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Using high-throughput phenotypic profiling to screen chemicals in a rainbow trout gill cell line (RTgill-W1)
Citation:
Harris, F., J. Nyffeler, S. Lasee, B. Blackwell, Dan Villeneuve, AND J. Harrill. Using high-throughput phenotypic profiling to screen chemicals in a rainbow trout gill cell line (RTgill-W1). Society of Toxicology 62nd Annual Meeting and ToxExpo 2023, Nashville, TN, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.22753823
Impact/Purpose:
Poster presented to the Society of Toxicology 62nd Annual Meeting and ToxExpo March 2023
Description:
High-throughput phenotypic profiling (HTPP) is a promising new approach method (NAM) for in vitro hazard assessment. “Cell Painting” is a current HTPP assay where cells are imaged after being labeled with fluorescent stains that bind to different cellular structures. Image processing software can calculate features for each fluorescent stain including intensity, texture, shape, and symmetry, which can then be used as quantitative information about cellular states at the time of sampling compared to control. Currently, most cell lines with HTPP data are human-derived, originating from various tissues. The RTgill-W1 cell line is a spontaneously immortalized rainbow trout gill cell line which has previously been utilized for toxicity testing. In January 2021, the Organization for Economic Co-operation and Development (OECD) released Test Guideline 249 (TG 249), an acute toxicity assay that uses three separate fluorescent dyes to measure metabolic activity, membrane integrity, and lysosomal activity in RTgill-W1 cells cultured in 24-well format. To increase the throughput of this test guideline, we have miniaturized it to run in 384-well format to match the conditions typically used for Cell Painting. In this study, we have used Cell Painting to profile RTgill-W1 cells exposed to 231 chemicals in concentration-response format. Chemicals were tested concurrently in 3 assays: the Cell Painting assay, an image-based cell viability assay, and the miniaturized OECD TG 249 cell viability assay. Chemicals tested included 110 tested previously in Cell Painting in human osteosarcoma cells (U 2-OS), 129 with in vivo rainbow trout data, 69 with in vitro rainbow trout data, and 29 detected in water samples from the Great Lakes. RTgill-W1 cells were seeded at 22.5k cells/well in a serum-containing medium, then switched to a serum-free minimal medium and exposed to 8 concentrations of test chemicals (0.05-100 µM) for 24 hours. Out of all chemicals tested, 119 had detectable changes in phenotype compared to vehicle control at sub-cytotoxic concentrations. 51 chemicals had detectable cytotoxicity in the plate reader and image-based assays, 10 showed detectable cytotoxicity in the plate reader assay but not the image-based assay, and 15 in the image-based but not plate reader assay. Of the 51 chemicals with cytotoxicity data from both assays, the cytotoxic concentrations were within one order of magnitude of each other. Ongoing work includes optimization of in vitro-to-in vivo extrapolation parameters and in vitro disposition measurements for a subset of chemicals to potentially improve the accuracy of the comparison to in vivo data. These data provide not only proof-of-concept for using Cell Painting to screen environmental chemicals in the RTgill-W1 cell line, but also an important point of comparison for both existing Cell Painting datasets and existing rainbow trout toxicity data. Due to the high dimensionality of the HTPP data there are many potential avenues for further study, including extrapolating mechanistic behavior and chemical profile similarity as well as inter-species comparison. This abstract does not reflect USEPA policy.
URLs/Downloads:
DOI: Using high-throughput phenotypic profiling to screen chemicals in a rainbow trout gill cell line (RTgill-W1)
FELIX SOT POSTER V8.PDF (PDF, NA pp, 1692.337 KB, about PDF)