Characterization of the Mercapturic Acid Pathway, an Important Phase II Biotransformation Route, in a Zebrafish Embryo Cell Line.

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high- throughput screening in toxicology and chemical hazard assessment.

Membrane protein interactions between different Arabidopsis thaliana MRS2-type magnesium transporters are highly permissive.

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.

Incorporation of sediment- and soil-specific aspects in the Criteria for Reporting and Evaluating Ecotoxicity Data (CRED).

In environmental risk assessment either for registration purposes or for retrospective assessments of monitoring data, the hazard assessment is predominantly based on effect data from ecotoxicity studies. Most regulatory frameworks require studies used for risk assessment to be evaluated for reliability and relevance. Historically, the Klimisch methodology was used in many regulatory procedures where reliability needed to be evaluated. More recently, the Criteria for Reporting and Evaluating Ecotoxicity Data (CRED) have been developed for aquatic ecotoxicity studies, providing more detailed guidance on the evaluation and reporting of not only the reliability but also the relevance of a scientific study. Here, we discuss the application of the CRED methodology for assessing sediment and soil ecotoxicity studies, addressing important sediment- and soil-specific criteria that should be included as part of the CRED evaluation system. We also provide detailed recommendations for the design and reporting of sediment and soil toxicity studies that can be used by scientists and researchers wishing to contribute ecotoxicological data for effect assessments carried out within regulatory frameworks. Integr Environ Assess Manag 2024;00:1-13. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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

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.

LC-APCI(-)-MS Determination of 1-Chloro-2,4-dinitrobenzene, a Model Substrate for Glutathione S-Transferases.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring the 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, nontoxic 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).

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio).

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.