| This research determined the induction effects and mechanism of ionophores, the inhibitory effects and mechanism of fluoquinolones and the inhibitory effects of classic inhibitors on CYPIA and 3A in broiler chicks. The purpose of research was to investigate pharmacological mechanism of clinical drug on chicken CYPIA and 3A enzyme, thus to provide a theoretical basis for clinical application. The details are divided into five parts as follows:1. To investigate the effects of three ionophore antibiotics on the enzyme activities of CYP1A and 3A in broiler chicks. Monensin, salinomycin and maduramycin at the dosage of 120,60 and 5 mg/kg were administrated in feed for 14 days. CYP1A and 3A activities were quantitated using cocktail probe drug including caffeine and dapsone in vivo and 0.5-20 mM phenacetin and 0.125-5 mM midazolam as substrates of microsome incubation in vitro. The results in. vivo indicated that three ionophores did not change the pharmacokinetics parameters of caffeine (P>0.05), but did decrease the AUC and t1/2 of dapsone and increase the total clearance, compared with control group, and the induction ability on dapsone metabolism is salinomycin> monensin> maduramycin. The results of microsome incubation trials indicated that three ionophores didn’t change the Vmax of acetaminophen production (P>0.05). Michaelis constant Km showed no significant difference with control group (P>0.05). Monensin significantly increased the production velocity of 1-hydroxy midazolam (P<0.01), while salinomycin and maduramycin increasd it with no significant difference (P>0.05). Three ionophore-treated groups had the similar Km value as control group (P>0.05). The similar results in vivo and in vitro suggest that monensin, salinomycin and maduramycin in feed for 14 days may have an induction effect on CYP3A enzyme activity of male AA broilers and maduramycin has the least induction effect, while they all don’t induce the CYP1A enzyme activity.2. To know the induction mechanism, the effect of three ionophore antibiotics on the CYP450, b5 content and the mRNA expression of hepatic CYP1A4,1A5 and 3A37 mRNA and the protein expression of CYP1A and 3A were investigated in broiler chicks. Content of CYP450 and b5 was measured by spectrophotometry. The mRNA levels of CYP1A4, CYP1A5 and CYP3A37 was detected by Real-time reverse transcriptase-polymerase chain reaction (Real-time PCR). The protein expression of CYP1A and CYP3A was detected by Western blot. No change was found in the CYP450 and b5 content, and CYP1A4, CYP1A5 and CYP3A37 mRNA expression in ionophore-treated groups compared with control group. Monensin and salinomycin increased the CYP3A protein expression with significant difference (P<0.05). However, maduramycin increased it with no significance difference (P>0.05). No change was observed in CYP1A protein expression. It is suggested that ionophore antibiotics have an induction effect on CYP3A expression and enzyme activity and such effects might be related to the post-transcriptional regulation of its protein expression.3. To investigate the inhibitory effects of three fluoquinolone antibiotics on the enzyme activities of CYP1A and 3A in broiler chicks. Enrofloxacin, sarafloxacin and marbofloxacin at the dosage of 20,8 and 5.5 mg/kg body weight were administrated for 7 days. Inhibitory effects of fluoquinolones on CYP1A and 3A activities were quantitated using cocktail probe drugs in vivo and microsome incubation in vitro. The results in vivo indicated that enrofloxacin and marbofloxacin increased the t1/2 of caffeine (P<0.05), and marbofloxacin increased the AUC, decreased the total clearance, compared with control group(p<0.05). Enrofloxacin increased AUC and t1/2 of dapsone metabolism (P<0.05), and decreased the total clearance (P<0.01). The results of microsome incubation trials indicated that all of three fluoquinolones decreased the Vmax of acetaminophen production (P<0.05). Enrofloxacin significantly decreased the production velocity of 1-hydroxy midazolam and the Km value (P<0.01), while sarafloxacin and marbofloxacin groups had the smaller Km value with no significant difference (P>0.05). The results in vivo and in vitro suggest that high dosage of enrofloxacin, sarafloxacin and marbofloxacin may have an inhibitory effects on CYP1A enzyme activity of broilers (marbofloxacin> enrofloxacin> sarafloxacin). Meanwhile, CYP3A activity could be inhibited by high dosage of enrofloxacin, but not sarafloxacin and marbofloxacin.4. To investigate the effect of three fluoquinolone antibiotics on the CYP450, b5 content, the CYP450 mRNA expression and CYPIA and 3A protein expression in broiler chicks. Enrofloxacin, sarafloxacin and marbofloxacin at twice the recommended dosage were administrated for 7 days. Content of cytochrome P450 and b5 was measured by spectrophotometry. The mRNA levels of CYP1A4, CYP1A5 and CYP3A37 was detected by Real-time PCR. The protein expressions of CYP1A and CYP3A was detected by Western blot. The results indicated that enrofloxacin and marbofloxacin decreased CYP450 content (P<0.05), while sarafloxacin didn’t change it. No change was found in the b5 content, and CYP1A4, CYP1A5 and CYP3A37 mRNA expression in fluquinolone-treated groups compared with control group. Enrofloxacin and marbofloxacin decreased the CYP1A protein expression with significant difference (P<0.05), meanwhile, enrofloxacin decreased the CYP3A protein expression (P<0.05). However, this high dosage of sarafloxacin did no effect on CYP1A and 3A protein expression. It is suggested that high dosage of enrofloxacin may inhibit CYPIA and 3A activities and protein expression, while marbofloxacin inhibit CYPIA activity and protein expression.5. Phenacetin O-deethylation and midazolam 1-hydroxylation activities were measured in chickens and rats. The inhibitory effects and mechanism of classic inhibitor a-napthtoflavone, ketoconazole and three fluoquinolone antibiotics were examined on phenacetin O-deethylation and midazolam 1-hydroxylation activities in chickens. We used mixed microsome incubation in vitro, and the formation velocity of acetaminophen and 1-hydroxy midazolam were referred as CYPIA and 3A activities. Both chicks and rats demonstrated Michaelis-Menten kinetics for the phenactin and midazolam metabolite. The Vmax for phenactin of chicks and rats was 0.089±0.053,0.671±0.288 nmol/mg/min and Km was 78.45±48.10,107.1±45.5μM, respectively. The Vmax for midazolam was 0.081±0.022,0.781±0.275μmol/mg/min and Km 5.33±2.56,1.85±0.67μM, respectively. a-napthtoflavone inhibited the phenacetin metabolism in a noncompetitive manner and IC50 = 0.1304μM, Ki= 0.580μM, while marbofloxacin with IC50= 93.79μM, Ki= 77.12μM, in a competitive-noncompetitive mixed manner. No inhibitory effect was examined of enrofloxacin and sarafloxacin. Ketoconazole inhibited the midazolam metabolism in the noncompetitive-uncompetitive mixed manner, as the same like three fluoquinolones. The IC50 value was 30.6,63.93,145.4 and 42.52μM, and Ki value was 93.01,70.76,150.84 and 64.24μM, respectively. Enrofloxacin, sarafloxacin and marbofloxacin showed time-dependent irreversible inhibitory effects on both phenacetin and midazolam metabolism. The result showed that the CYP1 A and 3A activities were markedly lower in chickens than in rats. a-napthtoflavone had strong.inhibitory effect on chicken CYP1 A activity, while ketoconazole had only weak inhibitory effect on chicken CYP3A activity like enrofloxacin, sarafloxacin and marbofloxacin. The time-dependent inhibitory may be the reason for the decreased CYP1A and 3A enzyme activities and protein expression, and the irreversible inhibitory would result in substantial drug-drug interaction in vivo. |
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