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>

<ol><li>Amaouche, N., Casaert Salome, H., Collignon, O., Santos, M. R., Ziogas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marketing+authorization+applications+submitted+to+the+European+Medicines+Agency+by+small+and+medium-sized+enterprises%3A+an+analysis+of+major+objections+and+their+impact+on+outcomes%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">Marketing authorization applications submitted to the European Medicines Agency by small and medium-sized enterprises: an analysis of major objections and their impact on outcomes.</a> Drug Discovery Today. 23 (10), 1801-1805 (2018).</li>
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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 genome25. Genetic pathways controlling the signal transduction and development are highly conserved between human and zebrafish26, and, therefore, chemical compounds are likely to have similar toxic effects in humans25.
Zebrafish are at the forefront of toxicological research and have already been extensively used to detect toxins in water samples and to study the mechanism of action of environmental toxins and their effects on the animal27. In this article, we show the use of developing zebrafish embryos to evaluate the toxicity of potential compounds at the early phase of drug discovery. Our rapid and multifaceted toxicity assessment is based on 1) changes in the phenotype of the organism (hatching, otolith sacs, pericardial edema, yolk sac utilization, notochord development, heartbeat, swim bladder development, movement) during embryonic development over a period of 5 days, and 2) determination of the lethal concentration&#160;(LC50). Reasonable hours of hands-on work (8-10 h divided over 8 days) is enough to determine the toxicity profile of a compound using this procedure. For newly synthesized compounds that are available in limited amounts, toxicity can be evaluated using a minimum of 3 mg of the compound. No special equipment is required. The procedure is, thus, a rapid and low-cost way to evaluate the toxic effects of any compound that can potentially be developed into the drug for human use.
The quality of the batch of embryos has a great impact on the outcome of the experiment. The quality of eggs (including the rate of fertilization) is not obvious immediately after collection from the breeding tanks (0-4 hpf). For obtaining only normally developing embryos for the experiments, before adding compounds, we allow the embryos to remain at 28.5 &#176;C for 24 h after the collection from the breeding tanks. After 24 hpf, any dead or unhealthy-looking embryos are discarded. In addition to this, it is advisable to have a mock-treated group of embryos in each experiment to further control for the quality of the batch of eggs used in the experiment as well as to see the baseline mortality to accurately determine LC50.
A mock-treated group is also needed to control for the toxicity of the vehicle of choice. Many chemicals are insoluble in water and DMSO is often used as the vehicle for the delivery of the test compounds. DMSO is generally well tolerated by the embryos in lower (0.1%) concentrations28. Sometimes, the compounds are not completely soluble even in DMSO and the solution appears cloudy making it difficult for the evaluation of the toxicity. In such cases, to get the stock solution of the compound completely soluble and clear, adding a drop of 0.1% NaOH will solve the problem. Appropriate control groups need to be set up for assessing the toxicity of the compound accurately. If other vehicles are used, their inherent toxicity might affect the experiment.
Each well of a 24-well plate contains from 1-10 embryos submerged in a total volume of 1 mL. If the test compound is available only in small amounts, it may be necessary to use up to 10 embryos per well for evaluating its toxicity. It is highly recommended that only 1 embryo per well should be used for each analysis16,24,29. The plate is stored at 28.5 &#176;C incubator for 5 days. Sometimes significant evaporation is observed causing a marked change in the concentration of the compound in a given well16. By sealing the 24-well plates with paraffin films from all sides, placing embryos only in the middle wells and filling the other wells along the rims with water, the problems caused by evaporation can be avoided. In our earlier studies, we did not observe any evaporation of the diluents of the chemicals from 24-well plates24,29. Also, if the compound in question is known to be unstable under ambient temperatures, daily changes of water with fresh compound will be needed for reliable results.
The swim pattern of zebrafish larvae is analyzed after the 5 days of exposure to the compound. The wells of a 24-well plate containing 5 dpf larvae are not ideal for the purpose due to the limited size of the well. Therefore, for accurate analysis of swim pattern, the larvae need to be transferred to a Petri dish containing 50 mL&#160;of E3 water and can settle for 2 min before the analysis. In our study, only two inhibitors showed the effect on swim pattern16 among the 52 &#945;-CA and &#946;-CA inhibitors screened for safety and toxicity, based on our experience this test can detect subtle changes including mild ataxic movement of the larvae.
This study demonstrated that the only limitation to the current method is physical dexterity. This concern can be overcome through repetition, as the skill of a person improves with practice. Once the expertise is achieved the evaluation of compounds using zebrafish embryos is ethical, easy, efficient and informative. We, therefore, expect that such a rapid assay using zebrafish embryos will become a popular tool for the in vivo toxicity screening in the early phase of drug development.

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

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 labeling at the bottom or sides of the plates is important to avoid mix-up later. &#160;If the compounds induce any phenotypic defects in the larvae exposed to different concentrations of inhibitors, the defects are recorded every 24 h over the period of 1-5 dpf (Figure 4A,B,C,D). The embryos treated with a known CA IX inhibitor at a concentration of 500 &#956;M did not show any apparent phenotypic changes in the 1-5 days of exposure to the chemical compound (Figure 4B). Figure 4C and Figure 4D shows the embryos treated with &#946;-CA inhibitors that induced various phenotypic defects unhatched embryos even at day 3 (arrow head), curved body structure (arrow), unutilized yolk sack and pericardial edema (arrow head) and absence of otolith sacs in the larvae at 5 days after treatment with chemical compound (arrow). In another study, the embryos treated with CA inhibitor (Figure 4E) shows the absence of otolith sacs and swim bladder (arrow heads). In our study, (Figure 4C,D), we documented phenotypic defects (fragile embryos and absence of pigmentation) even after day 1 of exposure to CA inhibitors. The phenotypic analyses showed that some of the inhibitor compounds are lethal and cannot be developed as drugs for human use (Figure 4C,D and Table 1).
The experiments identified a representative compound which induced minimal or no phenotypic changes during embryonic development and showed a high LC50 dose (Figure 5), suggesting that the compound is safe for further characterization and can be potentially developed into a drug candidate for human use16. Looking at still images of larvae exposed to the compound showed no phenotypic defects&#160;(Figure 4B). However, the same compound was found to be neurotoxic and induced ataxia in the larvae after 5-days of exposure to the CA inhibitor (Figure 6). This phenotype could only be detected by directly observing the swimming behavior of the zebrafish larvae under the microscope. These studies suggested that the neural development of zebrafish larvae is sensitive to the compound16,17.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59315/59315fig1v2.jpg" /> 
Figure 1: Time in hours&#160;required for the rapid screening of chemical compounds using zebrafish embryos: In one set of experiments, a person with hands-on experience can screen about 5 chemical compounds (each compound requiring a minimum of 6 dilutions) using zebrafish embryos in 24-well plates. In total, it takes about 8-10 h from the setting up of crosses to performing LC50 determination over a period of 8 days.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59315/59315fig2.jpg" /> 
Figure 2: Chart showing the breakdown of toxicity evaluation of chemical compounds. Toxicity screening of chemical compounds requires 8 days and can be broken down into five steps. (Day 1) Consists of setting up of zebrafish mating pairs in tanks. (Day 2) Involves collecting embryos from the mating tanks into Petri dishes followed by keeping them at a 28.5 &#176;C incubator for overnight. (Day 3) Examination of embryos using a stereomicroscope and cleaning the embryos. Distribution of the embryos into 24-well plates and adding the dilutions of the test compounds. (Day 4-8) Phenotypic analyses of larvae for developmental defects. Swim pattern study and LC50 determination was performed on the last day of exposure to compounds. <a href="https://www.jove.com/files/ftp_upload/59315/59315fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59315/59315fig3v2.jpg" /> 
Figure 3: Schematic presentation of experiments involved in toxicity evaluation. The toxicity evaluation consists of preparing tabulated chart containing information about chemical compounds, dilutions of the compounds and parameters to be assessed. Making the dilution of stock solutions to the desired concentrations in 15 mL centrifuge tubes (the dilution from one tube to be added to each row of the 24-well plate). A 24-well plate marked with a water proof marker with the name of the test compound, concentration in each row, and date of exposure. Examination and imaging of embryos by transferring the larvae onto a small Petri dish containing 3% high molecular weight methyl cellulose. <a href="https://www.jove.com/files/ftp_upload/59315/59315fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59315/59315fig4v3.jpg" /> 
Figure 4: Example of phenotypic analysis of the larvae in the control and test compound treated groups. (A) Phenotypic analyses of the control group larvae not treated with any compound showed no phenotypic defect under a microscope. (B) Larvae treated with 500 &#956;M CA inhibitor (known inhibitor of carbonic anhydrase IX showed no observable phenotypic defects. (C, D) The developing larvae treated with &#946;-CA inhibitor with concentrations of 250 &#956;&#924; and 125 &#956;M respectively. The compounds induced phenotypic defects such as curved body, pericardial edema, and unutilized yolk sac (arrows and arrow heads). Panels A and B has been modified from Aspatwar et al16. The panels C, D and E show previously unpublished images, which were obtained using a different microscope. <a href="https://www.jove.com/files/ftp_upload/59315/59315fig4v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59315/59315fig5v2.jpg" /> 
Figure 5: Determination of lethal concentration 50 (LC50) using 1-5 dpf zebrafish larvae. Toxicity assessment using zebrafish embryos allows researchers to determine the minimum lethal concentration of the chemical compound at the end of the experiment.&#160; The LC50 concentration of the compound (a known CA IX inhibitor) was 3.5 mM. The high LC50 concentration allows further characterization of the compound. This figure has been modified from Aspatwar et al.16. <a href="https://www.jove.com/files/ftp_upload/59315/59315fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59315/59315fig6v3.jpg" /> 
Figure 6: An example of swim pattern analysis in both untreated (A) and inhibitor treated (B) groups of larvae. The zebrafish larvae treated in panel B with 300 &#956;M test inhibitor compound (a known inhibitor of human carbonic anhydrase IX) showed an abnormal (ataxic) movement pattern (arrow heads), suggesting that the compound induces neurotoxicity in the developing embryos. The arrowheads point to the normal curvature of the tail during swimming. In panel A the arrows point to the normal curvature of the tail during swimming. This figure has been modified from &#160;Aspatwar et al.16. <a href="https://www.jove.com/files/ftp_upload/59315/59315fig6v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CA inhibitor</td>
			<td>Toxicity Screening&#160;</td>
			<td>LC50</td>
			<td>In vivo</td>
			<td>Toxic CAIs</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Carbamates</td>
			<td>24</td>
			<td>All</td>
			<td>1a</td>
			<td>3b</td>
			<td>24, Unpublished</td>
		</tr>
		<tr>
			<td>Coumarins</td>
			<td>10</td>
			<td>All</td>
			<td>-</td>
			<td>2</td>
			<td>Unpublished</td>
		</tr>
		<tr>
			<td>Nitroimidazoles</td>
			<td>2</td>
			<td>All</td>
			<td>2</td>
			<td>2c</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Sulfonamides</td>
			<td>5</td>
			<td>All</td>
			<td>-</td>
			<td>3</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>41</td>
			<td>All</td>
			<td>3</td>
			<td>10</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="6">aBased on the toxicity screening, the inhibitor was used for inhibition of Mycobacterium marinum in zebrafish model. bLethal inhibitor, cNeurotoxic above 300 &#956;M concentration</td>
		</tr>
	</tbody>
</table>
Table 1: A summary of the safety and toxicity of the compounds screened. The toxicity evaluation experiment helps the researcher to reach a conclusion about the safety of the tested chemical compounds. The LC50 concentration allows to define safe concentration for further characterization. A researcher can decide if the compound is lethal even after 24 h of exposure of embryos to the compound under investigation. In our example, a subtle effect on swimming due to neurotoxicity is the significant information, which is helpful for setting up further experiments. Accordingly, based on the toxicity screening, we were able to use safe concentrations of the CA inhibitor for further characterization in vivo24.

Watch this Scientific Journal Video about Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos at JoVE.com

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

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 ...

rapid-evaluation-toxicity-chemical-compounds-using-zebrafish

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.6K Views.  Tampere University. 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.

Video: Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

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

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

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.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

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.

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

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.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

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 are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Cancer Research

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

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.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Immunology and Infection

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

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

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.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

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

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

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.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

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

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

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

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

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

Source: Hao J. et al., <a href="https://www.jove.com/v/2243/" target="_blank">Large Scale Zebrafish-Based In vivo Small Molecule Screen</a>. J. Vis. Exp. (2010)
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.

Source: Hao J. et al., Large Scale Zebrafish-Based In vivo Small Molecule Screen. J. Vis. Exp. (2010)
In this article, zebrafish embryos are used for ...