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High-throughput ecotoxicology with a rainbow trout gill cell line (SETAC 2022)
Citation:
Nyffeler, J., F. Harris, S. Lasee, C. Willis, G. Byrd, C. Schaupp, J. Haselman, J. Nichols, B. Blackwell, K. Flynn, Dan Villeneuve, AND J. Harrill. High-throughput ecotoxicology with a rainbow trout gill cell line (SETAC 2022). Society of Environmental Toxicology and Chemistry (SETAC) Europe 32nd Annual Meeting, Copenhagen, Virtual, DENMARK, May 15 - 19, 2022. https://doi.org/10.23645/epacomptox.19657902
Impact/Purpose:
Poster presented to the Society of Environmental Toxicology and Chemistry (SETAC) Europe 32nd Annual Meeting May 2022.
Description:
The United States Environmental Protection Agency’s tiered hazard evaluation strategy is currently human-centric and does not include high-throughput (HT) in vitro assays for ecotoxicologically-relevant species. In 2021, the Organisation for Economic Co-operation and Development adopted test guideline 249 (OECD TG249) for cell viability testing in rainbow trout gill cells (RTgill-W1). Adjusting the assay results for in vitro disposition and conducting in vitro to in vivo extrapolation, excellent correlation to in vivo survival data was found. At present the assay is conducted in 24-well plates, which limits throughput. The goal of the present study was to miniaturize the assay to 384-well format while following OECD TG249 to the extent possible. Moreover, imaging-based HT phenotypic profiling (HTPP, ‘Cell Painting’) will be conducted in parallel to obtain information about sub-cytotoxic bioactivity of chemicals. RTgill-W1 cells were successfully expanded. One day after plating in 384-well format, media was exchanged, and cells treated with chemicals. After 24 h of exposure, viability stains (alamar blue, CFDA-AM, neutral red) were applied and measured using a plate reader. HTPP plates were labeled to visualize seven different cellular structures, followed by imaging and quantification. In a preliminary experiment, the positive control chemical 3,4-dichloroaniline affected cell viability at 500 uM, but phenotypic changes were already apparent at 5 uM. The established assay will be used to screen a set of 231 environmental chemicals. Of those, 128 have available rainbow trout in vivo toxicity data, 60 have been used in rainbow trout in vitro experiments, 110 have been tested in the HTPP assay in human U-2 OS cells and 29 were detected in the Great Lakes. For all chemicals, nominal in vitro points-of-departure (POD) will be estimated. Adjusting for in vitro disposition and applying HT toxicokinetics, this POD will be extrapolated to chemical concentrations in water used for in vivo exposures. For 12 compounds, chemical concentrations found in the different compartments will be analytically measured. To conclude, a low cost, HT test system suitable for screening large libraries of chemicals for cytotoxicity and phenotypic effects to fish gill cells was established. This expands the current first tier of the hazard evaluation strategy with inclusion of the first ecologically relevant species. This abstract does not reflect USEPA policy.
URLs/Downloads:
DOI: High-throughput ecotoxicology with a rainbow trout gill cell line (SETAC 2022)
NYFFELER_SETAC_RTGILL_V4.PDF (PDF, NA pp, 1075.695 KB, about PDF)