Comparative Glucocorticoid Receptor Agonism: In Silico, In Vitro, and In Vivo and Identification of Potential Biomarkers for Synthetic Glucocorticoid Exposure

Abstract

The glucocorticoid receptor (GR) is present in almost every vertebrate cell and is utilized in many biological processes. Despite an abundance of mammalian data, the structural conservation of the receptor and cross-species susceptibility, particularly for aquatic species, has not been well defined. Efforts to reduce, refine and/or replace animal testing have increased, driving the impetus to advance development of New Approach Methodologies (NAMs). Here we employed in silico, in vitro and in vivo methods to elucidate a greater understanding of receptor-mediated effects of synthetic glucocorticoid exposure in teleost fish. Evolutionary conservation of amino acid residues critical for transcriptional activation was confirmed in silico using Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS). Subsequent in vitro assays using zebrafish and human GR provided evidence of physiological congruence of GR agonism. Finally, adult fathead minnows (Pimephales promelas) were exposed in vivo to the synthetic glucocorticoids, dexamethasone (0.04, 400, 4000 µg/L) and beclomethasone dipropionate (130 µg/L), and GR agonism confirmed via digital PCR; in addition, EcoToxChip analyses identified potential mRNA biomarkers following glucocorticoid exposure. These findings support the use of NAMs to potentially reduce multi-species in vivo experimentation, while providing empirical evidence that expands the taxonomic domain of applicability (tDOA) for the GR agonism molecular initiating event within the broader GR agonism adverse outcome pathway network.