Assessment of xenoestrogen recognition by recombinant human estrogen receptors using ligand titration arrays

Endocrine disrupting compounds and xenoestrogens have emerged as a major public health issue due to their potentially disruptive effects through direct interaction with steroid hormone receptors. Determination of the extent of estrogen mimicry by compounds encountered in the environment is essential to estimate risk/benefit ratios. A novel biosensor (Endotech(TM)) was developed with laser-based fiber optics using fluorescent dye-labeled (Cy5) recombinant human estrogen receptor-alpha (hERalpha) as a probe (1-3). hERalpha was expressed in Saccharomyces cervisiae as ubiquitin fusion under control of a CUP1 promoter (4-5) and partially purified with heparin affinity chromatography in the unliganded state. Commercially available recombinant estrogen receptor-beta (hERbeta) was processed similarly. A ligand binding array was developed to evaluate candidate compounds, both naturally occurring and synthetic, for estrogen binding activity and affinity (6). Binding affinity of a suspected estrogen mimic was calculated over a range of [3H]estradiol-17beta concentrations, using hERalpha and hERbeta with Lundon OneSiteRTM and CompeteRTM software (Lundon Software, Inc.). beta-zearalenol, one of the ICCVAM validation substances for the in vitro estrogen receptor binding assays and transactivational assays (7) was employed as a model estrogen mimic to illustrate the approach methods and calculations using these techniques. The binding affinities of suspected estrogen mimics can then be used to calibrate the data generated from the biosensor using ligand-based optic fibers.