Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA.
Department of Public Health, New York Medical College, Valhalla, NY 10595, USA.
Toxicol Appl Pharmacol. 2022 Nov 15;455:116258. doi: 10.1016/j.taap.2022.116258. Epub 2022 Sep 26.
A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate HO, either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. HO production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched β-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 μM) for CYP1A2 and 4-methylpyrazole (32 μM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 μM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and HO generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.
细胞色素 P450（CYP）酶的一个特征是它们能够生成 HO，无论是直接还是间接通过超氧阴离子，这种反应被称为“NADPH 氧化酶”活性。CYP 产生的 HO 可导致细胞毒性活性氧的积累，从而损害细胞功能并导致组织损伤。在此，我们确定了选择性 CYP 抑制剂是否可以区分大鼠重组 CYP1A2、CYP2E1、CYP3A1 和 CYP3A2 以及 CYP1A1/2 富集的β-萘黄酮诱导的大鼠肝微粒体、CYP2E1 富集的异烟肼诱导的大鼠肝微粒体和 CYP3A 亚家族富集的地塞米松诱导的大鼠肝微粒体的单加氧酶和 NADPH 氧化酶活性。在存在 7,8-苯并黄酮（2.0 μM）用于 CYP1A2 和 4-甲基吡唑（32 μM）或 DMSO（16 mM）用于 CYP2E1 的情况下，两种重组酶和微粒体制剂的单加氧酶活性被阻断，而 NADPH 氧化酶活性不受影响。酮康唑（1.0 μM），一种选择性 CYP3A 亚家族酶的形式抑制剂，完全抑制大鼠重组 CYP3A1/3A2 和大鼠肝微粒体 CYP3A 亚家族的单加氧酶活性；它还部分抑制 NADPH 氧化酶活性。7,8-苯并黄酮是一种 I 型配体，与底物结合竞争，而 4-甲基吡唑和 DMSO 是 II 型血红素结合配体。血红素与这些 II 型配体的相互作用不足以干扰 NADPH 氧化酶活性所需的氧活化。酮康唑，一种已知与 CYP3A 亚家族酶上靠近血红素的多个位点结合的 II 型配体，也至少部分干扰了氧活化。这些数据表明，形式特异性抑制剂可用于区分 CYP1A2 和 CYP2E1 的单加氧酶反应和生成 HO 的 NADPH 氧化酶。酮康唑抑制 CYP3A NADPH 氧化酶的机制仍有待确定。
