Name: Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos (Video) | JoVE Journal
Uploaded: 2019-08-25T14:00:00.000+00:00
Duration: 7 s
Description: Learn rapid toxicity testing of chemical compounds using zebrafish embryos. This method detects subtle phenotypic changes and off-target effects with minimal compound usage, enabling efficient early-stage safety evaluation.

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5 minutes

The work was supported by grants from Sigrid Juselius Foundation (SP, MP), Finnish Cultural Foundation (AA, MH), Academy of Finland (SP, MP), Orion Farmos Foundation (MH), Tampere Tuberculosis Foundation (SP, MH and MP) and Jane and Aatos Erkko Foundation (SP and MP). We thank our Italian and French collaborators, Prof. Supuran, and Prof. Winum, for providing carbonic anhydrase inhibitors for safety and toxicity evaluation for anti-TB and anti-cancer drug development purposes. We thank Aulikki Lehmus and Marianne Kuuslahti for the technical assistance. We also thank Leena M&#228;kinen and Hannaleena Piippo for their help with the zebrafish breeding and collection of embryos. We sincerely thank Harlan Barker for critical evaluation of the manuscript and insightful comments.

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
<ol>
	<li>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).</li>
	<li>Set up several crosses to obtain enough embryos for assessing the toxicity of more than two chemical compounds. For the evaluation of toxicity, each concentration needs a minimum of 20 embryos23.</li>
	<li>To avoid handling stress to the animals, allow the animals to rest for 2 weeks before using the same individuals for breeding.</li>
</ol>
2. Collection of Embryos and Preparing Plates for Exposure to the Chemical Compounds
<ol>
	<li>Collect the embryos, the next day before noon, using a fine-mesh strainer and transfer them onto a Petri dish containing E3 embryo medium [5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% w/v Methylene Blue].</li>
	<li>Remove debris using a plastic Pasteur pipette (e.g., food and solid waste). Examine each batch of embryos under the stereomicroscope to remove the unfertilized/dead embryos (identified by their opaque appearance).</li>
	<li>Keep the embryos at 28.5 &#176;C in an incubator. Examine the embryos, the next morning, under a stereomicroscope and remove any unhealthy or dead embryos. Also, replace the old E3 medium with fresh E3 medium. 
	NOTE:&#160;Zebrafish embryos are always maintained at 28.5 &#176;C under laboratory conditions.&#160;</li>
	<li>Carefully transfer 1 embryo into each well of a 24-well plate containing enough E3 medium to cover the embryos.</li>
</ol>
3. Preparations of the Stock Solution of Chemical Compounds and Distribution of Diluted Compound into the Wells
<ol>
	<li>Take out the vials containing inhibitor compounds stored at 4 &#176;C. 
	NOTE: Depending on the properties of the compound, these are stored at different temperatures.</li>
	<li>Weigh the compound(s) using an analytical balance that can weigh a few milligrams (mg) of the compound accurately.</li>
	<li>Prepare at least 250 &#956;L (100 mM) of stock solution for each compound in an appropriate solvent (e.g., E3 water or Dimethyl sulfoxide (DMSO), based on the solubility properties of the compounds. 
	NOTE: The above steps can be done a day before the start of the experiment at a convenient time and stored at 4 &#176;C).</li>
	<li>Make serial dilutions of the stock solutions (e. g., 10 &#956;M, 20 &#956;M, 50 &#956;M, 100 &#956;M, 150 &#956;M, 300 &#956;M and 500 &#956;M) using E3 water in 15 mL centrifuge tubes. 
	NOTE: The concentrations and number of serial dilutions vary from one compound to another compound depending on their toxicity levels.</li>
	<li>From the 24 well plate containing embryos, remove E3 water from the wells using a Pasteur pipette and a 1 mL pipette (containing 1 dpf embryos) one row at a time.</li>
	<li>Distribute 1 mL of each diluent in each well (starting from lower and moving to higher concentration) into the wells of 24-well plate.</li>
	<li>Set up a control group from the same batch of embryos and add the corresponding amount of diluent.</li>
	<li>Label 24-well plates with the name and concentration of the compound and keep the plates at 28.5 &#176;C in an incubator.</li>
</ol>
4. Phenotypic Analysis and Imaging of the Embryos Using a Stereomicroscope
<ol>
	<li>Examine the embryos under a stereomicroscope for parameters 24 h after exposure to the chemical compounds.
	<ol>
		<li>Note the parameters such as mortality, hatching, heartbeat, utilization of yolk sack, swim bladder development, movement of the fish, pericardial edema, and shape of the body23.</li>
		<li>Take the larvae exposed to each concentration of the compound and lay them sideways in a small Petri dish containing 3% high molecular weight methyl cellulose using a metal probe. 
		NOTE: The 3% methyl cellulose (high molecular with) is a viscous liquid needed for embedding the fish with a required orientation for microscopic examination. For orienting the fish in this liquid, a metal probe is needed.</li>
		<li>Take the images using stereomicroscope attached to a camera. Save the images in a separate folder each day till the end of the experiment.</li>
		<li>Enter all the observations in a table each day either in an online table or on a printed sheet.</li>
		<li>If the compounds are neurotoxic, the 4 to 5 dpf larvae may show altered swim pattern, make a record of such changes either by capturing a short (30 s to 1 min) video of the larvae exhibiting abnormal movement pattern.</li>
		<li>After 5 days of exposure to the chemical compounds, note the concentration at which half of the embryos die for calculating the half maximal lethal concentration 50 (LC50) of each chemical. 
		NOTE: The LC50 is the concentration at which 50% of the embryos die at the end of 5 days of exposure to a chemical compound. Use a minimum of 20 embryos for testing the toxicity of each concentration of a compound23.</li>
		<li>Construct a curve for mortality of embryos for all the concentrations using a suitable program.</li>
	</ol>
	</li>
</ol>

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No potential conflict of interest was reported by the authors.

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<sup class="xref"...

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 evaluate the safety and toxicity of the compounds in a suitable model. There are several protocols that have been used for the toxicity screening of chemical compounds involving animal and cell culture models but there is not a single protocol that is validated and is in common use4,5. Existing protocols using zebrafish vary in length and have been used by individual researchers who evaluated the toxicity as per their convenience requirement6,7,8,9,10,11,12.
In the recent past, the zebrafish has emerged as a convenient model for the evaluation of the toxicity of chemical compounds during embryonic development6,7. The zebrafish has many in-built advantages for the evaluation of chemical compounds13. Even large-scale experiments are amenable, as a zebrafish female can lay batches of 200-300 eggs, which develop rapidly ex&#160;vivo, do not need external feeding for up to a week and are transparent. The compounds can be added directly into the water, where they can (depending on the nature of the compound) diffuse through the chorion, and after hatching, through the skin, gills and mouth of larvae. The experiments do not require copious amounts of chemical compounds14 due to the small size of the embryo. Developing zebrafish embryos express most of the proteins required to achieve the normal developmental outcome. Therefore, a zebrafish embryo is a sensitive model to assess whether a potential drug can disturb the function of a protein or signaling molecule that is developmentally significant. The organs of the zebrafish become functional between 2-5 dpf15, and compounds that are toxic during this sensitive period of embryonic development induce phenotypic defects in zebrafish larvae. These phenotypic changes can be readily detected using a simple microscope without invasive techniques11. Zebrafish embryos are widely used in toxicological research due to their much greater biological complexity compared to in vitro drug screening using cell culture models16,17. &#160;As a vertebrate, the genetic and physiologic makeup of zebrafish is comparable to humans and hence toxicities of chemical compounds are similar between zebrafish and humans8,18,19,20,21,22. Zebrafish is, thus, a valuable tool in the early phase of drug discovery for the evaluation of toxicity and safety of the chemical compounds.
In the present article, we provide a detailed description of the method used for evaluating the safety and toxicity of carbonic anhydrase (CA) inhibitor compounds using 1-5-day post fertilization (dpf) zebrafish embryos by a single researcher. The protocol involves exposing zebrafish embryos to different concentrations of chemical inhibitor compounds and studying the mortality and phenotypic changes during the embryonic development. At the end of the exposure to the chemical compounds, the LC50 dose of the chemical is determined. The method allows an individual to carry out efficient screening of 1-5 test compounds and takes about 8-10 h depending on the experience of the person with the method (Figure 1). Each of the steps required to assess the toxicity of the compounds is outlined in Figure 2. The evaluation of toxicity of CA inhibitors requires 8 days, and includes setting up of mating pairs (day 1); collection of embryos from breeding tanks, cleaning and transferring them to 28.5 &#176;C incubator (day 2); distribution of the embryos into the wells of a 24-well plate and addition of diluted CA inhibitor compounds (day 3); phenotypic analysis and imaging of larvae (day 4-8), and determination of LC50 dose (day8). &#160;This method is rapid and efficient, requires a small amount of the chemical compound and only basic facilities of the laboratory.

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

rapid evaluation of toxicity of chemical compounds using zebrafish embryos

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

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.

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...

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.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

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Research

JoVE Journal

Medicine

In this article, zebrafish embryos are used for chemical toxicity screening. Chemical toxicity is correlated with phenotype readouts. The sample protocol shows a methodological approach of performing the chemical toxicity screening.

Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially  devastating problems in perinatal medicine.  Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Learn rapid toxicity testing of chemical compounds using zebrafish embryos. This method detects subtle phenotypic changes and off-target effects with minimal compound usage, enabling efficient early-stage safety evaluation.

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

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