草鱼肝细胞中CYP2E1活性的诱导研究

INDUCTION OF CYP2E1 ACTIVITY IN CTENOPHARYNGODON IDELLUS HEPATOCYTE

CYP2E1为代谢大部分药物及环境中毒物的关键酶。以草鱼肝细胞(Ctenopharyngodon idellushepatocyte)为反应体系,选取氯唑沙宗(CZX)为底物,采用反相高效液相色谱(RP-HPLC)法测定其产物6-OH-氯唑沙宗(HCZX)的量,Lowry法测定肝细胞中蛋白的浓度从而反映CYP2E1活性,并采用该酶特异性诱导剂乙醇对其进行诱导,观察其酶活变化及CZX在细胞中代谢情况。结果表明,CZX在草鱼肝细胞中的基础代谢较低,经过对CYP2E1诱导条件的优化及筛选,得到最佳诱导剂剂量为4μg/mL、诱导时间为24h、底物浓度为50μg/mL并且孵育时间为1h时,其酶活达到最高,约为0.47μg/min.mg。对照组和诱导组的草鱼肝细胞中CZX的消除半衰期(t1/2)分别为202.10h和28.75h,差异极显著,表明乙醇诱导的CYP2E1能够加快底物的代谢。酶促反应动力学参数表明乙醇诱导的CYP2E1与底物的亲和力较高,酶促反应强度较大。该结果能够为CYP2E1代谢的药物及环境毒物的研究提供理论依据。

CYP2E1 can metabolize most of the drug and toxicant. To study the CYP2E1 activity, Ctenopharyngodon idellus hepatocyte was prepared and used as reaction system,selecting chlorzoxazone （CZX） as substrate and joining antipyrine as inner standard. RP-HPLC method was established to detect the product of 6-OH-chlorzoxazone （HCZX） and the Lowry method was used to measure the concentration of protein in cell, then the activity of CYP2E1 in cell was reflected. The medicine was extracted by dichloromethane from cell, and then the extracts were evaporated to dryness under 45℃ and were dissolved in mobile phase. The fat of the solute was degreased by hexane. CZX and HCZX analyses were performed by RP-HPLC. The mobile phase used for the analysis consisted of 0. 1 mol/L ammonium acetate, acetonitrile and tetrahydrofuran （72： 22.5： 5.5,v/v） delivered at a flow-rate of 1.0mL/min. The mobile phase was degassed and filtered through a 0.45μm membrane before being used. The column temperature was 40℃ and UV detection was measured at 280nm. Then the Ctenopharyngodon idellus hepatocyte was treated with ethanol in different dosage, which was specific inductor of CYP2E1 ,and the metabolism of CZX after induction in vitro was also studied. The result demonstrated that the RP-HPLC method established was simple and accurate for the determination of activity of CYP2E1. The mean recoveries for samples were all exceed 79.96% , Intra-day CV and inter-day CV were under 5.18% and 5.40% respectively. The elementary metabolism for CZX was low,while after the optimization and screening for induction condition, ethanol could significantly accelerate the metabolism of CZX in fish cell. When the cell was inducted by ethanol with dosage of 4μg/mL for 24h and incubated with CZX （50μg/mL） for lh, CYP2E1 activity achieved the highest level, which was 0. 47 μg/min/ mg. Elimination equations of CZX metabolism in control group and ethanol-related were Ct = 46.56e-0.038t（r2 = 0. 8370） and Ct = 44.54e -0052t（ r2 = 0. 8771 ） , respectively. The elimination half-lives were 202. 10h and 28.75h, respectively. The difference between them was conspicuous,and it indicated that ethanol could accelerate the metabolism of CZX. The enzymatic equations for control group and induction group were 1/V = 192.76 ×1/[ S ] ＋ 1.71 and 1/V = 52.44 × 1/[ S ] ＋ 0.90, respectively. Enzymatic parameters demonstrated CYP2E1 induced by ethanol in cell has higher affinity with substrate and stronger potency to catalyze CZX than the control. In conclusion,ethanol has big induction intensity to CYP2E1 in fish cell. The results of this study using an in vitro approach to clearly show the potential properties of P450 in fish drug residue and environmental toxicant.