Schweiger, N. | Search bioRxiv

bioRxiv2024

Leveraging zebrafish embryo phenotypic observations to advance data-driven analyses in toxicology

Zebrafish have emerged as a central model organism in toxicological research. Zebrafish embryos are exempt from certain animal testing regulations which facilitates their use in toxicological testing. Next to the zebrafish embryo acute toxicity test (ZFET) according to the OECD TG 236, fish embryos are used in mechanistic investigations, chemical screenings, in ecotoxicology, and drug development. However, inconsistencies in the applied test protocols and the monitored endpoints in addition to a lack of standardized data formats, impede comprehensive meta-analyses and cross-study comparisons. To address these challenges, we developed the Integrated Effect Database for Toxicological Observations (INTOB), a comprehensive data management tool that standardizes collection of metadata and phenotypic observations using a controlled vocabulary. By incorporating data from more than 600 experiments into the database and subsequent comprehensive data analyses, we demonstrate its utility in improving the comparability and interoperability of toxicity data. Our results show that the ZFET can detect toxicity spanning seven orders of magnitude at the scale of effect concentrations. We also highlight the potential of read-across analyses based on morphological fingerprints and their connection to chemical modes of action, provide information on control variability of the ZFET, and highlight the importance of time for mechanistic understanding in chemical exposure-effect assessments. We provide the full FAIR dataset as well as the analysis workflow and demonstrate how professional data management, as enabled with INTOB, marks a significant advancement by offering a comprehensive framework for the systematic use of zebrafish embryo toxicity data, thus paving the way for more reliable, data-driven chemical risk assessment. SynopsisThis article shows how a novel data management tool for zebrafish embryo toxicity data advances comparative chemical risk assessment by enhancing data and metadata standardization, comparability, and cross-study analyses.

pharmacology and toxicology↗

bioRxiv2024

Multi-behavioral phenotyping in zebrafish identifies a novel disruptor of non-associative learning with environmental and human relevance

BackgroundThe vertebrate nervous system is vulnerable to chemical toxicity and the widespread release of chemicals into the environment outstrips the capacity to assess their safety. The zebrafish (Danio rerio) is a powerful vertebrate model that can bridge the gap between in vitro and mammalian-based in vivo studies. However, the behavior-rich repertoire of larval zebrafish, a 3R-compliant model amenable to higher-throughput chemical screens, has yet to be fully deployed to identify and characterize chemical compounds that cause neurotoxicity. ObjectiveWe sought to establish a multi-behavioral phenotyping approach in larval zebrafish to identify and mechanistically elucidate neuroactive chemicals, with particular focus on chemical compounds that affect non-associative habituation learning. MethodsWe devised a battery of automated behavior assays in larval zebrafish. The battery captures stereotypical visual and acoustic behaviors including habituation, a form of non-associative learning. To elucidate mechanisms underlying exposure-induced behavioral alterations in zebrafish, in silico target predictions, pharmacological interventions, patch-clamp recordings in cultured mouse cortical neurons, and human multi-neurotransmitter (hMNR) assay in 3D BrainSpheres were used. ResultsKnown pharmacological modulators of habituation in zebrafish evoked distinct behavioral patterns. By screening chemicals positive for ex vivo N-methyl-D-aspartate receptor (NMDAR) modulation, we identified chlorophene, a biocide that caused sedation, paradoxical excitation, and reduced habituation in zebrafish. Using in silico target predictions and pharmacological interventions, we discovered that chlorophene acts via gamma-aminobutyric acid A receptors (GABAARs), a previously unknown target site. Orthogonal validation in cultured mouse cortical neurons and human stem cell-derived BrainSpheres confirmed chlorophenes interaction with GABAARs. Chlorophenes behavioral profile resembled that of flupirtine, a Kv7 potassium channel (M-current) activator, suggesting that habituation deficits stem from M-current rather than GABAAR modulation. ConclusionsThese studies combined a series of behavior assays in a phenotypically rich, rapid, and inexpensive non-mammalian vertebrate test system to screen chemicals for neurotoxicity. Together with in silico target predictions and mouse- and human-based models, our findings establish multi-behavioral phenotyping in zebrafish as a powerful toolkit for neurotoxicity testing and mechanism identification, with relevance for humans.

pharmacology and toxicology↗