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：（根据调查员的摘要改编）长期曝光 三氯乙烯（TE）引起大鼠的肾癌。 二氯乙炔 （DA）是TE的分解产物，既是肾毒性又是神经毒性。 TE和 DA被代谢为谷胱甘肽（GSH S-偶联和半胱氨酸 有毒的s-偶联物（= s-（1,2-二氯环烯基）-L-甲基，DCVC）。 DCVC对S3 Nephron节段造成选择性损害；线粒体是 易受伤害的。 半胱氨酸S-偶联β-裂解酸酯是吡啶还毒素 将DCVC和类似化合物转换为5'-磷酸依赖性酶 丙酮酸，氨和有毒碎片。 碎片通过 与大分子反应，耗尽细胞硫醇并刺激 过氧化损伤。 Kynureninase和胞质谷氨酰胺转氨酶K （CytgTK）具有半胱氨酸S-偶联β-酯活性。 然而， Kynureninase被DCVC灭活，Cytgtk的裂解酶活性为 由α-酮酸支撑，仅在体内存在于低水平。 高 分子量（MR）多生物裂解酶（最近由 研究者）存在于S3段，肾脏细胞质和 线粒体，并被生理水平灭活 α-Ketoglutarate。 因此，它可能是负责的主要酶 为了选择性肾脏损害。 当前建议的目的是净化 来自大鼠肾脏细胞质（以及线粒体）和克隆的高裂解酶 序列亚基。 将生成并用于确定抗体 酶/亚基的细胞/亚细胞位置以及 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 全酶中总体结构/催化功能的亚基。 LLC-PK1 缺乏线粒体和异源表达的细胞 线应提供有用的工具来研究高MR酶在 GSH/半胱氨酸S-偶联物的毒性。 拟议的工作将提供 洞悉高MR裂解酶与其他PLP酶的关系。 如果显示高MR裂解酶是生物活化的主要药物 肾毒性共轭结果将具有医学重要性。 TE是一个 常见污染物。 大量暴露于TE的人（或类似的风险） 化合物）可以通过抑制高MR裂解酶的方案来最小化。