title: A rapid and sensitive high throughput reporter gene assay for estrogenic effects of environmental contaminants.
abstract: We have developed an estrogen sensitive reporter gene assay and report here on its responsiveness to steroid hormones and several estrogenic contaminants, including pesticides and polychlorinated biphenyls (PCBs), GH4C1 rat pituitary cells were stably transfected with the reported gene ERE-TK-Luc which contains a luciferase gene under the transcriptional regulation of the Xenopus vitellogenin estrogen response element (ERE) and clones stably expressing ERE-TK-Luc were isolated. The sensitivity of the assay was determined by dose response with the endogenous ligand 17 beta-estradiol. An EC50 of 4.5 +/- 1.2 x 10(-11) M was determined for this compound. Natural and synthetic hormones such as dexamethasone, aldosterone, and thyroxine did not induce luciferase activity, whereas 10(-8) M diethylstilbestrol induced luciferase activity to a maximal equal to 17 beta-estradiol, indicating that transcription of the luciferase gene is primarily regulated through the endogenous estrogen receptors. The pesticide o,p'-DDT was found to be estrogenic at concentrations as low as 10(-7) M. Of the three PCBs assayed, 2',4',6'-trichlorobiphenyl and its hydroxylated metabolite 4O H-2',4',6'-tricholorbiphenyl were estrogenic at 10(-7) M, whereas 2,4,4'-trichlorobiphenyl did not induce luciferase activity. Lastly, the GH4C1 ERE-Luc reporter gene assay was modified to utilize a cell permeant luciferin and 96 well format suitable for multiple and complex samples and generation of detailed dose response data.
authors.affiliation: Marine Biotoxins Program, U.S. National Marine Fisheries Service, Medical University of South Carolina, Charleston 29412, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Edmunds
authors.forename: E R
authors.initials: ER
authors.lastname: Fairey
authors.forename: J S
authors.initials: JS
authors.lastname: Ramsdell
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004785:Environmental Pollutants / Q000633:toxicity*; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000633:toxicity*; D017930:Genes, Reporter / Q000187:drug effects*; D008156:Luciferases / Q000378:metabolism; D010575:Pesticides / Q000633:toxicity; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D011078:Polychlorinated Biphenyls / Q000633:toxicity; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D004785:Environmental Pollutants; D004967:Estrogens; D010575:Pesticides; D004958:Estradiol; D011078:Polychlorinated Biphenyls; D008156:Luciferases