Citation:

Deisenroth, C., D. DeGroot, T. Zurlinden, A. Eicher, J. McCord, M. Lee, P. Carmichael, AND R. Thomas. The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay with Metabolic Competence. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 178(2):281-301, (2020). https://doi.org/10.1093/toxsci/kfaa147

Impact/Purpose:

A challenge with interpreting high-throughput screen (HTS) data in toxicology is the limited metabolic capacity of the available molecular and cellular assays. The lack of metabolism in these systems could lead to false negative and false positive effects, as well as reduce confidence in the biological relevance of chemical perturbations in these assays. The Alginate Immobilization of Metabolic Enzymes (AIME) method developed in this study incorporates hepatic metabolism into high-throughput assays.The results demonstrate the utility of applying the AIME method for identification of false positive and false negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with a rodent bioassay. Integration of the AIME metabolism method may prove useful for future HTS applications.

Description:

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

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