评价卡巴扎兰 4-氧化和 -苯甲基鸟嘌呤 8-氧化作为人醛氧化酶的催化标志物：肝微粒体细胞质污染的影响。
Evaluation of Carbazeran 4-Oxidation and -Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes.
机构信息
Department of Pharmacy, Faculty of Science (J.X., N.F.S., S.C., S.Y.W., Z.P.Y., A.J.L.) and Department of Pharmacology, Yong Loo Lin School of Medicine (A.J.L.), National University of Singapore, Singapore; and NANO BIOTEC, LLC., Whippany, New Jersey (L.H.Z.).
Department of Pharmacy, Faculty of Science (J.X., N.F.S., S.C., S.Y.W., Z.P.Y., A.J.L.) and Department of Pharmacology, Yong Loo Lin School of Medicine (A.J.L.), National University of Singapore, Singapore; and NANO BIOTEC, LLC., Whippany, New Jersey (L.H.Z.)
出版信息
Drug Metab Dispos. 2019 Jan;47(1):26-37. doi: 10.1124/dmd.118.082099. Epub 2018 Oct 18.
The present study investigated the contribution of microsomal cytochrome P450 and cytosolic aldehyde oxidase-1 (AOX-1) to carbazeran 4-oxidation and -benzylguanine 8-oxidation in human liver microsomal, cytosolic, and S9 fractions. Incubations containing carbazeran and human liver microsomes with or without exogenously added NADPH yielded comparable levels of 4-oxo-carbazeran. -Benzylguanine 8-oxidation occurred in microsomal incubations, and the extent was increased by NADPH. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4-oxidation, whereas CYP1A2 was highly active in -benzylguanine 8-oxidation. 1-Aminobenzotriazole, a pan-cytochrome P450 inhibitor, decreased -benzylguanine 8-oxidation, but not carbazeran 4-oxidation, in microsomal incubations, whereas 1-aminobenzotriazole and furafylline (a CYP1A2-selective inhibitor) did not inhibit carbazeran 4-oxidation or -benzylguanine 8-oxidation in human liver S9 fraction. Carbazeran 4-oxidation in incubations containing human liver microsomes (from multiple donors and commercial suppliers) was attributed to microsomal preparations contaminated with AOX-1, as suggested by liver microsomal experiments indicating a decrease in carbazeran 4-oxidation by an AOX-1 inhibitor (hydralazine), and to detection of AOX-1 protein (at one-third the level of that in liver cytosol). Cytosolic contamination of liver microsomes was further demonstrated by the formation of dehydroepiandrosterone sulfate (catalyzed by cytosolic sulfotransferases) in liver microsomal incubations containing dehydroepiandrosterone. In conclusion, carbazeran 4-oxidation and -benzylguanine 8-oxidation are enzyme-selective catalytic markers of human AOX-1, as shown in human liver S9 fraction expressing cytochrome P450 and AOX-1. This study highlights the negative impact of cytosolic contamination of liver microsomes on the interpretation of reaction phenotyping data collected in an in vitro study performed in microsomal fractions.
本研究探讨了微粒体细胞色素 P450 和胞质醛氧化酶-1（AOX-1）在人肝微粒体、胞质和 S9 部分中对卡巴扎兰 4-氧化和 -苯甲基鸟嘌呤 8-氧化的贡献。含有卡巴扎兰和人肝微粒体的孵育物，无论是否外加 NADPH，都产生可比水平的 4-氧代卡巴扎兰。-苯甲基鸟嘌呤 8-氧化发生在微粒体孵育物中，并且 NADPH 增加了其程度。人重组 CYP1A2、CYP2C8、CYP2C9、CYP2C19、CYP2D6、CYP3A4 和 CYP3A5 均不能催化卡巴扎兰 4-氧化，而 CYP1A2 则在 -苯甲基鸟嘌呤 8-氧化中高度活跃。泛细胞色素 P450 抑制剂 1-氨基苯并三唑降低了微粒体孵育物中的 -苯甲基鸟嘌呤 8-氧化，但不降低卡巴扎兰 4-氧化，而 1-氨基苯并三唑和呋喃茶碱（CYP1A2 选择性抑制剂）不抑制人肝 S9 部分的卡巴扎兰 4-氧化或 -苯甲基鸟嘌呤 8-氧化。在含有人肝微粒体的孵育物中（来自多个供体和商业供应商）的卡巴扎兰 4-氧化归因于微粒体制剂中存在 AOX-1，这是因为肝微粒体实验表明 AOX-1 抑制剂（肼屈嗪）降低了卡巴扎兰 4-氧化，并且检测到 AOX-1 蛋白（水平为肝胞质的三分之一）。肝微粒体中细胞溶质的污染进一步通过含有脱氢表雄酮的肝微粒体孵育物中硫酸脱氢表雄酮的形成（由细胞溶质硫酸转移酶催化）得到证明。总之，卡巴扎兰 4-氧化和 -苯甲基鸟嘌呤 8-氧化是人类 AOX-1 的酶选择性催化标志物，如在表达细胞色素 P450 和 AOX-1 的人肝 S9 部分中所示。本研究强调了肝微粒体中细胞溶质污染对在微粒体部分进行的体外研究中收集的反应表型数据解释的负面影响。
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