| Oxolinic acid, ciprofloxacin and sulfadimidine are commonly used in aquaculture of antibiotic. Cytochrome P450is involved in drug metabolism in biology as the main enzymes, among which CYP3A can metabolize most types of drugs, including antibacterial drugs and antiviral drugs. The reasonable dosage regimen of the three drugs were established by studying their pharmacokinetics. In order to provide theoretical basis for clinical drug rationally, the relations of drug metabolism, CYP3A expression and enzymes activity were also explored in this paper. The research was divided into three parts:The pharmacokinetics of Oxolinic acid was studied in Scophthalmus maximus by using the high performance liquid chromatography (HPLC). The study was performed at(24.5±0.2)℃. All the health fish received a single oral administration of OA at a level of20mg/kg and intravenous administration of OA at a level of10mg/kg. The results showed that the hemolymph concentration-time course of OA can be described by a two-compartment open model with the first order absorption after a single oral administration, the pharmacokinetic equation was:Cpo=2.059e-0062t+0.645e-0023t-2.704e-0202t, the hemolymph concentration-time course of OA can be described by a two-compartment open model with the zero order absorption after a single intravenous administration, the pharmacokinetic equation was:Civ=12.284e-0.144t+0.284e-0.027t. The main pharmacokinetics parameters in plasma were those:The peak time,distribution and elimination half-lives (tmax,t1/2α and t1/2β) by intravenous administration were found to be0.083h,4.813h and25.441h which were shorter than6h,11.26h and30.212h of oral administration. The results indicated that the peak time, absorption and eliminate speed of intravenous administration were faster than oral administration. To the experimental results, the scheme of oxolinic acid was established to fish bacterial diseases, with the dose of21.41mg/kg at the interval of1d, continuous5-7days by oral administration.The pharmacokinetics of ciprofloxacin and sulfamethazine was studied in Scophthalmus maximus by using the high performance liquid chromatography (HPLC). The study was performed at(23.1±0.8)℃. All the health frish received a single oral administration of CIP and SM2at a level of20mg/kg and intravenous administration of CIP and SM2at a level of10mg/kg. The results showed that the hemolymph concentration-time course of CIP and SM2can be described by a two-compartment open model with the first order absorption after a single oral administration, the pharmacokinetic equations were:CCIP=14.811e-0337t+4.028e-0.063t-18.839e-0.616t、 CSM2=64.981e-0.141t+4.59e-0.004t-69.571e-0.19t; the hemolymph concentration-time course of CIP and SM2can be described by a two-compartment open model with the stepless absorption after a single intravenous administration, the pharmacokinetic equations were:CCIP=21.784e-1098t+1.514e-0.043t、CSM2=33.028e-5.687t+8.674e-0.013t. After the same dose oral drugs, the main pharmacokinetics parameters in plasma were those:the peak time, peak concentration, absorption, distribution and elimination half-lives (Tmax, Cmax, t1/2Ka, t1/2α and t1/2β) by ciprofloxacin administration were found to be6h,5.385μg/mL,1.125h,2.057h and11.028h which were shorter than8h,13.990μg/mL,3.647h,4.923h and173.407h of sulfamethazine administration,but its bioavailability60.57%were bigger than sulfamethazine administration which was47.13%. The results indicate that the peak time, absorption, distribution and eliminate speed of Ciprofloxacin administration were faster than sulfamethazine administration, ciprofloxacin was absorbed completely. To the experimental results, the scheme of ciprofloxacin and sulfamethazine was established to fish bacterial diseases, with the dose of28.01mg/kg and18.32mg/kg at the interval of Id, continuous3-5days by oral administration,respectively.The cytochrome P450is an important group of mixed function oxidases that play important roles in the metabolism of multiple exogenous substances. In this study, a cytochrome P4503A (CYP3A) gene was cloned from the turbot Scophthalmus maximus by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the CYP3A was of1969bp, containing a5’untranslated region (UTR) of34bp, a3’UTR of298bp with a poly (A) tail, and an open reading frame (ORF) of1637bp encoding a polypeptide of509amino acids with the predicted molecular weight of58.09kDa. The CYP3A amino acid sequence consists of a signal peptide and six conservative substrate recognition sites (SRS1-6). The conserved heme-binding motif of cytochrome P450monooxygenases (FXXGXXXCXG) identified in CYP3A suggested that the CYP3A belonged to the cytochrome P450subgroup. Sequence comparison showed that CYP3A of S. maximus shared66%,65%,65%,64%and64%identity with that of fishes Oryzias latipes, Micropterus salmoides, Fundulus heteroclitus, Salmo salar and Oncorhynchus mykiss, respectively. Quantitative real-time RT-PCR analysis revealed that S. maximus CYP3A transcript was detected in liver, kidney, gill, muscle, stomach, intestine, gallbladder and spleen. The expression level of CYP3A transcript in liver increased in the first24h after oral administration of sulfamethazine, as the administration progressed, the treated groups decreased to normal levels. The enzymes activity of ERND which is sign enzymes of CYP3A was detected by ELIAS A. The result was that the acticity of ERND was higher in experimental than in control group.ERND of high dose was bigger than low dose at the same time. Both the enzymes activity and expression level have the same change trend. These facts indicated that CYP3A transcript could be induced by sulfamethazine and might be involved in the drug-metabolic response of S. maximus. |
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