Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Summary

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Abstract

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC₅₀) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Introduction

Drug development is an expensive process. Before a single chemical compound is approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) several thousand compounds are screened at a cost of over one billion dollars¹. During the preclinical development, the largest part of this cost is required for the animal testing². To limit the costs, researchers in the field of drug development need alternative models for the safety screening of chemical compounds³. Therefore, in the early phase of the drug development, it would be very beneficial to use a method that can rapidly e....

Protocol

The zebrafish core facility at Tampere University has an establishment authorization granted by the National Animal Experiment Board (ESAVI/7975/04.10.05/2016). All the experiments using zebrafish embryos were performed according to the Provincial Government of Eastern Finland, Social and Health Department of Tampere Regional Service Unit protocol # LSLH-2007-7254/Ym-23.

1. Setting Up of Overnight Zebrafish Mating Tanks

1. Place 2-5 adult male zebrafish and 3-5 adult female zebrafish into mating tanks overnight. (Breeding is induced in the morning by automatic dark and light cycle overnight).
2. Set up several crosses to obt....

Results

The critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment. In the beginning, select the compounds for evaluation of toxicity, the number of concentrations to test for each compound, and accordingly, make a chart (Figure 3). We used a unique color for each compound to organize the samples (Figure 3). The use of solvent resistant marker and label.......

Discussion

In vitro toxicity test using cultured cells can detect survival and morphological studies of the cells providing limited information about the toxicity induced by the test compound. The advantage of toxicity screening of chemical compounds using zebrafish embryos is rapid detection of chemically induced phenotypic changes in a whole animal during embryonic development in a relevant model organism. Approximately 70% of protein-coding human genes have orthologs counterparts in the zebrafish genome

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Nunc	Thermo Scientific
Balance (Weighing scale)	KERN	PLJ3000-2CM
Balance (Weighing scale)	Mettler Toledo	AB104-S/PH
CaCl2	JT.Baker	RS421910024
Disecting Probe	Thermo Scientific	17-467-604
DMSO	Sigma Aldrich, Germany	D4540
Falcon tubes 15 mL	Greiner bio-one	188271
High molecular weight methylcellulose	Sigma Aldrich, Germany	M0262
Incubator for zebrafish larvae	Termaks	B8000
KCL	Merck	1.04936.0500
Methyl Blue	Sigma Aldrich, Germany	28983-56-4
MgSO4	Sigma Aldrich, Germany	M7506
Microcentrifuge tubes	Starlab	S1615-5500
NaCl	VWR Chemicals	27810.295
Paraffin Histoplast IM	Thermo Scientific	8331
Pasteur pipette	Sarstedt	86.1171
Petri dish	Thermo Scientific	101R20
Petri plates	Sarstedt	82.1473
Pipette (1 mL and 200 μL)	Thermo Scientific	4641230N, 4641210N
Plates 24-Well	Thermo Scientific	142485
Steriomicroscope/Camera	Zeiss	Stemi 2000-C/Axiocam 105 color
Vials (1.5 mL)	Fisherbrand	11569914
Zebrafish AB strains	ZIRC	ZL1