患者安全合理使用药物、规避不良反应提供科学依据。
一、细胞色素P450 2C9基因多态性研究
目的检测CYP2C9基因型,排除CYP2C9~*2和~*3突变等位基因对CYP2C9酶活性的影响,为下一步研究奠定基础。方法收集慢性HBV感染患者和无HBV感染者的肝组织和血液标本20例,以基因组DNA提取试剂盒提取血液基因组DNA,应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术对CYP2C9进行基因分型,并随机抽取数例样本进行TA克隆测序验证。结果提取的血液基因组DNA光密度值在1.8～1.9之间,含量为90ng/μl～230ng/μl,琼脂糖凝胶电泳提示基因组完整,PCR-RFLP分析发现20例均为野生型CYP2C9(~*1~*1),未检测到基因突变,TA克隆测序分析验证了PCR-RFLP分析结果。结论本实验中我们未检测到CYP2C9突变基因,CYP2C9基因多态性的种族差异与地域不均衡性是结果最可能的解释,但是为我们下一步检测疾病对CYP2C9酶活性的影响奠定了基础。
二、慢性乙型肝炎病毒感染对人肝细胞色素P450 2C9的影响
目的探讨慢性HBV感染对人肝脏细胞色素酶2C9表达的影响,为慢性HBV感染患者安全用药提供理论指导。方法慢性HBV感染患者和无HBV感染者的肝组织样本,匀浆后差速离心法制备肝脏微粒体酶混合物(S9),以甲苯磺丁脲为探针,用高效液相色谱(HPLC)的方法检测两组样本CYP2C9酶的活性,同时以RT-PCR和western blot测定两组肝组织样本中mRNA和蛋白表达的差异。结果高效液相色谱显示慢性HBV感染组Vmax为40.4±10.4 pmol·mg~(-1)·min~(-1),而无HBV感染对照组Vmax为52.6±13.4pmol·mg~(-1)·min~(-1),两组之间差异有统计学意义(P=0.0367),,慢性HBV感染组和对照组米氏常数(Km)分别为263.5±66.4μmol/L和284.6±85.9μmol/L,两组间差异无统计学意义(P=0.5471),RT-PCR和western blot显示慢性HBV感染组CYP2C9 mRNA和蛋白的表达也较对照组明显下降(0.39±0.28 vs 0.65±0.13,0.26±0.13 vs 0.60±0.19),差异均有统计学意义(P=0.0171,P=0.0002)。结论慢性HBV感染可以在mRNA和蛋白水平降低肝脏CYP2C9酶的表达,导致酶活性下降,慢性HBV感染对CYP2C9酶活性的抑制作用是非竞争性的,对酶的结构未造成影响。
三、乙型肝炎病毒X蛋白对细胞色素P450 2C9诱导的影响
目的探讨慢性HBV感染影响CYP2C9表达调控的机制,为临床合理用药提供理论依据。方法培养HepG2细胞,选用利福平作为诱导剂,以MTT法检测不同浓度的利福平对HepG2细胞的毒性作用,将乙型肝炎病毒X基因(HBX)插入pEGFP-N1中,构建了pEGFP-X真核表达质粒,将pEGFP-X和pEGFP-N1(转染对照)转染HepG2细胞,同时以利福平作为诱导剂,诱导HepG2细胞CYP2C9表达72小时,分别用RT-PCR和western blot方法,在mRNA和蛋白水平观察HBx对利福平诱导CYP2C9表达的影响。结果MTT法检检测提示5μmol/L、10μmol/L、25μmol/L和50μmol/L利福平对于HepG2细胞均无细胞毒性,利福平能很好诱导CYP2C9的表达,pEGFP-N1利福平诱导组和pEGFP-X利福平诱导组mRNA诱导表达量分别是空白细胞对照组的224%和163%,蛋白表达量分别是对照组的300%和245%,pEGFP-N1利福平诱导组和pEGFP-X利福平诱导组mRNA表达量分别为0.387±0.015和0.282±0.032,差异有统计学意义(P＜0.05);两组蛋白表达量分别为1.594±0.149和1.299±0.073,也有统计学差异(P＜0.05)。结论HBX可以干扰利福对HepG2细胞CYP2C9的诱导,提示慢性HBV感染导致的CYP2C9活性降低以及mRNA、蛋白表达的下降可能是由于HBX在人肝细胞中表达,干扰了CYP2C9表达所致。
小结:本课题通过收集慢性HBV感染患者和无HBV感染者的肝组织和血液标本,采用PCR-RFLP方法检测CYP2C9酶的基因多态性,以甲苯磺丁脲为探针底物,用HPLC方法检测两组肝组织样本中CYP2C9的酶活性。以RT-PCR和western印迹法测定了两组肝组织样本中mRNA和蛋白的表达差异,证实了慢性HBV感染患者CYP2C9酶活性较无HBV感染者明显下降,同时探讨了这一现象的机制,发现HBx可以干扰利福平对CYP2C9的诱导,提示HBX对CYP2C9的表达调控有抑制作用,这一发现对于临床患者安全合理用药、规避不良反应提供了一定的指导作用。
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 population
Objective: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 CYP2C9
Objective: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 cell
Objective: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.
肝脏是药物代谢的主要场所,肝脏进行药物代谢依赖于微粒体中的多种酶系,其中最重要的是细胞色素P450酶系。越来越多的证据表明,药物代谢过程所致肝损害与P450酶系密切相关,任何导致P450酶活性变化的因素都可以影响药物在体内的代谢,其中,遗传基因多态性和疾病的状况是公认的非常重要的因素。基因突变可以用来解释人们在服用药物时出现的药效学和药代学的个体差异,例如细胞色素P450亚家族CYP2C9酶,CYP2C9~*2和CYP2C9~*3突变基因能显著降低酶活性。另一个影响酶活性的因素是疾病的状况,对于慢性乙型肝炎患者而言,既往研究表明HBV可以对P450酶表达产生影响,但是HBV对P450酶各主要亚家族酶活力的影响是不一致的,而且先前的工作大部分都是在动物和细胞水平进行的,没有HBV感染影响人肝细胞色素P450表达的研究。
由于CYP2C9在人类肝脏中含量丰富,占总量的20%,仅次于CYP3A,是肝内含量第二的CYP亚家族酶,能代谢大约16%不同性质的临床药物,并在前致癌物、前毒物和致突变剂的活化中起一定作用。既然CYP2C9酶在临床药物代谢中有如此重要的作用,那么慢性HBV感染时,CYP2C9酶的活性是如何变化以及影响酶变化的机制是什么呢?这是我们关注的重点。本实验选取CYP2C9酶作为研究对象,收集临床肝脏标本,检测慢性HBV感染肝组织和无HBV感染的肝组织中CYP2C9酶的活性,并检测该酶在mRNA水平和蛋白水平变化情况,同时在细胞水平对临床研究结果进行机制的探讨,为慢性HBV感染患者安全合理使用药物、规避不良反应提供科学依据。
一、细胞色素P450 2C9基因多态性研究
目的检测CYP2C9基因型,排除CYP2C9~*2和~*3突变等位基因对CYP2C9酶活性的影响,为下一步研究奠定基础。方法收集慢性HBV感染患者和无HBV感染者的肝组织和血液标本20例,以基因组DNA提取试剂盒提取血液基因组DNA,应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术对CYP2C9进行基因分型,并随机抽取数例样本进行TA克隆测序验证。结果提取的血液基因组DNA光密度值在1.8～1.9之间,含量为90ng/μl～230ng/μl,琼脂糖凝胶电泳提示基因组完整,PCR-RFLP分析发现20例均为野生型CYP2C9(~*1~*1),未检测到基因突变,TA克隆测序分析验证了PCR-RFLP分析结果。结论本实验中我们未检测到CYP2C9突变基因,CYP2C9基因多态性的种族差异与地域不均衡性是结果最可能的解释,但是为我们下一步检测疾病对CYP2C9酶活性的影响奠定了基础。
二、慢性乙型肝炎病毒感染对人肝细胞色素P450 2C9的影响
目的探讨慢性HBV感染对人肝脏细胞色素酶2C9表达的影响,为慢性HBV感染患者安全用药提供理论指导。方法慢性HBV感染患者和无HBV感染者的肝组织样本,匀浆后差速离心法制备肝脏微粒体酶混合物(S9),以甲苯磺丁脲为探针,用高效液相色谱(HPLC)的方法检测两组样本CYP2C9酶的活性,同时以RT-PCR和western blot测定两组肝组织样本中mRNA和蛋白表达的差异。结果高效液相色谱显示慢性HBV感染组Vmax为40.4±10.4 pmol·mg~(-1)·min~(-1),而无HBV感染对照组Vmax为52.6±13.4pmol·mg~(-1)·min~(-1),两组之间差异有统计学意义(P=0.0367),,慢性HBV感染组和对照组米氏常数(Km)分别为263.5±66.4μmol/L和284.6±85.9μmol/L,两组间差异无统计学意义(P=0.5471),RT-PCR和western blot显示慢性HBV感染组CYP2C9 mRNA和蛋白的表达也较对照组明显下降(0.39±0.28 vs 0.65±0.13,0.26±0.13 vs 0.60±0.19),差异均有统计学意义(P=0.0171,P=0.0002)。结论慢性HBV感染可以在mRNA和蛋白水平降低肝脏CYP2C9酶的表达,导致酶活性下降,慢性HBV感染对CYP2C9酶活性的抑制作用是非竞争性的,对酶的结构未造成影响。
三、乙型肝炎病毒X蛋白对细胞色素P450 2C9诱导的影响
目的探讨慢性HBV感染影响CYP2C9表达调控的机制,为临床合理用药提供理论依据。方法培养HepG2细胞,选用利福平作为诱导剂,以MTT法检测不同浓度的利福平对HepG2细胞的毒性作用,将乙型肝炎病毒X基因(HBX)插入pEGFP-N1中,构建了pEGFP-X真核表达质粒,将pEGFP-X和pEGFP-N1(转染对照)转染HepG2细胞,同时以利福平作为诱导剂,诱导HepG2细胞CYP2C9表达72小时,分别用RT-PCR和western blot方法,在mRNA和蛋白水平观察HBx对利福平诱导CYP2C9表达的影响。结果MTT法检检测提示5μmol/L、10μmol/L、25μmol/L和50μmol/L利福平对于HepG2细胞均无细胞毒性,利福平能很好诱导CYP2C9的表达,pEGFP-N1利福平诱导组和pEGFP-X利福平诱导组mRNA诱导表达量分别是空白细胞对照组的224%和163%,蛋白表达量分别是对照组的300%和245%,pEGFP-N1利福平诱导组和pEGFP-X利福平诱导组mRNA表达量分别为0.387±0.015和0.282±0.032,差异有统计学意义(P＜0.05);两组蛋白表达量分别为1.594±0.149和1.299±0.073,也有统计学差异(P＜0.05)。结论HBX可以干扰利福对HepG2细胞CYP2C9的诱导,提示慢性HBV感染导致的CYP2C9活性降低以及mRNA、蛋白表达的下降可能是由于HBX在人肝细胞中表达,干扰了CYP2C9表达所致。
小结:本课题通过收集慢性HBV感染患者和无HBV感染者的肝组织和血液标本,采用PCR-RFLP方法检测CYP2C9酶的基因多态性,以甲苯磺丁脲为探针底物,用HPLC方法检测两组肝组织样本中CYP2C9的酶活性。以RT-PCR和western印迹法测定了两组肝组织样本中mRNA和蛋白的表达差异,证实了慢性HBV感染患者CYP2C9酶活性较无HBV感染者明显下降,同时探讨了这一现象的机制,发现HBx可以干扰利福平对CYP2C9的诱导,提示HBX对CYP2C9的表达调控有抑制作用,这一发现对于临床患者安全合理用药、规避不良反应提供了一定的指导作用。
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 population
Objective: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 CYP2C9
Objective: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 cell
Objective: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.
引文
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[3]Peyvandi F,Spreafico M,Siboni SM,et al.CYP2C9 genotypes and dose requirements during the induction phase of oral anticoagulant therapy.Clin Pharmacol Ther,2004,75(3):198-203.
[4]Scordo MG,Caputi AP,Arrigo CD,et al.Allele and genotype frequencies of CYP2C9,CYP2CI9 and CYP2D6 in an Italian population.Pharmacol Res,2004,50:195-200.
[5]励雁峰,吴万垠,张学理,等.HBV转基因小鼠的解毒活力变化的研究.肿瘤,1997,17(4):187-189.
[6]Chemin I,Ohgaki H,Chisari FV,et al.Altered expression of hepatic carcinogen metabolizing enzymes with liver injury in HBV transgenic mouse lineages expressing various amounts of hepatitis B surface antigen.Liver,1999,19(2):81-87.
[7]Chomarat P,Rice JM,Slagle BL,et al.Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes:the role of the HBx protein.Toxicol Lett,1998,28,102-103:595-601.
[8]李爽,缪晓辉,胡卓汉.慢性乙型肝炎病毒感染对人肝细胞色素酶P4503A4的影响.中华医学杂志,2006,10(17):2703-2706.
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[3]Kim JS,Nafziger AN,Gaedigk A,et al .Effects of oral Vitamin K on S-and R-warfarin pharmacokinetics and pharmacodynamics:enhanced safety of warfarin as a CYP2C9 probe..J Clin Pharmacol,2001,41(7):715-722.
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[5]Kirchheiner J,Brockmoller J.Clinical consequences of cytochrome P4502C9 polymorphisms.Clin Pharmacol Ther,2005,77(1):1-16.
[6]Miners JO,Birkett DJ.Cytochrome P450 2C9:an enzyme of major importance in human drug metabolism.Br J Clin Pharmacol,1998,45{6):525-538.
[7]Kirchheiner J,Meineke I,Muller G,et al.Influence of CYP2C9 and CYP2D6 polymorphisms on the pharmacokinetics of nateglinide in genotyped healthy volunteers.Clin Pharmacokinet, 2004,43(4):267-278.
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[11]Xie HG,Prasad HC,Kim RB,et al.CYP2C9 allelic variants: ethnic distribution and functional significance. Adv Drug Deliv Rev,2002,5410():1257-1270.
[12]Peyvandi F,Spreafico M,Siboni SM,et al. CYP2C9 genotypes and dose requirements during the induction phase of oral anticoagulant therapy. Clin Pharmacol Ther,2004,75(3): 198-203.
[13]Scordo MG, Caputi AP, Arrigo CD,et al. Allele and genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an Italian population.Pharmacol Res,2004,50:195-200.
[14]Dickmann LJ,Rettie AE,Kneller MB, et al. Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.Mol Pharmacol, 2001,60(2):382-387.
[15]Kidd RS,Curry TB,Gallagher S, et al. Identification of a null allele of CYP 2C9 in an African-American exhibiting toxicity to phenytoin. Pharmacogenet ics,2001,11(9):803-808.
[16]Wang SL,Huang J,Lai MD,et al.Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.Pharmacogenetics, 1995,5(1):37-42.
[17]Schwarz UI. Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur J Clin Invest, 2003,33(Suppl 2):23-30.
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[21]Imai J, Ieiri I, Mamiya K, Miyahara S,et al.Polymorphism of the cytochrome P450 (CYP) 2C9 gene in Japanese epileptic patients: genetic analysis of the CYP2C9 locus. Pharmacogenetics, 2000,10(1):85-89.
[22]Gaedigk A, Casley WL, Tyndale RF,et al.Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations. Can J Physiol Pharmacol,2001, 79(10):841-847.
[23]King B P, Khan T I, Aithal G P, et al. Up stream and coding region CYP2C9 polymorphisms:correlation with warfarin dose and metabolism.Pharm acogenetics, 2004,14 (12): 813-22.
[24]Ieiri I, Tainaka H, Morita T,et al. Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis. Ther Drug Monit, 2000,22(3):237-244.
[1]励雁峰,吴万垠,张学理.HBV转基因小鼠的解毒活力变化的研究.肿瘤,1997,17(4):187-189.
[2]Chemin I,Ohgaki H,Chisari FV,et al.Altered expression of hepatic carcinogen metabolizing enzymes with liver injury in HBV transgenic mouse lineages expressing various amounts of hepatitis B surface antigen.Liver,1999,19(2):81-87.
[3]Chomarat P,Rice JM,Slagle BL,et al.Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes:the role of the HBx protein.Toxicol Lett,1998,28,102-103:595-601.
[4]李爽,缪晓辉,胡卓汉.慢性乙型肝炎病毒感染对人肝细胞色素酶P4503A4的影响.中华医学杂志,2006,10(17):2703-2706.
[5]Nebert DW,Russell DW.Clinical importance of the cytochromes P450.Lancet,2002,360(9340):1155-1162.
[6]Kirchheiner J,Brockmoller J.Clinical consequences of cytochrome P4502C9polymorphisms.Clin Pharmacol Ther,2005,77(1):1-16.
[7]Lee CR,Goldstein JA,Pieper JA.Cytochrome P4502C9 polymorphisms:a co mprehensive review of the in-vitro and human data.Pharmacogenetics,2002,12(3):251-263.
[8]Lee CR,Pieper JA,Hinderliter AL,et al.Evaluation of cytochrome P4502C9metabolic activity with tolbutamide in CYP2C9 heterozygotes.Clin Pharmacol Ther.2002;72(5):562-271.
[9]Yang JQ,Morin S,Verstuyft C,et al.Frequency of cytochrome P450 2C9 al lelic variants in the Chinese and French populations.Fundam Clin Pharm acol,2003,17(3):373-376.
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[1]Nebert DW,Russell DW.Clinical importance of the cytochromes P450.Lancet,2002,360(9340):1155-1162.
[2]Miners JO,Birkett DJ.Cytochrome P450 2C9:an enzyme of major importance in human drug metabolism.Br J Clin Pharmacol,1998,45(6):525-538.
[3]Kirchheiner J,Meineke I,Muller G,et al.Influence of CYP2C9 and CYP2D6polymorphisms on the pharmacokinetics of nateglinide in genotyped healthy volunteers.Clin Pharmacokinet,2004,43(4):267-278.
[4]Vermeij P,Ferrari MD,Buruma OJ,et al.Inheritance of poor phenytoin para -hydroxylation capacity in an Dutch family.Clin Pharmacol Ther,1988,44(5):588-593.
[5]Sullivan-Klose TH,Ghanayem BI,Bell DA,et al.The role of the CYP2C9Leu359 allelic variant in the tolbutamide polymorphism.Pharmacogenetics,1996,6(4):341-349.
[6]Lee CR,Goldstein JA,Pieper JA.Cytochrome P450 2C9 polymorphisms:a comprehensive review of the in-vitro and human data.Pharmacogenetics,20 02,12(3):251-263.
[7]周宏灏主编.《遗传药理学》.北京,科学出版社,2001.
[8]Brandolese R,Scordo MG,Spina E,et al.Severe phenytoin intoxication in a subject homozygous for CYP2C9*3.Clin Pharmacol Ther,2001,70(4):391-394.
[9]Kim JS,Nafziger AN,Gaedigk A,et al.Effects of oral Vitamin K on S-and R-warfarin pharmacokinetics and pharmacodynamics:enhanced safety of warfarin as a CYP2C9 probe.J Clin Pharmacol,2001,41(7):715-722.
[10]Daly AK,King BP.Pharmacogenetics of oral anticoagulants.Pharmacogene tics,2003,13(5):247-252.
[11]Kirchheiner J,Brockmoller J.Clinical consequences of cytochrome P4502C9polymorphisms.Clin Pharmacol Ther,2005,77(1):1-16.
[12]Yang JQ,Morin S,Verstuyft C,et al.Frequency of cytochrome P450 2C9 allelic variants in the Chinese and French populations.Fundam Clin Pharmacol,2003,17(3):373-376.
[13]Xie HG,Prasad HC,Kim RB,et al.CYP2C9 allelic variants:ethnic distribution and functional significance.Adv Drug Deliv Rev,2002,5410():1257-1270.
[14]King BP,Khan TI,Aithal GP,et al.Up stream and coding region CYP2C9polymorphisms:correlation with warfarin dose and metabolism.Pharm acogenetics,2004,14(12):813-22.
[15]Wang SL,Huang J,Lai MD,et al.Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.Pharmacogenetics,1995,5(1):37-42.
[16]Ieiri I,Tainaka H,Morita T,et al.Catalytic activity of three variants(Ile,Leu,and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis.Ther Drug Monit,2000,22(3):237-244.
[17]Imai J,Ieiri I,Mamiya K,Miyahara S,et al.Polymorphism of the cytochrome P450 (CYP) 2C9 gene in Japanese epileptic patients: genetic analysis of the CYP2C9 locus.Pharmacogenetics, 2000,10(1):85-89.
[18]Gaedigk A, Casley WL, Tyndale RF,et al.Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations. Can J Physiol Pharmacol,2001, 79(10):841-847.
[19]Dickmann LJ, Rettie AE, Kneller MB,et al. Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.Mol Pharmacol,2001, 60(2):382-387.
[20]Schwarz UI.Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur J Clin Invest,2003,33(Suppl 2):23-30.
[21]Kidd RS,Curry TB,Gallagher S,et al.Identification of a null allele of CY P2C9 in an African-American exhibiting toxicity to phenytoin.Pharmaco genetics, 2001,11(9):803-808.
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[26]Gerbal-Chaloin S,Daujat M,Pascussi JM, el al.Transcriptional regulation of CYP2C9 gene.Role of glucocorticoid receptor and constitutive androstane receptor. J Biol Chem, 2002,277(1):209-217.
[27]Zhou HH,Anthony LB,Wood AJ,et al.Induction of polymorphic 4'-hydrox ylation of S-mephenytoin by rifampicin.Br J Clin Pharmacol, 1990,30(3): 471-475.
[28]Morel F,Beaune PH,Ratanasavanh D,et al.Expression of cytochrome P-450 enzymes in cultured human hepatocytes.Eur J Biochem,1990,191(2):437-444.
[29]Chang TK,Yu L,Maurel P,et al.Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines.Cancer Res,1997,57 (10):1946-1954.
[30]Gerbal-Chaloin S,Pascussi JM,Pichard-Garcia L,et al.Induction of CYP2C genes in human hepatocytes in primary culture.Drug Metab Dispos,200 1,29(3):242-251.
[31]Chen Y,Ferguson SS,Negishi M,et al.Induction of human CYP2C9 by rifampicin,hyperforin,and phenobarbital is mediated by the pregnane Xreceptor(PXR).J Pharmacol Exp Ther,2004,308(2):495-501.
[32]Bort R, Gomez-Lechon MJ, Castell JV,et al. Role of hepatocyte nuclear factor 3 gamma in the expression of human CYP2C genes. Arch Biochem Biophys, 2004,426(1):63-72.
[2]Xie HG,Prasad HC,Kim RB,et al.CYP2C9 allelic variants:ethnic distribution and functional significance.Adv Drug Deliv Rev,2002,54100:1257-1270.
[3]Peyvandi F,Spreafico M,Siboni SM,et al.CYP2C9 genotypes and dose requirements during the induction phase of oral anticoagulant therapy.Clin Pharmacol Ther,2004,75(3):198-203.
[4]Scordo MG,Caputi AP,Arrigo CD,et al.Allele and genotype frequencies of CYP2C9,CYP2CI9 and CYP2D6 in an Italian population.Pharmacol Res,2004,50:195-200.
[5]励雁峰,吴万垠,张学理,等.HBV转基因小鼠的解毒活力变化的研究.肿瘤,1997,17(4):187-189.
[6]Chemin I,Ohgaki H,Chisari FV,et al.Altered expression of hepatic carcinogen metabolizing enzymes with liver injury in HBV transgenic mouse lineages expressing various amounts of hepatitis B surface antigen.Liver,1999,19(2):81-87.
[7]Chomarat P,Rice JM,Slagle BL,et al.Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes:the role of the HBx protein.Toxicol Lett,1998,28,102-103:595-601.
[8]李爽,缪晓辉,胡卓汉.慢性乙型肝炎病毒感染对人肝细胞色素酶P4503A4的影响.中华医学杂志,2006,10(17):2703-2706.
[1]Nebert DW,Russell DW.Clinical importance of the cytochromes ?450.Lancet, 2002,360(9340): 1155-1162.
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[1]Nebert DW,Russell DW.Clinical importance of the cytochromes P450.Lancet,2002,360(9340):1155-1162.
[2]Miners JO,Birkett DJ.Cytochrome P450 2C9:an enzyme of major importance in human drug metabolism.Br J Clin Pharmacol,1998,45(6):525-538.
[3]Kirchheiner J,Meineke I,Muller G,et al.Influence of CYP2C9 and CYP2D6polymorphisms on the pharmacokinetics of nateglinide in genotyped healthy volunteers.Clin Pharmacokinet,2004,43(4):267-278.
[4]Vermeij P,Ferrari MD,Buruma OJ,et al.Inheritance of poor phenytoin para -hydroxylation capacity in an Dutch family.Clin Pharmacol Ther,1988,44(5):588-593.
[5]Sullivan-Klose TH,Ghanayem BI,Bell DA,et al.The role of the CYP2C9Leu359 allelic variant in the tolbutamide polymorphism.Pharmacogenetics,1996,6(4):341-349.
[6]Lee CR,Goldstein JA,Pieper JA.Cytochrome P450 2C9 polymorphisms:a comprehensive review of the in-vitro and human data.Pharmacogenetics,20 02,12(3):251-263.
[7]周宏灏主编.《遗传药理学》.北京,科学出版社,2001.
[8]Brandolese R,Scordo MG,Spina E,et al.Severe phenytoin intoxication in a subject homozygous for CYP2C9*3.Clin Pharmacol Ther,2001,70(4):391-394.
[9]Kim JS,Nafziger AN,Gaedigk A,et al.Effects of oral Vitamin K on S-and R-warfarin pharmacokinetics and pharmacodynamics:enhanced safety of warfarin as a CYP2C9 probe.J Clin Pharmacol,2001,41(7):715-722.
[10]Daly AK,King BP.Pharmacogenetics of oral anticoagulants.Pharmacogene tics,2003,13(5):247-252.
[11]Kirchheiner J,Brockmoller J.Clinical consequences of cytochrome P4502C9polymorphisms.Clin Pharmacol Ther,2005,77(1):1-16.
[12]Yang JQ,Morin S,Verstuyft C,et al.Frequency of cytochrome P450 2C9 allelic variants in the Chinese and French populations.Fundam Clin Pharmacol,2003,17(3):373-376.
[13]Xie HG,Prasad HC,Kim RB,et al.CYP2C9 allelic variants:ethnic distribution and functional significance.Adv Drug Deliv Rev,2002,5410():1257-1270.
[14]King BP,Khan TI,Aithal GP,et al.Up stream and coding region CYP2C9polymorphisms:correlation with warfarin dose and metabolism.Pharm acogenetics,2004,14(12):813-22.
[15]Wang SL,Huang J,Lai MD,et al.Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.Pharmacogenetics,1995,5(1):37-42.
[16]Ieiri I,Tainaka H,Morita T,et al.Catalytic activity of three variants(Ile,Leu,and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis.Ther Drug Monit,2000,22(3):237-244.
[17]Imai J,Ieiri I,Mamiya K,Miyahara S,et al.Polymorphism of the cytochrome P450 (CYP) 2C9 gene in Japanese epileptic patients: genetic analysis of the CYP2C9 locus.Pharmacogenetics, 2000,10(1):85-89.
[18]Gaedigk A, Casley WL, Tyndale RF,et al.Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations. Can J Physiol Pharmacol,2001, 79(10):841-847.
[19]Dickmann LJ, Rettie AE, Kneller MB,et al. Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.Mol Pharmacol,2001, 60(2):382-387.
[20]Schwarz UI.Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur J Clin Invest,2003,33(Suppl 2):23-30.
[21]Kidd RS,Curry TB,Gallagher S,et al.Identification of a null allele of CY P2C9 in an African-American exhibiting toxicity to phenytoin.Pharmaco genetics, 2001,11(9):803-808.
[22]de Morais SM,Schweikl H,Blaisdell J,et al.Gene structure and upstream regulatory regions of human CYP2C9 and CYP2C18.Biochem Biophys Res Commun,1993,194(1):194-201.
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[26]Gerbal-Chaloin S,Daujat M,Pascussi JM, el al.Transcriptional regulation of CYP2C9 gene.Role of glucocorticoid receptor and constitutive androstane receptor. J Biol Chem, 2002,277(1):209-217.
[27]Zhou HH,Anthony LB,Wood AJ,et al.Induction of polymorphic 4'-hydrox ylation of S-mephenytoin by rifampicin.Br J Clin Pharmacol, 1990,30(3): 471-475.
[28]Morel F,Beaune PH,Ratanasavanh D,et al.Expression of cytochrome P-450 enzymes in cultured human hepatocytes.Eur J Biochem,1990,191(2):437-444.
[29]Chang TK,Yu L,Maurel P,et al.Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines.Cancer Res,1997,57 (10):1946-1954.
[30]Gerbal-Chaloin S,Pascussi JM,Pichard-Garcia L,et al.Induction of CYP2C genes in human hepatocytes in primary culture.Drug Metab Dispos,200 1,29(3):242-251.
[31]Chen Y,Ferguson SS,Negishi M,et al.Induction of human CYP2C9 by rifampicin,hyperforin,and phenobarbital is mediated by the pregnane Xreceptor(PXR).J Pharmacol Exp Ther,2004,308(2):495-501.
[32]Bort R, Gomez-Lechon MJ, Castell JV,et al. Role of hepatocyte nuclear factor 3 gamma in the expression of human CYP2C genes. Arch Biochem Biophys, 2004,426(1):63-72.
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