Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations.

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 4 h 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.

Membrane proteins of the Arabidopsis thaliana MRS2 (MGT) family have been characterised as magnesium transporters. Like their bacterial CorA homologues, the plant MRS2 proteins are characterised by an invariable GMN tripeptide motif terminating the first of two closely spaced transmembrane domains at the carboxy-termini. The functional Mg(2+) transport channel is assembled as a pentamer in the case of CorA. However, in contrast to the single CorA genes of bacteria, plant genomes encode up to 10 highly divergent MRS2 proteins. To elucidate structure-function relationships and the possibility of plant MRS2 hetero-pentamer formation, we performed protein-protein interaction studies in the yeast mating-based split-ubiquitin system (mbSUS) and concomitant protein modelling using I-TASSER. Despite very restricted sequence similarities and variable polypeptide insertions all AtMRS2 proteins feature the key structural elements determined for the CorA crystal structure. The mbSUS setup conclusively demonstrates protein-protein interactions of any given AtMRS2 protein not only with itself but also highly permissive interactions to varying degrees among all AtMRS2 proteins. AtMRS2-3 seems particularly prone to non-selective, strong interactions with the other homologues. Deletion constructs show that six amino acids may be deleted from the carboxy-terminus and 27 (but not 41) from the amino-terminus of AtMRS2-7 without impairment of homologous or heterologous protein interactions. Despite significant diversification, the plant MRS2 proteins have obviously retained an ancient CorA/MRS2 core structure and the capacity for protein-protein interactions. Plant magnesium homeostasis may be influenced by hetero-oligomer channel formation where different plant MRS2 proteins meet in the same membrane naturally or in transgenic approaches.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabolic capacity of different test systems. To allow the quantification of CDNB at low, non-toxic concentrations, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negative ion mode. Gas phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/ml in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the common model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (Pac2).