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

Arianna Giusti,Deirdre Cabooter,Pieter Annaert,Noémie De Croze,Cecilia Ranieri,Annelii Ny,Johan Nicolaï,Marc Léonard,Peter De Witte,Xuan-Bac Nguyen,Stanislav Kislyuk,Marlies Oorts,Xiao Wu,Mélanie Mignot

doi:10.3390/ijms20071712

Arianna Giusti, Deirdre Cabooter + Show 12 more

Open Access

https://doi.org/10.3390/ijms20071712

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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.

Highlights

The process of drug discovery and development is characterized by several major steps including preclinical toxicity testing
MEofl.feSccit.s20o1f9,T2e0st Compounds Unexposed to rat liver microsomes (RLM) in Zebrafish Eleuthero Embryos
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Summary

Introduction

The process of drug discovery and development is characterized by several major steps including preclinical toxicity testing. Despite the combination of numerous in vitro and animal models, the toxicity of a subset of compounds still remains undetected [1]. In many cases, this situation has led to a withdrawal of compounds from the market, to loss of confidence in medicines by the public and major financial implications for the companies affected [2]. There is a sense of urgency to explore new approaches for identifying compounds with a potential toxic profile in the early stages of drug discovery and development (DDD) [1]. The capacity to metabolically convert xenobiotics is supposedly low and the effect of toxic metabolites formed in humans can go unobserved in zebrafish [18]

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