RESUMEN
Active ingredients of pesticides or biocides and industrial chemicals can negatively affect environmental organisms, potentially endangering populations and ecosystems. European legislation mandates that chemical manufacturers provide data for the environmental risk assessment of substances to obtain registration. Endocrine disruptors, substances that interfere with the hormone system, are not granted marketing authorization due to their adverse effects. Current methods for identifying disruptors targeting the thyroid hormone system are costly and require many amphibians. Consequently, alternative methods compliant with the 3R principle (replacement, reduction, refinement) are essential to prioritize risk assessment using reliable biomarkers at non-protected life stages. Our study focused on detecting robust biomarkers for thyroid-disrupting mechanisms of action (MoA) by analyzing molecular signatures in zebrafish embryos induced by deiodinase inhibitor iopanoic acid and thyroid peroxidase inhibitor methimazole. We exposed freshly fertilized zebrafish eggs to these compounds, measuring lethality, hatching rate, swim bladder size and transcriptomic responses. Both compounds significantly reduced swim bladder size, aligning with prior findings. Transcriptome analysis revealed specific molecular fingerprints consistent with the MoA under investigation. This analysis confirmed regulation directions seen in other studies involving thyroid disruptors and allowed us to identify genes like tg, scl2a11b, guca1d, cthrc1a, si:ch211-226h7.5, soul5, nnt2, cox6a2 and mep1a as biomarker genes for thyroid disrupting MoA in zebrafish embryos as per OECD test guideline 236. Future screening methods based on our findings will enable precise identification of thyroid-related activity in chemicals, promoting the development of environmentally safer substances. Additionally, these biomarkers could potentially be incorporated into legally mandated chronic toxicity tests in fish, potentially replacing amphibian tests for thyroid disruption screening in the future.
RESUMEN
In this study, we applied OMICs analysis to identify substance-specific biomarker candidates, which may act as early indicators for specific ecotoxic modes of actions (MoA). Zebrafish embryos were exposed to two sublethal concentrations of difenoconazole and metalaxyl according to a modified protocol of the OECD test guideline No. 236. At the end of exposure, total RNA and protein were extracted, followed by transcriptomics and proteomics analysis. The analysis of significantly differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) revealed a positive exposure-response correlation in all test concentrations for both fungicides. Similarly, also a positive correlation between the obtained transcriptome and proteome data was observed, highlighting the robustness of our approach. From the detected DEGs, candidate biomarkers specific for difenoconazole (apoa1b, gatm, mylpfb and acta1b) and metalaxyl (lgals2b, abat, fabp1b.1 and myh9a) were selected, and their biological functions were discussed to assess the predictive potential.
