Delineating mechanisms underlying azole-induced developmental toxicity using single cell transcriptomic approaches, genome editing tools, and alternative models

总结 唑类化合物是一种广泛应用于临床和农业的抗真菌药物。尽管有明显的暴露， 然而，对于人类而言，与怀孕期间接触唑类药物有关的发育健康风险仍未确定。 在脊椎动物模型中，唑类引起发育毒性，包括一系列先天性畸形。 虽然机制尚未解决，但唑类药物诱导胚胎发生类似于过量的变化， 全反式维甲酸（RA）的生物利用度由于在不良形态和分子生物学的相似性， 表型RA以时空依赖的方式调节数百个基因的转录， 几个已知的胚胎发育的基本功能。许多环境化学品 被怀疑通过破坏途径中不同点的RA信号传导而引起发育毒性。正如我们 向替代的、无动物的发育毒性试验方法过渡， 与关键信号通路（如RA）扰动相关的毒理学机制， 建立适当的体外和计算机测试模型，以识别化学危害。本课题 建议利用发育毒性试验的替代模型：大鼠全胚胎培养（WEC; Aim 1），斑马鱼（Zf; Aim 2）胚胎，和人类胚胎干细胞（hESC; Aim 3）模型和创新 分子工具（例如，单细胞RNA测序，CRISPR-Cas9），以研究与 在胚胎发生中预先确定的敏感窗口期间（早期）， 器官发生）。我们将确定保守的分子，细胞和形态学的变化，由于唑 在细胞增殖、分化和图案化中起作用的暴露和功能性靶点。结果将 用于描述唑类诱导的发育毒性的不良后果途径（AOP）。最后我们 这项研究将是实施单细胞转录组学和多基因编辑的首批研究之一， 将化学品接触与分子、细胞和生物体层面的不利发育结果联系起来。

Summary Azoles are antifungal agents widely-used in clinical applications and agriculture. Despite evident exposures in humans, the developmental health risks associated with azole exposures during pregnancy remains undefined. In vertebrate models, azoles cause developmental toxicity, including a spectrum of congenital malformations. While the mechanisms are unresolved, azoles induce changes in the embryo that resemble excess bioavailability of all-trans retinoic acid (RA) due to similarities in adverse morphological and molecular phenotypes. In a spatiotemporal-dependent manner, RA regulates the transcription of hundreds of genes, several with known essential functions for embryonic development. Many environmental chemicals are suspected to cause developmental toxicity by disrupting RA signaling at different points in the pathway. As we transition towards alternative, animal-free approaches for developmental toxicity testing, delineating toxicological mechanisms associated with perturbations in key signaling pathways such as RA is warranted to establish appropriate in vitro and in silico testing models for identifying chemical hazards. In this project, we propose to leverage alternative models for developmental toxicity testing: rat whole embryo culture (WEC; Aim 1), zebrafish (Zf; Aim 2) embryo, and human embryonic stem cell (hESC; Aim 3) models and innovative molecular tools (e.g., single-cell RNA sequencing, CRISPR-Cas9), to investigate mechanisms linked with azole-induced developmental toxicity during a predefined susceptible window in embryogenesis (early organogenesis). We will determine conserved molecular, cellular, and morphological changes due to azole exposure and functional targets with roles in cell proliferation, differentiation and patterning. Results will be used to delineate an adverse outcome pathway (AOP) of azole-induced developmental toxicity. Finally, our study will be one of the first investigations to implement single-cell transcriptomics and multi-gene editing to link chemical exposures to adverse developmental outcomes on molecular, cellular and organism levels.