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Alginate Immobilization of Metabolic Enzymes (AIME) Coupled to an Estrogen Receptor Transactivation Assay Detects Bioactivated and Bioinactivated Estrogen Receptor Agonists
Citation:
Degroot, D., T. Zurlinden, R. Thomas, M. Lee, P. Carmichael, AND C. Deisenroth. Alginate Immobilization of Metabolic Enzymes (AIME) Coupled to an Estrogen Receptor Transactivation Assay Detects Bioactivated and Bioinactivated Estrogen Receptor Agonists. Presented at Society of Toxicology annual meeting, Baltimore, MD, March 10 - 14, 2019. https://doi.org/10.23645/epacomptox.7868357
Impact/Purpose:
Characterization of chemical hazard is incomplete without evaluating the bioactivity of compounds processed through metabolic transformation and many high throughput screening (HTS) assays used for toxicity assessment lack xenobiotic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform is a HTS-compatible solution that retrofits existing in vitro assays with metabolic competence by attaching alginate-hepatic S9 microspheres to solid supports extending from custom microplate lids. We have previously demonstrated that the AIME platform can be coupled with the VM7Luc4E2 estrogen receptor (ER) transactivation assay (TA) using methoxychlor as a reference chemical for bioactivation to a more potent ER agonist. In this study, we describe a proof-of-principle chemical screen of 48 compounds using the AIME-VM7Luc4E2 ER TA, and show that the method can effectively detect bioactivated and bioinactivated ER compounds. Impact – This work supports US EPA efforts to enhance evaluation of potential chemical hazards by incorporating xenobiotic metabolism into existing high-throughput assays.
Description:
The Alginate Immobilization of Metabolic Enzymes (AIME) is a high-throughput screening (HTS)-compatible platform that retrofits existing in vitro assays with metabolic competence by attaching alginate-hepatic S9 microspheres to solid supports extending from custom microplate lids. We have previously demonstrated that the AIME platform can be coupled with the VM7Luc4E2 estrogen receptor (ER) transactivation assay using methoxychlor as a reference chemical for bioactivation to a more potent ER agonist. In this study, we selected 34 compounds reported to have more ER activity after biotransformation and 14 compounds reported to be unaffected by metabolism as a training set for evaluating the AIME approach (Pinto et al; DOI: 10.1021/acs.chemrestox.6b00079). All 48 compounds, plus assay-specific controls selected from OECD test guidelines, were screened using the AIME-VM7Luc4E2 coupled assay. AIME lids were prepared with either phenobarbital/5,6-benzoflavone-induced rat hepatic S9 (metabolism positive) or alginate-only (metabolism negative) microspheres and immediately added to 96-well plates containing test compounds in 10-point concentration-response in cell culture medium supplemented with an NADPH regeneration system. Following a 2-hour incubation period, the conditioned medium was transferred to estrogen-stripped VM7Luc4E2 cells and incubated for 24 hours to evaluate ER transactivation and cell viability. The Z’ scores for the AIME-coupled assay were 0.71 and 0.91 for metabolism positive and metabolism negative treatments, respectively. To capture efficacy and potency shifts resulting from metabolic activity, the difference in the fitted area under the curve for corresponding treatment groups (+/- metabolism) was calculated. Bioactivation was identified for 21 compounds while bioinactivation was observed for 18 compounds. Half-maximal active concentrations (AC50) for the majority of hits was below the compound-dependent cytotoxicity cutoff, indicating relevant bioactivity. These data demonstrate the metabolism coupled AIME-VM7Luc4E2 HTS platform can effectively detect bioactivated and bioinactivated ER compounds, and identifies new positive and negative reference compounds for metabolism-dependent ER assays. This abstract does not necessarily reflect the policy of the US EPA.
URLs/Downloads:
DOI: Alginate Immobilization of Metabolic Enzymes (AIME) Coupled to an Estrogen Receptor Transactivation Assay Detects Bioactivated and Bioinactivated Estrogen Receptor Agonists
DEGROOT_DEISENROTH_SOT_2019_V11_ POSTER FINAL.PDF (PDF, NA pp, 968.941 KB, about PDF)