Role of glutathione S-transferases and the mercapturic acid pathway in the biotransformation potential of two zebrafish (Danio rerio) test systems: early life stages and PAC2 cells

Zebrafish (Danio rerio) early life stages provide an important model for chemical risk assessment due to their genetic similarity to humans and the availability of well-established high-throughput techniques. Cells isolated from zebrafish conserve all these advantages and can, in addition, be multiplied in the laboratory to an unlimited extend. However, the biotransformation capacities of both systems have not been fully charac-terized. One biotransformation pathway, which is of utmost importance for the clearance of electrophilic compounds and phase I biotransformation products, is the mercapturic acid pathway. Its first step is the conjugation of the electrophile with glutathione, catalyzed by glutathione S-transferases (GSTs). The gluta-thione conjugates are usually further biotransformed within the mercapturic acid pathway by sequential re-moval of the glutamyl- and glycyl-moieties and an acetylation step followed by elimination of the mercap-turate. Considering the significance of this biotransformation route for the outcome of toxicological investi-gations, this thesis aims to close some major knowledge gaps regarding the protein expression of GSTs and the functionality of the mercapturic acid pathway in two test systems, zebrafish early life stages and the embryo-derived cell line PAC2. To investigate the repertoire of cytosolic GSTs and their expression dynamics, a targeted proteomics meth-od using electrospray ionization (ESI) was developed. Zebrafish embryos showed a basal expression of GST isoenzymes belonging to the classes alpha, mu, pi and rho as early as 4 hours post fertilization, indi-cating maternal transfer. After hatching, all cytosolic GST classes could be detected in the free-swimming larvae. The embryo-derived cell line PAC2 also expressed all cytosolic GST classes, except class alpha. Motivated by the abundant expression of GSTs in the zebrafish models, focus was set on the potential of GSTs to biotransform a model substrate (1-chloro-2,4-dinitrobenzene, CDNB) and to initiate its further bio-transformation within the mercapturic acid pathway. Since CDNB has no acid/base properties, an atmos-pheric pressure chemical ionization (APCI) method based on electron capture was developed for CDNB analysis at and below the nontoxic concentrations. For the determination of CDNB biotransformation prod-ucts of the mercapturic acid pathway, an ESI method was developed. In both test systems, the expression of cytosolic GSTs was not affected by non-toxic concentrations of the model substrate CDNB. Further-more, both test systems disclosed a fully functional mercapturic acid pathway with the first (glutathione conjugate) and last (mercapturate) biotransformation product being produced and excreted in zebrafish early life stages and PAC2 cells. To conclude, this thesis reveals the expression of a large GST repertoire and a functional mercapturic acid pathway in zebrafish early life stages and PAC2 cells. The presence of this important chemical activa-tion/deactivation and clearance route supports the application of these test systems as alternative models in toxicology and chemical hazard assessment.

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

Anita Orsolya Hidasi

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.

EPFL2016

Biotransformation Capacity of Zebrafish (Danio rerio) Early Life Stages: Functionality of the Mercapturic Acid Pathway

Kristin Schirmer, Alena Tierbach

Zebrafish (Danio rerio) early life stages offer a versatile model system to study the efficacy and safety of drugs or other chemicals with regard to human and environmental health. This is because, aside from the well-characterized genome of zebrafish and the availability of a broad range of experimental and computational research tools, they are exceptionally well suited for high-throughput approaches. Yet, one important pharmacokinetic aspect is thus far only poorly understood in zebrafish embryo and early larvae: their biotransformation capacity. Especially, biotransformation of electrophilic compounds is a critical pathway because they easily react with nucleophile molecules, such as DNA or proteins, potentially inducing adverse health effects. To combat such adverse effects, conjugation reactions with glutathione and further processing within the mercapturic acid pathway have evolved. We here explore the functionality of this pathway in zebrafish early life stages using a reference substrate (1-chloro-2,4-dinitrobenzene, CDNB). With this work, we show that zebrafish embryos can biotransform CDNB to the respective glutathione conjugate as early as 4h postfertilization. At all examined life stages, the glutathione conjugate is further biotransformed to the last metabolite of the mercapturic acid pathway, the mercapturate, which is slowly excreted. Being able to biotransform electrophiles within the mercapturic acid pathway shows that zebrafish early life stages possess the potential to process xenobiotic compounds through glutathione conjugation and the formation of mercapturates. The presence of this chemical biotransformation and clearance route in zebrafish early life stages supports the application of this model in toxicology and chemical hazard assessment.

OXFORD UNIV PRESS2020

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

Anita Orsolya Hidasi

EPFL2016