Skip Navigation


Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(2):363-370; doi:10.1093/carcin/bgm235
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/2/363    most recent
bgm235v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lehmann, L.
Right arrow Articles by Wagner, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lehmann, L.
Right arrow Articles by Wagner, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Soy isoflavones decrease the catechol-O-methyltransferase-mediated inactivation of 4-hydroxyestradiol in cultured MCF-7 cells

Leane Lehmann*, Ling Jiang and Jörg Wagner

Institute of Applied Biosciences, Section of Food Chemistry, University of Karlsruhe, Kaiserstraße 12, D-76131 Karlsruhe, Germany

* To whom correspondence should be addressed. Tel: +49 721 608 4177; Fax: +49 721 608 7255; Email: leane.lehmann{at}lmc.uni-karlsruhe.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The tissue concentrations of the female sex hormone 17β-estradiol (E2) and its reactive catechol metabolites such as 4-hydroxyestradiol (4-HO-E2) play important roles in hormonal carcinogenesis. They are influenced by the activity of local enzymes involved in the metabolic activation and inactivation of E2. In the mammary gland, catechol estrogens are predominately inactivated by catechol-O-methyltransferase (COMT). Food supplements containing the soy isoflavones genistein and daidzein are consumed because they are believed to protect from breast cancer; however, this proposed benefit is controversial. The aim of the present study was to investigate the influence of soy isoflavones on the gene expression and activity of COMT in cultured human mammary adenocarcinoma MCF-7 cells. Levels of COMT messenger RNA (mRNA) were determined by reverse transcription/competitive polymerase chain reaction and COMT activity was determined by high-performance liquid chromatography analysis of the methylation products of both the model substrate quercetin and the physiological relevant substrate 4-HO-E2. Our study demonstrates for the first time that soy isoflavones at hormonally active concentrations cause a significant reduction of both COMT mRNA levels and COMT activity as well as of the methylation of 4-HO-E2. Experiments using the estrogen receptor (ER) antagonist ICI 182,780 support a role of the ER in the isoflavone-induced down-regulation of COMT expression. Thus, this study not only demonstrates that hormonally active concentrations of soy isoflavones inhibit the detoxification of catechols in this human breast cancer cell line but also implies that diet might influence COMT activity to a greater extent than heretofore recognized.

Abbreviations: cDNA, complementary DNA; COMT, catechol-O-methyltransferase; CYP, cytochrome P450; DAI, daidzein; DMSO, dimethyl sulfoxide; E2, 17β-estradiol; ER, estrogen receptor; GC, gas chromatography; GEN, genistein; 4-HO-E2, 4-hydroxyestradiol; HPLC, high-performance liquid chromatography; HPRT, hypoxanthine-guanine phosphoribosyltransferase; ICI, ICI 182,780; MS, mass spectrometry; mRNA, messenger RNA; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Exposure to 17β-estradiol (E2; Figure 1) is commonly believed to be associated with an increased risk of breast cancer. Besides the induction of proliferation, which could favor tumor promotion, E2 can be metabolically activated to genotoxic metabolites (Figure 2). In the mammary gland, hydroxylation of E2 to the catechols 2- and 4-hydroxyestradiol (2- and 4-HO-E2) is catalyzed predominately by cytochrome P450 (CYP)1A1 and CYP1B1, respectively (1,2). In general, E2 as well as catechol estrogens are inactivated by conjugating reactions, such as glucuronidation, sulfonation and O-methylation and the inactivation of E2 catechols in the human mammary gland is predominately catalyzed by the ubiquitous enzyme catechol-O-methyltransferase (COMT; summarized in ref. 3). Both possible methylation products of 2-HO-E2 (i.e. 2-methoxy-E2 and 2-hydroxy-3-O-methyl-E2) are generated by rat and human COMT. In contrast, 4-methoxy-E2 (4-MeO-E2; Figure 1) seems to be preferably formed over 3-O-methyl-4-hydroxy-E2 (3-Me-4-HO-E2; Figure 1) by rat COMT and appears to represent the only methylation product of 4-HO-E2 formed by human COMT (4). Besides the inactivation of reactive molecules, the methylation products of E2 catechols exhibit distinct biological properties, which may exert further beneficial or detrimental effects in breast carcinogenesis, e.g. the suppression of growth of human breast cancer in mice by 2-MeO-E2 (5) and the induction of oxidative DNA damage by 4-MeO-E2 (6). If conjugation of catechol estrogens becomes deficient in the mammary gland, increased formation of E2-2,3-quinone and E2-3,4-quinone can occur, which may contribute to tumor initiation in estrogen-sensitive tissues (7).


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Chemical structures of E2, DAI, GEN, 4-HO-E2, 3-O-Me-4-HO-E2 and 4-MeO-E2.

 


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Schematic overview of the action and the metabolic fate of both E2 and its metabolites in MCF-7 cells. CYP, cytochrome-P450-dependent monooxygenase; GSH, glutathione; PO, peroxidase; QR, NADPH-quinone oxidoreductase; STS, steroidsulfatase; SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase.

 
The importance of E2 metabolism in breast cancer is emphasized by the finding that human breast tumors exhibit higher tissue concentration of 4-HO-E2, an abnormal ratio of 4- to 2-HO-E2 and less O-methylated products than normal breast tissue (8). Furthermore, CYP1B1 is more expressed and COMT is less expressed in breast carcinoma tissue as compared with normal female breast tissue (9,10). The activity of E2-metabolizing enzymes is determined by genetic polymorphisms (11) or modulated by the direct interaction with endogenous and exogenous compounds (12). Exogenous factors, e.g. diet and dietary supplements, may also affect the gene expression of such key enzymes of E2 metabolism.

Due to the lower breast cancer incidence of Asian women consuming a soy-based diet, the search for food constituents providing chemoprevention against breast cancer soon focused on isoflavones, especially genistein and daidzein (GEN and DAI; Figure 1; reviewed in ref. 13). Despite an abundance of studies concerning the effects of GEN and DAI in vivo and in vitro, the mechanisms of the putative chemoprevention by isoflavones remain elusive and the safety of high-dosed food supplements containing isoflavones still needs to be clarified. Protective [e.g. (14)] as well as adverse [e.g. (15–17)] effects of GEN have been observed in animal models. Adverse effects of GEN could be due to promotional effects caused by estrogen receptor (ER)-stimulated proliferation of estrogen-sensitive cell types. Therefore, it has been recommended that the impact of isoflavones on breast tissue should be evaluated at the cellular level in women at high risk for breast cancer (18). Whether GEN may also contribute to the initiation of tumors still remains unclear although GEN and, to a lesser extent, DAI are genotoxic in vitro (reviewed in ref. 19).

The question of whether soy isoflavones interact with the metabolism of E2 with beneficial or adverse outcome received hitherto little attention. The direct inhibitory effect of GEN on the enzyme activity of COMT has been investigated and concentrations up to 30 µM GEN did not affect COMT activity in the cytosolic fractions of healthy human mammary tissues from reduction mammoplasty (20). However, up to now the impact of isoflavones on the expression of the COMT gene has not been elucidated. Lower COMT activity in women compared with men and variations in COMT activity during the estrus cycle and pregnancy in animals indicate that the regulation of this gene is hormone sensitive (21–23). It has been demonstrated that COMT gene expression is down-regulated by E2 in vitro (24), estrogen-responsive elements have been identified in the promoter region of COMT and the binding of the activated ER to the DNA has been demonstrated in MCF-7 cells (25). Therefore, phytoestrogens such as DAI and GEN that represent selective ER modulators may also affect COMT expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Chemicals
E2, 4-HO-E2, GEN and DAI were purchased from Sigma (Taufkirchen, Germany), ICI 182,780 (ICI) from Tocris (Bristol, UK) and 4-MeO-E2 from Steraloids (Newport, RI). All other chemicals, media and medium supplements were obtained from Sigma or Roth (Karlsruhe, Germany) if not specified otherwise.

Cell culture
MCF-7 cells were cultured in complete medium: Dulbecco's modified eagle medium (MPBio, Illkirch, France) with phenol red, 100 U/ml penicillin and 100 µg/ml streptomycin, supplemented with 5% heat-inactivated fetal calf serum (Invitrogen, Karlsruhe, Germany). Experiments were carried out in steroid-deprived experimental medium: Dulbecco's modified eagle medium without phenol red, with 100 U/ml penicillin and 100 µg/ml streptomycin, supplemented with 5% charcoal/dextrane-treated fetal calf serum (Hyclone, South Logan, UT). Compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the medium to yield a final DMSO concentration of 0.1% (vol/vol). Control experiments were carried out with medium containing 0.1% DMSO.

Proliferation of MCF-7 cells
MCF-7 cells were seeded in 24-well plates (ICN Biochemicals, Eschwege, Germany; 2 x 104 cells per well) in experimental medium 24 h prior to incubation with E2, DAI and GEN (4–6 wells per compound and concentration). For co-treatment experiments, experimental medium containing DMSO, DAI or GEN together with ICI or E2 was prepared. After 6 days, medium was aspirated, 150 µl lysis buffer (Partec, Muenster, Germany) were added to each well and cells were lyzed for 5 min. Then, 750 µl staining solution (Partec) was added and the number of nuclei and the cell cycle distribution were determined by flow cytometry using a Ploidy Analyzer®-II (Partec).

Gene expression analysis
MCF-7 cells suspended in experimental medium were seeded in 6-well plates (Nunc, Wiesbaden, Germany; 1 x 106 cells per well) 24 h prior to incubation with compounds. After 24 h, total RNA was isolated using the GenElute total RNA Isolation Kit (Sigma). Contaminating traces of DNA were digested with DNase (Sigma) and 1 µg total RNA was reversely transcribed with 200 U murine lymphoma virus reverse transcriptase (Fermentas, St Leon-Rot, Germany) using oligo(dT)18 primer (Fermentas) according to the instruction of the manufacturer.

Complementary DNA (cDNA) mix (1.5 µl) was co-amplified in the presence of competitor DNA in a total volume of 25 µl in 75 mM Tris–HCl buffer, pH 8.0, containing 2.5 mM MgCl2, 20 mM (NH4)2SO4, 0.1% Tween 20, 200 µM deoxynucleoside triphosphate of each nucleotide and 0.4 µM each of the following forward and reverse primer (Hermann GBR, Freiburg, Germany): hypoxanthine-guanine phosphoribosyltransferase (HPRT) forward 5'-tgtaatgaccagtcaacaggg, HPRT reverse 5'-tggcttatatccaacacttcg; COMT forward 5'-ctgctcatgggtgacaccaag, COMT reverse 5'-tccaaccacaagggtgacctt and membrane-bound COMT forward 5'-accgccattgccgccatcgtcgt, membrane-bound COMT reverse 5'-acacagctgccaacagcagaggc. Amplification conditions were 94°C for 3 min; then 35 cycles: 94°C, 30 s; 61°C, 50 s and 72°C, 1 min and then 72°C for 5 min.

Polymerase chain reaction (PCR) products were prestained with SYBR®Green and separated on a 3% (HPRT) or 2% (COMT) agarose gel in Tris–acetic acid–ethylenediaminetetraacetic acid buffer. Electrophoresis was conducted at 5 V/cm for 1 h. DNA was visualized by excitation at 488 nm and the fluorescence intensities of the PCR product of the competitor (HPRT: 140 bp, COMT: 285 bp and membrane-bound COMT 169) and of the target (HPRT: 214 bp, COMT: 357 bp and membrane-bound COMT: 238) were quantified digitally (DIANA, Raytest, Straubenhardt, Germany) and analyzed with AIDA software (Raytest).

Competitor DNAs were generated by PCR as described by Anderson et al. (26). Briefly, cDNA was amplified with the normal set of primers, the PCR product was purified by agarose gel electrophoresis, isolated using a DNA gel isolation kit (Amersham, Freiburg, Germany) and used as template in the subsequent amplification reaction that was conducted with the respective forward primer and one of the following linker primers: 5'-tggcttatatccaacacttcg cctgcctgaccaaggaaag (HPRT), 5'-caagggtgaccttatggtgatgagcc (COMT) or 5'-tgccaacagcagaggctctggggtctcctct (membrane-bound COMT). Competitor DNAs were isolated and purified as described above and quantified fluorimetrically using SYBR® Green. The amount of target cDNA was determined by linear regression (for exemplary analysis, see Supplementary Figure 1, available at Carcinogenesis Online). In order to compensate for variation in RNA isolation and reverse transcription, results were standardized to the recommended (27,28) housekeeping gene HPRT.

COMT activity
Cell fractionation.
Since MCF-7 cells hardly proliferate in experimental medium, MCF-7 cells (3 x 106 cells per 175 cm2 flask) were initially cultured in complete medium for 72 h. Then, cells were washed with phosphate-buffered saline-(calcium- and magnesium-free) and kept in experimental medium for at least 24 h prior to treatment with E2, GEN, DAI or solvent (0.1% DMSO) alone for 48 h. At least 5 x 107 cells per treatment group were used for the isolation of cytosol: cells were washed with phosphate-buffered saline-(calcium- and magnesium-free) and lyzed by freezing at –80°C and subsequent homogenization in a buffer containing 60 mM KCl, 20 mM Tris and 0.4 mM ethylenediaminetetraacetic acid (pH 7.4) using a Dounce tissue grinder. Homogenates were centrifuged (9000g, 4°C, 10 min) and the supernatant was centrifuged again (100 000g, 4°C, 1 h) yielding the microsomal (pellet) and cytosolic (supernatant) fractions. The cytosol was used for the determination of COMT activity and of protein concentration by means of a modified Bradford protocol (29).

COMT activity and high-performance liquid chromatography analysis.
For the determination of COMT activity, 62.5 ng cytosolic protein was mixed with phosphate buffer (0.1 M, pH 7.4) containing 150 µM quercetin, 500 µM S-adenosyl methionine and 4 mM MgCl2 to give a final volume of 125 µl. Control experiments were performed with a reaction mixture without S-adenosyl methionine. After incubation at 37°C for up to 40 min, quercetin and its methylation products were extracted with ethyl acetate, the solvent was evaporated and the residue was dissolved in 40 µl methanol, separated by high-performance liquid chromatography (HPLC) (Jasco, Gross-Umstadt, Germany) using a Luna 5 µm C18(2) column (250 x 4.6 mm, Phenomenex, Aschaffenburg, Germany) with 7 volumes H2O (pH 3) + 3 volumes acetonitrile at 1 ml/min and detected by absorption at 375 nm. Methylation products eluted as a single peak (Supplementary Figure 3, available at Carcinogenesis Online) and the peak area was used for quantification. With each cytosol, relative conversion of quercetin to methylated products was determined at three time points and the formation of methylated quercetin per minute and milligrams protein was determined by linear regression (Supplementary Figure 2, available at Carcinogenesis Online).

Methylation of 4-HO-E2
MCF-7 cells were cultured, treated and homogenized and the cytosol was prepared as described in COMT activity. Then, the COMT assay was performed with 100 µM 4-HO-E2 as substrate. Since 4-HO-E2 is not as good as a substrate as quercetin, the detection of 4-HO-E2 by absorbance at 280 nm was less sensitive than that of quercetin at 375 nm, and the protein content could not be increased due to the poor proliferation of MCF-7 cells cultured in steroid-deprived medium; the incubation time for the enzymatic reaction was extended to 3 h. Then, 4-HO-E2 and its methylation products were extracted with ethyl acetate and the solvent was evaporated. The residue was dissolved in 40 µl methanol and separated by HPLC (see COMT activity) at 1 ml/min and detected by absorption at 280 nm. The eluent consisted of 6 volumes H2O (pH 3) + 4 volumes acetonitrile for 7 min; subsequently, the acetonitrile proportion was increased until 1 volume H2O + 9 volumes acetonitrile were reached at 15 min and 100% acetonitrile at 18 min.

Peak identification by gas chromatography/mass spectrometry.
The compounds eluting at 8.1, 13.7 and 16.3 min were collected, the mobile phase was evaporated and the remainder derivatized to the trimethylsilyl ethers by reaction with N,O-bis(trimethylsilyl) trifluoroacetamide. For gas chromatography (GC)/mass spectrometry (MS) analysis, a Finnigan GCQ capillary gas chromatograph equipped with a 30 m x 0.25 mm intradermally, 0.25 µm, DB-5 fused-silica column (Supelco, Bellefonte, PA), coupled to an ion-trap detector, was operated with electron impact ionization at 70 eV (Thermo Finnigan, Austin, TX). Oven temperature was programmed from 60 (1 min hold) to 250°C at a rate of 30°C/min and subsequently to 295°C at a rate of 3°C/min (5 min hold). Temperatures of injector, transfer line and ion source were 275, 275 and 250°C, respectively. Samples (1 µl) were injected using programmed temperature vaporization splitless injection with helium as carrier gas at a flow rate of 40 cm/s. Mass spectra were scanned from m/z 50 to 650 at a rate of 0.5 s per scan. Samples obtained from the HPLC peaks eluting at 13.7 and 16.3 min (methylation products of 4-HO-E2) were further analyzed by MS/MS using the molecule ion (m/z = 446) as precursor ion.

Statistical methods
Cells were treated with the compounds and either cytosol was prepared or total RNA was isolated from control and estrogen-treated cells simultaneously. Treatments and isolations were repeated at least two times with a new batch of cells and fresh solutions yielding three independent experiments. Data were expressed (i) as mean ± standard deviation of the individual experiments or (ii) the treatment groups were standardized to the solvent control (100%) in each experiment prior to calculation of means and standard deviations in order to facilitate the comparability of the endpoints and whenever a column with its variation bar is <100% the effect was observed in each experiment. In order to assess statistical significance, in both cases, Student's paired t-test of significant differences was conducted with the untransformed data using the Origin® program of MicrocalTM Software. Co-incubation experiments were analyzed by ANOVA using WinSTAT® software (Fitch Software, Bad Krozingen, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Effect of estrogens on the expression of COMT
Proliferation.
In experiments used for the determination of COMT expression, the absolute cell number after treatment of MCF-7 cells with the solvent (0.1% DMSO) for 6 days was 44 ± 9 x 103 cells per well. Most cells were in G1/G0 phase of the cell cycle (89 ± 3%, data not shown). As expected, 10 pM to 1 nM E2 significantly induced the proliferation of MCF-7 cells and the maximum effect was observed at 100 pM (1025 ± 116% of the cell number of solvent-treated cells; Figure 3). Likewise, DAI and GEN significantly induced the proliferation of MCF-7 cells, with maximum stimulation at 1 µM (1004 ± 114%, DAI, and 850 ± 128%, GEN). The cytotoxicity of GEN at concentrations >1 µM precluded the use of higher concentrations, whereas up to 100 µM DAI did not affect cell viability (data not shown).


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Relative proliferation (top panel), relative mRNA expression COMT (middle panel) and relative COMT activity (bottom panel) of cultured MCF-7 cells after treatment with various concentrations of E2 (left panel), DAI (middle panel) and GEN (right panel) for 6 days (proliferation), 24 h (COMT mRNA) and 48 h (COMT activity). Each individual experiment was standardized to the solvent control (0.1% DMSO, 100%, black line). Data represent means + standard deviations of the results of at least three independent experiments (see Statistical methods). Statistical significance was determined using Student’s paired t-test with the untransformed data. Levels of significance: *0.05; **0.01 and ***0.001.

 
Messenger RNA level.
For the assessment of COMT messenger RNA (mRNA) levels in MCF-7 cells, cells were treated with 10 pM to 1 nM E2, 100 nM to 10 µM DAI and 10 nM to 1 µM GEN for 24 h prior to determination of relative mRNA levels. Initial experiments demonstrated the presence of 1% or less cDNA specific for the membrane-bound COMT compared with both COMT isozymes (data not shown), demonstrating a minor role, if any, of the membrane-bound COMT. Therefore, mRNA levels of total COMT are shown in the following paragraphs.

In cDNA derived from solvent-treated cells, the number of copies of HPRT and COMT was 3.7 ± 1.04 and 1.0 ± 0.04 fmol/µg total RNA, respectively. The average COMT:HPRT ratio of the cDNA derived from solvent-treated MCF-7 cells of all experiments was 0.28 ± 0.07 (data not shown). Treatment of MCF-7 cells with 10 pM to 1 nM E2 for 24 h significantly decreased the relative mRNA level compared with that of the solvent control, reaching maximum effect at 100 pM E2 (reduction to 24 ± 12.2% of that of the solvent control; Figure 3). DAI and GEN also significantly decreased the expression of COMT in MCF-7 cells at hormonally active concentrations (Figure 3), with the maximum effect observed at the highest dose tested (1–10 µM DAI, reduction to 28 ± 4.3%, and 1 µM GEN, reduction to 35 ± 7.6%, respectively).

COMT activity.
In order to assess the consequence of reduced COMT mRNA levels on the activity of the COMT protein, MCF-7 cells were treated with E2, DAI or GEN for 48 h prior to isolation of the cytosolic soluble COMT enzyme. Then, the methylation of the standard substrate quercetin was determined using HPLC. The reaction yielded linear kinetics with a conversion of <16% quercetin (Supplementary Figure 2, available at carcinogenesis Online). Since quercetin concentration was sufficiently high [150 µM versus a Km value of 5.3 µM for the methylation of quercetin by human COMT (30)] and not significantly reduced during the reaction period, the product formation was considered proportional to the amount of enzyme. Using these conditions, the cytosol derived from solvent-treated MCF-7 cells formed quercetin methylation products at a rate of 1118 ± 211 pmol/min/mg cytosolic protein (data not shown). In contrast, treatment with E2 (100 pM and 1 nM), DAI (1 µM and 10 µM) and GEN (1 µM) significantly decreased the formation of methylated quercetin to 50 ± 8% (E2), 54 ± 10% (DAI) and 68 ± 15% (GEN) of that of the solvent control (Figure 3), reflecting a decrease in COMT enzyme levels.

Co-treatment.
Depending on their diet, humans might be co-exposed to E2 and soy isoflavones. In order to investigate the impact of such a combined exposure on COMT expression, MCF-7 cells were treated either with concentrations of E2 plus isoflavones that each induced maximum proliferation or with concentrations that each induced submaximum proliferation.

Interestingly, when MCF-7 cells were co-exposed to 10 pM E2 (expected induction of ~60% of maximum proliferation) and 100 nM DAI (~25% of maximum induction), the effect of E2 was not modified significantly (Figure 4, upper panel). Likewise, the combination of 10 pM E2 and 100 nM GEN (60% of maximum stimulation) resulted in a small, non-significant increase of cell numbers (Figure 4). Moreover, maximum proliferation induced by 100 pM E2 was not significantly inhibited by non-toxic concentrations of DAI (1 µM) and GEN (1 µM), which induced maximum proliferation in the absence of E2. Relative mRNA levels of COMT were inversely correlated with the proliferative effect of the estrogens, reaching minimum levels when proliferation showed a maximum (Figure 4).


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Proliferation (top panel) and mRNA expression of COMT (bottom panel) after treatment of cultured MCF-7 cells with 10 and 100 pM E2 in the absence (bars with stripes) and presence (bars with diamonds) of the isoflavones (IF) DAI (left panel) and GEN (right panel) for 24 h (mRNA) and 6 days (proliferation). Each individual experiment was standardized to the solvent control (0.15% DMSO, 100%). Data represent means + standard deviations of at least three independent experiments. Statistical significance was determined using analysis of variance with the untransformed data (see Statistical methods). Bars with different characters (a–d, DAI, and a–c, GEN) are statistically different at P < 0.05.

 
Estrogen-mediated down-regulation of COMT and methylation of 4-HO-E2
In order to assess the impact of a reduction of COMT activity on the deactivation of 4-HO-E2, the methylation of 4-HO-E2 by cytosolic COMT derived from MCF-7 cells treated with 100 pM E2, 10 µM DAI or 1 µM GEN for 48 h was determined. In order to ensure that the methylation was exclusively dependent on COMT concentration and not on substrate availability, a substrate concentration of ~10-fold the Km value for the methylation of 4-HO-E2 by human COMT [~10 µM; (31)] was chosen and the reaction was stopped after ~20% 4-MeO-E2 formation (3 h). Initial experiments verified that the reaction exhibited linear kinetics for up to 5 h at least (Supplementary Figure 3, available at Carcinogenesis Online). Furthermore, virtually the same total peak area of 4-HO-E2 plus methylated products was determined by HPLC when extracted from a reaction mixture prior to and after incubation at 37°C for up to 5 h (data not shown), indicating no apparent loss of 4-HO-E2 or reaction products, e.g. due to oxidation. Experiments using incubation times that yielded up to 80% product formation revealed a third peak (data not shown). Whereas 4-HO-E2 and 4-MeO-E2 could be unambiguously identified using the commercially available standard compounds, the third peak was collected and analyzed by GC/MS and GC/MS/MS for identification. By means of co-chromatography, it was verified that it exhibited different gas chromatographic properties than 4-MeO-E2 (Supplementary Figure 4, available at Carcinogenesis Online). Mass spectra of both compounds exhibited the same molecule ion (data not shown) and qualitatively similar but quantitatively different MS/MS fragmentation patterns were obtained when using the molecule ion as precursor ion (Supplementary Figure 4, available at Carcinogenesis Online), suggesting the formation of 3-O-Me-4-HO-E2. Yet, due to the small proportion of 3-O-Me-4-HO-E2, it was not included in the quantification.

Cytosolic COMT derived from solvent-treated cells catalyzed the formation of 356 ± 12 pmol 4-MeO-E2/min/mg cytosolic protein (Figure 5). Using cytosol derived from MCF-7 cells previously treated with 100 pM E2, a reduction of COMT activity to 39 ± 6% of that of the solvent control was observed. Likewise, treatment with 10 µM DAI and 1 µM GEN resulted in a reduction of the methylation of 4-HO-E2 to 40 ± 1% and 61 ± 4% of that of the solvent-treated cells, respectively.


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Methylation of 4-HO-E2 by cytosolic COMT derived from MCF-7 cells treated with 100 pM E2, 10 µM DAI or 1 µM GEN for 48 h. Data represent means + standard deviations of three independent experiments. Statistical significance was determined using Student’s paired t-test (see Statistical methods). Levels of significance: *0.05; **0.01 and ***0.001.

 
Role of the ER in the expression of COMT
The role of the ER in the down-regulation of COMT expression was investigated using the ER antagonist ICI. When MCF-7 cells were treated with 100 pM E2, 10 µM DAI or 1 µM GEN in the absence of ICI, the expected maximum proliferation as well as reduction of COMT levels was observed (Figure 6). Cell numbers in the ICI-only group were 17 ± 3 x 103 cells per well, indicating that the ER antagonist ICI completely prevented the proliferation of the 20 x 103 initially seeded and solvent-treated cells. Usually in the absence of ICI, ~2-fold increase of the number of seeded cells is observed after treatment with solvent only (data not shown), suggesting the presence of small amounts of residual steroid in the experimental medium. Likewise, the proliferation of the cells treated with 100 pM E2 was completely prevented by a 100-fold excess of ICI (Figure 6, upper panel), which is consistent with the comparable binding affinities of ICI and E2 to ERs (32–34). Moreover, the E2-induced reduction of COMT mRNA levels was not only prevented by ICI but also increased to the level of ICI-only treated cells (136 ± 30%; Figure 6). Despite the theoretically possible complete prevention of proliferation based on ER-binding affinities (32), 10 nM ICI did not completely prevent the stimulation of the proliferation induced by 10 µM DAI and 1 µM GEN but the cell numbers were merely increased to 129 ± 30% (DAI) and 284 ± 23% (GEN) of that of the solvent control (Figure 6, upper panel). Accordingly, in the presence of ICI, a significant increase of COMT levels to approximately that of the solvent-treated cells was observed after treatment of MCF-7 cells with DAI (109 ± 31%) and GEN (88 ± 27%), respectively (Figure 6).


Figure 6
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Proliferation (upper panel) and relative mRNA expression of the COMT (lower panel) of cultured MCF-7 cells after treatment with solvent (DMSO), E2, DAI and GEN at the indicated concentrations in the absence (bars with stripes) and presence (bars with squares) of 10 nM ICI 182,780 (ICI) for 24 h (mRNA) or 6 days (proliferation). Each individual experiment was standardized to the solvent control (0.15% DMSO, 100%). Data represent means + standard deviations of at least three independent experiments. Statistical significance was determined using analysis of variance with the unmodified data (see Statistical methods). Columns with different characters are statistically different at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
The present study demonstrates the down-regulation of COMT by the isoflavones DAI and GEN. Very recently, a microarray study revealed COMT to be among the genes down-regulated by GEN in MCF-7 cells (35). Yet, the present study is the first to demonstrate that reduced expression of COMT by both isoflavones has significant impact on both COMT activity and the methylation of 4-HO-E2. Moreover, it suggests a role for the ER in the regulation of COMT. A decrease of COMT mRNA (24) levels and COMT activity (25) in MCF-7 cells has been observed previously for E2. However, both studies needed up to 1000-fold higher concentrations to obtain the same effect as observed in our study. Differences in the MCF-7 sub-cell line and culture conditions may be the reasons for this discrepancy. It is known that the MCF-7 cell sublines differ in their sensitivity to estrogens and that the MCF-7 BUS sub-cell line represents the most sensitive variant (36,37).

An average maximum activity for the methylation of 4-HO-E2 of 1000 pmol/min/mg protein [human liver cytosol (31)], 2000 pmol/min/nmol COMT [recombinant enzyme (4)] and 200 pmol/min/nmol COMT [MCF-7 cytosol (4)] was observed and using a modified enzyme-linked immunosorbent assay technique a COMT concentration of 8.15 µg COMT per mg cytosolic protein was determined (4). Regarding the many factors (normal tissue versus cultured tumor cell line, liver versus mammary gland), we observed a comparable COMT activity (356 ± 12 pmol/min/mg protein) and COMT content (estimated 2 µg COMT per mg cytosolic protein) in our MCF-7 cells.

It has been known for a long time that COMT activity in female rats varies with the estrous cycle (23), suggesting a role of endogenous hormones in the regulation of the gene expression of COMT. In concordance with the findings of Jiang et al. (25), the E2-induced decrease in COMT activity was prevented by an ER antagonist in the present study. Jiang et al. (25) also demonstrated that nuclear extracts of MCF-7 cells, which had been pre-treated with E2, bound to the proximal and distal promoter regions of COMT. A similar molecular mechanism for the interference of the soy isoflavones DAI and GEN with COMT expression was suggested by (i) the close correlation of the induction of proliferation and the reduction of both COMT mRNA and activity and (ii) the prevention of both effects by the ER antagonist ICI. Furthermore, ~10 000-fold higher concentrations of isoflavones were needed to achieve the same effect as with E2, which roughly correlates with ER-binding affinities (32).

When MCF-7 cells were exposed to a combination of E2 and isoflavones at concentrations that would have stimulated maximum proliferation alone, proliferation did not differ from that of cells treated with E2 alone. Based on an ER-mediated mechanism, more than maximum stimulation cannot be expected, and the lack of decreased proliferation makes anti-estrogenic properties of the isoflavones unlikely. However, combination of E2 with the isoflavones at concentrations each inducing about half maximum stimulation also did not (DAI) or hardly (GEN) increase cell proliferation. Due to the low concentration of the isoflavones (0.1 µM), interference of cytotoxic with estrogenic properties seems to be unlikely. It has been proposed that GEN and DAI are selective ER modulators rather than pure ER agonists. Therefore, they might not be able to induce the same response as E2 when bound to the ER. GEN, in particular, is known to interfere with multiple cellular signaling pathways thus further modulating the cellular response to E2 (summarized in ref. 18). However, this modulation seemed to be insufficient to reduce E2-induced proliferation and the estrogenic property of the isoflavones might dominate the effect of low E2/high isoflavone combinations as suggested by an initial experiment (Supplementary Figure 5, available at Carcinogenesis Online).

Besides the formation of the expected metabolite 4-MeO-E2, we also surprisingly detected a second methylation product 3-O-Me-4-HO-E2. With MCF-7 cell cytosol and recombinant COMT protein, only 4-MeO-E2 had been detected so far (4). Since 4-MeO-E2 was the major product and 3-O-Me-4-HO-E2 was only detectable when 50% or more 4-HO-E2 had been converted, the reaction time in the cited study was probably too short for the detection of 3-O-Me-4-HO-E2. It should be noted that alike E2, GEN and DAI are subject to oxidative metabolism yielding metabolites hydroxylated at positions 3' or 6 (GEN) and 3', 6, or 8 (DAI), respectively (38), some of which also exhibit a weak genotoxic and/or estrogenic potential in vitro (39). Since these hydroxylated metabolites are catechols, they are probably to be substrates of COMT. However, up to now, the methylated catecholic oxidation products of GEN and DAI remain elusive.

Our findings raise the question of the relevance of a reduction of COMT activity in humans. Up to now, it is commonly hypothesized that the individual activity of COMT may be one of the etiological factors in estrogen-induced carcinogenesis, supported by in vitro, in vivo and epidemiological studies: E2-induced formation of 8-oxyguanine was observed in MCF-7 cells after induction of CYP expression by 2,3,7,8-tetrachlorodibenzo(p)dioxin and inhibition of COMT activity by a COMT inhibitor (40). Moreover, inhibition of COMT activity increased the cell-transforming activity and related genetic effects of catechol estrogens in Syrian hamster embryo cells (41). Evidence for the consequence of an interference with the inactivation of catechol estrogens on estrogen-related carcinogenesis has been derived from studies using the male Syrian hamster kidney model: exposure to the COMT inhibitor quercetin, which reduced the O-methylation of 2- and 4-HO-E2 by 34 and 22%, respectively, not only resulted in an increased level of catechol estrogens in the hamster kidney but also enhanced E2-induced renal carcinogenesis (12,42). Epidemiological studies on the correlation of polymorphisms of the COMT gene with breast cancer incidence are still controversial: whereas studies among Taiwanese and Caucasian women detected an increased breast cancer risk associated with a low activity of COMT (43–46), studies in other Asian populations found no major associations of breast cancer risk with CYP1B1 and/or COMT polymorphisms (47–49).

Dietary intake of ~40 mg isoflavone per day with soy protein resulted in an increase of follicular phase length and/or delayed menstruation and altered plasma estradiol concentrations (50,51). These doses were much lower than the possible intake via food supplement, which provide up to 0.5 g soy isoflavones per capsule. Our data therefore suggest that isoflavones might prevent the cycling of COMT activity, making the following scenarios possible: normally, without the intake of isoflavones, COMT expression and activity reaches its minimum after the E2 peak during the estrus cycle and increases again when E2 levels decline. In contrast, after consumption of hormonally active concentrations of soy isoflavones, COMT levels will stay low during the whole estrous cycle preventing the normal increase of COMT expression at low E2 concentrations. The resulting permanently low COMT expression, instead of the cycling COMT activity, may have implications for a range of biological effects and for hormonal carcinogenesis.

The MCF-7 cell line is a widely used model system for hormonal carcinogenesis, allowing the study of ER-mediated mechanisms as they may also occur in non-transformed cells. MCF-7 cells bear the low activity COMT allele (4). Studies on the COMT activity of other breast cell lines are scarce. ZR-75 cells expressing the wild-type COMT allele displayed 2- to 3-fold higher COMT activity than MCF-7 cells (4). Although the high and low activity COMT phenotypes were shown to be slightly differentially expressed in brain as well as in lymphoblasts (52), all COMT alleles might be regulated by E2 in the same way. Thus, besides genetic predisposition, diet may also modulate COMT activity complicating studies of COMT phenotypes and cancer risk.

Our study indicates that estrogen-active concentrations of GEN and DAI, which can be reached by consumption of isoflavone-enriched food, may reduce the detoxification of catechols in estrogen-responsive cells. Thus, besides the tumor-promoting potential of GEN, a second possibly adverse property of GEN in hormonal carcinogenesis has been revealed. Our study further demonstrates that besides genetic polymorphisms of the COMT gene, diet may be a stronger influence on COMT activity than hitherto recognized. The role of diet may well be the basis for the contradictory outcomes of studies of COMT phenotypes and breast cancer risk.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary material can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Deutsche Forschungsgemeinschaft (Me574/21-1 and Le1329/7-1).


   Acknowledgments
 
The authors thank Doris Honig and Renate Loske for their help with the HPLC and GC/MS analysis. Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Badawi AF, et al. Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16alpha-hydroxylation of 17beta-estradiol. Metabolism (2001) 50:1001–1003.[CrossRef][Web of Science][Medline]
  2. Zhu BT, et al. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis (1998) 19:1–27.[Abstract/Free Full Text]
  3. Ball P, et al. Catecholoestrogens (2-and 4-hydroxyoestrogens): chemistry, biogenesis, metabolism, occurrence and physiological significance. Acta Endocrinol. Suppl. (Copenh) (1980) 232:1–127.[Medline]
  4. Dawling S, et al. Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol estrogens: comparison of wild-type and variant COMT isoforms. Cancer Res. (2001) 61:6716–6722.[Abstract/Free Full Text]
  5. Klauber N, et al. Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol. Cancer Res. (1997) 57:81–86.[Abstract/Free Full Text]
  6. Cheng Y, et al. 4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells. Toxicol. Appl. Pharmacol. (2007) 220:271–277.[CrossRef][Medline]
  7. Cavalieri E, et al. Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochim. Biophys. Acta (2006) 1766:63–78.[Medline]
  8. Rogan EG, et al. Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: potential biomarkers of susceptibility to cancer. Carcinogenesis (2003) 24:697–702.[Abstract/Free Full Text]
  9. Singh S, et al. Relative imbalances in the expression of estrogen-metabolizing enzymes in the breast tissue of women with breast carcinoma. Oncol. Rep. (2005) 14:1091–1096.[Medline]
  10. Wen W, et al. Expression of cytochrome P450 1B1 and catechol-O-methyltransferase in breast tissue and their associations with breast cancer risk. Cancer Epidemiol. Biomarkers Prev. (2007) 16:917–920.[Abstract/Free Full Text]
  11. Mitrunen K, et al. Molecular epidemiology of sporadic breast cancer. The role of polymorphic genes involved in oestrogen biosynthesis and metabolism. Mutat. Res. (2003) 544:9–41.[CrossRef][Web of Science][Medline]
  12. Zhu BT, et al. Inhibition of catechol O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by quercetin. Possible role in estradiol-induced tumorigenesis. J. Biol. Chem. (1996) 271:1357–1363.[Abstract/Free Full Text]
  13. Adlercreutz H, et al. Phytoestrogens, cancer and coronary heart disease. Biofactors (2004) 22:229–236.[Medline]
  14. Lamartiniere CA, et al. Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J. Nutr. (2002) 132:552S–558S.[Abstract/Free Full Text]
  15. Allred CD, et al. Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein. Carcinogenesis (2001) 22:1667–1673.[Abstract/Free Full Text]
  16. Day JK, et al. Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice. Nutr. Cancer (2001) 39:226–232.[CrossRef][Web of Science][Medline]
  17. Hsieh CY, et al. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res. (1998) 58:3833–3838.[Abstract/Free Full Text]
  18. Messina M, et al. Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings. J. Natl Cancer Inst. (2006) 98:1275–1284.[Abstract/Free Full Text]
  19. Stopper H, et al. Genotoxicity of phytoestrogens. Mutat. Res. (2005) 574:139–155.[Medline]
  20. van Duursen MB, et al. Phytochemicals inhibit catechol-O-methyltransferase activity in cytosolic fractions from healthy human mammary tissues: implications for catechol estrogen-induced DNA damage. Toxicol. Sci. (2004) 81:316–324.[Abstract/Free Full Text]
  21. Boudikova B, et al. Human liver catechol-O-methyltransferase pharmacogenetics. Clin. Pharmacol. Ther. (1990) 48:381–389.[Web of Science][Medline]
  22. Jori A, et al. Homovanillic acid levels in rat striatum during the oestrous cycle. J. Endocrinol. (1973) 58:341–342.[Abstract/Free Full Text]
  23. Parvez S, et al. Activity of catechol-o-methyltransferase in brain regions and adrenal gland during the oestrus cycle. J. Neural Transm. (1978) 42:305–312.[CrossRef][Medline]
  24. Xie T, et al. Characterization and implications of estrogenic down-regulation of human catechol-O-methyltransferase gene transcription. Mol. Pharmacol. (1999) 56:31–38.[Abstract/Free Full Text]
  25. Jiang H, et al. Human catechol-O-methyltransferase down-regulation by estradiol. Neuropharmacology (2003) 45:1011–1018.[CrossRef][Web of Science][Medline]
  26. Anderson KM, et al. Rapid generation of homologous internal standards and evaluation of data for quantitation of messenger RNA by competitive polymerase chain reaction. J. Pharmacol. Toxicol. Methods (1997) 38:133–140.[CrossRef][Web of Science][Medline]
  27. de Kok JB, et al. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab. Invest. (2005) 85:154–159.[Web of Science][Medline]
  28. Rey JM, et al. Semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression patterns of genes involved in the oestrogen pathway. J. Mol. Endocrinol. (2000) 24:433–440.[Abstract]
  29. Brogdon WG, et al. A microassay system for measuring esterase activity and protein concentration in small samples and in high-pressure liquid chromatography eluate fractions. Anal. Biochem. (1983) 131:499–503.[CrossRef][Medline]
  30. Gugler R, et al. Inhibition of human liver catechol-O-methyltransferase by flavonoids. Naunyn Schmiedebergs Arch. Pharmacol. (1973) 276:223–233.[CrossRef][Medline]
  31. Nagai M, et al. Strong inhibitory effects of common tea catechins and bioflavonoids on the O-methylation of catechol estrogens catalyzed by human liver cytosolic catechol-O-methyltransferase. Drug Metab. Dispos. (2004) 32:497–504.[Abstract/Free Full Text]
  32. Kuiper GG, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology (1998) 139:4252–4263.[Abstract/Free Full Text]
  33. Wakeling AE, et al. ICI 182,780, a new antioestrogen with clinical potential. J. Steroid Biochem. Mol. Biol. (1992) 43:173–177.[CrossRef][Web of Science][Medline]
  34. Wakeling AE, et al. A potent specific pure antiestrogen with clinical potential. Cancer Res. (1991) 51:3867–3873.[Abstract/Free Full Text]
  35. Lavigne JA, et al. Concentration-dependent effects of genistein on global gene expression in MCF-7 breast cancer cells: an oligo microarray study. Breast Cancer Res. Treat. (2007) in press.
  36. Payne J, et al. Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens. Sci. Total Environ. (2000) 248:51–62.[CrossRef][Medline]
  37. Rasmussen TH, et al. Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen). Biomarkers (2002) 7:322–336.[CrossRef][Web of Science][Medline]
  38. Kulling S, et al. Oxidative metabolism and genotoxic potential of major isoflavone phytoestrogens. J. Chromatagr. B (2002) 777:211–218.[CrossRef]
  39. Lehmann L, et al. Estrogenic and genotoxic potential of equol and two hydroxylated metabolites of daidzein in cultured human Ishikawa cells. Toxicol. Lett. (2005) 158:72–86.[CrossRef][Medline]
  40. Lavigne JA, et al. The effects of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells. Cancer Res. (2001) 61:7488–7494.[Abstract/Free Full Text]
  41. Hirose Y, et al. Effects of a catechol-O-methyltransferase inhibitor on catechol estrogen-induced cellular transformation, chromosome aberrations and apoptosis in Syrian hamster embryo cells. Int. J. Cancer (2007) 120:1627–1633.[CrossRef][Medline]
  42. Zhu BT, et al. Quercetin increases the severity of estradiol-induced tumorigenesis in hamster kidney. Toxicol. Appl. Pharmacol. (1994) 125:149–158.[CrossRef][Web of Science][Medline]
  43. Gaudet MM, et al. Catechol-O-methyltransferase haplotypes and breast cancer among women on Long Island, New York. Breast Cancer Res. Treat. (2006) 99:235–240.[CrossRef][Medline]
  44. Gaudet MM, et al. Comprehensive assessment of genetic variation of catechol-o-methyltransferase and breast cancer risk. Cancer Res. (2006) 66:9781–9785.[Abstract/Free Full Text]
  45. Huang CS, et al. Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility. Cancer Res. (1999) 59:4870–4875.[Abstract/Free Full Text]
  46. Lavigne JA, et al. An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer. Cancer Res. (1997) 57:5493–5497.[Abstract/Free Full Text]
  47. Hamajima N, et al. Limited association between a catechol-O-methyltransferase (COMT) polymorphism and breast cancer risk in Japan. Int. J. Clin. Oncol. (2001) 6:13–18.[CrossRef][Medline]
  48. Hong CC, et al. Val158Met Polymorphism in catechol-O-methyltransferase gene associated with risk factors for breast cancer. Cancer Epidemiol. Biomarkers Prev. (2003) 12:838–847.[Abstract/Free Full Text]
  49. Wen W, et al. Cytochrome P450 1B1 and catechol-O-methyltransferase genetic polymorphisms and breast cancer risk in Chinese women: results from the shanghai breast cancer study and a meta-analysis. Cancer Epidemiol. Biomarkers Prev. (2005) 14:329–335.[Abstract/Free Full Text]
  50. Cassidy A, et al. Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. Am. J. Clin. Nutr. (1994) 60:333–340.[Abstract/Free Full Text]
  51. Watanabe S, et al. Effects of isoflavone supplement on healthy women. Biofactors (2000) 12:233–241.[Medline]
  52. Zhu G, et al. Differential expression of human COMT alleles in brain and lymphoblasts detected by RT-coupled 5' nuclease assay. Psychopharmacology (Berl) (2004) 177:178–184.[CrossRef][Medline]
Received July 25, 2007; revised October 3, 2007; accepted October 18, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
B. J. Fuhrman, R. Pfeiffer, X. Xu, A. H. Wu, L. Korde, M. H. Gail, L. K. Keefer, T. D. Veenstra, R. N. Hoover, and R. G. Ziegler
Soy Intake is Associated with Increased 2-Hydroxylation and Decreased 16{alpha}-Hydroxylation of Estrogens in Asian-American Women
Cancer Epidemiol. Biomarkers Prev., October 1, 2009; 18(10): 2751 - 2760.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
L. M. Scott, X. Xu, T. D. Veenstra, J. A. Tooze, C. E. Wood, T. C. Register, N. D. Kock, and J. M. Cline
Past Oral Contraceptive Use and Current Dietary Soy Isoflavones Influence Estrogen Metabolism in Postmenopausal Monkeys (Macaca fascicularis)
Cancer Epidemiol. Biomarkers Prev., October 1, 2008; 17(10): 2594 - 2602.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/2/363    most recent
bgm235v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lehmann, L.
Right arrow Articles by Wagner, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lehmann, L.
Right arrow Articles by Wagner, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?