Legler J, van den Brink C E, Brouwer A, Murk A J, van der Saag P T, Vethaak A D, van der Burg B
Department of Food Technology and Nutritional Sciences, Agricultural University, Wageningen, The Netherlands.
Toxicol Sci. 1999 Mar;48(1):55-66. doi: 10.1093/toxsci/48.1.55.
Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.
通过在多个细胞系中稳定转染荧光素酶报告基因，尝试开发一种雌激素受体介导的化学激活荧光素酶报告基因表达（ER-CALUX）检测方法。在MCF-7乳腺癌细胞系和Hepa.1c1c7小鼠肝癌细胞系中稳定转染嵌合型Gal4雌激素受体和荧光素酶基因构建体，以及在ECC-1人子宫内膜细胞系中转染新构建的荧光素酶报告基因pEREtata-Luc，均产生了组成型、非雌二醇诱导型克隆。然而，在T47D乳腺癌细胞系中稳定转染pEREtata-Luc，得到了一种极其敏感、反应性高的细胞系。在暴露于雌二醇（E2）24小时后，稳定转染的T47D.Luc细胞的检测限为0.5 pM，半数有效浓度（EC50）为6 pM，相对于溶剂对照的最大诱导倍数为100倍。在延长的培养期（50代）内未发现反应性明显降低。抗雌激素药物ICI 182,780、四氯二苯并二恶英（TCDD）和他莫昔芬抑制了雌二醇介导的荧光素酶诱导。染料木黄酮、壬基酚和邻，对'-滴滴涕是该系统中测试的最有效的（拟）雌激素（EC50分别为100、260和660 nM）。对（拟）雌激素壬基酚、邻，对'-滴滴涕、氯丹、硫丹、狄氏剂和甲氧滴滴涕的相互作用效应的测定表明，与3 pM E2联合时，（拟）雌激素具有相加作用。在3至6 microM范围内测试的狄氏剂和硫丹组合发现有略高于相加的效应（小于2倍）。在这些浓度下，硫丹和氯丹的组合表现出相加相互作用。用T47D细胞进行的ER-CALUX检测可提供一个灵敏、反应性高且快速的体外系统，用于检测和测量具有潜在（抗）雌激素活性的物质。
