Zebrafish Cell Lines and High-Throughput Transcriptomics: Advancing In vitro and Bioinformatics Methods for Supporting Environmental Risk Assessment

Historic animal-based toxicity testing methods cannot keep pace with the need for prioritizing new and existing chemicals for comprehensive risk assessment. New approach methodologies such as high-throughput in vitro transcriptomics screening have emerged to address this challenge. However, most in vitro methods were developed using mammalian cell lines, including human, and may not adequately represent environmental species, potentially limiting the utility of this methodology for supporting environmental risk assessment. The objective of this study was to evaluate whether zebrafish cell lines can generate biologically meaningful chemical effects data in a high-throughput transcriptomics pipeline that is protective of toxicologically relevant aquatic apical endpoints. Forty-two test chemicals were screened in two commercially available zebrafish cell lines (ZFL liver and ZEM2S embryonic fibroblast) using the TempO-Seq zS1500+ platform. Transcriptomic points of departure (tPODs) were derived using two methods: Gene-level analysis (tPODgenes) with BMDExpress software and biological pathway altering concentrations (BPACs/tPODsignatures) from signature-based dose-response analysis. When converted to predicted external water concentrations using quantitative in vitro-in vivo extrapolation models, tPODs were generally protective of aquatic in vivo endpoints from the ECOTOX Knowledgebase. Differential gene expression and biological pathway analysis revealed potential cell-type specific effects for several chemicals, highlighting the value of using multiple cell types for capturing tissue-specific responses. Lastly, the biological pathway information was used to extrapolate the chemical effects data across species through an integration of protein-protein interaction network analysis and the Sequence Alignment to Predict Across Species Susceptibility tool, which has significant implications for improving the ecological relevance of these methods.