In vitro bioactivity of 17α-estradiol

• Published in: The Journal of steroid biochemistry and molecular biology 2004-12, Vol.92 (5), p.455-463
• Main Authors: Sievernich, André, Wildt, Ludwig, Lichtenberg-Fraté, Hella
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Saccharomyces cerevisiae; Vertebrates: endocrinology
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2004.09.004

A miniaturised short-term in vitro assay based on the activation of the human estrogen receptor α and genetically modified yeast ( Saccharomyces cerevisiae) cells was performed to explore the capacity of this system to monitor the bioactivity of estrogenic compounds, particularly 17α- and 17β-estradiol. Together with the human estrogen receptor (hER)-α plasmid, the reporter plasmid containing a yeast-optimised version of the green fluorescent protein (yEGFP) linked to three repeats of the cis-acting estrogen hormone-responsive element (ERE) were expressed in a strain being deleted in the pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1, known to facilitate efflux of organic compounds including steroids and chemotherapeutics. Agonists that bind to hER in vitro trigger estrogen receptor-mediated transcriptional activation of the GFP reporter gene monitored by fluorescence emission at 535 nm. The sensitivity of the assay was tested with various 17α- and 17β-estradiol concentrations, yielding a detection limit of 5 pg/ml (0.018 nM) for the agonist 17β-E2 in solvent and in human charcoal-stripped serum using a S. cerevisiae pdr5, snq2 and yor1 mutant strain. For 17α-estradiol only, at approximately 1500 pg/ml a similar fluorescence response compared to 100 pg/ml 17β-E2 was observed implicating a much weaker potency of this stereoisomer. The specificity of the system was tested by expression of a truncated hER lacking the ligand-binding domain E and by administration of the androgen, 4-androsten 3,17 dione. Both controls did not yield an increase in fluorescence emission. This fluorescence emission assay enables detection of estrogenic biological activity induced by direct agonists, such as 17β-E2 at concentrations similar to those found in human sera or by estrogen-like chemicals.