Halogenated Alkenes and Microsomal GSH-transferases

描述(申请人提供)：附近的卤代烯是在许多超级基金地点常见的有毒物质。在超级基金网站检测到的30种最常见的毒物中，有5种是肾毒性的邻近卤代烯。与其他卤代烃不同，邻位卤代烯通过破坏近端小管细胞而独特地损害肾脏，并导致肾癌。据认为，邻位卤代烯烃的肾毒性和肾癌作用源于它们在肝微粒体中被微粒体谷胱甘肽转移酶-1转化为谷胱甘肽S结合物，再被转运到肠道，然后转化为相应的半胱氨酸S结合物。然后，这些半胱氨酸S结合物被转运到肾脏，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中的线粒体。这一假设是有争议的，因为有相互竞争的理论，既不包括肝脏的作用，也不包括MGST1的作用。由于多个器官系统之间复杂的相互作用以及缺乏体外模型，这一假说很难得到证实(或反证)。使问题复杂化的是最近的一项发现，即存在多个能够结合卤代烃的人类微粒体谷胱甘肽转移酶。我们的目标是[1]明确地确定MGST1在调节这些超级基金邻域卤代烯烃污染物的毒性中的作用。这将通过培育MGST1过度表达的动物和两种类型的MGST1缺陷动物(完全MGST1缺失型与仅肝虚型)来实现，并确定它们对原型邻近卤代烯烃三氯乙烯(TCE)和六氯丁二烯(HCBD)的敏感性/抵抗力。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，也将确定MGST1的缺失是否会导致其他胞质和微粒体GST亚型以及选定的抗氧化系统的代偿性变化。我们还将通过检测由不同起始点产生的mRNA转录的变化，并监测不同器官中MGST1蛋白含量的变化，来研究压力诱导的MGST1的调节。[2]确定MGST家族的其他成员是否能够结合邻近的卤代烯，从而在这些毒素的生物激活中发挥潜在的作用。[3]确定重组MGST蛋白是否有助于HCBD的生物修复。[4]通过利用人MGST1和MGST2缺失细胞的研究，确定MGST1和MGST2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.