Glucocorticoid receptor in fish: assessing receptor binding, cellular and metabolic effects
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Abstract
Various stressors, such as chemical exposure and starvation, can affect fish performance in aquatic environments. Among the chemicals detected in surface waters are synthetic glucocorticoids (GCs), which are pharmaceutical compounds activating the hypothalamic-pituitary-interrenal endocrine axis in fish by binding to the glucocorticoid receptor (GR). While short-term and long-term effects of certain GCs on teleost fish have been investigated using different biological endpoints, our understanding of the metabolic effects of GCs on fish remains limited. In this multidisciplinary study, the adverse effects of chronic and acute exposure to the GC dexamethasone (DEX) in fish have been studied at different biological levels, from the molecular interactions to organism levels by using in silico, in vitro, ex vivo, and in vivo experiments. To assess the molecular interaction between Atlantic cod GR and DEX, and compare the results with zebrafish and human GR, three-dimensional structures of the GR ligand binding domain from these species were generated using both the AlphaFold2 and the SWISS-MODEL software. Molecular dynamic simulations revealed that DEX exhibits high stability when bound to the hGR, followed by the cGR and zfGR. This was confirmed by the in vitro luciferase reporter gene assay, showing that DEX produced a higher efficacy and was more potent towards hGR in comparison to GR from cod and zebrafish. Furthermore, a novel protocol to monitor metabolic shifts after DEX exposure of both fish liver cells and ex vivo liver tissue was established by using the Agilent Seahorse XFe24 analyzer. Findings from the ex vivo experiment indicate that acute DEX exposure could lead to a metabolic shift in Atlantic cod liver. In the in vivo investigation of the current study, Atlantic cod were exposed to environmentally relevant concentrations of DEX, as well as concentrations 10 and 100 times higher, for two weeks under both satiety and hunger conditions. These experiments revealed that exposure to DEX does not significantly disrupt gluconeogenesis and glycolytic pathways in the liver at the gene transcription level, even at concentrations 100 times higher than those found in the environment. However, a high correlation between the transcription of foxo1a and a gluconeogenesis pathway gene, pck1, was observed, which could suggest a regulatory role of FOXO1a on the gluconeogenesis pathway in fish. Moreover, no significant changes in body mass, fork length, or hepatosomatic index (HSI) compared to controls were found. Nevertheless, starved fish exposed to environmentally relevant concentrations of DEX experienced significant mortality, potentially due to the detrimental impact of DEX on organs such as the liver, since higher liver atrophy was observed in starved fish under exposure conditions compared to control. Although different DEX concentrations negatively impacted the condition index under hunger conditions, no differences in plasma biochemical parameters (cortisol and glucose) were observed between the treated and control subgroups, suggesting negative feedback to reach homeostasis after two weeks of exposure. The main observation in glucose measurement was hyperglycemia in starved fish. Additionally, an initial quantitative adverse outcome pathway (qAOP) to predict the multi-level effects of DEX in Atlantic cod was suggested, which underscores the complexity of GR responses to varying DEX concentrations at different biological levels. The GR response magnitude to DEX is also influenced by exposure duration and species-specific factors. The findings presented here lay the foundation for assessing acute and chronic GC exposure in aquatic environments through future comparative investigations.