ToxSci Advance Access originally published online on August 29, 2006
Toxicological Sciences 2006 94(1):46-56; doi:10.1093/toxsci/kfl092
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Published by Oxford University Press 2006.
Nature of the Binding Interaction for 50 Structurally Diverse Chemicals with Rat Estrogen Receptors
* Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Mail-drop 72, Research Triangle Park, North Carolina 27711
Physiology-Pharmacology Department, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157
Received May 30, 2006; accepted August 22, 2006
This study was conducted to characterize the estrogen receptor (ER)–binding affinities of 50 chemicals selected from among the high production volume chemicals under the U.S. EPA's (U.S. Environmental Protection Agency's) Toxic Substances Control Act inventory. The chemicals were evaluated using the rat uterine cytosolic (RUC) ER-competitive binding assay, with secondary analysis using Lineweaver-Burk plots and slope replots to confirm true competitive inhibition and to determine an experimental Ki. Data from these ER-competitive binding assays represent the types of competitive binding curves that can be obtained when screening chemicals with broad structural diversity. True competitive inhibition was observed in 17 of 50 chemicals. Binding affinities were much lower than that of estradiol (E2) with Ki concentrations ranging from 0.6 to 373µM as compared with that of E2 (0.77nM). Other chemicals that appeared to displace radiolabeled E2 binding to ER were, in fact, found not to be competitive inhibitors in the secondary Ki experiments. These seven chemicals likely altered the stability of the assay by changing the buffer pH, denaturing ER, or disrupting the ER-binding kinetics. Thus, several conditions that may confound interpretation of RUC ER-binding assay data are illustrated. For another group of eight chemicals, neither an IC50 nor Ki could be determined due to solubility constraints. These chemicals exhibited slight (20–40%) inhibition at concentrations of 10–100µM, suggesting that they could be competitors at very high concentrations, yet Ki experiments were not possible as the limit of chemical solubility in the aqueous assay buffer was well above the IC50. An additional 18 of the 50 chemicals were classified as nonbinders because in concentrations up to 100µM they produced essentially no displacement of radiolabeled E2. These results show that although the ER-competitive binding assay is a valuable tool for screening chemicals, secondary tests such as a double reciprocal Lineweaver-Burk experiment with slope replot should be used to confirm true competitive inhibition. This information will be useful for the ongoing validation of the RUC ER-competitive binding assay under the U.S. EPA's Endocrine Disruptor Screening Program, as well as to support research efforts to develop computational models designed to identify chemicals with the ability to bind to ER.
Key Words: estrogen receptors; rat uterine cytosol; environmental chemicals; Ki.