Molecular Toxicology in Human Kidney Cells

描述（由申请人提供）：各种环境和职业化学品都是肾毒性物质。研究这些化学物质的相互作用很重要，因为人类通常一次接触一种以上的化学物质。作为这些相互作用的模型，该提案将研究两种环境化学品三氯乙烯（TRI）和无机汞（Hg）的影响，这两种化学品都将肾脏作为主要靶器官。三碘甲状腺原氨酸对实验室动物和人类都有致癌性，而汞则具有急性细胞毒性。这两种化学物质也与谷胱甘肽（GSH）相互作用。TRI的肾毒性和肾致癌性归因于其通过GSH缀合的生物活化和随后加工成倒数第二个毒性代谢物S-（1，2-二氯乙烯基）-L-半胱氨酸（DCVC），其通过半胱氨酸缀合物β-裂解酶或含黄素的单加氧酶代谢以形成反应性物质。与此相反，谷胱甘肽在汞的处理和肾毒性中的作用更为复杂，它既有保护作用，又有抑制作用。本提案将研究TRI及其代谢产物DCVC与汞在相同孵育条件下人近端肾小管（hPT）细胞和大鼠PT（rPT）细胞融合原代培养物中的相互作用。本研究将对以下四个假设进行检验：1）低浓度亚毒性汞预暴露hPT和rPT细胞，通过增强TRI和/或DCVC代谢相关酶的表达和活性，增强TRI和DCVC的生物活化; 2）将hPT和rPT细胞预暴露于低，亚毒性浓度的汞由于TRI和/或DCVC生物活化的增加而增强TRI和DCVC的细胞毒性; 3）将hPT和rPT细胞预暴露于低至中等毒性浓度的TRI或DCVC通过耗尽GSH而增强Hg诱导的细胞毒性; 4）通过预暴露于另一种化学物质来调节hPT和rPT细胞中的一种化学物质的细胞毒性与细胞因子、生长因子和/或热休克蛋白的表达的变化相关。从这些研究的结果将提供信息的两个重要的环境毒物在人类和大鼠肾细胞模型的相互作用机制，并将使我们能够确定的因素，有助于物种的易感性差异。

DESCRIPTION (provided by applicant): A diverse array of environmental and occupational chemicals are nephrotoxicants. It is important to study the interaction of these chemicals because humans are generally exposed to more than one chemical at a time. As a model for these interactions, this proposal will study the effects of two environmental chemicals, trichloroethylene (TRI) and inorganic mercury (Hg), that both have the kidneys as a primary target organ. TRI is carcinogenic in both laboratory animals and in humans while Hg is acutely cytotoxic. Both chemicals also interact with glutathione (GSH). The nephrotoxicity and nephrocarcinogenicity of TRI is attributed to its bioactivation by GSH conjugation and subsequent processing to the penultimate toxic metabolite S-(1 ,2-dichlorovinyl)-L-cysteine (DCVC), which is metabolized by the cysteine conjugate beta-lyase or flavin-containing monooxygenase, to form reactive species. In contrast, the role of GSH in the handling and nephrotoxicity of Hg is more complex, with GSH having both a protective and an intoxifying role. This proposal will study the interactions between TRI, its metabolite DCVC, and Hg in confluent primary cultures of human proximal tubular (hPT) cells and rat PT (rPT) cells under identical incubation conditions. Four hypotheses will be tested: 1) Pre-exposure of hPT and rPT cells to low, subtoxic concentrations of Hg enhances the bioactivation of both TRI and DCVC by enhancement of expression and activity of enzymes involved in TRI and/or DCVC metabolism; 2) pre-exposure of hPT and rPT cells to low, subtoxic concentrations of Hg enhances cytotoxicity of both TRI and DCVC as a consequence of increases in TRI and/or DCVC bioactivation; 3) pre-exposure of hPT and rPT cells to low- to moderately-toxic concentrations of TRI or DCVC enhances Hg-induced cytotoxicity by depletion of GSH; 4) modulation of cytotoxicity of one chemical in hPT and rPT cells by pre-exposure to another chemical is associated with changes in expression of cytokines, growth factors, and/or heat shock proteins. Results from these studies will provide information on the mechanism of interaction of two important environmental toxicants in a model of human and rat kidney cells and will allow us to determine factors that contribute to species differences in susceptibility.