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Cameron Q. Sheeler, Mark W. Dudley,^* and Sohaib A. Khan

Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

Abstract

We used three yeast genetic systems to investigate the estrogen-like activity of octylphenol (OP) , bisphenol-A (BPA) , o,p´-DDT, and o,p´-DDE to induce human estrogen receptor (hER) dimerization and transcriptional activation. We have demonstrated that OP, BPA, and o,p´-DDT can induce hER ligand-dependent dimerization using a yeast two-hybrid assay. All three xenoestrogens, plus estradiol, enhanced estrogen response element (ERE) -dependent transcriptional activation of hER. In the presence of receptor interacting protein 140 (RIP140) , ERE-dependent activity was dramatically amplified by 100-fold for estradiol, OP, BPA, and o,p´-DDT. A yeast whole-cell [³H]estradiol binding assay was developed to determine the site of interaction on the hER. We determined nonspecific binding by parallel incubations run in the presence of 5 µM unlabelled estradiol in PCY2 yeast. At the concentrations tested, unlabeled estradiol, OP, and BPA displaced [³H]estradiol in this binding assay, whereas the concentrations of o,p´-DDT and o,p´-DDE tested were insufficient to inhibit binding. Incubating yeast in the presence of increasing concentrations of estradiol and OP (1 µM) or BPA (1 µM) neither blocked nor altered the effect of estradiol on hER activity. We observed no agonistic activity of o,p´-DDE in any of the yeast models used. These results suggest that OP, BPA, and o,p´-DDT exert their estrogen-like activity through the ER in a manner similar to that of estradiol, and the coactivator RIP140 markedly potentiates this activity. Key words: environmental estrogens, estrogen receptor, ligand binding, receptor dimerization, RIP140, xenoestrogens. Environ Health Perspect 108:97-103 (2000) . [Online 23 December 1999]

http://ehpnet1.niehs.nih.gov/docs/2000/108p97-103sheeler/ abstract.html

Address correspondence to S.A. Khan, Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0521 USA. Telephone: (513) 558-7224. Fax: (513) 558-4454. E-mail: sohaib.khan@uc.edu

*Current address: Quintiles Inc., Kansas City, MO 64137.

We are grateful to M.G. Parker (Imperial Cancer Research Fund) for his generous gift of the coding region of RIP140 cDNA in pEF-BOS, T.R. Butt (Gene Transcription Technologies, Inc., Philadelphia, PA) for his generous gift of YEpE12, and H.Wang (University of Cincinnati College of Medicine) for subcloning RIP140 into pPC62. We also thank K.P. Nephew (Indiana University School of Medicine) , R. Morris, M. Kaminski, and E.L. Cardell (University of Cincinnati College of Medicine) for critically reading this manuscript.

This work was supported by funds from NIH/NICHD training grant (T32 HD07463-05) , NIH grant CA-72039-03, and American Cancer Society grant RPG-96-075-03-CNE.

Received 27 April 1999 ; accepted 19 August 1999.