MOLECULAR TOXICOLOGY IN HUMAN KIDNEY CELLS

人肾细胞的分子毒理学

• 批准号: 6382219
• 负责人: LAWRENCE H. LASH
• 金额: $ 16.83万
• 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 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382219
• 关键词: apoptosis; chemical conjugate; cysteine; cytotoxicity; glutathione; human tissue; kidney cell; kidney metabolism; renal toxin; renal tubule; species difference; tissue /cell culture; toxin metabolism; trichloroethylene

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.

描述：（改编自研究者摘要）三氯乙烯（Tri） 是一种主要的环境污染物，并且是一种确定的动物致癌物。 Tri的人类健康风险评估是困难的，因为它具有明显的性别特征， 代谢、毒性和靶器官的种属依赖性差异 的特异性Tri在肾脏中的毒性和致癌作用是由于 其代谢通过谷胱甘肽结合，随后代谢为 半胱氨酸结合、S-（1，2-二氯乙烯基）-L-半胱氨酸（DCVC）和代谢 DCVC通过半胱氨酸缀合物b-裂解酶裂解以形成反应性化合物。大鼠 最易受Tri-induced肾毒性影响的种属， 关于肾脏作为Tri在人类中的靶器官的分歧。这 提案将使用人近端肾小管（hPT）的融合原代培养物 细胞作为模型，以确定可能导致相对较低的 人类对Tri-induced肾毒性的易感性。将获得细胞 通过胶原酶消化从新鲜的人肾组织中分离并培养 在无血清的、血液学限定的条件下。这些细胞维持着 在培养过程中几种近端肾小管功能的表达。该提案 将解决三个假设：1）较低的代谢率和/或运输 说明hPT细胞对DCVC的敏感性相对较低; 2）DCVC 通过坏死和凋亡在hPT细胞中产生肾小管细胞死亡; DCVC诱导的线粒体功能改变是其致病的原因。 与hPT细胞凋亡相关。以前的工作定义了 Tri在人肝和肾组织中的谷胱甘肽结合，并表明， 代谢途径的这一初始步骤不能解释 人类肾脏组织对Tri-induced毒性的易感性。此外，委员会认为， 已知DCVC是Tri通过该途径的倒数第二个毒性代谢产物。 因此，这些研究将使用DCVC作为主要试验药物。第一 将通过测量b-裂解酶的DCVC代谢来解决这一假设 和N-乙酰基转移酶，其形成巯基尿酸盐，N-乙酰基-S-（1，2或 2，2-二氯乙烯基）-L-半胱氨酸（NAcDCVC）。NAcDCVC脱乙酰化的速率将 也可以量化。DCVC和NAcDCVC通过基底外侧和 将在生长的hPT细胞中定量和表征刷状缘膜 在过滤器插入物上。第二个假设将通过定义精确 DCVC产生坏死的暴露条件（时间、浓度） 或凋亡。将通过胞质酶的释放来测量坏死 而细胞凋亡将通过几种测定法来评估，包括细胞周期 流式细胞术分析、DNA片段化和膜联蛋白染色、细胞色素 c释放和bcl-2表达。第三个假设将通过以下方式进行检验： DCVC诱导的各种线粒体功能指标变化的相关性 与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如何在人类肾脏中产生肾细胞损伤， 应作为一种模式，分析物种的差异， 其他肾毒性化学品，并应提高我们的能力，以评估人类 对化学性肾损伤的易感性。