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

描述(申请人提供)：左心发育不全是一种严重的先天性疾病，如果不治疗，通常是致命的，需要多次手术才能存活。最近，在巴尔的摩-华盛顿地区的儿童中发现了一群HLH，这与二恶英和多氯联苯(PCbs)的意外释放有关。2004年在北卡罗来纳州东部发生的一起无关事件中，三名外来务工人员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畸形，第三名婴儿缺乏生殖结构，出生后不久死亡；所有出生缺陷都与过量接触农药有关。值得注意的是，多氯联苯诱导的高促黄体生成素和农药相关的出生缺陷可能与多氯联苯和一些农药通过相同的机制干扰细胞有关：激活芳烃受体(AHR)信号通路，导致组织特异性细胞周期停滞。最近，我们建立了一种新的毒素诱导斑马鱼HLH的模型，其中AHR激动剂PCB-126诱导出与HLH相同的室缩和流出道缺陷的线条心脏；值得注意的是，PCB-126上调了p53，下调了心脏特异转录因子tbx5，这与Holt-Oram综合征和HLH有关。我们还发现，常见的有机磷杀虫剂毒死蜱，既是一种发育神经毒素，也是一种AHR激动剂，导致心房扩大和脑室缩小，这与多氯联苯处理的斑马鱼相似。事实上，多氯联苯-126和毒死蜱的共同心脏毒性可能反映了这一事实，即两者都是AHR激动剂。我们建议在斑马鱼中创建荧光报告菌株，当AHR激动剂干扰心血管发育时，这些菌株将在活胚胎中发出信号，并将使用这种方法来评估杀虫剂对发育心脏的毒性，特别是致畸协同作用。在目标1中，我们通过遗传上位性和DNA微阵列分析，验证了以下假设：在心脏中，PCB126或毒死蜱激活AHR导致tbx5下调，随后p53介导的细胞周期停滞。在目标2中，将在活的斑马鱼心脏中构建荧光报告，然后验证其用于发出暴露于多氯联苯-126和毒死蜱的心脏毒性信号。然后，这些报道菌株将被用于筛选农业中使用的杀虫剂，按照生长季节的典型组合和序列进行筛选。我们预计，我们的研究将提供一种新的测试范式，以更准确地预测EPA批准的毒素的风险，因为它们在现实生活中经常遇到。我们寻求保护我们社会中最容易受到毒素暴露的人，生活在贫困中的人，卫生保健不足的人，以及因职业原因过度接触农药的外来农场工人。左心发育不良是一种严重的先天性疾病，如果不治疗，通常是致命的，需要多次手术才能存活。最近，在巴尔的摩-华盛顿地区的儿童中发现了一群HLH，这与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建立了一个高促黄体生成素的模型，其中AHR激动剂PCB-126诱导出与患有高促黄体生成素的儿童相同的、具有减少的脑室和流出道缺陷的细长心脏。我们建议在斑马鱼中创造荧光报告菌株，当AHR激动剂干扰心血管发育时，将在活胚胎中发出信号，并将使用这种方法来评估杀虫剂对发育的心脏毒性；我们寻求保护我们社会中最容易受到毒素暴露的人，生活在贫困中的人，卫生保健不足的人，以及职业过度暴露于杀虫剂的外来农场工人。

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.