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

项目摘要 出生缺陷造成巨大的个人、家庭和社会负担， 生物学和病因学的复杂性阻碍了预防战略。多因素的示例 出生缺陷被认为是基本上由环境调节的是口面裂（OFC）， 前脑无裂畸形（HPE），常见的人类面部和大脑畸形。两者的发病机制 在动物模型中，OFC和HPE与Sonic hedgehog（Shh）信号的胚胎破坏直接相关， 支持以路径为基础的调查环境对出生缺陷病因学的贡献。那个嘘 信令包括多步骤过程，该多步骤过程固有地对跨其多个步骤的调制敏感。 信号级联使得该途径特别适合于检查共同暴露对 病因复杂的多因素疾病。本申请中提出的研究旨在测试 中心假设，结构上不同的Shh抑制剂协同作用，以减少途径活性， 细胞水平和加剧Shh相关颅面畸形。为了验证这个假设，我 利用NIEHS和EPA支持的高通量化学筛选， 环境相关的，假定的Shh通路干扰物，并开发了一种新的Shh通路完整细胞 培养系统，是敏感的抑制整个Shh途径。我将利用这个系统和其他 基于机制的体外试验，以验证真正的途径拮抗剂，描绘分子靶点， Shh途径，并评估由共同暴露于化合物引起的加和和协同相互作用 不同途径的靶点。然后将检查这些化合物的发育毒性， 单独和组合地，通过靶向给药至Shh途径活性的关键时期， 在颅面发育过程中。已知的Shh拮抗剂环巴胺、维莫德吉和胡椒基 在整个拟定研究中，将使用丁醇作为途径特异性效应的阳性对照。这些 严格设计的实验有望揭示环境因素的不利影响， 开发和阐明促进协同相互作用的Shh途径破坏机制。在 完成建议的课程后，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，并有效地传播科学概念和结果。这些 作品也将促进申请人的目标，成为一个独立的调查员在政府机构 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.