Molecular Toxicology in Human Kidney Cells

描述（由申请人提供）：三氯乙烯（TRI）是一种主要的环境污染物，是一种确定的动物致癌物，被国家毒理学计划和国际癌症研究机构认为是“可能的人类致癌物”。肾脏是TRI的靶器官之一，其肾毒性和肾致癌作用是由与谷胱甘肽（GSH）结合产生的代谢物介导的。随后对半胱氨酸缀合物S-（1,2-二氯乙烯基）- l -半胱氨酸（DCVC）的代谢产生倒数第二的毒性代谢物。是半胱氨酸偶联酶或含黄素的单加氧酶对DCVC的代谢产生最终的反应性和毒性物质。大多数先前描述TRI和DCVC的代谢和潜在作用模式的研究都是在啮齿动物或啮齿动物组织中进行的。虽然这些研究（其中一些是由PI进行的）提供了许多有用的见解，但在使用从啮齿动物获得的数据进行人类健康风险评估方面存在问题。对于卤化溶剂，如TRI，尤其如此，因为它们在代谢、运输和对毒性的总体敏感性方面存在明显的物种差异。我们之前的研究表明，DCVC可以引起人近端小管细胞（hPT）原代培养细胞的凋亡或坏死，这取决于暴露的浓度和时间。研究结果还表明，DCVC对应激反应和细胞生长调节相关蛋白的表达有影响。本应用以hPT细胞原代培养物为实验模型，研究暴露于中等毒性浓度DCVC的hPT细胞进行修复和再生的能力，DCVC通过非基因毒性机制诱导细胞增殖的潜力，以及暴露于毒性事件过程中线粒体毒性的要求。该应用程序包括三个具体目标。特异性目标1解决了暴露于中等毒性浓度DCVC的hPT细胞是否进行修复和再生的问题。几个修复标记将被评估和精确的条件和潜在的机制，修复和再生反应诱导将被研究。特异性目标2将解决DCVC是否能刺激hPT细胞不受控制的增殖的问题。研究不同条件下DCVC暴露对细胞周期和细胞周期信号分子的影响。最后，Specific Aim 3将解决线粒体毒性是否对hPT细胞中dcvc诱导的毒性是充分和必要的问题。尽管先前的研究表明线粒体是DCVC的早期和潜在的细胞内靶点，但目前尚不清楚线粒体毒性是否是DCVC暴露后发生的事件进展的必要步骤，也不清楚是否有其他独立于线粒体的途径可以介导肾细胞损伤。这些目标的实现应该建立在我们之前对人类肾脏细胞的研究基础上，并对其进行扩展，以更全面地了解DCVC影响人类肾脏的各种复杂方式

DESCRIPTION (provided by applicant): Trichloroethylene (TRI) is a major environmental contaminant, is an established animal carcinogen, and is considered a "probably human carcinogen" by the National Toxicology Program and the International Agency for Research on Cancer. The kidneys are one target organ for TRI and its nephrotoxic and nephrocarcinogenic effects are mediated by metabolites derived from conjugation with glutathione (GSH). Subsequent metabolism to the cysteine conjugate S- (1,2- dichlorovinyl)-L-cysteine (DCVC) generates the penultimate toxic metabolite. It is metabolism of DCVC by either the cysteine conjugate ¿-Iyase or the flavin-containing monooxygenase that generates the ultimate reactive and toxic species. Most of the previous research that has delineated the metabolism and potential modes of action for TRI and DCVC has been performed in rodents or with tissue from rodents. While these studies, some of which have been done by the PI, have provided much useful insight, there are problems in using data obtained from rodents for human health risk assessment. This is particularly true for halogenated solvents such as TRI, because of marked species differences in metabolism, transport, and overall sensitivity to toxicity. Previous studies of ours showed that DCVC can cause both apoptosis or necrosis in primary cultures of human proximal tubular (hPT) cells, depending on concentration and time of exposure. Findings also suggested effects of DCVC on expression of proteins related to stress response and regulation of cell growth. This application uses primary cultures of hPT cells as the experimental model and will investigate the ability of hPT cells exposed to moderately toxic concentrations of DCVC to undergo repair and regeneration, the potential for DCVC to induce cell proliferation by non-genotoxic mechanisms, and the requirement for mitochondrial toxicity in the course of events leading from exposure to toxicity. The application comprises three Specific Aims. Specific Aim 1 addresses the question of whether hPT cells exposed to moderately toxic concentrations of DCVC undergo repair and regeneration. Several markers of repair will be assessed and precise conditions and potential mechanisms by which the repair and regeneration response are induced will be investigated. Specific Aim 2 will address the question of whether DCVC can stimulate uncontrolled proliferation of hPT cells. Effects on cell cycle and cell cycle signaling molecules under various conditions of DCVC exposure will be studied. Finally, Specific Aim 3 will address the question of whether mitochondrial toxicity is sufficient and necessary for DCVC-induced toxicity in hPT cells. Although previous work has shown that mitochondria are early and potently affected intracellular targets of DCVC, it is not known whether mitochondrial toxicity is an obligatory step in the progression of events that occur after DCVC exposure or whether other pathways that are independent of mitochondria can mediate renal cell injury. Achievement of these aims should build on our previous work in human kidney cells and extend it to provide a much more complete understanding of the various and complex ways in which DCVC affects the human kidney