Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples

• Published in: Aquatic toxicology Vol. 208; pp. 157 - 167
• Main Authors: Bakos, Katalin, Kovacs, Robert, Balogh, Erna, Sipos, Dora Kanaine, Reining, Marta, Gyomorei-Neuberger, Orsolya, Balazs, Adrienn, Kriszt, Balazs, Bencsik, Dora, Csepeli, Andrea, Gazsi, Gyongyi, Hadzhiev, Yavor, Urbanyi, Bela, Mueller, Ferenc, Kovacs, Balazs, Csenki, Zsolt
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: Endocrine disrupting chemicals; In vivo; Transgenic; Vitellogenin; YES; Transgenic; In vivo; Vitellogenin; YES; Endocrine disrupting chemicals
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2019.01.008

•Tg(vtg1:mCherry) expresses mCherry under the control of a 3357 bp promoter of the zebrafish vtg1and carries 26 natural EREs.•Endogenous vtg and mCherry are produced only in the left hepatic lobe of 5 dpf zebrafish embryos and show similar spatial expression.•Tg(vtg1:mCherry) embryos detect E2 from 100 ng/L, EE2 from 1 ng/L, ZEA from 100 ng/L and BPA from 1 mg/L.•Tg(vtg1:mCherry) adults are more sensitive, with detectable transgene activity from 5 ng/L E2.•Tg(vtg1:mCherry) is suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.