Human Estrogen Receptors-α & β as Probes in a Biosensor for Detecting Therapeutic and Environmental Estrogen-Mimics

Estrogens are the focus of health concerns currently, and a multitude of naturallyoccurring and synthetic compounds may exhibit endocrine-disrupting activities. We and IA, Inc. (Ann Arbor, MI) developed an evanescent fiber optic sensor (Patent No. US6,300,082B1) for screening compounds for estrogen-like activity, utilizing CY5- labeled recombinant human estrogen receptors (hER) as probes. The hypothesis is that receptor isoforms hERα and hERβ may be used to distinguish diverse estrogen mimics by their ligand binding specificities. hERα and hERβ were incubated independently with [3 H]estradiol-17β in the presence of increasing concentrations of an unlabeled putative estrogen mimic using a titration array (Raffelsberger & Wittliff, J Clin Ligand Assay 18:211, 1995). Results were analyzed for specific binding capacity (pmol/mg protein), apparent dissociation constant (Kd, M), IC50 and Ki values. Using [3H]estradiol-17β, hERα exhibited Kd = 2-6 x 10-10 M compared to hERβ, which gave Kd = 1-7 x 10-9 M indicating a lower affinity for this potent, naturally-occurring estrogen. Of 22 compounds studied, zearalenone and α-zearalenol associated with hERα and hERβ with similar affinities. However, hERβ bound the phytoestrogens, daidzein (Kd = 2-6 x 10-9 M) and genistein (Kd = 1-5 x 10-9 M), with 10 to 20-fold higher affinities than observed for hERα. Biochanin A was recognized by hERβ (Kd = 2-3 x 10-7 M) with low affinity but not by hERα. Ethynylestradiol, bound to hERα with a 8-fold greater affinity (Kd = 3-7 X 10-10 M) than hERβ. Nafoxidine also recognized hERα with a higher affinity than hERβ. Tamoxifen recognized hERα and hERβ similarly with affinities of 1-6 x 10-8 M, while 4- hydroxytamoxifen also bound to hERα and hERβ with similar affinities 2-8 x 10-8 M. These results and others indicate that hERα and hERβ isoforms may be employed as biosensor probes to distinguish estrogen mimics with a broad range of affinities