Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals.
Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV)2-luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p'-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast cancer cells with an estrogen response element-luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties.
Institute of Food Safety and Toxicology, Division of Biochemical and Molecular Toxicology, Danish Veterinary and Food Administration, Morkhoj Bygade 19, Soborg, 2860, Denmark. amv@vfd.dk
A M
AM
Vinggaard
E C
EC
Joergensen
J C
JC
Larsen
D000726:Androgen Antagonists / Q000633:toxicity*; D000818:Animals; D001943:Breast Neoplasms; D016466:CHO Cells; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004785:Environmental Pollutants / Q000633:toxicity*; D004965:Estrogen Antagonists / Q000633:toxicity*; D017930:Genes, Reporter; D006801:Humans; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D010714:Phosphatidylethanolamines / Q000235:genetics / Q000378:metabolism; D011944:Receptors, Androgen / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D000726:Androgen Antagonists; D004785:Environmental Pollutants; D004965:Estrogen Antagonists; D007202:Indicators and Reagents; D010714:Phosphatidylethanolamines; D011944:Receptors, Androgen; C020888:1,2-dielaidoylphosphatidylethanolamine; D008156:Luciferases