BIOCHEMICAL MECHANISMS OF CYCLOPHOSPHAMIDE TOXICITY

我们研究环磷酰胺的整体目标是：(A) 对CP的代谢和相互作用有详细的了解 CP代谢产物与组织大分子，特别是DNA，RNA和 蛋白质；(B)阐明CP毒性的化学和生化基础 由于活化的CP代谢产物之间的相互作用而产生 和组织大分子；以及(C)应用从上述 研究开发生物化学干预措施以避免(改善) CP的一些不良毒性及其几乎不受其干扰 化疗活性。[~3H-氯乙基]-环磷酰胺(~3H-CP)的代谢 [14C-CP]-CP(14C-CP)对大分子结合代谢产物和细胞毒作用的研究 和其他代谢产物，由纯化的细胞色素P-450形成，肝细胞核 部分和前列腺素合成酶，将在体外进行研究。新的 代谢物，以及偏高岭石-大分子加合物， 与世隔绝的，有特点的。这些加合物的体内形成及其相互作用 在器官特异性毒性，特别是肝和肺毒性中的作用 将对CP及其活性类似物(ASTZ-Z-7557和PS-CP)进行研究。 将进行研究，以确定特定的 生化改变(脂质过氧化、谷胱甘肽耗竭、 抑制抗氧化剂防御、DNA损伤、形成和 CP和Cp诱导的代谢物-大分子加合物的持久性 CP的某些特定毒性，如肝和肺毒性。 许多拟议的工作将被执行：(A)在体外用14C-CP， ~3H-CP和CP的一些标记代谢物(例如4-羟基-CP， 二氯二羟基-CP、磷酰胺芥末)、核酸(DNA、RNA、 嘌呤和嘧啶核苷聚合物)和酶制剂 (细胞色素P-450、前列腺素合成酶和肝细胞核) 和小鼠；(B)用标记的CP在小鼠和大鼠体内测定 环磷酰胺的体内大分子结合及其与毒性的关系； 在大鼠和小鼠体内阐明CP产生的机制 特定的生化效应，例如谷胱甘肽耗竭和脂质 过氧化。具体使用的方法包括：放射化学 技术，薄层层析，高压液相色谱， 柱层析、有机分析技术和其他化学物质， 生化和酶学方法。

The overall objectives of our studies on cyclophosphamide (CP) are to: (a) obtain a detailed understanding of the metabolism of CP and the interaction of CP metabolites with tissue macromolecules, especially DNA, RNA and proteins; (b) elucidate the chemical and biochemical basis of CP toxicity arising as a consequence of interaction between activated CP metabolites and tissue macromolecules; and (c) apply information derived in the above studies to develop biochemical interventions for obviating (ameliorating) some of the undesirable toxicities of CP with little interference of its chemotherapeutic activity. Metabolism of [3H-chloroethyl]-CP(3H-CP) and [14C-CP]-CP (14C-CP) to macromolecular-binding metabolites and to cytotoxic and other metabolites, formed by purified cytochrome P-450, hepatic nuclear fractions and prostaglandin synthetase, will be investigated in vitro. New metabolites, and metagolite-macromolecular adducts will be identified, isolated and characterized. In vivo formation of these adducts and their role in organ specific toxicities, especially liver and lung toxicity, of CP and its activated analogs (ASTZ-Z-7557 and PS-CP) will be investigated. Studies will be undertaken to define relationships between specific biochemical alterations (lipid peroxidation, glutathione depletion, inhibition of antioxidant defenses, DNA damage, and formation and persistance of CP metabolite - macromolecular adducts) induced by CP and certain specific toxicities of CP, such as liver and lung toxicity. Much of the proposed work will be performed: (a) in vitro with 14C-CP, 3H-CP and some labeled metabolites of CP (e.g. 4-hydroxy-CP, didechlorodihydroxy-CP, phosphoramide mustard), nucleic acids (DNA, RNA, purine and pyrimidine-nucleoside polymers) and enzyme preparations (cytochromes P-450, prostaglandin synthetase and hepatic nuclei) from rats and mice; (b) in vivo with labeled-CP in mice and rats to determine the in vivo macromolecular binding of CP and its relation to toxicity; and (c) in vivo in rats and mice to elucidate the mechanism by which CP produces specific biochemical effects, e.g. glutathione depletion and lipid peroxidation. Particular methods to be used include: radiochemical techniques, thin layer chromatography, high pressure liquid chromatography, column chromatography, organic analytical techniques, and other chemical, biochemical and enzymological methods.