MECHANISM OF GLUTATHIONE CONJUGATE DEPENDENT TOXICITY

谷胱甘肽结合物依赖性毒性机制

• 批准号: 6178519
• 负责人: Arthur Joseph Cooper
• 金额: $ 25.37万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178519
• 关键词: Xenopus oocyte; alkynes; chemical conjugate; environmental toxicology; enzyme structure; glutathione; laboratory rabbit; laboratory rat; lyase; protein purification; renal toxin; toxin metabolism; trichloroethylene

DESCRIPTION: (Adapted from the Investigator's Abstract) Long-term exposure to trichloroethylene (TE) causes renal cancer in rats. Dichloroacetylene (DA), a breakdown product of TE, is both nephrotoxic and neurotoxic. TE and DA are metabolized to the glutathione (GSH S-conjugate and cysteine S-conjugate (= S-(1,2-dichlorovinyl)-L-cysteine, DCVC) which are toxic. DCVC causes selective damage to the S3 nephron segment; mitochondria are vulnerable. Cysteine S-conjugate beta-lyases are pyridoxal 5'-phosphate-dependent enzymes that convert DCVC and similar compounds to pyruvate, ammonia and a toxic fragment. The fragment kills renal cells by reacting with macromolecules, depleting cellular thiols and stimulating peroxidative damage. Kynureninase and cytosolic glutamine transaminase K (cytGTK) have cysteine S-conjugate beta-lyase activity. However, kynureninase is inactivated by DCVC and the lyase activity of cytGTK is supported by alpha-keto acids present in vivo only at low levels. A high molecular weight (Mr) multi-subunit lyase (recently discovered by the investigator) is present in the S3 segment and in kidney cytosol and mitochondria, and is inactivated by physiological levels of alpha-ketoglutarate. Therefore, it may be the principal enzyme responsible for selective renal damage. The aim of the current proposal is to purify the high Mr lyase from rat kidney cytosol (and mitochondria) and clone and sequence the subunits. Antibodies will be generated and used to determine the cellular/subcellular location of the enzyme/subunits and contribution of the enzyme to total beta-lyase activity of the kidney. The Xenopus oocyte expression system will be used to determine the relative importance of each subunit to overall structure/catalytic function in the holoenzyme. LLC-PK1 cells lacking mitochondria and heterologous expression in a mammalian cell line should provide useful tools to study the role of the high Mr enzyme in the toxicity of GSH-/cysteine S-conjugates. The proposed work will provide insights into the relationship of the high Mr lyase to other PLP enzymes. If the high Mr lyase is shown to be the principal agent for bioactivation of nephrotoxic conjugates the results will have medical importance. TE is a common pollutant. Risks to humans heavily exposed to TE (or similar compounds) may be minimized by regimens that inhibit the high Mr lyase.

描述：（改编自研究者摘要）长期暴露 三氯乙烯（TE）导致大鼠肾癌。 二氯乙炔 (DA)TE的分解产物，具有肾毒性和神经毒性。 TE和 DA代谢为谷胱甘肽（GSH S-结合物和半胱氨酸 S-共轭物（= S-（1，2-二氯乙烯基）-L-半胱氨酸，DCVC），具有毒性。 DCVC导致S3肾单位段的选择性损伤; 脆弱 半胱氨酸S-共轭β-裂解酶是吡哆醛 5 '-磷酸依赖性酶，其将DCVC和类似化合物转化为 丙酮酸氨和有毒碎片 碎片杀死肾细胞， 与大分子反应，消耗细胞硫醇并刺激 过氧化损伤 犬尿氨酸酶和胞浆谷氨酰胺转氨酶K （cytGTK）具有半胱氨酸S-缀合物β-裂解酶活性。 然而，在这方面， 犬尿氨酸酶被DCVC灭活，并且cytGTK的裂解酶活性被 由仅以低水平存在于体内的α-酮酸支持。 高 分子量（Mr）多亚基裂解酶（最近由 研究者）存在于S3节段和肾细胞质中， 线粒体，并被生理水平的 α-酮戊二酸 因此，它可能是负责 选择性肾损伤 目前提案的目的是净化 从大鼠肾细胞质（和线粒体）中分离高分子量裂解酶， 对亚基进行测序。 将生成抗体并用于确定 酶/亚基的细胞/亚细胞位置和 酶与肾脏总β-裂解酶活性的比值。 非洲爪蟾卵母细胞 表达系统将被用来确定每个的相对重要性， 在全酶中亚基到整体结构/催化功能。 LLC-PK1 在哺乳动物细胞中缺乏线粒体和异源表达的细胞 线应该提供有用的工具来研究高Mr酶的作用， GSH-/半胱氨酸S-缀合物的毒性。 拟议的工作将提供 深入了解高Mr裂解酶与其他PLP酶的关系。 如果高Mr裂解酶被证明是生物激活的主要因素， 肾毒性缀合物的结果将具有医学重要性。 TE是一个 常见污染物 严重暴露于TE（或类似物质）的人类风险 化合物）可以通过抑制高Mr裂解酶的方案来最小化。