DEVELOPMENTAL TOXICITY OF ORGANOPHOSPHATE-BASED FLAME RETARDANTS

2005年，被称为五溴二苯醚的商用多溴二苯醚混合物-一种广泛使用的 溴化阻燃剂（FR）-在美国被自愿淘汰，由于担心 持久性、生物累积性和毒性。由于五溴二苯醚替代品在低密度环境中的使用增加， 由于许多产品中含有聚氨酯泡沫，现在已检测到有机磷酸酯基FR（OPFR）， 浓度与室内灰尘中的多溴二苯醚总浓度相当，在某些情况下高于室内灰尘中的多溴二苯醚总浓度， 这表明，人类长期接触这些替代阻燃剂后， 最终用途产品在美国很常见。以斑马鱼为模型，我们的长期目标是 确定外源性物质介导的途径，有助于在早期胚胎发育不良的结果， 发展与这一长期目标相一致，本申请的总体目标是继续 揭示了两种高产量OPFR的发育毒性机制， 在室内环境中检测到高浓度。我们的中心假设是，tris（1，3- 二氯-2-丙基）磷酸（TDCPP，一种氯化磷酸酯）在DNA甲基化过程中破坏DNA甲基化。 分裂，因此，延迟从囊胚后期到原肠胚的外延进展，而三苯基 磷酸（TPP，一种未取代的芳基磷酸酯）激活过氧化物酶体增殖物激活受体γ (PPARγ）-TPP诱导的结合和激活的主要靶点-在发育中的胚胎心脏内 在咽期间，导致正常视黄酸受体（RAR）/类维生素A X受体（RXR）的破坏- 介导的信号传导和抑制心脏循环。根据我们实验室在2000年至2005年期间进行的研究， 在过去的五年里，这些假设将通过追求两个全面的具体目标进行测试：1）确定如何 TDCPP在卵裂过程中诱导的DNA甲基化破坏延迟了晚期囊胚的外胚发育 2）确定TPP诱导的PPARγ激活如何破坏RAR-RXR信号传导并阻断 心脏形态发生过程中的心脏循环。这项研究是创新的，因为我们将（1）利用 斑马鱼胚胎模型的功能和多功能性;（2）利用我们广泛的专业知识， 图像采集和分析;（3）首次依靠亚硫酸氢盐扩增子测序和整体封片 甲基化特异性荧光原位杂交检测斑马鱼DNA甲基化动态 胚胎;（4）第一次，开发一个稳定的转基因报告斑马鱼系，这将使我们和 其他研究人员在体内鉴定潜在的PPARγ配体。这一贡献是重要的，因为它（1） 开始解决有关发育OPFR毒性机制的关键不确定性;（2）有助于优先考虑 使用啮齿动物产前发育毒性研究进行有针对性的、以机制为重点的评价， 人群中的流行病学研究;（3）提出了关于以下潜在健康风险的问题： 两种广泛使用的OPFR对发育中的人类胚胎造成长期和普遍的暴露。

In 2005, the commercial polybrominated diphenyl ether (PBDE) mixture known as PentaBDE – a widely used brominated flame retardant (FR) – was voluntarily phased out in the United States due to concerns about persistence, bioaccumulation, and toxicity. Due to increased use as PentaBDE replacements for low-density polyurethane foam in numerous products, organophosphate-based FRs (OPFRs) have now been detected at concentrations comparable to and, in some cases, higher than total PBDE concentrations within indoor dust, suggesting that chronic human exposure to these alternative flame retardants following migration from treated end-use products is common within the United States. Using zebrafish as a model, our long-term goal is to identify xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Consistent with this long-term goal, the overall objective of this application is to continue uncovering the mechanism of developmental toxicity for two high-production volume OPFRs commonly detected at elevated concentrations within indoor environments. Our central hypotheses are that tris(1,3- dichloro-2-propyl) phosphate (TDCPP, a chlorinated phosphate ester) disrupts DNA methylation during cleavage and, consequently, delays epiboly progression from late-blastula through gastrula, whereas triphenyl phosphate (TPP, an unsubstituted aryl phosphate ester) activates peroxisome proliferator-activated receptor γ (PPARγ) – a major target for TPP-induced binding and activation – within the developing embryonic heart during pharyngula, resulting in disruption of normal retinoic acid receptor (RAR)/retinoid X receptor (RXR)- mediated signaling and inhibition of cardiac looping. Based on studies conducted within our laboratory over the last five years, these hypotheses will be tested by pursuing two comprehensive specific aims: 1) Identify how TDCPP-induced disruption of DNA methylation during cleavage delays epiboly progression from late-blastula through gastrula; and 2) Identify how TPP-induced PPARγ activation disrupts RAR-RXR signaling and blocks cardiac looping during heart morphogenesis. The proposed research is innovative because we will (1) leverage the power and versatility of the zebrafish embryo model; (2) leverage our extensive expertise with automated image acquisition and analysis; (3) for the first time, rely on bisulfite amplicon sequencing and whole-mount methylation-specific fluorescence in situ hybridization to assess DNA methylation dynamics within zebrafish embryos; and (4) for the first time, develop a stable transgenic reporter zebrafish line that will allow us and other investigators to identify potential PPARγ ligands in vivo. This contribution is significant because it (1) begins to address key uncertainties about mechanisms of developmental OPFR toxicity; (2) helps prioritize targeted, mechanism-focused evaluations using prenatal developmental toxicity studies within rodents and epidemiological studies within human populations; and (3) raises questions about the potential health risks of two widely used OPFRs to developing human embryos resulting from chronic and ubiquitous exposure.