* Molecular Toxicology, Institute of Toxicology, Merck KGaA, Darmstadt, Germany; Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and
Institute of Food Chemistry and Toxicology, University of Karlsruhe, Karlsruhe, Germany
Received January 9, 2004; accepted April 5, 2004
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ABSTRACT |
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Key Words: ER; ERß; xenoestrogen; endocrine disruptors; Ishikawa; coactivator recruitment.
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INTRODUCTION |
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Most phytoestrogens exert pleiotropic effects involving kinase inhibition, cell cycle regulation, and antioxidative properties that are likely to contribute to the beneficial effects of phytoestrogens (Basly et al., 2000; Cappelletti et al., 2000
). Nevertheless, estrogenic and/or antiestrogenic activities of phytoestrogens like resveratrol and genistein may reduce but also stimulate estrogen-dependent tumor growth depending on dose and timing of exposure (Allred et al., 2001
; Bhat et al., 2001
; Cotroneo et al., 2002
).
So, we have analyzed a selection of phytoestrogens (Fig. 1), with known or suspected estrogenic action (reviewed in Cassidy et al., 2000; Mazur and Adlercreutz, 2000
; Mueller, 2002
), in a series of cellular assays for dose-dependent agonistic and antagonistic activity on ER
and ERß. One major drawback of cellular assays is that the cell lines used are incapable of metabolizing xenobiotics. Therefore, we have evaluated some of the major human metabolites of enterolactone and daidzein for their estrogenic activities (Fig. 1; Jacobs et al., 1999
; Niemeyer et al., 2000
). Several reports have been published that characterized selected features of phytoestrogen action including receptor binding, ER transactivation, or target gene expression (reviewed in Mueller, 2002
). However, a comprehensive analysis of the potencies of several phytoestrogens in one comparable system for both ER
and ERß activities is lacking. Thus, we have employed a human endometrial Ishikawa cell line that stably expresses human ER
or ERß (Mueller et al., 2003a
) to assess the estrogenic and antiestrogenic potencies of the selected phytoestrogens (Fig. 1). Furthermore, binding affinities for ER
and ERß were analyzed. To elucidate the mechanism of ER activation or inhibition by phytoestrogens in more detail, we evaluated the recruitment of coactivators to ER
and ERß by selected phytoestrogens in mammalian two-hybrid assays. By using this approach, we were able to rank and characterize the analyzed phytoestrogens for their estrogenic and/or antiestrogenic properties in human cells.
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MATERIALS AND METHODS |
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Cell culture. Human endometrial Ishikawa cells with stable expression of human ER (Ishikawa-hER
) or human ERß (Ishikawa-hERß; Mueller et al., 2003a
) were cultured in phenol redfree Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 10% DCC/FBS, as described previously (Mueller et al., 2003a
). Human hepatoma HepG2 cells were maintained in DMEM/F12 supplemented with 10% FBS, sodium pyruvate, and antibiotics. Cells were cultured at 37°C/5% CO2 in air in a humidified atmosphere.
Ligand-binding studies by fluorescence polarization. Ligand-binding affinities to purified human ER and ERß were determined by competition binding of the test compounds against fluorescein-labeled estradiol (ES2) using an ER-screening kit according to the manufacturer's protocol (Panvera, Göttingen, Germany). Recombinant human ER
or ERß (10 nM) were incubated with the test compounds for 2 h in the presence of 1 nM ES2 (Panvera). Fluorescence polarization was measured using a Polarion multiwell plate reader (Tecan, Crailsheim, Germany). Ligand concentration yielding 50% inhibition of binding of fluorescently labeled ES2 to ER (IC50) was derived by nonlinear curve-fitting using ORIGIN software (Microcal Software, Northhampton, MA) from competition binding curves. The IC50 were determined from at least three independent experiments and are given as mean ± standard deviation.
Transient transfection and transactivation assay. Ishikawa-hER and Ishikawa-hERß cells were seeded on 24-well plates 15 h prior to transfection in phenol redfree DMEM/F12 supplemented with 10% DCC/FBS. The plasmids were transfected in phenol redfree DMEM/F12 supplemented with 5% DCC/FBS using Fugene 6 (Roche, Mannheim, Germany), according to the manufacturer's protocol. Each well received 0.5 µg of reporter plasmid and 0.01 µg pRL-CMV (Renilla luciferase for normalization; Promega, Madison, WI). A firefly luciferase reporter driven by three copies of the vitellogenin estrogen response element (3 x ERE-Luc) and a reporter containing the human complement 3 gene (C3) promoter (reporter kindly provided by D. P. McDonnell, Duke University, Durham, NC) were used to measure ER transcriptional activity. Test compounds (final concentration of vehicle ethanol 1% v/v) in 200 µl phenol redfree DMEM/F12 supplemented with 10% DCC/FBS were added to the cells after transfection and incubated for 22 h. Luciferase assays were performed using the dual-luciferase reporter assay system according to the manufacturer's protocol (Promega). Each value was normalized to the Renilla luciferase control and each data point obtained represents the average of duplicate determinations. All experiments were repeated at least three times. EC50 values (ligand concentration yielding half-maximal activation) or IC50 values (ligand concentration yielding half-maximal inhibition of DES-mediated activity) were derived by nonlinear curve-fitting using ORIGIN software (Microcal Software) from transactivation curves and are given as mean ± standard deviation of at least three independent experiments.
Mammalian two-hybrid assays. For mammalian two-hybrid assays, HepG2 cells were plated in 24-well plates 15 h prior to transfection. The plasmids were transfected in phenol redfree DMEM/F12 supplemented with 5% DCC/FBS using Fugene 6 (Roche), as described previously (Mueller et al., 2003a). In standard transfections, 0.5 µg of reporter 5 x -Gal4-TATA-Luc, containing five binding sites for the yeast Gal4 transcription factor, 0.09 µg of receptor (either pVP16-hER
[GenBank accession M12674] or pVP16-hERß [GenBank accession AF051427, "short" form of ERß]), 0.5 µg of Gal4 DNA binding domain fused to the nuclear receptor interaction domain (NR-box) of each coactivator (pM-SRC-1 [NR box] or pM-GRIP1 [NR box]; plasmids kindly provided by D. P. McDonnell), and 0.01 µg of the pRL-CMV Renilla luciferase normalization vector were used for each well. Test compounds in 200 µl phenol redfree DMEM/F12 supplemented with 10% DCC/FBS were then added to the cells and incubated for 22 h. Luciferase assays were performed using the dual-luciferase reporter assay system according to the manufacturer's protocol. Each value was normalized to the Renilla luciferase control, and each data point generated is the average with range of duplicate determinations. All experiments were performed three times.
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RESULTS |
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The dose-response transactivation curves of the tested phytoestrogens on the 3 x ERE reporter are shown in Figure 2. For comparison of the estrogenic potencies of phytoestrogens with that of DES and E2, we calculated the EC50 values for ER transactivation. Also, the efficacy, i.e., the maximum-fold induction over control observed, was also determined (Fig. 3). The overall ranking of the potency was consistent with the observed binding affinities (Table 1). ZEA and COUM were the most potent phytoestrogens on ER, and COUM, GEN, and equol preferentially activated ERß (Fig. 3) in accordance with published reports (reviewed in Mueller, 2002
). In contrast, ZEA had higher potencies on ER
than on ERß (Fig. 3). RESV and ENL showed very low potency to induce ER
or ERß activity; 6OH-ENL and the daidzein metabolites 347-IF and 467-IF displayed the lowest potencies to activate ER
or ERß (Fig. 3).
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Another measure of estrogenic activity is the efficacy. ZEA, COUM, GEN, equol, and RESV had efficacies that were comparable to DES and E2 (Fig. 3). Therefore, these compounds were considered full agonists. ENL and 6OH-ENL showed markedly lower efficacies than DES and were considered partial agonists (Fig. 3). More interestingly, the daidzein metabolites 347-IF and 467-IF showed distinctively higher-fold induction than DES or E2 (Figs. 2 and 3). These high efficacies were observed at doses of 10 µM and higher only. These compounds resembled superagonists but with very weak potencies.
Next to the agonistic activity, the antagonistic activity on ER is important for the characterization of endocrine-active compounds. All phytoestrogens (Fig. 1) were tested for their potency to inhibit DES-induced, ER-mediated activity in Ishikawa-hER and Ishikawa-hERß. As expected, the partial agonists ENL and 6OH-ENL acted as partial antagonists (2040% reduction of DES activity, data not shown). Distinct antagonistic activities were observed for RESV and ZEA only (Fig. 4). All other phytoestrogens tested did not inhibit ER activity at doses up to 100 µM (data not shown). RESV has been identified as a mixed ER
agonist/antagonist (Bhat et al., 2001
; Bowers et al., 2000
). We analyzed in more detail its antagonistic properties with regard to ER
and ERß. In contrast to ZEA that showed dose-dependent antagonistic activity on ER
and ERß, RESV displayed an inverted U-shaped dose response (Fig. 4). At low doses, RESV increased the DES-induced activity of ER
and ERß, but at high doses it inhibited activity of ER
and ERß (Fig. 4). We then compared the antiestrogenic potencies of RESV and ZEA with the pure antiestrogen ICI (Wakeling and Bowler, 1992
). The IC50 values for the inhibition of DES-induced activity are shown in Table 2. ZEA showed a higher antiestrogenic potency than RESV; both RESV and ZEA showed similar potencies on ER
and ERß in the µM range, whereas ICI was antiestrogenic at nM doses (Table 2).
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DISCUSSION |
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In an attempt to provide a comprehensive analysis and potency comparison of the (anti-) estrogenicity of phytoestrogens, we evaluated several abundant phytoestrogens for ER binding, activation/inhibition, and coactivator recruitment in human cells. For this, we used a cell line with defined expression of either human ER or ERß (Mueller et al., 2003a
). Since this cell line lacks activity to metabolize xenobiotics, we also analyzed some major human metabolites. The physiological effects of estrogens are mediated predominantly by ER
(Couse and Korach, 1999
; Mueller and Korach, 2001a
), whereas ERß may counteract ER
action (Hall and McDonnell, 1999
; Liu et al., 2002
) and has an important role at least in ovarian development (Couse and Korach 1999
; Couse et al., 1999
). Therefore, it is crucial for the evaluation of the (anti)-estrogenicity of phytoestrogens to analyze both ER
and ERß. Kuiper and colleagues were the first ones to compare several xenoestrogens for ER
versus ERß binding and activity (Kuiper et al., 1998
). However, no study has yet compared the potencies and characteristics of various phytoestrogens and their metabolites for ER
and ERß activity in a comprehensive manner.
Table 3 summarizes our results on the estrogenic characteristics of the phytoestrogens tested. In the former study, GEN and COUM displayed a preference for ERß (Kuiper et al., 1998). We confirmed that GEN, COUM, and equol have a distinctively higher binding affinity but only slight preference for transactivation of ERß compared to ER
. However, the physiological relevance, if any, for this ERß propensity remains elusive. The potencies of these phytoestrogens were 10- to 100-fold less than that of DES. In contrast, ZEA was the most potent phytoestrogen tested and activated preferentially ER
. All other phytoestrogens tested including RESV and the daidzein and ENL metabolites were weak to very weak ER agonists (Table 3). The daidzein metabolites 347-IF and 467-IF exhibited an interesting and rather surprising feature. Both isoflavones were very weak ER agonists, eliciting no activity up to a dose of 1 µM, but were superagonists compared to DES at doses of 10 to 100 µM. The pure antiestrogen ICI completely blocked 347-IF and 467-IFinduced activity (data not shown), indicating that the measured ER superagonism is due to ligand-dependent activation.
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Beneficial effects on estrogen-related diseases are often attributed to antiestrogenic activities of phytoestrogens (Bingham et al., 1998). Analysis of antagonistic activity on ER
and ERß revealed that only RESV and ZEA displayed apparent inhibitory properties on both ER
and ERß transactivation. The partial agonists ENL and 6OH-ENL were also partial antagonists in accordance with basic pharmacological principles. Bowers and colleagues reported that RESV showed slight antagonistic properties on ER
but not ERß depending on the ERE sequence (Bowers et al., 2000
). We observed an increase of DES-induced activity on the consensus ERE by both ER
and ERß at doses up to 10 µM for RESV. At 100 µM, RESV repressed activity of both ER
and ERß. The apparently contrasting results observed with ERß are likely due to the lower maximum dose of 50 µM tested in the former study and/or the use of different ERE sequences (Bowers et al., 2000
). The ER
antagonistic properties of RESV were confirmed in mammary tumor models as well as in Ishikawa cells with endogenous ER
expression (Bhat et al., 2001
; Bhat and Pezzuto, 2001
). Taken together, the published reports and our data support that RESV has the potential to act as a mixed agonist/antagonist in an ERE- and dose-dependent fashion, and this may, next to its nonestrogenic effects (Dong, 2003
), account for RESV's beneficial effects. ZEA was a more potent antiestrogen than RESV and lacked any additive agonistic effects with DES on ER
and ERß, indicating its potential to act as a pure ER antagonist at high doses.
Recruitment of coactivators to ER and ERß is a prerequisite for ER action, and ER coactivators are also important determinants for tissue-specific estrogen action, since coregulators show a tissue-specific expression profile (reviewed in Tremblay and Giguere, 2002
). ER ligands regulate recruitment of coactivators by inducing a distinct conformation of the ligand binding and activation domains of the ERs (Paige et al., 1999
). Thus, it is reasonable to assume that a conformational change induced by different phytoestrogens caused the observed distinct transactivation of ER
and ERß (Hall et al., 2002
). We showed that all ER agonists tested enhanced recruitment of coactivators at doses similar to that which induced transactivation. This confirmed that coactivator recruitment is necessary for ER action and that a two-hybrid coactivator assay could serve as surrogate marker for ER activation (Nishikawa et al., 1999
).
Previous studies analyzed coactivator recruitment to ER and ERß in the presence of phytoestrogens using glutathione-S-transferase pull-down or yeast two-hybrid assays (An et al., 2001
; Nishikawa et al., 1999
; Routledge et al., 2000
). These studies showed that ERß-selective phytoestrogens like genistein were more potent to recruit coactivators to ERß compared to ER
. We also observed maximum recruitment of coactivators to ERß at lower doses of GEN and COUM, albeit with lower efficacy, compared to ER
. All the rather potent phytoestrogens we studied enhanced preferentially recruitment of GRIP1 to ER
, confirming the report by Nishikawa and colleagues (1999)
. This phytoestrogen-induced GRIP1 propensity of ER
was similar to that induced by E2, indicating that the molecular mechanism of ER
agonist activity of the phytoestrogens tested is comparable to potent estrogens. In contrast, the weakly potent superagonists 347-IF and 467-IF did not preferentially recruit one coactivator. For ERß, no distinct preference for one coactivator was apparent and the overall association was less pronounced than for ER
. This confirmed the generally observed lower efficacy of ERß compared to ER
regardless of the higher ERß affinity and potency of some phytoestrogens tested. The ER antagonists ZEA and RESV repressed GRIP1 association to ER
to a greater extent than for SRC-1, confirming their agonist profiles. Interestingly, RESV displayed the typical inverted U-shaped dose response for ERß antagonism for SRC-1 association but not for GRIP1. Also, GRIP1 association with ERß was diminished at low, agonistic doses of RESV. Taken together, these findings indicate that SRC-1 rather than GRIP1 may contribute to RESV's weak agonistic activity on ERß at lower doses.
The reported agonistic and antagonistic activity of the phytoestrogens we studied can aid in the evaluation of potential hazards or benefits of these compounds. Enterolactones are highly abundant in flaxseeds and lentils, food more common in a western diet, while isoflavones found in soy and tofu are generally considered part of an eastern diet (Cassidy et al., 2000). Due to their abundance (Cassidy et al., 2000
; Munro et al., 2003
) and potencies, the soy-derived GEN, COUM, the daidzein metabolites equol and the superagonists 347-IF and 467-IF, RESV found mainly in grapes, and the mycotoxin ZEA seem to be relevant as far as potential risk/benefit of their (anti)-estrogenic effects. The isoflavones showed all pure ER agonistic activity. Thus, these compounds should be regarded as potentially estrogenic and, consequently, as potential endocrine disruptors that may cause elevated cell proliferation leading to estrogen-dependent tumor promotion (Allred et al., 2001
; Cotroneo et al., 2002
; Hilakivi-Clarke et al., 1999a
; Newbold et al., 2001
) and that may also induce adverse developmental effects (Delclos et al., 2001
). The beneficial effects associated with soy intake are likely due in part to non-ER-mediated effects as described above. But, with regard to the adverse and beneficial ER-mediated effects, the timing of exposure is important (Bouker and Hilakivi-Clarke, 2000
; Cotroneo et al., 2002
). In contrast, RESV's very weak agonistic properties together with its antagonistic properties are likely to be major contributors to the beneficial effects attributed to RESV (Bhat et al., 2001
). ZEA might act as an endocrine disruptor at low doses, but its ER antagonistic activity at high doses might contribute to the observed reduction of mammary tumors (Hilakivi-Clarke et al., 1999b
). Next to the direct effects on ER activity that were analyzed in this study, phytoestrogens might also affect the formation of endogenous estrogens like 17ß-estradiol. One well-characterized and potential target that regulates estradiol formation is aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Indeed, indirect evidence for antiestrogenic effects due to inhibition of the formation of 17ß-estradiol has been provided for biochanin A but not genistein at low doses (Almstrup et al., 2002
).
In conclusion, the risks and benefits of estrogenic or antiestrogenic effects depend highly on the target tissue as well as the timing and level of exposure. These latter two factors along with further research on the potential tissue-specific effects of phytoestrogens should aid in the assessment of the real risks and benefits of phytoestrogen-containing diets.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Institute of Toxicology, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany. Fax: + 49. E-mail: stefan.o.mueller{at}merck.de.
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