Molecular Toxicology in Human Kidney Cells

说明(申请人提供)：三氯乙烯(TRI)是一种主要的环境污染物，是公认的动物致癌物质，被国家毒理学计划和国际癌症研究机构视为“可能的人类致癌物质”。肾脏是TRI的一个靶器官，其肾毒性和肾癌作用是通过与谷胱甘肽(GSH)结合而产生的代谢产物介导的。半胱氨酸偶合物S-(1，2-二氯乙烯)-L-半胱氨酸的后续代谢产生倒数第二个有毒代谢物。DCVC的代谢是通过半胱氨酸偶联酶或含有黄素的单加氧酶来产生最终的活性和毒性物种。以前的大多数研究描绘了TRI和DCVC的新陈代谢和潜在的作用模式，这些研究都是在啮齿动物身上进行的，或者是用啮齿动物的组织进行的。虽然这些研究提供了许多有用的见解，但使用从啮齿动物获得的数据进行人类健康风险评估仍存在问题。这对于卤化溶剂来说尤其如此，因为在新陈代谢、运输和对毒性的总体敏感性方面存在显著的物种差异。我们以前的研究表明，DCVC可以引起原代培养的人近端小管(HPT)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对与应激反应和细胞生长调节相关的蛋白质的表达有影响。这项应用使用原代培养的HPT细胞作为实验模型，并将研究中等毒性浓度的DCVC对HPT细胞进行修复和再生的能力，DCVC通过非遗传毒性机制诱导细胞增殖的潜力，以及在暴露于毒性事件过程中对线粒体毒性的要求。该申请包括三个具体目标。具体目标1涉及暴露于中等毒性浓度的DCVC的HPT细胞是否进行修复和再生的问题。将评估修复的几个标志物，并将研究诱导修复和再生反应的准确条件和潜在机制。具体目标2将解决DCVC是否能刺激HPT细胞不受控制的增殖的问题。将研究不同条件下DCVC暴露对细胞周期和细胞周期信号分子的影响。最后，具体目标3将解决线粒体毒性对于DCVC诱导的HPT细胞毒性是否充分和必要的问题。尽管先前的工作表明线粒体是DCVC早期和有效的细胞内靶点，但尚不清楚线粒体毒性是否是DCVC暴露后发生的事件发展的必需步骤，也不知道其他独立于线粒体的途径是否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在人类肾脏细胞上的工作的基础上，并将其扩展到对DCVC影响人类肾脏的各种复杂方式的更全面的理解

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