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

Abstract

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.

The chemical defensome of five model teleost fish

“…In a previous study, zebrafish cyp3a65 appear to have bimodal expression 49 , but this was not observed in the set of developmental stages included in our study. For the glutathione transferases, we found substantial variations in expression between different genes, which is also previously shown at the protein expression and enzyme activity level 80 , 81 . Thus, although it has been demonstrated that genes regulated by common transcription factors tend to be located spatially close in the genome sequence and thus facilitate a concerted gene expression 82 , we did not observe such patterns in our datasets.…”

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

“…This adherence-dependent cell line was originally derived from 24 h post fertilization (hpf) zebrafish embryos via spontaneous immortalization; 20 we selected it under the premise that, based on its origin, it is a suitable representative of zebrafish embryo tissue explored in our prior work. 21 The cells were routinely cultured in Leibovitz L-15 medium supplemented with 10% FBS at 26 ± 2 °C in ambient atmosphere in an incubator in the dark. The cells were subcultured every 5−10 days at a ratio of 1:2 to 1:3 after the formation of a confluent monolayer.…”

“…Subsequently, proteins in each sample were digested with trypsin at 37 °C for 16 h. The samples were desalted using reversed-phase cartridges (Sep-Pak Vac tC18, Waters, United States) and stored at 4 °C until targeted proteomics analysis on the TSQ Vantage (Thermo Scientific, United States), carried out as described in Tierbach et al 27 Analysis of CDNB Biotransformation Products in PAC2 Cells and Exposure Medium. Prior to all sample preparations, cell and medium samples were spiked with 2.5 μL of a reference standard, 2,4dinitrotoluene-S-glutathione (DNT-SG), as described in ref 21. The extraction of CDNB biotransformation products from the cells was performed according to the method established in ref 21, with some modifications.…”

“…The samples were eluted with 800 μL of 80% aqueous acetonitrile solution with 0.1% formic acid, evaporated using a vacuum centrifuge at 30 °C, redissolved in 25 μL of nanopure water with 0.1% formic acid, and stored at 4 °C. The LC-HRMS analysis of CDNB biotransformation products in cells and in medium samples was performed as described in detail in ref 21. In some cases, we observed the generation of DNP-CG through gasphase reactions in the ESI source, clearly distinguishable from the biological transformation product DNP-CG by its longer retention time (further details are presented in the Supporting Information "Generation of DNP-CG through gas-phase reactions in the ESI source").…”

“…In cell viability assays, the background fluorescence of CDNB in the absence of cells was subtracted from data obtained with the three fluorescent dyes (AlamarBlue, CFDA-AM and Neutral Red) and normalized to a CDNB-free solvent control. The EC 50 and NtC were calculated based on the nominal and measured concentrations with the algorithm described by Stadnicka-Michalak et al 28 The analysis of mass spectrometric data of CDNB biotransformation products was performed with Thermo Xcalibur 3.0.63 (Thermo Fisher Scientific Inc.) as described in ref 21.…”