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Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments
Citation:
Nyffeler, J., C. Willis, F. Harris, M. Foster, B. Chambers, M. Culbreth, R. Brockway, S. Davidson-Fritz, D. Dawson, I. Shah, K. Paul-Friedman, D. Chang, L. Everett, J. Wambaugh, G. Patlewicz, AND J. Harrill. Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, 468:116513, (2023). https://doi.org/10.1016/j.taap.2023.116513
Impact/Purpose:
This sub-product is a journal article summarizing the results from high-throughput phenotypic profiling (HTPP) of hundreds of ToxCast chemicals in the U-2 OS Cell model. This sub-product is a follow-on from sub-product 1.4.2.1. The US EPA Center for Computational Toxicology and Exposure has research programs focused on developing the tools, approaches and data needed to accelerate the pace of chemical risk assessment and foster incorporation of non-traditional toxicity testing data into regulatory decision-making processes. High-throughput phenotypic profiling (HTPP) with Cell Painting is a promising technology for comprehensive and cost-effective bioactivity screening of chemicals. We have developed robust laboratory and bioinformatics workflows for generating HTPP data on thousands of chemicals in concentration-response. Information gleaned from these assays includes molecular point of departures representing the threshold for perturbation of cellular biology and phenotypic profiles that can be used to identify chemicals that act through similar mechanisms of action. This information would be of interest to scientists using or contemplating the use of new approach methodologies (NAMs) for next generation risk assessment (NGRA). This publication summarizes the results of an HTPP screen of approximately 1200 chemicals from the ToxCast collection in human-derived U-2 OS cells, the “gold standard” cell line for this assay.
Description:
‘Cell Painting’ is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.
URLs/Downloads:
DOI: Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments
https://pubmed.ncbi.nlm.nih.gov/37044265/