Toxicogenomic fin(ger)prints for thyroid disruption AOP refinement and biomarker identification in zebrafish embryos.

Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the hazard assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here, we used transcriptomics and proteomics for identifying molecular signatures of interference with thyroid hormone signaling preceding physiological effects in zebrafish embryos. For this, we analyzed the thyroid hormone 3,3',5-triiodothyronine (T3) and the thyroid peroxidase inhibitor 6-propyl-2-thiouracil (6-PTU) as model substances for increased and repressed thyroid hormone signaling in a modified zebrafish embryo toxicity test. We identified consistent gene expression fingerprints for both modes-of-action (MoA) at sublethal test concentrations. T3 and 6-PTU both significantly target the expression of genes involved in muscle contraction and functioning in an opposing fashion, allowing for a mechanistic refinement of key event relationships in thyroid-related adverse outcome pathways in fish. Furthermore, our fingerprints identify biomarker candidates for thyroid disruption hazard screening approaches. Perspectively, our findings will promote the AOP-based development of in vitro assays for thyroidal ED assessment, which in the long term will contribute to a reduction of regulatory animal tests.

Fast Multiplex real time PCR method for sex-identification of medaka (Oryzias latipes) by non-invasive sampling.

Medaka fish (Oryzias latipes) has been widely used in fish screening and multi-generation tests to provide relevant data to assess impacts of endocrine disrupting chemicals (EDCs) in fish populations. The genotypic differentiation of Medaka sex allows diagnosing the sex reversal, and is required in current test guidelines (e.g. OECD TG 240, 2015). DNA isolation for genetic sex-identification requires sample collection, which has been normally conducted using invasive (fish sacrifice) or semi-invasive (fin-clip) procedures, which conflicts with the need for a fast, simple, and stress-free method. Swabbing skin mucus to collect DNA has been adopted in ecological studies of larger fish, however for smaller fish, it has to be established. To handle larger number of samples, real-time PCR represents a faster and sensitive method compared to conventional PCR. In this study, we aimed to develop a multiplex real-time PCR method for Medaka genetic sex-identification, using DNA sampled by swabbing as less invasive technique. In this approach, the male-determining gene DMY was used in combination with the cytochrome b housekeeping gene. •The method developed is a robust, rapid and a sensitive multiplex real-time PCR for Medaka genetic sex-identification.•This method allows the use of DNA isolated from fish by swabbing, as non-invasive sampling method.