title: Assessing the estrogenicity of environmental chemicals with a stably transfected lactoferrin gene promoter reporter in HeLa cells.
abstract: Lactoferrin is an important marker protein of the estrogens. In mice, lactoferrin expression is stimulated in the uterus by ligand-bound estrogen receptors (ERs). With this study we aimed to evaluate the effect of different environmental estrogenic chemicals on the mouse lactoferrin gene expression in a cell-based assay. We constructed a reporter that contains the firefly luciferase gene under hormone-inducible control of a 1.1kbp fragment of the mouse lactoferrin gene promoter. In an attempt to study the promoter regulation in a chromatin context, we stably transfected the construct (pGL3-mLF-Luc) into HeLa cells, and a stable clone (HeLa-mLF-Luc) incorporating the construct was subsequently generated. Transient transfection of HeLa-mLF-Luc cells with ERα and ERβ expression plasmids showed that both 17β-estradiol (E2) and diethylstilbestrol (DES) at 10(-7)M significantly increased luciferase expression via ERα and ERβ. Xenoestrogens such as bisphenol A, 4-octylphenol, 4-nonylphenol and the phytoestrogen genistein when used at increasing concentrations (10(-8) to 10(-5)M) revealed varying magnitudes of activation (1.96-8-fold). The environmental estrogens showed similar magnitudes of luciferase induction when acting through ERα and ERβ-mediated pathways. Also, in the absence of ERs, the xenoestrogens could not induce luciferase expression thereby reflecting receptor dependency. Taken together, the results indicate a significant responsiveness of the stably transfected mouse lactoferrin promoter to endogenous estrogen and environmental estrogenic compounds through ERs. This cell-based transcription assay system may be useful in understanding the susceptibility of estrogen target gene expression by these chemicals at the chromatin level.
authors.affiliation: Gene Regulation Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Harmit S
authors.initials: HS
authors.lastname: Ranhotra
authors.forename: Christina T
authors.initials: CT
authors.lastname: Teng