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恩诺沙星在异育银鲫肝微粒体中代谢及对P450主要亚酶影响的初步研究

2024-04-26 来源:爱问旅游网
恩诺沙星在异育银鲫肝微粒体中代谢及对

P450主要亚酶影响的初步研究

摘 要

细胞色素P450酶系(Cytochrome P450,简称CYP或者P450)是参与药物代谢最主要的一组同工酶。P450介导了药物在生物体内的代谢过程,而药物可能对参与其代谢的P450相关酶活产生诱导或抑制作用。本试验选取我国普遍养殖的淡水品种异育银鲫(Carasius auratus gibelio)为研究对象,以第三代氟喹诺酮类药物恩诺沙星(Enrofloxacin)为主要研究药物,考察恩诺沙星在其肝微粒体的代谢情况及对主要P450亚型酶活影响进行初步研究。以期为该类药物对鱼类P450的进一步深入研究,指导临床合理应用提供理论基础。

应用差速离心法提取银鲫微粒体,以CO还原差示光谱法测定肝、肾、鳃、肠、肌肉微粒体的细胞色素P450及b5含量。结果显示,细胞色素P450及b5含量均以肝微粒体最高,其P450及b5含量分别为0.312±0.008 nmol/mg,0.142±0.010 nmol/mg,其次为肾、鳃、肠微粒体,肌肉最低,其P450及b5含量分别为0.008±0.032 nmol/mg,0.0015±0.033 nmol/mg。用紫外分光光度法测定CYP2B型氨基比林N-脱甲基酶(Aminopyrine N-demethylase,APD)、CYP3A型红霉素N-脱甲基酶(Erythromycin N-demethylase,ERND)及CYP2E型苯胺-4-羟化酶(4-aniline-hydroxylase,AH)活性。APD及ERND活性在上述组织中分布差异性类似,均表现为肝微粒体中最高,分别为1.668±0.104 nmol/min/mg和0.941±0.061 nmol/min/mg,其次为肾、鳃、肠微粒体,肌肉微粒体中最低,两者活性分别为0.245±0.011 nmol/min/mg和0.078±0.019 nmol/min/mg;AH活性以肝微粒体最高,为0.052±0.009 nmol/min/mg,其次为鳃微粒体,为0.044±0.007 nmol/min/mg,肌肉微粒体不能检出。以7-乙氧基异吩噁唑酮-O-脱乙基酶(7-ethoxyresorufin-O-deethylase,EROD)活性及7-乙氧基香豆素-O-脱乙基酶(7-ethoxycoumarin-O-deethylase,ECOD)活性反映CYP1A的活性,用荧光分光光度法测定两者活性在肝微粒体中最高,分别为0.064±0.008 nmol/min/mg 和0.037±0.006 nmol/min/mg,其次为肾和鳃微粒体,肠微粒体含量最低,而肌肉微粒体不能检出。提示银鲫主要组织微粒体具有上述P450亚型活性,且它们在银鲫体内的分布和活性存在组织和器官差异性,各酶活性均以肝微粒体中含量最高。

采用电喷雾离子阱质谱法在正离子检测方式下,对异育银鲫口灌恩诺沙星给药后血淋巴中的恩诺沙星和其代谢产物进行定性分析。对其中恩诺沙星及代谢产物的质谱裂解行为进行碰撞诱导解离分析,结果表明恩诺沙星在异育银鲫体内的代谢反应以脱乙基反应为主,还有羟基化反应。应用RP-HPLC法检测其主要代谢产物环丙沙星(Ciprofloxacin, CF),以间接反映恩诺沙星N-脱乙基酶的活性。结果显示,采用二氯甲烷提取、正已烷去脂提取有效成分,样品回收率均大于81.4%,含CF或EF的肝微粒体样品能在4 ℃下稳定至少72 h,日内变异系数不超过3.51%,日间变异系数不超过3.71%,EF和CF准确度分别小于8.1%、6.5%,检测限(信超比为3)分别为0.15 µmol/L、0.3 µmol/L。EF在银鲫肝微粒体中代谢符合一级消除方程

C140e0.072t,消除速率常数(K)为0.072 /h,消除半衰期(T1/2)为9.627 h。用Origin

Lab软件的Hyperbl模型进行拟合,以方程yP1x/(P2x)表示米氏常数Vmax、Km两者的关系,求得Vmax为0.2602±0.0147 nmol/(mg·min),Km为53.8985±10.2257 µmol/L,内在清除率(Clint)约为0.0048 mL/min·mg。据此,推测EF-N-脱乙基酶与底物结合力不强,酶促反应强度处于中等水平,EF在银鲫肝微粒体中的代谢较缓慢。

分别采用腹腔注射单次给药3 mg/kg、3 mg/kg连续给药三天及30 mg/kg连续给药三天,30 mg/kg口灌给药三天,各组均于末次给药24 h后对银鲫肝微粒体上述五个主要药酶进行活性测定,并对各试验组进行组间差异性比较分析。结果表明腹腔注射给药组的恩诺沙星对银鲫鱼肝微粒体主要酶活的抑制具有不同程度的剂量-时间效应,而口灌组恩诺沙星对银鲫肝微粒体酶活抑制强度均普遍高于腹腔注射给药组。体外实验表明10 µM恩诺沙星均能不同程度抑制APD、ERND、EROD、ECOD及AH活性,特别是对依赖于3A亚型的ERND抑制效果最为显著,抑制率达到0.40左右,AH酶受抑制最小。提示EF对银鲫肝微粒体上述主要药酶具有广泛的抑制作用,水产养殖临床应用此药需慎重。

关键词:药物代谢酶;细胞色素P450;异育银鲫;肝微粒体;恩诺沙星

Preliminary Studies on Metabolism and Impact on

Main P450 Isoenzymes of Enrofloxacin in Carasius auratus gibelio liver microsome

Abstract

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 gibelio 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 dichloromethane 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 0.3 µmol/L. The data of the metabolism studies were fitted into first-order elimination equation

C140e0.0072t, the elimination rate constant (K) and elimination

half life (T1/2) were 0.072 /h and 9.627 h, respectively. The Michaelis-Menten parameters such as

Km and

Vmaxwere estimated by analyzing Eadie-Hofstee plots using the

software Origin Lab ,the equation relationship between

Km

yP1x/(P2x)was applied to demonstrate the

and

Vmax. The maximum reaction rate (Vmax)was reached to

0.2602 ±0.0147 nmol/(mg·min), with a michaelis constant (Km) of 53.8985±10.2257 µmol/L and the internal clearance rate(Clint)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 10 µM 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.

Key words: drug metabolism enzymes; P450; Carasius auratus gibelio; liver microsome;

enrofloxacin

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