You are here:
High-throughput phenotypic profiling within the NAMs-based, tiered hazard evaluation strategy at the U.S. Environmental Protection Agency (EuroTox 2021)
Citation:
Nyffeler, J., C. Willis, M. Culbreth, Richard E. Brockway, L. Everett, G. Patlewicz, I. Shah, D. Chang, J. Wambaugh, K. Paul-Friedman, AND J. Harrill. High-throughput phenotypic profiling within the NAMs-based, tiered hazard evaluation strategy at the U.S. Environmental Protection Agency (EuroTox 2021). EUROTOX congress 2021, virtual, NC, September 26 - October 01, 2021. https://doi.org/10.23645/epacomptox.16838164
Impact/Purpose:
Presentation to the EUROTOX Congress 2021 Virtual meeting Sept 2021
Description:
The U.S. Environmental Protection Agency (USEPA) has outlined a tiered testing strategy for human hazard evaluation based on new approach methodologies (NAMs) [1]. The first tier consists of two high-throughput profiling assays: High-throughput transcriptomics (HTTr) and image-based high-throughput phenotypic profiling (HTPP). In HTPP, cultured cells are exposed to chemicals, followed by fluorescent labeling of various cell organelles and high content imaging [2]. Using automated image analysis, the distribution and intensity of the different labels are quantified, resulting in 1300 features per cell [3]. The collected data can then be used for two applications: First, to derive for each chemical an in vitro potency estimate, i.e., a lower bound for bioactivity [4]. This value can be used to compare to legacy in vivo data, or to human exposure estimates [5], in order to prioritize chemicals for higher-tier testing. Second, to predict mechanisms of toxicity. For this purpose, the phenotypic profiles of chemicals are compared to profiles of chemicals with annotated mechanisms. To date, we have screened > 1200 ToxCast chemicals at multiple concentrations in U-2 OS osteosarcoma cells, with approximately 40% of chemicals being active in HTPP. For ~ 300 chemicals, the in vitro potencies were used in reverse dosimetry to compare to in vivo effect values. While HTPP estimates were higher than other NAMs (ToxCast assays, threshold of toxicological concern), the values were closer to the in vivo point-of-departure (POD), and for 78% of chemicals within two orders of magnitudes of the in vivo POD. Moreover, for 49% of chemicals bioactivity occurred only at concentrations 1000x higher than the estimated exposure. Profile comparison of all active chemicals showed that structurally related chemicals tend to result in similar phenotypic profiles. However, phenotypic similarity was also observed for structurally unrelated chemicals in some cases. A subset of chemicals induced highly specific phenotypes, characteristic for glucocorticoids and retinoids, respectively. However, approximately half of the active chemicals resulted in a non-specific phenotype, probably indicative of general cell stress. Thus, this shows that HTPP can be used both to identify specific mode-of-actions, as well as general cell stress. Overall, this work shows how HTPP could be used as an integral part of USEPA’s tiered hazard evaluation strategy and help in prioritization of chemicals. In future efforts, this HTPP assay will be applied to diverse cell types [6] to increase biological coverage. This abstract does not reflect USEPA policy.
URLs/Downloads:
DOI: High-throughput phenotypic profiling within the NAMs-based, tiered hazard evaluation strategy at the U.S. Environmental Protection Agency (EuroTox 2021)
EUROTOX_NYFFELER_V4_TAKE1-M.PDF (PDF, NA pp, 2258.457 KB, about PDF)