Effect Of Quinolones On Cytochrome P450 In Crucian Carp (Carassius Auratus Gibelio) And Cloning Of CYP3A Gene

Effect of quinolones on cytochrome P450 in Crucian Carp (Carassius auratus gibelio) and cloning of CYP3A gene Hu Xiao (Aquaculture) Directed by Dr. Wen-hong FangThe whole dissertation was divided into two parts. Part one was to investigate the effects of two fluoroquinolones, flumequine and enrofloxacin on cytochrome P450 of Crucian Carp(Carassius auratus gibelio). In this part, fish administered with flumequine was used to study the enzymes, protein and mRNA of CYPs in different tissue; In the second group, time course and dose-response of inhibition by enrofloxacin was studied at enzymes, protein and mRNA level. In part two, cloning, sequencing and tissue expression of the cytochrome CYP3A gene in Crucian Carp (Carassius auratus gibelio) was present, using RACE and semiquantitative RT-PCR assay.1. Effect of flumequine and enrofloxacin on cytochrome P450 in Crucian CarpThe effects of flumequine on microsomal cytochrome P450 monooxygenases in Crucian Carp(Carassius auratus gibelio) were examined during this study. After 24h treatment, flumequine, administered to fish with a single intraperitoneal injection at a dose of 35mg/kg, resulted in a significant induction in the liver of the P4501A-linked ethoxyresorufin-O-deethylase (EROD) activity (54.33±5.42 pmol/mg-min) compared to control values (34.00±5.87 pmol/mg-min) (p<0.01). Erythromycin N-demethylase (ERND), aminopyrine N-demethylase (APD) and ethoxycoumarin O-deethylase (ECOD) were not affected by flumequine. Liver has the highest P450 enzymes activities except for ERND in kidney. Western blotting indicated that CYP1A protein level was induced (about 2 fold), which is consistent with the EROD activity in the experimental group. Semiquantitative RT-PCR analysis showed CYP1A mRNA were all expressed in liver, kidney and intestine, however, there is no significant differences between control and experimental groups. In vitro assay, various concentrations of flumequine were incubated with microsomes, no dose-and time-dependent manner was observed. The lack of induction speculated that flumequine caused an activation of CYP1A, occured at post-translational level, probably by protein stabilisation.Enrofloxacin exhibited a potent inhibition (40%) on CYP3A-linked erythromycin N-demethylase (ERND) after a single intraperitoneal injection at a dose of 10mg/kg. ERND activity was decreased within 48h (306.93±19.30 pmol/mg·min) and retrun to pre-treatment level at 8d (496.76±46.04 pmol/mg-min). As for CYP1A, ethoxyresorufin-O-deethylase (EROD) activity was maximal inhibited at 24h (85.79±6.86/71.50±7.38 pmol/mg·min, for control and experimental group, respectively), but not as much as that in CYP3A. Enrofloxacin caused a slight increase in CYP1A protein level, while CYP3A protein was markedly decreased in 4d. Semiquantitative RT-PCR analysis showed that CYP1A mRNA and CYP3A mRNA were all decreased at 24h and 48h respectively, but have not returned to control level after 8d. This suggest a long-term inhibition of CYP1A, CYP3A by enrofloxacin. In the dose-response study, different concentrations of enrofloxacin (0mg/kg,3mg/kg,10mg/kg,30mg/kg,60mg/kg) were administered. Fish were sampled 24h after treatment, the results showed a dose-response reduction of hepatic CYP1A and CYP3A activity. The maximal inhibition of EROD and ERND was 48%,52%, respectively. Western blotting indicated that CYP1A protein level was not affect at dose of 3mg/kg,10mg/kg, but was significantly decreased (about 50%) at a higher dose. CYP3A protein content was remain at pre-treatment level after various concentration of enrofloxacin injection. Semiquantitative RT-PCR assay suggested that the changes of CYP1A mRNA is correlated well with that in EROD activity. The content of CYP3A mRNA was still unchanged. In vitro experiment, enrofloxacin, when preincubated with Crucian Carp liver microsomes, inhibited ERND activity in a dose-and time-dependent manner, however, the CYP1A-linked EROD activity was not affected. Taken overall, the inhibition of enrofloxacin on CYP1A probably occured in vivo, while enrofloxacin has been also demonstrated to be a powerful mechanism-based inhibitor primarily of P450 3A isoform in Crucian Carp.2. Cloning, sequencing and tissue expression of the cytochrome P450 3A gene in Crucian CarpDegenerate primers derived from conserved CYP3A sequences of four kind of fish were used to clone Crucian Carp CYP3A. A cDNA sequence (GenBank accession number: GU998964) was isolated and identified from the total RNA using RT-PCR and RACE assay. The sequence of CYP3A is 1770bp which contains a 5'UTR of 30bp and 3'UTR of 195bp, and an open reading frame (ORF) with 1545 bp that encodes a protein of 515 amino acids. The amino acid sequence of Crucian Carp shared 73%-75% of residue identity with the CYP3A subfamily in human, mouse and rat, and shared 92% of residue identity with Gobiocypris rarus. Molecular phylogenetic analysis of Crucian Carp CYP3A with other CYP3A forms from mammals and teleosts indicated that Crucian Carp CYP3A amino acid sequence has the highest identity with cyprinid fishes (Cyprinidae). Semiquantitative RT-PCR analysis showed that mRNA expression of CYP3A can be detected in liver, kidney, intestine, gill, muscle and spleen. Liver was found to have the highest CYP3A mRNA expression, followed by intestine, kidney and gill.