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Quantitative High-Throughput Screening and Confirmation Studies for Identification of Compounds that Activate the Aryl Hydrocarbon Receptor Pathway (SETAC)
Citation:
Franzosa, J., K. Mansouri, J. Hsieh, S. Ferguson, X. Chang, N. Kleinstreurer, J. Hamm, Dan Villeneuve, R. Judson, R. Thomas, R. Huang, M. Xia, S. Kullman, M. Denison, AND K. Houck. Quantitative High-Throughput Screening and Confirmation Studies for Identification of Compounds that Activate the Aryl Hydrocarbon Receptor Pathway (SETAC). Presented at SETAC, Vancouver, BC, CANADA, November 09 - 12, 2014.
Impact/Purpose:
Quantitative High-Throughput Screening and Confirmation Studies for Identification of Compounds that Activate the Aryl Hydrocarbon Receptor Pathway
Description:
The aryl hydrocarbon receptor (AhR) is a transcription factor that mediates adaptive responses to known environmental pollutants, such as aromatic hydrocarbons, through regulation of Phase I and II xenobiotic metabolizing enzymes as well as important growth and differentiation pathways. The AhR is activated by a wide range of structurally diverse chemicals that include naturally occurring, synthetic, and environmental chemicals. As part of the U.S. Tox21 collaboration, an approximately 8.5K chemical library was profiled in a quantitative high-throughput screening (qHTS) platform in a 1536-well plate format with an AhR luciferase reporter gene assay in human HepG2 cells at 15 concentrations run in triplicate. Results from the qHTS data identified 738 compounds as potential AhR ligands; of which ~300 demonstrated activity at less than 10 µM. Artificial neural networks were used to train supervised and unsupervised self-organizing maps to identify structural features associated with potency and efficacy of AhR activity. A subset of the Tox21 library (~1800 compounds) and 24 surface water samples collected from various locations across the U.S. were also screened through the EPA ToxCast Program in the Attagene Factorial assay, which evaluates the activity of transcription factors using reporter DNA constructs in HepG2 cells. The in vitro library data combined with in silico (ADMET-Predictor) predictions of metabolic clearance rates and literature, were used to guide the selection of 50 compounds with a range of potencies, efficacies, structures and clearance rates to be tested in orthogonal in vitro and in vivo assays. Results of DNA binding in vitro (GRAB bioassay), and apical and transcriptional evaluations from teleost models, were used to confirm the qHTS data. Taken together, these data allow for increased confidence in the use of robotic qHTS data to predict chemical hazards. In addition, many of the environmental water samples elicited AhR activity along with various other endpoints measured. These results demonstrate the ability to directly test relevant environmental samples with HTS assays and link potential exposures directly to molecular initiating events, such as AhR activation, and facilitate the assessment and characterization of chemical induced toxicity. This abstract does not necessarily reflect US EPA policy.