title: Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening.
abstract: The discovery of xenobiotics that interfere with androgen activity has highlighted the need to assess chemicals for their ability to modulate dihydrotestosterone (DHT)-receptor binding. Previous test systems have used cells transfected with plasmid containing a reporter gene. Here we report the use of transduction for gene delivery and assessment of the modulation of DHT-induced gene activation. Transduction, the ability of replication-defective viruses to deliver biologically competent genes, is a well understood biological process, which has been utilized to repair defective genes in humans as well as to express exogenous genes in rodent models. Human breast carcinoma cells (MDA-MB-453) containing endogenous copies of the androgen (hAR) and glucocorticoid (GR) receptors were transduced with replication-defective human adenovirus type 5 containing the luciferase (Luc) reporter gene driven by the AR- and GR-responsive glucocorticoid-inducible hormone response element found with the mammary tumor virus LTR (Ad/MLUC7). In a second set of experiments, CV-1 cells were transduced as above with MMTV-luc and also hAR. Cells were subcultured in 96-well plates, transduced with virus, exposed to chemicals, incubated for 48 h, lysed, and assayed for luciferase. Luc gene expression was induced in a dose-dependent manner by DHT, estradiol, and dexamethasone (MDA only) and inhibited by AR antagonist hydroxyflutamide (OHF), hydroxy-DDE, HPTE (2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane), a methoxychlor metabolite, and M1 and M2 (vinclozolin metabolites). The transduced cells responded to AR agonists and antagonists as predicted from our other studies, with a very robust and reproducible response. Over all replicates, 0.1 nM DHT induced luc expression by about 45-fold in CV-1 cells (intra-assay CV = 20%) and 1micromolar OHF inhibited DHT by about 80%. In the transduced MDA cells, 0.1 nM DHT induced luc by about 24-fold (intra-assay CV = 33%), which was inhibited by OHF by about 85%. DHT-induced luciferase activity peaked in both cell lines between 1 and 100 nM, displaying about 64- and 115-fold maximal induction in the CV-1 and MDA 453 cells, respectively. For agonists, a two-fold induction of luc over media control was statistically significant. For AR antagonists, a 25-30% inhibition of DHT-induced luc expression was typically statistically significant. Comparing the two assays, the transduced CV-1 cells were slightly more sensitive to AR-mediated responses, but the transduced MDA 453 cells were more responsive to GR agonists. In summary, these assays correctly identified the endocrine activity of all chemicals examined and displayed sensitivity with a relatively low variability and a high-fold induction over background. Adenovirus transduction for EDC screening has the potential to be employed in a high-throughput mode, and could easily be applied to other cell lines and utilized to deliver other receptors and reporter genes.
authors.affiliation: Reproductive Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. hartig.phillip@epa.gov
authors.forename: P C
authors.initials: PC
authors.lastname: Hartig
authors.forename: K L
authors.initials: KL
authors.lastname: Bobseine
authors.forename: B H
authors.initials: BH
authors.lastname: Britt
authors.forename: M C
authors.initials: MC
authors.lastname: Cardon
authors.forename: C R
authors.initials: CR
authors.lastname: Lambright
authors.forename: V S
authors.initials: VS
authors.lastname: Wilson
authors.forename: L E
authors.initials: LE
authors.lastname: Gray
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000726:Androgen Antagonists / Q000494:pharmacology; D059002:Androgen Receptor Antagonists; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000494:pharmacology; D005485:Flutamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D010575:Pesticides / Q000494:pharmacology; D011374:Progesterone / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011944:Receptors, Androgen / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D000728:Androgens; D005938:Glucocorticoids; D010575:Pesticides; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D013196:Dihydrotestosterone; C014290:hydroxyflutamide; D011374:Progesterone; D005485:Flutamide; D003907:Dexamethasone; D008156:Luciferases