DEVELOPMENTAL TOXICITY OF ORGANOPHOSPHATE-BASED FLAME RETARDANTS

描述（由申请人提供）：2005年，被称为五溴二苯醚（PentaBDE）的商用多溴二苯醚（PBDE）混合物——一种广泛使用的溴化阻燃剂（FR）——由于对持久性、生物蓄积性和毒性的担忧，在美国自愿逐步淘汰。由于作为五溴二苯醚替代品的使用增加，现在已经检测到基于有机磷酸酯的FRs （OPFRs）的浓度与家庭粉尘中多溴二苯醚的总浓度相当，在某些情况下，甚至高于家庭粉尘中多溴二苯醚的总浓度，这表明人类慢性接触在美国很常见。使用斑马鱼作为模型，我们的长期目标是确定在早期脊椎动物发育过程中导致不良结果的外源性介导途径。作为实现这一长期目标的一步，本应用程序的目标是揭示室内环境中常见的两种高产量opfr的发育毒性机制。我们的中心假设是，两大类OPFRs——氯代磷酸酯（cpe）和芳基磷酸酯（ape）——广泛用作FRs和增塑剂，在胚胎发生过程中表现出不同的作用模式。具体来说，我们的工作假设是：(1)CPE三（1,3-二氯-2-丙基）磷酸（TDCPP）抑制DNA甲基转移酶（DNMT）活性，从而延迟胚胎早期的合子基因组甲基化；(2)APE三苯基磷酸（TPP）通过异常激活视黄酸受体（RAR）依赖途径阻止正常的心脏环。这些假设是根据申请人实验室的初步数据制定的。中心假设将通过追求两个特定目标来验证：1)确定在卵裂期间tdcpp诱导的合子基因组甲基化延迟的表观遗传机制；2)确定RAR异常激活在心脏形态发生过程中tpp诱导的环损伤中的作用。对于第一个目标，我们将依赖于早期胚胎暴露，荧光DMNT活性测定，甲基化DNA亲和力捕获以及高通量下一代测序（MethylCap-seq）的组合。对于第二个目标，我们将依赖于高含量筛选（HCS）测定、反向遗传学、实时PCR和体外人类RAR报告基因测定的组合。这项研究是创新的，因为我们将首次利用(1)MethylCap-seq的力量来揭示化学诱导对斑马鱼胚胎甲基组的影响；(2)我们现有的384孔HCS试验结合反向遗传学来确定RAR激活在化学诱导对斑马鱼胚胎心血管发育的影响中的作用。这一贡献是重要的，因为它(1)开始解决关于发育性OPFR毒性机制的关键不确定性；(2)利用基于人类细胞系的检测和啮齿动物的产前发育毒性研究，帮助确定有针对性的、以机制为重点的评估；(3)提出了关于两种广泛使用的OPFRs对发育中的人类胚胎造成长期和普遍接触的潜在健康风险的问题。

DESCRIPTION (provided by applicant): 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, organophosphate-based FRs (OPFRs) have now been detected at concentrations comparable to and, in some cases, higher than total PBDE concentrations in household dust, suggesting that chronic human exposure is common within the US. Using zebrafish as a model, our long-term goal is to identify xenobiotic-mediated pathways that contribute to adverse outcomes during early vertebrate development. As a step toward this long-term goal, the objective of this application is to uncover mechanisms of developmental toxicity for two high-production volume OPFRs commonly detected within indoor environments. Our central hypothesis is that two major classes of OPFRs - chlorinated phosphate esters (CPEs) and aryl phosphate esters (APEs) - widely used as FRs and plasticizers exhibit distinct modes of action during embryogenesis. Specifically, our working hypotheses are that (1) the CPE tris(1,3-dichloro-2-propyl) phosphate (TDCPP) inhibits DNA methyltransferase (DNMT) activity and, as a result, delays zygotic genome methylation during early embryogenesis and (2) the APE triphenyl phosphate (TPP) prevents normal cardiac looping through aberrant activation of a retinoic acid receptor (RAR)-dependent pathway. These hypotheses were formulated based on preliminary data from the applicant's laboratory. The central hypothesis will be tested by pursuing two specific aims: 1) Identify the epigenetic mechanism responsible for TDCPP-induced delays in zygotic genome methylation during cleavage; and 2) Identify the role of aberrant RAR activation in TPP-induced looping impairments during heart morphogenesis. For the first aim, we will rely on a combination of early embryonic exposures, fluorometric DMNT activity assays, and methylated DNA affinity capture coupled with high- throughput next-generation sequencing (MethylCap-seq). For the second aim, we will rely on a combination of high-content screening (HCS) assays, reverse genetics, real-time PCR, and in vitro human RAR reporter assays. The proposed research is innovative because, for the first time, we will leverage the power of (1) MethylCap-seq to reveal chemically-induced effects on the zebrafish embryonic methylome and (2) our existing 384-well-based HCS assays coupled with reverse genetics to identify the role of RAR activation in chemically-induced effects on cardiovascular development within zebrafish embryos. 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 human cell line-based assays and prenatal developmental toxicity studies within rodents; and (3) raises questions about the potential health risks of two widely used OPFRs to developing human embryos resulting from chronic and ubiquitous exposure.