| CytochromeP450enzymes (cytochrome450, CYP), also called mixed function oxidase and monooxygenase, is a superfamily of hemoproteins which plays a critical role in metabolism of endogenous and exogenous compounds. CYP2A6and CYP2C9not only can account for about3%and15%clinical drug metabolism repectively, but also metabolize and/or activate a large variety of drugs, pre-carcinoges, pre-toxins and mutagens.Now various drug metabolic enzyme content and enzyme activity exhibit significant polymorphism in inter-individual and inter-ethnic, which in turn shows individual differences in sensitivity for clinical medication, environmental chemicals and carcinogens toxicity.In general,it seems that individual differences are mainly attributed to gene polymorphisms of CYP enzyme.CYP2A6and CYP2C9gene are highly polymorphic, previous research were mostly developed in recombinant enzyme system and in human. Though the former can better reflect one single enzyme activity, it is very different from human environment. The latter is close to human environment, but involved in lots of links about drug disposition which cannot reflect the single metabolic process. Both of them could not completely reflect the metabolism and activity of CYP. In a word, human liver metabolism is the most likely human body in vitro platform.Our study was designed to investigate the effects of gene polymorphisms on CYP2A6and CYP2C9activity using coumarin (COH) and tolbutamide (TOB) in normal human liver microsomes, in order to provide theoretical basis for rational clinical use of drugs metabolized by CYP2A6and CYP2C9.Methods1Human liver samplesHuman liver samples with normal liver function were obtained from108patients including36male samples and72female samples. The age ranged from20to75years(Median:47years). Smoking history was positive for96donors, negative for12donors. All subjects wrote informed consent and this study was approved by the ethics committee of Zhengzhou University.2Genotyping for CYP2A6and CYP2C9geneGenomic DNA was isolated with DNA extraction kit from human liver tissues.The subjects were genotyped using polymerase chain reaction (PCR) and direct sequence analysis of polymorphisms for CYP2A6and CYP2C9.3Preparation of human liver microsomesHuman liver microsomes (HLMs) were prepared by differential centrifugation and microsome protent level was determined by Bradford method. Coumarin and tolbutamide were selected as the in vitro probe substrates for CYP2A6and CYP2C9, respectively.4Determination of probe drug and metabolitesHuman liver microsome incubation mixtures contained probe, phosphate buffer, NAPDH and so on. After pre-incubation, Incubations were initiated by the addition of NADPH. Then they were stopped by addition of perchioric acid cooling on ice. After a series of concentration and time trials, the perfect incubation time and microsomal protein were determined. The sensitive and specific HPLC-UV methods were establised for determination of both probes and metabolites. The linear range, precision, recovery and stability were examined according to the guidance of determination of biological specimen.5Detemination of enzyme kinetic parameters in human liver microsomesSubstrate concentrations were examined over the following ranges:0.15625～20μM for coumarin,31.25～2000μM for tolbutamide. The concentration of protein for CYP2A6and CYP2C9was0.3mg·ml-1and0.5mg·ml-1, respectively. The optimal incubation time was30min and60min for CYP2A6and CYP2C9respectively. The Km, Vmax and CLint were determined in105HLMs and108HLMs for CYP2A6and CYP2C9, respectively.6Statistical analysisCalculation of enzyme kinetic parameters and statistical analysis was performed by GraphPad Prism5.0and SPSS17.0software, respectively. All data were routinely tested for normality of distribution and equal variance, and for these tests failure, nonparametric methods of statistical analysis were used. A value of P<0.05was set as statistically significant.Results1Effect of gene polymorphisms on CYP2A6activity in HLMs1.1The enzyme kinetics of coumarin in HLMsThe median of Km.Vmax and CLint in HLMs (n=105)were2.33(0.78～10.09) μM,354.4(3.7-1430.0) pmol-min-1·mg-1protein,144.5(1.2～544.7)μl-min-1·mg-1protein. The data displayed a12.9,386.5and453.9-fold inter-individual varation for Km,Vmax and CLint, respectively.It revealed that there were great inter-individual differences in CYP2A6metabolic activity towards coumarin.1.2The effects of genotype of CYP2A6*1B on enzyme kinetics of coumarin The frequency of CYP2A6*1B was53.3%. The data displayed there were no differences on the enzyme kinetic parameters among these three genotypes. It revealed that the genotype of CYP2A6*1B had no effects on enzyme kinetic parameters of CYP2A6(P>0.05).1.3The effects of genotype of CYP2A6*4on enzyme kinetics of coumarinAmong the105samples studied,90were genotyped as*1/*1, and15were*1/*4,. The frequency of CYP2A6*4was7.1%. The values of Km, Vmax and CLint of subjects with CYP2A6*1/*1was higer than that of*1/*4genotype with2.44(0.87～10.09) vs1.18(0.78～7.88)μM,374.5(49.7～1430.0) vs42.5(3.7～495.1) pmol·min-1·mg"1protein,147.4(27.41～544.74) vs43.2(1.2～209.7)μl.min-1·mg-1prote-in (P<0.05).It revealed that the genotype of CYP2A6*4significantly reduced its drug metabolic activity.1.4The effects of genotype of CYP2A6*9on enzyme kinetics of coumarinThe frequency of CYP2A6*9was23.3%. The Km was higher in*1/*1of2.72(0.98～10.09)μM than that in*1/*9of2.09(0.87-6.18)μM and*9/*9of1.27(0.89～1.96) μM.*9/*9genotype showed a lower Vmax with179.2(117.4～248.9) pmol·min-1mg-1protein than that of*1/*1genotype of417.2(49.7～1430.0) pm-ol·min-1·mg-1protein (P<0.05). It revealed that the genotype of CYP2A6*9significantly reduced its drug metabolic activity.2Effect of gene polymorphisms on CYP2C9activity in HLMs2.1The enzyme kinetics of tolbutamide in HLMsThe median of Km, Vmax and CLint in HLMs (n=108) were230.4(101.2～555.3) μM,254.1(82.4～454.8) pmol·min-1·mg-1protein,1.11(0.17～4.18)μl·min-1·mg-1protein.The data displayed a5.5-fold inter-individual varation for both Km and Vmax, and24.6-fold inter-individual varation for CLint. It revealed that there were great inter-individual differences in CYP2C9metabolic activity towards tolbutamide.2.2The effects of genotype of CYP2C9*3on enzyme kinetics of tolbutamideAmong the108samples studied.102were genotyped as*1/*1,6were*1/*3. The frequency of CYP2C9*3was2.78%. The values of Km of subjects with CYP*1/*3genotype was higher than that of*1/*1genotype with324.7(266.3-510.9) vs217.9(101.2-555.3) μM. But the values of Vmax and CLint of CYP*1/*3genotype was lower than that of*1/*1genotype with172.6(82.4～207.0) vs257.1(83.8～454.8) pmol-min-1·mg-1protein,0.47(0.30-0.78) vs1.18(0.17～4.18)μl·min-1·mg-1protein (P<0.05). It revealed that the genotype of CYP2C9*3significantly reduced its drug metabolic activity.Conclusions1. There is great inter-individual difference in the drug metabolic activity by CYP2A6and CYP2C9.1. The genotypes of CYP2A6*4and CYP2A6*9have lower CYP2A6metabolic activity. The polymorphism of*1B has no significant effects on enzyme kinetic parameters of CYP2A6.2. The genotype of CYP2C9*3has significantly lower CYP2C9metabolism activity.3. There is marked inter-individual varation among the same genotype of CYP2A6and CYP2C9. |
PDF Full Text Request