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

When screening chemicals for toxicity, a current experimental challenge is the determination of toxic effects in vivo, taking into account the complexity of a whole organism, and to do this in a rapid, high throughput manner. By using a behavioral readout that monitors the escape response of zebrafish embryos to a vibration stimulus, our system allows us to identify compounds that interfere with neural or muscular function. Because dead embryos don't move, we also capture compounds that cause lethality by unspecific toxicity.

The system we present can be built for a modest price and is customizable. It is also easy to maintain and all pieces can be replaced. We are currently employing the startle assay system within the PrecisionTox Consortium to determine a chemical compound dose for OMICS data acquisition.

The data are generated across five model organisms and human cell lines. They will be used to derive toxicity pathways and biomarkers for human toxicity prediction. To begin, collect embryos in cleavage stages, specifically from two-cell to eight-cell stage from natural spawning in a 10-centimeter Petri dish.

Remove unfertilized eggs, debris, and scales from the Petri dish to clean it. Place 60 embryos per one Petri dish containing 15 milliliters of E3 medium. Position all dishes containing embryos in a humidified chamber prepared with water-soaked paper towels.

Then incubate them 72 hours post fertilization in an incubator set at 28.5 degrees Celsius. Next, retrieve the desired chemical stock solutions from the minus 20 degrees Celsius freezer and allow it to thaw. Prepare serial dilutions of each chemical in E3 medium in a glass bottle.

Inspect the solution for precipitation, and if present, record it, then further dilute to achieve the next highest concentration. Check and repeat until there is no precipitation. Then check and record the pH of the exposure solution.

If it falls outside the range of pH 7.0 to 8.5, adjust it to 7.4 with hydrochloric acid or sodium hydroxide. Dispose of any unused exposure media in accordance with local regulations. To begin, collect the zebrafish embryo and raise them until 72 hours post fertilization in an incubator at 28.5 degrees Celsius.

Simultaneously prepare the chemicals to be tested at the desired concentration. Examine the 72-hour post fertilization embryos. Remove dead or unhatched embryos.

Place 10 embryos in each six-centimeter tissue culture dish with nine milliliters of E3 medium. These are called exposure plates. Label each exposure plate with the compound name, exposure concentration, and replicate number.

Add one milliliter of exposure solution to each plate, starting with the lowest concentration, and gently swirl the plate. For compounds with low solubility, replace the entire 10 milliliters of the medium with the exposure solution. Record the sequence in which the compound solutions were added to the embryos.

Then incubate the plates in the humidified chamber within an incubator at 28.5 degrees Celsius for 48 hours. To begin the vibration startle assay, turn on the computer and the vibration device. Create a spreadsheet for the configuration file.

Include exposure information for each of the five plate positions, specifying compound, concentration, and replicate. Open the general user interface or GUI program for the vibration startle assay kit. Select different positions in the GUI program to check the camera movement and observe the camera's response.

Remove the sample plates from the incubator. Place them in the five designated positions and let the embryos settle for several minutes. In the GUI program, click Record and the new window will appear.

In this window, select the configuration file prepared earlier. Verify that the sample description matches the samples on each position. Observe the LED turning on when the sound pulse is activated by the program.

After recording, the camera will return to position one and the software begins compressing the files. Replace the measured samples with the next set and proceed with the next run as demonstrated. Then collect the exposure solutions from all measured plates using a sieve to simultaneously retain the embryos.

To initiate the data analysis, open the video data with VirtualDub or another viewing software. Visually score the number of embryos responding to the sound pulse. Record the compound name, the replicate, the concentration of the compound, and the percentage of immotile embryos in the spreadsheet.

Conduct the benchmark concentration analysis using a KNIME workflow with embedded R scripts. An assessment of immotility in untreated wild-type embryos showed an average immotility of 14.33%when subjected to vibration stimulus with variations across different clutches. The benchmark concentration calculations for tricaine effects on motility showed a reduction in motility at 1%concentration, ceasing activity above 2.5%with a benchmark concentration 50 of 164.9 micromolar.

A suboptimal assay example showed inconsistencies in embryo responses, implying developmental issues affecting the robustness of the startle response.