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

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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.