Ng S F, Waxman D J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Cancer Res. 1990 Feb 1;50(3):464-71.
Oxidative metabolism of the polyfunctional alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N''-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (Km (app) 19 microM; Vmax (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by kinactivation = 0.096 min-1, KI = 24 microM, and a partition ratio of 136 +/- 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-hydroxysteroid:NADP+ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
为阐明药物氧化途径并确定各个细胞色素P-450(P-450)酶对这种化疗药物生物活化的可能作用,在离体大鼠肝微粒体以及纯化、重组的细胞色素P-450(P-450)酶系统中研究了多功能烷化剂N,N',N''-三乙烯硫代磷酰胺(硫代替派)的氧化代谢。发现大鼠肝微粒体在P-450依赖性反应中催化硫代替派转化为其氧代代谢产物N,N',N''-三乙烯磷酰胺(替派),该反应在体内预先用肝P-450亚家族IIB(苯巴比妥)的药物诱导剂处理后会受到显著刺激,但用P-450亚家族IA(β-萘黄酮)或IIE(异烟肼)的诱导剂预处理则不会。苯巴比妥诱导的肝微粒体催化的硫代替派消耗和替派形成均被与P-450 PB-4(基因产物IIB1)选择性反应的抗体抑制90%以上,P-450 PB-4是苯巴比妥诱导的大鼠肝微粒体中主要的P-450形式,但不被对其他7种大鼠肝P-450有抑制作用的抗体抑制。在用细胞色素与NADPH P-450还原酶在脂质环境中重组后,纯化的P-450 PB-4(表观Km为19微摩尔;表观Vmax = 11摩尔硫代替派代谢/分钟/摩尔P-450 PB-4)也催化硫代替派氧化为替派。P-450 PB-4对硫代替派的代谢与细胞色素的自杀性失活有关,其特征为失活常数kinactivation = 0.096分钟-1,抑制常数KI = 24微摩尔以及分配比为136±28(标准差)摩尔硫代替派代谢/摩尔P-450失活。然而,硫代替派的代谢产物替派不会使细胞色素失活,也不会发生进一步可检测到的代谢。在微粒体孵育中,硫代替派的代谢导致P-450 PB-4(类固醇16β-羟化酶)以及P-450 IIIA相关酶(类固醇6β-羟化酶)和不依赖P-450的酶类固醇17β-羟类固醇:NADP+ 17-氧化还原酶失活,这通过使用P-450形式选择性的甾体底物雄甾-4-烯-3,17-二酮得以证明。相比之下,未观察到微粒体P-450 IIA相关酶(类固醇7α-羟化酶)或微粒体NADPH P-450还原酶有明显失活。(摘要截短至400字)