DEVELOPMENTAL TOXICITY OF ORGANOPHOSPHATE-BASED FLAME RETARDANTS

描述(申请人提供)：2005年，商业多溴二苯醚(PBDE)混合物，即五溴二苯醚--一种广泛使用的溴化阻燃剂(FR)--出于对持久性、生物积累和毒性的担忧，在美国被自愿淘汰。由于作为五溴二苯醚替代品的使用量增加，现已检测到与家庭粉尘中多溴二苯醚总浓度相当的浓度，在某些情况下甚至高于家庭粉尘中的多溴二苯醚总浓度，这表明慢性人类暴露在美国很常见。以斑马鱼为模型，我们的长期目标是识别在脊椎动物早期发育过程中导致不利结果的异物介导的途径。作为迈向这一长期目标的一步，这项应用的目标是揭示两种通常在室内环境中检测到的高产量OPFR的发育毒性机制。我们的中心假设是，两类主要的OPFR-氯代磷酸酯(CPE)和芳基磷酸酯(APE)-被广泛用作FR和增塑剂，在胚胎发育过程中表现出不同的作用模式。具体地说，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)的活性，从而延迟合子基因组在胚胎发育早期的甲基化；(2)APE三苯基磷酸(TPP)通过异常激活视黄酸受体(RAR)依赖的途径来防止正常的心脏循环。这些假设是基于申请人实验室的初步数据提出的。中心假说将通过追求两个具体目标来检验：1)确定TDCPP在卵裂过程中导致合子基因组甲基化延迟的表观遗传学机制；2)确定RAR异常激活在TPP诱导的心脏形态发生过程中的环状损伤中的作用。对于第一个目标，我们将依赖于早期胚胎暴露、荧光DMNT活性分析和甲基化DNA亲和捕获与高通量下一代测序(MethylCap-seq)的组合。对于第二个目标，我们将依靠高含量筛选(HCS)分析、反向遗传学、实时聚合酶链式反应和体外人类RAR报告分析的组合。这项拟议的研究具有创新性，因为我们将首次利用(1)甲基帽-序列的力量来揭示化学诱导对斑马鱼胚胎甲基组的影响，以及(2)我们现有的基于384良好基础的HCS分析结合反向遗传学来确定RAR激活在化学诱导的斑马鱼胚胎心血管发育中的作用。这一贡献具有重大意义，因为它(1)开始解决发育中OPFR毒性机制的关键不确定性；(2)利用基于人类细胞系的分析和啮齿动物产前发育毒性研究，帮助确定有针对性的、侧重于机制的评估的优先次序；(3)提出了关于两种广泛使用的OPFR对人类胚胎发育的潜在健康风险的问题，这些风险是由于长期和无处不在的暴露造成的。

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.