Innovative Research for a Sustainable Future www.epa.gov/research Danica DeGroot l degroot.danica@epa.gov l 919-541-2482 Chemical Screening for Bioactivated Electrophilic Metabolites Using Alginate Immobilization of Metabolic Enzymes (AIME) 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 hepatic S9 in alginate microspheres, cytotoxicity and assay interference associated with direct addition of S9 is reduced. Here we describe deployment strategies used with the AIME platform and present our data from three different assay deployments to illustrate the advantages and disadvantages of each strategy. Chemicals – All chemicals were purchased from Sigma-Aldrich and stock solutions were prepared in DMSO. Alginate Immobilization of Metabolic Enzymes (AIME) – Human hepatic S9 (pooled, mixed gender) or Aroclor-induced male rat hepatic S9 was encapsulated in alginate microspheres using a modification of a cell encapsulation protocol by Lee et al. (1). References 1. Lee et al. (2013) Sens. Actuators, B 177: 78-85 2. McCallum et al. (2013) J. Biomol. Screening, 18(6): 705-713 3. Rogers and Denison (2000) In Vitro Mol. Toxicol. 13(1): 67-82 This poster does not necessarily reflect EPA policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. • We have successfully produced functional AIME microspheres on lids compatible with 96- and 384-well microplates. • Three strategies for deployment of the AIME platform include the All-in-One method, conditioned medium transfer (CMT) and concentrated reagent addition (CRA). • The All-in-One method is preferred for retrofitting existing HTS assays due to its speed and ease of deployment; however, assays utilizing buffers or other reagents which are incompatible with xenobiotic metabolizing enzymes may dictate use of an alternative method. • Conditioned Medium Transfer (CMT) has been successful used to deploy the AIME platform to a cell-based estrogen receptor transactivation assay. • Using Concentrated Reagent Addition (CRA), the AIME platform was coupled to the MSTI Fluorescence-Based Thiol-Reactive Assay™. However, AIME- mediated metabolic activity had limited effect on enhancing the electrophilicity of compounds screened with this assay. • For several compounds (2,2’,4’-trichloroacetophenone, 1,4-dihydroxy-2- naphthoic acid, and diquat bromide) AIME-mediated metabolic activity resulted in a suppression of electrophilicity that was not due to protein binding, suggesting a possible detoxification. • Overall, coupling the AIME platform to the MSTI assay did not identify any novel electrophiles in the tested chemical library. Possible explanations include assay incompatibility with AIME, low formation of metabolites, and/or low electrophile capture due to the highly-reactive nature of these compounds. • Encapsulation of human hepatocytes and/or Supersomes to increase the metabolic capacity of the AIME platform • Identification and quantitation of metabolites using LC-MS/MS to quantify metabolic output of the AIME platform. This will provide information needed to determine the minimum metabolite levels