Choi Dalwoong, Leininger-Muller Brigitte, Kim Young Chul, Leroy Pierre, Siest Gerard, Wellman Maria
Thiols et fonctions celluloizes, INSERM U525, Equipe 4, Faculty of Pharmacy, Université Henri Poincaré Nancy 1, France.
Free Radic Res. 2002 Aug;36(8):893-903. doi: 10.1080/1071576021000005339.
We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 >> acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.
我们评估了“弱”CYP2E1结合剂（乙醇、丙酮和甘油）、“紧密”CYP2E1结合剂（4-甲基吡唑、咪唑、异烟肼和吡啶）以及CCl4（CYP2E1的自杀底物）对NADPH依赖性微粒体活性氧（ROS）生成、脂质过氧化（LPO）以及随后微粒体和CYP2E1蛋白修饰的影响。2',7'-二氯荧光素二乙酸酯（DCFHDA）的氧化用作微粒体ROS和LPO衍生活性物质形成的指标。微粒体LPO通过丙二醛（MDA）的HPLC测定来确定。向大鼠肝微粒体中添加NADPH引发DCFHDA氧化和MDA形成，导致微粒体蛋白的进一步选择性修饰以及CYP2E1蛋白的蛋白酶非依赖性降解。铁螯合剂可阻止这些过程，而羟基自由基清除剂的作用较弱，表明LPO起重要作用。在测试的CYP2E1结合剂中，只有异烟肼强烈抑制NADPH依赖性DCFHDA氧化、LPO和微粒体蛋白修饰。其他CYP2E1结合剂对这些过程的抑制作用较弱。关于NADPH依赖性CYP2E1蛋白修饰，除甘油外，所有测试的CYP2E1结合剂均以不同效力阻止了这一过程（异烟肼>4-甲基吡唑=咪唑=吡啶3>>丙酮>乙醇）。“紧密”结合剂比“弱”结合剂更有效。CCl4刺激DCFHDA氧化、LPO和CYP2E1蛋白修饰。在测试的CYP2E1结合剂中，只有异烟肼能有效清除2,2-二苯基-1-苦基肼基（DPPH）自由基。在从CYP2E1转染的HepG2细胞中分离的微粒体中，异烟肼抑制CYP2E1依赖性DCFHDA氧化，而其他CYP2E1结合剂虽然强烈抑制CYP2E1活性，但并未抑制该反应。本研究表明，CYP2E1结合剂和异烟肼在NADPH依赖性微粒体反应中对LPO催化的CYP2E1蛋白氧化修饰具有不同的抑制作用。似乎CYP2E1结合剂主要通过与酶的活性位点结合来保护CYP2E1免受氧化修饰，而不是通过阻断活性物质的产生。异烟肼的强保护作用可归因于其清除自由基的能力。CYP2E
