Studies on the mechanism of denaturation of cytochrome P-450 by cyclophosphamide and its metabolites.
Studies on the mechanism of denaturation of cytochrome P-450 by cyclophosphamide and its metabolites.
复制标题
DOI：
10.1016/s0021-9258(19)68462-0
复制
发表时间：
1981-11
期刊：
影响因子：
--
通讯作者：
H. Gurtoo;A. Marinello;R. Struck;B. Paul;R. Dahms
中科院分区：
文献类型：
--
作者：
H. Gurtoo;A. Marinello;R. Struck;B. Paul;R. Dahms
为了了解环磷酰胺（一种重要的抗癌和免疫抑制剂）体内抑制大鼠肝微粒体混合功能氧化酶的机制，我们进行了几方面的研究。与[氯乙基-3H]环磷酰胺的3H相比，[4-14C]环磷酰胺的14C与微粒体蛋白的结合基本上是排他性的代谢依赖性，表明与代谢物丙烯醛的结合。在所测试的环磷酰胺的各种代谢物和类似物（不含过氧基或氢过氧基）中，只有丙烯醛和4-羟基环磷酰胺（在溶液中释放丙烯醛）在体外引起微粒体细胞色素P-450变性；这种变性与巯基试剂产生的变性相同。在所测试的各种化学物质中，只有那些含有游离氨基（赖氨酸除外）和/或游离巯基（例如氨基脲、半胱氨酸、甘氨酸、葡萄糖胺）的化学物质才能有效阻断（40-80%）14C的结合，并防止丙烯醛诱导的细胞色素 P-450 变性。这些数据进一步表明环磷酰胺代谢物与蛋白质中的游离氨基和/或游离巯基相互作用。然而，与通过形成席夫碱与游离蛋白质氨基相互作用的[3H]黄曲霉毒素B2a相比，清楚地归因于14C优先结合这些蛋白质中的半胱氨酸巯基。对丙烯醛和半胱氨酸之间反应的化学模型的研究也支持了这一建议。当从代谢[4-14C]环磷酰胺的培养物中分离出的微粒体进行凝胶电泳时，放射自显影检测到的主要放射性条带与55,000道尔顿的细胞色素P-450条带相关，这是苯巴比妥在大鼠中诱导的主要条带。所有这些结果综合在一起强烈表明丙烯醛可能是导致混合功能氧化酶活性抑制的环磷酰胺代谢物。丙烯醛很可能通过细胞色素 P-450 活性位点中的巯基的烷基化来产生这种效果。
Several lines of investigation were pursued to understand mechanisms involved in the in vivo depression of rat hepatic microsomal mixed function oxidase by cyclophosphamide, an important anti-cancer and immunosuppressive agent. Essentially exclusive metabolism-dependent binding to microsomal proteins of 14C from [4-14C]cyclophosphamide, compared with 3H from [chloroethyl-3H]cyclophosphamide, suggests the binding of the metabolite acrolein. Of the various metabolites and analogs of cyclophosphamide tested (which did not contain a peroxy or a hydroperoxy group), only acrolein and 4-hydroxycyclophosphamide (which releases acrolein in solution) caused denaturation of microsomal cytochrome P-450 in vitro; this denaturation was identical with that produced by sulfhydryl reagents. Of the various chemicals tested, only those which contained either a free amino group (except lysine) and/or a free sulfhydryl group (e.g. semicarbazide, cysteine, glycine, glucosamine) effectively blocked (40-80%) the binding of 14C as well as protected against acrolein-induced denaturation of cytochrome P-450. These data further suggested interaction of cyclophosphamide metabolite with free amino and/or free sulfhydryl groups in proteins. However, comparison with [3H]aflatoxin B2a which interacts with free protein amino groups via the formation of Schiff bases, clearly attributed the preferential binding of 14C to cysteine sulfhydryl groups in these proteins. Studies on chemical models derived from reaction between acrolein and cysteine also supported this suggestion. When microsomes isolated from incubations metabolizing [4-14C]cyclophosphamide were subjected to gel electrophoresis, the major radioactive band detected by autoradiography was associated with a cytochrome P-450 band at 55,000 daltons, the major band induced by phenobarbital in the rat. All these results taken together strongly point to the possibility that acrolein is the cyclophosphamide metabolite responsible for the depression of the mixed function oxidase activities. Acrolein most likely produces this effect by alkylation of the sulfhydryl group(s) in the active site of cytochrome P-450.