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(398) Determination of estrogenic activity in environmental matrices using in vivo transgenic zebrafish and in vitro ER-CALUX reporter gene assays . Legler, Juliette1,2, Lanser, Peter1, De Voogt, Pim3, Vethaak, Dick4, Brouwer, Abraham5, Murk, Tinka2, Van Der Burg, Bart1, 1 2 3 4 5 ABSTRACT- Functional in vitro and in vivo reporter gene assays were recently developed to measure biological exposure to (xeno-)estrogens. The in vitro estrogen receptor (ER)-mediated chemical activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced, allowing for prediction of estrogenic effects in target tissues and critical life stages of zebrafish. Considerable differences were found in the relative potencies of various (xeno-) estrogens in vitro and in vivo. Ethynylestradiol (EE2) was the most potent of the (xeno-)estrogens tested and was 100 times more potent than E2 in the transgenic zebrafish assay whereas in the ER-CALUX assay, EE2 was only slightly (1.2 times) more potent than E2. Although the xeno-estrogens o,p-DDT, nonylphenol, bisphenol A were full estrogen agonists in the ER-CALUX assay, only o,p'DDT demonstrated dose-related estrogenic activity in vivo. Di-(2-ethylhexyl)phthalate (DEHP) was only weakly estrogenic in both assays at concentrations above 1000 nM. Estrogenic potency determined in mixtures of (xeno-)estrogenic compounds in domestic wastewater treatment plant effluent revealed levels of (xeno-)estrogens that exceed threshold values for estrogenic effects in fish. Biological validation of the ER-CALUX and transgenic zebrafish assays is currently ongoing in order to determine critical levels of reporter gene induction that correspond with ecologically relevant effects (i.e. on gonad differentiation and reproduction) in the zebrafish. Key words: (xeno-)estrogens, bioassays, effluent |
