Identification of Molecular Signatures for Azole Fungicides Toxicity in Zebrafish Embryos by Integrating Transcriptomics and Gene Network Analysis

Azoles control fungal growth by inhibiting sterol biosynthesis in fungi according to the fungicide resistance action committee. Furthermore, previous studies have highlighted several effects of azole fungicides in fish including endocrine disruption. In this study, we analysed the transcriptome responses of zebrafish embryos exposed to azole fungicides to identify gene expression fingerprints indicating toxic effects such as endocrine disruption induced by sterol biosynthesis inhibition. Firstly, a modified zebrafish embryo toxicity test was conducted following the OECD 236 guideline, exposing embryos to difenoconazole, epoxiconazole, and tebuconazole. After 96 hours, RNA was extracted for transcriptome analysis, which revealed concentration-dependent responses for each fungicide. Additionally, over representation analysis of significantly differentially expressed genes revealed biological functions related to sterol biosynthesis and endocrine disruption. Gene set with specific expression patterns was was identified as molecular signature for indicating adverse effects induced by sterol biosynthesis inhibitors in zebrafish embryos such as endocrine disruption. After further validation, the gene expression fingerprints and biomarkers identified in this study may be used in the future to identify endocrine activity of substances under development in a pre-regulatory screening using the zebrafish embryo model.