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NATURE OF BINDING INTERACTION OF SELECTED CHEMICALS WITH RAT ESTROGEN RECEPTORS
Citation:
ELDRIDGE, J. C., S. YAVANHXAY, R. L. COOPER, AND S. C. LAWS. NATURE OF BINDING INTERACTION OF SELECTED CHEMICALS WITH RAT ESTROGEN RECEPTORS. Presented at Society of Toxicology, San Diego, CA, March 05 - 09, 2006.
Description:
The US EPA is currently validating a rat uterine estrogen receptor (ER) binding assay as part of the Tier 1 Screening Battery for the Endocrine Disruptor Program. An eventual goal is to use interactive data to create computerized structure-activity models. However, more information is needed regarding true competitive interaction at the ER hormone binding domain (HBD), particularly for chemicals with very poor affinity for ER and/or poor solubility in the aqueous assay system. For the present studies, batch cytosols were prepared from 12-15 uteri of ovariectomized Harlan S-D rats, and stored @ -80? in small aliquots. Thawed aliquots were pooled for each assay and diluted to a protein conc of ~ 3 mg/ml. Assay buffer was 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, pH 7.4. Tracer was 3H-estradiol, 170 Ci/mmol. Bound and free were separated by dextran-charcoal. Chemicals were selected by the EPA and were assayed at concentrations up to 0.1 mMolar. Results fell into 3 groups: Group A, dislacing 0-20% tracer; Group B, displacing 20-60% tracer; Group C, displacing > 60% tracer. For Group C compounds, re-analysis of competitive properties by a series of Lineweaver-Burk (L-B) plots revealed that nearly all were true competitive inhibitors at the HBD. For Group B, L-B plots succeeded for some and failed for others. For Group A, re-analysis with L-B plots was impossible because an IC-50 could not be observed at the solubility limit. In summary, true competitive inhibition of ER binding can be observed for some compounds, even when the EC-50 is as much as 5 orders of magnitude higher than that of estradiol. Importantly, L-B plot analyses revealed that some tracer-displacing compounds were not competitive at the HBD. Still other compounds could not demonstrate competition due to poor solubility, thus pointing to a need to modify competitive assay conditions. Supported by US EPA, 3D-5967-NTEX. This abstract does not necessarily reflect EPA policy.