Submission Number: VIC-4-34-23
Abstract Number: 346
STANDARDIZATION AND VALIDATION OF A NOVEL CELL LINE THAT STABLY EXPRESSES THE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS.
Vickie S Wilson 1,2, K Bobseine 2, C Lambright 2 and L Earl Gray Jr* 2
NCSU/USEPA Cooperative Training Program, Raleigh, NC. 1
USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC. 2
Abstract:
The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. Our overall objective was the development of a stable cell line to detect AR agonists and antagonists. To this end, MDA-MB-453 cells, which contain both endogenous AR and glucocorticoid receptor (GR), were stably transfected with the MMTV.neo.luciferase gene construct. The specific goal of this study was to characterize the specificity, accuracy, and sensitivity of this cell line. Since both GR and AR can act through the same response element, compounds that act through either receptor should activate the luciferase reporter. We have tested the AR agonists, dihydrotestosterone (DHT) and medroxyprogesterone acetate, and GR agonists, dexamethasone, corticosterone, and aldosterone. Cells display appropriate sensitivity to agonists. For example, DHT produces consistent 3 to 9 fold induction over background at concentrations from 0.1 to 10 nM. To distinguish between AR and GR ligands, chemicals that increase luciferase activity were assayed concurrently with hydroxyflutamide which blocks AR but not GR ligands. The AR antagonists, hydroxyflutamide, vinclozolin, vinclozolin metabolites M1 and M2, p,p'-DDE, and linuron, inhibited DHT induced luciferase expression. Some chemicals, as previously published, displayed mixed agonist/antagonist activity depending on their concentration. This assay has several advantages over transient transfection systems and is relatively rapid and produces consistent reproducible results. In summary, we have developed a cell line that can be used to screen chemicals not just for AR mediated activity but GR activity as well. DISCLAIMER: This is an abstract of a proposed presentation and does not necessarily reflect USEPA policy. .
Keywords: androgen, stable cell line, endocrine disruption
This abstract is being presented at: 8:00 AM in session:
Toxicology II