Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos
Scope of the method
- Translational - Applied Research
- In vivo
- Animal derived cells / tissues / organs
- zebrafish eleuthero embryos
- Drug discovery
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 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.
- - UHPLC system,
- - Mass spectrometer,
- - Stereomicroscope.
- Published in peer reviewed journal
Pros, cons & Future potential
It is uncertain whether zebrafish eleuthero embryos have a sufficient capacity to metabolically convert xenobiotics in an adequate way, and little is known regarding the identity of the metabolites formed. Since metabolism might increase or reduce the toxic profile of compounds dramatically, the use of eleuthero embryos might result in false negative or positive results. We successfully developed a platform combining the high-throughput of an in vivo zebrafish-based toxicity test with a mammalian pre-metabolism step.
Although validating the platform with many more compounds is necessary to further confirm its applicability, we believe that the comprehensive methodology developed herein is promising in identifying compounds that are converted into toxic/non-toxic metabolites via phase I metabolism, thereby increasing the possibility to detect harmful chemicals in an early drug discovery phase.
References, associated documents and other information
Contact personMarie Pardon
OrganisationsKatholieke Universiteit Leuven (KUL)
Pharmaceutical and Pharmacological Sciences