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Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.
Citation:
RIDER, C. V., P. C. HARTIG, M. C. CARDON, AND V. S. WILSON. Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems. ENVIRONMENTAL SCIENCE AND TECHNOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 28(10):2175-2181, (2009).
Impact/Purpose:
The Food Quality Protection Act passed in 1996 mandated that the US EPA test pesticides and environmental chemicals for endocrine disrupting potential. Several in vitro assays were proposed to identify whether chemicals could bind to estrogen or androgen receptor. These proposed in vitro assays utilize mammalian receptors. However, it is not clear whether mammalian receptors are appropriate surrogates for other vertebrate classes. In the current study, we compared chemical binding to full length human and fathead minnow recombinant estrogen receptors alpha in two different in vitro binding systems. We found that fathead minnow estrogen receptor alpha (fhERα) was temperature sensitive and lost the ability to bind to 17β-estradiol at 37°C. This has implications for using fhERα in some in vitro assays that require 37°C incubation (transcriptional activation assays in mammalian cell systems). Although only modest binding differences were observed among the two receptors, we found that there was a tendency for some chemicals to have a higher affinity for fhERα than human estrogen receptor alpha (hERα). There was good reproducibility in both the whole cell and cell-free binding assays. Overall, our findings support the use of mammalian receptors in vitro estrogen receptor binding assays and indicate that in vitro assays must be optimized for use with non-mammalian estrogen receptors.
Description:
Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell binding assay and a cell-free binding assay utilizing insect cell lysate infected with receptor expressed in baculovirus. We found that fhERα, and not hERα, displayed a significant (p=0.03) reduction in total ligand binding of 73% at a higher temperature (37°C) as compared to room temperature. Several of the chemicals we tested displayed higher affinity for fhERα than hERα. The phthalates, dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) completely displaced 17β-estradiol from fhERα, but not from hERα (fhERα DBP IC50 = 19.8 μM and BBP IC50 = 8.6 μM). In general, there was good agreement between the whole cell binding assay and the cell-free binding assay; however, in the cell-free assay DBP did not completely displace 17β-estradiol from either receptor. The functional difference between fhERα and hERα in temperature tolerance and modest binding differences warrant further investigation to determine if there are significant differences in the affinity of xenoestrogens for ERα of species from different vertebrate classes.