Changes Of Cytochrome P450 Activity In Liver For Patients With Hepatocellular Carcinoma Associated With Fibrosis

Liver fibrosis is a chronic inflammatory state in liver cells,and it is developed associated with the external factors（long term exposure to carcinogens aflatoxin,persistent infection of hepatitis B virus,long-term alcohol intake）and intrinsic metabolic diseases（diabetes,obesity）and other factors.The disease which showed a body of inflammatory damage repair response to external stimuli at the early time,is early cirrhosis.This process is reversible.When the long-term stimulation of the outside world,the degree of fibrosis continues to progress and eventually form cirrhosis（Hepacullar cirosis）.Previous epidemiological studies have shown that the majority of liver cancer is developed in the context of chronic inflammation,usually through the development of chronic liver disease to liver fibrosis,cirrhosis,and finally deterioration of liver cancer.The liver is the largest metabolic organ in the human body,and the change of metabolic enzyme activity will affect our clinical rational use of drugs.The aim of the study is to provide the reference for clinical rational use of drugs,and to provide individualized medication advice,so as to reduce the occurrence and development of diseases.Some of our research showed that in the condition of in cirrhosis and liver cancer,some changes had occurred in the liver metabolism such as CYP1A2,CYP2A8,CYP2C19 and CYP3A4/5 were decreased,the activity of CYP2A6,CYP2B6,CYP2C9,CYP2D6 were unchanged,the activity of CYP2E1 was increased.However,the changes of drug metabolizing enzymes are different in different disease states.The present study which focuses on the HCC or liver cirrhosis is ignore the the important process of hepatocellular carcinoma from hepatic fibrosis to cirrhosis.It have an important significance to research the change of liver metabolism in the fibrosis.In addition,our previous research method which establish 10 main metabolic model of CYP enzyme in liver in vitro condition（liver microsomes were prepared on the basis of determination of the corresponding metabolic value）is at the micro level,and it is not consistent with the report which demonstrate the active of CYP is decreased in HCC.This phenomenon may be due to the fact that we ignore the differences in our liver tissue.Therefore,the expression of the enzyme activity at the liver level is likely to be the most realistic response to the actual liver condition.Because of we cannot get to the actual size of the liver weight,we could only use some corresponding reports of body weight and other related physiological data to calculated the corresponding levels of liver enzymes by the microsomal content per gram of liver（MPPGL）.In this study,we collected 78 cases of normal liver samples and 53 cases of patients with hepatocellular carcinoma associated with fibrosis,in order to investigate the changes of drug metabolism in patients with liver cancer associated with fibrosis,so as to support the clinical rational use of drugs.The metabolic activity of CYP1A2,CYP2A6,CYP2C6 C,CYP2C9,CYP2D6,CYP2E1 and CYP3A4/5 in the liver microsomal were investigated in our study.The concentration of microsomal protein in the liver was measured.The content of microsomal protein in the liver was measured.The metabolic activity of 10 kinds of CYP enzymes in liver（CLL）was calculated.The effect of liver fibrosis on the enzyme activity was analyzed and the effect of demographic factors on the enzyme activity was analyzed.From these factors in order to find the liver fibrosis in patients with individual differences in drug metabolism and influencing factors for clinical individualized drug to provide a theoretical basis.Method 1 Liver samplesThe normal liver samples of this project were collected from Henan Provincial People’s Hospital and Henan Provincial Tumor Hospital.All of them were normal liver tissues,which were mainly hepatic hemangioma,gallstones and gallbladder lesions.The liver function was normal without HBV or HCV infection,and were confirmed by histopathology as normal liver specimens.Hepatic fibrosis samples are also derived from the two hospitals,with HBV or HCV infection,all liver fibrosis specimens are histopathologically confirmed with liver fibrosis.All specimens were resected and stored within 60 minutes after treatment in liquid nitrogen.The liver tissue was divided into four stages according to the Scheuer classification criteria,which were S0-S3 stage,the stage of S1-S3 was liver fibrosis,and the degree of pathological changes was low.2 Microsomes prepared and protein content determinedThe liver microsomes were prepared by differential centrifugation and the liver microsomal protein concentration was determined by Bradford method.3 Metabolic activity of 10 CYP enzymes determinedThe subject of 10 enzymes are that CYP2A6 coumarin,CYP2B6 bupropion,CYP2C8 paclitaxel,CYP2C9 toluenesulfonylurea,CYP2C19 omeprazole,CYP2D6 dextromethorphan,CYP2E1 chloride Oxazolone,CYP3A4/5 midazolam.All metabolites were analyzed by high^performance liquid chromatography（HPLC）combined with UV（UV）or fluorescence（FLD）;the reaction rate was expressed as the degree of biotransformation（TR）of the substrate;Graph Pad Prism 5.0 software K_m and V_max,the clearance rate CL is the ratio of V_max to K_m.4 Statistical analysisStatistical analysis was performed by SPSS 17.0 statistical software.Graphic Prism 5.0 software was used to calculate the enzyme kinetic parameters and the enzyme activity data.The nonparametric data were analyzed by nonparametric test.The test level is 0.05.Result 1 Liver samplesIn normal tissue,there were 27 males and 51 females,.51 samples were age> 45 years old,27 patients aged ≤45 years,15 in smokers,63 in non smokers,14 in alcohol and 64 in non-drinkers.In fibrosis tissue,there were 45 males and 8 females,42 samples were age> 45 years old,11 patients were age≤45 years,27 smokers and 26 non-smokers,17 drinkers and 17 non-drinkers.CYP enzyme inhibitors and inducers was not used in the history of medication.In the classification of liver fibrosis,S0 = 3 cases,S1 = 5 cases,S2 = 19 cases,S3 = 26 cases.2 The amount of liver microsomal protein per gram（MPPGL）The median MPPGL of 78 normal liver was 34.0 mg·g-1,the range（9.9¹28）mg·g-1,95% confidence interval（14.2¹16.8）mg·g-1.The median of MPPGL in 53 cases of fibrosis were 28.6 mg·g-1,the range（7.6⁹3.6）mg·g-1,95% confidence interval（9.4⁸2.2）mg·g-1.MPPGL was approximately 13 times in normal human liver,and about 12.3 times in hepatic fibrosis.The MPPGL in liver fibrosis was significantly lower than that in normal liver（P<0.01）.3 The metabolic activity of 10 CYPs enzymes3.1 The metabolic activity of microsomes levelThe enzyme activity of the liver microsomes was measured as per milligram liver microsomal protein.In the normal tissue,the activity of CYP showed great difference.The CLM of CYP2C19 and CYP2A6 showed biggest difference and the fold were166 and 454,followed by CYP3A4/5 > CYP2C8 > CYP2B6 > CYP2D6 > CYP1A2>CYP2E1> C YP2C9（The fold is73,46,40,33,21,12,5）.In the fibrosis tissue,the activity of CYP also showed great difference.The CLM of CYP2A6 and CYP1A2 showed biggest difference and the fold were 284 and 142,followed by CYP2D6 > CYP2C8 > CYP2B6 > CYP2C9 > CYP2C19 > CYP3A4/5 > CYP2E1（The fold is 109,100,56,48,20,6,4）.In the microsomal level,compared with the normal control group,hepatic fibrosis group CLM of CYP1A2,CYP2C8 are reduced;CYP2A6,CYP2B6,CYP2C19 are not changed;CYP2C9,CYP3A4/5,CYP2E1 are elevated.Compared with the normal control group,hepatic fibrosis group V_max of CYP1A2,CYP2C8 are reduced;CYP2C19 are not changed;CYP2A6,CYP2B6,CYP2C9,CYP2E1,CYP3A4/5 are elevated.3.2 The metabolic activity of liver levelThe enzyme activity of liver tissue was calculated according to the content of MPPGL in liver tissue,and the activity of the enzyme was calculated by the activity of microsomal enzymes.The liver clearance rate（CLL）represented the metabolic activity of the enzymeIn the normal tissue,the activity of CYP showed great difference.The CLL of CYP2B6 and CYP2C19 showed biggest difference and the fold were 740 and 110,followed by CYP3A4/5 > CYP2C8 > CYP1A2 > CYP2A6 = CYP2C8 > CYP2C9 = CYP2E1（The fold is90,66,51,29,29,24,24）.In the fibrosis tissue,the activity of CYP also showed great difference.The CLL of CYP2A6 and CYP1A2 showed biggest difference and the fold were 2546 and 266,followed by CYP2D6 > CYP2C8 > CYP2B6 > CYP2C9 > CYP2C19 > CYP2E1 > CYP3A4/5（The fold is 241,137,74,49,29,20,14）.In the liver,compared with normal control group,hepatic fibrosis group CLL of CYP1A2,CYP2B6,CYP2C8,CYP2C19 are reduced,;CYP2C9,CYP2D6,CYP2A6,CYP3A4/5 are not changed;CYP2E1 are elevated.Compared with the normal control group,hepatic fibrosis group V_max of CYP1A2,CYP2C8 are decreased;CYP2A6,CYP2C9,CYP2C19,CYP2D6,CYP3A4/5 are not changed;CYP2E1 are elevated.4.The correlation of enzyme kinetics between the two level4.1 The correlation of enzyme kinetics on V_max between the two level4.1.1 The correlation of enzyme kinetics on V_max between the two level in normal tissuesUse Spearman inspection of the enzyme kinetics of the two level in normal tissues on V_max,the correlation of CYP3A4/5 was the highest in R=0.684,and the correlation between and CYP2C9 was the lowest in R=0.330.The correlation of CYP1A2 was R=0.667,CYP2A6 was R=0.652 CYP2B6 was R=0.532 correlation,CYP2C8 was R=0.530,CYP2C9 was R=0.330,CYP2C19 was R=0.588,CYP2D6 was R=0.562,CYP2E1 was R=0.608,P<0.01.CYP3A4/5 was R= 0.684.The correlation between the two levels of the V_max of the 10 enzymes was P<0.01.4.1.2 The correlation of enzyme kinetics on V_max between the two level in fibrosis tissuesUse Spearman inspection of the enzyme kinetics of the two level infibrosis tissues on V_max,the correlation of CYP3A4/5 was the highest in R=0.684,and the other correlation was 0.528（CYP1A2）,0.681（CYP2A6）,0.711（CYP2B6）,0.589（CYP2C8）,0.565（CYP2C9）,0.585（CYP2C19）,0.671（CYP2D6）,0.581（CYP2E1）.The correlation between the two levels of the V_max of the 10 enzymes was P<0.01.4.2 The correlation of enzyme kinetics on CL between the two level4.2.1 The correlation of enzyme kinetics on CL between the two level in normal tissuesUse Spearman inspection of the enzyme kinetics of the two level in norm al tissues on CL,the correlation was 0.554（CYP1A2）,0.565（CYP2A6）,0.642（CYP2B6）,0.579（CYP2C8）,0.564（CYP2C9）,0.574（CYP2C19）,0.708（CYP2D6）,0.482（CYP2E1）,0.553（CYP3A4/5）.The correlation between the two levels of t he CLM of the 10 enzymes was P<0.01.4.2.2 The correlation of enzyme kinetics on V_max between the two level in fibrosis tissuesUse Spearman inspection of the enzyme kinetics of the two level in fibro sis tissues on CL,the correlation was 0.122（CYP1A2）,0.138（CYP2A6）,0.073（CYP2B6）,0.103（CYP2C8）,0.138（CYP2C9）,0.046（CYP2C19）,0.087（C YP2D6）,0.073（CYP2E1）.5.The effect of degree of fibrosis on CYP enzymes activityThe K_m and V_max of CYP1A2 in fibrosis are significate raise than normal tissue,P<0.01.But the CL reduced more than normal tissue.The V_max and K_m of CYP2A6 in S2 and S3 group are raised than normal tissue,P<0.05.But CL in normal and fibrosis groups are not change.V_max of CYP2B6 in S1 and S3 groups are raised than normal tissue,P<0.01.The K_m of CYP2B6 in S3 group is higher than normal tissue,P<0.01.But the CL in normal and fibrosis groups are not changed.The V_max of CYP2C8 in fibrosis group is lower,K_m is high and the CL is lower than normal group,P<0.01.The V_max and CL of CYP2C19 in S3 group is lower than normal tissue,P<0.01.But K_m in tow groups is not changed.CL of CYP2D6 in S3 group is high than normal tissue,P<0.05.V_max and K_m in normal and fibrosis groups are not changed.The V_max,K_m and CL of CYP2E1 in S1,S2 and S3 groups are higher than normal tissue,P<0.01.V_max and K_m of CYP3A4/5 in S1,S2 and S3 groups is higher than normal tissue,P<0.01.But CL is not changed.6 The effect of demography factor on CYP enzyme activityIn microsomes level,the V_max and CL of normal tissue of CYP1A2 in male is high than in female,P<0.05.V_max of CYP1A2 and CYP2C9 of normal tissue in smoker is high than non-smoker,P<0.05.CL of CYP2D6 in smoker is high than non-smoker,P<0.01.The V_max and CL of CYP2C9 in drinker is high than non-drinker,P<0.01.In microsomes level age is not effect on enzymes activity.In liver level,the V_max in male is high than female,P<0.05.Age,smoking and drinking are not effect on enzymes activity.In fibrosis tissue and in microsomes level,the K_m of CYP2A6 in drinker is low than non-drinker,P<0.05.CL of CYP2B6 in male is high than female,P<0.05.Age and smoking are not effect on the enzymes activity.In liver level,the CL of CYP2B6 in male is high than female,P<0.05.Age,smoking and drinking have not effect on enzymes activity.Conclusion1.The MPPGL of human liver had individual difference.The difference of MPPGL was about 13 times in normal tissue,and the difference of MPPGL was about 12.3 times in fibrosis tissue.The MPPGL of fibrosis group was significantly lower compared with the normal group（P<0.01）;2.The CYP activity in fibrosis had changed,and there are large individual differences;Compared with normal group,different enzyme varied differently;3.The activity of the CYP enzymes are great individual difference at the two level of CYP activity,the CYP activity in liver was more significant than that of microsomal;4.The demography factor have not great effect on CYP activity.