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

Abstract

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.

Refinement of the zebrafish embryo developmental toxicity assay

“…Depending on this outcome, we will increase the concentrations of carbamazepine in our metabolic activation system (up to substrate inhibition) first (as depicted in step 18 of our mZEDTA protocol) and when the ZEDTA remains negative, we will concentrate our test solution (see step 19 of the mZEDTA) by using a volatile solvent, such as methanol, followed by evaporation when higher concentrations of carbamazepine 10,11-epoxide are needed. This latter procedure has recently been proven successful for chemicals when using zebrafish embryos/larvae of 72 hpf [13] .

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Spatiotemporal Imaging and Pharmacokinetic of Fluorescent Compounds in Zebrafish Eleuthero-Embryos After Different Routes of Administration

“…In this way, we guaranteed that the body and tissue distribution of the compounds was determined by their intrinsic chemical characteristics only, and not by their reactivity towards biomolecules like peptides and proteins. Moreover, since the metabolic capacity of zebrafish eleuthero-embryos is very limited [ 15,16 ], a straightforward correlation can be assumed between the intrabody fluorescence observed and the amount of compound present. We initially selected compounds with cLogD values that varied from low to high lipophilicity (calculated by ChemDraw v18).…”

“…Some methodological advances have been developed to address this issue. For instance, protocols have been established that allow zebrafish eleuthero-embryos to be exposed to compounds after prior in vitro metabolism by rat liver microsomes [ 15,16 ].…”

A robust human norovirus replication model in zebrafish larvae

“…Consequently, a 100 mg stool aliquot (with a high virus titer) is sufficient to inject about 300,000 larvae, yielding 30,000+ data points. The ability to generate large and homogenous datasets is essential for large-scale efforts such as the development of therapeutics [36–38]. The zebrafish larvae fit in 96- and 384-well plates and small molecules can be simply added to the swimming water.…”