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

使用单细胞转录组学方法、基因组编辑工具和替代模型描述唑类诱导的发育毒性的机制

• 批准号: 10584486
• 负责人: Joshua Frederick Robinson
• 金额: $ 71.57万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-04 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584486
• 关键词: Agriculture; Animals; Antifungal Agents; Azoles; Biological Availability; Biological Models; Branchial arch structure; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cell Wall; Cells; Chemical Exposure; Chemicals; Classification; Congenital Abnormality; Custom; Cytochrome P450; Data; Data Set; Defect; Dermal; Development; Dysmorphology; Embryo; Embryonic Development; Endocrine; Event; Exposure to; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Health; Homeobox Genes; Human; Impairment; In Vitro; Intravenous; Investigation; Laboratories; Libraries; Ligands; Link; Lipids; Maps; Medicine; Metabolism; Modeling; Molecular; Molecular Target; Morphology; Neural Tube Development; Nuclear; Oral; Organism; Organogenesis; Outcome; Pathway interactions; Pattern; Phenotype; Predisposition; Pregnancy; Pregnant Women; Proliferating; RNA; Rattus; Risk; Rodent; Role; Route; Safety; Signal Pathway; Signal Transduction; Sterols; System; Teratogens; Testing; Time; Toxic effect; Toxicity Tests; Toxicogenomics; Toxicology; Tretinoin; Validation; Vertebrates; Zebrafish; adverse outcome; blastomere structure; clinical application; craniofacial; data integration; design; developmental toxicity; developmental toxicology; differential expression; embryo cell; embryo culture; environmental chemical; enzyme pathway; flusilazole; genetic signature; genome editing; hazard; hindbrain; human embryonic stem cell; in silico; innovation; insight; malformation; molecular phenotype; nerve stem cell; neural; novel; single-cell RNA sequencing; spatiotemporal; stem cell model; steroid metabolism; tool; transcriptome; transcriptome sequencing; transcriptomics

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.

概括 唑类是广泛用于临床应用和农业的抗真菌剂。尽管有明显的暴露 对于人类来说，与怀孕期间接触唑类相关的发育健康风险仍不清楚。 在脊椎动物模型中，唑类会引起发育毒性，包括一系列先天畸形。 虽然机制尚未解决，但唑类药物会引起胚胎发生类似过量的变化。 由于不良形态和分子的相似性，全反式视黄酸（RA）的生物利用度 表型。 RA 以时空依赖性方式调节数百个基因的转录， 一些已知的胚胎发育必需功能。许多环境化学品 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、无动物的发育毒性测试方法过渡，描述 与关键信号通路（如 RA）扰动相关的毒理学机制有必要 建立适当的体外和计算机测试模型来识别化学危害。在这个项目中，我们 提议利用替代模型进行发育毒性测试：大鼠全胚胎培养（WEC；目的 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 在胚胎发生的预定易感窗口期间（早期），唑类诱导的发育毒性 器官发生）。我们将确定唑类引起的保守分子、细胞和形态变化 暴露和在细胞增殖、分化和模式化中发挥作用的功能靶标。结果将是 用于描述唑类诱导的发育毒性的不良结果途径（AOP）。最后，我们的 该研究将是首批实施单细胞转录组学和多基因编辑的研究之一 将化学物质暴露与分子、细胞和生物体水平上的不良发育结果联系起来。