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and ßbeta; receptors
* IZMB, AG Molekulare Bioenergetik, Universität Bonn, Bonn, Germany; and
Klinik für gynäkologische Endokrinologie und Sterilität, Universität Innsbruck, Innsbruck, Austria
1Correspondence: IZMB, Universität Bonn, Kirschallee 1, Bonn 53115, Germany. E-mail: h.lichtenberg{at}uni-bonn.de
SPECIFIC AIMS
Need and relevance for (anti) estrogenic bioactivity testing
In view of the growing concern about the potential link between certain compounds (natural and synthetic) to observed and suspected adverse effects on reproduction in both wildlife and humans, rapid and reliable high-throughput assays are required to more accurately evaluate the estrogenic effects of a large number of chemicals. In addition, given the current distrust in postmenopausal hormone replacement therapy and the importance for investigating total estrogenic bioactivity of ex vivo samples from women before and under therapy, systems are required to appropriately classify total estrogenicity.
Sensitized recombinant Saccharomyces cerevisiae hosts
The human estrogen receptors 
(hER) and ßbeta; (hERßbeta;), differentially expressed and localized in various tissues and cell types, mediate transcriptional activation of target genes encoding a variety of physiological reproductive and nonreproductive functions involved in energy metabolism, salt balance, immune system, development, and differentiation. As a step toward developing a screening assay for the use in applications where significant numbers of compounds or complex matrices need to be tested for (anti) estrogenic activity, the hER and hERßbeta; receptors were expressed in a genetically modified S. cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and YOR1). Such transport activities have so far received limited attention by other (yeast) assays but may confound attempts to develop bioassays for estrogens or related compounds, since continuous removal of the relevant compound from the cell potentially compromises the sensitivity of any test.
Reporter gene assay: Functional analysis of transcriptional activation
Functional studies were carried out by investigation of receptor-mediated transcriptional activation of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of estrogen responsive elements (ERE). The bioactivity assay was conducted on 17 natural or synthetic compounds. Among the natural compounds were the endogenous estrogens estradiol-17ßbeta; (E2) and estriol (E3); the phyto- or mykoestrogens genistein, coumestrol, and zearalenone; and the synthetic compounds with clinical application ethinylestradiol, 17-estradiol, mestranol, estradiol-3-benzoate, 17-hydroxyprogesterone, norethisteron, tamoxifen, 3-hydroxytamoxifen, and tibolone with its metabolites, or without (current) clinical application as the nonsteroidal estrogen diethylstilbestrol. As example for a complex matrix, the blood serum of two volunteers was investigated for estrogenic bioactivity.
PRINCIPAL FINDINGS
1. Specificity
The specificity of the assay was confirmed by the nil transactivation response on application of the synthetic gestagens 17-hydroxyprogesteron and norethisteron and the ER receptor antagonists tamoxifen and 3-OH-tamoxifen with both the hER and hERßbeta; receptor.
2. Sensitivity
The sensitivity of the assay is expressed by calculated EC50 values as obtained from the fit of the Hill function by nonlinear regression. Evidence for the advantage of the genetically modified (
pdr5, yor2, and snq1) yeast host for the transactivation assay was obtained by an increased sensitivity (3- to 240-fold) compared with the wild type on application of all tested compounds except ethinyl-estradiol and mestranol. Thus most of the tested natural and synthetic compounds appear to be substrates for either or all of Pdr5p, Snq2p, or Yor1p. Side chains like the ethinyl group (ethinyl-estradiol and mestranol) and the additional 3-methyl-ether of mestranol may render them poor efflux substrates enabling cellular uptake.
In humans and other mammalians, the response of different organs, tissues, and cell types on estradiol is dependent on the differential expression of the two estradiol receptors (and coactivators) and their intrinsic (binding) properties. Subtle differences were observed between the hER and ßbeta; receptor mediated responses for the endogenous hormones E2 and E3 for which in this assay approximately twofold different and opposed EC50 values with 0.22 vs. 0.41 nM (E2) and 3.46 vs. 1.87 nM (E3) for hER and hERßbeta;, respectively, were determined. Obtained values were in good agreement with other yeast assays and appeared considerably more sensitive than receptor binding assay results but less sensitive than the E-screen-assay. For the stereo isomer 17-estradiol, generally considered as being devoid of classical biological estrogen activity and only rarely detected in human serum or urine, the threefold lower EC50 (1.7 nM) of hERßbeta; compared with hER (5.5 nM) emphasizes the different receptor affinities.
The most remarkable difference between hER and hERßbeta; was, among the natural compounds, detected for the isoflavone genistein with 17-fold higher sensitivity of hER than hERßbeta; (4.47 vs. 76.4 nM). For another phytoestrogen, coumestrol, related EC50 values (2.5 vs. 3.9 nM) and only for the mycotoxin zearalenone a twofold lower hERßbeta; EC50 value (5.3 vs. 2.3 nM) were determined. Since these data are in tendency comparable with other in vitro data, the notion of greater affinity of the phytoestrogens for hERßbeta; cannot be confirmed, at least by our bioactivity assay. For the synthetic estrogens, except ßbeta;-estradiol-3-benzoate, hERßbeta; was found more sensitive whereby the strongest difference was observed for DES (12-fold higher sensitivity, 0.07 nM) followed by mestranol (5-fold, 2.8 nM) and 17-ethinylestradiol (2.7-fold, 0.2 nM). ßbeta;-Estradiol-3-benzoate, which itself does not bind to the receptor, requires metabolic conversion. The rather low EC50 value of 
3.9 nM for both receptors is indicative of metabolic conversion by yeast esterases. Except for genistein all compounds tested in this assay displayed lower relative potencies (to E2) with the hER than with the hERßbeta;. Taken together, such spectrum of estrogenic activity may characterize the estrogen receptor subtypes, potentially indicative for the structural differences within the C-terminal ligand binding (59% homology) domain.
3. Tibolone and its metabolites
As synthetic steroids, the selective tissue estrogenic activity regulator (STEAR) tibolone and its 3- and 3ßbeta;-hydroxyl-metabolites were tested. All three compounds induced similar maximum transactivation responses with both the hER- and ßbeta;-receptors up to 50,000 pg/ml. Interestingly, approximately twofold differences in sensitivity of hERßbeta; over hER were observed for 3-OH-tibolone (EC50 values of 45.4 vs. 88.7 nM) and 3ßbeta;-OH-tibolone (61.6 vs. 108.4 nM). Tibolone (EC50 value of 105.1 nM) and the 3ßbeta;-OH metabolite (108.4 nM) induced similar responses with hER. With hERßbeta;, the tibolone EC50 (34 nM) was approximately threefold lower compared withthe hER EC50 and twice as low as with the 3ßbeta;-OH metabolite (61.6 nM) indicative of lower efficacy of this metabolite. Since no specific 3-/3ßbeta;-hydroxysteroid dehydrogenase activity has yet been reported in yeast and kinetic fluorescence development analysis (1 and 15 min interval resolution) revealed almost identical inductions for tibolone and the 3-OH/3ßbeta;-OH-metabolites, we hypothesized that conversion into the proposed active derivatives is not necessary to exert transcriptional activation in the yeast assay. Tibolone and its metabolites exhibited clear estrogenic effects, though considerably less potent than all other natural (500 fold lower than those of E2 for hER and 100-fold lower for hERßbeta;) and synthetic compounds.
4. Ex vivo samples
To compare human serum total estrogenic bioactivity with E2 content, parallel investigations were performed. Estrogenic bioactivity was determined with our S. cerevisiae reporter gene assay on complex human serum samples (premenopausal) using a E2 calibration in charcoal-stripped serum and by standard receptor immune determination. Sample 1 corresponded to day 19 of the menstrual cycle (luteal phase) and was determined as of 239 pg/ml E2 [5.9 pg/ml follicle-stimulating hormone (FSH)] by receptor immune assay, sample 2 corresponded to day 4 of the cycle (follicular phase) and was determined as of 46 pg/ml E2 (15.1 pg/ml FSH). In contrast, with the use of our yeast assay, for sample 1 total estrogenic bioactivity was calculated to correspond to 650 pg/ml E2 and for sample 2 to 610 pg/ml E2, based on triplicate results.
5. Subcellular localization
Subcellular localization of the GFP-tagged hER in the yeast cells revealed an even cytosolic fluorescence pattern as could be expected for multicopy, constitutive CUP promoter driven expression of the fusion protein, indicative of 1) no predominant retrograde transport or premature degradation of the heterologously expressed human hER cDNA, and 2) correct yeast chaperone molecular interaction necessary to mediate the fit of hormones to the binding pocket and production of complexes to exert the transactivation effects.
CONCLUSIONS AND SIGNIFICANCE
Human ER and ERßbeta; mediate the physiological effects of both endogenous and synthetic estrogens. The addressed basic biological question concerns the specificity and sensitivity of a S. cerevisiae based estrogenic bioactivity assay. Since yeast do not contain endogenous steroid receptors, the indicator strains expressing the full functional hERs enable quantification of both the DNA binding and transcriptional activation function because the receptors are estrogen-induced and bind their own response element. We provided evidence for the advantage of xenobiotic transport sensitized yeast hosts, which may have an effect on investigating pharmaceutical compounds. In this regard, the most remarkable result was that tibolone exerted its effects to the same extent as its 3- and 3ßbeta;-OH metabolites. This is (at least in our view) the first time that an in vitro assay reveals this result and is in contrast to the belief that only the main metabolites exert estrogenic effects, which may have further implications in related research or in clinical investigations related to hormone replacement therapy.
We could empirically demonstrate compound specific differences in the hER and hERßbeta; receptor mediated reporter responses, and our investigations may thus support the available data pool for characterization of the hERßbeta;, which has received considerable attention in view of further cardiovascular implications.
The potential role of the yeast test in routine laboratory diagnostics was investigated by correlation of total estrogenic bioactivity and E2 content in human serum samples. The E2 levels well reflected the luteal and follicular phases of the menstrual cycle. However, the equivalent total estrogenic bioactivities at both time points were 2.7 and 13.2 times higher, respectively, and with only little variation. Whether such high and rather constant estrogenicity is common remains to be determined. Clinically, such information may be useful in the future both for monitoring total estrogenicity of women before and within postmenopausal hormone replacement therapy, which in return bear the potential to improve true replacement and prevent adverse effects due to the commonly applied pharmaceutical treatment and for pre/post endocrinological disease treatment.
The simple and robust assay protocol together with a higher throughput and lower compound consumption can provide an effective (anti) estrogenic bioactivity screening system for routine testing.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5413fje
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