Madan A, Parkinson A
Department of Pharmacology, Toxicology, and Therapeutics, Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City 66160-7417, USA.
Drug Metab Dispos. 1996 Dec;24(12):1307-13.
Activation of halothane to trifluoroacetyl halide, followed by covalent binding to proteins (neoantigen formation) has been proposed to be the mechanism by which halothane causes immune hepatitis. The aim of this study was to identify the cytochrome P450 (CYP) enzyme primarily responsible for the NADPH-dependent covalent binding of [14C]halothane to human liver microsomes. Human liver microsomes were incubated in the absence and presence of NADPH with various concentrations of halothane (from 4.6 to 3,300 microM) to examine the effects of substrate concentration on the nonspecific and specific (NADPH-dependent) binding of [14C]halothane to microsomal protein. As a function of substrate concentration, the specific binding of [14C]halothane to human liver microsomes was biphasic, suggesting that the activation of halothane is catalyzed by a high-affinity enzyme(s) at low substrate concentrations (<150 microM) and by a low-affinity enzyme(s) at high substrate concentrations (>150 microM). For the high-affinity enzyme, the apparent KM for the covalent binding of [14C]halothane was approximately 10 microM, and Vmax was approximately 32 pmol equivalents of halothane bound/mg protein/min under conditions where covalent binding was directly proportional to incubation time and protein concentration. Ten individual samples of human liver microsomes were incubated with a low concentration of halothane (35 microM) to determine the sample-to-sample variation in the specific binding of [14C]halothane to microsomal protein. Covalent binding ranged from 10 to 40 pmol equivalents of halothane bound/mg protein/min and was highly correlated (r2 = 0.93) with the sample-to-sample variation in chlorzoxazone 6-hydroxylase activity, which reflects the levels of CYP2E1. These results suggest that CYP2E1 is the high-affinity enzyme in human liver microsomes responsible for activating halothane to a reactive metabolite. This is supported by the observation that 4-methylpyrazole, a CYP2E1 inhibitor, inhibited the NADPH-dependent binding of [14C]halothane to microsomal protein. The sample-to-sample variation in the covalent binding of [14C]halothane at high substrate concentrations did not correlate with any known CYP enzyme activity. This suggests that several enzymes catalyze the oxidation of halothane at higher substrate concentrations. In conclusion, at pharmacologically relevant concentrations, the covalent binding of halothane to human liver microsomes is primarily catalyzed by CYP2E1.
氟烷被激活生成三氟乙酰卤，随后与蛋白质共价结合（形成新抗原），这一过程被认为是氟烷引发免疫性肝炎的机制。本研究的目的是确定主要负责[14C]氟烷与人类肝微粒体进行NADPH依赖的共价结合的细胞色素P450（CYP）酶。将人类肝微粒体在不存在和存在NADPH的情况下，与不同浓度的氟烷（4.6至3300 microM）一起孵育，以研究底物浓度对[14C]氟烷与微粒体蛋白的非特异性和特异性（NADPH依赖）结合的影响。作为底物浓度的函数，[14C]氟烷与人类肝微粒体的特异性结合呈双相性，这表明在低底物浓度（<150 microM）下，氟烷的激活由高亲和力酶催化，而在高底物浓度（>150 microM）下，由低亲和力酶催化。对于高亲和力酶，[14C]氟烷共价结合的表观KM约为10 microM，在共价结合与孵育时间和蛋白浓度成正比的条件下，Vmax约为32 pmol当量的氟烷结合/mg蛋白/分钟。将十个单独的人类肝微粒体样品与低浓度的氟烷（35 microM）一起孵育，以确定[14C]氟烷与微粒体蛋白特异性结合的样品间差异。共价结合范围为10至40 pmol当量的氟烷结合/mg蛋白/分钟，并且与反映CYP2E1水平的氯唑沙宗6-羟化酶活性的样品间差异高度相关（r2 = 0.93）。这些结果表明，CYP2E1是人类肝微粒体中负责将氟烷激活为反应性代谢物的高亲和力酶。这一观点得到了以下观察结果的支持：CYP2E1抑制剂4-甲基吡唑抑制了[14C]氟烷与微粒体蛋白的NADPH依赖结合。在高底物浓度下，[14C]氟烷共价结合的样品间差异与任何已知的CYP酶活性均无相关性。这表明在较高底物浓度下，几种酶催化氟烷的氧化。总之，在药理学相关浓度下，氟烷与人类肝微粒体的共价结合主要由CYP2E1催化。
