Evaluation of a rapid in vitro androgen receptor transcriptional activation assay using AR-EcoScreen cells.
An accurate and reliable in vitro assay system has been needed for first tier screening of endocrine disrupting chemicals. For the purpose, we previously developed stable AR-EcoScreen cell lines to assess androgen receptor (AR)-mediated transcriptional activation. In this report, we evaluated AR-EcoScreen cell lines as the phase I of prevalidation study by determining the intra-laboratory reproducibility, assay stability, and overall protocol performance of AR-EcoScreen assays. Forty compounds recommended by the ICCVAM were tested for AR agonist and antagonist activity. The mean coefficient of variation (CV) for intra-assay reproducibility in the AR agonist assay was 4.35% for 5alpha-dihydrotestosterone (DHT), and that for the antagonist assay was 5.51% for hydroxyflutamide. The detection limit of the agonist assay was 2.3x10(-11) M for 5alpha-dihydrotestosterone. Furthermore, we examined the overall performance of the method by comparing the predicted result with the ICCVAM classification. Thus, the overall sensitivity, specificity, and accuracy of the agonist assay were 89%, 94%, and 91%, respectively. For the antagonist assay, these values were 94%, 100%, and 96%, respectively. In summary, we concluded that AR-EcoScreen method was ready to proceed to the phase II prevalidation study to asses the inter-laboratory variability and transfer of the protocol.
Araki
Naohiro
N
EcoScreen R&D Section, Endocrine Disrupting Chemical Analysis Center, Otsuka Life Science Initiative, Otsuka Pharmaceutical Co. Ltd., 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima 771-0195, Japan.
Ohno
Ken
K
Takeyoshi
Masahiro
M
Iida
Mitsuru
M
D000726:Androgen Antagonists / Q000145:classification / Q000494:pharmacology*; D059002:Androgen Receptor Antagonists; D000728:Androgens / Q000145:classification / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000187:drug effects; D011944:Receptors, Androgen / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015533:Transcriptional Activation / Q000187:drug effects*; D015262:Xenobiotics / Q000145:classification / Q000494:pharmacology*
D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D000728:Androgens; D011944:Receptors, Androgen; D015262:Xenobiotics