| Hepatocellular carcinoma(HCC) is the most common form of liver cancer and most common liver malignancy in China, which with high degree of malignancy, strong invasiveness, fast progress, poor prognosis, and high mortality.Cytochrome P450(CYP) is a large group of heme-thiolate protein superfamily that localize to the endoplasmic reticulum and play crucial roles in the metabolism of endogenous and exogenous molecules. Studies have confirmed that nitrosamine, aflatoxin exposure, tabacco smoking and heavy alcohol consumption have been documented as HCC risk factors which were metabolized by CYPs. Therefore, in-depth study of CYP activity associated with liver cancer is an important topic in today’s international research.To data there is no report concerning CYP metabolic activity in patients with HCC. In order to assess the individual variation in drug-metabolizing of patients with liver cancer, here we performed an in vitro study of liver microsomes from 102 HCC patient liver samples and 105 normal liver samples that focused on activity changes of 10 CYPs. Meanwhile, we measured the liver microsomal protein level. Moreover, we investigated the effect of accompanying disease(i.e., cirrhosis and fibrosis) and genetic polymorphisms on CYP activity, which could be associated with hepatocarcinogenesis. We wish to find individual variation of drug metabolism of patients with HCC and the impact factors to support their optimal use in personalized treatmeat of HCC.Materials and methods1. Ethic statementApprovals for tissue collection and in vitro xenobiotic metabolism studies were obtained from the Medical Ethics Committee of Zhengzhou University. Informed written consent was obtained from all participants.2. Human liver samplesAll non-tumor liver samples were obtained from members of the Chinese Han population who underwent surgical resection in the Henan Provincal People’s Hospital and Henan Provincal Tumor Hospital between March 2012 and July 2014. HBV- or HCV-infected liver tissues(102 samples) were from patients with HCC that were classified as having fibrosis or cirrhosis according to histological diagnosis. Liver tissue samples(105 samples) were also collected from subjects with liver hemangioma, metastatic carcinoma, cholelithiasis, or gallbladder cancer. These subjects had normal liver function and negative serum HBV and HCV markers, and the liver tissue samples were found to be normal according to histological diagnosis. These samples were used as normal liver controls. Liver tissue samples were stored in liquid nitrogen within 30 min of resection.In HCC group, there are 86 males, 16 females, 45 smokers and 37 drinkers which the mean age(± standard deviation; SD) was 53 ± 11(range: 29~75) years. In controls, there are 37 males, 68 females, 12 smokers and 12 drinkers which the mean age(± standard deviation; SD) was 48 ± 10(range: 20~75) years. All subjects only used regular anesthetics and had no history of exposure to known CYP-inducing or-inhibiting agents.HCC liver tissues were fixed with 10% dampen formaldehyde solution, buried by paraffin, and HE and Masson strained. Liver samples were assigned four stage of S1 to S4 depending on fibrotic degree according to Scheuer’s grading standard. Stage S1 to S3 conrespond the fibrosis, and cirrhosis refers to stage S4.3. Preparation of liver microsomes and microsomal protein determinationHuman liver microsomes(HLMs) were prepared according to the hypothermal differential centrifugation method. Microsomal protein concentration was determined according to the Bradford method. Total CYP content and cytochrome b5 content was determined by carbon monoxide differential method. The P450 oxidoreductase(POR) activity assay was based on the method of electron acceptor surrogate cytochrome c. The ration of activity of POR as measured in homogenates and microsomes produced from the same liver tissue sample was used to estimate liver recovery. Microsomal protein per gram of liver(MPPGL) was calculated by microsomal protein concentration×volume/liver sample pure weight/liver recovery.4. GenotypingPolymorphisms in CYP isoforms with frequencies of more than 1% in the Chinese sample set were investigated. Twenty-four CYP allelic mutations and 16 POR SNPs were identified. CYP2E1*1C*1D were genotyped by measured the product fragment length in one-step PCR. The remaining mutant sites were determined by mass spectrometry performed.5. CYP enzyme metabolic activity of probe drugSubstrate metabolites were determined by HPLC-UV or HPLC-FLD. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion 4-hydroxylation, paclitaxel 6-hydroxylation, tolbutamide 4-hydroxylation, omeprazole 5-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1-hydroxylation were used as activity indicators of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A/5, respectively. The biotransformation(TR) was used as velocity. The Michaelis constant(Km) and maximum velocity(Vmax) values were determined using Graph Pad Prism 5.0. In vitro intrinsic clearance(CLint) was calculated from the ratio of Vmax to Km. The linear range, precision, relative recovery and stability were all examined according to the guidance of determination of biological specimen.For biotransformation, seven or eight substrate concentrations were examined. 6.25~800 μM for phenacetin, 0.156~20 μM coumarin, 7.8~500 μM bupropion,2.5~80 μM paclitaxel, 31.25~2000 μM tolbutamide, 3.9~500 μM omeprazole, 2.5~640 μM dextromethorphan, 7.8~500 μM chlorzoxazone, and 0.39~50 μM midazolam. Incubation mixtures contained HLMs(0.3 mg·ml-1 for CYP1A2, CYP2A6 and CYP2E1; 0.2 mg·ml-1 for CYP2D6 and CYP3A4/5; 0.5 mg·ml-1 for CYP2B6, CYP2C8, CYP2C9 and CYP2C19), 100 m M phosphate buffer(p H 7.4), and different concentrations of substrate with 1 m M NADPH. The mixture was pre-incubated for 5 min at 37°C. Optimal incubation times for each substrate were as follows: 30 min for phenacetin O-deethylation, coumarin 7-hydroxylation and chlorzoxazone 6-hydroxylation; 60 min for bupropion 4-hydroxylation, and tolbutamide 4-hydroxylation; 90 min for omeprazole 5-hydroxylation; 120 min for paclitaxel 6-hydroxylation; 20 min for dextromethorphan O-demethylation; and 5 min for midazolam 1′-hydroxylation. Reactions were terminated by adding 20 μl ice-cold acetonitrile or 1 ml ethylacetate or perchloric acid.6. Statistical analysisSince most data sets were not normally distributed, nonparametric methods were generally used for statistical analysis. SPSS statistics17.0 software was used for data management and statistical analyses. The Mann-Whitney U test was used for pairwise comparisons. Genotype distributions between two groups were compared using chi-square test. A P-value < 0.05 was considered statistically significant(two-tailed).Results1. The histological classification of liver tissuesOut of 102 cases of HCC liver samples, there were 8, 19, 27 and 48 cases corresponding S1, S2, S3 and S4 stage of, respectively. There were 54 cases of fibrotic stage and 48 cases of cirrhotic stage.2. Liver microsomal protein levelMicrosomal protein parameters were expressed as median and range. In HCC group, total CYP content, cytochrome b5 content and MPPGL were 134.0(33.5~468.9) pmol·mg-1, 293.2(109.7~506.3) pmol·mg-1 and 28.9(7.7~93.6) mg·g-1, respectively. POR activity was 44.6(17.8~74.3) pmol·min-1·mg-1.Compared with controls, total CYP content, MPPGL and POR activity were all lower in HCC patients.3. CYP enzyme activity3.1 The enzyme activity of HLMs levelThe median individual metabolic parameters(Km, Vmax, CLint) of liver microsomes from HCC patients were determined. CYP1A2(70.0, 33.2~687.8) μM,(521.5, 68.6~2605.0) pmol·min-1·mg-1 and(7.8, 0.29~42.8) μl·min-1·mg-1. The data displayed 20~147 fold inter individual variation. CYP2A6(3.6, 0.58~17.6) μM,(522.8, 0.73~2880.0) pmol·min-1·mg-1 and(145.4, 0.26~260.0) μl·min-1·mg-1. The data displayed 30~3945 fold inter individual variation. CYP2B6(108.7, 46.9~1198.0) μM,(78.0, 15.0 ~ 237.9) pmol·min-1·mg-1 and(0.71, 0.049 ~ 4.5) μl·min-1·mg-1. The data displayed 15~91 fold inter individual variation. CYP2C8(23.4, 9.5~235.5) μM,(13.5,4.7~112.3) pmol·min-1·mg-1 and(0.63, 0.035~4.0) μl·min-1·mg-1. The data displayed 23~114 fold inter individual variation. CYP2C9(204.6, 60.1~773.1) μM,(319.1, 61.9~903.2) pmol·min-1·mg-1 and(1.5, 0.080~4.5) μl·min-1·mg-1. The data displayed 12~56 fold inter individual variation. CYP2C19(71.6,28.8~275.5) μM,(101.8, 21.5~268.4) pmol·min-1·mg-1 and(1.4, 0.22~3.9) μl·min-1·mg-1. The data displayed 9~17 fold inter individual variation. CYP2D6(30.7, 7.8~273.5) μM,(172.4, 37.8~560.4) pmol·min-1·mg-1 and(6.9, 0.49~54.7) μl·min-1·mg-1. The data displayed 14~111 fold inter individual variation. CYP2E1(60.3, 24.3~91.3) μM,(1238.0, 151.0~4858.0) pmol·min-1·mg-1 and(20.9, 4.1~53.2) μl·min-1·mg-1. The data displayed 3 ~ 32 fold inter individual variation. CYP3A4/5(2.6, 1.1~8.0) μM,(1205.5, 332.4~4602.0) pmol·min-1·mg-1protein and(524.8, 73.8~1099.8) μl·min-1·mg-1. The data displayed 7~14 fold inter individual variation.Compared with controls, the Km values for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1 and CYP3A4/5 were higher in HCC patients. Meanwhile, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4/5 had higher Vmax values in HCC patients, while the Vmax values of CYP1A2 and CYP2C8 were significantly reduced relative to control samples. The most notable difference between the two groups was the Vmax of CYP2E1, which was increased by 2.3-fold in HCC patients relative to controls. The CLint values for CYP2C9, CYP2D6 and CYP2E1 were increased in HCC patients, while CYP1A2, CYP2C8 and CYP2C19 values were lower in HCC patients than in controls. The CLint values for CYP2A6, CYP2B6 and CYP3A4/5 were unchanged between the two groups. The most prominent change was the CLint of CYP2C8, which in HCC patients was 77% that of the value for the controls.3.2 The enzyme activity of liver levelIn HCC group, the median individual metabolic parameters(Vmax and CLint) of per gram liver level were determined according to MPPGL. CYP1A2(13551.5, 1110.7 ~ 158520.7) pmol·min-1·mg-1 and(199.6, 13.5 ~ 2436.9) μl·min-1·mg-1. CYP2A6(14988.9, 18.4~114723.7) pmol·min-1·mg-1 and(3760.4, 8.2~13285.9) μl·min-1·mg-1. CYP2B6(2159.1, 286.6~14008.3) pmol·min-1·mg-1 and(19.7, 0.63~98.9) μl·min-1·mg-1. CYP2C8(408.4, 63.0~2915.4) pmol·min-1·mg-1 and(17.4, 0.80~149.4) μl·min-1·mg-1. CYP2C9(9037.1, 816.2~37935.6) pmol·min-1·mg-1 and(43.6, 3.0 ~ 178.9) μl·min-1·mg-1. CYP2C19(2816.3, 260.3 ~ 11678.2) pmol·min-1·mg-1 and(39.1,1.7~127.3) μl·min-1·mg-1. CYP2D6(5300.4, 332.3~28727.7) pmol·min-1·mg-1 and(191.0, 4.6~1711.0) μl·min-1·mg-1. CYP2E1(34690.2, 1155.1 ~ 113794.2) pmol·min-1·mg-1 and(604.6, 31.1 ~ 2024.8) μl·min-1·mg-1. CYP3A4/5(31264.6, 2666.7~143812.5) pmol·min-1·mg-1 and(14644.2, 1167.6~51938.0) μl·min-1·mg-1.Compared with controls, the Vmax values of CYP1A2, CYP2C8 and CYP2C19 were lower, while the Vmax of CYP2E1 was higher. For Clint, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19 and CYP3A4/5 values were decreased, Clint of CYP2E1 was increased in HCC patients.3.3 The enzyme activity of CYP levelIn HCC group, the median individual metabolic parameters(Vmax, CLint) of CYP level were determined according to total CYP content. CYP1A2(3.9, 0.30~31.3) pmol·min-1·mg-1 and(0.06, 0.002~0.52) μl·min-1·mg-1. CYP2A6(3.7, 0.004~43.0)pmol·min-1·mg-1 and(0.90, 0.002~4.2) μl·min-1·mg-1. CYP2B6(0.58, 0.03~3.3) pmol·min-1·mg-1 and(0.005, 0.0003~0.03) μl·min-1·mg-1. CYP2C8(0.10, 0.02~0.83) pmol·min-1·mg-1,(0.005, 0.0001~0.04) μl·min-1·mg-1. CYP2C9(2.3, 0.33~12.5) pmol·min-1·mg-1protein and(0.01, 0.001~0.08) μl·min-1·mg-1. CYP2C19(0.62, 0.046 ~ 3.6) pmol·min-1·mg-1 and(0.0099, 0.00099 ~ 0.05) μl·min-1·mg-1. CYP2D6(1.2, 0.14~7.7) pmol·min-1·mg-1 and(0.044, 0.001~0.49) μl·min-1·mg-1. CYP2E1(8.3, 1.1~48.4) pmol·min-1·mg-1 and(0.15, 0.023~0.67) μl·min-1·mg-1. CYP3A4/5(9.1, 1.1~57.6) pmol·min-1·mg-1 and(3.5, 0.49~13.5) μl·min-1·mg-1.Compared with controls, for Vmax, CYP2C8 was lower in HCC patients. Meanwhile, values of CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4/5 were higher in HCC group relative to control subjects. The CLint values of CYP1A2 and CYP2C8 were decreased, and CLint values of CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4/5 were increased in HCC group relative to controls.4. CYP allelic frequency distributionThe allelic frequency of CYP2D6*10(100C>T) significantly differed between the control and HCC groups. CYP2D6*10 mutant homozygote TT frequency was lower(16.7% vs. 46.7%, P < 0.01) and the heterozygote CT frequency was significantly higher(46.9% vs. 21.9%, P < 0.01) in HCC groups relative to control subjects. Among control group subjects, the highest genotype frequency was TT genotype, which had the lowest frequency in the HCC group.Moreover, the allelic frequency of CYP2D6*10 was significantly reduced in HCC patients with fibrosis or cirrhosis relative to control subjects. Both in fibrosis and cirrhosis subgroups, mutant homozygote TT frequency were higher than in control subjects. In contrast, the frequency of CT was increased both in fibrosis and cirrhosis subgroups than in control group.In addition, the frequency of mutant homozygote TT was significantly reduced in cirrhosis subgroup relative to fibrosis subgroup(8.9% vs. 23.5%, P < 0.01).5. The effect of CYP genetic polymorphism on enzyme activityThere was four CYP polymorphisms had significant effects on enzyme activity in HCC patients. The Km values of the CYP2D6*10(100C>T) mutant homozygote TT(125.1 μM) and heterozygote CT(30.4μM) were significantly higher than that for wild type CC(17.4 μM). The pattern for Vmax values differed from that for Km values, with CC being the highest and TT being the lowest, while CT had intermediate activity. The CLint values of TT(0.7 μl·min-1·mg-1) and CT(6.9 μl·min-1·mg-1) were remarkably lower than that for CC(13.2 μl·min-1·mg-1). The most significant change in CLint was for TT was decreased by 95% compared with CC.The Km values of CYP3A5*3 mutant homozygote *3*3(2.3 μM) and mutant heterozygote *1*3(2.8 μM) were significantly lower than in wild type *1*1(3.9 μM). The Vmax showed the same differences as Km(*3*3<*1*3<*1*1). Similarly, the CLint of *3*3(490.8 μl·min-1·mg-1) and *1*3(542.0 μl·min-1·mg-1) both were significantly lower than *1*1(762.9 μl·min-1·mg-1).The CYP2C9*3 mutant homozygote *3*3 was not detected in HCC patients. The Vmax of the mutant heterozygote *1*3 was lower than that for wild type *1*1(223.2 vs. 326.5 pmol·min-1·mg-1, P < 0.01). Meanwhile, the CLint of *1*3(1.1 pmol·min-1·mg-1) was lower than for *1*1(1.6 pmol·min-1·mg-1, P < 0.05).In addition, the CLint and Vmax of CYP2B6(516G>T) mutant homozygote TT were lower than that of wild type GG. There was no prominent influence on enzyme activity by remaining CYP polymorphisms.In control group, similar result was observed. The results indicated that gene mutations in CYP2D6*10, CYP3A5*3 and CYP2C9*3 had definite effects on enzyme activities in both HCC group and controls which can cause enzyme activities decreased.6. The effect of POR genetic polymorphism on CYP enzyme activityCYP activity values expressed as intrinsic clearance(CLint). POR polymorphisms can affect the CLint of CYPs including CYP1A2, CYP2A6, CYP2C8, CYP2D6 and CYP2E1. The CYP2E1 CLint of POR(rs10954732) heterozygote GA was lower than wild type GG. POR(rs2286823) had the similar effect on CYP2E1 which heterozygote GA had lower activity than that for wild type GG. In addition, the CYP2D6 CLint of POR(rs4732516) heterozygote CG was higher than those for wild type CC. Meanwhile, the activity of mutant homozygote GG was lower relative to CG carriers. For CYP1A2, POR(rs1057870) heterozygote GA had higher CLint compared to wild type GG carriers.In controls, the POR polymorphisms cause CYP activities changed(incliding CYP2B6, CYP2C8 and CYP2E1). POR rs1057868 heterozygote CT had lower CLint values of CYP2B6 and CYP2C8 than wild type CC. POR rs3823884 heterozygote AC had decreased CYP2E1 CLint than those for wild type AA. POR rs2286823 heterozygote CT and homozygote TT both had lower CYP2E1 activity than wild type CC carriers.The activities of CYP2C8 and CYP2E1 were impacted by POR polymorphisms both in HCC patients and control subjects. Compared with control subjects, more CYPs were affected by POR polymorphisms in HCC patients such as CYP1A2, CYP2A6 and CYP2D6.7. The effect of fibrotic progression on CYP enzyme activityIn order to further evaluate the effect of disease progression on CYP activity, HCC patients were categorized into two subgroups that included patients with fibrosis or cirrhosis according to histological diagnosis. The median individual metabolic parameters(Km, Vmax, CLint) of liver microsomes from fibrosis and cirrhosis samples were determined.The Km values of most CYP enzymes did not differ between the fibrosis and cirrhosis groups with the exception of CYP2C8 and CYP2D6. The Km of CYP2C8 was higher, but the value of CYP2D6 was lower in cirrhosis group relative to fibrosis group. The CLint of CYP2D6 was higher in cirrhosis group than in fibrosis group(8.4 vs. 3.9 μl·min-1·mg-1, P < 0.05).Both in fibrosis and cirrhosis subgroups, the CLint values of CYP1A2, CYP2C8 and CYP2C19 were lower than in controls, while CYP2C9 and CYP2E1 values were increased. However, the presence of cirrhosis significantly affected CYP2D6 activity, which the Km was decreased(21.4 vs. 28.9 μM,P < 0.05) and CLint was increased(8.4 vs. 3.5 μl·min-1·mg-1, P < 0.01) in patients with cirrhosis relative to control subjects.8. The effect of demographic factor on CYP enzyme activityIn order to further assess the influence of lifestyle-related factor, a non-parametric test was used to analyze sex, tobacco smoking and alcohol consumption. Females with HCC had higher CYP2A6 CLint values than male HCC patients(164.1 vs. 142.1 μl·min-1·mg-1, P < 0.05). The CLint for CYP2D6 was higher for non-smoking HCC patients than for HCC patients who smoke(9.0 vs. 5.3 μl·min-1·mg-1, P < 0.05). The CLint of CYP2D6 for HCC patients who did not drink was higher than for those HCC patients who did drink(8.5 vs. 5.0 μl·min-1·mg-1, P < 0.05).For control subjects, there were no statistically significant differences in the Km, Vmax and CLint of 10 CYPs as a function of gender, smoking status, drinking habit with CYP2D6 exception. History of smoking was significantly associated with differences in CLint of CYP2D6(3.0 vs. 5.7 μl·min-1·mg-1, P < 0.05).Smoker had lower CYP2D6 CLint in HCC patients but had higher CYP2D6 CLint in controls relative to nosmoker. Gender and alcohol consumption causes HCC patients enzyme activity change, but had no significant effect on normal population.Conclusions1. Ten tested CYP isoforms have significant activity changes in HCC patients as evidence by increased CLint for CYP2C9, CYP2D6 and CYP2E1, While CYP1A2, CYP2C8 and CYP2C19 CLint values decrease, and CYP2A6, CYP2B6 and CYP3A4/5 activity are unchanged in HCC patients relative to controls. Meanwhile, there are large inter-invidual variations in CYP metabolic activity out of HCC patients.2. CYP2D6*10(100C>T) allelic frequency differs in HCC patients. The mutant homozygote TT frequency is lower, and the heterozygote CT frequency is higher in HCC group than in controls.3. The total CYP level, MPPGL, and POR activity are reduced in HCC liver samples.The reduction of liver microsomes content cause the change of enzyme activity of liver level.4. CYP2D6*10, CYP2C9*3, and CYP3A5*3 polymorphisms have definite effects on enzyme activities which cause those decreased.5. There is no influence by disease degree on CYP activity with CYP2D6 exception, which has higher CLint values in cirrhosis subgroup than in fifrosis subgroup and controls. |
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