title: Sensitive in vitro test systems to determine androgenic/antiandrogenic activity.
abstract: We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS-7 African green monkey kidney cells. Three plasmids were co-transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVbeta. Transcriptional activation was measured by luciferase-mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen-induced expression of prostate-specific antigen (PSA), the other based on the androgen-repressed expression of testosterone repressed message 2 (TRPM-2). PSA protein was measured by enzyme-linked immunosorbent assay (ELISA), TRPM-2 m-RNA by reverse transcriptase polymerase chain reaction (RT-PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p'-dichlorodiphenylethene (p,p'-DDE) as antagonists. The PSA assay was the most sensitive test system with an EC50 of 0.7 nM for DHT-induced response. The transient transactivation assay in COS-7 cells was less sensitive with an EC50 of 9.7 nM DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC30 = 0.02 microM) than p,p'-DDE (IC30 = 0.9 microM). In the transient transactivation assay in COS-7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.
authors.affiliation: Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Kaiserslautem, Germany.
authors.forename: Sabine E
authors.initials: SE
authors.lastname: Guth
authors.forename: Sonja
authors.initials: S
authors.lastname: Böhm
authors.forename: Bernd H
authors.initials: BH
authors.lastname: Mussler
authors.forename: Gerhard
authors.initials: G
authors.lastname: Eisenbrand
mesh_terms: D000726:Androgen Antagonists / Q000032:analysis / Q000494:pharmacology*; D000728:Androgens / Q000032:analysis / Q000494:pharmacology*; D000818:Animals; D001943:Breast Neoplasms; D019556:COS Cells; D002522:Chlorocebus aethiops; D051152:Clusterin; D004777:Environment; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017430:Prostate-Specific Antigen / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011944:Receptors, Androgen / Q000235:genetics; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; C495242:CLU protein, human; C495243:Clu protein, mouse; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D011944:Receptors, Androgen; D008156:Luciferases; D017430:Prostate-Specific Antigen