AS-GSH CONJUGATION LIMITS AS AVAILABILITY & TOXICITY

AS-GSH 结合限制了可用性

• 批准号: 6382356
• 负责人: Michael Lieberman
• 金额: $ 29.9万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-05 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382356
• 关键词: arsenic; bile; cell line; chemical conjugate; clearance rate; detoxification; environmental toxicology; enzyme deficiency; glutamyltransferase; glutathione; laboratory mouse; laboratory rat; liquid chromatography mass spectrometry; urinalysis

The overall goal of this work is to learn more about the detoxification and clearance of arsenic (As) and to examine the role that As-protein conjugates play in toxicity. The major hypothesis to be tested is that clearance of As in vivo is dependent on two separate pathways, a major one involving formation and transport of As-GSH conjugates and a minor one dependent on transport of As ions; further, failure to reduce the reactivity of As b GSH conjugation allows the formation of metal-protein thiols that are toxic to the cell. The project takes advantage of recent advances in mass spectrometry and molecular genetics to address these issues. Although it is widely postulated that GSH conjugates of As are essential for its detoxification, there is no convincing demonstration of their existence in vivo. The investigators have synthesized arsenic triglutathione (ATG) and methyl arsenic diglutathione (MADG) and identified them in the urine of gamma-glutamyl transpeptidase (GGT)-deficient mice and the bile of wild type mice by use of liquid chromatography/mass spec (LCMS) and LC/inductive coupled MS (LC/ICP-MS). Other preliminary data using multidrug resistance-associated protein 2 (MRP2)-deficient rats have shown that As excretion into bile is dependent on this gene. They have also used gamma-glutamyl cysteine synthetase (GGCS)-deficient embryos to develop cells that lack the ability to synthesize GSH and demonstrated that these cells are As-sensitive. They will extend these studies and develop methods to study other As-thiol conjugates in vivo. They will test the hypothesis that formation of As-GSH conjugates and their As-cysteine derivatives are quantitatively the most important pathway in As clearance. They will use rodents deficient in MDR proteins and MRPs to test the hypothesis that these proteins function in As-GSH excretion. They have determined that yeast deficient in the ubiquitin/proteasome pathway for protein degradation is sensitive to As. They will use these, GGCS-deficient and GSH-overproducing mammalian cells, and mice deficient in ubiquitin/proteasome function to test the hypothesis that failure to form As-GSH conjugates allows formation of As-protein thiol conjugates that are toxic to the cell.

这项工作的总体目标是更多地了解排毒 和砷 (As) 的清除，并检查 As 蛋白的作用 缀合物发挥毒性。要检验的主要假设是 砷在体内的清除依赖于两个独立的途径，一个是主要途径 涉及 As-GSH 缀合物和次要缀合物的形成和运输 依赖于砷离子的传输；此外，未能降低反应性 由于 b GSH 结合可形成金属-蛋白质硫醇， 对细胞有毒。该项目利用了大规模技术的最新进展 光谱测定法和分子遗传学来解决这些问题。虽然它是 人们普遍认为 As 的 GSH 结合物对于其作用至关重要 解毒，没有令人信服的证据证明它们的存在 体内。研究人员合成了砷三谷胱甘肽（ATG）和 甲基砷二谷胱甘肽 (MADG) 并在尿液中鉴定出它们 γ-谷氨酰转肽酶 (GGT) 缺陷小鼠和野生型的胆汁 使用液相色谱/质谱 (LCMS) 和 LC/感应耦合对小鼠进行 质谱（LC/ICP-MS）。使用与多重耐药性相关的其他初步数据 蛋白质 2 (MRP2) 缺陷的大鼠表明，As 排泄到胆汁中 依赖这个基因。他们还使用了γ-谷氨酰半胱氨酸合成酶 (GGCS) 缺陷胚胎发育出缺乏合成能力的细胞 GSH 并证明这些细胞对 As 敏感。他们将扩展这些 研究并开发在体内研究其他 As-硫醇缀合物的方法。他们 将检验 As-GSH 缀合物的形成及其作用的假设 As-半胱氨酸衍生物在数量上是 As 中最重要的途径 清除。他们将使用缺乏 MDR 蛋白和 MRP 的啮齿动物来测试 假设这些蛋白质在 As-GSH 排泄中发挥作用。他们有 确定酵母缺乏蛋白质的泛素/蛋白酶体途径 降解对 As 敏感。他们将使用这些缺乏 GGCS 且 谷胱甘肽过量产生的哺乳动物细胞和缺乏泛素/蛋白酶体的小鼠 功能来检验未能形成 As-GSH 缀合物的假设 形成对细胞有毒的As-蛋白质硫醇缀合物。