Molecular Toxicology in Human Kidney Cells

人肾细胞的分子毒理学

• 批准号: 6781240
• 负责人: LAWRENCE H. LASH
• 金额: $ 23.29万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6781240
• 关键词: DNA damage; DNA repair; apoptosis; cell proliferation; chemical conjugate; cytotoxicity; flow cytometry; human tissue; kidney cell; kidney metabolism; mitochondria; mitochondrial membrane; regeneration; renal toxin; renal tubule; tissue /cell culture; toxin metabolism; trichloroethylene; western blottings

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

描述（由申请人提供）：三氯乙烯 (TRI) 是一种主要的环境污染物，是一种已确定的动物致癌物，并且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，其肾毒性和肾癌作用是由谷胱甘肽 (GSH) 结合产生的代谢产物介导的。随后代谢为半胱氨酸结合物 S-(1,2-二氯乙烯基)-L-半胱氨酸 (DCVC)，产生倒数第二个有毒代谢物。 DCVC 通过半胱氨酸结合酶或含黄素单加氧酶进行代谢，产生最终的反应性和毒性物质。先前描述 TRI 和 DCVC 代谢和潜在作用模式的大部分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 完成的）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。对于 TRI 等卤化溶剂尤其如此，因为不同物种在代谢、运输和总体毒性敏感性方面存在显着差异。我们之前的研究表明，DCVC 可导致人近端肾小管 (hPT) 细胞原代培养物凋亡或坏死，具体取决于浓度和暴露时间。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表达有影响。本申请使用 hPT 细胞的原代培养物作为实验模型，将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，以及在暴露于毒性导致的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 解决了暴露于中等毒性浓度 DCVC 的 hPT 细胞是否会进行修复和再生的问题。将评估几种修复标记，并研究诱导修复和再生反应的精确条件和潜在机制。具体目标 2 将解决 DCVC 是否可以刺激 hPT 细胞不受控制的增殖的问题。将研究不同 DCVC 暴露条件下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 细胞毒性是否充分且必要的问题。尽管之前的研究表明线粒体是 DCVC 早期且有效影响的细胞内靶标，但尚不清楚线粒体毒性是否是 DCVC 暴露后发生的事件进展中的必然步骤，或者独立于线粒体的其他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细胞方面的工作的基础上，并将其扩展以提供对 DCVC 影响人类肾脏的各种复杂方式的更全面的了解