Halogenated Alkenes and Microsomal GSH-transferases

描述（由申请人提供）：邻位卤代烯烃是在许多超级基金场所常见的有毒物质。在超级基金站点检测到的30种最常见的有毒物质中，有5种是肾毒性邻位卤代烯烃。与其他卤代烃不同，邻位卤代烯烃通过破坏近端小管细胞而独特地损害肾脏并诱发肾癌。据信，邻位卤代烯烃的肾毒性和肾致癌作用源于它们在肝微粒体中通过酶微粒体谷胱甘肽转移酶-1（MGST 1）转化为GSH S-缀合物，GSH S-缀合物被转运到肠，然后转化为相应的半胱氨酸S-缀合物。然后这些半胱氨酸S-缀合物被转运至肾脏并被肾半胱氨酸β-裂解酶裂解以形成毒性卤代烷基硫醇，其损害肾近端小管细胞中的线粒体。这一假设是有争议的，因为有竞争的理论不包括肝脏或MGST 1的作用。由于多器官系统之间的复杂相互作用和缺乏体外模型，这一假设的证实（或反驳）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结合卤代烃。我们的目标是[1]明确确定MGST 1在调节这些超级基金邻位卤代烯烃污染物毒性中的作用。这将通过生产MGST 1过度表达动物和两种类型的MGST 1缺陷动物（完全没有MGST 1和只有肝脏缺陷），并确定它们对原型邻位卤代烯烃三氯乙烯（TCE）和六氯丁二烯（HCBD）的敏感性/抗性来实现。我们的研究将确认MGST 1的组织和亚细胞分布，这也是有争议的，并确定是否删除MGST 1的结果在其他细胞溶质和微粒体GST亚型和选择的抗氧化系统的代偿性变化。我们还将通过检查替代起始位点产生的mRNA转录物的变化，并监测各种器官中MGST 1蛋白含量的变化，来研究应激诱导的MGST 1调节。[2]确定MGST家族的其他成员是否能够结合邻位卤代烯烃，从而在这些毒素的生物活化中发挥潜在作用。[3]确定重组MGST蛋白是否有助于对六氯丁二烯进行生物修复。[4]通过使用人MGST 1和MGST 2缺失细胞的研究，确定MGST 1和MGST 2对细胞抗氧化能力的相对贡献。

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.