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Chemical Screening for Bioactivated Electrophilic Metabolites Using Alginate Immobilization of Metabolic Enzymes (AIME) (SOT)
Citation:
DeGroot, D., R. Thomas, AND Steve Simmons. Chemical Screening for Bioactivated Electrophilic Metabolites Using Alginate Immobilization of Metabolic Enzymes (AIME) (SOT). Presented at SOT Annual Meeting, Baltimore, Maryland, March 12 - 16, 2017. https://doi.org/10.23645/epacomptox.5181259
Impact/Purpose:
Existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization of chemical hazard if the parent compound is detoxified or bioactivated in vivo. The Alginate Immobilization of Metabolic Enzymes (AIME) platform is an HTS-compatible, retrofit solution that addresses this problem by attaching metabolically active alginate-hepatic S9 microspheres to solid supports extending from microplate lids.
Description:
The US EPA's ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization of chemical hazard if the parent compound is detoxified or bioactivated in vivo. The Alginate Immobilization of Metabolic Enzymes (AIME) platform is an HTS-compatible, retrofit solution that addresses this problem by attaching metabolically active alginate-hepatic S9 microspheres to solid supports extending from microplate lids. As an initial proof-of-concept study, we coupled the AIME platform with the MSTI Fluorescence-Based Thiol-Reactive Assay™ (MSTI) to identify electrophiles formed by metabolic bioactivation. Eighty compounds were acoustically dispensed into 96-well plates to which phosphate buffer containing an NADPH regeneration system was subsequently added. AIME lids containing active human hepatic S9, heat-inactivated S9 or alginate-only (empty) microspheres were immediately added and allowed to incubate with the test compounds, each at a single concentration, for 1 hour at 37°C. The MSTI probe was then added to each plate to detect the formation of electrophiles. The Z’ scores were 0.66, 0.82, and 0.77 for the active, heat-inactivated, and empty microsphere plates, respectively indicating HTS screening compatibility of the MSTI assay with the AIME platform. Nine compounds demonstrated increased electrophilicity in the presence of the active S9 AIME lid (5-12% over the heat-inactivated control). In an effort to increase the sensitivity of the AIME-MSTI coupled assay and the capture of potentially electrophilic metabolites, the experiment was repeated with the MSTI probe included during the AIME incubation. The resulting Z’ scores (<0.5) indicated that this procedure was unacceptable for this HTS assay. Overall, these results indicate the potential for retrofitting chemical assays with the AIME platform, but also highlight a major challenge -- the determination of assay conditions that not only support the activity of xenobiotic metabolizing enzymes but are compatible with the recipient assay. This abstract does not necessarily reflect the policy of the US EPA.
URLs/Downloads:
DOI: Chemical Screening for Bioactivated Electrophilic Metabolites Using Alginate Immobilization of Metabolic Enzymes (AIME) (SOT)