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Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow
Citation:
U.S. EPA, C. V. RIDER, P. C. HARTIG, M. C. CARDON, AND V. S. WILSON. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow. Presented at Society of Environmental Toxicology and Chemistry Meeting (SETAC), Tampa, FL, November 16 - 20, 2008.
Impact/Purpose:
We have developed a simple 96-well plate estrogen receptor binding assay that is compatible with estrogen receptors contained in baculovirus expression vectors. The binding of potential endocrine disrupting chemicals to estrogen receptors from different species can be directly compared in this in vitro system. This approach has the potential to decrease use of animals. We can use this assay to determine whether mammalian receptors offer adequate surrogates for other vertebrate species in proposed endocrine disruptor screening assays.
Description:
In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach allowed for a limitless supply of receptor without the use of animals. Hormone binding to estrogen receptors from human (hERα), quail (qERα), alligator (aERα), salamander (sERα), and fathead minnow (fhERα) was compared in saturation and competitive binding assays. The 17β-estradiol (E2) dissociation constants (Kd) generated from saturation binding experiments were 0.20 ± 0.024nM for hERα, 0.25 ± 0.042nM for sERα, 0.44 ± 0.039nM for aERα, 0.55 ± 0.061nM for fhERα, and 0.87 ± 0.16nM for qERα. Binding specificity to each of the receptors was evaluated using the steroid hormones E2, dihydrotestosterone (DHT) and corticosterone (C) and the synthetic steroid ethynylestradiol (EE). E2 and EE were strong binders with IC50’s for E2 ranging from 0.65nM with hERα to 1.01nM with sERα and for EE ranging from 0.68nM with sERα to 1.20nM with qERα. As expected, DHT was a weak binder with IC50’s ranging from 7.0μM with qERα to 18.6μM with fhERα and C did not bind any of the estrogen receptors at concentrations up to 100μM. Our future plans include using these competitive binding assays to perform targeted binding comparisons to evaluate and/or corroborate reported differences in chemical binding affinity for estrogen receptors from different species. Funding was provided by the NCSU/EPA Cooperative Training Program CT833235-01-0. Disclaimer: This is an abstract of a proposed presentation and does not necessarily reflect USEPA policy.