The zebrafish embryo as a potential 3R-compliant model to identify endocrine activity across estrogenic, androgenic and steroidogenic (EAS) modalities.

Endocrine disrupting compounds (EDCs) are substances that can interfere with hormonal systems, posing risks to wildlife and human health. For wildlife, a substance must exhibit endocrine activity at a relevant target (receptor or enzyme) and, as a direct consequence, cause population-relevant adverse effects in the exposed organism or its offspring to be classified as an EDC. Current regulatory frameworks utilize various in vivo assays to identify potential EDCs related to estrogenic, androgenic and steroidogenesis (EAS) modalities. However, ethical concerns have generated increasing momentum to align regulatory approaches with the 3R´s principle (Refinement, Reduction, Replacement) to reduce dependency on animal-based test systems. This study explores the potential of transcriptomics in non-transgenic fish embryos as a 3R-aligned complementary screening approach and proposes a conceptual framework for the identification of endocrine activity across all three EAS modalities. We compiled an overview of known gene networks associated with EAS modalities and mapped publicly available literature data on gene expression and functionality during early embryonic development. We found that during the first 96 h post fertilization, zebrafish embryos naturally express many key genes coding for enzymes and receptors involved in sex hormone biosynthesis, estrogen and androgen receptor signaling pathways. Additionally, zebrafish embryos express genes in EAS-associated pathways, namely mineralocorticoid, glucocorticoid, progesterone and glucuronosylation pathways. While available transcriptomic and functional data suggests that zebrafish embryos could in principle generate transcriptional responses indicative of endocrine activity, the currently available information is insufficient to identify robust, specific and mechanistically informed biomarkers associated with endocrine activity in general or specific for EAS modalities. To resolve this, we propose a systematic empirical assessment of transcriptomic responses to additional endocrine-active substances of EAS modalities.