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

Scope of the method

The Method relates to
  • Other
Safety Assessment of Compounds after In Vitro Metabolic Conversion
The Method is situated in
  • Translational - Applied Research
Type of method
  • In vivo
Used species
Targeted organ system or type of research
General toxicity


Method keywords
  • microsomes
  • zebrafish eleuthero embryos
Scientific area keywords
  • Drug discovery
  • toxicity
  • microsomes
  • metabolism
Method description

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.

Lab equipment
  • - UHPLC system,
  • - Mass spectrometer,
  • - Stereomicroscope.
Method status
  • 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


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

Contact person

Marie Pardon


Katholieke Universiteit Leuven (KUL)
Pharmaceutical and Pharmacological Sciences