The FASEB Journal • FJ Express Full-length Article

Estrogenic effects of natural and synthetic compoundsincluding tibolone assessed in Saccharomyces cerevisiaeexpressing the human estrogen � and � receptors

Guido Hasenbrink,* Andre Sievernich,* Ludwig Wildt,† Jost Ludwig,*and Hella Lichtenberg-Frate*,1

*IZMB, AG Molekulare Bioenergetik, Universitat Bonn, Bonn, Germany; and †Klinik furgynakologische Endokrinologie und Sterilitat, Universitat Innsbruck, Innsbruck, Austria

ABSTRACT The human estrogen receptors (hER)�and hER�, differentially expressed and localized invarious tissues and cell types, mediate transcriptionalactivation of target genes. These encode a variety ofphysiological reproductive and nonreproductive func-tions involved in energy metabolism, salt balance, im-mune system, development, and differentiation. As a steptoward developing a screening assay for the use in appli-cations where significant numbers of compounds or com-plex matrices need to be tested for (anti) estrogenicbioactivity, hER� and hER� were expressed in a geneti-cally modified Saccharomyces cerevisiae strain, devoid ofthree endogenous xenobiotic transporters (PDR5, SNQ2,and YOR1). By using receptor-mediated transcriptionalactivation of the green fluorescent protein optimized forexpression in yeast (yEGFP) as reporter 17 natural, com-prising estrogens and phytoestrogens or synthetic com-pounds among which tibolone with its metabolites, gesta-gens, and antiestrogens were investigated. The reporterassay deployed a simple and robust protocol for the rapiddetection of estrogenic effects within a 96-well microplateformat. Results were expressed as effective concentra-tions (EC50) and correlated to other yeast based and cellline assays. Tibolone and its metabolites exerted clearestrogenic effects, though considerably less potent thanall other natural and synthetic compounds. For the bloodserum of two volunteers, considerable higher total estro-genic bioactivity than single estradiol concentrations asdetermined by immunoassay was found. Visualization of ahER�/GFP fusion protein in yeast revealed a sub cellularcytosolic localization. This study demonstrates the versa-tility of (anti) estrogenic bioactivity determination usingsensitized S. cerevisiae cells to assess estrogenic exposureand effects.—Hasenbrink, G., Sievernich, A., Wildt, L.,Ludwig, J., and Lichtenberg-Frate, H. Estrogenic effectsof natural and synthetic compunds including tiboloneassessed in Saccharomyces cerevisiae expressing the humanestrogen � and � receptors. FASEB J. 20, E861–E870 (2006)

Key Words: S. cerevisiae in vitro assay � hER� � hER�

Estrogens are an important class of signaling mole-cules, regulating a diverse range of physiological pro-

cesses in animals and humans. The actions of thesehormones are mediated by intracellular receptor pro-teins (ERs) that on dimerization act as ligand-activatedtranscription factors by binding of the DNA-bindingdomain (DBD) to specific DNA sequences [estrogenresponsive elements (ERE)] in the regulatory regionsof target gene promoters. Both quality (which genes)and quantity (degree) of transcriptional stimulation ina certain cell depend on the kind and concentration ofthe activated estrogen receptor(s) and several complexregulating factors, e.g., coactivators. The human estro-gen receptors � (hER�) and � (hER�) belong to thesteroid-thyroid-retinoic acid super family of nuclearreceptors. The hER� receptor was identified a decadeago (1) and found to be expressed in the same tissuesas hER� except in the liver (hER�) and the gastroin-testinal tract (hER�). However, the two receptors areoften localized to different cell types though het-erodimers of ER� and ER� have been found in vitro(2). Receptor-mediated gene regulation also involvesligand dependent interaction with other nuclear factorssuch as activating protein-1 with, however, differentialactivation of the two receptors (3). Together with struc-tural, such as N-terminal and ligand-binding pocket do-main, differences between hER� and hER�, these func-tional differences suggested a potential significant role forER� in brain (4) and cardiovascular functions amongother important physiological control systems.

Saccharomyces cerevisiae cells do not contain membersof the steroid-thyroid-retinoic acid receptor super fam-ily but have been used to study respective receptorexpression (5) and structure-function relationships(6–8) and for the identification of signaling pathwaycomponents (9–11). Such approaches used that byexpression of the receptor proteins and on ligandexposure the receptor-mediated signaling can be re-constituted and measured using a reporter gene drivenby DNA responsive elements. In the absence of hor-mone, steroid receptors are found in a complex withmolecular chaperones, such as Hsp90, in the cytoplasm

1Correspondence: IZMB, Universitat Bonn, Kirschallee 1,Bonn 53115, Germany. E-mail: h.lichtenberg@uni-bonn.de

doi: 10.1096/fj.05-5413fje

E8610892-6638/06/0020-0861 © FASEB

or nucleus (12). Yeast Hsp82 and YDF1 as counterpartsof mammalian molecular chaperones function to main-tain steroid receptors in a hormone-binding conforma-tion and to assist activation of the downstream pathway(10). The introduction of S. cerevisiae based screens forestrogenic activity (13–18) has also demonstrated theapplicability of this type of assay in such diverse disci-plines as environmental monitoring and analysis offood components. Besides classical hormone testingsuch assays served to detect estrogenic activity of natu-ral (e.g., phytoestrogens and synthetic compounds, thelatter widely referred as to xenoestrogens among theendocrine disruptors) (19–23). Many of the yeast assaysdeploy the Escherichia coli lacZ reporter connected totwo or three copies of the DNA estrogen responsiveelement to determine estrogenic activity. More re-cently, utilization of a yeast-optimized green fluores-cent protein (yEGFP; 24) fused to the cis-acting EREtook advantage of the direct fluorescence read outopportunity (17, 18) and thus avoided cell lysis proce-dures.

Previously, we (18) have described the constructionand preliminary characterization of the hER�-receptorERE-GFP-reporter test system in S. cerevisiae. Here, wereport on the extension of the transactivation assaytoward hER�-receptor expression coupled to the ERE-GFP-reporter in a genetically modified yeast strain,devoid of three endogenous xenobiotic transporters(PDR5, SNQ2, and YOR1), and comparative analysis ofnatural and synthetic (anti) estrogenic compounds.Among the natural compounds were the endogenousestrogens estradiol-17� (E2) and estriol (E3); the phyto-or mykoestrogens genistein, coumestrol, andzearalenone; and the synthetic compounds with clinicalapplication ethinylestradiol, 17�-estradiol, mestranol,estradiol-3-benzoate, 17�-hydroxyprogesterone, nore-thisteron, tamoxifen, 3-hydroxytamoxifen, and tibo-lone with its metabolites, or without (current) clinicalapplication as the nonsteroidal estrogen diethylstilbes-trol. Transcriptionally activated ERE-GFP fluorescenceemissions were normalized to cell growth determinedby absorption and correlated to internal referencestandards. Obtained dose-response curves served forEC50 value calculation. Assay protocol optimizationcomprised conditions like agitation, temperature, andpH value to determine the most practical test handlingconditions. Finally, the blood serum of two volunteerswas also investigated under the optimized test condi-

tions revealing significant differences between the es-trogenic bioactivity and E2 concentrations as deter-mined by standard methods.

MATERIALS AND METHODS

Plasmids and yeast strains

The plasmid pcDNA3-hER�, harboring the human beta re-ceptor, was kindly provided by Dr. K. Korach (NationalInstitutes of Health). For expression in S. cerevisiae, the hER�receptor was excised as 1508 bp BamHI-XhoI fragment withthe protruding XhoI end filled with T4-DNA polymerase (MBIFermentas) and gel purified. The episomal vector backbonewas the high copy Escherichia coli/yeast shuttle vectorpYEX-BX (Clontech, Palo Alto, CA) in which the URA cassettewas replaced with the TRP selection marker. After EcoRIlinearization, filling in of the protruding end and subsequentBamHI digest both fragments were ligated to yield the 8.8 kbpYEX-hER� plasmid. Plasmid pERE-CYC1-GFP was describedbefore (18). Plasmid YEpE12 (hER�) was a gift from Dr. A.Jungbauer (University of Vienna). For microscopic visualiza-tion, the complete 2012 bp ub-ER fragment was amplifiedfrom the YEpE12 DNA template by polymerase chain reaction(PCR) with Pwo polymerase (Novagen) using primers corre-sponding to the nucleotides 5�-gagaggatccgaattcattatgca-gatcttcg-3� and 5�-gagaactagttcagactgtggcagggaaacc-3� contain-ing primer-encoded BamHI and SpeI restriction sites. Thefragment was then ligated to the BglII and SpeI restriction sitesof the modified pYEX-GFP to yield the fusion constructpYEX-GFP/hER�.

For the SNQ2 gene deletion cassette, the pUG6 plasmidwith the kanamycin marker gene (25) was used as template.For the deletion cassettes of the S. cerevisiae YOR1 and PDR5genes, the pUG6 plasmid (25) was modified by exchange ofthe kanamycin with the LEU2 marker. The isopropyl-malatedehydogenase gene was amplified with S. cerevisiae wild-type(S288C) genomic DNA as template by PCR using the oligo-nucleotides 5�-gagaagatctgagttcgaatctcttagcaacc-3� and 5�-gagagagctccaaattaggaatcgtagtttcatg-3� with primer encoded Bg-lII and SacI restriction sites. The PCR fragment was cleavedwith BglII/SacI and ligated with the BglII/SacI-digested pUG6to yield plasmid pUG6(LEU). PDR5 and YOR1 gene replace-ment cassettes were PCR generated comprising the LEU2(PDR5 and YOR1) or kanamycin resistance (SNQ2) geneflanked by �500 bp gene specific homologous 5�- and 3�-targeting regions. The oligonucleotides used for the SNQ2,YOR1, and PDR5 gene replacement cassettes are listed inTable 1. All gene replacement cassettes were subcloned topBSK (SmaI). All recombinant plasmids recovered from trans-formed E. coli XL1-blue cells were mapped by restrictionanalysis and confirmed by sequencing (GeneART). Com-

TABLE 1. Oligonucleotides

Oligonucleotide Sequence (5´-3´)

PvuII-PDR5 5´Target-s gagacagctgcttctacgccgtggtacgatatcSalI-PDR5 5´Target-as gagagtcgacttgtctaaagtctttcgaacgagcgSpeI-PDR5 3´Target-s gagaactagtgaattttgaatttggttaagaaaagaaacSacII-PDR5 3´Target-as gagaccgcggctatatccattgcgtcctttctttttaPvuII-YOR1 5´Target-s gagacagctgccgcgaggcggaaatggcacaSalI-YOR1 5´Target-as gagagtcgacattcgtatatagcaacggctttactcSpeI-YOR1 3´Target-s gagaactagttttatattatttgttgcatgatttttctcSacII-YOR1 3´Target-as gagaccgcggctctttaaacaagaatggctttttcc

E862 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal

puter analysis of nucleotide and amino acid sequences wasperformed using the Vnti software (Informax).

The yeast strains used throughout this study are summa-rized in Table 2. The S. cerevisiae pdr5 snq2 yor1 disruptionstrain was derived from PLY232 (26), using the Cre-loxP-recombination system described by Guldener et al. (25),except that for PDR5 and YOR1 the LEU2 marker was used forreplacement. After yeast transformation, in LEU2 positive orG-418 (200 mg/l) resistant single cell derived colonies theORF::loxP-kanMX/LEU2-loxP introduced markers were res-cued on expression of the Cre recombinase with plasmidpSH47. Final pSH47 plasmid loss was achieved by growth onYPD medium for 3 days. The eventually obtained triple S.cerevisiae pdr5 snq2 yor 1 mutant strain served as host for expres-sion of the hER�-ERE-GFP, hER�-ERE-GFP, and pYEX-GFP/hER� plasmids. The plasmids were obtained by standard DNAmanipulations according to Sambrook et al. (27) and used totransform S. cerevisiae pdr5 snq2 yor1 cells to tryptophan, uracil,and leucine prototrophy by standard methods (28).

Media and growth conditions

All yeast cells were grown aerobically at 30°C. Nutritionalrequirements appropriate for selection and maintenance ofmutants and plasmids in the transformed strain were scoredon either liquid complete synthetic SDAP medium (29) plus0.5% d-glucose or minimal YNB media consisting of 0.67%yeast nitrogen base (YNB) with (NH4)2SO4, amino acids, and0.5% d-glucose without uracil, tryptophan, and leucine (thelatter to use the LEU2-d function of the pYEX-BX vector toincrease for plasmid copy numbers) adjusted to pH 6.4 or 4.5with NaOH and, where indicated, buffered with 50 mM citricacid monohydrate.

Assay conditions and fluorescence monitoring

For quantitative assessment of growth phenotypes and fluo-rescence development, logarithmic growing cells (70% bud-ding) were diluted to a start optical density (OD)600 of 0.4(Pharmacia Ultrospec 2000 Spectrophotometer) correspond-ing to 3.25 � 106 cells/ml. For each tested compound, at leastthree tests were carried out on different days. Each experimentconsisted of six replica test cultures with respective controls andminimum four test concentrations. Test compounds were dis-solved in DMSO and added to the test cultures of yeast strainstransformed with the respective expression-reporter system in atotal volume of 200 �l. The final concentration of the DMSOsolvent did not exceed 0.5%. For serum samples, 20% charcoal-stripped serum (CSS; provided by Dr. Daxenbichler, Universityof Innsbruck) was used to dissolve E2 as positive control. Thegrowth was estimated by end point OD600 measurements after16.5 h incubation in transparent 96-well microtitre plates usinga microplate reader (Tecan, Spectrofluor Plus). Tests wereconsidered as valid when the turbidity of the control cultures

increased at least fivefold during the incubation period. Forfluorescence development read-outs, the excitation wavelengthwas adjusted to 485 nM and emission was observed at 535 (25nM bandwith).

Data capture and evaluation

After 16.5 h incubation, obtained end point fluorescence(FL) values (corrected for blanks) were divided by growthdetermined as OD 600 (corrected for blanks, OD) for eachreplica well to normalize fluorescence for cell number (FL/OD). To increase the reproducibility of results, the FL/ODvalues obtained for a test compound at a given concentrationwere expressed as fractional values of the maximal responseof a saturating concentration of the reference compound E2,applied in a concentration range from 0–150 pg/ml. Thefractional values (response relative to the maximal E2(Rrel�O) at a given concentration of test compound (ct) werecalculated according to Rrel�O(ct) � (FL/OD(ct)�bottom)/(top�bottom), with “top” corresponding to the fitted FL/ODat saturating E2 concentration and “bottom” to the fittedFL/OD for the negative (solvent) control. The top andbottom values were obtained by Hill equation fit:

y(x) � bottom �(top�bottom)

110(LEC50�x)*hill_slope with y(x) � FL/OD

at the actual compound concentration, x � the decadicallogarithm of compound concentration, LEC50 � decadicallogarithm of EC50, top � fitted maximal FL/OD at saturatingconcentrations, bottom � fitted maximal FL/OD of negativecontrol and hill_slope as the hill steepness parameter to theFL/OD values for each E2 concentration using the R functionnls (The R Foundation for Statistical Computing, http://www.r-project.org/). Dose-response data for test compoundswere obtained from the Rrel�O(ct) for each test compoundusing the analogous fitting algorithm. EC50, EC20, and EC90values were calculated from the fitted dose-response curves,confidence intervals were determined using the R functionconfint. Relative potencies (E2�1) were determined as ratio ofEC50 of E2 and the compound EC50.

Fluorescence microscopy

Microscopic images were obtained with a Leica TCS 4Dconfocal microscope (Leica, Wetzlar, Germany) with an 40 �1.4 oil objective. The excitation source was an argon/kryptonlaser providing spectral lines for excitation at 488 nM (GFP)and 568 nM [nuclear stain with propidium iodide (PI)].Fluorescence emission was filtered by a 530 nM band passfilter for GFP and a 590 nM long pass filter for PI.

RESULTS

A triply PDR5, SNQ2, and YOR1 deleted S. cerevisiaeyeast strain was used as host for the heterologous

TABLE 2. Haploid yeast strains

Genotype Source

PLY232 MATa his3-200 leu2–3,112 trp1-901 ura3–52 suc2-9

Bertl et al., 2003

SCTD-10/04 MATa his3-200 leu2–3,112 trp1-901 ura3–52 suc2-9 pdr5-1::loxP snq2::loxP yor1–1::loxP

This study

SCTD-10/04 hER� SCTD-10/04 �pERE-CYC1-gfp� �YEpE12� This studySCTD-10/04 hER� truncated SCTD-10/04 �pERE-CYC1-gfp� �YEpE12 1184–1793� Sievernich et al., 2004SCTD-10/04 hERß SCTD-10/04 �pERE-CYC1-gfp� �pYEX-hERß� This studySCTD-10/04 hER�-GFP SCTD-10/04 �pYEX-GFP/hER�� This study

E863YEAST (ANTI) ESTROGEN BIOACTIVITY TESTING

expression of hER� and hER�. The expression de-ployed a two plasmid strategy, the episomal expressionof the individual receptors whereby the receptor cDNAwas set under control of the constitutive CUP1 pro-moter and the episomal expression of the cis-actingestrogen hormone-responsive element (ERE) fused tothe yeast-optimized green fluorescent protein (yEGFP;ref 24). The advantage of using a S. cerevisiae mutantdevoid of the three major transporters conferring pleio-tropic drug resistance and thus inability to efficientlyexport small, hydrophobic molecules can be derivedfrom the experiments given in Fig. 1. Wild-type and thetriply deleted mutant S. cerevisiae cells expressing thehER� were challenged with increasing tibolone con-centrations (Fig. 1A). The transactivation assay revealeda fivefold increased response and significantly highersensitivity on application of tibolone (up to 50,000pg/ml) with the genetically modified host. With wild-type expressed hER� similar observations were ob-tained for 3�-OH-tibolone (3-fold decrease in sensitiv-ity), the phyto- and mykoestrogens (6- to 240-folddecrease, Fig. 1B), and for E3 with the EC50 value of67.8 vs. 3.46 nM (�20-fold) in comparison to themutant. A minor difference was observed for estradiol-3-benzoate with the EC50 value of 9.0 nM (wild-type) vs.3.98 nM (mutant) and no difference for ethinyl-estra-diol and mestranol (Fig. 1C).

For visual inspection of subcellular localization anddistribution, the hER�-receptor was N terminally fused toGFP. The multicopy GFP tagged hER� was then investi-gated in S. cerevisiae pdr5 snq2 yor1 cells (Fig. 2) revealingan evenly cytosolic distributed fluorescence labeling ex-cept for the nucleus. No accumulated vacuolar labelingwas observed, indicative of stable receptor fusion proteinsnot subjected to premature vacuolar degradation.

Test conditions

For dose-response curves, fluorescence readouts wereobtained by measuring emission at 535 nM and nor-malization to cell growth on 16.5 h compound expo-sure in liquid media. Thereby, possible strain-specificand compound-induced altered growth characteristicswith variations of maximum cell density in the station-ary phase were taken into account, enabling consistentcurve fits. Blank and negative control cultures served tocorrect for the accumulation of oxidized flavines in thelate growth phase. Test validity criteria of at leastfivefold increase in turbidity with distinguishable fluo-rescence emission were investigated by different E2concentrations on hER� in complete synthetic SDAP-and YNB-minimal medium (data not shown). Compar-ative data set evaluation revealed the latter as mostsuitable, which was used for all further investigations.Two different external pH values were investigated, pH4.5 as the optimal yeast growth condition and pH 6.4 asrelated to complex, native (e.g., environmental origin orblood serum) samples. E2 exposure at pH 4.5, 6.4, and in50 mM citric-acid buffered YNB growth medium (pH 6.4)did not alter the steroid induced response (data not

Figure 1. A) Dose responses of S. cerevisiae wild-type (E) and pdr5snq2 yor1 (f) strains expressing the hER� receptor and ERE-GFP reporter constructs on exposure to increasing tiboloneconcentrations. Fluorescence emission was measured (excita-tion at 485 nM) at 535 nM and normalized to OD (OD600).Tibolone exerted its effects much stronger in the triply PDRtransporter deleted strain resulting in a 5-fold increase inmaximum transactivation response. B, C) Dose–response curvefits of S. cerevisiae wild type expressing hER�. B) For genistein(�, EC50 of 1.1 �M), coumestrol (▫, EC50 of 29.3 nM) andzearalenone (Œ, EC50 of 33.4 nM) EC50 values of mutant wereconsiderably lower with 4.5, 2.5, and 5.3 nM, respectively. C) Forethinyl-estradiol (�, EC50 of 0.25 nM) and mestranol (�, EC50of 7.3 nM) PDR-mediated efflux appeared not relevant, wild-type responded within a comparable concentration range as themutant (cf. also Table 3, Fig. 4). Values are mean of quadruplicatesamples of 6 (A) and 4 (B, C) independent experiments se.

E864 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal

shown), the latter was thus routinely adopted. In contrast,by investigation of incubation temperature and cultureshaking (room temperature: 25°C vs. 30°C with andwithout shaking) conditions, constant shaking of themicroplates turned out to be an essential factor. Routineapplication may thus be performed at room temperatureor 30°C whereby incubation shaking is indispensable.

Comparison of compound mediated (anti) estrogenicbioactivity on hER� and hER�

Several natural and synthetic compounds were ana-lyzed for their (anti) estrogenic bioactivity. The sensi-tivity of the hER�- and �-bioactivity assay, representedby the calculated EC50 values as obtained from the fit of

the Hill function by nonlinear regression, is given incomparison to other test systems in Table 3. Among thenatural hormonal compounds for E2 as perfect ligand,EC50 values of 0.22 nM and 0.41 nM with the hER�- and�-receptor, respectively, were determined. The stereoisomer 17�-estradiol, previously shown as of 15-foldweaker potency compared with E2 with the hER�-receptor (18), induced a similar weak response with thehER�-receptor (Fig. 3 and Table 3). For the secondmain E2 metabolite E3, different sensitivities with EC50values of 3.46 and 1.87 were determined for the hER�-and �-receptor, respectively.

Among the plant secondary metabolites for genisteinas isoflavone, EC50 values of 4.47 vs. 76.4 nM wereobtained with hER� and hER�, respectively. Coumes-trol as a member of the coumestanes was comparedwith the Fusarium spp. produced mycotoxin zearalenone,and only small differences in EC50 values between thereceptors were observed (Table 3). Of the synthetichormones, 17�-ethinylestradiol exhibited the strongestestrogenic bioactivity, followed by diethylstilbestrol(DES), �-estradiol-3-benzoate, mestranol, and tiboloneand its derivatives with the hER�-receptor. Interest-ingly, the hER�-receptor exhibited a fivefold highersensitivity to mestranol in comparison to hER� withEC50 values of 2.85 vs. 15.8 (Fig. 4). The syntheticgestagens 17�-hydroxyprogesterone and norethisterondid with neither the hER� nor the hER� inducetranscriptional activation of the fluorophore; corre-spondingly, no EC values could be determined as forthe ER-receptor antagonists tamoxifen and 3-hy-droxytamoxifen. All experiments were paralleled by thenonfunctional hER control strain (SCTD-10/04, trun-cated hER�; ref. 18) lacking most of the C-terminalligand binding domain E and not responding to com-pound induced transactivation (data not shown).

As the synthetic steroid, the selective tissue estro-genic activity regulator (STEAR) tibolone and its 3�-and 3�-OH metabolites were tested with the yeast assay.As shown in Fig. 5 all three compounds, the originsubstance as well as the active metabolites inducedsimilar transactivation responses with both the hER�-and �-receptors. EC50 values between 88.4 nM for3�-hydroxyl-tibolone and �107 nM for both tiboloneand 3�-hydroxyl-tibolone with hER� were however inmarked contrast to approximately threefold lower EC50values of tibolone (34 nM) and 3�-hydroxyl-tibolone(45.4 nM) with hER� (Table 3). The EC50 value for3�-hydroxyl-tibolone was with 61.6 nM approximatelytwofold higher than for tibolone. The relative potenciesof tibolone and its metabolites were �500-fold lowerthan E2 for hER� and �100-fold lower for hER�.

Ex vivo samples

To compare human serum total estrogenic bioactivitywith E2 content, parallel investigations were performed.Estrogenic bioactivity was determined with the S. cerevi-siae pdr5 snq2 yor1 hER�/ERE-GFP indicator strain assaycontaining 50% complex human serum samples (pre-

Figure 2. Subcellular localization and distribution of gfp-taggedhER�. Upper panel) S. cerevisiae pdr5 snq2 yor1 expressing multi-copy plasmid pYEX-GFP/hER� with even cytosolic fluorescencedistribution except nucleus, as shown in by DNA PI stain (middlepanel) and, the overlay image (lower panel) indicative for cyto-plasmic receptor localization and no accumulation in intracel-lular compartments (retrograde transport) and particular thevacuole as for premature degradation of overexpressed heterol-ogous proteins.

E865YEAST (ANTI) ESTROGEN BIOACTIVITY TESTING

TA

BL

E3.

EC5

0va

lues

test

edan

dre

lativ

epo

tenc

yof

ER-a

goni

stic

com

poun

dsin

com

pari

son

toot

her

dete

ctio

nsy

stem

s

hE

R�

hE

Rß

Oth

erSt

udie

s

Tra

nsa

ctiv

atio

nas

says

Cel

llin

esor

rece

ptor

bin

din

g

Cla

ssC

ompo

und

EC

50

(nM

)E

C20–

EC

90

Rel

ativ

epo

ten

cyh

ER

�E

C50

(nM

)

EC

20-

EC

90

(nM

)

Rel

ativ

epo

ten

cyh

ER

ßh

ER

�EC

50

(nM

)h

ER

ßEC

50

(nM

)E

-sre

en-a

ssay

EC50

(nM

)R

B(h

ER

�)

EC50

(nM

)R

B(h

ER

ß)EC

50(n

M)

Nat

ural

estr

ogen

17ß-

estr

adio

l0.

221

0.41

0.08

–5.

321

0.44

0.

15(3

2)

0.06

–0.2

5(33)

0.00

53

0.00

05(3

2)

7.4

1.

3(32)

5.6

0.

6(32)

0.5–

1.0(3

3)

0.00

5(34)

3.5(3

4)

65(3

4)

0.45

(16)

17�

-est

radi

ol5.

50.

041.

710.

56–

10.1

0.24

7(33)

2.8(3

3)

Est

riol

3.46

0.06

1.87

0.52

–10

.40.

2212

0(3

3)

12(3

3)

0.07

(33)

50(3

3)

250(3

3)

1(16)

Nat

ural

phyt

oes

trog

enG

enis

tein

4.47

0.04

976

.45.

94–

1950

0.00

530.

002(3

3)

8(33)

40(3

2)

35�

10(3

32)

2�

10(3

32)

900(1

6)

Cou

mes

trol

2.49

0.89

–12

.70.

093.

951.

00–

35.2

0.1

0.01

4(33)

3(33)

45(3

4)

3�

10(3

34)

3�

10(3

34)

(2.3

1–2.

70)

9.6(1

6)

Nat

ural

myk

oes

trog

enZ

eara

len

one

5.28

(4.0

8–9.

30)

1.93

–26

.10.

042

2.30

0.63

–17

.70.

180.

013(3

3)

20(3

3)

29(1

6)

Syn

thet

ices

trog

ens

Phar

mac

euti

cals

17�

-eth

inyl

es-

trad

iol

0.54

0.18

–3.

170.

410.

200.

043

–2.

282.

050.

56

0.08

(32)

0.12

(33)

0.01

1(32)

2.4

0.

3(32)

1.1

0.

4(32)

0.5(3

3)

0.00

4(34)

3(34)

45(3

4)

0.17

(16)

Mes

tran

ol15

.80.

014

2.85

1.03

–14

.20.

149.

1(33)

100(3

3)

ß-es

trad

iol-3

-be

nzo

ate

3.98

1.04

–36

.20.

055

3.95

1.02

–34

.70.

170

(33)

30(3

3)

Tib

olon

e10

5.1

0.00

2134

.0(2

8.2–

41.2

)12

.4–

168.

90.

012

8.7(3

7)

7.0(3

7)

190(3

7)

475(3

7)

3-al

pha-

OH

-T

ibol

one

88.7

(71.

0–11

4.9)

32.2

–43

7.0

0.00

2445

.421

.0–

153.

60.

009

0.6(3

7)

10.6

(37)

38(3

7)

158(3

7)

3-be

ta-O

H-T

ibol

one

108.

435

.7–

631.

10.

0020

61.6

20.7

–34

7.0

0.00

670.

7(37)

21.1

(37)

50(3

7)

950(3

7)

Syn

thet

ices

trog

enD

ieth

ylst

il-be

stro

l0.

90(0

.83–

0.97

)0.

32–

4.61

0.24

0.07

5.85

8.0

2.

2(32

)0.

06(3

3)

0.07

5(32)

39

5(32)

51(3

2)

0.6(3

3)

0.00

2(34)

2(34)

50(3

4)

Syn

thet

icge

stag

en17

�-h

ydro

xypr

o-ge

ster

one

n.d

.n

.d.

-n

.d.

n.d

.-

Nor

eth

iste

ron

n.d

.n

.d.

-n

.d.

n.d

.-

Syn

thet

ican

tago

nis

tT

amox

ifen

n.d

.n

.d.

-n

.d.

n.d

.-

200

–30

0(16)

700(3

4)

1.5(3

4)

1.5(3

4)

3-h

ydro

xyta

mo-

xife

nn

.d.

n.d

.-

n.d

.n

.d.

-15

0(34)

1.2

�10

(34)

n.d

.�

No

resp

onse

dete

cted

,n

umbe

rsin

pare

nth

eses

belo

wth

eE

C50

valu

esre

fer

to95

%co

nfi

den

cein

terv

als.

(32)

Sch

ulti

san

dM

etzg

er;

(33)

Bov

eeet

al.;

(16)

Jun

gbau

eran

dB

eck;

(34)

Gut

endo

rfan

dW

este

ndo

rf,

(37)

deG

ooye

ret

al.,

wh

ere

num

bers

corr

espo

nd

tore

fere

nce

s.

E866 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal

menopausal) against a E2 calibration in 20% CSS andby standard receptor immune determination. Sample 1corresponded to day 19 of the menstrual cycle (lutealphase) and was determined as 239 pg/ml E2 (5.9 pg/mlFSH) by receptor immune assay. Sample 2 corre-sponded to day 4 of the cycle (follicular phase), withreceptor immune assay determined as 46 pg/ml E2(15.1 pg/ml FSH). In contrast, for sample 1 totalestrogenic bioactivity was, based on triplicate results,calculated to correspond to 650 pg/ml E2 and forsample 2 to correspond 610 pg/ml E2.

DISCUSSION

Current investigations focused on sensitized S. cerevisiaestrains expressing either the hER� or hER� receptor

and pERE-GFP reporter plasmids to identify and assess(anti) estrogenic bioactivity. Since yeast do not containendogenous steroid receptors, the indicator strainsexpressing the full functional hERs enable quantifica-tion of both the DNA binding and transcriptionalactivation function because the receptors are estrogen-induced and bind their own response element. Investi-gations comprised both sensitivity and specificity of thesystem. Implementation of a S. cerevisiae host straindevoid of three endogenous xenobiotic-transportingplasma membrane ATPases enhanced the sensitivity forsteroids (Fig. 1) except ethinyl-estradiol and mestranol(Fig. 1C). Thus most of the tested natural and syntheticcompounds appear to be substrates for either or all ofPdr5p, Snq2p, or Yor1p. However, for ethinyl-estradioland mestranol, access to the cell interior appears to benot limited by PDR export. Since all tested compounds

Figure 3. Dose-response curve fit of S. cerevisiaepdr5,snq2,yor1 hER� receptor and ERE-gfp reporter express-ing strain by 17�-estradiol (E) and E2 (f) induction. Thealpha stereoisomer is of considerable weaker potency to thehER� receptor with 0.24 relative potency compared with theperfect ligand E2 (cf. also Table 3). Calculation of fractionalresponses was performed as described in Materials and Meth-ods. Values are mean of quadruplicate samples of 8 indepen-dent experiments se.

Figure 4. Dose-response curve fit of mestranol on hER�mediated transactivation. As pharmaceutical steroid mestra-nol ranked with an EC50 value of 4900 pg/ml medium amongthe synthetic compounds with the hER� (E) and with anEC50 value of 1102 pg/ml in the range of the natural phyto-and mykoestrogenes coumestrol and zearalenone with thehER� (f). Values are mean of 6 equal sample concentrationsin 4 independent experiments se.

Figure 5. Tibolone (f, fitted curveOO) and its 3�- (�, fittedcurve ����) and 3�-OH (E, fitted curve O O O) metabolitesdose-dependent in vitro bioactivity with the hER� (A) andhER� (B) receptors. Tested concentrations (up to 2.5�105

pg/ml) were applied in buffered YNB medium, pH 6.4supplemented with 20% charcoal stripped serum. This upperlimit is �E2 lower than recommended daily dose of 2.5mg/50 kg. The parent compound, tibolone exerted with bothreceptors similar maximum transactivation responses as me-tabolites. Values are mean of quadruplicate (hER�) andduplicate (hER�) samples of eight and six independentexperiments se.

E867YEAST (ANTI) ESTROGEN BIOACTIVITY TESTING

are between 260 and 320 kDa and zearalenone aslargest is clearly a substrate for the PDRs (Fig. 1B), sizealone may not be the relevant substrate determiningparameter. Side chains like the ethinyl group (ethinyl-estradiol and mestranol) and the additional 3-methyl-ether of mestranol may render them poor efflux sub-strates and enable cellular uptake. However, generallyincreased sensitivity of the S. cerevisiae mutant may assistadoption of this short term in vitro assay for environ-mental or ex vivo test settings.

Subcellular localization of the GFP-tagged hER� inthe yeast cells revealed an even cytosolic fluorescencepattern similar to the data of Kousteni et al. (30) ascould be expected for multicopy, constitutive CUPpromoter driven expression of the fusion construct(Fig. 2), indicative of 1) no predominant retrogradetransport or premature degradation of the heterolo-gously expressed hER� cDNA, and 2) correct yeastchaperone molecular interaction (10) necessary tomediate the fit of hormones to the binding pocket andproduction of complexes to exert the transactivationeffects.

Applicability

Various incubation conditions were investigated toidentify the most suitable and practical protocol for thedifferent requirements of (e.g., yeast growth, detectionlimits, handling, and robustness). Growth in YNB metthe defined validity criteria as fitness parameter. Highspeed culture shaking (950 rpm) was identified as anessential factor. serving both oxygen supply and evencell distribution within the well and thus eventuallygrowth and, if induced, GFP production. The yeastplasma membrane H-ATPase Pma1 (31) is the majorsource for cytosolic proton extrusion and generation ofthe electrochemical gradient (proton motive force)across the cellular membrane. In logarithmically grow-ing yeast cultures the external pH can thus declinedown to values of 3 or 3.5 in unbuffered media. Sinceno significant differences of E2 induced hER� re-sponses at external pH values of 4.5, 6.4, and buffered6.4 were observed, we conclude the robustness of theassay.

Specificity and sensitivity

The specificity of the assay was confirmed by the niltransactivation response on application of the syntheticgestagens 17�-hydroxyprogesteron and norethisteronand the ER receptor antagonists tamoxifen and 3-OH-tamoxifen with both hER� and hER� (Table 3). Inhumans and other mammalians the response of differ-ent organs, tissues, and cell types on estradiol is depen-dent on the differential expression of the two estradiolreceptors (and coactivators) and their intrinsic (bind-ing) properties. Subtle differences were observed be-tween the hER� and -� mediated responses for theendogenous hormones E2 and E3, for which in thisassay approximately twofold different and opposed

EC50 values with 0.22 vs. 0.41 nM (E2) and 3.46 vs. 1.87nM (E3) for hER� and hER�, respectively, were deter-mined. Obtained values were in good agreement withother yeast assays (16, 32, 33) and appeared consider-ably more sensitive than receptor binding assay results(34) but less sensitive than the E-screen-assay withMCF-7 cells (34). For 17�-estradiol, generally consid-ered as being devoid of classical biological estrogenactivity and only rarely detected in human serum orurine (35), the threefold lower EC50 of the hER�compared with the hER� receptor (Table 3), alsoobserved by Bovee et al. (33), emphasizes the differentreceptor affinities.

The most remarkable difference was among thenatural compounds detected for genistein with 17-foldhigher sensitivity of hER� than hER�, similar to otheryeast and receptor binding tests (33, 34). Since forcoumestrol related and only for zearalenone, twofoldlower hER� EC50 values were determined and thesedata are in tendency comparable to other in vitro data(33, 34 for coumestrol), the notion of greater affinity ofthe phytoestrogens for hER� (36) can, at least by yeasttests, not be confirmed.

Among the synthetic estrogens hER� was, except for�-estradiol-3-benzoate, found more sensitive wherebythe strongest difference was observed for DES (12-foldhigher sensitivity) followed by mestranol (5-fold) and17�-ethinylestradiol (2.7-fold). �-Estradiol-3-benzoate,which itself does not bind to the receptor, requiresmetabolic conversion. The rather low EC50 value of�3.9 nM for both receptors is indicative of metabolicconversion by yeast esterases.

Tibolone and its metabolites

For the STEAR tibolone and its 3�- and 3�-OH metab-olites, clear estrogenic effects, though considerably lesspotent than all other natural and synthetic compounds,were detected. Interestingly, twofold differences insensitivity of hER� over hER� were observed similar toreceptor binding tests (37). In contrast to other in vitrotests (37), tibolone and the 3�-OH metabolite inducedsimilar transactivation activity with hER� but with hER�the tibolone EC50 was twice as low as with the 3�-OHmetabolite (Table 3) indicative of lower efficacy of thismetabolite. The 4-isomer was, due to its androgenicproperties, not tested. The known estrogenic effects oftibolone on brain, bone, and vagina are mediated bythe two major free 3�- and 3�-OH metabolites as aresult of metabolic conversion by 3�- and 3�-hydroxys-teroid dehydrogenase (3�-/3�HSD) conversion of theparent compound (38). 17�- and 20�HSD activity hasbeen reported for S. cerevisiae (39) but not yet for3�-/3�HSD activity. The observed transactivation re-sponse might thus be either due to a so far uncharac-terized specific 3�-/3�HSD activity, unspecific activityof yeast HSDs, or at least partial binding of tibolone tothe estradiol receptors. Kinetic fluorescence develop-ment analysis (1 and 15 min interval resolution, datanot shown) revealed almost identical inductions for

E868 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal

tibolone and the 3�-OH/3�-OH-metabolites, indicativethat conversion into the proposed active derivatives isnot necessary to exert transcriptional activation in theyeast bioactivity assay.

Relative potencies

Based on the relative potencies (Table 3, Fig. 6), aranking of compounds with the hER� was determinedas E2, 17�-ethinylestradiol, DES, coumestrol, E3, �-es-tradiol-3-benzoate, genistein, zearalenone, 17�-estra-diol, mestranol, 3�-OH-tibolone, tibolone, and 3�-OH-tibolone.

For hER�, the compound ranking was DES, 17�-ethinylestradiol, E2, 17�-estradiol, E3, zearalenone,mestranol, coumestrol, �-estradiol-3-benzoate, tibo-lone, 3�-OH-tibolone, 3�-OH-tibolone, and genistein,revealing considerable differences between the recep-tor affinities and thus transactivation activity. Except forgenistein all compounds tested in this assay displayedlower relative potencies (relative to E2) with hER� thanwith hER� (Fig. 6). Taken together, such spectrum ofestrogenic activity may characterize the estrogen recep-tor subtypes, potentially indicative for the structuraldifferences within the C-terminal ligand binding (59%homology) domain.

Ex vivo samples

The potential role of the yeast test in routine laboratorydiagnostics was investigated by correlation of totalestrogenic bioactivity and E2 content in human serumsamples. The E2 levels well reflected the luteal andfollicular phases of the menstrual cycle. However, theequivalent total estrogenic bioactivity at both timepoints was 2.7 and 13.2 times higher, respectively, andwith only little variation. Whether such high and rather

constant estrogenic activity is common remains to bedetermined. Clinically, such information may be usefulin the future both for monitoring total estrogenicity ofwomen before and within menopausal hormone re-placement therapy and pre- and postendocrinologicaldisease treatment.

The results of the functional assay for estrogenicbioactivity, based on a receptor/reporter simple opticalreadout assay, demonstrated the versatility of applying a“simple” single-celled eukaryote for analyses of com-pounds belonging to different chemical classes as wellas ex vivo samples.

The authors thank Dr. H. J. Kloosterboer (N. V. Organon,The Netherlands) for the gift of tibolone and derivatives andcritical reading of the manuscript and B. Kirberg for excellenttechnical assistance. The work was in part funded by EUQLK3-CT-2001–00401.

REFERENCES

1. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., andGustafsson, J. A. (1996) Cloning of a nocel estrogen receptorexpressed in rat prostate and ovary. Proc. Natl. Acad. Sci. U.S.A.93, 5925–5930

2. Pettersson, K., Grandien, K., Kuiper, G. G., and Gustafsson, J. A.(1997) Mouse estrogen receptor beta forms estrogen responseelement-binding heterodimers with estrogen receptor alpha.Mol. Endocrinol. 11, 1486–1496

3. Paech, K., Webb, P., Kuiper, G. G., Nilsson, S., Gustafsson, J.,Kushner, P. J., and Scanlan, T. S. (1997) Differential ligandactivation of estrogen receptors ERalpha and ERbeta at AP1sites. Science 277, 1508–1510

4. Gustafsson, J. A. (1999) Estrogen receptor beta- a new dimen-sion in estrogen mechanism of action. J. Endocrinol. 163, 379–383

5. McEwan, I. J. (1999) Investigation of steroid receptor functionin the budding yeast Saccharomyces cerevisiae. FEMS Microbiol.Lett. 176, 1–9

6. Wrenn, C. K., and Katzenellenbogen, B. S. (1993) Structure-function analysis of the hormone binding domain of the humanestrogen receptor by region-specific mutagenesis and pheno-typic screening in yeast. J. Biol. Chem. 268, 24089–24098

7. Graumann, K., Wittliff, J. L., Raffelsberger, W., Miles, L., Jung-bauer, A., and Butt, T. R. (1996) Structural and functionalanalysis of N-terminal point mutants of the human estrogenreceptor. J. Steroid Biochem. Mol. Biol. 57, 293–300

8. Almlof, T., Gustafsson, J. A., and Wright, A. P. (1997) Role ofhydrophobic amino acid clusters in the transactivation activity ofthe human glucocorticoid receptor. Mol. Cell. Biol. 17, 934–945

9. Kimura, Y., Yahara, I., and Lindquist, S. (1995) Role of theprotein chaperone YDJ1 in establishing Hsp90-mediated signaltransduction pathways. Science 268, 1362–1365

10. Caplan, A. J. (1997) Yeast molecular chaperones and themechanism of steroid hormone action. Trends Endocrinol. Metab.8, 271–276

11. vom Baur, E., Harbers, M., Um, S. J., Benecke, A., Chambon, P.,and Losson, R. (1998) The yeast Ada complex mediates theligand-dependent activation function AF-2 of retinoid X andestrogen receptors. Genes Dev. 12, 1278–1289

12. DeFranco, D. B. (1999) Regulation of steroid receptor subcel-lular trafficking. Cell Biochem. Biophys. 30, 1–24

13. Arnold, S. F., Robinson, M. K., Notides, A. C., Guilette, L. J., andMcLachlan, J. A. (1996) A yeast estrogen screen for examiningthe relative exposure of cells to natural and xenoestrogens.Environ. Health Perspect. 104, 544–548

14. Shiau, P., Glasebrook, A., Hardikar, S. D., Yang, N. N., andHershberger, C. L. (1996) Activation of the human estrogenreceptor by estrogenic and antiestrogenic compounds in Sac-

Figure 6. Relative potencies (REP, relative to E2) of naturalestrogens E2 (f), 17�-estradiol (�), and E3 (f), the syntheticestrogens 17�-ethinylestradiol (}), mestranol ({), �-estradi-ol-3-benzoate (}), tibolone (Œ), 3-alpha-OH-tibolone (‚)and 3-beta-OH-tibolone (Œ) and natural phyto-/mykoestro-gens genistein (�), coumestrol (�) and Zearalenone () forhER� plotted vs. hER�. Dashed line with a slope of 1 indicatesdifferences in relative potency of single compounds betweenhER� and hER�.

E869YEAST (ANTI) ESTROGEN BIOACTIVITY TESTING

charomyces cerevisiae: a positive selection screen. Gene 179,205–210

15. Chen, C. W., Hurd, C., Vorojeikina, D. P., Arnolds, S. F., andNotides, A. C. (1997) Transcriptional activation of the humanestrogen receptor by DDT isomers and metabolites in yeast andMCF-7 cells. Biochem. Pharmacol. 53, 1161–1172

16. Jungbauer, A., and Beck, V. J. (2002) Yeast reporter system forrapid determination of estrogenic activity. Chromatogr. B Analyt.Technol. Biomed. Life Sci. 777, 167–78

17. Bovee, T. F., Helsdingen, R. J., Koks, P. D., Kuiper, H. A.,Hoogenboom, R. L., and Keijer, J. (2004) Development of arapid yeast estrogen bioassay, based on the expression of greenfluorescent protein. Gene 325, 187–200

18. Sievernich, A., Wildt, L., and Lichtenberg-Frate, H. (2004) Invitro bioactivity of 17alpha-estradiol. J. Steroid Biochem. Mol. Biol.92, 455–463

19. Graumann, K., Breithofer, A., and Jungbauer, A. (1999) Moni-toring of estrogen mimics by a recombinant yeast assay: synergybetween natural and synthetic compounds? Sci. Total Environ.225, 69–79

20. Payne, J., Rajapakse, N., Wilkins, M., and Kortenkamp, A. (2000)Prediction and assessment of the effects of mixtures of fourxenoestrogens. Environ. Health Perspect. 108, 983–987

21. Agradi, E., Fico, G., Cillo, F., Francisci, C., and Tome, F. (2001)Estrogenic activity of phenolic compounds from Nigella dama-scena evaluated using a recombinant yeast screen. Planta Med.67, 553–555

22. De Boever, P., Demare, W., Vanderperren, E., Cooreman, K.,Bossier, P., and Verstraete, W. (2001) Optimization of a yeastestrogen screen and its applicability to study the release ofestrogenic isoflavones from a soygerm powder. Environ. HealthPerspect. 109, 691–697

23. Witters, H. E., Vangenechten, C., and Berckmans, P. (2001)Detection of estrogenic activity in Flemish surface waters usingan in vitro recombinant assay with yeast cells. Water Sci. Technol.43, 117–123

24. Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene173, 33–38

25. Guldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hege-mann, J. H. (1996) A new efficient gene disruption cassette forrepeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524

26. Bertl, A., Ramos, J., Ludwig, J., Lichtenberg-Frate, H., Reid, J.,Bihler, H., Calero, F., Martinez, P., and Ljungdahl, P. O. (2003)Characterization of potassium transport in wild-type and iso-genic yeast strains carrying all combinations of trk1, trk2 andtok1 null mutations. Mol. Microbiol. 47, 767–780

27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) MolecularCloning, Cold Spring Harbor Laboratory Press, New York

28. Rothstein, R. (1991) Targeting, disruption, replacement, andallele rescue: Integrative DNA transformation in yeast. In:

Methods in Enzymology (Guthrie, C., and Fink, G.R., eds) vol. 194,pp. 281–302, Academic Press, New York

29. Rodriguez-Navarro, A., and Ramos, J. (1984) Dual system forpotassium transport in Saccharomyces cerevisiae. J. Bacteriol.159, 940–945

30. Kousteni, S., Bellido, T., Plotkin, L. I., O’Brien, C. A., Bodenner,D. L., Han, L., Han, K., DiGregorio, G. B., Katzenellenbogen,J. A., Katzenellenbogen, B.S., et al. (2001) Nongenotropic,sex-nonspecific signaling through the estrogen or androgenreceptors: dissociation from transcriptional activity. Cell 104,719–30

31. Serrano, R., Kielland-Brandt, M., and Fink, G. R. (1986) Yeastplasma membrane H-ATPase is essential for growth and hashomology with (Na-K)-, K- and Ca2-ATPases. Nature 319,689–693

32. Schultis, T., and Metzger, J. W. (2004) Determination ofestrogenic activity by LYES-assay (yeast estrogen screen-assayassisted by enzymatic digestion with lyticase). Chemosphere 57,1649 –1655

33. Bovee, T. F., Helsdingen, R. J., Rietjens, I. M., Keijer, J., andHoogenboom, R. L. (2004) Rapid yeast estrogen bioassays stablyexpressing human estrogen receptors alpha and beta, andgreen fluorescent protein: a comparison of different com-pounds with both receptor types. J. Steroid Biochem. Mol. Biol. 91,99–109

34. Gutendorf, B., and Westendorf, J. (2001) Comparison of anarray of in vitro assays for the assessment of the estrogenicpotential of natural and synthetic estrogens, phytoestrogens andxenoestrogens. Toxicology 166, 79–89

35. Hobe, G., Schon, R., Goncharov, N., Katsiya, G., Koryakin, M.,Gesson-Cholat, I., Oettel, M., and Zimmermann, H. (2002)Some new aspects of 17alpha-estradiol in man. Steroids 67,883–893

36. Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe,S. H., van der Saag, P. T., van der Burg, B., and Gustafsson,J. A. (1998) Interaction of estrogenic chemicals and phytoestro-gens with estrogen receptor beta. Endocrinology 139, 4252–4263

37. de Gooyer, M. E., Deckers, G. H., Schoonen, W. G. E. J.,Verheul, H. A. M., and Kloosterboer, H. J. (2003) Receptorprofiling and endocrine interactions of tibolone. Steroids 68,21–30

38. Kloosterboer, H. J. (2004) Tissue-selective effects of tibolone onthe breast. Maturitas 49, 5–15

39. Vico, P., Cauet, G., Rose, K., Lathe, R., and Degryse, E. (2002)Dehydroepiandrosterone (DHEA) metabolism in Saccharomy-ces cerevisiae expressing mammalian steroid hydroxylaseCYP7B: Ayr1p and Fox2p display 17�-hydroxysteroid dehydro-genase activity. Yeast 19, 873–886

Received for publication November 30, 2005.Accepted for publication February 27, 2006.

E870 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal

The FASEB Journal • FJ Express Summary

Estrogenic effects of natural and synthetic compoundsincluding tibolone assessed in Saccharomyces cerevisiaeexpressing the human estrogen � and � receptors

Guido Hasenbrink,* Andre Sievernich,* Ludwig Wildt,† Jost Ludwig,*and Hella Lichtenberg-Frate*,1

*IZMB, AG Molekulare Bioenergetik, Universitat Bonn, Bonn, Germany; and †Klinik furgynakologische Endokrinologie und Sterilitat, Universitat Innsbruck, Innsbruck, Austria

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5413fje

SPECIFIC AIMS

Need and relevance for (anti) estrogenicbioactivity testing

In view of the growing concern about the potential linkbetween certain compounds (natural and synthetic) toobserved and suspected adverse effects on reproduc-tion in both wildlife and humans, rapid and reliablehigh-throughput assays are required to more accuratelyevaluate the estrogenic effects of a large number ofchemicals. In addition, given the current distrust inpostmenopausal hormone replacement therapy andthe importance for investigating total estrogenic bioac-tivity of ex vivo samples from women before and undertherapy, systems are required to appropriately classifytotal estrogenicity.

Sensitized recombinant Saccharomyces cerevisiae hosts

The human estrogen receptors � (hER�) and �(hER�), differentially expressed and localized in vari-ous tissues and cell types, mediate transcriptional acti-vation of target genes encoding a variety of physiolog-ical reproductive and nonreproductive functionsinvolved in energy metabolism, salt balance, immunesystem, development, and differentiation. As a steptoward developing a screening assay for the use inapplications where significant numbers of compoundsor complex matrices need to be tested for (anti)estrogenic activity, the hER� and hER� receptors wereexpressed in a genetically modified S. cerevisiae strain,devoid of three endogenous xenobiotic transporters(PDR5, SNQ2, and YOR1). Such transport activities haveso far received limited attention by other (yeast) assaysbut may confound attempts to develop bioassays forestrogens or related compounds, since continuous re-moval of the relevant compound from the cell poten-tially compromises the sensitivity of any test.

Reporter gene assay: Functional analysis oftranscriptional activation

Functional studies were carried out by investigation ofreceptor-mediated transcriptional activation of thegreen fluorescent protein optimized for expression inyeast (yEGFP3) under control of estrogen responsiveelements (ERE). The bioactivity assay was conductedon 17 natural or synthetic compounds. Among thenatural compounds were the endogenous estrogensestradiol-17� (E2) and estriol (E3); the phyto- ormykoestrogens genistein, coumestrol, and zearalenone;and the synthetic compounds with clinical applicationethinylestradiol, 17�-estradiol, mestranol, estradiol-3-benzoate, 17�-hydroxyprogesterone, norethisteron, ta-moxifen, 3-hydroxytamoxifen, and tibolone with itsmetabolites, or without (current) clinical application asthe nonsteroidal estrogen diethylstilbestrol. As examplefor a complex matrix, the blood serum of two volun-teers was investigated for estrogenic bioactivity.

PRINCIPAL FINDINGS

1. Specificity

The specificity of the assay was confirmed by the niltransactivation response on application of the syntheticgestagens 17�-hydroxyprogesteron and norethisteronand the ER receptor antagonists tamoxifen and 3-OH-tamoxifen with both the hER� and hER� receptor.

2. Sensitivity

The sensitivity of the assay is expressed by calculatedEC50 values as obtained from the fit of the Hill function

1Correspondence: IZMB, Universitat Bonn, Kirschallee 1,Bonn 53115, Germany. E-mail: h.lichtenberg@uni-bonn.de

doi: 10.1096/fj.05-5413fje

1552 0892-6638/06/0020-1552 © FASEB

by nonlinear regression. Evidence for the advantage ofthe genetically modified (�pdr5, yor2, and snq1) yeasthost for the transactivation assay was obtained by anincreased sensitivity (3- to 240-fold) compared with thewild type on application of all tested compounds exceptethinyl-estradiol and mestranol. Thus most of the testednatural and synthetic compounds appear to be sub-strates for either or all of Pdr5p, Snq2p, or Yor1p. Sidechains like the ethinyl group (ethinyl-estradiol andmestranol) and the additional 3-methyl-ether of mestra-nol may render them poor efflux substrates enablingcellular uptake.

In humans and other mammalians, the response ofdifferent organs, tissues, and cell types on estradiol isdependent on the differential expression of the twoestradiol receptors (and coactivators) and their intrin-sic (binding) properties. Subtle differences were ob-served between the hER� and � receptor mediatedresponses for the endogenous hormones E2 and E3 forwhich in this assay approximately twofold different andopposed EC50 values with 0.22 vs. 0.41 nM (E2) and 3.46vs. 1.87 nM (E3) for hER� and hER�, respectively, weredetermined. Obtained values were in good agreementwith other yeast assays and appeared considerably moresensitive than receptor binding assay results but lesssensitive than the E-screen-assay. For the stereo isomer17�-estradiol, generally considered as being devoid ofclassical biological estrogen activity and only rarelydetected in human serum or urine, the threefold lowerEC50 (1.7 nM) of hER� compared with hER� (5.5 nM)emphasizes the different receptor affinities.

The most remarkable difference between hER� andhER� was, among the natural compounds, detected forthe isoflavone genistein with 17-fold higher sensitivityof hER� than hER� (4.47 vs. 76.4 nM). For anotherphytoestrogen, coumestrol, related EC50 values (2.5 vs.3.9 nM) and only for the mycotoxin zearalenone atwofold lower hER� EC50 value (5.3 vs. 2.3 nM) weredetermined. Since these data are in tendency compa-rable with other in vitro data, the notion of greateraffinity of the phytoestrogens for hER� cannot beconfirmed, at least by our bioactivity assay. For thesynthetic estrogens, except �-estradiol-3-benzoate,hER� was found more sensitive whereby the strongestdifference was observed for DES (12-fold higher sensi-tivity, 0.07 nM) followed by mestranol (5-fold, 2.8 nM)and 17�-ethinylestradiol (2.7-fold, 0.2 nM). �-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 ofmetabolic conversion by yeast esterases. Except forgenistein all compounds tested in this assay displayedlower relative potencies (to E2) with the hER� thanwith the hER�. Taken together, such spectrum ofestrogenic activity may characterize the estrogen recep-tor subtypes, potentially indicative for the structuraldifferences within the C-terminal ligand binding (59%homology) domain.

3. Tibolone and its metabolites

As synthetic steroids, the selective tissue estrogenicactivity regulator (STEAR) tibolone and its 3�- and3�-hydroxyl-metabolites were tested. All three com-pounds induced similar maximum transactivation re-sponses with both the hER�- and �-receptors up to50,000 pg/ml. Interestingly, approximately twofold dif-ferences in sensitivity of hER� over hER� were ob-served for 3�-OH-tibolone (EC50 values of 45.4 vs. 88.7nM) and 3�-OH-tibolone (61.6 vs. 108.4 nM). Tibolone(EC50 value of 105.1 nM) and the 3�-OH metabolite(108.4 nM) induced similar responses with hER�. WithhER�, the tibolone EC50 (34 nM) was approximatelythreefold lower compared withthe hER� EC50 and twiceas low as with the 3�-OH metabolite (61.6 nM) indica-tive of lower efficacy of this metabolite. Since nospecific 3�-/3�-hydroxysteroid dehydrogenase activityhas yet been reported in yeast and kinetic fluorescencedevelopment analysis (1 and 15 min interval resolu-tion) revealed almost identical inductions for tiboloneand the 3�-OH/3�-OH-metabolites, we hypothesizedthat conversion into the proposed active derivatives isnot necessary to exert transcriptional activation in theyeast assay. Tibolone and its metabolites exhibited clearestrogenic effects, though considerably less potent thanall other natural (�500 fold lower than those of E2 forhER� and �100-fold lower for hER�) and syntheticcompounds.

4. Ex vivo samples

To compare human serum total estrogenic bioactivitywith E2 content, parallel investigations were performed.Estrogenic bioactivity was determined with our S. cerevi-siae reporter gene assay on complex human serumsamples (premenopausal) using a E2 calibration incharcoal-stripped serum and by standard receptor im-mune determination. Sample 1 corresponded to day 19of the menstrual cycle (luteal phase) and was deter-mined as of 239 pg/ml E2 [5.9 pg/ml follicle-stimulat-ing hormone (FSH)] by receptor immune assay, sam-ple 2 corresponded to day 4 of the cycle (follicularphase) and was determined as of 46 pg/ml E2 (15.1pg/ml FSH). In contrast, with the use of our yeast assay,for sample 1 total estrogenic bioactivity was calculatedto correspond to 650 pg/ml E2 and for sample 2 to 610pg/ml E2, based on triplicate results.

5. Subcellular localization

Subcellular localization of the GFP-tagged hER� in theyeast cells revealed an even cytosolic fluorescence pat-tern as could be expected for multicopy, constitutiveCUP promoter driven expression of the fusion protein,indicative of 1) no predominant retrograde transportor premature degradation of the heterologously ex-pressed human hER� cDNA, and 2) correct yeastchaperone molecular interaction necessary to mediate

1553YEAST (ANTI) ESTROGEN BIOACTIVITY TESTING

the fit of hormones to the binding pocket and produc-tion of complexes to exert the transactivation effects.

CONCLUSIONS AND SIGNIFICANCE

Human ER� and ER� mediate the physiological effectsof both endogenous and synthetic estrogens. The ad-dressed basic biological question concerns the specific-ity and sensitivity of a S. cerevisiae based estrogenicbioactivity assay. Since yeast do not contain endogenoussteroid receptors, the indicator strains expressing thefull functional hERs enable quantification of both theDNA binding and transcriptional activation functionbecause the receptors are estrogen-induced and bindtheir own response element. We provided evidence forthe advantage of xenobiotic transport sensitized yeasthosts, which may have an effect on investigating pharma-ceutical compounds. In this regard, the most remarkableresult was that tibolone exerted its effects to the sameextent as its 3�- and 3�-OH metabolites. This is (at least inour view) the first time that an in vitro assay reveals thisresult and is in contrast to the belief that only the mainmetabolites exert estrogenic effects, which may have fur-ther implications in related research or in clinical investi-gations related to hormone replacement therapy.

We could empirically demonstrate compound spe-

cific differences in the hER� and hER� receptor medi-ated reporter responses, and our investigations maythus support the available data pool for characteriza-tion of the hER�, which has received considerableattention in view of further cardiovascular implications.

The potential role of the yeast test in routine labora-tory diagnostics was investigated by correlation of totalestrogenic bioactivity and E2 content in human serumsamples. The E2 levels well reflected the luteal andfollicular phases of the menstrual cycle. However, theequivalent total estrogenic bioactivities at both timepoints were 2.7 and 13.2 times higher, respectively, andwith only little variation. Whether such high and ratherconstant estrogenicity is common remains to be deter-mined. Clinically, such information may be useful inthe future both for monitoring total estrogenicity ofwomen before and within postmenopausal hormonereplacement therapy, which in return bear the poten-tial to improve true replacement and prevent adverseeffects due to the commonly applied pharmaceuticaltreatment and for pre/post endocrinological diseasetreatment.

The simple and robust assay protocol together with ahigher throughput and lower compound consumptioncan provide an effective (anti) estrogenic bioactivityscreening system for routine testing.

Figure 1. Schematic Diagram.

1554 Vol. 20 July 2006 HASENBRINK ET AL.The FASEB Journal