This work was supported by the National Research, Development and Innovation Office (NKFIH) from the National Research, Development and Innovation Fund (NKFIA); Grant Agreement: NVKP_16-1-2016-0003, EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union, and the Thematic Excellence Program NKFIH-831-10/2019 of Szent Istv&#225;n University, awarded by Ministry for Innovation and Technology.

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
<ol>
	<li>Maintain Tg(vtg1:mCherry) zebrafish at 25.5 &#177; 0.5 &#176;C, pH = 7 &#177; 0.2, conductivity between 525 &#177; 50 &#956;S/m, oxygen level &#8805;80% of saturation, and 14 h light and 10 h dark cycle.</li>
	<li>Fill the mating tanks with system water and set up the...

<ol>
	<li>Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+Disrupters+in+the+Aquatic+Environment+:+An+Overview.">Endocrine Disrupters in the Aquatic Environment : An Overview.</a> Acta Hydrochimica et Hydrobiologica. 33 (1), 9-16 (2005).</li><li>Routledge, E. J., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+activity+of+surfactants+and+some+of+their+degradation+products+assessed+using+a+recombinant+yeast+screen.">Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen.</a> Environmental Toxicology and Chemistry. 15 (3), 241-248 (1996).</li><li>Sanseverino, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Saccharomyces+cerevisiae+BLYES+Expressing+Bacterial+Bioluminescence+for+Rapid,+Sensitive+Detection+of+Estrogenic+Compounds.">Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds.</a> Applied and Environmental Microbiology. 71 (8), 4455-4460 (2008).</li><li>Fetter, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect-directed+analysis+for+estrogenic+compounds+in+a+fluvial+sediment+sample+using+transgenic+cyp19a1b-GFP+zebrafish+embryos.">Effect-directed analysis for estrogenic compounds in a fluvial sediment sample using transgenic cyp19a1b-GFP zebrafish embryos.</a> Aquatic Toxicology. 154, 221-229 (2014).</li><li>Gorelick, D. A., Halpern, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+estrogen+receptor+transcriptional+activation+in+zebrafish.">Visualization of estrogen receptor transcriptional activation in zebrafish.</a> Endocrinology. 152 (7), 2690-2703 (2011).</li><li>Rider, C. V., Hartig, P. C., Cardon, M. C., Wilson, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+competitive+binding+assay+system+with+recombinant+estrogen+receptors+from+multiple+species.">Development of a competitive binding assay system with recombinant estrogen receptors from multiple species.</a> Toxicology Letters. 184 (2), 85-89 (2009).</li><li>Gunnarsson, L., Kristiansson, E., F&#246;rlin, L., Nerman, O., Larsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+gene+expression+changes+in+fish+exposed+to+estrogen+-+a+microarray+approach.">Sensitive and robust gene expression changes in fish exposed to estrogen - a microarray approach.</a> BMC Genomics. 8 (149), 1-9 (2007).</li><li>Vander Ven, L. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitellogenin+expression+in+zebrafish+Danio+rerio+evaluation+by+histochemistry,+immunohistochemistry,+and+in+situ+mRNA+hybridisation.">Vitellogenin expression in zebrafish Danio rerio evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridisation.</a> Aquatic Toxicology. 65 (1), 1-11 (2003).</li><li>Bakos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+and+estrogenic+potency+of+zearalenone+in+zebrafish+(Danio+rerio).">Developmental toxicity and estrogenic potency of zearalenone in zebrafish (Danio rerio).</a> Aquatic Toxicology. 136-137, 13-21 (2013).</li><li>Str&#228;hle, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+as+an+alternative+to+animal+experiments+-+A+commentary+on+the+definition+of+the+onset+of+protected+life+stages+in+animal+welfare+regulations.">Zebrafish embryos as an alternative to animal experiments - A commentary on the definition of the onset of protected life stages in animal welfare regulations.</a> Reproductive Toxicology. 33 (2), 128-132 (2012).</li><li>Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+:+A+Tool+for+Chemical+Screens.">Zebrafish : A Tool for Chemical Screens.</a> Birth Defects Research, Part C. 90 (3), 185-192 (2010).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicological Sciences. 86 (1), 6-19 (2005).</li><li>Lee, C., Na, J. G., Lee, K., Park, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choriogenin+mRNA+induction+in+male+medaka,+Oryzias+latipes+as+a+biomarker+of+endocrine+disruption.">Choriogenin mRNA induction in male medaka, Oryzias latipes as a biomarker of endocrine disruption.</a> Aquatic Toxicology. 61 (3-4), 233-241 (2002).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+fluorescent+transgenic+zebrafish+for+detection+of+environmental+estrogens.">Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens.</a> Aquatic Toxicology. 96 (1), 53-61 (2010).</li><li>Bakos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+sensitive+liver+transgenic+zebrafish+(Danio+rerio)+line+(Tg(vtg1:mCherry))+suitable+for+the+direct+detection+of+estrogenicity+in+environmental+samples.">Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples.</a> Aquatic Toxicology. 208, 157-167 (2019).</li><li>Abdelmoneim, A., Clark, C., Mukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+reporter+zebrafish+line+for+estrogenic+compound+screening+generated+using+a+CRISPR/Cas9-mediated+knock-in+system.">Fluorescent reporter zebrafish line for estrogenic compound screening generated using a CRISPR/Cas9-mediated knock-in system.</a> Toxicological Sciences. 173 (2), 336-346 (2019).</li><li>Tong, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyp19a1b-GFP+(aromatase+B)+transgenic+zebrafish+line+that+expresses+GFP+in+radial+glial+cells.">A cyp19a1b-GFP (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells.</a> Genesis. 47 (2), 67-73 (2009).</li><li>Segner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+as+a+model+organism+for+investigating+endocrine+disruption.">Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption.</a> Comparative Biochemistry and Physiology, Part C: Toxicology and Pharmacology. 149 (2), 187-195 (2009).</li><li>Klinge, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+interaction+with+estrogen+response+elements.">Estrogen receptor interaction with estrogen response elements.</a> Nucleic Acids Res. 29 (14), 2905-2919 (2001).</li><li>Faisal, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+beta-cyclodextrins+vs.+zearalenone-induced+toxicity+in+HeLa+cells+and+Tg(vtg1:mCherry)+zebrafish+embryos.">Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish embryos.</a> Chemosphere. 240, 1-11 (2020).</li><li>Kolpin, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceuticals,+hormones,+and+other+organic+wastewater+contaminants+in+U.S.+streams,+1999-2000:+A+national+reconnaissance.">Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: A national reconnaissance.</a> Environmental Science and Technology. 36 (6), 1202-1211 (2002).</li><li>Kuch, H. M., Ballschmiter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+endocrine-disrupting+phenolic+compounds+and+estrogens+in+surface+and+drinking+water+by+HRGC-(NCI)-MS+in+the+picogram+per+liter+range.">Determination of endocrine-disrupting phenolic compounds and estrogens in surface and drinking water by HRGC-(NCI)-MS in the picogram per liter range.</a> Environmental Science and Technology. 35 (15), 3201-3206 (2001).</li><li>Lundgren, M. S., Novak, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+phytoestrogens+in+industrial+waste+streams.">Quantification of phytoestrogens in industrial waste streams.</a> Environmental Toxicology and Chemistry. 28 (11), 2318-2323 (2009).</li><li>Masoner, J. R., Kolpin, D. W., Furlong, E. T., Cozzarelli, I. M., Gray, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landfill+leachate+as+a+mirror+of+today&#8217;s+disposable+society:+Pharmaceuticals+and+other+contaminants+of+emerging+concern+in+final+leachate+from+landfills+in+the+conterminous+United+States.">Landfill leachate as a mirror of today&#8217;s disposable society: Pharmaceuticals and other contaminants of emerging concern in final leachate from landfills in the conterminous United States.</a> Environmental Toxicology and Chemistry. 35 (4), 906-918 (2016).</li><li>Panel on Additives and Products or Substances used in Animal Feed (FEEDA). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFSA+Statement+on+the+establishment+of+guidelines+for+the+assessment+of+additives+from+the+functional+group+&#8216;substances+for+reduction+of+the+contamination+of+feed+by+mycotoxins'+1+EFSA.">EFSA Statement on the establishment of guidelines for the assessment of additives from the functional group &#8216;substances for reduction of the contamination of feed by mycotoxins' 1 EFSA.</a> EFSA Journal. 8 (7), 1-8 (2010).</li><li>Braunbeck, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+alternative+for+the+acute+fish+LC(50)+test+in+chemical+assessment:+the+fish+embryo+toxicity+test+goes+multi-species+-+an+update.">Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species - an update.</a> Altex. 22 (50), 87-102 (2005).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Ober, E. A., Field, H. A., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endoderm+formation+to+liver+and+pancreas+development+in+zebrafish.">From endoderm formation to liver and pancreas development in zebrafish.</a> Mechanisms of Development. 120 (1), 5-18 (2003).</li><li>Tao, T., Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+development+in+zebrafish+(Danio+rerio).">Liver development in zebrafish (Danio rerio).</a> Journal of Genetics and Genomics. 36 (6), 325-334 (2009).</li><li>Shier, W. T., Shier, A. C., Xie, W., Mirocha, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationships+for+human+estrogenic+activity+in+zearalenone+mycotoxins.">Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins.</a> Toxicon. 39 (9), 1435-1438 (2001).</li><li>Panel, E., Chain, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriateness+to+set+a+group+health-based+guidance+value+for+zearalenone+and+its+modified+forms+EFSA+Panel+on+Contaminants+in+the+Food+Chain+(CONTAM).">Appropriateness to set a group health-based guidance value for zearalenone and its modified forms EFSA Panel on Contaminants in the Food Chain (CONTAM).</a> EFSA Journal. 14, 4425 (2016).</li><li>Binder, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+the+risk+of+mycotoxins+in+modern+feed+production.">Managing the risk of mycotoxins in modern feed production.</a> Animal Feed Science and Technology. 133 (1-2), 149-166 (2007).</li><li>Risa, A., Krifaton, C., Kukolya, J., Kriszt, B., Cserh&#225;ti, M., T&#225;ncsics, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxin+B1+and+Zearalenone-Detoxifying+Profile+of+Rhodococcus+Type+Strains.">Aflatoxin B1 and Zearalenone-Detoxifying Profile of Rhodococcus Type Strains.</a> Current Microbiology. 75 (7), 907-917 (2018).</li></ol>

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...

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, too1. This problem can be solved by effect-detecting methods, such as the use of biomonitor/bioindicator organisms that show estrogenic effects2,3,4,5.
Recently, a variety of cell line6 and yeast-based test systems2,3 have been developed to detect estrogenic effects. However, these are generally only able to detect the binding of the substance to the estrogen receptor2,3. In addition, they are unable to model complex physiological processes in the organism, or to detect hormone-sensitive phases of life stages; thus, they often lead to false results.
It is known that certain genes react sensitively to estrogen in living organisms7. The detection of gene products by molecular biology methods is also possible at the protein or mRNA level8,9, but usually involves animal sacrifice. Animal protection laws have become stricter, and there is a growing demand for alternative test systems that minimize the number and suffering of animals used in experiments or the replacement of the animal model with another model system10. With the discovery of fluorescent proteins and the creation of biomarker lines, transgenic technologies provide a good alternative11. With these lines, the activation of an estrogen-sensitive gene can be tested in vivo.
Among vertebrates, the potential of fish in environmental risk assessment is outstanding. They offer many advantages over mammalian models: being aquatic organisms, they are able to absorb pollutants through their entire body, produce a large number of offspring, and some of their species are characterized by short generation time. Their endocrine system and physiological processes show great similarities with other vertebrates and even with mammals, including humans12.
Several genes for the detection of estrogenic effects in fish are also known. The most important are the estrogen receptors aromatase-b, choriogenin-H, and vitellogenin (vtg)7,13. Recently, several estrogen-producing biosensor lines have also been created from fish models used in the laboratory, such as from zebrafish (Danio rerio)4,5,14,15,16,17. The main advantage of zebrafish in creating biosensor lines is the transparent body of the embryos and larvae, because the fluorescent reporter signal can then be easily studied in vivo without sacrificing the animal10. In addition to animal protection, it is also a valuable feature as it allows for studying the reaction of the same individual at different times of the treatment18.
These experiments use a vitellogenin reporter transgenic zebrafish line15. The transgene construct used for the development of Tg(vtg1:mCherry) has a long (3.4 kbp) natural vitellogenin-1 promoter. The estrogen receptor (ER) is an enhancer protein activated by ligands that is a representative of the steroid/nuclear receptor superfamily. ER binds to specific DNA sequences called estrogen response elements (EREs) with high affinity and transactivates gene expression in response to estradiol and other estrogenic substances, so the more ERE in the promoter causes a stronger response19. There are 17 ERE sites in the promoter region of the Tg(vtg1:mCherry) transgene construct and they are expected to mimic the expression of the native vtg gene15. There is a continuous expression of the fluorescent signal in sexually matured females. However, in males and embryo the expression in the liver is only visible upon treatment with estrogenic substances (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60462/60462fig01.jpg" /> 
Figure 1: Red fluorescent signal in the liver of vtg1:mCherry transgenic adult zebrafish and 5 dpf embryos, following 17-&#223;-estradiol (E2) induction.&#160;In female and in male treated with E2 (25 &#181;g/L exposure time:48hrs) strong fluorescence of the liver is visible even through the pigmented skin. No fluorescent signal is visible in untreated male (A). Following E2 induction (50 &#181;g/L exposure time: 0-120 hpf), a red fluorescent signal in the liver of 5 dpf embryos can also be observed, which is not visible in control embryos (B). While the fluorescent signal is continuously present in adult females, primarily males and embryos of the line are suitable for detecting estrogenic effects. (BF: bright field, mCherry: red fluorescent filter view, single plain images, Scale bar A: 5mm, scale bar B: 250 &#181;m) <a href="https://www.jove.com/files/ftp_upload/60462/60462fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similar to the endogenous vitellogenin, the mCherry reporter is only expressed in the liver. Because vitellogenin is only produced in the presence of estrogen, there is no fluorescent signal in the controls. Because the expression is only in the liver, the evaluation of the results is much easier15.
The sensitivity and usability of this line&#39;s embryos have been investigated on various estrogenic compound mixtures and also on environmental samples15,20, and in most cases dose-response relationships were documented (Figure 2). However, in the case of highly toxic, mainly hepatotoxic, substances (e.g., zearalenone), only a very weak fluorescent signal may be visible in the liver of treated embryos and the maximum intensity fluorescent signal caused can be reached within a very small concentration range, which makes it difficult to establish dose-effect relationships20.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60462/60462fig02.jpg" /> 
Figure 2: Dose-response diagram (A) and fluorescent images (mCherry) of the liver (B) exposed to 17-&#945;-ethynilestradiol (EE2), in 5 dpf vtg1:mCherry larvae.&#160;Results are expressed as integrated density generated from the signal strength and the size of the affected area (&#177;SEM, n = 60). 100% refers to the observed maximum. Fluorescent signal intensity increased gradually with concentration. Scale bar = 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60462/60462fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several estrogenic substances present in the environment, such as 17-&#946;-estradiol (environmental concentration: 0.1&#8211;5.1 ng/L)21, 17-&#945;-ethynylestradiol (environmental concentration: 0.16&#8211;0.2 &#181;g/L)22, zearalenone (environmental concentration: 0.095&#8211;0.22 &#181;g/L)23, bisphenol-A (environmental concentration: 0.45&#8211;17.2 mg/L)24. When testing these substances in a pure active form with the help of mCherry transgenic embryos, the lowest observed effect concentrations (LOEC) for fluorescent sign detection were 100 ng/L for 17-&#223;-estradiol, 1 ng/L for 17-&#945;-ethynilestradiol, 100 ng/L for zearalenone, and 1 mg/L for bisphenol-A (96&#8211;120 hpf treatment), which is very close to or within the range of environmental concentrations of the substances15. The Tg(vtg1:mCherry) transgenic line can help detect estrogenicity in wastewater samples after direct exposure. The line is as sensitive as the commonly used yeast estrogen test, the bioluminiscent yeast estrogen (BLYES) assay15. With the help of this line, the protective effects of beta-cyclodextrins against zearalenone-induced toxicity has been confirmed using chemical mixtures20.
In a recent report, the in vivo use of the transgenic line was demonstrated with the help of two estrogenic zearalenone (ZEA) metabolites, &#945;- and &#946;-zearalenol (&#945;-ZOL and &#946;-ZOL)25. The protocol baseline is appropriate to study the estrogenic effects of several compounds or environmental samples on Tg(vtg1:mCherry) embryos.

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

using tg(vtg1:mcherry) zebrafish embryos to test the estrogenic effects of endocrine disrupting compounds

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, &#945;- and &#946;-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.

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...

Watch this Scientific Journal Video about Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds at JoVE.com

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

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).

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

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

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.

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6.1K visualizações.  Szent István University. Há um alto número de produtos químicos EDC em nosso ambiente, principalmente substâncias estrogênicas. A diversidade química das substâncias pertencentes ao grupo dificulta seus testes, pois diferentes métodos são necessários para sua detecção. Com base na estrutura química, é realmente difícil determinar se uma substância é realmente capaz de funcionar como um estrogênio.Além disso, essas substâncias nunca estão presentes de forma pura no ambiente, de modo que seu...