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Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents
Author(s) -
Ackermann Gabriele E.,
Brombacher Eva,
Fent Karl
Publication year - 2002
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1002/etc.5620210914
Subject(s) - reporter gene , estrogen receptor , ethinylestradiol , nonylphenol , estrogen , medicine , rainbow trout , chemistry , endocrinology , complementary dna , agonist , effluent , gene expression , biology , microbiology and biotechnology , receptor , biochemistry , environmental chemistry , gene , fish <actinopterygii> , fishery , population , cancer , environmental engineering , environmental health , engineering , research methodology , breast cancer
This study reports on the development and application of a fish‐specific estrogen‐responsive reporter gene assay. The assay is based on the rainbow trout ( Oncorhynchus mykiss ) gonad cell line RTG‐2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERα cDNA) in the presence of an estrogen‐dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG‐2 cells were stably transfected with the rtERα cDNA expression vector, and clones responsive to 17β‐estradiol (E 2 ) were selected. The estrogenic activity of E 2 , 17α‐ethinylestradiol, 4‐nonylphenol, nonylphenoxy acetic acid, 4‐ tert ‐octylphenol, bisphenol A, o,p ′‐DDT, p,p ′‐DDT, o,p ′‐2,2‐bis(chlorophenyl)‐1,1‐dichloroethylene ( o,p ′‐DDE), p,p ′‐DDE, o,p ′‐2,2‐bis(chlorophenyl)‐1,1‐di‐chloroethane ( o,p ′‐DDD), p,p ′‐DDD, and p,p ′‐2,2‐bis(chlorophenyl)acetic acid ( p,p ′‐DDA) was assessed at increasing concentrations. All compounds except o,p ′‐DDT, p,p ′‐DDE, and p,p ′‐DDA showed logistic dose‐response curves, which allowed the calculation of lowest‐observed‐effect concentrations and the concentrations at which half‐maximal reporter gene activities were reached. To check whether estrogen‐responsive RTG‐2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E 2 . Dose‐response curves of nonylphenol, octylphenol, bisphenol A, and o,p ′‐DDD revealed that the RTG‐2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E 2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed.