Novel Zebrafish Models to Study Toxicity of PCBs and Pesticides During Pregnancy

描述（由申请人提供）：左心发育不全（HLH）是一种严重的先天性疾病，如果不治疗，通常是致命的，需要多次手术才能生存。最近在巴尔的摩-华盛顿地区的儿童中发现了一组HLH，这与二恶英和多氯联苯（PCBs）的意外释放有关。2004年在北卡州东部发生的一起无关事件中，三名移徙工人生下了患有严重先天性缺陷的婴儿，其中一名婴儿出生时没有四肢，另一名婴儿患有严重的面部缺陷和相关的心脏异常，第三名婴儿缺乏生殖结构，出生后不久死亡；所有出生缺陷都与农药过量接触有关。引人注目的是，多氯联苯诱导的HLH和农药相关的出生缺陷可能是相关的，因为多氯联苯和一些农药通过相同的机制破坏细胞：激活芳烃受体（AHR）信号通路，导致组织特异性细胞周期阻滞。最近，我们在斑马鱼中创建了一种新的毒素诱导HLH模型，其中AHR激动剂PCB-126诱导具有心室缩小和流出道缺陷的带状心脏，与HLH相同；值得注意的是，PCB-126上调p53，下调与Holt-Oram综合征和HLH有关的心脏特异性转录因子tbx5。我们还发现，常见的有机磷农药毒死蜱，既是一种发育神经毒素，也是一种AHR激动剂，诱导心房扩大和心室缩小，这让人想起在pcb处理的斑马鱼中看到的。的确，多氯联苯-126和毒死蜱共同的心脏毒性可能反映了两者都是AHR激动剂的事实。我们建议在斑马鱼中创建荧光报告菌株，当AHR激动剂破坏心血管发育时，它们会在活胚胎中发出信号，并将使用这种方法评估农药的发育性心脏毒性，特别是致畸协同作用。在Aim 1中，我们通过遗传上位和DNA微阵列分析验证了pcbs -126或毒死蜱激活AHR导致tbx5下调以及p53介导的心脏细胞周期停滞的假设。在Aim 2中，将在活斑马鱼心脏中构建荧光报告基因，然后验证其用于暴露于多氯联苯-126和毒死蜱的心脏毒性信号。然后，报告菌株将用于筛选农业中使用的农药，以典型生长季节的组合和顺序。我们希望我们的研究将提供一个新的测试范式，以更准确地预测环境保护局批准的毒素的风险，因为它们在现实生活中经常遇到。我们力求保护我们社会中最容易接触毒素的人——生活贫困的人、保健服务不足的人以及因职业原因过度接触农药的移徙农场工人。左心发育不全（HLH）是一种严重的先天性疾病，如果不治疗，通常是致命的，需要多次手术才能生存。最近在巴尔的摩-华盛顿地区的儿童中发现了一组HLH，这与被禁用的二恶英和多氯联苯（PCBs）的意外释放有关。我们已经在斑马鱼中创建了HLH模型，其中AHR激动剂PCB-126诱导具有心室缩小和流出道缺陷的弓形心脏，与儿童HLH中所见的相同。我们建议在斑马鱼中创建荧光报告菌株，当AHR激动剂破坏心血管发育时，它们将在活胚胎中发出信号，并将使用这种方法评估农药的发育性心脏毒性；我们力求保护我们社会中最容易接触毒素的人——生活贫困的人、保健服务不足的人以及因职业原因过度接触农药的移徙农场工人。

DESCRIPTION (provided by applicant): Hypoplastic left heart (HLH) is a grave congenital condition, uniformly fatal if untreated and requiring multiple surgeries for survival. Recently a cluster of HLH was discovered in children in the Baltimore-Washington area that correlated with accidental release of dioxins and polychlorinated biphenyls (PCBs). In an unrelated incident in Eastern NC during 2004, three migrant workers gave birth to infants with severe congenital defects, including one baby born without limbs, another with severe facial defects and associated heart abnormalities, and a third lacking reproductive structures who died shortly after birth; all birth defects were associated with pesticide overexposure. Strikingly, the PCB- induced HLH and pesticide associated birth defects may be related because PCBs and some pesticides disrupt cells by the same mechanism: activation of the aryl hydrocarbon receptor (AHR) signaling pathway which leads to tissue specific cell cycle arrest. Recently we created a novel model for toxin induced HLH in zebrafish where the AHR agonist PCB-126 induces a stringy heart with a reduced ventricle and out flow tract defects identical to those in HLH; remarkably, PCB-126 up regulates p53 and down regulates a heart specific transcription factor tbx5, implicated in Holt-Oram Syndrome and in HLH. We also found that the common organophosphate pesticide chlorpyrifos, both a developmental neurotoxin and AHR agonist, induces an enlarged atrium and a diminished ventricle reminiscent of that seen in PCB-treated zebrafish. Indeed, the cardiotoxicity common to PCB-126 and chlorpyrifos may reflect the fact that both are AHR agonists. We propose to create fluorescent reporter strains in zebrafish that will signal in living embryos when an AHR agonist disrupts cardiovascular development, and will use this approach to evaluate pesticides for developmental cardiotoxicity, particularly for teratogenic synergy. In Aim 1, we test the hypothesis that PCB-126 or chlorpyrifos activated AHR leads to down regulation of tbx5 followed by p53 mediated cell cycle arrest in the heart by using genetic epistasis and DNA microarray analysis. In Aim 2, fluorescent reporters in living zebrafish hearts will be constructed and then validated for use in signaling cardiotoxicity from exposure to PCB-126 and chlorpyrifos. The reporter strains then will be used to screen pesticides used in agriculture, in combinations and sequence typical of a growing season. We expect that our studies will provide a new testing paradigm to more accurately predict risk to EPA approved toxins as they are encountered routinely in real life. We seek to protect those in our society most vulnerable to toxin exposure those living in poverty, those with inadequate health care and migrant farm workers suffering occupational over exposure to pesticides.Hypoplastic left heart (HLH) is a grave congenital condition, uniformly fatal if untreated and requiring multiple surgeries for survival. Recently a cluster of HLH was discovered in children in the Baltimore-Washington area that correlated with accidental release of banned dioxins and polychlorinated biphenyls (PCBs). We have created a model for HLH in zebrafish where the AHR agonist, PCB-126, induces a stringy heart with a reduced ventricle and out flow tract defects identical to those seen in children with HLH. We propose to create fluorescent reporter strains in zebrafish that will signal in living embryos when an AHR agonist disrupts cardiovascular development and will use this approach to evaluate pesticides for developmental cardiotoxicity; we seek to protect those in our society most vulnerable to toxin exposure those living in poverty, those with inadequate health care and migrant farm workers suffering occupational over exposure to pesticides.