Citation:

Nyffeler, J., C. Willis, Danielle Harris, L. Taylor, R. Judson, L. Everett, AND J. Harrill. Combining phenotypic profiles and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: retinoic acid as an example. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, 444:116032, (2022). https://doi.org/10.1016/j.taap.2022.116032

Impact/Purpose:

This manuscript describes the optimization of the U-2 OS cell model for parallel-track screening using high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP).  Both of these are Tier 1 screening assays implemented as part of the US EPA Blueprint for Computational Toxicology (Thomas et al. 2019 PMID: 30835285).  This manuscript is foundational for subsequent large scale chemical hazard screening activities underway at US EPA. The manuscript also explores the linkages between retinoic acid receptor (RAR) activation and emergent morphological phenotypes in the U-2 OS cell model.  These data may be of interest to those considering the use of animal-free New Approach Methods (NAMs) in chemical risk assessment. This sub-product is a journal article that describes optimization of the U-2 OS cell model for concurrent chemical screening using HTPP and HTTr. It characterizes the effects of a small collection of phenotypic reference chemicals on gene expression and cell morphology. It also explores the linkage between pharmacological activation of the RAR and RXR nuclear receptors with changes in cell morphology.

Description:

The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.

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