INDUCTION OF CYP2E1 ACT IVITY IN CTENOPHARYNGODON IDELLUS HEPATOCYTE

CYP2E1 can m etabolizem ost of the drug and tox icant1To study the CYP2E1 act iv ity, Ctenopharyngodon idellus hepatocyte was prepared and used as react ion system, selecting chlorzoxazone (CZX) as substrate and joining ant ipyrine as inner standard RP-H PLC m ethod was established to detect the product of 6-OH-chlorzoxazone (HCZX) and the Lowry method was used tom easure the concentration of protein incel,l then the act ivity of CYP2E1 in cell was reflected1The medicine was extracted by dichlorom ethane from cel,l and then the extracts were evaporated to dryness under 45e and were dissolved in mobile phase1The fat of the solute was degreased by hexane1CZX andHCZX analyses were performed by RP-HPLC1Them obile phase used for the analys is consisted of 011mo l/L ammon ium acetate, acetonitrile and tetrahydrofuran(72B2215B515, v/v) delivered at a flow-rate of 110mL/m in1The mob ile phase was degassed and filtered through a0145um membrane before be ing used1The co lumn temperature was 40e and UV detection wasm easured at 280nm Then the Ctenopharyngodon idellus hepatocyte was treated w ith ethano l in d ifferent dosage, which was specific inductor of CYP2E1, and them etabo lism of CZX after induction in vitro was also studied1The result demonstrated that the RP-HPLC method established was smi ple and accurate for the determ ination o f activity ofCYP2E1 Them ean recoveries for samples were all exceed 79196%, Intra-day CV and inter-day CV were under 5118% and 5140% respectively1T he elementarym etabolism for CZX was low, while after the optmi ization and screening for induct ion condition, ethanol could significantly accelerate them etabolism of CZX in fish cell1When the cellwas inducted by ethano lw ith dosage of 4Lg/mL for 24h and incubated with CZX (50Lg/mL) for 1h, CYP2E1 activity achieved the h ighest leve,l which was 0147Lg/m in/mg1E lmi ination equations of CZX metabo lism in contro l group and ethano-l related were Ct=46156e-01038t (r2=018370)and Ct=44154e-01052t (r2=018771), respectively1The elmi inat ion hal-f liveswere 202110h and 28175h, respectively The difference betw een them was consp icuous, and it indicated that ethano l could accelerate themetabolism of CZX1The enzym at icequations for control group and induct ion group were 1/V=192176 @ 1/ S+1171 and 1/V=52144 @ 1/ S+0190, respectively1Enzym atic param eters dem onstrated CYP2E1 induced by ethanol in cell has h igher affin ity w ith substrate and stronger potency to catalyze CZX than the control1 In conclusion, ethano l has big induction intensity to CYP2E1 in fish cell1T he results of th is study using an in v itro approach to clearly show the potential properties of P450 in fish drugres idue and environmental tox icant1