Preliminary Studies On Metabolism And Impact On Main P450 Isoenzymes Of Enrofloxacin In Carasius Auratus Gibelio Liver Microsome

Cytochrome P450(to abbreviate CYP or P450) is a large superfamily for the drug metabolism.P450 participates in the metabolism of drug transformation,on the contrary, there may exist induction and inhibition of drug toward relative drug-relay-P450 isozymes.In this article,Carasius auratus gibelio which cultivate generally in our country and enrofloxacin which is one of the third fluoroquiolones agents were the study object,preliminary study on the drug metabolism in Carasius auratus gibelio liver microsome and the influence on activity of main cytochrome P450 isoenzymes.In order to provide theoretical foundation for further research on fishery P450 and guide clinical application of the kind of drugs in aquiculture.Liver microsomes were prepared by differential ultracentrifugation.Microsomal P450 and b5 were determined by the method of CO differential spectrum display in liver,kidney, gill,intestine and muscle microsomes.The activities of CYP3A,CYP2B and CYP2E were reflected by erythromycin N-demethylase(ERND),aminopyrine N-demethylase(APD), 4-aniline-hydroxylase(AH) with spectrophotometry,respectively.The activities of CYP1A were reflected by 7-ethoxyresorufin-O-deethylase(EROD) and 7-ethoxycoumarin-O-deethylase(ECOD) with fluorospectrophotometry.The results suggested that both cytochrome P450 and cytochrome b5 contents were the highest expressed level in liver microsome,followed by kidney,gill,intestine microsomes.There was the lowest content in muscle microsome.The P450 content and b5 content of liver microsome were 0.312±0.008 nmol/mg,0.142±0.010 nmol/mg,respectively,However,the P450 content and b5 content of muscle microsome were 0.008±0.032 nmol/mg, 0.0015±0.033 nmol/mg,respectively.The activities of APD and ERND had similar distribution variability,which were 1.668±0.104 nmol/min/mg,0.941±0.061 nmol/min/mg in liver microsome,and also were the lowest in muscle microsome,0.245±0.011 nmol/min/mg and 0.078±0.019 nmol/min/mg,respectively.The activity of AH was 0.052±0.009 nmol/min/mg in liver microsome,followed by 0.044±0.007 nmol/min/mg in gill microsome,however,not determinded in muscle microsome.EROD and ECOD were highest expressed level in liver microsome,which were 0.064±0.008 nmol/min/mg and 0.037±0.006 nmol/min/mg,respectively,followed by the contents in kidney and gill microsomes.There were very low expressed level in intestine microsome,however,not determinded in muscle microsome.The aboved results showed that the above-mentioned P450 subforms had certain variability of distribution and activity in main tissular microsoms from Carasius auratus gibelio.The enrofloxacin and its metabolites in the hemolymph from Carasius auratus gibelio following oral administration were analyzed using positive ion electrospray ion trap mass spectrometry in a multi-stage MS full scan mode.The multi-stage mass spectral information was obtained following the mass spectrometric analysis of enrofloxacin and its metabolites by collision induced dissociation.The result showed that enrofloxacin was biotransformed to the metabolites in the hemolymph,which the peak area of ciprofloxacin is the biggest among the metabolites,the second is hydroxylate enrofloxacin.An enzymatic reaction system with enrofloxacin(EF) in Carasius auratus gibeIio liver microsome was established.A reliable method for the determination of EF-N-deethylase activity using major metabolite ciprofloxacin(CF) by reversed phase high performance liquid chromatography(RP-HPLC) was established.The medicine was extracted by dichioromethane and the liver microsome was degreased by N-hexane. Average recoveries were higher than 81.4%,CF or EF were stable at 4℃for at least 72 h in spiked liver microsomes samples.The inter-day precision and intra-day precision were lower than 3.51%and 3.71%,respectively.The accuracy of EF and CF was lower than 8.1%and 6.5%,the detection limit for EF and CF(signal-to-noise ratio of 3) was 0.15 and 0.3μmol/L.The data of the metabolism studies were fitted into first-order elimination equation C=140 e^-0.0072t,the elimination rate constant(K) and elimination half life(T_1/2) were 0.072/h and 9.627 h,respectively.The Michaelis-Menten parameters such as K_m and V_max were estimated by analyzing Eadie-Hofstee plots using the software Origin Lab,the equation y=P₁·x/(P₂+x)was applied to demonstrate the relationship between K_m and V_max.The maximum reaction rate(V_max)was reached to 0.2602±0.0147 nmol/(mg·min),with a michaelis constant(K_m) of 53.8985±10.2257μmol/L and the internal clearance rate(C_Iint)of 0.0048 mL/(min·mg) could be calculated. According to the results,the affinity between EF-deethylase and the substrate was moderate.The intensity for enzymatic reaction and the metabolism for EF was low.A reliable method was developed for evaluating vitro and vivo effects of EF on the aboved P450 isoenzymes of Carasius auratus gibelio liver microsome.Carasius auratus gibelio pretreated with a single dose of EF(3 mg/kg i.p.),three daily doses of EF(3 mg/kg i.p.),three daily doses of EF(30 mg/kg i.p.) and with three daily doses of EF(30 mg/kg i.o.),respectively.Activities of main drug metabolism enzymes were determined after 24 h from the last administration.The results suggested that there was different dosage-time effect in according to the inhibition of i.p.EF on main drug enzymes with different dosage and time.And the inhibition of i.o.EF on main drug enzymes was greater than the i.p.EF. In vitro experiments showed that EF at 10μM inhibited the activities of APD,EROD, ECOD,and ERND at different degree,in particular,the erythromycin N-demethylase (ERND),likely dependant on a P450 3A subform,the inhibition rate is about 0.40,while the inhibition rate of AH is lowest.In conclusion,the EF seems to be a powerful inhibitor of P450s in the Carasius auratus gibelio liver microsome.Therefore,the clinical use of this antibiotic in aquaculture has to be considered with caution.