Multifactorial environmental inhibition of Sonic hedgehog signaling: Impact of chemical interactions on pathway activity and craniofacial development

项目总结 出生缺陷造成巨大的个人、家庭和社会负担，并有针对性地发展 生物和病原学的复杂性阻碍了预防策略的实施。多因素的典范 被认为在很大程度上受环境影响的出生缺陷是口面部裂(OFCs)和 无前脑畸形(HPE)是人类常见的面部和大脑畸形。两者的发病机制 在动物模型中，OFCS和HPE直接与Sonic Hedgehog(Shh)信号的胚胎干扰有关， 支持对环境对出生缺陷病因的贡献进行基于途径的调查。那个嘘 信令包括对ITS的多个步骤中的调制固有地敏感的多步骤过程 信号级联使该通路特别适用于检验联合暴露对 病因复杂，多因素疾病。本申请中提出的研究旨在测试 中心假设结构不同的Shh抑制剂协同作用降低信号转导通路的活性 细胞水平和加重Shh相关的头面部畸形。为了检验这一假设，我有 利用NIEHS和EPA支持的高通量化学筛选来创建按优先顺序排列的 与环境相关的假定Shh途径干扰物并开发了一种新的Shh途径-完整细胞 在整个Shh途径中对抑制敏感的培养系统。我将利用这个系统和其他系统 基于机制的体外试验验证真正的途径拮抗剂，描绘分子靶点 Shh途径，并评估因共同接触化合物而产生的相加和协同作用 具有不同的途径目标。然后将检查这些化合物的发育毒性，两者 单独和组合，通过靶向Shh途径活性的关键期给药 在头面部发育过程中。已知的Shh拮抗剂环丙胺、vismodegib和胡椒基 在整个拟议的研究中，丁醚将被用作特定途径效应的阳性对照。这些 经过严格设计的实验有望揭示对环境产生不利影响的环境因素。 发展和阐明Shh途径破坏促进协同作用的机制。在……里面 完成拟议的学业后，申请人的专业发展将通过 建立熟练的实验设计，评估混合物的毒性，利用动物 发展毒理学模型，并有效地交流科学概念和成果。这些 工作还将促进申请者成为政府机构独立调查员的目标 在美国境内。

PROJECT SUMMARY Birth defects cause tremendous individual, familial, and societal burdens, and the development of targeted prevention strategies has been stymied by biological and etiological complexity. Exemplary of multifactorial birth defects thought to be substantially modulated by the environment are orofacial clefts (OFCs) and holoprosencephaly (HPE), common human malformations of the face and brain. The pathogenesis of both OFCs and HPE is directly linked to embryonic disruption of Sonic hedgehog (Shh) signaling in animal models, supporting a pathway-based investigation of environmental contributions to birth defect etiology. That Shh signaling comprises a multi-step process inherently sensitive to modulation across multiple steps of its signaling cascade makes the pathway especially germane for examining the impact of co-exposures on etiologically complex, multifactorial disease. The studies proposed in this application are designed to test the central hypothesis that structurally diverse Shh inhibitors synergistically interact to reduce pathway activity at the cellular level and exacerbate Shh-associated craniofacial malformations. To test this hypothesis, I have leveraged NIEHS- and EPA-supported high-throughput chemical screens to create a prioritized list of environmentally relevant, putative Shh pathway disruptors and developed a novel Shh pathway-complete cell culture system that is sensitive to inhibition throughout the Shh pathway. I will utilize this system and other mechanism-based in vitro assays to validate bona fide pathway antagonists, delineate molecular targets within the Shh pathway, and evaluate additive and synergistic interactions resulting from co-exposure to compounds with diverse pathway targets. The developmental toxicity of these compounds will then be examined, both individually and in combination, by targeting dose administration to critical periods of Shh pathway activity during craniofacial development. The known Shh antagonists cyclopamine, vismodegib, and piperonyl butoxide will be used throughout the proposed studies as positive controls for pathway-specific effects. These rigorously designed experiments are expected to reveal environmental factors that adversely impact development and elucidate mechanisms of Shh pathway disruption that promote synergistic interactions. In completing the proposed studies, the applicant’s professional development will be advanced through the establishment of proficiency in experimental design, evaluating the toxicity of mixtures, utilizing animal models of developmental toxicology, and effectively communicating scientific concepts and results. These works will also facilitate the applicant’s goal of becoming an independent investigator at a government agency within the United States.