喹噁啉类在大鼠、猪和鸡的比较代谢研究
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摘要
喹噁啉类是化学合成的具有抗菌、促生长作用的动物专用药,该类的卡巴氧和喹乙醇已被证明有较强的毒副作用,并被禁止或限制用作饲料药物添加剂。我国相继批准了乙酰甲喹和喹烯酮的使用,并正在开发安全的喹赛多等同类产品。国内外研究证明,卡巴氧和喹乙醇的毒性与其代谢密切相关,喹噁啉类原形在组织中无残留,但有其代谢物残留。目前,国内外主要确定了喹乙醇、卡巴氧及喹赛多的还原代谢物和残留标识物,对许多存在的未知代谢物尚未鉴定,对乙酰甲喹和喹烯酮的代谢研究也未报道,同时,喹赛多的代谢存在种属差异,而其它喹噁啉类是否也存在代谢种属差异还尚不明确。作为食品生产动物用药,其安全性已成为食品加工科技领域原料控制质量的一个研究热点,而作为影响食品安全性的主要因素之一的兽药残留,其代谢研究更是避免残留的重要科技基础。因此,本课题以喹噁啉类为研究对象,采用液相与离子阱飞行时间质谱(LC/MS-ITTOF)联用技术,比较喹乙醇在大鼠、猪和鸡肝微粒体与肝细胞中的代谢,且在此基础上,研究其它喹噁啉类在肝微粒体的比较代谢,并全面系统研究喹乙醇在体内的比较代谢,进一步探讨喹乙醇还原、氧化和N-脱羟乙基化代谢酶的种属差异,旨在阐明喹噁啉类在不同动物的代谢特点以及结构与代谢的关系,以全面揭示喹乙醇的生物转化及种属差异原因。
1.受试动物体外肝代谢系统建立
用差速离心法制备大鼠、猪和鸡肝微粒体,测定细胞色素P450(CYP)的CO差示光谱,考察探针药物香豆素在微粒体中的活性;用改良的胶原酶灌注法分离肝细胞,对肝细胞进行计数和活率测定,考察香豆素在肝细胞中的代谢。结果表明,CYP在450nm处有最大吸收,香豆素在肝微粒体与肝细胞中均能代谢成7-羟基香豆素,从而证实了肝微粒体与肝细胞具有代谢酶的活性。
用探针药物香豆素进一步考察猪肝微粒体CYP2A的酶促动力学,评价5种人CYP同工酶常用的选择性抑制剂对猪CYP2A活性的抑制效果。结果显示,猪肝微粒体中香豆素7-羟化酶(CYP2A)的酶促动力学参数K_m和V_(max)分别是1μmol/L和0.26nmol/mg/min,磺胺苯吡唑(SUL)、奎尼丁(QUI)和醋竹桃霉素(TAO)对CYP2A很少或无抑制作用(K_i＞200μmol/L);α-萘黄酮(ANF)预反应对猪肝微粒体CYP2A的抑制无增强作用,其抑制常数K_i为32μmol/L:8-甲氧补骨脂素(MOP)无预反应时对猪肝微粒体CYP2A的抑制常数K_i为1.1μmol/L,当预反应3min后,其抑制常数K_i降为0.1μmol/L。由此表明,MOP可作为猪肝微粒体CYP2A的一个基于机制失活的特异性非竞争性抑制剂。
2.喹乙醇在离体肝系统的比较代谢研究
选择已建立的肝微粒体和肝细胞系统,研究喹乙醇或其代谢物脱二氧喹乙醇在肝系统的比较代谢,用高灵敏、高质量精度、高分辨率和多级质谱的LC/MS-ITTOF可快速、准确地鉴定其代谢物。结果显示,喹乙醇在大鼠肝微粒体中可代谢成13种代谢物,而猪和鸡只检出7种;同时,除了已报道的3种还原代谢物(O1、O2、O9)和2种羧酸衍生物(O8、O10)外,首次鉴定了5种羟化物(O3～O7)、2种N-去乙醇代谢物(O11、O12)和1种醛中间代谢物(O13);并且,O2是喹乙醇在猪和鸡的主要代谢物,而O1、O2和O9是大鼠的主要代谢物。脱二氧喹乙醇在大鼠肝微粒体可代谢成6种代谢物,且鉴定为O2、O10、O12、O13和2种羟化代谢物(O14、O15);在猪和鸡肝微粒体中均能检出除O15外的其它5种代谢物;O10是脱二氧喹乙醇在三种动物的主要代谢物。喹乙醇在猪和大鼠肝细胞中可生成5种代谢物,分别是O1、O2、O5、O8和O9;鸡肝细胞中除O8外,其它4种都检出。本试验结果表明,喹乙醇的主要代谢途径是N→O基团还原和羟基氧化,大鼠的N→O基团还原和羟化能力最强,鸡的氧化能力最强,喹乙醇N→O基团还原和N-氧化可相互转化,O13、O15和3种还原代谢物是潜在的毒性代谢物。
3.乙酰甲喹、喹烯酮、卡巴氧和喹赛多在肝微粒体的比较代谢研究
为阐明喹噁啉类各自的代谢特点以及结构与代谢的关系,本研究比较了其在肝微粒体中的代谢。结果显示,乙酰甲喹在鸡肝微粒体中能代谢成14种代谢物,且鉴定为3种N→O基团还原代谢物(M1、M2、M6)、5种羰基还原代谢物(M10～M14)、6种羟化代谢物(M3、M4、M5、M7、M9、M8);在大鼠能检出10种代谢物,分别是M1、M2、M3、M4、M6、M7、M10、M11、M12和M14;而在猪肝微粒体中,除检出大鼠的10种外,还检出了M13;所有这些乙酰甲喹代谢物都是首次报道,并且,M2、M10和M12是猪的主要代谢物,M2、M6和M12是鸡的主要代谢物,M2和M6是大鼠的主要代谢物。喹烯酮在大鼠肝微粒体可代谢成27种代谢物(Q1～Q27);鸡肝微粒体中能代谢成23种,其中Q1～Q27中无Q8、Q11、Q14、Q15、Q19、Q25、Q26、Q27,但检出Q28～Q31;猪肝微粒体代谢成25种,其中Q1～Q27中无Q8、Q9、Q10、Q11、Q14,但检出Q28、Q32和Q33;这些代谢物除脱二氧喹烯酮(Q6)外,其它都是首次鉴定,Q2和Q4是大鼠的主要代谢物,Q2、Q4、Q17和Q20是鸡的主要代谢物,Q2和Q20是猪的主要代谢物。卡巴氧在大鼠肝微粒体中能生成6种代谢物,且鉴定为3种N→O基团还原物(Cb1～Cb3)和3种羟化代谢物(Cb4～Cb6);猪和鸡都只检出Cb1、Cb2、Cb3和Cb4,其中Cb1和Cb4是猪的主要代谢物,Cb1、Cb3和Cb4是鸡的主要代谢物,Cb1是大鼠的主要代谢物;除Cb3外,其它5种代谢物也属首次鉴定。喹赛多在猪肝微粒体中可代谢成7种代谢物,且鉴定为3种N→O基团还原物(Cy1、Cy2、Cy4)、1种羟化代谢物(Cy3)和3种水解代谢物(Cy5～Cy7),除代谢物Cy7外,其它6种代谢物都在大鼠和鸡肝微粒体中检出,其中Cy1、Cy4、Cy5和Cy6是猪的主要代谢物,Cy1是鸡的主要代谢物,Cy1和Cy4是大鼠的主要代谢物;除Cy2和Cy4外,其它5种代谢物仍属首次鉴定。
喹噁啉类共同的主要代谢途径是N→O基团还原,其次是羟化,且其毒性与其N→O基团还原有关。不同化合物由于侧链不一样,其代谢途径存在明显差异,同一化合物在不同动物的代谢途径基本相似,但主要代谢物和代谢物的量存在明显的种属差异。大鼠对喹噁啉类的N→O基团还原和羟化能力最强,猪对其羰基还原和酰胺水解能力最强,鸡则对其羟基氧化能力最强。
4.喹乙醇在大鼠、猪和鸡体内的比较代谢研究
大鼠、猪和鸡分别按10、5和30mg/kg b.w.单次口服给药后,对其尿液、血浆、粪便、肌肉、肝脏、肾脏和胃肠内容物中的代谢物进行检测。结果显示,在大鼠尿液中共检出18种代谢物,其中O1和O2是雌性大鼠的主要代谢物,O2、O9和O13是雄性大鼠的主要代谢物;雌性大鼠粪便、盲肠内容物、肌肉、肾脏和肝脏中分别检出6种(O2、O9、O10、O13、O16和O19)、3种(O10、O13和O19)、2种(O9和O19)、2种(O9和O19)和3种(O9、O12和O19),雄性大鼠粪便中也能检出上述6种代谢物,其它组织都只检出O9。猪尿液中检测到16种代谢物,其中O2和O9是猪的主要代谢物;雌性猪粪便、盲肠内容物、肌肉、肾脏和肝脏中分别检出6种(O2、O9、O10、O13、O16和O19)、1种(O13)、2种(O9和O19)、2种(O9和O19)和3种(O9、O12和O19)代谢物,雄性猪粪便中同样能检出上述6种,而其它组织都只检出O9。鸡给药2h后,血浆中能检测出15种代谢物,其中O2、O8和O9是雌性鸡的主要代谢物,O8和O13是雄性鸡的主要代谢物;其粪中除上述15种代谢物外,还检出O16、O25和O26;雌性鸡肌肉、肝脏和肾脏分别检出1种(O9)、3种(O9、O12和O19)和6种(O2、O8、O9、O10、O11和O12),雄性鸡的肌肉中也只检出O9,而肝脏中能检出O10、O12和O19,肾脏中可检出O8、O9、O10、O11、O12、O19和O25。
除了3种还原代谢物、4种羧酸衍生物和3-甲基喹嚅啉-2-羧酸(O19)外,其它15种代谢物都是在体内首次报道。同时,除大鼠肝微粒体中的O6和O7外,其它所有在肝微粒体和肝细胞中检测到的代谢物在体内均可检出;喹乙醇在不同动物的主要代谢途径和代谢方式相同,但次要代谢物和代谢速率存在种属差异,喹乙醇及其代谢物在大鼠体内的排泄速度最快,鸡最慢;喹乙醇的代谢存在性别差异,雄性猪和大鼠的代谢和排泄速度比雌性快,但鸡却正好与之相反。
5.喹乙醇还原、氧化和N-脱羟乙基化代谢酶的种属差异研究
为深入了解喹乙醇代谢机制和解释种属差异,本研究比较喹乙醇N1与N4还原在大鼠、猪、鸡肝微粒和胞液中的还原机制,探讨不同动物催化脱二氧喹乙醇氧化和N-脱羟乙基化的CYP与非CYP。
肝微粒体或胞液与NAD(P)H在低氧条件下孵育后,喹乙醇能还原成O2,氧气和一氧化碳(CO)对其还原活性有抑制作用,核黄素对其活性有增强作用,煮沸的肝微粒体和胞液同样存在N1还原活性;甲萘醌能显著增加猪和大鼠胞液中O2的生成,而对鸡肝胞液中O2的生成无影响,且它在猪胞液中能直接还原喹乙醇,而在大鼠和鸡肝胞液中无此活性;以上结果证明,喹乙醇在肝微粒体和胞液的N1还原可经酶和非酶催化,其还原机制在肝微粒体无种属差异,但胞液存在酶的种属差异。苯甲醛与肝胞液在低氧条件下孵育后的结果显示,苯甲醛能显著增强大鼠和猪肝胞液中N4还原活性,也能增强大鼠肝胞液中N1还原活性;醛氧化酶(AO)和黄嘌呤氧化酶(XO)的抑制剂对大鼠N4还原活性有抑制作用,AO抑制剂对N1还原活性有抑制作用,提示大鼠AO和XO部参与喹乙醇N4还原,AO参与喹乙醇N1还原;AO抑制剂对猪肝胞液N4还原活性有抑制作用,表明猪AO催化喹乙醇的N4还原;鸡肝胞液不存在苯甲醛增强的作用,提示鸡可能缺乏AO和XO。
用不同浓度的脱二氧喹乙醇分别与大鼠、猪和鸡肝微粒体孵育,结果显示,O10在大鼠肝微粒体中受单一CYP催化,猪和鸡肝微粒体中至少受两种CYP催化,O12在三种动物均至少受两种CYP催化;O10的内在清除率在鸡最高,其次是猪,最后是大鼠,O12的内在清除率顺序正好相反。用选择性抑制剂探讨参与羟基氧化和N-脱羟乙基化反应的CYP的种属差异时,发现MOP、4-甲基吡唑(MP)和ANF抑制3种动物肝微粒体O10的生成,MOP预反应对其抑制无显著增强作用;而且,二乙基二硫代氨基甲酸酯(DDTC)和双硫仑(DIS)能抑制猪和鸡肝微粒体O10的生成,TAO和QUI能抑制剂鸡肝微粒体O10的生成;MOP、MP、ANF、DDTC和DIS都能抑制三种动物肝微粒体中O12的生成,其中DDTC和DIS的预反应对O12的抑制有增强作用;TAO和QUI对猪和大鼠肝微粒体O12的生成无抑制,但其预反应对鸡肝微粒体中O12的生成有显著抑制作用。上述研究表明,大鼠CYP1A催化羟基氧化,猪CYP1A和CYP2E可能催化羟基氧化,大鼠CYP1A和CYP2E催化N-脱羟乙基化反应,猪CYP2A和CYP2E参与其反应,鸡的多种CYP同工酶可能都参与这两种反应。
用化学抑制剂MP、DIS、DDTC、氯丙醛(CPZ)、异丙醛(PZ)、甲萘醌(MEN)和7-羟基香豆素(HCO)分别和脱二氧喹乙醇在大鼠、猪和鸡肝胞液中孵育,结果发现,大鼠胞液中,除HCO外的其它抑制剂对O10都有抑制作用,提示其胞液中醇脱氢酶(ADH)、醛脱氢酶(ALDH)和AO参与羟基氧化;猪胞液中,MP、CPZ和MEN对O10有抑制作用,表明其胞液中ADH和AO参与羟基的氧化;鸡胞液中,所有的抑制剂都对O10无抑制,提示抑制剂对其肝胞液中的酶无抑制或其它酶可能参与羟基氧化。
综上所述,本课题以比较代谢研究为中心,首次应用了LC/MS-ITTOF对代谢物进行定性与相对定量分析,阐明了喹噁啉类在不同动物的代谢途径和特点,鉴定了数十种未报道的代谢物,分析了结构与代谢的关系,揭示了喹乙醇在不同动物体内的命运,比较了体内与体外代谢的相关性,并深入探讨了喹乙醇代谢酶的种属差异。其研究结果证明了LC/MS-ITTOF是鉴定代谢物结构的快速、有效和可靠的分析工具,充分完善了喹乙醇的代谢及其代谢机制,为喹乙醇可能代谢部位确定、药动学特征及种属差异原因解释提供了重要的理论基础,对其毒副作用机制研究、残留监控和残留标识物确定具有十分重要的指导意义,为其它喹噁啉类N→O基团还原机制、代谢酶和体内代谢研究提供了重要思路。
Quinoxalines are animal special drugs of chemical synthesis which are widely used as antibacterial growth promoters. Carbadox and olaquindox are the well-known members, which have been banned or limited to be used in food animal for their potential side effects. Mequindox and quinocetone have been successively sanctioned to be used in food animals in China, and the safer medicines such as cyadox which belongs to quinoxalines family are being developed. Studies have demonstrated that the toxicities of carbadox and olaquindox are closely associated with their metabolism. Some metabolites of quinoxalines have been detected in various tissues, but parent drugs have not been. Currently, the major identified metabolites of olaquindox, carbadox and cyadox are their reduced metabolites and residue markers; most of their metabolites have not yet been identified. The metabolism of mequindox and quinocetone has not been investigated till now. Moreover, it has been demonstrated that the metabolism of cyadox has species difference. But, it is unknown whether the metabolism of other quinoxalines has also the species difference As drug for food producing animals, food safety has become a hot problem in the filed of food processing science. Drug residue is one of the main factors to endanger food safety, while drug metabolism is the scientific groundwork to avoid drug residue. Therefore, the quinoxalines were used as objects in this study. The comparative metabolism of olaquindox in rat, swine and chicken liver microsomes and hepatoctyes was investigated using high-performance liquid chromatography combined with IT/TOF mass spectrometry (LC/MS-ITTOF). Based on these results, the metabolic profiles of other quinoxalines in liver microsomes of the three species were compared. The metabolism of olaquindox in rats, swine and chicken was also examined in detail. In addition, the species differences of metabolic enzymes involved in the N→O group reduction, hydroxyl oxidation and N-dehydroxyethylation were studied. The aims of these studies were to clarify the metabolic characteristics and relationship between structure and metabolism of quinoxalines. Moreover, the biotransformation and reasons of species difference of olaquindox were revealed comprehensively in the study.
1. The establishment of in vitro liver systems of animal subject
Liver microsomes from rats, swine and chicken were prepared using differential centrifugation. The enzymatic activities of microsomes were evaluated by the carbon monoxide (CO) difference spectrum and the metabolism of coumarin as probe drug. Hepatocytes from rats, swine and chicken were separated by a modification of a collagenase perfusion method. The metabolic activities of hepatocytes were measured by the metabolism of coumarin after cell counting and cell viability assay. The results showed a maximum absorption peak of microsomes at 450nm. Coumarin could be metabolized to 7-hydroxycoumarin in liver microsomes and hepatocytes. Therefore, the in vitro liver systems had the activities of drug metabolic enzymes.
The enzymatic kinetic of CYP2A activity in swine liver microsomes was further examined using coumarin as a probe drug. The inhibitory effects on swine CYP2A activity were also evaluated using five commonly used human CYP inhibitors. The results demonstrated that the K_m and V_(max) for coumarin 7-hydroxylase (CYP2A) in swine liver microsomes were estimated to be 1μmol/L and 0.26nmol/mg/min, respectively. The following human CYP inhibitors caused litter or no inhibition of CYP2A as defined by a K_i>200umol/L: quinidine (QUI), troleandomycin (TAO), and sulfaphenazole (SUL).α-Naphthoflavone (ANF) inhibited the 7-hydroxylation of coumarin with a K_i value of 32μmol/L, which did not increase ability to inhibit CYP2A when ANF was preincubated with swine liver microsomes for 3 min. In the absence of a preincubation period, 8-methoxypsoralen (MOP) inhibited the 7-hydroxylation of coumarin with a K_i value of 1.1μmol/L, which decreased to 0.1μmol/L when MOP was preincubated with swine liver microsomes for 3 min. These results indicated that MOP may be used as a specific uncompetitive inhibitor of mechanism-based inactive of swine CYP2A in liver microsomes.
2. Comparative metabolism of olaquindox in vitro liver systems
The purpose of this study was to compare the metabolic pathways of olaquindox and its metabolite desoxyolaquindox (DES) using reliable systems of liver microsomes and hepatocytes. The metabolites were characterized rapidly and accurately using LC/MS-ITTOF with MS~n capability, high sensitivity, high resolution and mass accuracy. Thirteen metabolites of olaquindox were detected in rat liver microsomes. However, only seven metabolites were formed in swine and chicken liver microsomes. Among the identified metabolites, beside three reduced metabolites (01, 02, 09) and two carboxylic acid derivatives (08, 010) were consistent with the early report, five hydroxylation metabolites (O3～O7), two N-dehydroxyethylation metabolites (O11,O12) and a aldehyde metabolite (O13) were found for the first time in liver microsomes. O2 was the major metabolite in swine and chicken, but O1, O2 and O9 were the major metabolites in rats. Six metabolites of DES were found in rat liver microsomes and identified as O2, O10, O13, O12 and two hydroxylation metabolites (O14, O15). Except metabolite O15, other five metabolites were also measured in swine and chicken liver microsomes. O10 was the major metabolite of DES in the three species. Five metabolites were detected in rat and swine hepatocytes, and identified as O1, O2, O5, O8 and O9. Except metabolite O8, other four metabolites were observed in chicken hepatocytes. The present results revealed that N→O group reduction and oxidation of hydroxyl group were the main metabolic pathways of olaquindox in all three species. The ability of N→O group reduction and hydroxylation in rat was higher than other two species, but the oxidation ability in chicken was highest among the three species. The N→O group reduction and N-oxidation of olaquindox could interconvert in liver microsomes. The results also suggested that the O13, O15 and three reduced metabolites were related to the toxicity of olaquindox.
3. Comparative metabolism of mequindox, quinocetone, carbadox and cyadox in liver microsomes
To investigate the metabolic characteristics of quinoxalines and reveal the relation between structure and metabolism of quinoxalines, the metabolism of other quinoxalines in liver microsomes from the three species was compared in this study. The results showed that fourteen metabolites of mequindox were firstly detected in chicken liver microsomes, including three reduction metabolites (M1, M2, M6), five carbonyl reduction metabolites (M10～M14) and six hydroxylation metabolites (M3, M4, M5, M7～M9). Ten metabolites (M1～M4, M6, M7, M10～M12, M14) were found in rat and swine liver microsomes. Moreover, a additional metabolite M13 was detected in swine liver microsomes. M2, M10 and M12 were the major metabolites in swine. M2, M6 and M12 were the major metabolites in chicken, but M2 and M6 were the major metabolites in rats.
A total of twenty-seven metabolites (Q1～Q27) of quinocetone were tentatively identified in the rat liver microsomes. Twenty-three and twenty-five metabolites were found in chicken and swine liver mircrosomes, respectively. Compared with metabolites found in rat liver microsmes, except Q8, Q11, Q14, Q15, Q19, Q25, Q26 and Q27, other metabolites were detected in chicken liver microsomes. Four additional metabolites (Q28～Q31) were observed in chicken liver microsomes. Compared with metabolites found in rat liver microsmes, except metabolites Q8, Q9, Q10, Q11, and Q14, other metabolites were also deteced in swine liver microsomes. Three additional metabolites (Q28, Q32 and Q33) were measured in swine liver microsomes. Except metabolite Q6, other metabolites were firstly found and identified in liver microsomes. Q2 and Q4 were the major metabolites in rats. Q2, Q4, Q17 and Q20 were the major metabolites in chicken, but Q2 and Q6 were the major metabolites in swine.
Six metabolites of carbadox were detected in rat liver microsomes, and identified as three reduced metabolites (Cb1～Cb3), three hydroxylation metabolites (Cb4～Cb6). Only four metabolites (Cb1～Cb4) were measured in chicken and swine liver microsomes. Cb1 was the major metabolites in rats. Cb1, Cb3 and Cb4 were the major metabolites in chicken, but Cb1 and CM were the major metabolites in swine. Among the identified metabolites, beside Cb3 was consistent with the early report, other five metabolites of carbadox were found for the first time in liver microsomes.
Except metabolite Cy7 only formed in swine liver microsomes, other six metabolites (Cy1～Cy6) were detected in rat, swine and chicken liver microsmoes. Metabolites were identified as three reduced metabolites (Cy1, Cy2, Cy4), a hydroxylation metabolite (Cy3) and three hydrolysis metabolites on the amide bond (Cy5～Cy7). Cy1, Cy4, Cy5 and Cy6 were the major metabolites in swine. Cy1 and Cy4 were the major metabolites in rats, but only Cy1 was the major metabolite in chicken. Except Cy2 and Cy4, other five minor metabolites were firstly found in liver microsomes.
The metabolic studies of quinoxalines in different species liver microsomes show that the N→O group reduction and hydroxlation are the same two metabolic pathways of quinoxalines. Moreover, the toxicities of quinoxlaines are related to the N→O group reduction. Drugs have obvious metabolism differences owing to the differences on the side chain of quinoxalines. Almost no qualitative species difference in the metabolic pathways of quinoxalines is measured. There are metabolic rate and minor metabolites species difference in the metabolism of quinoxalines among the three species due to enzymes difference. The abilities of N→O group reduction and hydroxylation in rat are higher than other two species, while the oxidation of hydroxyl in chicken and the carbonyl reduction and hydrolysis of amide bond in swine are highest among the three species, respectively.
4. Comparative metabolism of olaquindox in rats, swine and chicken
Rats, swine and chicken were administered olaquindox oral gavages with a single dosage of 10, 5 and 30 mg/kg b.w, respectively. The method using LC/MS-ITTOF had been developed for the analysis olaquindox and its metabolites in urine, plasma, feces, muscle, liver, kidney and contents of intestinum cecum from female and male rats, swine and chicken. A total of eighteen metabolites in rat urine were detected after administration of olaquindox. O1 and O2 were the major metabolites in female rats, but O2, O9 and O13 were the major metabolites in male rat. Six (O2, O9, O10, O13, O16 and O19), three (O10, O13, O19), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female rat feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were also found in male swine feces, only O9 was detected in male rat tissues.
A total of sixteen metabolites in swine urine were detected after administration of olaquindox. O2 and O9 were the major metabolites in swine. Six (O2, O9, O10, O13, O16 and O19), one (O13), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female swine feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were found in male swine feces, only O9 was detected in male swine tissues.
A total of fifteen metabolites in female and male chicken were deteced at 2h after administration of olaquindox. O2, O8 and O9 were the major metabolites in female chicken, but O8 and O13 were the major metabolites in male chicken. Beside all metabolites detected in plasma, O16, O25 and O26 were also found in chicken feces. One (O9), six (O2, O8, O9, O10, O11, O12) and three (O9, O12 and O19) metabolites were found in female chicken muscle, kidney and liver, respectively. One (O9), seven (O2, O8, O9, O10, O11, O12, O19) and four (O9, O10, O12 and O19) metabolites were found in male chicken muscle, kidney and liver, respectively.
In conclusion, except three reduced metabolites (O1, O2 and O9), four carboxylic acid derivatives (O8, O10, O20 and O21) and MQCA (O19) were consistent with the early report, other fifteen metabolites of olaquindox were found for the first time in vivo. These results demonstrate that, except O6 and O7, all other metabolites of olaquindox formed in liver microsomes and hepatocytes are also detected in vivo. The results show that the major metabolic pathways are similar to the three species, but the minor metabolites have obvious species difference, which are closely with enzymes difference. The excretion rate of olaquindox in three species is: rat> swine > chicken. The rates of excretion and metabolism of olaquindox in male rat and swine are faster than those in female's, but gender difference is opposite in chicken.
5. Species differences of metabolic enzymes involved in the reduction, oxidation and N-dehydroxyethylation of olaquindox
To understand the metabolic mechanism and species difference of olaquindox, the reductive mechanisms of N1 and N4 reduction of olaquindox in liver microsomes and cytosol for rats, swine and chicken were investigated. Then, the CYP and non-CYP enzymes involved in the oxidation and N-dehydroxyethylation of DES in the three species were also examined.
Olaquindox was reduced to O2 by liver microsomes and cytosol from rats, swine and chicken under hypoxic conditions. The N1 -reducing activity was inhibited in the presence of NADH by air and CO. However, the activity was stimulated by the addition of riboflavin under hypoxic conditions. When the liver microsomes and cytosol were boiled, these activities were not abolished, but were enhanced in the presence of NADH and riboflavin. The NADH-linked activities of the rat and swine live cytosol were enhanced significantly by the addition of menadione, but chicken liver cytosol did not. Swine liver cytosol exhibited a significant reductase activity toward N1-reduction of olaquindox to form O2 in the presence of menadione under hypoxic conditions, but rat and chicken did not. These results suggest that the N1 -reduction of olaquindox may be enzymatic and non-enzymatic involvement in liver microsomes and cytosol of the three species. The enzymatic reduction of olaquindox in liver microsomes has not species difference, but the enzymatic reduction of olaquindox in liver cytosol has obvious species difference. Rat and swine liver cytosol exhibited N4-reducing activity toward olaquindox to form N4-reduced olaquindox (O1) in the presence of benzaldehyde (BZA) under hypoxic conditions. The N4-reducing activity was inhibited in rat and swine liver cytosol by inhibitors of aldehyde oxidase. The N1 -reducing activity was inhibited in rat liver cytosol by xanthine oxidase inhibitor, but swine liver cytsol did not. These results suggest that N4-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase and xanthine oxidase. The N4-reducing activity may be catalyzed by aldehyde oxidase in swine cytosol. However, N1-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase. Chicken liver cytosol had not the N4-reducing activity toward olaquindox to form O1 in the presence of BZA, suggesting chicken may be devoid of the activities of aldehyde oxidase or xanthine oxidase.
In this study, various concentrations of DES were incubated with rat, swine and chicken liver microsomes, respectively. The results showed that the formation of O10 in rat liver microsomes exhibited a single CYP isoform involved, but chicken and swine liver microsomes would be involved in this metabolic pathway by at least two CYP isoforms. The formation of O12 in all species microsomes exhibited at least two CYP isoforms involvement in the N-dehydroxyethylation of DES. The rank orders of intrinsic clearance rates for O10 and O12 with liver microsomes obtained from the three species were chicken>swine>rat and rat>swine>chicken, respectively. Species difference of CYP enzymes involved in the oxidation of hydroxyl group and N-dehydroxyethylation of DES were investigated using selective chemical inhibitors. The results indicated that MOP, MP and ANF inhibited the formation of O10 in all species liver microsomes. However, the degree to MOP inhibited O10 was not markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. DDTC and DIS inhibited the formation O10 in swine and chicken liver microsomes. In addition, TAO and QUI could inhibit the formation of O10 in chicken liver micrsomes. MOP, MP, ANF, DDTC and DIS inhibited the N-dehydroxyethylation of DES to O12 in all species liver microsomes. However, the degree to DDTC and DIS inhibited O12 were markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. TAO and QUI inhibited the formation O12 in chicken liver microsomes, but rat and swine liver microsomes did not. The results suggest that CYP1A may involve in the formation of O10 in rat liver microsomes, CYP1A and CYP2E may involve in the formation of O10 in swine liver microsomes. The formation of O12 may be catalyzed by CYP1A and CYP2E in rat liver microsomes. CYP2A and CYP2E may involve in the formation of O12 in swine liver microsomes. The results suggested chicken various CYP isoforms may catalyze the two reactions.
DES was incubated with MP, DIS, DDTC chlorpromazine (CPZ), promethazine (PZ), 7-hydroxycoumarin (HCO) in rat, swine and chicken liver cytosol, respectively. In rat liver cytosol, except HCO, other inhibitors all inhibited the formation of O10. MP, CPZ and MEN inhibited the formation of O10 in swine liver cytosol. However, all inhibitors did not inhibit the formation of O10 in chicken liver cytosol. The results indicate that alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and aldehyde oxidase (AO) may all involve in the oxidation of alcohol of DES in rat liver cytosol. The formation of O10 may be carried out by ADH and AO in swine liver cytosol. However, it is possible that the inhibitors have not selectively in chicken liver cytosol or other enzymes may involve in the reaction.
In conclusion, the metabolic pathways and characteristics of quinoxalines in different species were illustrated for the first time circled the comparative metabolism. The application of LC/MS-ITTOF in the qualitative and relative quantitative metabolites was developed firstly. Many new metabolites of quinoxalins were identified, and the relationship between structure and metabolism of quinoxalines were revealed in this study. At the same time, the metabolic fate of olaquindox in vivo from various species was investigated in detail. The species differences of metabolic enzymes involved in the metabolism of olaquindox were examined in depth for the first time in this study. The results demonstrate that the use of LC/MS-ITTOF approach in structural characterization of drug metabolites appears rapid, efficient and reliable. These results will provide comprehensive data to clarify the metabolism of olaquindox, and give scientific guidance for the identification of metabolic locations, the explanation of pharmacokinetics and cause of species difference of olaquindox. The study will contribute valuable information to the following mechanism study of toxicity of olaquindox, the development of analytical methods of residues and identification of residue marker of olaquindox. Moreover, the study will also give useful information for the studies of N→O group reduced mechanism, metabolic enzymes and in vivo metabolism of other quinoxalines.
1.受试动物体外肝代谢系统建立
用差速离心法制备大鼠、猪和鸡肝微粒体,测定细胞色素P450(CYP)的CO差示光谱,考察探针药物香豆素在微粒体中的活性;用改良的胶原酶灌注法分离肝细胞,对肝细胞进行计数和活率测定,考察香豆素在肝细胞中的代谢。结果表明,CYP在450nm处有最大吸收,香豆素在肝微粒体与肝细胞中均能代谢成7-羟基香豆素,从而证实了肝微粒体与肝细胞具有代谢酶的活性。
用探针药物香豆素进一步考察猪肝微粒体CYP2A的酶促动力学,评价5种人CYP同工酶常用的选择性抑制剂对猪CYP2A活性的抑制效果。结果显示,猪肝微粒体中香豆素7-羟化酶(CYP2A)的酶促动力学参数K_m和V_(max)分别是1μmol/L和0.26nmol/mg/min,磺胺苯吡唑(SUL)、奎尼丁(QUI)和醋竹桃霉素(TAO)对CYP2A很少或无抑制作用(K_i＞200μmol/L);α-萘黄酮(ANF)预反应对猪肝微粒体CYP2A的抑制无增强作用,其抑制常数K_i为32μmol/L:8-甲氧补骨脂素(MOP)无预反应时对猪肝微粒体CYP2A的抑制常数K_i为1.1μmol/L,当预反应3min后,其抑制常数K_i降为0.1μmol/L。由此表明,MOP可作为猪肝微粒体CYP2A的一个基于机制失活的特异性非竞争性抑制剂。
2.喹乙醇在离体肝系统的比较代谢研究
选择已建立的肝微粒体和肝细胞系统,研究喹乙醇或其代谢物脱二氧喹乙醇在肝系统的比较代谢,用高灵敏、高质量精度、高分辨率和多级质谱的LC/MS-ITTOF可快速、准确地鉴定其代谢物。结果显示,喹乙醇在大鼠肝微粒体中可代谢成13种代谢物,而猪和鸡只检出7种;同时,除了已报道的3种还原代谢物(O1、O2、O9)和2种羧酸衍生物(O8、O10)外,首次鉴定了5种羟化物(O3～O7)、2种N-去乙醇代谢物(O11、O12)和1种醛中间代谢物(O13);并且,O2是喹乙醇在猪和鸡的主要代谢物,而O1、O2和O9是大鼠的主要代谢物。脱二氧喹乙醇在大鼠肝微粒体可代谢成6种代谢物,且鉴定为O2、O10、O12、O13和2种羟化代谢物(O14、O15);在猪和鸡肝微粒体中均能检出除O15外的其它5种代谢物;O10是脱二氧喹乙醇在三种动物的主要代谢物。喹乙醇在猪和大鼠肝细胞中可生成5种代谢物,分别是O1、O2、O5、O8和O9;鸡肝细胞中除O8外,其它4种都检出。本试验结果表明,喹乙醇的主要代谢途径是N→O基团还原和羟基氧化,大鼠的N→O基团还原和羟化能力最强,鸡的氧化能力最强,喹乙醇N→O基团还原和N-氧化可相互转化,O13、O15和3种还原代谢物是潜在的毒性代谢物。
3.乙酰甲喹、喹烯酮、卡巴氧和喹赛多在肝微粒体的比较代谢研究
为阐明喹噁啉类各自的代谢特点以及结构与代谢的关系,本研究比较了其在肝微粒体中的代谢。结果显示,乙酰甲喹在鸡肝微粒体中能代谢成14种代谢物,且鉴定为3种N→O基团还原代谢物(M1、M2、M6)、5种羰基还原代谢物(M10～M14)、6种羟化代谢物(M3、M4、M5、M7、M9、M8);在大鼠能检出10种代谢物,分别是M1、M2、M3、M4、M6、M7、M10、M11、M12和M14;而在猪肝微粒体中,除检出大鼠的10种外,还检出了M13;所有这些乙酰甲喹代谢物都是首次报道,并且,M2、M10和M12是猪的主要代谢物,M2、M6和M12是鸡的主要代谢物,M2和M6是大鼠的主要代谢物。喹烯酮在大鼠肝微粒体可代谢成27种代谢物(Q1～Q27);鸡肝微粒体中能代谢成23种,其中Q1～Q27中无Q8、Q11、Q14、Q15、Q19、Q25、Q26、Q27,但检出Q28～Q31;猪肝微粒体代谢成25种,其中Q1～Q27中无Q8、Q9、Q10、Q11、Q14,但检出Q28、Q32和Q33;这些代谢物除脱二氧喹烯酮(Q6)外,其它都是首次鉴定,Q2和Q4是大鼠的主要代谢物,Q2、Q4、Q17和Q20是鸡的主要代谢物,Q2和Q20是猪的主要代谢物。卡巴氧在大鼠肝微粒体中能生成6种代谢物,且鉴定为3种N→O基团还原物(Cb1～Cb3)和3种羟化代谢物(Cb4～Cb6);猪和鸡都只检出Cb1、Cb2、Cb3和Cb4,其中Cb1和Cb4是猪的主要代谢物,Cb1、Cb3和Cb4是鸡的主要代谢物,Cb1是大鼠的主要代谢物;除Cb3外,其它5种代谢物也属首次鉴定。喹赛多在猪肝微粒体中可代谢成7种代谢物,且鉴定为3种N→O基团还原物(Cy1、Cy2、Cy4)、1种羟化代谢物(Cy3)和3种水解代谢物(Cy5～Cy7),除代谢物Cy7外,其它6种代谢物都在大鼠和鸡肝微粒体中检出,其中Cy1、Cy4、Cy5和Cy6是猪的主要代谢物,Cy1是鸡的主要代谢物,Cy1和Cy4是大鼠的主要代谢物;除Cy2和Cy4外,其它5种代谢物仍属首次鉴定。
喹噁啉类共同的主要代谢途径是N→O基团还原,其次是羟化,且其毒性与其N→O基团还原有关。不同化合物由于侧链不一样,其代谢途径存在明显差异,同一化合物在不同动物的代谢途径基本相似,但主要代谢物和代谢物的量存在明显的种属差异。大鼠对喹噁啉类的N→O基团还原和羟化能力最强,猪对其羰基还原和酰胺水解能力最强,鸡则对其羟基氧化能力最强。
4.喹乙醇在大鼠、猪和鸡体内的比较代谢研究
大鼠、猪和鸡分别按10、5和30mg/kg b.w.单次口服给药后,对其尿液、血浆、粪便、肌肉、肝脏、肾脏和胃肠内容物中的代谢物进行检测。结果显示,在大鼠尿液中共检出18种代谢物,其中O1和O2是雌性大鼠的主要代谢物,O2、O9和O13是雄性大鼠的主要代谢物;雌性大鼠粪便、盲肠内容物、肌肉、肾脏和肝脏中分别检出6种(O2、O9、O10、O13、O16和O19)、3种(O10、O13和O19)、2种(O9和O19)、2种(O9和O19)和3种(O9、O12和O19),雄性大鼠粪便中也能检出上述6种代谢物,其它组织都只检出O9。猪尿液中检测到16种代谢物,其中O2和O9是猪的主要代谢物;雌性猪粪便、盲肠内容物、肌肉、肾脏和肝脏中分别检出6种(O2、O9、O10、O13、O16和O19)、1种(O13)、2种(O9和O19)、2种(O9和O19)和3种(O9、O12和O19)代谢物,雄性猪粪便中同样能检出上述6种,而其它组织都只检出O9。鸡给药2h后,血浆中能检测出15种代谢物,其中O2、O8和O9是雌性鸡的主要代谢物,O8和O13是雄性鸡的主要代谢物;其粪中除上述15种代谢物外,还检出O16、O25和O26;雌性鸡肌肉、肝脏和肾脏分别检出1种(O9)、3种(O9、O12和O19)和6种(O2、O8、O9、O10、O11和O12),雄性鸡的肌肉中也只检出O9,而肝脏中能检出O10、O12和O19,肾脏中可检出O8、O9、O10、O11、O12、O19和O25。
除了3种还原代谢物、4种羧酸衍生物和3-甲基喹嚅啉-2-羧酸(O19)外,其它15种代谢物都是在体内首次报道。同时,除大鼠肝微粒体中的O6和O7外,其它所有在肝微粒体和肝细胞中检测到的代谢物在体内均可检出;喹乙醇在不同动物的主要代谢途径和代谢方式相同,但次要代谢物和代谢速率存在种属差异,喹乙醇及其代谢物在大鼠体内的排泄速度最快,鸡最慢;喹乙醇的代谢存在性别差异,雄性猪和大鼠的代谢和排泄速度比雌性快,但鸡却正好与之相反。
5.喹乙醇还原、氧化和N-脱羟乙基化代谢酶的种属差异研究
为深入了解喹乙醇代谢机制和解释种属差异,本研究比较喹乙醇N1与N4还原在大鼠、猪、鸡肝微粒和胞液中的还原机制,探讨不同动物催化脱二氧喹乙醇氧化和N-脱羟乙基化的CYP与非CYP。
肝微粒体或胞液与NAD(P)H在低氧条件下孵育后,喹乙醇能还原成O2,氧气和一氧化碳(CO)对其还原活性有抑制作用,核黄素对其活性有增强作用,煮沸的肝微粒体和胞液同样存在N1还原活性;甲萘醌能显著增加猪和大鼠胞液中O2的生成,而对鸡肝胞液中O2的生成无影响,且它在猪胞液中能直接还原喹乙醇,而在大鼠和鸡肝胞液中无此活性;以上结果证明,喹乙醇在肝微粒体和胞液的N1还原可经酶和非酶催化,其还原机制在肝微粒体无种属差异,但胞液存在酶的种属差异。苯甲醛与肝胞液在低氧条件下孵育后的结果显示,苯甲醛能显著增强大鼠和猪肝胞液中N4还原活性,也能增强大鼠肝胞液中N1还原活性;醛氧化酶(AO)和黄嘌呤氧化酶(XO)的抑制剂对大鼠N4还原活性有抑制作用,AO抑制剂对N1还原活性有抑制作用,提示大鼠AO和XO部参与喹乙醇N4还原,AO参与喹乙醇N1还原;AO抑制剂对猪肝胞液N4还原活性有抑制作用,表明猪AO催化喹乙醇的N4还原;鸡肝胞液不存在苯甲醛增强的作用,提示鸡可能缺乏AO和XO。
用不同浓度的脱二氧喹乙醇分别与大鼠、猪和鸡肝微粒体孵育,结果显示,O10在大鼠肝微粒体中受单一CYP催化,猪和鸡肝微粒体中至少受两种CYP催化,O12在三种动物均至少受两种CYP催化;O10的内在清除率在鸡最高,其次是猪,最后是大鼠,O12的内在清除率顺序正好相反。用选择性抑制剂探讨参与羟基氧化和N-脱羟乙基化反应的CYP的种属差异时,发现MOP、4-甲基吡唑(MP)和ANF抑制3种动物肝微粒体O10的生成,MOP预反应对其抑制无显著增强作用;而且,二乙基二硫代氨基甲酸酯(DDTC)和双硫仑(DIS)能抑制猪和鸡肝微粒体O10的生成,TAO和QUI能抑制剂鸡肝微粒体O10的生成;MOP、MP、ANF、DDTC和DIS都能抑制三种动物肝微粒体中O12的生成,其中DDTC和DIS的预反应对O12的抑制有增强作用;TAO和QUI对猪和大鼠肝微粒体O12的生成无抑制,但其预反应对鸡肝微粒体中O12的生成有显著抑制作用。上述研究表明,大鼠CYP1A催化羟基氧化,猪CYP1A和CYP2E可能催化羟基氧化,大鼠CYP1A和CYP2E催化N-脱羟乙基化反应,猪CYP2A和CYP2E参与其反应,鸡的多种CYP同工酶可能都参与这两种反应。
用化学抑制剂MP、DIS、DDTC、氯丙醛(CPZ)、异丙醛(PZ)、甲萘醌(MEN)和7-羟基香豆素(HCO)分别和脱二氧喹乙醇在大鼠、猪和鸡肝胞液中孵育,结果发现,大鼠胞液中,除HCO外的其它抑制剂对O10都有抑制作用,提示其胞液中醇脱氢酶(ADH)、醛脱氢酶(ALDH)和AO参与羟基氧化;猪胞液中,MP、CPZ和MEN对O10有抑制作用,表明其胞液中ADH和AO参与羟基的氧化;鸡胞液中,所有的抑制剂都对O10无抑制,提示抑制剂对其肝胞液中的酶无抑制或其它酶可能参与羟基氧化。
综上所述,本课题以比较代谢研究为中心,首次应用了LC/MS-ITTOF对代谢物进行定性与相对定量分析,阐明了喹噁啉类在不同动物的代谢途径和特点,鉴定了数十种未报道的代谢物,分析了结构与代谢的关系,揭示了喹乙醇在不同动物体内的命运,比较了体内与体外代谢的相关性,并深入探讨了喹乙醇代谢酶的种属差异。其研究结果证明了LC/MS-ITTOF是鉴定代谢物结构的快速、有效和可靠的分析工具,充分完善了喹乙醇的代谢及其代谢机制,为喹乙醇可能代谢部位确定、药动学特征及种属差异原因解释提供了重要的理论基础,对其毒副作用机制研究、残留监控和残留标识物确定具有十分重要的指导意义,为其它喹噁啉类N→O基团还原机制、代谢酶和体内代谢研究提供了重要思路。
Quinoxalines are animal special drugs of chemical synthesis which are widely used as antibacterial growth promoters. Carbadox and olaquindox are the well-known members, which have been banned or limited to be used in food animal for their potential side effects. Mequindox and quinocetone have been successively sanctioned to be used in food animals in China, and the safer medicines such as cyadox which belongs to quinoxalines family are being developed. Studies have demonstrated that the toxicities of carbadox and olaquindox are closely associated with their metabolism. Some metabolites of quinoxalines have been detected in various tissues, but parent drugs have not been. Currently, the major identified metabolites of olaquindox, carbadox and cyadox are their reduced metabolites and residue markers; most of their metabolites have not yet been identified. The metabolism of mequindox and quinocetone has not been investigated till now. Moreover, it has been demonstrated that the metabolism of cyadox has species difference. But, it is unknown whether the metabolism of other quinoxalines has also the species difference As drug for food producing animals, food safety has become a hot problem in the filed of food processing science. Drug residue is one of the main factors to endanger food safety, while drug metabolism is the scientific groundwork to avoid drug residue. Therefore, the quinoxalines were used as objects in this study. The comparative metabolism of olaquindox in rat, swine and chicken liver microsomes and hepatoctyes was investigated using high-performance liquid chromatography combined with IT/TOF mass spectrometry (LC/MS-ITTOF). Based on these results, the metabolic profiles of other quinoxalines in liver microsomes of the three species were compared. The metabolism of olaquindox in rats, swine and chicken was also examined in detail. In addition, the species differences of metabolic enzymes involved in the N→O group reduction, hydroxyl oxidation and N-dehydroxyethylation were studied. The aims of these studies were to clarify the metabolic characteristics and relationship between structure and metabolism of quinoxalines. Moreover, the biotransformation and reasons of species difference of olaquindox were revealed comprehensively in the study.
1. The establishment of in vitro liver systems of animal subject
Liver microsomes from rats, swine and chicken were prepared using differential centrifugation. The enzymatic activities of microsomes were evaluated by the carbon monoxide (CO) difference spectrum and the metabolism of coumarin as probe drug. Hepatocytes from rats, swine and chicken were separated by a modification of a collagenase perfusion method. The metabolic activities of hepatocytes were measured by the metabolism of coumarin after cell counting and cell viability assay. The results showed a maximum absorption peak of microsomes at 450nm. Coumarin could be metabolized to 7-hydroxycoumarin in liver microsomes and hepatocytes. Therefore, the in vitro liver systems had the activities of drug metabolic enzymes.
The enzymatic kinetic of CYP2A activity in swine liver microsomes was further examined using coumarin as a probe drug. The inhibitory effects on swine CYP2A activity were also evaluated using five commonly used human CYP inhibitors. The results demonstrated that the K_m and V_(max) for coumarin 7-hydroxylase (CYP2A) in swine liver microsomes were estimated to be 1μmol/L and 0.26nmol/mg/min, respectively. The following human CYP inhibitors caused litter or no inhibition of CYP2A as defined by a K_i>200umol/L: quinidine (QUI), troleandomycin (TAO), and sulfaphenazole (SUL).α-Naphthoflavone (ANF) inhibited the 7-hydroxylation of coumarin with a K_i value of 32μmol/L, which did not increase ability to inhibit CYP2A when ANF was preincubated with swine liver microsomes for 3 min. In the absence of a preincubation period, 8-methoxypsoralen (MOP) inhibited the 7-hydroxylation of coumarin with a K_i value of 1.1μmol/L, which decreased to 0.1μmol/L when MOP was preincubated with swine liver microsomes for 3 min. These results indicated that MOP may be used as a specific uncompetitive inhibitor of mechanism-based inactive of swine CYP2A in liver microsomes.
2. Comparative metabolism of olaquindox in vitro liver systems
The purpose of this study was to compare the metabolic pathways of olaquindox and its metabolite desoxyolaquindox (DES) using reliable systems of liver microsomes and hepatocytes. The metabolites were characterized rapidly and accurately using LC/MS-ITTOF with MS~n capability, high sensitivity, high resolution and mass accuracy. Thirteen metabolites of olaquindox were detected in rat liver microsomes. However, only seven metabolites were formed in swine and chicken liver microsomes. Among the identified metabolites, beside three reduced metabolites (01, 02, 09) and two carboxylic acid derivatives (08, 010) were consistent with the early report, five hydroxylation metabolites (O3～O7), two N-dehydroxyethylation metabolites (O11,O12) and a aldehyde metabolite (O13) were found for the first time in liver microsomes. O2 was the major metabolite in swine and chicken, but O1, O2 and O9 were the major metabolites in rats. Six metabolites of DES were found in rat liver microsomes and identified as O2, O10, O13, O12 and two hydroxylation metabolites (O14, O15). Except metabolite O15, other five metabolites were also measured in swine and chicken liver microsomes. O10 was the major metabolite of DES in the three species. Five metabolites were detected in rat and swine hepatocytes, and identified as O1, O2, O5, O8 and O9. Except metabolite O8, other four metabolites were observed in chicken hepatocytes. The present results revealed that N→O group reduction and oxidation of hydroxyl group were the main metabolic pathways of olaquindox in all three species. The ability of N→O group reduction and hydroxylation in rat was higher than other two species, but the oxidation ability in chicken was highest among the three species. The N→O group reduction and N-oxidation of olaquindox could interconvert in liver microsomes. The results also suggested that the O13, O15 and three reduced metabolites were related to the toxicity of olaquindox.
3. Comparative metabolism of mequindox, quinocetone, carbadox and cyadox in liver microsomes
To investigate the metabolic characteristics of quinoxalines and reveal the relation between structure and metabolism of quinoxalines, the metabolism of other quinoxalines in liver microsomes from the three species was compared in this study. The results showed that fourteen metabolites of mequindox were firstly detected in chicken liver microsomes, including three reduction metabolites (M1, M2, M6), five carbonyl reduction metabolites (M10～M14) and six hydroxylation metabolites (M3, M4, M5, M7～M9). Ten metabolites (M1～M4, M6, M7, M10～M12, M14) were found in rat and swine liver microsomes. Moreover, a additional metabolite M13 was detected in swine liver microsomes. M2, M10 and M12 were the major metabolites in swine. M2, M6 and M12 were the major metabolites in chicken, but M2 and M6 were the major metabolites in rats.
A total of twenty-seven metabolites (Q1～Q27) of quinocetone were tentatively identified in the rat liver microsomes. Twenty-three and twenty-five metabolites were found in chicken and swine liver mircrosomes, respectively. Compared with metabolites found in rat liver microsmes, except Q8, Q11, Q14, Q15, Q19, Q25, Q26 and Q27, other metabolites were detected in chicken liver microsomes. Four additional metabolites (Q28～Q31) were observed in chicken liver microsomes. Compared with metabolites found in rat liver microsmes, except metabolites Q8, Q9, Q10, Q11, and Q14, other metabolites were also deteced in swine liver microsomes. Three additional metabolites (Q28, Q32 and Q33) were measured in swine liver microsomes. Except metabolite Q6, other metabolites were firstly found and identified in liver microsomes. Q2 and Q4 were the major metabolites in rats. Q2, Q4, Q17 and Q20 were the major metabolites in chicken, but Q2 and Q6 were the major metabolites in swine.
Six metabolites of carbadox were detected in rat liver microsomes, and identified as three reduced metabolites (Cb1～Cb3), three hydroxylation metabolites (Cb4～Cb6). Only four metabolites (Cb1～Cb4) were measured in chicken and swine liver microsomes. Cb1 was the major metabolites in rats. Cb1, Cb3 and Cb4 were the major metabolites in chicken, but Cb1 and CM were the major metabolites in swine. Among the identified metabolites, beside Cb3 was consistent with the early report, other five metabolites of carbadox were found for the first time in liver microsomes.
Except metabolite Cy7 only formed in swine liver microsomes, other six metabolites (Cy1～Cy6) were detected in rat, swine and chicken liver microsmoes. Metabolites were identified as three reduced metabolites (Cy1, Cy2, Cy4), a hydroxylation metabolite (Cy3) and three hydrolysis metabolites on the amide bond (Cy5～Cy7). Cy1, Cy4, Cy5 and Cy6 were the major metabolites in swine. Cy1 and Cy4 were the major metabolites in rats, but only Cy1 was the major metabolite in chicken. Except Cy2 and Cy4, other five minor metabolites were firstly found in liver microsomes.
The metabolic studies of quinoxalines in different species liver microsomes show that the N→O group reduction and hydroxlation are the same two metabolic pathways of quinoxalines. Moreover, the toxicities of quinoxlaines are related to the N→O group reduction. Drugs have obvious metabolism differences owing to the differences on the side chain of quinoxalines. Almost no qualitative species difference in the metabolic pathways of quinoxalines is measured. There are metabolic rate and minor metabolites species difference in the metabolism of quinoxalines among the three species due to enzymes difference. The abilities of N→O group reduction and hydroxylation in rat are higher than other two species, while the oxidation of hydroxyl in chicken and the carbonyl reduction and hydrolysis of amide bond in swine are highest among the three species, respectively.
4. Comparative metabolism of olaquindox in rats, swine and chicken
Rats, swine and chicken were administered olaquindox oral gavages with a single dosage of 10, 5 and 30 mg/kg b.w, respectively. The method using LC/MS-ITTOF had been developed for the analysis olaquindox and its metabolites in urine, plasma, feces, muscle, liver, kidney and contents of intestinum cecum from female and male rats, swine and chicken. A total of eighteen metabolites in rat urine were detected after administration of olaquindox. O1 and O2 were the major metabolites in female rats, but O2, O9 and O13 were the major metabolites in male rat. Six (O2, O9, O10, O13, O16 and O19), three (O10, O13, O19), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female rat feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were also found in male swine feces, only O9 was detected in male rat tissues.
A total of sixteen metabolites in swine urine were detected after administration of olaquindox. O2 and O9 were the major metabolites in swine. Six (O2, O9, O10, O13, O16 and O19), one (O13), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female swine feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were found in male swine feces, only O9 was detected in male swine tissues.
A total of fifteen metabolites in female and male chicken were deteced at 2h after administration of olaquindox. O2, O8 and O9 were the major metabolites in female chicken, but O8 and O13 were the major metabolites in male chicken. Beside all metabolites detected in plasma, O16, O25 and O26 were also found in chicken feces. One (O9), six (O2, O8, O9, O10, O11, O12) and three (O9, O12 and O19) metabolites were found in female chicken muscle, kidney and liver, respectively. One (O9), seven (O2, O8, O9, O10, O11, O12, O19) and four (O9, O10, O12 and O19) metabolites were found in male chicken muscle, kidney and liver, respectively.
In conclusion, except three reduced metabolites (O1, O2 and O9), four carboxylic acid derivatives (O8, O10, O20 and O21) and MQCA (O19) were consistent with the early report, other fifteen metabolites of olaquindox were found for the first time in vivo. These results demonstrate that, except O6 and O7, all other metabolites of olaquindox formed in liver microsomes and hepatocytes are also detected in vivo. The results show that the major metabolic pathways are similar to the three species, but the minor metabolites have obvious species difference, which are closely with enzymes difference. The excretion rate of olaquindox in three species is: rat> swine > chicken. The rates of excretion and metabolism of olaquindox in male rat and swine are faster than those in female's, but gender difference is opposite in chicken.
5. Species differences of metabolic enzymes involved in the reduction, oxidation and N-dehydroxyethylation of olaquindox
To understand the metabolic mechanism and species difference of olaquindox, the reductive mechanisms of N1 and N4 reduction of olaquindox in liver microsomes and cytosol for rats, swine and chicken were investigated. Then, the CYP and non-CYP enzymes involved in the oxidation and N-dehydroxyethylation of DES in the three species were also examined.
Olaquindox was reduced to O2 by liver microsomes and cytosol from rats, swine and chicken under hypoxic conditions. The N1 -reducing activity was inhibited in the presence of NADH by air and CO. However, the activity was stimulated by the addition of riboflavin under hypoxic conditions. When the liver microsomes and cytosol were boiled, these activities were not abolished, but were enhanced in the presence of NADH and riboflavin. The NADH-linked activities of the rat and swine live cytosol were enhanced significantly by the addition of menadione, but chicken liver cytosol did not. Swine liver cytosol exhibited a significant reductase activity toward N1-reduction of olaquindox to form O2 in the presence of menadione under hypoxic conditions, but rat and chicken did not. These results suggest that the N1 -reduction of olaquindox may be enzymatic and non-enzymatic involvement in liver microsomes and cytosol of the three species. The enzymatic reduction of olaquindox in liver microsomes has not species difference, but the enzymatic reduction of olaquindox in liver cytosol has obvious species difference. Rat and swine liver cytosol exhibited N4-reducing activity toward olaquindox to form N4-reduced olaquindox (O1) in the presence of benzaldehyde (BZA) under hypoxic conditions. The N4-reducing activity was inhibited in rat and swine liver cytosol by inhibitors of aldehyde oxidase. The N1 -reducing activity was inhibited in rat liver cytosol by xanthine oxidase inhibitor, but swine liver cytsol did not. These results suggest that N4-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase and xanthine oxidase. The N4-reducing activity may be catalyzed by aldehyde oxidase in swine cytosol. However, N1-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase. Chicken liver cytosol had not the N4-reducing activity toward olaquindox to form O1 in the presence of BZA, suggesting chicken may be devoid of the activities of aldehyde oxidase or xanthine oxidase.
In this study, various concentrations of DES were incubated with rat, swine and chicken liver microsomes, respectively. The results showed that the formation of O10 in rat liver microsomes exhibited a single CYP isoform involved, but chicken and swine liver microsomes would be involved in this metabolic pathway by at least two CYP isoforms. The formation of O12 in all species microsomes exhibited at least two CYP isoforms involvement in the N-dehydroxyethylation of DES. The rank orders of intrinsic clearance rates for O10 and O12 with liver microsomes obtained from the three species were chicken>swine>rat and rat>swine>chicken, respectively. Species difference of CYP enzymes involved in the oxidation of hydroxyl group and N-dehydroxyethylation of DES were investigated using selective chemical inhibitors. The results indicated that MOP, MP and ANF inhibited the formation of O10 in all species liver microsomes. However, the degree to MOP inhibited O10 was not markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. DDTC and DIS inhibited the formation O10 in swine and chicken liver microsomes. In addition, TAO and QUI could inhibit the formation of O10 in chicken liver micrsomes. MOP, MP, ANF, DDTC and DIS inhibited the N-dehydroxyethylation of DES to O12 in all species liver microsomes. However, the degree to DDTC and DIS inhibited O12 were markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. TAO and QUI inhibited the formation O12 in chicken liver microsomes, but rat and swine liver microsomes did not. The results suggest that CYP1A may involve in the formation of O10 in rat liver microsomes, CYP1A and CYP2E may involve in the formation of O10 in swine liver microsomes. The formation of O12 may be catalyzed by CYP1A and CYP2E in rat liver microsomes. CYP2A and CYP2E may involve in the formation of O12 in swine liver microsomes. The results suggested chicken various CYP isoforms may catalyze the two reactions.
DES was incubated with MP, DIS, DDTC chlorpromazine (CPZ), promethazine (PZ), 7-hydroxycoumarin (HCO) in rat, swine and chicken liver cytosol, respectively. In rat liver cytosol, except HCO, other inhibitors all inhibited the formation of O10. MP, CPZ and MEN inhibited the formation of O10 in swine liver cytosol. However, all inhibitors did not inhibit the formation of O10 in chicken liver cytosol. The results indicate that alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and aldehyde oxidase (AO) may all involve in the oxidation of alcohol of DES in rat liver cytosol. The formation of O10 may be carried out by ADH and AO in swine liver cytosol. However, it is possible that the inhibitors have not selectively in chicken liver cytosol or other enzymes may involve in the reaction.
In conclusion, the metabolic pathways and characteristics of quinoxalines in different species were illustrated for the first time circled the comparative metabolism. The application of LC/MS-ITTOF in the qualitative and relative quantitative metabolites was developed firstly. Many new metabolites of quinoxalins were identified, and the relationship between structure and metabolism of quinoxalines were revealed in this study. At the same time, the metabolic fate of olaquindox in vivo from various species was investigated in detail. The species differences of metabolic enzymes involved in the metabolism of olaquindox were examined in depth for the first time in this study. The results demonstrate that the use of LC/MS-ITTOF approach in structural characterization of drug metabolites appears rapid, efficient and reliable. These results will provide comprehensive data to clarify the metabolism of olaquindox, and give scientific guidance for the identification of metabolic locations, the explanation of pharmacokinetics and cause of species difference of olaquindox. The study will contribute valuable information to the following mechanism study of toxicity of olaquindox, the development of analytical methods of residues and identification of residue marker of olaquindox. Moreover, the study will also give useful information for the studies of N→O group reduced mechanism, metabolic enzymes and in vivo metabolism of other quinoxalines.
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