MECHANISM OF GLUTATHIONE CONJUGATE DEPENDENT TOXICITY

谷胱甘肽结合物依赖性毒性机制

• 批准号: 2749706
• 负责人: Arthur Joseph Cooper
• 金额: $ 23.92万
• 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 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749706
• 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) 会导致大鼠患肾癌。 二氯乙炔 (DA) 是 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-结合物（= S-(1,2-二氯乙烯基)-L-半胱氨酸，DCVC）有毒。 DCVC对S3肾单位段造成选择性损伤；线粒体是 易受伤害的。 半胱氨酸 S-缀合物 β-裂合酶是吡哆醛 5'-磷酸盐依赖性酶，可将 DCVC 和类似化合物转化为 丙酮酸、氨和有毒片段。 该片段通过以下方式杀死肾细胞 与大分子发生反应，消耗细胞硫醇并刺激 过氧化损伤。 犬尿氨酸酶和胞质谷氨酰胺转氨酶 K (cytGTK) 具有半胱氨酸 S-缀合物 β-裂合酶活性。 然而， 犬尿氨酸酶被 DCVC 灭活，并且 cytGTK 的裂解酶活性为 由体内仅存在低水平的α-酮酸支持。 一个高 分子量 (Mr) 多亚基裂解酶（最近由 研究者）存在于 S3 段和肾细胞质中 线粒体，并被生理水平失活 α-酮戊二酸。 因此，它可能是负责的主要酶。 用于选择性肾损伤。 当前提案的目的是净化 来自大鼠肾细胞质（和线粒体）的高 Mr 裂解酶并克隆和 对亚基进行排序。 将生成抗体并用于确定 酶/亚基的细胞/亚细胞位置和贡献 肾脏总β-裂解酶活性的酶。 爪蟾卵母细胞 表达系统将用于确定每个的相对重要性 亚基对全酶整体结构/催化功能的影响。 LLC-PK1 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 酶在 GSH-/半胱氨酸S-结合物的毒性。 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 裂合酶被证明是生物活化的主要作用剂 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）的人类面临的风险 化合物）可以通过抑制高Mr裂解酶的方案来最小化。