Innovative Research for a Sustainable Future www.epa.gov/research Danica DeGroot l degroot.danica@epa.gov l 919-541-2482 Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays Danica E. DeGroot, Russell S. Thomas, Steven O. Simmons National Center for Computational Toxicology, U.S. EPA, Research Triangle Park, NC Objective Materials & Methods Results & Conclusions Future Directions To develop a platform to retrofit existing high-throughput screening assays with metabolic competence. Introduction The EPA’s ToxCast program utilizes a wide variety of high-throughput screening assays (HTS) to assess chemical perturbations of molecular and cellular endpoints. A key limitation of many HTS assays used for toxicity assessment is the lack of xenobiotic metabolism which precludes the detoxification as well as toxic bioactivation of chemicals tested in vitro, thereby mischaracterizing the potential hazard posed by these chemicals. To address this deficiency, we have developed the AIME platform to retrofit existing HTS assays with extracellular xenobiotic metabolism. By encapsulating human hepatic S9 in alginate microspheres, cytotoxicity and assay interference associated with direct addition of S9 is reduced. Here we demonstrate the deployment of the AIME platform to the MSTI Fluorescence-Based Thiol-Reactive Assay™. Chemicals – All chemicals were purchased from Sigma-Aldrich and stock solutions were prepared in DMSO. Alginate Immobilization of Metabolic Enzymes (AIME) – Human hepatic S9 (BioreclamationIVT; Lot OUL or TOF) was encapsulated in alginate microspheres using a modification of a cell encapsulation protocol by Lee et al. (1). In Figure 1, AIME microspheres are shown attached to a prototype lid for 384-well plates. In Figures 2-4, microspheres were made using either 10% or 20% S9 and attached to commercially available Nunc polystyrene replicators (for 96-well plates). Cytochrome P450 (CYP) Enzyme Activity – CYP enzyme activity was quantified using Promega P450-Glo kits with the following modifications: assays were performed in DMEM/1% FBS and substrate incubations were standardized to 1 hour. All assays included an NADPH regeneration system per the manufacturer's protocol. AIME Coupled MSTI Fluorescence-Based Thiol-Reactive Assay – The MSTI Fluorescence-Based Thiol-Reactive Assay™(2) was used to identify electrophilic compounds with and without metabolic competence. Eighty test compounds (prepared at their solubility limit in DMSO) were acoustically dispensed into triplicate black, 96-well plates followed by the addition of phosphate buffer (pH 7.4) containing an NADPH regeneration system (DMSO 0.1% (v/v) final concentration). AIME lids containing active human hepatic S9, heat-inactivated S9 or an empty microsphere (no S9) were added to the plates and allowed to incubate with the test compounds for 1 hour at 37ᵒC. The lids were removed and a 2X MSTI (30 µM final concentration) was added. Acetyl-MSTI was used as a positive control. The plates were incubated at room temperature for 30 minutes followed by quantitation of the fluorescent signal (excitation: 510 nm; emission 650 nm). An increase in electrophilicity was detected as a decrease in the fluorescent signal. CYP1A2 CYP2B6 CYP2C9 CYP3A4 0 10 20 30 Fold Activity/Free S9 (1 µg) Hours Percent Activity 0 1 2 3 4 5 0 50 100 150 CYP1A2 CYP2B6 CYP2C9 CYP3A4 Figure 2: Encapsulated human hepatic S9 maintains measurable cytochrome P450 activity over time AIME microspheres were incubated at 37oC in DMEM/1% FBS for the indicated time. CYP-specific pro-luciferin substrates were then added and the reaction was allowed to proceed for 1 hour prior to detection of the luciferin metabolite. Data are normalized to activity at t=0. Inset: relative CYP activity at t=0. Values represent the mean ± SEM of three measurements. -3 -2 -1 0 1 2 3 0 50 100 150 CYP1A2 [Compound] log µM Percent Activity -3 -2 -1 0 1 2 3 0 50 100 150 CYP2B6 [Compound] log µM Percent Activi