Metabolic Activity Of Human Liver CYP450 And Correlation Between CYP2E1 And Hepatocellular Carcinom

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 ?m-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.