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20191010 - High-throughput phenotypic profiling for bioactivity screening of environmental chemicals (EUSAAT)
Citation:
Nyffeler, J., C. Willis, K. Friedman, I. Shah, J. Wambaugh, AND J. Harrill. 20191010 - High-throughput phenotypic profiling for bioactivity screening of environmental chemicals (EUSAAT). European Society for Alternatives to Animal Testing (EUSAAT) annual conference, Linz, N/A, AUSTRIA, October 10 - 13, 2019. https://doi.org/10.23645/epacomptox.9924614
Impact/Purpose:
Abstract for submission to the European Society for Alternatives to Animal Testing (EUSAAT) annual conference in October 2019. The USEPA is currently exploring two potential applications of high-throughput phenotypic profiling (HTPP). (I) Estimating the threshold for chemical bioactivity for comparison with human exposure estimates. (II) Using phenotypic profiles to discern putative mechanisms-of-action to guide further bioactivity testing. Here, we adapted an existing assay that evaluates changes in organelle morphology for use in high-throughput bioactivity screening of environmental chemicals.
Description:
The United States Environmental Protection Agency (USEPA) is exploring the use of new approach methodologies (NAMs) for rapid bioactivity screening and hazard evaluation. The first tier of the screening strategy specifies the use of high-throughput profiling methods, i.e. high-throughput transcriptomics and high-throughput phenotypic profiling (HTPP). Phenotypic profiling is an imaging-based screening method that combines automated microscopy and image analysis to measure a large variety of morphological features at the single cell level. Here, we adapted an existing assay that evaluates changes in organelle morphology for use in high-throughput bioactivity screening of environmental chemicals. The USEPA is currently exploring two potential applications of HTPP. (I) Estimating the threshold for chemical bioactivity for comparison with human exposure estimates. (II) Using phenotypic profiles to discern putative mechanisms-of-action to guide further bioactivity testing. In these studies, U-2 OS cells were plated in 384-well plate format and after 24 h treated with 8 concentrations (1/2 log10 spacing, n=3) of test chemicals, with reference chemicals run on each plate. After 24 h of exposure, cells were labeled with MitoTracker (mitochondria), fixed, permeabilized and labeled with Hoechst-33342 (nuclei), SYTO14 (nucleoli) and fluorescent conjugates of concanavalin A (ER), phalloidin (actin cytoskeleton), and wheat germ agglutinin (Golgi/plasma membrane). Confocal images were acquired using an Opera Phenix HCS system and analyzed using Harmony software, yielding ~1300 features per cell. Cell-level data were normalized to DMSO controls using median absolute deviation and benchmark concentration (BMC) modeling was performed on well-level median values with BMDExpress. The threshold for in vitro bioactivity was defined as the median BMC of the most sensitive category of morphological features. In vitro potency estimates were converted to administered equivalent doses (AED) using the httk R package and compared to in vivo oral toxicity data available in the ToxValDB database and to high-throughput exposure estimates from the Systematic Empirical Evaluation of Models (SEEM3) framework. A set of 462 unique chemicals of mostly bioactive chemicals was screened, and 441 (95%) were active in the HTPP assay. For 68% of chemicals (286/420), the estimated AED was comparable or lower than the in vivo toxicity estimate. While for most chemicals the bioactivity estimate was orders of magnitude above the estimated exposure, for 17/433 (4%) chemicals, the lower bound of the bioactivity estimate (AED 95th) was below the upper bound of the exposure estimate (SEEM 95th). This overlap indicates a potential for humans to be exposed to bioactive concentrations of these environmental chemicals. Moreover, it was observed, that chemicals with similar chemical structure produced similar phenotypic profiles. Similar profiles were also observed for chemicals with the same putative mechanism-of-action. Overall, these findings indicate that the phenotypic profiling assay could be used for bioactivity screening of environmental chemicals and provision of NAMs data for potential use in chemical safety assessment. This abstract does not necessarily reflect USEPA policy.
URLs/Downloads:
DOI: 20191010 - High-throughput phenotypic profiling for bioactivity screening of environmental chemicals (EUSAAT)
NYFFELER_EUSAAT_POSTER_V4.PDF (PDF, NA pp, 4504.157 KB, about PDF)