MOLECULAR TOXICOLOGY IN HUMAN KIDNEY CELLS

描述：(改编自调查人员摘要)三氯乙烯(Tri) 是一种主要的环境污染物，也是公认的动物致癌物质。 TRI的人类健康风险评估很困难，因为有明显的性行为-以及 新陈代谢、毒性和靶器官的物种差异 专一性。Tri对肾脏的毒性和致癌作用是由于 它的代谢通过谷胱甘肽结合，随后代谢到 半胱氨酸偶联、S-(1，2-二氯乙烯)-L-半胱氨酸与代谢 通过半胱氨酸偶联b-裂解酶结合DCVC形成活性化合物。老鼠是 对三致肾毒性最敏感的物种但有很多 关于肾脏是否为人类TRI的靶器官存在分歧。这 建议使用融合的人近端肾小管原代培养(HPT) 以细胞为模型，确定可能导致相对较低的因素 人类对三聚氰胺所致肾毒性的易感性。将获得单元格 用胶原酶消化新鲜的人肾组织，并将其培养 在无血清、荷尔蒙定义的条件下。这些细胞维持着 培养过程中近端肾小管功能的表达。这项建议 将解决三个假设：1)新陈代谢和/或运输速度较低 HPT细胞对DCVC的敏感性相对较低；2)DCVC 在HPT细胞中通过坏死和凋亡导致肾小管细胞死亡； (3)DCVC引起的线粒体功能改变是 与HPT细胞的凋亡有关。以前的工作定义的比率 人肝肾组织中Tri的谷胱甘肽结合 代谢途径的这一初始步骤不能解释 人肾组织对三氯甲烷毒性的敏感性。此外， DCVC被认为是Tri的倒数第二个毒性代谢物。 因此，这些研究将使用DCVC作为主要测试剂。第一 假说将通过用b-裂解酶测量DCVC新陈代谢来解决。 和N-乙酰基转移酶，它形成硫代-S-(1，2或 2，2-二氯乙烯-L-半胱氨酸(NAcDCVC)。NAcDCVC脱乙酰化率将 也是可以量化的。DCVC和NAcDCVC的基底侧向和横向输送 在培养的HPT细胞中将对刷状缘膜进行定量和表征 在过滤器插入件上。第二个假设将通过定义精确的 暴露条件(时间、浓度)，使DCVC产生其中一种坏死 或者是细胞凋亡。坏死将通过释放一种胞浆酶来衡量。 而细胞凋亡将通过几种方法进行评估，包括细胞周期。 流式细胞术、细胞色素分析、DNA断裂和膜联蛋白染色 C释放，bc1-2表达。第三个假设将通过以下方式进行检验 DCVC诱导的不同线粒体功能指标变化的相关性 与细胞凋亡的发生和严重程度有关。这些研究将加强我们的 了解DCVC如何引起人肾脏和肾脏的细胞损伤 应作为分析物种差异反应的模型 其他肾毒性化学物质，并应增强我们评估人类 对化学性肾损伤的易感性。

DESCRIPTION: (Adapted from the Investigator's Abstract) Trichloroethylene (Tri) is a major environmental contaminant and is an established animal carcinogen. Human health risk assessment for Tri is difficult because of marked sex- and species-dependent differences in metabolism, toxicity, and target organ specificity. Toxic and carcinogenic effects of Tri in the kidneys are due to its metabolism by glutathione conjugation, subsequent metabolism to the cysteine conjugation, S-(1,2 dichlorovinyl)-L-cysteine (DCVC), and metabolism of DCVC by the cysteine conjugate b-lyase to form reactive compounds. Rats are the most susceptible species to Tri-induce kidney toxicity but there is much disagreement about the kidney as a target organ for Tri in humans. This proposal will use confluent primary cultures of human proximal tubular (hPT) cells as a model to determine factors that may contribute to the relatively low susceptibility of humans to Tri-induced kidney toxicity. Cells will be obtained from fresh human kidney tissue by collagenase digestion and will be cultured under serum-free, hormonally-defined conditions. These cells maintain expression of several proximal tubular functions during culture. The proposal will address three hypotheses: 1) Lower rates of metabolism and/or transport account for the relatively low susceptibility of hPT cells to DCVC; 2) DCVC produces renal tubular cell death in hPT cells by both necrosis and apoptosis; and 3) DCVC-induced alteration in mitochondrial function are causally associated with apoptosis in hPT cells. Previous work defined rates of glutathione conjugation of Tri in human liver and kidney tissue and showed that this initial step of the metabolic pathway cannot account for differences in susceptibility of human kidney tissue to Tri-induced toxicity. Furthermore, DCVC is known to be the penultimate toxic metabolite of Tri by this pathway. Accordingly, these studies will use DCVC as the primary test agent. The first hypothesis will be addressed by measurement of DCVC metabolism by the b-lyase and the N-acetyltransferase, which forms the mercapturate, N-acetyl-S-(1,2 or 2,2-dichlorovinyl)-L-cysteine (NAcDCVC). Rates of NAcDCVC deacetylation will also be quantitated. Transport of DCVC and NAcDCVC across by basolateral and brush-border membranes will be quantitated and characterized in hPT cells grown on filter inserts. The second hypothesis will be addressed by defining precise exposure conditions (time, concentration) whereby DCVC produces either necrosis or apoptosis. Necrosis will be measured by release of a cytosolic enzyme whereas apoptosis will be assessed by several assays, including cell cycle analysis, DNA fragmentation, and annexin staining by flow cytometry, cytochrome c release, and bcl-2 expression. The third hypothesis will be tested by correlating DCVC-induced changes in various measurers of mitochondrial function with the onset and severity of apoptosis. These studies will enhance our understanding of how DCVC produces renal cell injury in the human kidney and should serve as a model for analysis of species differences in responses to other nephrotoxic chemicals and should enhance our ability to evaluate human susceptibility to chemically induced renal injury.