Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Summary

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Abstract

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, α- and β-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Introduction

There is a significant number of endocrine disrupting compounds (EDC) that are among the most hazardous substances in our environment. These are mainly estrogenic compounds that contaminate water from natural resources. The chemical diversity of the substances belonging to the group makes testing for their presence difficult, as different analytical methods are required for their detection. Based on their chemical structure it is very difficult to determine whether a substance is actually able to act as an estrogen. In addition, these substances are never present in a pure form in the environment, so their effects may be affected by other compounds, too

Protocol

The Animal Protocol was approved under the Hungarian Animal Welfare Law and all studies were completed before the treated individuals would have reached the free feeding stage.

1. Embryo harvest and treatment

1. Maintain Tg(vtg1:mCherry) zebrafish at 25.5 ± 0.5 °C, pH = 7 ± 0.2, conductivity between 525 ± 50 μS/m, oxygen level ≥80% of saturation, and 14 h light and 10 h dark cycle.
2. Fill the mating tanks with system water and set up the fish for mating the afternoon before harvesting eggs.
3. Place the male and female fish into the tank and separate them with the help of a divider.
4. Remove the divi....

Results

In the experiment presented in this manuscript, the effects of two estrogenic substances were tested at five concentrations starting at fertilization for 5 days on Tg(vtg1:mCherry) zebrafish embryos. We investigated whether fluorescent signals appeared in the liver of fish by the end of the exposure time because of the substances and whether there were differences in the estrogenicity of the two substances. Results were evaluated on the basis of the fluorescent images and integrated density values. In general, b.......

Discussion

The use of biomonitors/bioindicators for estrogenic effects has been spreading in toxicological studies. In vivo models play an outstanding role, because unlike in vitro tests, they not only provide information about the response of a cell or a receptor, but also allow the investigation of complex processes in the organism. Several transgenic lines for studying estrogenic effects have been produced from zebrafish, one of which Tg(vtg1:mCherry) was used for these studies. The method described here illustrates a p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 well tissue culture plate	Jet Biofil	TCP011024
Calcium-chloride (CaCl2)	Reanal Laborvegyszer Ltd.	16383-0-27-39
GraphPad Prism 6.01 software	GraphPad Software Inc.
ImageJ software	National Institutes of Health, USA		Public access software, downloadable from: http://imagej.nih.gov/
Leica Application Suite X calibrated software	Leica Microsystems GmbH.		We used the softver described in the experiments, but any photographic software complies with the tests
Leica M205 FA stereomicroscope, Leica DFC 7000T camera	Leica Microsystems GmbH.		We used the equipments described in the experiments, but any fluorescent stereomicroscope is suitable for the tests
Magnesium-sulphate (MgSO4)	Reanal Laborvegyszer Ltd.	20342-0-27-38
mCherry filter	Leica Microsystems GmbH.
Mehyl-cellulose	Sigma Aldrich Ltd.	274429
Microloader pipette tip	Eppendorf GmbH.	5242956003
Pasteur pipette	VWR International LLC.	612-1684
Petri-dish	Jet Biofil	TCD000060
Potassium-chloride (KCl)	Reanal Laborvegyszer Ltd.	18050-0-01-33
Sodium-chloride (NaCl)	Reanal Laborvegyszer Ltd.	24640-0-01-38
Tricane-methanesulfonate (MS-222)	Sigma Aldrich Ltd.	E10521