Molecular Toxicology in Human Kidney Cells

人肾细胞的分子毒理学

• 批准号: 7046832
• 负责人: LAWRENCE H. LASH
• 金额: $ 22.11万
• 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/7046832
• 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（由申请方提供）：三氯乙烯（TRI）是一种主要的环境污染物，是一种既定的动物致癌物，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的一个靶器官，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），产生倒数第二个毒性代谢产物。DCVC通过半胱氨酸缀合物裂解酶或含黄素的单加氧酶代谢，产生最终的活性和毒性物质。大多数以前的研究已经描绘了TRI和DCVC的代谢和潜在的作用模式，已经在啮齿动物或啮齿动物组织中进行。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TRI等卤代溶剂尤其如此，因为物种在代谢、运输和对毒性的总体敏感性方面存在显着差异。我们以前的研究表明，DCVC可以导致细胞凋亡或坏死的原代培养的人近端肾小管（hPT）细胞，这取决于浓度和暴露时间。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的影响。本申请使用hPT细胞的原代培养物作为实验模型，并将研究暴露于中等毒性浓度的DCVC的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细胞是否经历修复和再生的问题。将评估修复的几个标志物，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细胞的不受控制的增殖的问题。将研究DCVC暴露在各种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将解决线粒体毒性是否足以和必要的DCVC诱导的hPT细胞毒性的问题。尽管先前的工作已经表明线粒体是DCVC的早期和有效影响的细胞内靶点，但尚不清楚线粒体毒性是否是DCVC暴露后发生的事件进展中的强制性步骤，或者不依赖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以前在人肾细胞中的工作基础上，并将其扩展到对DCVC影响人肾的各种复杂方式提供更全面的理解