There is a high number of EDC chemicals in our environment, mainly estrogenic substances. The chemical diversity of the substances belonging to the group makes their testing difficult, as different methods are required for their detection. Based on the chemical structure, it is really difficult to determine if a substance is actually able to work as an estrogen.
In addition, these substances are never present in a pure form in the environment, so their effects may be influenced by other compounds, too. This problem is solved by effect dialecting methods such as the use of biomonitor, bioindicator organisms that show estrogen effect. It is known that certain genes react sensitively to estrogen in living organisms.
The detection of gene products by molecular methods is also possible at protein or mRNA level, but usually involves animal sacrifice. Animal protection laws have been becoming stricter, and there is a growing demand for alternative test systems. Cross-genetic technologies represent the possibility with this development and with the discovery of fluorescent proteins, the way for the creation of biomarker have been paved.
By the help of such the activation of estrogen sensitive genes can be tested in vivo. In this video, a possible method for use of vtg:1mCherry transgene zebrafish embryos is being shown for the testing of estrogenic substances with the help of two compounds. The protocol can serve as a background for studying the estrogen effect of different chemical or environmental samples on biomarker embryos.
The zebrafish line used in our experiments is a vitellogenin reporter transgenic zebrafish line. The line was created by using the Tol2 Transposon system. The transgene construct used for the development carried the long natural vitellogenin-1 promoter sequence, with a high number of ERI sides.
The expression of the fluorescent signal in the sexually mature female is continuous. However, in males in embryos, it only appears by the action or presence of estrogenic substances, so the latter are more suitable for testing. The sensitivity and usability of the embryos of the line have been tested on several estrogenic compounds as well as on environmental samples.
All experiments on live animals were performed according to the Hungarian Animal Welfare Law and all studies were completed before the animals reached free feeding stage. Fill the mating tanks with system water and set up the fish for mating the before harvesting eggs. Place male and female fish into the tank, and separate them with the help of a divider.
Remove the divider from the tanks as the light switches on next morning. Check the mating tanks for eggs every 15 to 20 minutes. Harvest all embryos using a tea strainer, or densely woven, fine mesh, and combine them into one large Petri dish with E3 buffer, or clear system water.
Place the embryos in the incubators set to 25.5 degrees Celsius. One to one and a half hours after, remove and discard unfertilized, or inadequately divided eggs with a plastic transfer pipette under a dissecting microscope. Place the selected embryos in the treatment vessels, for example in Petri dishes or tissue culture plates, which have already been labeled and filled up with different concentrations of the test substance.
Incubate the embryos at 25.5 degrees Celsius until the end of the experiment. Refresh the test solution if it is necessary in order to maintain the treatment concentration. Be careful when changing the test solution, in order to avoid damaging the embryos.
Prepare four percent methoseladose with ms 222 beforehand. Place the five day old larvae into a five centimeter Petri dish per treatment group with a plastic pipette. Remove the treatment solution from the larvae with a plastic pipette, then fill the Petri dish with two milliliters of 0.02 percent ms 222 anesthetic solution.
Fill each square of a specially designed 10 centimeter Petri dish with four percent methoseladose ms 222. Transfer the anesthetized larvae to methoseladose with a little water in one of the two squares. From the first square, transfer the larvae into the second square.
In the second square, rotate and orient larvae to their left side and gently press them down to the bottom of the with a microloader pipette tip cut up to two centimeters. In order to evaluate the signal of the expressed reporter, image the embryos in the same view and settings. Place the Petri dish on the stage of the microscope.
Focus on the liver of the embryo, and capture a bright field image using the associated software. Switch the microscope to the mCherry filter, and take a florescent image of the liver under fluorescent light using the associated software. Repeat the previous two steps until you have captured all the embryos in the experiment.
Open ImageJ, then upload the fluorescent image to be analyzed by either dragging and dropping the image, or clicking on File Open. Click on Image, Color, Split Channels to split the image made by the florescent filter according to the RGB color chart. Work with the red channel image spectrum.
Close the other channels. Designate a similarly sized elliptical area in the image, so full signals do not interfere with the evaluation. Using the oval tools draw an ellipse over the highlighted liver area as accurately as possible.
If the signal is weak, use light microspic images, the bright field pair of the fluorescent image, to determine the location of the liver. Click on Analyze, Measure, to determine the signal strength and the size of the effected area. The integrated density value is automatically calculated by the software column in the chart.
Continue the analysis by repeating the previous steps until you analyze all the fluorescence images of all embryos in the treatment group. Save the data, and then analyze the integrated density values. In the experiment, estrogen sensitive embryos of the biomarker zebrafish line were treated with 0.5 and eight micromole concentrations of alpha-and beta-Zearalenol, or ZOL, for fertilization up to the age of five days.
We investigated whether florescent signals appear in the liver of the fish by the end of the experiment due to substances, and whether there are differences in estrogenicity of the two substances. Results were evaluated on the basis of fluorescent images and integrated density values. In the case of alpha-ZOL, at the highest test concentration, all the individuals died.
So in this case the fluorescent signal could not be examined. At lower concentrations, a strong fluorescent signal can be observed in the embryo's liver. With no significant difference in the strength of fluorescent signal and size of illuminated areas.
There was also no significant difference between the integrated density values and the treatments. There was no mortality during the treatment with beta-ZOL. The substance induced transgene activity in all treatment concentrations.
The increase of fluorescent signal intensity, and the size of the illuminated area can be observed in the fluorescence images as the concentration increases. By examining the integrated values of beta-ZOL, this change can also be discovered. The average integrated density value almost doubles between the lowest and the highest treatment concentrations.
However, in the case of beta-ZOL, we did not find significant difference between the integrated density values of individual concentrations. By examining the integrated density values obtained from the same treatment concentrations of the two substances, it can be said that alpha-ZOL gave higher integrated density averages in each case relative to beta-ZOL, which is consistent with the differences between signal strength observed in fluorescent images. This differences with significant at all concentrations.
The use of bioindicators for estrogen effects has been spreading in toxicological studies. Several transgenic lines showing estrogen effects have also been produced from zebrafish, of which vtg:1mCherry was used in our studies. The method described here illustrates a possible protocol for the testing of embryos of this line, in order to allow in vivo detection of estrogen activity in case of pure agents.
It is important to mention that in the case of embryos, the expression of the transgene, similarly to the production of endogenous vitellogenin, shows a large dispersion, and differences in individual sensitivity should be taken into account when designing experiments. An important aspect in determining treatment concentrations is that cells of embryos, including liver cells, can be damaged by higher concentrations of highly toxic substances, which can lead to a decline in vitellogenin induction. Therefore tests should be performed at concentrations below L-C 10.
This protocol can be altered in many points according to the planned test endpoints, and to the samples which are going to be tested. It can be completed with other test methods, for example molecular methods. Thus, we hope that the use of the vtg:1mCherry line will appear as a model of estrogenicity tests and may also may be a model for standardized testing methods.
