622
Chem. Res. Toxicol. 1998, 11, 622-629
Detection of Weak Estrogenic Flavonoids Using a Recombinant Yeast Strain and a Modified MCF7 Cell Proliferation Assay Vibeke Breinholt* and John Christian Larsen Institute of Food Safety and Toxicology, Division of Biochemical and Molecular Toxicology, The Danish Veterinary and Food Administration, Mørkhøj Bygade 19, 2860 Søborg, Denmark Received September 17, 1997
A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids. Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 µM. Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 µM, whereas the remaining flavonoids were devoid of activity. The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A. With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4′-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin. The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17β-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation. Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavoestrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay applicable as a large-scale screening tool for estrogenic compounds.
Introduction Flavonoids are naturally occurring in all plant tissues. They are members of the large flavonoid family which share with steroidal estrogens the ability to bind to the estrogen receptor and mediate transcription of estrogenresponsive genes. Several of the most potent phytoestrogens known belong to the group of isoflavonoids. The most frequently occurring isoflavonoids in food plants are genistein and daidzein. Several studies have shown that isoflavonoids are capable of mediating an estrogenic response in vitro (1-6) as well as in vivo in experimental animals (7-10) and livestock (11-13), thereby interfering with various reproductive parameters. The isoflavonoids, however, are characteristic of only a few edible plants, of which the predominant source is soya beans. Exposure to this group of dietary estrogens is particularly high in most Asian countries. The potential clinical benefits of dietary phytoestrogens have been reported in studies showing that Asian populations, who share a high consumption of soy-based foodstuffs, have markedly lower incidences of breast and prostate cancers (14-16) than, for instance, their American counterparts (16, 17) * To whom all correspondence should be addressed. E-mail:
[email protected]. Fax: +45 33 95 66 96.
who consume fewer soy products. These observations have suggested that dietary estrogens can act as protective factors in hormone-dependent cancers, although the specific mechanism(s) of protection remains to be clarified. The frequency of red flushes and other menopausal symptoms is also substantially lower in, for instance, Japanese women compared to American or Finnish women (18). These observations strongly suggest that phytoestrogens reach the target tissue at sufficiently high concentrations after oral intake of isoflavonoid-rich foodstuffs to exert an estrogenic or estrogen antagonistic effect in humans. In addition to mimicking endogenous estrogens, several of the isoflavonoids are capable of altering various enzymatic processes associated with tumor growth and recurrence, such as inhibition of topoisomerase (19, 20) and protein kinase activity (2123). The proposed mechanisms of protection by isoflavonoids against hormone-dependent cancers might therefore arise not only from estrogen receptor-mediated activities but also from alteration of signal transduction or other estrogen-independent pathways involved in cancer development. Recent data by Miksicek (4, 5) indicate that other members of the flavonoid family, besides the isoflavonoids, exhibit estrogenic properties. The non-isofla-
S0893-228x(97)00170-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/27/1998
Estrogenic Activity of Dietary Flavonoids
vonoid flavonoids with estrogenic activity, designated flavo-estrogens, are widely distributed in edible food plants. The dietary intake of flavonoids, including flavoestrogens, has been estimated to range from 0.1 to 1.1 g/day in the United States (24) and to 25 mg/day in The Netherlands (25); thus the flavo-estrogens potentially contribute significantly to our daily load of estrogenic substances. This might be taken to indicate that the commonly occurring flavo-estrogens, at least in part, account for the protective activity toward hormonedependent cancers, associated with a high intake of vegetables and fruits (26-29). The present study was initiated to systematically examine a large series of plant flavonoids in order to evaluate their estrogenic potentials and determine the structural requirements for their estrogenic activity. A newly developed and highly sensitive recombinant yeast (Saccaromyces cerevisiae) strain (30), in which the human estrogen receptor has been integrated into the genome of the yeast, was used. The estrogenic potential of the isoflavonoids and flavo-estrogens was further assayed in mammalian MCF7 cells, which require estrogens or estrogen-mimicking compounds for growth. Data are presented showing that several commonly occurring flavonoids exhibit estrogenic activity in the same order of magnitude as the well-known isoflavonoid phytoestrogens. The MCF7 cell proliferation assay was improved in the present study by using cellular reduction of the vital dye Alamar Blue as a measure of cell growth and adapting the assay to 96-well microtiter plates.
Experimental Procedures Chemicals. The chemicals used in the present study were obtained from the following sources: Sigma Chemical Co. (St. Louis, MO)smorin, (+)-catechin, taxifolin, hesperetin, phloretin, genistein, 17β-estradiol (17βE2); Aldrich Chemical Co. (Milwaukee, WI)schrysin, apigenin, fisetin, quercetin, (-)-epicatechin, naringenin, caffeic acid, chlorogenic acid, ferulic acid; Apin Chemicals Ltd. (Abingdon, Oxon, U.K.)smyricetin, luteolin, naringin, rutin, kaempferol; Roth (Karlsruhe, FRG)s isorhamnetin, tangeretin, eriodictyol; ICN (Costa Mesa, CA)s daidzein. The compounds were between 95% and 99% pure and used without further purification. The exact concentration of the compound was corrected for in the assay. Cell Cultures, Culture Media, and Supplements. MCF7 cells (batch F-11967) were purchased from the American Type Culture Collection (Rockville, MA). The recombinant yeast strain was provided with permission by Prof. John Sumpter (Brunel University, U.K.), who obtained it from Glaxo Wellcome (Stevenage, Herts, U.K.). Culture medium, antibiotics, serum, microtiter plates, and other cell culture supplements were from Life Technologies (Roskilde, Denmark), except for 96-well fluorescence plates with clear bottoms, which were from Corning Costar Corp. (Cambridge, MA). The yeast growth medium was prepared as described by Routledge and Sumpter (30). Methods. (A) Determination of Estrogenic Activity in the Recombinant Yeast Screen. The yeast screen was conducted as described by Routledge and Sumpter (30). The yeast, in addition to expressing the human estrogen receptor, contains expression plasmids carrying the reporter gene Lac-Z immediately preceded by an estrogen-responsive element (ERE1) placed in a very strong promoter region (PGK promoter). Binding of the receptor-ligand complex to the ERE results in 1 Abbreviations: 17βE2, 17β-estradiol; ERE, estrogen-responsive element; EC50, concentration of compound required to induce 50% of maximum estrogenic effect; AB, Alamar Blue; FCS, fetal calf serum; DES, diethylstilbestrol; SD, standard deviation; CYP, cytochrome P450; PBS, phosphate-buffered saline.
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 623 expression of the reporter gene and subsequent secretion of β-galactosidase into the medium. When present in the medium β-galactosidase metabolizes the substrate, chlorophenol red β-galactopyranoside, into chlorophenol red which exhibits an absorption maximum at 540 nm. Yeast growth was measured as turbidity at 630 nm. The absorbance was measured in a Titretek Multiscan PLUS ELISA reader. An estrogenic substance will thus result in red-coloring of the growth medium. The relative potency of the test compounds was assessed by comparing with the endogenous estrogen 17βE2. The yeast screen is at present one of the most sensitive methods for detecting estrogenic compounds. The natural estrogen 17βE2 was used as standard and tested in concentrations ranging from 1.0 pM to 1.0 nM. A 17βE2 standard curve was obtained from each individual microtiter plate. All test compounds were dissolved in ethanol and tested at concentrations between 0.01 and 350 µM, as saturation of all dose-response curves were observed within this concentration range. The plates were read at days 3, 5, and 7, and all compounds were assayed between 3 and 6 times. The solvent was evaporated off prior to addition of the yeast. After evaporation of the solvent, yeast growth medium was added and the plates were mixed vigorously on a Heidolph Titramax 100 microtiter plate shaker (Struers KEBO Lab, Albertslund, Denmark) for 5 min. [See Routhledge and Sumpter (30) for further details on the yeast screen.] To ensure that the chemicals went completely back into solution, the plates were analyzed under a light microscope to verify that no crystals remained at the bottom of the well. Additionally the concentration in the medium of the two relatively unpolar flavonoids, chrysin and tangeretin, and two of the more polar flavonoids, genistein and myricetin, was assayed in the medium after shaking the plates for 5 min, to verify that the compounds were completely dissolved. The cell protein was precipitated with 4 volumes of methanol followed by centrifugation at 9000g for 5 min; 3 mL of the supernatant was transferred to glass vials and evaporated to dryness in vacuo. The dry residues were reconstituted in 150 µL of 25% acetonitrile containing 1% formic acid, of which 100 µL was injected and analyzed by HPLC on a 30min linear gradient from 25% to 100% acetonitrile using 1% formic acid as the aqueous phase and a flow rate of 1 mL/min. The HPLC analysis was conducted on a Hewlett-Packard 1100 system with diode-array detector (Waldbron, Germany) using a 5-µm Purospher RP-18 column (250 × 4.0 mm; HewlettPackard). Column temperature was kept constant at 35 °C. For all compounds investigated (triplicates) 80-90% of the compounds could be found in the medium (data not shown). (B) Determination of Estrogenic Activity in MCF7 Cells. MCF7 breast cancer cells were grown in RPM1 1640 medium without phenol red in 75-cm2 culture flasks supplemented with 5% fetal calf serum (FCS), 5 mL of MEM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 15 mM HEPES, 10 ng/mL insulin, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells were subcultured every 5 days at a split ratio of 1:3 corresponding to an approximate cell density of 2 × 106 cells/mL. Prior to subculturing, the cell layer was washed twice with phosphate-buffered saline (PBS), treated with 1 mL of trypsin (0.5 g/L) at 37 °C for 1 min, and left at room temperature for 2-3 min or until the cells had detached. FCS medium depleted of steroids (DCC medium) was prepared by heating the serum for 45 min at 56 °C followed by a 45-min incubation, likewise at 56 °C with 1% dextran-coated charcoal under continuous stirring. The serum was distributed into sterile 50-mL polypropylene tubes and centrifuged at 4000g for 15 min at 4 °C. After centrifugation the above procedure was repeated on the supernatant. After the final centrifugation the serum was filter-sterilized through a 0.22-µm Millipore filter, distributed into sterile 50-mL polypropylene tubes, and kept at -20 °C until use. Four days prior to conducting an MCF7 cell proliferation assay, the cells were trypsinized and transferred to a new 75-cm2 culture flask containing 5% DCC medium. For the time-response studies cells were seeded at a density of 20 000 cells/well in 6-well microtiter plates in a total of 2.5 mL.
624 Chem. Res. Toxicol., Vol. 11, No. 6, 1998 In the time-response studies, which were conducted in triplicates for all compounds, a fixed concentration of 1 µM was used for all flavonoids tested and 17βE2 was applied at a single concentration of 10 nM. In this set of experiments the cell number was determined in a Coulter counter after trypsinizing the rinsed cell layer with 500 µL of trypsin (0.5 g/L) followed by dilution of the cell suspension with up to 5 volumes of 5% DCC medium. The medium was renewed every second day after day 3, concurrently with addition of fresh test compound. For the dose-response studies the cells were seeded at 1500 cells/ well in a total volume of 300 µL in 96-well microtiter plates. Exposure to the test compounds for both protocols was initiated 24 h after seeding. All compounds tested were dissolved in ethanol and added every second day in fresh medium. The compounds were tested at concentrations between 0.01 and 350 µM, and 3-6 individual determinations were conducted for each compound. All compounds tested exhibited full dose-response curves within this concentration range. The plates were read every second day until day 9. In the dose-response studies 17βE2 was tested at concentrations between 1.0 pM and 100 nM. The exposure was done in 5% DCC medium without supplementation with insulin. In the dose-response studies the cell number was determined by measuring the reduction of Alamar Blue (AB) as described by the manufacturer (Serotec, Kidlington, U.K.) with modifications. The assay which is based on metabolic reduction of the AB dye into a fluorescent species, which can easily be detected after excitation of the reduced AB dye at 560 nm and subsequent emission at 580 nm, was adapted to 96-well fluorescence plates with clear bottomed wells, which allowed direct reading of the plates without sample transfer. AB reduction was measured every second day, by removal of the growth medium and substituting it with AB-containing medium. Reduction of AB was determined at 2 h after addition of the AB substrate, using a total volume of 300 µL of a 1:20 diluted AB stock solution per well. Fluorescence was measured at a slit width of 5 nm for both excitation and emission using a Perkin-Elmer luminescence spectrophotometer LS 50B, equipped with a microtiter plate reader. The fluorescence intensity was found to be linear for up to 4 h after addition of the AB-containing medium, with cell numbers exceeding 20 000/well (unpublished data) as determined by Coulter counting. Furthermore, AB reduction correlated with the cell number in both treated and nontreated cells, indicating that the test compounds did not alter the metabolic pathways involved in AB reduction (data not shown). The correlation coefficient between cell count and fluorescent intensity was >0.97 for all tested cell concentrations between 500 and 10 000 cells/well, indicating that AB reduction is a very reliable measure for MCF7 cell proliferation. After reading the plates, the AB-containing medium was removed, the cells were rinsed twice with PBS, and fresh DCC medium containing the test compounds was added to the plates. The proliferative activity is presented as arbitrary fluorescence units. Statistical Procedures. Statistical analyses were conducted using SAS version 6.08 (SAS Institute, Inc., Cary, NC). Statistical significance in estrogenic response between compounds was evaluated by one-way analysis of variance. A P value less than 0.01 was used as the level of significance.
Results Twenty-three flavonoids of dietary relevance (Chart 1) were tested in the recombinant yeast assay. Approximately two-thirds of the flavonoids exhibited significant estrogenic activity. The estrogenic potency evaluated on the basis of EC50 values ranged from 0.1 to 102 µM, when assayed in the yeast estrogen screen (Table 1). The remaining compounds tested, catechin, epicatechin, fisetin, taxifolin, eriodictyol, quercetin, luteolin, tangeretin, rutin, and naringin, were devoid of estrogenic activity. A common feature of the most estrogenic compounds
Breinholt and Larsen Table 1. Estrogenic Activity of Flavonoids in the Yeast Screen max A540/630 nma
fold inductionb
17βE2
1.76 (0.07)
12.6
solvent equol naringenin phloretin genistein apigenin daidzein biochanin A kaempferol morin hesperetin myricetin chrysin isorhamnetin
0.14 (0.08) 1.76 (0.08) 1.76 (0.07) 1.70 (0.07) 1.65 (0.08) 1.35 (0.08) 1.32 (0.06) 1.30 (0.06) 0.70 (0.05) 0.73 (0.05) 0.63 (0.10) 0.65 (0.06) 0.35 (0.06) 0.29 (0.06)
1.0 12.6 12.6 12.1 11.8 9.6 9.4 9.3 5.0 5.2 4.5 4.6 2.5 2.1
compound
EC50 (µM)c 2.3 × 10-5 (4.1 × 10-6) 0.1 (0.04)e 0.3 (0.06)e 25.0 (3.61) 0.5 (0.13)f 10.0 (2.08)g 8.5 (0.91)g 9.2 (0.72)g 20.4 (4.16) 97.2 (5.91)i 89.6 (2.72)h,i 98.3 (6.65)i 84.0 (6.03)i 102.0 (2.45)i
% of 17βE2d 100 2.3 × 10-2 7.8 × 10-3 9.4 × 10-5 4.5 × 10-3 2.4 × 10-4 2.8 × 10-4 2.5 × 10-4 1.1 × 10-4 2.4 × 10-5 2.6 × 10-5 2.4 × 10-5 2.8 × 10-5 2.3 × 10-5
a The presented data are means of 3-6 individual experiments. The standard deviation (SD) on the raw data is given in parentheses. Plate readings are from day 5. max A540/630: maximum induction level, measured as peak absorbance, corrected for yeast growth measured at 630 nm. b Fold induction is expressed as the ratio of the maximum A540/630 of the test compound relative to the solvent control (0.14) under the conditions of the assay. c EC50 values are calculated from the linear part of dose-response curves obtained at day 5. 17βE2 and flavonoids were added as a single dose at day 1. d Percentage EC50 of 17βE2 relative to EC50 of test compounds. e Significantly different from the remaining compounds tested (P < 0.01). f Significantly more estrogenic (P < 0.01) than daidzein and biochanin A. g The three compounds are not statistically different from each other (P > 0.01). h Significantly more estrogenic than isorhamnetin (P < 0.01). i Compounds are significantly less estrogenic than the remaining compounds tested.
tested was found to be the presence of a 4′-hydroxyl group at the para-position of the flavonoid B-ring comparable to the 4-position of the B-ring in the open-ringed chalcone phloretin (Chart 1). The most potent flavonoids tested were the flavanone naringenin and the isoflavone equol which exhibited EC50 values of 0.3 and 0.1 µM, respectively, and under the conditions of the assay induced gene transcription in the yeast system by close to 13-fold. For comparison the EC50 of 17βE2 in the present study was estimated to be 0.023 nM. The isoflavone genistein was slightly lower in estrogenic potency than equol with an EC50 of 0.5 µM but significantly more potent than the structurally similar compounds daidzein and biochanin A (P < 0.01) (Table 1). The flavone apigenin, the flavonol kaempferol, and the chalcone phloretin were of even lower potencies, exhibiting EC50 values between 10 and 25 µM. The flavanone hesperetin (3′,5,7-trihydroxy-4′methoxyflavanone) was found to induce a weak estrogenic response in the yeast screen (see Table 1), whereas the structural analogues eriodictyol and taxifolin were devoid of activity (Chart 1, Table 1). Hesperetin, in contrast to eriodictyol and taxifolin, is methoxylated in the 4′-position, similar to biochanin A, which also exhibited significant estrogenic activity. Isorhamnetin, which is methoxylated in the 3′-position, also induced a weak estrogenic effect, although significantly lower (P < 0.01) than observed for hesperetin. This observation suggests that the exact position of the methyl group on the B-ring is important in eliciting an estrogenic response, with the 4′-position being the more favorable. Morin, which has a 2′,4′- hydroxylated B-ring (see Chart 1), was also found to be weakly estrogenic, giving rise to a 5-fold increase in gene expression relative to the control, despite the lack of a p-hydroxyl-substituted B-ring. The two remaining
Estrogenic Activity of Dietary Flavonoids
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 625
Chart 1. Chemical Structures of Flavonoid Compoundsa
a OH, OCH3, and OGLY signify addition of hydroxyl groups, methoxy groups, or sugars to the flavan nucleus. Glu and Rham are abbreviations for glucose and rhamnose, respectively.
compounds that exhibited estrogenic activity in the yeast screen were the two flavones myricetin and chrysin, which have three and zero hydroxyl groups on the B-ring, respectively (Chart 1, Table 1). Representative doseresponse curves for selected flavonoids are given in Figure 1. Characteristic of the majority of the compounds
that were devoid of estrogenic activity was the presence of a catechol moiety on the B-ring, as seen in quercetin, luteolin, fisetin, taxifolin, eriodictyol, catechin, and epicatechin. Tangeretin, which is methoxylated on both the A- and B-rings (see Chart 1), and the glycosides of quercetin and naringenin, named rutin and naringin,
626 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Breinholt and Larsen
Figure 1. Representative dose-response curves for 17βE2 and selected flavonoids in the yeast screen. The data are given as the A540/630 ratio from readings at day 5 following a single estrogen treatment. Each data point represents the mean ( SD of 3-6 individual determinations of the estrogenic response. A: 2, 17βE2; b, naringenin; O, daidzein; 1, morin; 3, chrysin; 9, isorhamnetin. B: 2, 17βE2; b, genistein; O, apigenin; 1, phloretin; 3, kaempferol; 9, hesperetin.
Figure 2. Time-response curves for 17βE2 and selected flavonoids in the MCF7 cell proliferation assay. The concentration of 17βE2 in the medium was 10 nM and the concentration of the flavonoids 1 µM. Both 17βE2 and the flavonoids were added fresh to the medium every second day throughout the experiment. A: 0, 17βE2; 1, apigenin; 3, daidzein; 9, chrysin; O, cells + ethanol; b, cells only. B: 0, 17βE2; 1, genistein; 3, kaempferol; 9, luteolin; O, cells + ethanol; b, cells only. Each data point represents the mean ( SD of triplicate determinations of the cell number.
were also devoid of estrogenic activity. None of the simple phenolics tested, caffeic acid, ferulic acid, or chlorogenic acid, were found to be estrogenic (results not shown). The same set of flavonoid compounds was tested in the mammalian MCF7 cell assay. The cell number was found to increase with time, as shown in Figure 2. Between days 9 and 11 an increased number of detached cells were found floating around in the medium, in both the flavonoid- and the 17βE2-treated cells. The cell numbers given in Figure 2 for day 11 are thus associated with an increased degree of inaccuracy, compared to the other data points. From other time-response studies conducted in this laboratory with MCF7 cells, it has been observed that repetitious trypsination of the cells decreases the ability of the cells to adhere to the bottom of
the wells. The mechanism responsible for this is not known but could be due to inactivation by trypsin of enzymes involved in cell surface attachment, as a small amount of trypsin was retained in the medium after each trysination. Dose-response studies on all the flavonoid compounds were also conducted, to determine the EC50 for the flavonoids in the MCF7 cell proliferation assay. Representative dose-response curves are shown in Figure 3. Seven compounds were found to stimulate cell growth of the estrogen-dependent cell line between 4- and 7-fold under the conditions of the assay (Table 2), with corresponding EC50 values between 0.3 and 1.1 µM. Nine other compounds elicited 3-5-fold increases in cellular proliferation (EC50: 1.3-8.0 µM). The remaining seven compounds, fisetin, quercetin, hesperetin, myricetin,
Estrogenic Activity of Dietary Flavonoids
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 627 Table 2. Estrogenic Activity of Flavonoids in MCF7 Cells max F580 nma
fold inductionb
EC50 (µM)c
17βE2
0.65 (0.05)
8.1
7.7 × 10-5 (7.1 × 10-6)
100
solvent equol naringenin genistein apigenin daidzein biochanin A kaempferol chrysin phloretin morin catechin epicatechin eriodictyol isorhamnetin luteolin taxifolin
0.08 (0.01) 0.44 (0.04) 0.46 (0.07) 0.46 (0.11) 0.53 (0.06) 0.30 (0.06) 0.37 (0.07) 0.38 (0.06) 0.25 (0.05) 0.17 (0.05) 0.15 (0.04) 0.19 (0.02) 0.20 (0.02) 0.22 (0.07) 0.15 (0.04) 0.25 (0.05) 0.20 (0.07)
1.0 5.5 5.8 5.8 6.6 3.8 4.6 4.8 3.1 2.1 1.9 2.4 2.5 2.8 2.8 3.1 2.5
0.6 (0.30)e 1.0 (0.32)e 0.3 (0.16)f 0.5 (0.09)f 0.7 (0.11)e 0.7 (0.14)e 1.1 (0.30)e 3.2 (0.74)g 3.1 (0.71)g 8.0 (1.17)h 4.0 (0.97)g 3.0 (0.70)g 5.6 (0.27)h 4.2 (0.65)g 1.3 (0.17)h 4.2 (0.76)g
1.3 × 10-2 7.7 × 10-3 2.6 × 10-2 1.5 × 10-2 1.1 × 10-2 1.1 × 10-2 7.0 × 10-3 2.4 × 10-3 2.5 × 10-3 9.6 × 10-4 1.9 × 10-3 2.6 × 10-3 1.5 × 10-3 1.8 × 10-3 5.9 × 10-3 1.8 × 10-3
compound
Figure 3. Dose-response curves for selected flavonoids in the MCF7 cell proliferation assay. The degree of cell proliferation is given as arbitrary fluorescence units (see text for more details). The presented plate readings are from day 5: b, genistein; 0, apigenin; O, naringenin; 1, chrysin; 9, quercetin; 3, morin. The drop in the response curve for quercetin and genistein at high doses is due to cell toxicity (see text for further details). Each data point represents the mean ( SD of triplicate determinations of the cell-mediated reduction of AB.
tangeretin, naringin, and rutin, did not give rise to any proliferative events in the MCF7 cell line. The measured and calculated parameters for estrogenic activity in the MCF7 cell proliferation assay are reported in Table 2. In the present set of experiments, the EC50 for 17βE2 in the MCF7 cell proliferation assay was estimated to be 0.077 nM. The decrease in fluorescence observed for genistein and quercetin at concentrations exceeding 1 µM was due to toxicity, verified by an increased number of detached, dead cells. At higher concentrations, all the tested flavonoids were cytotoxic, although to greatly varying extents (31). From Tables 1 and 2 it is evident that the structural requirements for estrogenic activity in the MCF7 estrogen screen and the yeast system are similar. The most potent flavonoid estrogens were thus almost identical for the two assays, although differences in the rankings of the flavonoids were observed. As flavonoids are known to be reducing agents, the ability of the flavonoids to reduce AB to a fluorescent species in the absence of MCF7 cells was investigated in a separate study. From this set of experiments it was evident that none of the flavonoids under the employed conditions were capable of reducing AB in the absence of MCF7 cells. The reduction of AB can thus be regarded as a cell-specific event.
Discussion The recombinant yeast assay, which in the present study was adapted to 96-well microtiter plates, requires only a single treatment of the indicator cells with the estrogenic substance to induce an estrogenic response and furthermore does not require sample processing. These features make the yeast estrogen screen ideal for large-scale screening purposes, in addition to being one of the most sensitive estrogen screens available. In the present study the recombinant yeast screen was capable
% of 17βE2d
a The presented data are means of triplicate experiments. The SD for the raw data is given in parentheses. max F580: maximum induction level, measured as peak fluorescence, using an excitation wavelength of 560 nm. b Fold induction is expressed as the ratio of the maximum F580 of the test compound relative to the solvent control (0.08) under the conditions of the assay. c EC50 values are calculated from the linear part of dose-response curves. The doseresponse curves were obtained at day 8, preceded by estrogen or flavonoid treatment every second day. d Percentage EC50 of 17βE2 relative to EC50 of test compounds. e The five compounds are not statistically different from each other (P > 0.01). f The two compounds are not statistically different from each other (P > 0.01), but each individual compound differs significantly from biochanin A and kaempferol (P < 0.01). g The six compounds are not statistically different from each other (P > 0.01), but each individual compound differs significantly from the remaining compounds tested (P < 0.01). h The three compounds are statistically different from each other (P < 0.01).
of detecting a number of weakly estrogenic flavonoids after a single addition of the flavonoid. The potency of the most active flavonoid estrogens was estimated to be 4 000-4 000 000 times lower than that observed for 17βE2, when based on calculated EC50 values. The estrogenic activity of some of the flavo-estrogens, such as naringenin, apigenin, and kaempferol, was found to be in the same order of magnitude as that of the isoflavonoid estrogens, which have been suggested as cancer protective substances in foodstuffs. As the flavoestrogens are much more abundant than the isoflavonoids in commonly consumed fruits and vegetables, it may be tempting to suggest that the flavo-estrogens might also play a cancer protective role, by virtue of their estrogenic properties. The flavo-estrogens were also active in the mammalian MCF7 cell proliferation assay. The estrogenic potencies of the flavonoids in this assay were found to be in accord with the results from the yeast screen (see Tables 1 and 2). The individual ranking of the flavonoids, however, differed somewhat between the two systems. In the MCF7 cell proliferation assay, genistein and apigenin were equally potent in inducing an estrogenic response, when evaluated on the basis of EC50 values, whereas in yeast cells apigenin was 20-fold less potent than genistein. In the MCF7 cell proliferation assay, compounds such as catechin, epicatechin, eriodictyol, luteolin, and taxifolin were found to be weakly estrogenic, whereas these compounds were devoid of activity in the yeast screen. In contrast, myricetin and hesperetin which were weakly estrogenic in the yeast screen had no activity in MCF7
628 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
cells. Some of the observed discrepancies between the two test systems are thought, at least partly, to be due to differences in biotransformation capacity between the yeast and mammalian cells. Preliminary data from this laboratory indicate that MCF7 cells are capable of extensively metabolizing flavonoids, whereas no metabolites were detected in parallel exposures of yeast cells to flavonoids. Furthermore Gaido et al. (32) observed a lower activity or lack of estrogenicity of several DDT analogues in the yeast system as compared to testing in MCF7 cells, which is similar to what we found for some of the flavonoid compounds. These observations might result from the ability of yeast cells to actively transport specific compounds out of the cell (33), a feature that the mammalian cells do not exhibit. In some cases the yeast assay may thus underestimate the actual potency of a compound, if the compound being tested is undergoing active transport out of the yeast cell. These observations stress the importance of testing potential estrogenic substances in several test systems and not solely evaluating estrogenic potency on the basis of results from one assay system. In the present study it was decided to report the EC50 values of the tested flavonoids, as this is the most commonly used way of reporting potencies of estrogenic substances. As no valid method for comparing full agonists with partial agonists is available at present, we furthermore compared the obtained EC50 values for the flavonoid compounds with the employed estrogen standard, irrespective of the fact that the shape of several of the flavonoid dose-response curves differed from that of 17βE2 and that most of the flavonoid compounds tested were only partial agonists. Several improvements of the MCF7 cell proliferation assay were implemented in order to provide a mammalian estrogen assay system that can accommodate a large number of samples and as such can be used as a fast screening tool like the yeast screen, but with the specific biochemical features of a mammalian cell. In contrast to the yeast assay, where only a single addition of the estrogenic principle was required to elicit an estrogenic response, the MCF7 cell proliferation assay required multiple additions of the estrogenic compound to affect the cell number. As humans are continuously exposed to environmental and dietary estrogens, it can be argued that the multiple exposure regimen employed in the MCF7 cell proliferation assay might be the more appropriate way of testing the estrogenic potency of a compound. If only a single dose of the estrogenic compound was added, no increase in cell number could be detected, even with the 17βE2 standard. The failure of the MCF7 cells to detect a single dose of an estrogenic substance in contrast to the yeast assay presumably reflects differences in absorption and metabolism between the two systems, as well as differences in the level of the estrogen receptor among the two systems. As control of cell proliferation is a complex phenomenon involving multiple factors, in addition to steroid hormone binding to nuclear and cytosolic receptors, the differential response of the flavonoid estrogens in the yeast and mammalian assay systems might also reflect inherent differences in enzymatic activities and the level of growth factors of associated growth-regulating pathways. The estrogenic response can, for instance, be dramatically increased by protein kinases and likewise decreased by the action of phosphatases (34, 35), enzyme activities that
Breinholt and Larsen
are affected by several flavonoid compounds (21-23). It is presently under investigation whether the flavonoid estrogens act exclusively as estrogen receptor ligands or whether they also function as activators of the mitogenactivated protein kinase pathway. The yeast and the mammalian MCF7 cell line have potential to be employed in a large number of chemical screenings, so a more thorough investigation of the mechanisms responsible for the differences between the two estrogen screens is warranted to ensure that assay conditions mimic, as much as possible, the situation in the mammalian organism. When the structures of the flavonoids are related to their estrogenic activity in the two assays, it is evident that some flexibility exists in the structural features required for eliciting an estrogenic response. From the results it can be concluded that the major determinant for estrogenic activity of the flavo-estrogens and the isoflavonoids is the presence of a p-hydroxyl group on the B-ring, whereas changes in the A- or C-ring hydroxylation pattern are of minor importance. The fact that simple phenolics, such as caffeic, ferulic, or chlorogenic acids, with only one aromatic ring, were not able to induce an estrogenic response in either assay suggests that the diaryl ring structure, common to the flavonoids, greatly facilitates binding to the estrogen receptor and subsequent binding of the receptor-ligand complex to the EREs. Phloretin, an open-ringed chalcone, on the other hand, also induced an estrogenic response, despite the fact that the two aromatic ring structures in this chalcone are separated by a 3-carbon-long interconnecting ring system. Estrogenic activity of compounds without adjacent aromatic rings has been observed for other groups of compounds, such as the alkylphenols, which are regarded as environmental estrogens. Several of the alkylphenols were found to be weakly estrogenic in MCF7 cells (36) and in the yeast screen (37). Despite the fact that the presently investigated flavonoids are weak estrogens in the two employed test systems, it is speculated that the flavonoid estrogens will be stronger estrogens relative to, for instance, 17βE2 when administered in vivo. In animals or humans, endogenous estrogens are bound to transport proteins in the blood (38), and only a small fraction of the estrogen is free and thus bioavailable. Some of the flavonoid estrogens along with the synthetic estrogen diethylstilbestrol (DES), on the other hand, bind very poorly to serum hormone-binding proteins (ref 39; unpublished findings), and thus the major portion of the flavoestrogens and DES remains bioavailable in the circulatory system. The phytoestrogen coumestrol, which in vitro is several-thousand-fold less potent than 17βE2, exhibits an in vivo effect on uterine weight gain, when administered postnatally at days 1-5, that is similar to that of 17βE2 (40). Therefore, although the isoflavonoids and members of the flavo-estrogens are only weakly estrogenic in vitro, these compounds have the potential to exert an estrogenic effect in vivo, after entry into the circulatory system. The recent finding of estrogenic compounds within the flavonoid family, to which humans are extensively exposed via fruits and vegetables, raises a number of questions regarding the possible beneficial and hazardous potential of these dietary components and warrants a more thorough investigation of the cancer protective and/ or endocrine disrupting activities of this group of com-
Estrogenic Activity of Dietary Flavonoids
pounds. Short- and long-term cancer and reproductive studies are currently being initiated in our laboratory, employing some of the isoflavonoids and, in particular, the more commonly occurring flavo-estrogens that are present ubiquitously in a westernized diet.
Acknowledgment. We would like to acknowledge Glaxo Wellcome who developed the yeast screen, which, with permission, was given to us by Prof. John Sumpter, Brunel University, U.K. An extra special thank you is given to John Sumpter for letting Vibeke Breinholt stay in his laboratory for an extended period of time to learn the yeast screen. We also thank Katrin Christiansen for excellent technical assistance.
References (1) Markiewicz, L., Garey, J., Adlerkreutz, H., and Gurpide, E. (1993) In vitro bioassays of nonsteroidal phytoestrogens. J. Steroid. Biochem. Mol. Biol. 45 (5), 399-405. (2) Mayr, U., Butsch, A., and Schneider, S. (1992) Validation of two in vitro test systems for estrogenic activities of zearalenone, phytoestrogens and cereal extracts. Toxicology 74, 135-149. (3) Welshons, W. V., Murphey, C. S., Koch, R., Calaf, G., and Jordan, V. C. (1987) Stimulation of breast cancer cells in vitro by the environmental estrogen enterolactone and the phytoestrogen equol. Breast Cancer Res. Treat. 10, 169-175. (4) Micsicek, R. J. (1993) Commonly occurring plant flavonoids have estrogenic activity. Mol. Pharmacol. 44, 37-43. (5) Micsicek, R. J. (1994) Interaction of naturally occurring nonsteroidal estrogens with expressed recombinant human estrogen receptor. J. Steroid. Biochem. Biol. 49 (2/3), 153-160. (6) Martin, P. M., Horwitz, K. B., Ryan, D. S., and McGuire, W. L. (1978) Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology 103, 1860-1867. (7) Faber, K. A., and Hughes, C. L. (1993) Dose-response characteristics of neonatal exposure to genistein on pituitary responsiveness to gonadotropin releasing hormone and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postpubertal castrated female rats. Reprod. Toxicol. 7, 35-39. (8) Farmakalidis, E., Hathcock, J. N., and Murphy, P. A. (1985) Estrogenic potency of genistein and daidzein in mice. Food Chem. Toxicol. 23, 741-745. (9) Levy, J. R., Faber, K. A., Ayyash, L., and Hughes, C. L. (1995) The effect of prenatal exposure to the phytoestrogen genistein on sexual differentiation in rats. Proc. Soc. Exp. Biol. Med. 208 (1), 60-66. (10) Kitts, W. D., Newsome, F. E., and Runeckles, V. C. (1983) The estrogenic and antiestrogenic effects of coumestrol and zearalenol on the immature rat uterus. Can. J. Anim. Sci. 63, 823-824. (11) Shutt, D. A. (1976) The effect of plant estrogens on animal reproduction. Endeavour (Oxford) 35, 110-113. (12) Bennetts, H. W., Underwood, E. J., and Shier, F. L. A. (1946) Breeding problem in sheep in the South-West Division of Western Australia. J. Agric. West. Aust. 23, 1-12. (13) Cox, R. I., and Braden, A. W. (1974) The metabolism and physiological effects of phytoestrogens in Livestock. Proc. Aust. Soc. Anim. Prod. 10, 122-129. (14) Tominaga, S. (1985) Cancer incidence in Japanese in Japan, Hawaii, and Western United States. Natl. Cancer Inst. Monogr. 69, 83-92. (15) Messina, M., and Barnes, S. (1991) The role of soy products in reducing risk of cancer. J. Natl. Cancer Inst. 83, 541-546. (16) Dunn, J. E. (1975) Cancer epidemiology in populations of the United States with emphasis on Hawaii and California and Japan. Cancer Res. 35, 3240-3245. (17) Adlercreutz, H. (1990) Western diet and Western diseases: some hormonal and biochemical mechanisms and associations. Scan. J. Clin. Lab. Invest. 50 (Suppl. 201), 3-23. (18) Adlercreutz, H., Hamalainen, E., Gorbach, S., and Goldin, N. (1992) Dietary phytoestrogens and the menopause in Japan. Lancet 339, 1233. (19) Okura, A., Arakawa, H., Oka, H., Yoshinari, T., and Monden, Y. (1988) Effect of genistein on topoisomerase activity and on the
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 629
(20)
(21)
(22)
(23)
(24) (25)
(26) (27) (28) (29) (30)
(31) (32)
(33)
(34) (35) (36)
(37) (38) (39)
(40)
growth of [val 12] Ha-ras-transformed NIH-3T3 cells. Biochem. Biophys. Res. Commun. 157, 183-189. Markovits, J., Linassier, C., Fosse, P., Couprie, J., JaqueminSablon, A., Pecq, J. B., and Larson, A. K. (1989) Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian topoisomerase II. Cancer Res. 49, 5111-5117. Linassier, C., Pierre, M., Lepecq, J. B., and Pierre, J. (1990) Mechanisms of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity. Biochem. Pharmacol. 39, 187-193. Akiayama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S.-I., Itoh, N., Shibuya, M., and Fukami, Y. (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262, 5592-5595. Makishma, M., Homa, Y., Hozumi, M., Sampi, K., Hattori, M., Motoyoshi, K., and Saitama, K. (1991) Effects of inhibitors of protein kinase activity and/or phosphatidylinositol turnover on differentiation of some human myelomonocytic leukemia cells. Leuk. Res. 15, 701-709. Ku¨hnau. (1976) The flavonoids. A class of semi-essential food components, Their role in human nutrition. World Rev. Nutr. Diet. 24, 117-191. Herzog, M. G. L., Feskens, E. J. M., Hollmann, P. C. H., Katan, M. B., and Kromhout, D. (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342, 1007-1011. Doll, R., and Peto, R. (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst. 66, 1191-1308. Doll, R. (1990) An overview of the epidemiological evidence linking diet and cancer. Proc. Nutr. Soc. 49, 119-131. Faber, E. (1982) Chemical carcinogenesis. A biological perspective. Am. J. Pathol. 106, 271-296. Hirayama, T. (1985) Mortality in Japanese with life-styles similar to Seventh-Day-Adventists strategy for risk reduction by lifestyle. Natl. Cancer Inst. Monogr. 69, 143-153. Routledge, E., and Sumpter, J. P. (1996) Estrogenic activity of surfactants and some of their degradation products using a recombinant yeast screen. Environ. Toxicol. and Chem. 15, 241248. Breinholt, V., and Dragsted, L. O. (1998) Structure-cytotoxicity relationships for dietary flavonoids. In Vitro Toxicol., in press. Gaido, K. W., Leonard, K. S., Lovell, S., Gould, J. C., Babai, D., Portier, C. J., and Mcdonnell, D. P. (1997) Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 143, 205-212. Kralli, A., Bohen, S. P., and Yamamoto, K. R. (1995) LEM1, an ATP-binding-cassette tansporter, selectively modulates the biological potency of steroid hormones. Proc. Natl. Acad. Sci. U.S.A. 92 (10), 4701-4705. Wakeling, A. E. (1995) Use of pure antiestrogens to elucidate the mode of action of estrogens. Biochem. Pharmacol. 49 (11), 15451549. Freiss, G., and Vignon, F. (1994) Antiestrogens increase protein tyrosine phosphatase activity in human breast cancer cells. Mol. Endocrinol. 8 (10), 1389-1396. Soto, A. M., Sonnenschein, C., Chung, K. L., Fernandez, M. F., Olea, N., and Serrano, F. O. (1995) The E-screen assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environ. Health Perspect. 103 (Suppl. 7), 113-122. Routledge, E., and Sumpter, J. P. (1997). Structural features of alkylphenolic chemicals associated with estrogenic activity. J. Biol. Chem. 272 (6), 3280-3288. Mendel, C. M. (1989) The free hormone hypothesis: A physiologically based mathematical model. Endocrinol. Rev. 10, 232-343. vom Saal, F. S., Nagel, S. C., Palanza, P., Boechler, M., Parmigiani, S., and Welshons, W. V. (1995) Estrogenic pesticides: Binding relative to estradiol in MCF7 cells and effects of exposure during fetal life on subsequent territorial behavior in male mice. Toxicol. Lett. 77, 343-350. Sheehan, D. M., Branham, W. S., and Medlock, K. L. (1995) An estrogen developmental potency and toxicity assay for environmental estrogens. Teratology 51 (3), 196-197.
TX970170Y
