Halogenated Alkenes and Microsomal GSH-transferases

卤代烯烃和微粒体 GSH 转移酶

• 批准号: 7141476
• 负责人: MICHAEL J KELNER
• 金额: $ 32.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-08 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7141476
• 关键词: alkenes; antioxidants; biological transport; biotransformation; chemical conjugate; cysteine; enzyme activity; gene deletion mutation; gene expression; genetically modified animals; glutathione transferase; halogenation; human tissue; kidney neoplasms; laboratory mouse; lyase; microsomes; natural killer cells; protein localization; protein structure function; recombinant proteins; renal toxin; toxicology

DESCRIPTION (provided by applicant): The vicinal haloalkenes are toxicants commonly found at many Superfund sites. Of the 30 most common toxicants detected at Superfund sites, five are nephrotoxic vicinal haloalkenes. Unlike other halogenated hydrocarbons, vicinal haloalkenes uniquely damage the kidney by destroying proximal tubule cells and induce renal carcinomas. It is believed that the nephrotoxic and nephrocarcinogenic effects of vicinal haloalkenes stems from their conversion in hepatic microsomes by the enzyme microsomal glutathione transferase-1 (MGST1) to GSH S-conjugates, which are transported to intestine and then converted to the corresponding cysteine S-conjugates. These cysteine S-conjugates are then transported to the kidney and cleaved by renal cysteine beta-lyases to form toxic haloalkylthiols that damage mitochondria in renal proximal tubular cells. This hypothesis is controversial as there are competing theories that do not include a role for either the liver or for MGST1. Confirmation (or disproof) of this hypothesis has been difficult due to the complex interaction between multiple organ systems and a lack of in vitro models. Complicating the issue is the recent finding that there are multiple human microsomal glutathione transferases capable of conjugating halogenated hydrocarbons. Our objectives are [1] To definitively determine the role of MGST1 in modulating the toxicity of these Superfund vicinal haloalkene contaminates. This will be accomplished by producing both MGST1 overexpressing animals and two types of MGST1 -deficient animals (complete MGST1 nulls versus liver-deficient only) and determining their sensitivity/resistance to the prototype vicinal haloalkenes trichloroethylene (TCE) and hexachlorobutadiene (HCBD). Our studies will confirm the tissue and subcellular distribution of MGST1, which is also controversial, and also determine if deletion of MGST1 results in compensatory changes in other cytosolic and microsomal GST isoforms and in select antioxidant systems. We will also investigate the stress-induced regulation of MGST1 by examining variation in mRNA transcripts that are produced by alternative start sites, and monitoring changes in MGST1 protein content in various organs. [2] To determine if other members of the MGST family are capable of conjugating vicinal haloalkenes and thereby have a potential role in bioactivation of these toxins. [3] To determine whether recombinant MGST proteins could assist in bioremediation of HCBD. [4] To determine the relative contribution of MGST1 and MGST2 to cellular antioxidant capacity through studies utilizing human MGST1 and MGST2 null cells.

描述（由申请人提供）：邻近卤代烃是在许多超级基金遗址中常见的有毒物质。在超级基金检测到的30种最常见的毒物中，有5种是具有肾毒性的卤代烃。与其他卤代烃不同，邻代卤代烃通过破坏近端小管细胞和诱发肾癌来损害肾脏。人们认为，邻代卤代烃的肾毒性和肾致癌作用源于它们在肝微粒体中通过微粒体谷胱甘肽转移酶-1 （MGST1）转化为谷胱甘肽s -缀合物，后者被转运到肠道，然后转化为相应的半胱氨酸s -缀合物。这些半胱氨酸s -缀合物随后被运送到肾脏，并被肾脏半胱氨酸β -裂解酶裂解，形成有毒的卤代烷基硫醇，损害肾近端小管细胞的线粒体。这个假设是有争议的，因为有一些相互竞争的理论没有包括肝脏或MGST1的作用。由于多器官系统之间复杂的相互作用和缺乏体外模型，证实（或反驳）这一假设一直很困难。使问题复杂化的是，最近发现有多种人类微粒体谷胱甘肽转移酶能够偶联卤化烃。我们的目标是明确确定MGST1在调节这些超级基金附近卤代烃污染物的毒性中的作用。这将通过生产MGST1过表达动物和两种类型的MGST1缺陷动物（完全MGST1缺失与仅肝缺陷）来实现，并确定它们对原型邻代卤代烯三氯乙烯（TCE）和六氯丁二烯（HCBD）的敏感性/耐药性。我们的研究将证实MGST1的组织和亚细胞分布（这也是有争议的），并确定MGST1的缺失是否会导致其他细胞质和微粒体GST亚型以及某些抗氧化系统的代偿性变化。我们还将通过检测由不同起始位点产生的mRNA转录物的变化，以及监测不同器官中MGST1蛋白含量的变化，来研究应激诱导的MGST1调控。[2]目的是确定MGST家族的其他成员是否能够偶联邻近的卤代烯，从而在这些毒素的生物活化中发挥潜在作用。[3]目的探讨重组MGST蛋白是否能辅助HCBD的生物修复。[4]通过对人MGST1和MGST2空细胞的研究，确定MGST1和MGST2对细胞抗氧化能力的相对贡献。