【作者】 高洁；

【导师】 乔海灵；

【作者基本信息】 郑州大学 ， 分子药理学， 2018， 博士

【摘要】 肝脏细胞色素P450氧化酶(CYP450)参与70%～80%临床药物的代谢。CYP450活性的个体差异是导致药物代谢、药物效应及毒性存在较大个体差异的主要原因。肝细胞癌(HCC)患者生理、生化(肝脏功能、肝血流量、具有功能的肝脏大小、血浆蛋白结合率等)的改变会影响CYP450的活性,进而导致药物清除率发生变化。因此,确定正常人及HCC患者CYP450的活性、个体差异及影响因素是个体化用药的重要前提。HCC是我国常见的恶性肿瘤之一,死亡率位居恶性肿瘤前列,发现时大多在中晚期,至今尚缺乏有效的治疗方案。亚硝胺类化合物是目前公认的前致癌物,经CYP2E1代谢活化生成致癌物而引发肿瘤。食物中亚硝胺类的含量与HCC的发生密切相关,但同样食用高亚硝胺含量的食物,有人发展成HCC,有人却很安全。推测CYP2E1代谢活性存在较大个体差异是HCC易感性表现出显著个体差异的原因。本研究以正常肝标本为研究对象,探究人肝10种CYP450生理活性的相关性及个体内差异;以CYP2D6为例,阐明其不同水平活性表型间的关系及基因型对不同水平活性表型的影响。以HCC患者非肿瘤肝组织为研究对象,分析10种CYP450不同水平的活性及影响因素;测定CYP2E1代谢二乙基亚硝胺的活性,考察CYP2E1活性、临床指标对HCC切除术患者生存期的影响。以DEN诱导的大鼠肝癌模型为研究对象,探究CYP2E1先天活性及CYP2E1抑制活性与HCC的相关性。通过上述研究,以求为正常人及HCC患者个体化用药提供更精准的指导;以期发现HCC防治新靶点,为HCC的早防早治提供新的策略。1方法1.1人肝标本收集105例肝功能正常人肝标本及102例HCC患者非肿瘤肝标本。研究方案经郑州大学生命伦理审查委员会审查通过,肝标本供体签署知情同意书。1.2肝微粒体蛋白浓度及含量测定低温差速离心法制备人肝微粒体(HLMs)。Bradford法测定HLMs蛋白浓度。肝微粒体蛋白含量(MPPGL)通过测定肝组织匀浆及HLMs中细胞色素P450氧化还原酶(POR)的活性确定。1.3 CYP450活性测定孵育体系包含HLMs、不同浓度的探针药物、磷酸盐缓冲液及NADPH。高效液相色谱法测定 10 种 CYP450(CYP1A2、CYP2A6、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4/5)代谢产物的生成量。通过非线性回归分析方法(GraphPad Prism 5.04)计算CYP450的肝微粒体水平清除率(CLM)。采用体外-体内外推法推算CYP450其他水平的清除率(肝组织水平,CLLT;肝脏水平,CLL;整体水平,CLH)。1.4 CYP450CLM的相关性采用Pearson相关分析2种CYP450CLM间的相关性。采用层次聚类分析10种CYP450 CLM间的相关性。采用多重线性回归分析1种CYP450 CLM与其余CYP450 CLM之间的数量依存关系。1.5 CYP2D6蛋白含量测定将稳定同位素标记的CYP2D6特异性肽段整合在一个QconCAT蛋白序列中,采用稳定同位素稀释内标-多反应监测-质谱技术(SID-MRM-MS)对其定量。1.6 CYP450探针药物的人体药代动力学数据收集在PubMed数据库中检索自1975年至2016年关于CYP450探针药物的人体药代动力学的文献。1.7 CYP450基因多态性测定通过SNP MassARRAY方法检测中国人群的10种CYP450发生频率大于1%的基因多态性位点。1.8 HCC患者术后随访采用电话随访为主、登门随访为辅的方式进行随访。自切除术后,每隔6个月随访一次,随访历时42～54个月。1.9大鼠肝癌模型建立实验开始前4周,每周给予大鼠腹腔注射两次二乙基亚硝胺(DEN,50 mg/kg),第5到14周每周给予大鼠腹腔注射一次DEN(50 mg/kg)。喂养到第19周处死全部大鼠,记录并测量肝脏表面结节(≥ 5 mm)个数、大小。留取部分肝脏标本固定于福尔马林溶液中,剩余肝标本冻存于液氮中备用。1.10大鼠CYP2E1先天活性及抑制活性测定CYP2E1先天活性系指肝癌模型建立前(第0周)健康大鼠CYP2E1代谢DEN的活性;CYP2E1抑制活性系指肝癌模型建立前(第0周)健康大鼠腹腔注射CYP2E1抑制剂氯美噻唑(CMZ)后CYP2E1代谢DEN的活性。测定大鼠腹腔注射DEN(50 mg/kg)的毒代动力学,确定大鼠CYP2E1的先天活性;腹腔注射CMZ后立即腹腔注射DEN(50 mg/kg),测定大鼠DEN的毒代动力学,确定大鼠CYP2E1的抑制活性。毒代动力学参数采用药物和统计学DAS 3.2.6软件(中国药理学会数学药理专业委员会,上海)处理。1.11组织病理及免疫组化分析分别用HE和Masson对切片染色,根据Ishak评分系统对HE染色的病理结果进行评分。通过免疫组化方法,分别用Anti-Ki67抗体及Anti-PCNA抗体检测肝组织中Ki67+细胞数及PCNA+细胞数。1.12统计学分析采用 SPSS17.0 software package(SPSS Inc.,Chicago,IL,USA)进行统计分析,P<0.05认为存在统计学意义。采用Adobe Photoshop CC 2014(Adobe,San Jose,CA,USA)和 GraphPad Prism 5.04 software package(GraphPad Inc.,La Jolla,CA,U.S.A.)进行作图。2 结果2.1人肝10种CYP450生理活性2.1.1 CYP450 CLM 的相关性两种CYP450 CYP1A2及CYP2B6分别与4种CYP450存在相关性;CYP2C8、CYP2C9 及 CYP2E1 分别与 3 种 CYP450 存在相关性;CYP2C19 及CYP2D6分别与2种CYP450存在相关性;CYP3A4/5仅与CYP2C19存在相关性;CYP2A6与其他CYP450均不存在相关性。其中CYP2C8与CYP2C9的相关性最好(r = 0.485,P=1.62E-07),其次为 CYP1A2 与 CYP2C9(r = 0.400,P= 2.39E-05)。多种CYP450若将10种CYP450聚成2类,则第一类包括:CYP2C8、CYP2C9、CYP1A2 和 CYP2B6,第二类包括:CYP2A6、CYP2D6、CYP2C19、CYP2E1和CYP3A4/5;若将10种CYP450聚成4类,则第一类包括:CYP2C8和CYP2C9,第二类包括:CYP1A2和CYP2B6,第三类包括:CYP2A6和CYP2D6,第四类包括:CYP2C19、CYP2E1 和 CYP3A4/5。根据 10 种 CYP450 CLm 间存在的数量依存关系,可以通过CYP1A2、CYP2B6、CYP2C9和CYP2C19的CLm推算出 CYP2A6、CYP2C8、CYP2D6、CYP2E1 和 CYP3A4/5 的 CLM。2.1.2 CYP450CLM的个体内差异个体内差异10种CYP450的CLM存在较大的个体内差异(变异系数,ICV),不同个体的ICV不同。其中ICV较大者(ICV>100%)5例,占比4.8%;ICV 中等者(50%<ICV≤100%)55 例,占比 52.8%;ICV 较小者(ICV<50%)45例,占比42.9%。影响因素性别、饮酒对个体内差异无显著影响;年龄通过影响CYP3A4/5的CLM而影响个体内差异;吸烟通过影响CYP1A2、CYP2C9和CYP2D6的CLM而影响个体内差异;CYP2A6*4、CYP2B6 15631G>T、CYB618053A>G、CYP2C942614A>C、CYP2D6 100C>T、1661G>C 和CYP2D6 2850G>A 通过影响其CLM而影响个体内差异。2.1.3 CYP2D6基因型及其活性表型基因型正常人CYP2D6 100CC、CYP2D6 100CT和CYP2D6 100TT的基因频率分别为 31.1%、22.2%和 46.7%。CYP2D6 1661GG、CYP2D6 1661GC和CYP2D6 1661CC 的基因频率分别为 50.6%、37.1%和 12.3%。基因型对不同水平活性表型的影响不同水平的活性表型系指分子活性表型(CLP)、亚细胞活性表型(CLM)、组织活性表型(CLLT)、器官活性表型(CLL)、整体活性表型(CLH)。一般,CYP2D6 100C>T突变导致各个水平的活性表型降低,而CYP2D61661G>C突变导致各个水平的活性表型升高。与100CC个体相比,100TT个体的CLP降低30.0%。与1661GG个体相比,1661GC个体的CLP增加2.00倍,而1661CC个体的CLP增加1.66倍。与100CC个体相比,100CT个体的CLM、CLLT、CLL均降低约20.0%,100TT个体的CLM、CLLLT、CLL均降低约70.0%。与1661GG个体相比,1661GC个体的CLM、CLLLT、CLL均增加约3.15 倍,而1661CC 个体的 CLm、CLLT和 CLL 分别增加 3.10、3.75 和 4.17 倍。与100CC个体相比,100CT个体的CLH降低18.6%,100TT个体的CLH降低70.4%。与166GG个体相比,1661GC个体的CLH增加3.28倍,而1661CC个体的CLH增加3.69倍。2.2 HCC患者10种CYP450不同水平的清除率2.2.1清除率改变与对照组相比,3种CYP450(CYP2C9、CYP2D6及CYP2E1)的CLM显著增高,3 种 CYP450(CYP1A2、CYP2C8 及 CYP2C19)的 CLM 显著降低,而 4种 CYP450(CYP2A6、CYP2B6 及 CYP3A4/5)的 CLm 基本不变。10 种 CYP450 CLLT、CLL及CLH的变化趋势一致,但变化率不同。仅CYP2E1的CLLT、CLL及 CLH 显著增高,7 种 CYP450(CYP1A2、CYP2A6、CYP2B6、CYP2C8、CYP2C19及 CYP3 A4/5)的 CLLT、CLL 及 CLH 显著降低,2 种 CYP450(CYP2C9 及 CYP2D6)的CLLLT、CLL及CLH基本不变。2.2.2影响因素肝微粒体水平女性CYP2A6的CLM显著高于男性;年龄对CLM无显著影响;吸烟者及饮酒者CYP2D6的CLM均显著降低;CYP1A2-163C>A、CYP2C942614A>C、CYP2D6100C>T、CYP2D61846G>A 及 CYP3A5 6986A>G 突变使其CLm显著降低。肝组织水平性别及年龄对CLLT无显著影响;吸烟者及饮酒者CYP2D6的CLLT均显著降低;CYP2C9 42614A>C、CYP2D6 100>T、CYP2T6 1846G>A、CYP2E及-333T>A及CYP3A5 698>G突变使其 CLLT 显著降低。肝脏水平性别及年龄对CLL无显著影响;吸烟者及饮酒者CYP2D6的CLL均显著降低CYP2C9 42614A>C、CYP2D6 100C>T、CYP2D6 1846G>A、CYP2E1-333T>A及CYP3A5 6986A>G突变使其CLL显著降低。整体水平性别及年龄对CLH无显著影响;吸烟者及饮酒者CYP2D6的CLL均显著降低;CYP2C942614A>C、CYP2D6100C>T、CYP2D61846G>A、CYP2E1-333T>A及CYP3A56986A>G突变使其CLH显著降低。2.3 CYP2E1与HCC相关性2.3.1 CYP2E1 代谢 DEN 活性与 HCC活性改变以CYP2E1代谢DEN的转化率(VDEN)表示CYP2E1代谢DEN的活性。HCC患者VDEN显著升高,增加了 109.4%。采用百分位数法(单侧,95%)确定正常参考值。与正常参考值相比,30(42.8%)例HCC患者的VDEN为阳性。影响因素性别、年龄及临床病理特征[甲胎蛋白(AFP)、肝硬化、最大肿瘤直径、病灶个数、肿瘤分级及门静脉癌栓]对VDEN无显著影响;CYP2E1-333T>A基因多态性位点显著影响其VDEN,-333AA个体较-333TA个体的VDEN显著升高。2.3.2 CYP2E1活性与生存期总体生存时间死亡患者平均生存时间为397天,95%置信区间为319～475天;中位生存时间为306天,95%置信区间为237～365天。影响生存期的单因素性别、年龄、AFP(20ng/mL)、肝硬化、最大肿瘤直径、病灶个数、肿瘤分级、门静脉癌栓及CYP2E1基因多态性位点均对生存时间无显著影响;VDEN阴性者较VDEN阳性者生存期显著延长,延长了 114.4%;AFP≤600ng/mL者较>600ng/mL者生存期显著延长,延长了 69.8%。影响生存期的独立危险因素VDEN和最大肿瘤直径是影响HCC患者生存的独立风险因素。HCC患者的VDEN越高、肿瘤越大,其生存期越短。2.3.3 CYP2E1先天活性与DEN所致大鼠肝癌的相关性对肝癌发生率的影响低半衰期(t1/2)组与高t1/2组的肝癌发生率无显著差异(77.8%vs52.6%);低时间曲线下面积(AUC0-t)组较高AUC0-t组的肝癌发生率显著升高(83.3%vs47.4%);高表观清除率(CL/F)组较低CL/F组的肝癌发生率显著升高(84.2%vs 44.4%)。对肝癌严重程度的影响具有较短t1/2大鼠的最大肿瘤直径及累积肿瘤直径比具有较长t1/2大鼠的显著增大,且t1/2与上述指标均呈负相关;具有较低AUC0-t大鼠的最大肿瘤直径、累积肿瘤直径、最大肿瘤体积及累积肿瘤体积比具有较高AUC0-t大鼠的显著增大,且AUC0-t与上述指标均呈负相关;具有较高CL/F大鼠的结节个数、最大肿瘤直径、累积肿瘤直径、最大肿瘤体积及累积肿瘤体积比具有较低CL/F大鼠的显著增多(增大),且CL/F与上述指标均呈正相关。2.3.4 CYP2E1抑制活性与DEN所致大鼠肝癌的相关性对肝癌发生率的影响模型组、CMZ低剂量组和CMZ高剂量组肝癌发生率分别为64.9%、58.0%和10.0%,CMZ高剂量组的肝癌发生率显著低于模型组。对肝癌严重程度的影响CMZ高剂量组的结节个数、最大肿瘤直径、累积肿瘤直径、最大肿瘤体积、累积肿瘤体积、Ki67+细胞数和PCNA+细胞数均较模型组显著降低。结论1.通过CYP1A2、CYP2B6、CYP2C9和CYP2C19的肝微粒体水平清除率可推算出 CYP2A6、CYP2C8、CYP2D6、CYP2E1 和 CYP3A4/5 的清除率。2.正常人肝微粒体10种CYP450的清除率存在较大的个体内差异。年龄、吸烟、CYP2A6*4、CYP2B615631G>T、CYP2B618053A>G、CYP2C942614A>C、CYP2D6100C>T、CYP2D61661G>C、CYP2D62850G>A可显著影响个体内差异。3.CYP2D6特定基因型显著影响其在不同水平(分子、亚细胞、组织、器官、整体)的活性表型,且影响程度不同。4.肝癌患者不同水平的CYP2E1清除率均较正常人显著升高,且肝癌患者CYP2E1活性与生存期呈负相关。5.大鼠CYP2E1先天活性与二乙基亚硝胺诱发的肝癌呈因果关系,CYP2E1非特异性抑制剂可降低肝癌发生率。更多还原

【Abstract】 CYP450 located in liver play an important role in metabolism of 70%～80%of all drugs used in clinical.Individual variation in the activities of CYP450 is the main reason for individual variation in metabolism,differences in the response to and toxicity of drugs.HCC often results in biochemical and physiological changes,such as altered hepatic function,hepatic blood flow,functional liver size,and plasma protein binding.These changes can result in changed clearance compared with what is observed in subjects with normal hepatic function.Therefore,determining the CYP450 activities and its individual variation for normal subjects and HCC patients is an important prerequisite for personalized medicine.HCC is one of the most common types of primary liver cancer.The mortality rate of HCC is in the forefront of malignant tumors.Early stage HCC is difficult to detect and there is lack of efficient therapeutic methods in clinic,therefore,it is necessary to search for additional novel therapeutic targets for HCC.N-alkylnitrosamines can induce various cancers in animals and maybe even in humans after metabolic activation mediated by CYP2E1.Although diet contributes greatly to nitrosamines exposure,only a handful of people will develop HCC.Given there exist individual variation in CYP2E1 activity,the discrepancy between exposure and induction of cancer may be associated with CYP2E1 activity.In this study,using the normal human liver samples,the correlation and intra-individual variation of 10 CYP450 activities were explored;taking CYP2D6 as an example,the relationship between different phenotypes and the effects of genotypes on different phenotypes were clarified.Using the non-tumor liver samples from HCC patients,the activities of 10 CYP450 activities at different levels and its influencing factor were analyzed;the CYP2E1-mediated metabolic activities for diethylnitrosamine was determined,and the impact of CYP2E1 activity and clinical characteristics on survival time were investigated.Using the rat liver tumor model induced by diethylnitrosamine,the correlation between CYP2E1 innate activity and hepatocarcinogenesis was examined,the effect of inhibition of CYP2E1 activity on hepatocarcinogenesis was investigated.Through the above research,we hope to provide more precise guidance for clinical individualized medication in normal subjects and HCC patients;we hope to find new targets for HCC prevention and treatment,which might very helpful for early prevention and early treatment of HCC.1 Methods1.1 Human liver samplesWe collected 105 human liver samples with normal function and 102 non-tumor liver samples of HCC patients.The Medical Ethics Committee of Zhengzhou University,Zhengzhou,China approved the study protocol,and patients provided written informed consent for the use of all surgically-removed tissue specimens used in this study.1.2 Concentration of HLMs and MPPGLHLMs were prepared by differential centrifugation.Concentration of HLMs was measured by the Bradford method.MPPGL was estimated using the POR activity in homogenates and HLMs.1.3 Activity of CYP450The incubation mixtures contained HLMs,probe substrate,phosphate buffer,and NADPH.After optimal incubation times,reaction was terminated by adding terminating reagent.The amount of production of metabolites was measured by high performance liquid chromatography.The CLM was determined by nonlinear regression analysis using GraphPad Prism 5(GraphPad Inc.,La Jolla,CA,U.S.A.),The clearance of CYP450 at other different levels(CLLT,CLL,and CLH)was calculated using in vitro-in vivo extrapolation methods.1.4 Correlation between CLM of CYP450Pearson correlation analysis was performed to determine the correlation between the CLM of 2 CYP450.Hierarchical cluster analysis was performed to analyze the correlation among the CLM of 10 CYP450.Multiple liner regression was used to explore the quantitative dependency relationship between the CLM of one CYP450 and the CLM of other 9 CYP450.1.5 Content of CYP2D6Stable isotope-labeled peptides of CYP2D6 was integrated in a QconCAT protein.SID-MRM-MS was used to determine the content of CYP2D6.1.6 Collection of in vivo dataAPUBMED search for articles published from 1975 to 2016 was performed to collect information on the pharmacokinetics of probe drugs for CYP450 in humans.1.7 Genotypes of CYP450Polymorphisms in 10 CYP450 with frequencies of more than 1%in the Chinese sample set were determined using SNP MassARRAY.1.8 Follow-up of HCC patientsTelephone follow-up was mainly used.After surgical resection,every 6 months was followed up,and the follow-up time was continued for 42～54 months.1.9 The rat liver tumor modelSix-week-old male rats were injected intraperitoneally with DEN(50 mg/kg)twice a week in the first four weeks and once a week over the next ten weeks.At the 19th week,the animals were killed and liver nodules(≥ 5 mm)were counted and measured,and the liver was sectioned and fixed in 10%formalin.The remaining portions of the liver were instantly frozen and stored in liquid nitrogen until use.1.10 Constitutive and inhibited activities of CYP2E1The CYP2E1 constitutive activity of each rat was determined by measuring toxicokinetic parameters of DEN(50 mg/kg)which was administered by intraperitoneal injection to rats at week 0.The CYP2E1 inhibited activity in the presence of CMZ was determined by measuring the toxicokinetic parameters of DEN metabolism(50 mg/kg)which was injected intraperitoneally immediately after CMZ to rats at week 0.The Drug and Statistics DAS 3.2.6(DAS 3.2.6,Mathematical Pharmacology Professional Committee of China,Shanghai,China)was used to calculate toxicokinetics parameters using a one-compartment model.1.11 Histology and immunohistochemistryFormalin-fixed samples were embedded in paraffin,cut into 5 μm-thick sections and stained with hematoxylin and eosin and Masson’s trichrome according to standard procedures.Trichrome-stained sections were analyzed to score fibrosis staging according to the method of Ishak.Additional sections were stained with antibodies specific for Ki67 and PCNA.After staining for Ki67 and PCNA,positive cells were morphometrically quantified with image processing software.1.12 Statistical analysis and toxicokinetics parametersStatistical analysis was performed using SPSS 17.0 software(SPSS Inc.,Chicago,IL,USA).P<0.05 was considered statistically significant.All graphs were generated using the Adobe Photoshop CC 2014(Adobe,San Jose,CA,USA)and GraphPad Prism 5.04 software package(GraphPad Inc.,La Jolla,CA,U.S.A.).2 Results2.1 Physiological activities of 10 CYP4502.1.1 Correlation of CYP450 CLMTwo CYP450 CYP1A2 and CYP2B6 were correlated with 4 CYP450,respectively;CYP2C8,CYP2C9,and CYP2E1 were correlated with 3 CYP450,respectively;CYP2C19 and CYP2D6 were correlated with 2 CYP450,respectively;CYP3 A4/5 was only correlated with CYP2C19;CYP2A6 was not correlated with other 9 CYP450.Multiple CYP450 Adopting hierarchical cluster analysis for the values of relative CLM,the 10 CYPs were divided into two groups;the first group included CYP2C8,CYP2C9,CYP1A2,and CYP2B6,the second included CYP2A6,CYP2D6,CYP2C19,CYP2E1,and CYP3A4/5.There was the highest correlation and similarity between CYP2C8 and CYP2C9.High correlation and similarity also held in CYP2C19 and CYP2E1.CYP2A6 showed the weakest correlation and similarity to CYP2C8.Measuring the values of CLM for CYP1A2,CYP2B6,CYP2C9,and CYP2C19 allowed us to accurately predict the values of CLM for CYP2A6,CYP2C8,CYP2D6,CYP2E1,and CYP3A4/5.2.1.2 Intra-individual variation of CYP450 CLmIntra-individual variation There exist huge intra-individual variation in the 10 CYP450 CLM.In the 105 samples,the number(percentage)of high,moderate and low ICV for the values of CLM was 5(4.76%),55(52.83)and 45(42.86%).Impact factors Gender and drinking had no effect on intra-individual variation;age had effect on intra-individual variation by influencing CLM of CYP3A4/5;smoking had effect on intra-individual variation by influencing the values of CLM for CYP1A2,CYP2C9 and CYP2D6;CYP2A6*4,CYP2B6 15631G>T,CYP2B6 18053A>G,CYP2C9 42614A>C,CYP2D6 100C>T,CYP2D6 1661G>C,and CYP2D6 2850G>A had effect on intra-individual variation by influencing its CLM.2.1.3 Genotype and phenotype of CYP2D6Genotype The frequency of CYP2D6 100CC,CYP2D6 100CT,and CYP2D6100TT was 31.1%,22.2%,and 46.7%,respectively.The frequency of CYP2D61661GG,CYP2D6 1661GC,and CYP2D6 1661CC was 50.6%,37.1%,and 12.3%,respectively.Impact of genotype on phenotypes at different levels Generally speaking,the CYP2D6 100C>Tsubstitution led to decreased clearance,and the CYP2D6 1661G>C substitution led to increased clearance at each level.Compared with 100CC,the changes in CLp for 100TT showed an approximate decrease of 30.0%.Compared with 1661GG,the CLp of 1661GC increased to about 2-fold,while the 1661CC increased to 1.66-fold.For CLM,CLLT,and CLL,changes were basically the same for 100CT,100TT,and 1661 GC individuals,demonstrating a change of 0.80,0.30,and 3.15-fold,respectively.The changes in CLM(3.10-fold),CLLT(3.75-fold),and CLL(4.17-fold)for 1661CC were different.The CLH of 100CT and 100TT were reduced by 18.6%and 70.4%,respectively.The CLH of 1661GC and 1661CC increased by 3.28-and 3.69-fold,respectively.2.2 Clearance of 10 CYP450 at different levels in HCC patlients2.2.1 Changes of clearanceAt each level,changes in the clearance values for CYP450 differed.Notably,at each level(microsome,liver tissue,liver,and in vivo),CYP2E1 clearance was significantly increased,while the clearance values for CYP1A2,CYP2C8,and CYP2C19 were significantly reduced.The clearance values for CYP2A6,CYP2B6,and CYP3A4/5 showed no change at the microsomal level,although the clearance values at the other three levels were significantly reduced.The clearance values for CYP2C9 and CYP2D6 at the microsomal level were markedly increased,while those at the other three levels showed no change.2.2.2 Impact factorsMicrosomal level Female had higher CLM than male;age had no effect CLM;smokers and drinkers had lower CLM for CYP2D6;CYP1A2-163C>A,CYP2C942614A>C,CYP2D6 100C>T,CYP2D61846G>A,and CYP3A5 6986A>G mutations reduced its CLM.Liver tissue level Female and age had no effect CLLT;smokers and drinkers had lower CLLT for CYP2D6;CYP2C9 42614A>C,CYP2D6 100C>T,CYP2D61846G>A,CYP2E1-333T>A,and CYP3A5 6986A>G mutations reduced its CLLT.Liver level Female and age had no effect CLL;smokers and drinkers had lower CLL for CYP2D6;CYP2C9 42614A>C,CYP2D6 100C>T,CYP2D6 1846G>A,CYP2E1-333T>A,and CYP3A5 6986A>G mutations reduced its CLL.Organismal level Female and age had no effect CLH;smokers and drinkers had lower CLH for CYP2D6;CYP2C9 42614A>C,CYP2D6 100C>T,CYP2D61846G>A,CYP2E1-333T>A,and CYP3A5 6986A>G mutations reduced its CLH.2.3 Correlation between CYP2E1 and HCC2.3.1 VDEN and HCCChange of VDEN The VDEN was significantly increased by 109.4%in HCC patients.Because VDEN in control subjects was not normally distributed,the one-sided 95%reference value was calculated by adopting the percentile method.The data showed that CYP2E1 positivity in 42.8%of the HCC patients analyzed.Impact factors Gender,age,clinical characteristics(AFP,cirrhosis,maximum tumor diameter,number of lesions,tumor grade,and portal vein tumor thrombus)had no effect on VDEN.CYP2E1-333AA had higher VDEN than CYP2E1-333TA.2.3.2 CYP2E1 activity and overall survivalOverall survival The mean and median overall survival were 397 days(95%Cl:319-475 days)and 306 days(95%Cl:237-365 days),respectively.Single factors of overall survival Gender,age,AFP level(20 ng/mL),cirrhosis,maximum tumor diameter,number of lesions,tumor grade,portal vein tumor thrombus,and polymorphisms of CYP2E1 had no effect on overall survival;the mean overall survival of CYP2E1 activity-negative HCC patients was longer than CYP2E1 activity-positive HCC patients;the mean overall survival of HCC patients with lower AFP level(≤ 600 ng/mL)was longer than HCC patients with higher AFP level(>600 ng/mL).Independent risk factor of overall survival CYP2E1 activity and largest tumor size were determined as independent risk factor for overall survival.HCC patients with higher CYP2E1 activity and larger tumor size had shorter overall survival.2.3.3 CYP2E1 constitutive activity and hepatocarcinogenesis induced by DENImpact on incidence of HCC The incidence of HCC for rats with lower t1/2 and higher t1/2 was not significantly different(77.8%vs 52.6%);the incidence of HCC for rats with lower AUC0-t and higher AUC0-t was significantly different(83.3%vs 47.4%);the incidence of HCC for rats with lower CL/F and higher CL/F was significantly different(44.4%vs 84.2%).Impact on severity of HCC The largest tumor diameter and cumulative tumor diameter for rats with lower t1/2 and higher t1/2 was significantly different and there exist negative correlation between t1/2 and the above characteristics;the largest tumor diameter,cumulative tumor diameter,largest tumor volume,and cumulative tumor volume for rats with lower AUC0-t and higher AUC0-t was significantly different and there exist negative correlation between AUC0-t and the above characteristics;the nodule numbers,largest tumor diameter,cumulative tumor diameter,largest tumor volume,and cumulative tumor volume for rats with lower CL/F and higher CL/F was significantly different and there exist positive correlation between CL/F and the above characteristics.2.3.4 CYP2E1 inhibited activity and hepatocarcinogenesis induced by DENImpact of on incidence of HCC The incidence of HCC for model groups,CMZ(10 mg/kg)group,and CMZ(100 mg/kg)group was 64.9%,58.0%,and 10.0%,respectively.The incidence of HCC was dramatically reduced in CMZ(100 mg/kg)as compared with model group.Impact of on severity of HCC The nodule numbers,largest tumor diameter,cumulative tumor diameter,largest tumor volume,cumulative tumor volume,Ki67+cells,and PCNA+ cells was dramatically reduced in CMZ(100 mg/kg)as compared with model group.Conclusions1.Measuring the values of CLM for CYP1A2,CYP2B6,CYP2C9 and CYP2C19 allowed us to predict the values of CLM for CYP2A6,CYP2C8,CYP2D6,CYP2E1,and CYP3A4/5.2.There exist huge intra-individual variation in CLM of 10 CYP450.The intra-individual variation was influenced by age,smoking,CYP2A6*4,CYP2B615631G>T,CYP2B6 18053A>G,CYP2C9 42614A>C,CYP2D6 100C>T,CYP2D6 1661G>C,and CYP2D6 2850G>A.3.Specific genotypes of CYP2D6 significantly affected its molecular,sub-cellular,tissue,organ,and organismal phenotypes with different degrees.4.Clearance of CYP2E1 at different levels was significantly increased in HCC patients,and CYP2E1 activity negatively correlated with overall survival.5.CYP2E1 constitutive activity had causal relationship with hepatocarcinogenesis and inhibition of CYP2E1 activity prevented hepatocarcinogenesis in rat liver tumor model induced by DEN.更多还原