Project 3: Mitochondrial and cellular mechanisms of neurotoxicity of Superfund chemical co-exposures

摘要 在该项目正在进行的工作中，我们发现发育过程中暴露于线粒体毒物会导致 秀丽隐杆线虫的神经毒性结果，包括神经元的形态变化 行为，从长远来看，增加了神经变性的易感性。为了与环保局保持一致，我们描述了 所有这些都被称为“发育神经毒性(DNT)”，因为它们是由发生在 发展。DNT的两个重要和最主要的机制是：1)改变神经发生，导致 改变细胞命运、形态和连通性(“硬连线”)，以及2)功能的持续变化 形态上看起来正常的神经元(表观遗传学的“编程”)。区别它们的是 挑战性；我们提出了一种新颖而强大的方法来评估每种可能性。我们将从体内开始 产量相对较高且经济的模式，线虫。线虫还提供了一个额外的关键好处： 发育性神经发生通常是不变的，也允许清楚地识别硬连接的变异。 作为行为和应激反应的变化，而不需要形态改变(编程)。在C++中工作。 在优雅之后，将在人类神经干细胞(HNSCs)中进行测试，允许与人类相关的DNT 测试，以及识别特定性别差异和表观遗传修饰的机会。 对DNT进行严格评估的化学品相对较少。信息的匮乏就更是如此 尽管事实是联合暴露是现实，但化学联合暴露是明显的。这种缺乏 混合物测试的结果部分是由于监管政策，部分是由于实验室测试中的技术挑战 共同曝光的证据。我们的体内-体外联合方法将使我们能够严格测试由 既有复杂的环境混合物，也有来自定义的个别超级基金化学品的组合，我们 将评估非相加效果。我们将测试典型的发育神经毒剂的效果 已知环境规定的单一或组合的PB、Cd和多环芳烃 浓度。我们将把我们在线虫和hNSCs上的结果与其他项目在 鱼、老鼠和人。证明线虫可以可靠地用于研究混合物DNT将增加一个 强大的环境混合物测试和监管新模式。 最后，我们将测试线粒体功能障碍的程度，线粒体功能障碍是神经发育的关键，通过 这些典型的化学物质。这些化学物质有多个分子靶点，包括但不限于 不同的线粒体大分子。这些化学物质各自都会影响线粒体和 神经元，但通过不同的机制，是我们预测协同作用的原因。然而，虽然线粒体 是这些化学物质的已知靶标，线粒体毒性在多大程度上驱动它们的DNT尚不清楚。 我们的工作将确定线粒体功能障碍在单一和联合化学DNT中的贡献， 告知不良后果途径和干预措施的发展情况。

Abstract In ongoing work by this Project, we have found that developmental exposures to mitochondrial toxicants cause neurotoxic outcomes in Caenorhabditis elegans, including morphological alterations in neurons, altered behavior, and, in the long term, increased susceptibility to neurodegeneration. In line with the EPA, we describe all of these as “developmental neurotoxicity (DNT),” because they result from exposures that occur during development. Two important and overarching mechanisms of DNT are 1) changes to neurogenesis resulting in altered cell fate, morphology, and connectivity (“hardwiring”), and 2) persistent changes to the function of neurons that appear to be morphologically normal (epigenetic “programming”). Distinguishing these is challenging; we propose a novel and powerful way to assess each possibility. We will begin with an in vivo yet relatively high-throughput and economic model, C. elegans. C. elegans offers an additional, key benefit: developmental neurogenesis is normally invariant, permitting clear identification of variation in hardwiring as well as behavioral and stress-responsive changes without morphological alteration (programming). Work in C. elegans will be followed by testing in human neuronal stem cells (hNSCs) that permit human-relevant DNT testing, plus the opportunity to identify sex-specific differences and epigenetic modifications. Relatively few chemicals have been rigorously evaluated for DNT. The paucity of information is even more pronounced for chemical co-exposures, despite the fact that combined exposures are the reality. This lack of testing of mixtures results partly from regulatory policy, and partly from technical challenges in laboratory testing of co-exposures. Our combined in vivo-in vitro approach will permit us to rigorously test for DNT resulting from both complex environmental mixtures, and from defined combinations of individual Superfund chemicals that we will evaluate for non-additive effects. We will test the effects of the prototypical developmental neurotoxicants Pb, Cd, and polycyclic aromatic hydrocarbons, singly and in combinations dictated by known environmental concentrations. We will compare our outcomes in C. elegans and hNSCs, to those obtained by other Projects in fish, rats, and people. Demonstration that C. elegans can be reliably used to investigate mixture DNT will add a powerful new model for testing and regulation of environmental mixtures. Finally, we will test the degree to which mitochondrial dysfunction, key to neurodevelopment, drives DNT by these prototypical chemicals. These chemicals have multiple molecular targets, including but not limited to different mitochondrial macromolecules. The fact that these chemicals individually all affect mitochondria and neurons, but by different mechanisms, is why we predict synergistic interactions. However, while mitochondria are known targets of these chemicals, the extent to which mitochondrial toxicity drives their DNT is not known. Our work will establish the contribution of mitochondrial dysfunction in single and combined chemical DNT, informing development of adverse outcome pathways and intervention efforts.