Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos.

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at -20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau's medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.

Development of a sensitive and quantitative capillary LC-UV method to study the uptake of pharmaceuticals in zebrafish brain.

The present study explores the potential of 10-day-old zebrafish (Danio rerio) as a predictive blood-brain-barrier model using a set of 7 pharmaceutical agents. For this purpose, zebrafish were incubated with each of these 7 drugs separately via the route of immersion and the concentration reaching the brain was determined by applying a brain extraction procedure allowing isolation of the intact brain from the head of the zebrafish larvae. Sample analysis was performed utilizing capillary ultra-high performance liquid chromatography (cap-UHPLC) on a Pepmap RSLC C18 capillary column (150 mm × 300 µm, dp = 2 µm) coupled to a variable wavelength UV detector. Gradient separation was performed in 28 min at a flow rate of 5 µL/min and the optimal injection volume was determined to be 1 µL. The brain extraction procedure was established for the zebrafish strain TG898 exhibiting red fluorescence of the brain, allowing control of the integrity of the extracted parts. Quantitative experiments carried out on pooled samples of six zebrafish (n = 6) demonstrated the selective semipermeable nature of the blood-brain barrier after incubating the zebrafish at the maximum tolerated concentration for the investigated pharmaceuticals. The obtained brain-to-trunk ratios ranged between 0.3 for the most excluded compound and 1.2 for the pharmaceutical agent being most accumulated in the brain of the fish. Graphical abstract Workflow of brain extraction to study the uptake of pharmaceuticals in the brain of zebrafish larvae.

Development of a sensitive and quantitative UHPLC-MS/MS method to study the whole-body uptake of pharmaceuticals in zebrafish.

An analytical procedure to measure the whole-body uptake of pharmaceuticals in zebrafish has been developed using state-of-the-art methodologies. A sample preparation procedure for 9 pharmaceuticals displaying a variety in physicochemical properties was developed using 10-day old zebrafish (TG898). For an efficient homogenization of the samples and subsequent recovery of the compounds of interest, different amounts of organic solvents in combination with acidic modifiers were added to zebrafish samples. Samples were subsequently processed using a powerful bath sonicator and centrifuged. Supernatant was then removed and evaporated in a vacuum oven before being reconstituted in a mobile phase-like solvent. Samples were analyzed using ultra-high performance liquid chromatography (UHPLC) on an Acquity BEH C18 column (100 × 2.1mm, dp=1.7µm) coupled to a Waters Xevo TQ-S mass spectrometer. For this purpose, a generic gradient was run, wherein the percentage of acetonitrile was varied from 3% to 82% in 10.5min at a flow rate of 0.41mL/min. Linearity of the method was demonstrated for all compounds (R2 > 0.997) in a practically relevant concentration range. Matrix effects were between 81% and 106%, except for amitriptyline (51%). Using this method, it was demonstrated that a sample pretreatment using 1:2 (v/v) water:methanol in combination with 0.1% formic acid resulted in acceptable recoveries between 74% and 100% for all compounds. Together with the obtained lower limits of quantification of the analytical method (between 0.005 and 1.5ng/mL), this allowed the use of a single zebrafish to study the whole-body uptake of a particular drug, after incubating zebrafish at the maximum tolerated concentration for this drug.

Cell Imaging Counting as a Novel Ex Vivo Approach for Investigating Drug-Induced Hepatotoxicity in Zebrafish Larvae.

Drug-induced liver injury (DILI) is the most common reason for failures during the drug development process and for safety-related withdrawal of drugs from the pharmaceutical market. Therefore, having tools and techniques that can detect hepatotoxic properties in drug candidates at an early discovery stage is highly desirable. In this study, cell imaging counting was used to measure in a fast, straightforward, and unbiased way the effect of paracetamol and tetracycline, (compounds known to cause hepatotoxicity in humans) on the amount of DsRed-labeled hepatocytes recovered by protease digestion from Tg(fabp10a:DsRed) transgenic zebrafish. The outcome was in general comparable with the results obtained using two reference methods, i.e., visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images. In addition, our study shows that administering compounds into the yolk is relevant in the framework of hepatotoxicity testing. Taken together, cell imaging counting provides a novel and rapid tool for screening hepatotoxicants in early stages of drug development. This method is also suitable for testing of other organ-related toxicities subject to the organs and tissues expressing fluorescent proteins in transgenic zebrafish lines.