Abstract
A three-tiered approach was developed to determine the influence of a chemically-diverse group of compounds exhibiting estrogen mimicry using recombinant human estrogen receptor (rhER) activity to calibrate a receptor protein-based biosensor. In the initial tier, a ligand competition array was developed to evaluate compounds inhibiting [3H]estradiol-17β binding to rhER. Each of six different concentrations of [3H]estradiol-17β was mixed with increasing concentrations of an unlabeled putative mimic. Each of these mixtures was incubated with a constant amount of rhERα and then receptorbound [[3H]estradiol-17β was measured. This array protocol analyzes ligand binding affinities of hERα with a potential inhibitor over the entire range of receptor protein saturation.
When either hERα or hERβ binds to an estrogenic ligand, the receptor monomer forms both homo- and hetero-dimers. Then the ligand-receptor dimer complex activates transcription by associating with an estrogen response element (ERE), which is a specific DNA sequence located upstream of estrogenresponsive genes. The second tier for ligand evaluation utilized an electrophoretic mobility shift assay (EMSA), which was performed with an ERE sequence labeled with [α[32]P]dATP and incubated with rhER in the presence or absence of unlabeled ligand. ERE-hER complexes were separated by electrophoresis and analyzed using phosphor imaging technology.
To assess biological effects of an estrogen mimic on expression of an ER-target gene, a yeast cell-based bioassay was constructed with recombinant DNA technology using Saccharomyces cerevisiae. Each of these engineered yeast cells contained a rhERα expression plasmid (YEpE12) and a separate reporter plasmid (YRG2) containing an ERE sequence upstream of a β-galactosidase reporter gene. Incubation of these yeast cells with an estrogenic compound allows formation of ligand-hERα complexes, which recognize the ERE sequence regulating β-galactosidase expression. Estrogenic compounds, which were evaluated as calibrators for ligand-based and EREbased biosensors, elicit varying responses in each of the three tiers of the protocol.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
J. L. Wittliff and W. Raffelsberger, Mechanisms of Signal Transduction: Sex Hormones, Their Receptors, and Clinical Utility, J. Clin. Ligand Assay 18(4), 211–235 (1995).
J. L. Wittliff, R. Pasic, K. I. Bland, In: The Breast: Comprehensive Management of Benign and Malignant Diseases, edited by K. I. Bland and E. M. Copeland III (W.B. Saunders Co., Philadelphia, 1998), pp. 458–498.
T. Colburn, C. Clement, Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection (Princeton Sci. Publ. Co., 1992), pp. 403.
B. Gutendorf and J. Westendorf, Comparison of an Array of in vitro Assays for the Assessment of the Estrogenic Potential of Natural and Synthetic Estrogens, Phytoestrogens and Xenoestrogens, Toxicology 166(1–2), 79–89 (2001).
R. H. Smith, W. J. Lemon, J. L. Erb, J. R. Erb-Downward, J. G. Downward, O. E. Ulrich, and J. L. Wittliff, Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor, Clin. Chem. 45(9), 1683–1685 (1999).
E. A. E. Garber, J. L. Erb, J. G. Downward, E. M. Priuska, J. L. Wittliff, W. Feng, J. Magner, and G. L. Larsen, Biosensor, ELISA, and Frog Embryo Teratogenesis Assay: Xenopus (FETAX) Analysis of Water Associated with Frog Malformations in Minnesota, Proc. Soc. Photo-Optical Instrumentation Engineers (SPIE) 4206, 147–158 (2001).
J. L. Erb, E. A. E. Garber, J. G. Downward IV, and E. M. Priuska, Data from an Estrogen Receptor-based Biosensor Correlates with Evidence of Frog Malformation and Demonstrates a Differential Response of hERα & β to Beneficial and Harmful Estrogenic Compounds, In: Proc. 2nd Intl. Conf. Pharmaceuticas & Endocrine Disrupting Chemicals in Water, p. 203–217, Westerville, OH, The National Ground Water Association (2001).
J. L. Wittliff, L. L. Wenz, J. Dong, Z. Nawaz, and T. R. Butt, Expression and Characterization of an Active Estrogen Receptor as a Ubiquitin Fusion Protein from Escherichia coli, J. Biol. Chem. 265(35), 22016–22025 (1990).
K. Graumann, J. L. Wittliff, W. Raffelsberger, L. Miles, A. Jungbauer, and T.R. Butt, Structural and Functional Analysis of N-terminal Point Mutants of the Human Estrogen Receptor, J. Steroid Biochem. Mol. Biol. 57(5–6), 292–300 (1996).
W. Raffelsberger and J. L. Wittliff, A Novel Approach for Comparing Ligand Binding Results from Titration and Competition Analyses to Study Hormone Mimics, J. Clin. Ligand Assay 20(4), 275–280 (1997).
C. R. Lyttle, P. Damian-Matsumura, H. Juul, and T. R. Butt, Human Estrogen Receptor Regulation in a Yeast Model System and Studies on Receptor Agonists and Antagonists, J. Steroid Biochem. Mol. Biol. 42(7), 77–685 (1992).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Springer Science+Business Media, LLC
About this paper
Cite this paper
Andres, S.A. et al. (2008). A Three-tiered Approach for Calibration of a Biosensor to Detect Estrogen Mimics. In: Kang, K.A., Harrison, D.K., Bruley, D.F. (eds) Oxygen Transport to Tissue XXIX. Advances In Experimental Medicine And Biology, vol 614. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-74911-2_34
Download citation
DOI: https://doi.org/10.1007/978-0-387-74911-2_34
Publisher Name: Springer, Boston, MA
Print ISBN: 978-0-387-74910-5
Online ISBN: 978-0-387-74911-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)