Identity, mechanisms and early life impacts of transporter interfering compounds

项目摘要 产前暴露于环境化学品已被证明会对以后的健康产生不利影响， 通常涉及生殖功能障碍。本研究的总体目标是了解 控制胚胎中环境化学物质积累的机制，这样我们就可以预测和 减轻这些风险的负面影响。在本提案中，我们解决了两个关键问题， 外源性物质在胚胎中的积累，特别关注外源性物质转运蛋白在胚胎发育过程中的作用。 原始生殖细胞（PGC）形成。首先，我们要问的是，发展计划如何导致 外源性转运蛋白表达，从而产生对外源性转运蛋白的敏感性或抗性的窗口。 积累第二，我们问如何现实世界的化学混合物，同时含有底物和抑制剂， 转运蛋白，影响这种保守的保护系统的功效。Aim 1使用了一种强大的体外分子 进化技术，快速进化，验证和使用称为纳米抗体的抗体样结合剂， 表征人PGCs样细胞（PGLC）和模式生物胚胎中的外源性转运蛋白 (sea海胆和斑马鱼）。目的2应用生物化学和细胞方法来确定相关的 环境配体的人和模型系统外源性转运蛋白，并利用了强大的 分子结构测定管道来剖析这些相互作用的分子机制。目标3 使用目标1和2的模型和分子靶点来检验PGC易受 环境化学物质对转运蛋白防御系统的干扰作用，以及这种干扰的破坏， 系统导致异生物质挑战后生殖适合度下降。这一结果将提供新的 深入了解环境和发展因素如何共同作用，以管理的易感性， 新生的胚胎生殖细胞对致畸剂的反应。

PROJECT SUMMARY Prenatal exposures to environmental chemicals have been shown to cause adverse later life health effects, often involving disorders of reproductive dysfunction. The overall goal of this research is to understand the mechanisms governing accumulation of environmental chemicals in the embryo, so that we can predict and mitigate the negative effects of these exposures. In this proposal, we address two key questions with regard to xenobiotic accumulation in the embryo, with a specific focus on the role of xenobiotic transporters during primordial germ cell (PGC) formation. First, we ask how the program of development leads to changes in xenobiotic transporter expression, and thus generates windows of susceptibility or resistance to xenobiotic accumulation. Second, we ask how real-world chemical mixtures, containing both substrates and inhibitors of transporters, impact the efficacy of this conserved, protective system. Aim 1 uses a powerful in vitro molecular evolution technology to rapidly evolve, validate, and use antibody-like binders called nanobodies to characterize xenobiotic transporter proteins in human PGC-like cells (PGLCs) and in model organism embryos (sea urchin and zebrafish). Aim 2 applies biochemical and cellular approaches to determine relevant environmental ligands of human and model system xenobiotic transporters, and takes advantage of a powerful molecular structure determination pipeline to dissect the molecular mechanisms of these interactions. Aim 3 uses models and molecular targets from Aims 1 and 2 to test the hypothesis that PGCs are vulnerable to the interfering effects of environmental chemicals on the transporter defense system, and that disruption of this system leads to decreased reproductive fitness after xenobiotic challenge. This results will provide new insights into how environmental and developmental factors act in combination to govern the susceptibility of the nascent embryonic germ line to teratogens.