Citation:

Blackwell, B., Dan Villeneuve, G. Ankley, C. LaLone, AND J. Doering. Novel in vitro methods for ecological species: Evaluating cross-species differences in nuclear receptor-ligand interactions. EPA Board of Scientific Councilors (BOSC) Virtual Meeting, Duluth,MN, February 02 - 05, 2021. https://doi.org/10.23645/epacomptox.13619237

Impact/Purpose:

High throughput screening assays are increasingly employed to evaluate chemical safety. However, most in vitro screening assays to date utilize human or mammalian receptors, enzymes, etc. The peroxisome proliferator activated receptors (PPAR), glucocorticoid receptor (GR), and retinoid X receptor beta (RXRb) represent several examples for which both the sensitivity and selectivity for activation by contaminants can vary substantially across species. The current research examined 14 reference chemicals and over 60 complex mixtures extracted from surface waters to evaluate how concordant results of a human based screening assay for PPAR, GR, or RXRb activity were with those of four other vertebrates representing some of the major structural diversity in these receptors. Results inform the need to strategically develop screening assays for additional species in order to assure that ecological hazards are adequately accounted for in high throughput screening programs. Results of this work are expected to help OCSPP and the Regions meet their mandate to assure the safety of chemicals for both human health, and the environment.

Description:

Extracts of surface water from the Laurentian Great Lakes basin, and various locations across the United States have been shown to activate human peroxisome proliferator activated receptors (PPARs), retinoid X receptor beta (RXRb), and glucocorticoid receptor (GR). At present, identification of compounds responsible and their ecotoxicological relevance is complicated by reported differences between fish and humans regarding sensitivity to certain PPAR and GR agonists. The aim of the present study was to evaluate cross-species differences in the in vitro sensitivity of vertebrate PPAR (α and γ), RXRb, and GR to selected ligands as well as environmental extracts. First, EPA’s SeqAPASS tool was used to strategically identify surrogate species representing the diversity of amino acid substitutions found in critical positions within the ligand binding domains of the proteins. For example, five types of PPARγ were identified, namely Type 1 (mammals; represented by Homo sapiens), Type 2 (birds, reptiles, amphibians, ancient fish; represented by Xenopus laevis), Type 3 (most fish; represented by Oryzias latipes), Type 4 (salmonids; represented by Oncorhynchus mykiss), and Type 5 (Danio rerio). A novel, multiplexed assay was then developed to allow for simultaneous screening of the ability of a chemical or mixture to interact with the PPARα, PPARγ, GR, or RXRb of these five species. The assay was employed to screen 14 reference chemicals and over 60 environmental samples. The O. latipes-type PPARα was notably less sensitive to designed PPARα agonists (e.g., rosiglitazone and gemfibrozil). Type 3, 4, 5 PPARγ receptors showed reduced sensitivity and differential selectivity compared with human PPARγ. For RXRb sensitivity varied among species but selectivity was broadly similar. Finally, while all species showed similar responsiveness to the reference GR agonist dexamethasone, the X. laevis type GR was considerably more responsive to a GR active surface water extract than the other species. The results suggest there are intrinsic differences in in vitro sensitivity and/selectivity of vertebrate GR and PPARs across species. Strategic selection of surrogate species that represent known diversity in receptor structure may be an efficient way to account for this variability when selecting in vitro screening assays for ecological hazard characterization or effects-based monitoring.

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