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

摘要 唑类化合物是一种广泛应用于临床和农业的抗真菌药物。尽管明显暴露在 对于人类来说，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，氮唑会导致发育毒性，包括一系列先天性畸形。 虽然机制尚未解决，但氮唑类药物会在胚胎中诱导类似过剩的变化 全反式维甲酸(RA)的生物利用度与不良形态和分子相似 表型。RA以一种时空依赖的方式调控数百个基因的转录， 其中几种具有已知的胚胎发育所必需的功能。许多环境化学品是 被怀疑通过在途径的不同点扰乱RA信号而导致发育毒性。因为我们 向替代的、无动物参与的发育毒性试验方法过渡 与RA等关键信号通路的扰动相关的毒理学机制是有根据的 建立适当的体外和电子计算机测试模型，以识别化学危险。在这个项目中，我们 建议利用替代模型进行发育毒性测试：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 在胚胎发育的预定义敏感窗口(早期)中，唑类药物引起的发育毒性 器官发生)。我们将确定由唑类引起的保守的分子、细胞和形态变化。 暴露和功能靶点在细胞增殖、分化和图案化中的作用。结果将是 用于描述唑类发育毒性的不良结局途径(AOP)。最后，我们的 这项研究将是实施单细胞转录和多基因编辑的首批研究之一 在分子、细胞和生物体水平上将化学暴露与不利的发育后果联系起来。

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.