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 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 use^4,5. Existing protocols using zebrafish vary in length and have been used by individual researchers who evaluated the toxicity as per their convenience requirement^{6,7,8,9,10,11,12}.

In the recent past, the zebrafish has emerged as a convenient model for the evaluation of the toxicity of chemical compounds during embryonic development^6,7. 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 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 models^16,17.  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 humans^{8,18,19,20,21,22}. 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 °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).  This method is rapid and efficient, requires a small amount of the chemical compound and only basic facilities of the laboratory.

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 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.
3. To avoid handling stress to the animals, allow the animals to rest for 2 weeks before using the same individuals for breeding.

2. Collection of Embryos and Preparing Plates for Exposure to the Chemical Compounds

1. 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].
2. 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).
3. Keep the embryos at 28.5 °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: Zebrafish embryos are always maintained at 28.5 °C under laboratory conditions. 
4. Carefully transfer 1 embryo into each well of a 24-well plate containing enough E3 medium to cover the embryos.

3. Preparations of the Stock Solution of Chemical Compounds and Distribution of Diluted Compound into the Wells

1. Take out the vials containing inhibitor compounds stored at 4 °C.
   NOTE: Depending on the properties of the compound, these are stored at different temperatures.
2. Weigh the compound(s) using an analytical balance that can weigh a few milligrams (mg) of the compound accurately.
3. Prepare at least 250 μL (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 °C).
4. Make serial dilutions of the stock solutions (e. g., 10 μM, 20 μM, 50 μM, 100 μM, 150 μM, 300 μM and 500 μM) 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.
5. 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.
6. Distribute 1 mL of each diluent in each well (starting from lower and moving to higher concentration) into the wells of 24-well plate.
7. Set up a control group from the same batch of embryos and add the corresponding amount of diluent.
8. Label 24-well plates with the name and concentration of the compound and keep the plates at 28.5 °C in an incubator.

4. Phenotypic Analysis and Imaging of the Embryos Using a Stereomicroscope

1. Examine the embryos under a stereomicroscope for parameters 24 h after exposure to the chemical compounds.
   1. 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.
   2. 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.
   3. Take the images using stereomicroscope attached to a camera. Save the images in a separate folder each day till the end of the experiment.
   4. Enter all the observations in a table each day either in an online table or on a printed sheet.
   5. 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.
   6. 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.
   7. Construct a curve for mortality of embryos for all the concentrations using a suitable program.

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

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