Detection of environmental glucocorticoids and investigation of their effects using the zebrafish embryo as a model

Synthetic glucocorticoids (GCs) are widely used anti-inflammatory drugs. They mimic cortisol, the natural stress hormone in humans and in fish. Cortisol binds and activates the glucocorticoid receptor (GR), which regulates genes governing development, immune response, osmoregulation, and behavior. Therefore, the presence of GCs in the aquatic environment may cause endocrine disruption through impairing these essential physiological processes in fish. The aim of this thesis was to assess the effects of environmentally relevant concentrations of GCs in teleost fish, focusing on the inflammatory response and molecular mechanisms of GC action on different levels, from mRNA to proteins to metabolites. To confirm the environmental presence of GCs, GC activity was determined along a Swiss freshwater stream impacted by a wastewater treatment plant, using effect-directed analysis. The effluent samples showed the highest potency to activate the GR, as determined by the GR-CALUX® assay. The chemical analysis found clobetasol propionate (CP), a highly potent GC, to be responsible for 63% of the total GR activity in the sample. CP was selected as a model chemical to study GC effects in fish. Embryos of zebrafish (Danio rerio) aged until 5 days post fertilization (dpf) were used as a model organism; working with embryonic stages is a refinement of animal experiments. We demonstrated that CP is readily taken up by the embryos. Bacterial lipopolysaccharides (LPS) induce one of the main inflammatory cascades. As observed in LPS challenge assays, the inflammatory response of the embryos was significantly reduced after exposure to ¿0.1 nM CP. We showed that mRNA expression of several LPS mechanism- and GC action-related genes was regulated by CP. Among them, Annexin a1b (anxa1b) was significantly down-regulated after exposure to ¿0.05 nM CP. Sensitive regulation of anxa1b by the non-steroidal anti-inflammatory drug diclofenac (DCF) suggested that this gene product is a potential biomarker of steroidal and non-steroidal immunosuppressive effects. An LC-MS/MS-based targeted proteomics technique was developed to measure proteins, as it is these molecules that actually carry out cellular functions. 12 out of 40 GC action-related protein targets were detectable in control embryo digests and were subsequently monitored after CP, DCF and grab water sample exposures. We found that the muscle protein Myhz2 was regulated only after DCF exposure, while I¿B¿, an anti-inflammatory protein, was regulated only by CP, in agreement with mRNA level data. Global and targeted metabolomics analyses were performed in zebrafish embryos exposed to CP from 4 to 5 dpf, in collaboration with M. Lamoree (VU Amsterdam). Several of the detected compounds showed significant response to CP: lysine, nicotinamide, choline, hypoxanthine, tyrosine and tryptophan. To conclude, this thesis demonstrates that GCs are present in the aquatic environment and may suppress the inflammatory response of fish at environmentally relevant concentrations.

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