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 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.