Molecular Toxicology in Human Kidney Cells

描述(申请人提供)：各种环境和职业化学物质都是肾毒物。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的化学物质不止一种。作为这些相互作用的模型，这项提议将研究两种环境化学品--三氯乙烯(Tri)和无机汞(Hg)--的影响，这两种化学品都以肾脏为主要靶器官。三碘对实验动物和人类都是致癌的，而汞具有强烈的细胞毒性。这两种化学物质也与谷胱甘肽(GSH)相互作用。Tri的肾毒性和致癌作用归因于其通过谷胱甘肽偶联被生物激活，然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，该代谢物被半胱氨酸偶联β-裂解酶或含黄素的单加氧酶代谢形成活性物种。相比之下，GSH在汞的处理和肾毒性中的作用更为复杂，GSH既有保护作用，也有毒性作用。这项建议将研究在相同培养条件下，人近端肾小管(HPT)细胞和大鼠肾小管(RPT)细胞融合原代培养中Tri、其代谢物DCVC和汞之间的相互作用。将检验四个假设：1)HPT和RPT细胞预先暴露在低、亚毒性浓度的汞中，通过增强参与TRI和/或DCVC代谢的酶的表达和活性来增强TRI和DCVC的生物活性；2)预先暴露于低浓度、亚毒性汞的HPT和RPT细胞，由于TRI和/或DCVC的生物激活增加，从而增强了TRI和DCVC的细胞毒性；3)预先暴露于低到中等毒性浓度的TRI或DCVC，通过耗尽GSH而增强汞诱导的细胞毒性；4)一种化学物质对HPT和RPT细胞毒性的调节与细胞因子、生长因子和/或热休克蛋白表达的变化有关。这些研究的结果将提供两种重要环境毒物在人和大鼠肾细胞模型中相互作用的机制的信息，并将使我们能够确定导致物种敏感性差异的因素。

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.