709Reprod. Nutr. Dev. 45 (2005) 709–720© INRA, EDP Sciences, 2005DOI: 10.1051/rnd:2005055

Original article

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Katie M. EDMUNDSa, Alison C. HOLLOWAYa, Denis J. CRANKSHAWa, Sanjay K. AGARWALb, Warren G. FOSTERa*

a Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Hamilton, Ontario, USA

b Department of Reproductive Medicine, University of California, San Diego, California, USA

(Received 14 January 2005; accepted 26 May 2005)

Abstract – Dietary phytoestrogens have been reported to inhibit aromatase activity in placentalmicrosomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limitingenzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in theendometrium of women with endometriosis and is thought to play a role in the pathophysiology ofthis disease. Therefore, the objective of this study was to screen dietary phytoestrogens for theirability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potentialnovel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity bydirect interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin wastested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultureswere also examined. Genistein and daidzein were inactive in the human recombinant aromatase assaywhereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM)stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositivearomatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures.Taken together, our data suggest that genistein can increase aromatase activity in ESC likely viaincreased enzyme expression.

phytoestrogens / endometriosis / aromatase / genistein / endometrium

1. INTRODUCTION

Cytochrome P450 aromatase (P450AROM)is the rate limiting enzyme that catalyzes theconversion of androstenedione and testo-sterone to estrone and 17β-estradiol,respectively. While the ovaries are the pri-mary source of estrogen in the body, localproduction of estrogen by other tissues hasalso been demonstrated in estrogen depend-

ent diseases such as breast cancer [1, 2] andendometriosis [3–5]. Endometriosis is acommon gynecologic disorder that is char-acterized by the presence of endometrialglands and stroma outside of the uterinecavity. Endometriosis is an estrogen depend-ent disease [7, 8] that affects approximately14% of all women of reproductive age, and30–50% of infertile women [9]. Local pro-duction of estrogens by ectopic endometrial

* Corresponding author: fosterw@mcmaster.ca

Article published by EDP Sciences and available at http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2005055

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implants in women with endometriosis mayexplain treatment failures and the persist-ence of recalcitrant endometriosis in post-menopausal women [6]. Therefore targetedinhibition of local estrogen production inendometriotic lesions by inhibition of aro-matase activity may have a place in themanagement of this disease. As endometri-osis is the leading cause of hospitalizationfor gynecologic surgery [10], thus novel,safe and effective treatment options areurgently needed.

Phytoestrogens are a class of plant estro-gens that include isoflavones, flavones, fla-vonones and several mycotoxins such ascoumestrol and zeralanone. Phytoestrogensare thought to have health benefits such asproviding protection against breast cancerdevelopment [11, 12] and are potentiallyuseful in the management of menopausalsymptoms [13, 14]. Dietary factors such asphytoestrogens have been shown to inhibitaromatase activity [15, 16] without alteringplasma estrogen concentrations [17]. There-fore, therapeutic use of phytoestrogens maybe of benefit to women with endometriosis.

Soy-based foods have high phytoestro-gen content of which genistein is the dom-inant isoflavone [18]. Chrysin, a flavonefound in the plant Passiflora coerula andnaringenin, a flavonone found in citrusfruits, have been shown to inhibit aromataseactivity in hepatocytes and placental micro-somes in vitro [19, 20]. However, theeffects of these compounds on aromataseexpression are unknown. Furthermore, aro-matase expression is regulated via differentpromoter regions in a tissue specific man-ner [21] and thus the effects of phytoestro-gens on endometrial aromatase expressionand activity are also unknown. Therefore,the objective of this study was to screen sev-eral phytoestrogens for their ability todirectly inhibit aromatase activity and todetermine the effect of dietary phytoestro-gens on aromatase expression and activityin human endometrial stromal cells. Genis-tein and daidzein, the dominant phytoestro-gens in the diet, together with chrysin and

naringenin, two phytoestrogens previouslyshown to inhibit aromatase activity wereselected as the test compounds for thisstudy. We hypothesized that phytoestro-gens will inhibit aromatase activity inendometrial stromal cell cultures and thuspotentially provide a novel therapeuticoption that is both natural and effective inthe management of endometriosis.

2. MATERIALS AND METHODS

2.1. Cell-free assay

The ability of the test compounds tointeract directly with the enzyme to alteraromatase activity was investigated in a cellfree assay by modification of the fluores-cence assay described previously [22],using human recombinant aromataseexpressed in insect cell microsomes (CYP19suprasomes BD Gentest Biosciences,Woburn, USA) and 0.25 µM dibenzylfuo-rescein (BD Gentest Biosciences, Woburn,USA) as the substrate. The ability of testcompounds (1 pM–100 µM in 0.1 M potas-sium phosphate buffer pH 7.4) to inhibitaromatase enzyme activity (0.4 pmol aro-matase/well was examined by incubation inthe presence of cofactors (40 µM NADP,100 µM Glucose-6-phosphate, 100 µMMgCl2) and DMSO (1%). Assays were per-formed in a 96-well black walled cultureplate (Becton Dickinson, Franklin LakesUSA) in a total volume of 202 µL. Reac-tions were started by addition of 50 µL ofprewarmed (37 °C) enzyme to the pre-warmed plates. Blank wells contained50 µL of buffer in place of the enzyme. Theplate was incubated at 37 ºC for 1 h and pre-liminary experiments showed that enzymeactivity was linear up to 90 min. The reac-tion was stopped by the addition of 75 µLof 2 M NaOH to each well. Fluorescencewas measured using a PerkinElmer HTS7000 Bio Assay Reader at an excitationwavelength = 485 nm and emission = 535 nm.Fluorescein (Sigma Aldrich, Oakville,Canada) was used as the standard.

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Non-linear least-squares regression analy-sis was used to fit inhibition curves to theequation:

where Emax and Emin are the maximum andminimum effects of the test compound,respectively. pIC50 is the negative log of themolar concentration of the compound thatproduces 50% inhibition of enzyme and logC is the molar concentration of the com-pound that produces the effect E. IC50 val-ues were converted to Kis using the ChengPrusoff equation [23]:

Ki = IC50/(1 + S/Km))

where S is the substrate concentration andKm is the Michaelis constant for theenzyme. The Km and maximum velocity(Vmax) of the enzyme reactions were deter-mined under the same conditions asdescribed for inhibition experiments exceptthat the substrate concentration variedbetween 0 and 0.4 µM. Data for these exper-iments were fit by non-linear least-squaresregression to:

V = (Vmax × S)/(Km + S)

where V is the reaction velocity at substrateconcentration S.

2.2. Endometrial stromal cell culture

Endometrial biopsies were obtainedfrom eighteen women aged 27–44 (mean(± SD) of 38.3 ± 6.0 years) undergoingbenign gynecologic surgery at McMasterUniversity Medical Centre. Informed con-sent was obtained from each patient by aresearch nurse and all procedures were car-ried out in accordance with approval of theMcMaster University Research EthicsBoard. Among the eighteen patients includedin this study, eleven had a laparoscopicdiagnosis of endometriosis and seven didnot have any evidence of pelvic endometri-osis. None of the study subjects hadreceived endocrine therapy in the previoussix months before surgery. Endometrial tis-

sue (1–2 g) obtained at hysterectomy wasrinsed in Hanks’ balanced salt solution (HBSS)containing 200 units·mL–1 penicillin,0.2 mg·mL–1 streptomycin and 0.5 µg·mL–1

amphotericin B (Sigma Aldrich, OakvilleCanada) to remove blood and debris. Sep-aration of the endometrial stromal cells wasperformed as previously described [24].Briefly, the tissue was minced into 1 mm3

fragments and digested for 2.5 h at 37 °Cin medium containing collagenase type IA(2 mg·mL–1, Sigma-Aldrich, Oakville,Canada). After digestion, the remaining tis-sue fragments were mechanically dispersedand the dispersed cells were filteredthrough a 100 µm and subsequently a40 µm cell strainer (Becton Dickson, FranklinLakes, USA). Centrifugation (10 min, 725 ×g) was used to pellet the cells after whichtime they were resuspended in 3 mL of plat-ing media [DMEM:F12, 4% FBS, 1% ITS+and 1% antibiotic antimycotic solution(100 units·mL–1 penicillin, 0.1 mg·mL–1

streptomycin and 0.25 g·mL–1 amphoter-icin B (Sigma Aldrich, Oakville, Canada)].Red blood cells were removed by layeringthe cell suspension over 3 mL of Ficoll-Paque PLUS (Amersham Biosciences,Uppsala, Sweden) in a sterile 15 mL poly-propylene tube. The solution was centri-fuged for 10 min at 400 × g. The media/Ficoll interface layer containing the stromalcells was plated into 48 well Falcon tissueculture plates (Becton Dickson, FranklinLakes, USA) at a density of 200 000 cells/well/0.5 mL. Media was changed after 48 hand the cells were treated after 96 h in cul-ture, when the cells were near confluence.Purity of the cell preparation was confirmedby immunostaining for vimentin (mesen-chymal cell marker) and cytokeratin (epi-thelial cell marker) as described below.

2.3. Cell treatment and aromatase activity assay

Cells were washed twice in HBSS andincubated for a minimum of 1 h in serum-freeDMEM-F12 containing 100 units·mL–1

penicillin, 0.1 mg·mL–1 streptomycin and

E Emin +=

Emax Emin–( )/ 1 10pIC50 Clog––

+( )

712 K.M. Edmunds et al.

0.25 µg·mL–1 amphotericin B (Sigma Aldrich,Oakville, Canada) prior to treatment for 24 hwith increasing log concentrations (10–9–10–4 M) of genistein, daidzein, naringeninor chrysin (Sigma Aldrich, Oakville, Can-ada) diluted in serum free media. To exam-ine the role of estrogen receptor mediatedeffects, the cells were also treated with gen-istein in the presence of a non-selectiveestrogen receptor antagonist (ICI 182,780;Tocris, Ellisville, USA). After 24 h, thetreatment media was removed and replacedwith 500 µL of [1β-3H]-androstenedione[2.5 µCi·mL–1] (Perkin Elmer, Boston, USA)in DMEM-F12 (containing 100 units·mL–1

penicillin, 0.1 mg·mL–1 streptomycin and0.25 µg·mL–1 amphotericin B) for 4 h at37 °C.

Aromatase activity was assayed using aradiometric technique that quantifies theincorporation of tritium from [1β-3H]-androstenedione into 3H-labeled water aspreviously described [25]. Briefly, aro-matase activity was determined by transfer-ring 300 µL of the incubation medium toglass tubes, adding 300 µL of dextrancoated activated charcoal (250 mg·mL–1,BD Biosciences, Oakville, Canada) to eachtube and incubating for 2 h at 4° C. Thesamples were then centrifuged (15 min,2500 × g) and the tritiated water content wasdetermined by counting the supernatant in5 mL of scintillation fluid (Aqueous Count-ing Scintillant, Amersham, England) in aliquid scintillation counter. To control forvariation in the number of cells in each well,the aromatase activity was normalized tothe cell protein content in each well asdetermined by the Bradford method. Due tovariation in basal aromatase activity betweenpatients, normalized aromatase activity wasconverted to a percentage of the controllevel for each culture. The aromatase assayis based on the release of tritiated water andthe specificity of the assay was determinedby co-incubation with 4-hydroxyandros-tendione an irreversible inhibitor of the cat-alytic activity of aromatase [26] to block theformation of tritiated water.

2.4. Immunocytochemistry

Cells were seeded into 8 well Lab-Tekchamber slides (BD Biosciences, Oakville,Canada) at a density of 200 000 cells/well/0.5 mL. Media was changed after 48 h andthe cells were treated with genistein (10–6)after 96 h in culture. After 24 h of treatment,the cells were fixed in 10% neutral bufferedformalin, washed in PBS, and endogenousperoxidase activity was quenched by incu-bating the cells in 3% hydrogen peroxide (inmethanol) for 5 min. The cells were washedin PBS, incubated with the primary anti-bodies (Dako Diagnostics, Mississauga,Canada) for cytokeratin (1:50), and vimen-tin (1:50) for 1 h at room temperature andimmunostaining was identified usingEnVision (Dako Diagnostics, Mississauga,Canada) with diaminobenzidine (Sigma-Aldrich, Oakville, Canada) as the chro-mogen. The cells were counterstained withCarazzi hematoxylin. For negative controls,the cells were incubated with non-immuneserum in place of the primary antibodies. Tostain for the presence of aromatase in thegenistein treated cultures and untreatedcontrols, immunohistochemistry was per-formed on the chamberslides using a pri-mary monoclonal mouse antibody againsthuman aromatase (1:50 Serotec, Raleigh,USA). Immunostaining was identified withthe avidin-biotin-peroxidase technique usingthe Vectastain kit (Vector Laboratories,Burlington, Canada) with diaminobenza-dine as the chromogen and Carazzi hema-toxylin as a counter stain.

2.5. Statistical analyses

Data were analyzed for equal varianceand normal distribution. An effect of treat-ment on ESC aromatase activity was testedusing a one-way analysis of variance(ANOVA) and differences between doseswere determined using the Tukey multiplecomparison method. A p value < 0.05 wasconsidered to be statistically significant forall procedures used.

Genistein induced increased aromatase activity 713

3. RESULTS

3.1. Recombinant human aromatase activity

Aromatase activity in the presence ofincreasing substrate yielded a Km of 0.26 µM(pKm = 6.6 ± 0.2) and a Vmax of 2.2 ± 1 pmolfluorescein released per mol enzyme perminute (Fig. 1). Naringenin (Ki = 0.3 µM)and chrysin (Ki = 1 µM) were potent inhib-itors of recombinant human aromatasewhereas genistein and daidzein were weak(Ki > 50 µM) inhibitors (Fig. 2).

3.2. Aromatase activity after phytoestrogen treatment of endometrial stromal cells

Immunocytochemical staining for cellsof mesenchymal origin and epithelial cellsillustrated that our cultures consisted of

Figure 1. Michaelis-Menten plot of the diben-zyfluorescein deakylase activity of recombinanthuman aromatase determined as described inmaterials and methods. Points represent meansand standard errors of triplicates within a singleexperiment. Km and Vmax values from this expe-riment were 0.22 µM and 1.4 pmol/pmol/min,respectively.

Figure 2. Human recombinant aromatase activity as indicated by fluorimetrically quantified DBFdealkylase after treatment with naringenin, chrysin, genistein and dadizein. Each data point is themean (± SEM) from three separated experiments. Naringenin and chrysin were effective inhibitorsof the enzyme with a Ki = 0.3 and 1.0 µM, respectively, while genistein and daidzein were ineffectiveas shown by a Ki > 50 µM.

714 K.M. Edmunds et al.

greater than 99% endometrial stromal cells(data not shown).

Phytoestrogen treatment did not attenu-ate aromatase activity in ESC from womenwith endometriosis (n = 11) at any concen-tration tested (Fig. 3). However, genistein(10–9–10–6 M) treatment of ESC fromwomen without endometriosis (n = 7) resultedin a significant increase in aromatase activ-ity (P < 0.05) to approximately 150% abovethe activity observed in untreated ESC fromthe same patient (Fig. 4), whereas daidzein,naringenin and chrysin treatment had noeffect. Furthermore, the genistein inducedincrease in aromatase activity was not atten-uated by co-treatment with the estrogenreceptor antagonist ICI 182,780 (P > 0.1,Fig. 5).

3.3. Immunocytochemistry

Immunopositive aromatase staining wasevident as a diffuse brown cytoplasmic pre-

cipitate (Fig. 6) that was absent in controlcultures where the primary antibody wassubstituted with non-immune serum. Immu-nopositive staining was focally present insome but not all genistein (10–6 M) treatedESC from eutopic endometrium of womenwithout endometriosis. Moreover, no immu-noreactive aromatase staining was visible inthe untreated ESC taken from the samepatient.

4. DISCUSSION

The objective of the current study was toscreen dietary phytoestrogens for their abil-ity to inhibit human recombinant aromataseactivity and to determine the effect of die-tary phytoestrogens on endometrial stromalcell aromatase activity in culture. Althoughnaringenin and chrysin inhibited aromatasein our cell-free assay, they were ineffectivein endometrial stromal cell cultures from

Figure 3. Aromatase activity was unchanged in genistein treated endometrial stromal cell culturesfrom women with endometriosis (n = 11). The control bar represents the aromatase activity fromthe vehicle treated cells from each of the patients and the data bars represent the aromatase activityof the cells following treatment with genistein represented as percent of control. The control valuehas arbitrarily been set to 100% and data are presented as the mean ± SEM.

Genistein induced increased aromatase activity 715

Figure 4. The effects of genistein treatment for 24 h on aromatase activity in endometrial stromalcells obtained from eutopic endometrium of women without endometriosis (n = 7). The control barrepresents the aromatase activity from the untreated cells from each of the patients and the data barsrepresent the aromatase activity of the cells following treatment with genistein represented as percentof control. The results are the mean (± SEM) from seven different cultures. Values with differentsuperscripts are significantly (P < 0.05) different.

Figure 5. The effects of 10–6 M genistein (GEN) alone and in combination with 10–6 M ICI 182 780(ICI), and non-selective estrogen receptor antagonist, on aromatase activity obtained from the euto-pic endometrium of women without endometriosis (n = 3). The results are the mean (± SEM) fromthree different cultures. Means identified with a different letter were significantly different (P =0.008).

716 K.M. Edmunds et al.

women with and without endometriosis andthus are unlikely to have any potential ther-apeutic benefit in the management ofendometriosis. In contrast, genistein, thedominant isoflavone found in soy-basedfoods, was inactive in the cell-free assay butto our surprise increased aromatase activityin endometrial stromal cells of womenwithout endometriosis. These data suggestthat the observed effects of genistein are notmediated through direct effects of genisteinon enzyme activity but indirectly viaenhanced aromatase expression in endome-trial stromal cells or via intermediates onaromatase activity. This point is supportedby evidence of immunocytochemical stain-ing for aromatase in genistein treated butnot untreated cells. In our study, the con-centrations of genistein that were used totreat the human endometrial stromal cells(1 nM to 10 mM) correspond to the serumconcentrations of both Asian and Cauca-sian women who are consuming soy-basedfoods [27–29] and thus are considered to bephysiologically relevant. Taken together,our results suggest that while phytoestro-gens may have health benefits such as theproposed protection against breast cancerdevelopment [11, 12], genistein is unlikely

to have any therapeutic value in the man-agement of endometriosis and more impor-tantly may increase aromatase activity inthe endometrium and thus could be animportant factor in the pathobiology of thisenigmatic disease.

In the present study, aromatase was notdetected by immunohistochemistry in con-trol cultures of endometrial stromal cellsfrom women without endometriosis. Inaddition, aromatase activity of vehicletreated endometrial cells was at backgroundlevels for the assay and thus supports theview that aromatase is either absent orinhibited in the endometrium from womenwithout endometriosis. Our findings are inagreement with prior studies in which aro-matase cytochrome P450 has been reportedto be expressed in the endometrium ofwomen with endometriosis but is eitherabsent [4], or expressed at low levels in theendometrium of women without endome-triosis [30]. Therefore, the patients in thecurrent study were grouped into two cate-gories: endometriotic and non-endometri-otic. None of the phytoestrogens testedinhibited aromatase activity of the ESCfrom women with endometriosis. However,in the current study, genistein-treatment of

Figure 6. Immunocytochemical staining for aromatase in untreated cells (A) and cells treated with10–6 M genistein (B) reveals positive staining in the treated cells (arrows).

Genistein induced increased aromatase activity 717

ESC from women without endometriosisinduced an increase in aromatase activity to150% of the untreated controls similar tothe findings using adrenocortical carci-noma cell lines treated with herbicides [31,32]. Furthermore, our results are harmoni-ous with the previous finding that genistein(30 µM) increased aromatase activity 3 foldin the H295R human adrenocortical carci-noma cell line [33]. Hence, genistein treat-ment-induced changes in aromatase activitycould lead to increased local levels of estro-gens in the endometrium. However, thefunctional significance of genistein inducedchanges in aromatase activity is unknown.A previous study has demonstrated thatgenistein treatment increased cell prolifer-ation and was weakly estrogenic inendometrial stromal cell and Ishikawa cellcultures [34]. However, genistein treatmentantagonized the effects of estradiol in thesecultures suggesting that genistein is a com-petitive antagonist of estradiol. Therefore,a genistein induced increase in aromataseactivity and local estrogen production in theendometrium could be relevant in hypoes-trogenic states such as menopause. Whilegenistein treatment was without effect onthe endometrium of macaque monkeyswith surgically induced menopause [35],our proposal is supported by the observa-tion that endometrial hyperplasia was sig-nificantly more prevalent in postmenopausalwomen receiving soy tablets vs. a referencegroup that received a placebo [36]. Moreo-ver, a recent study [37] has also shown thathigh dose phytoestrogens can reverse theantiestrogenic effects of clomiphene citrateon the endometrium. Hence, we proposethat the effects of soy isoflavones, includinggenistein on the endometrium is complexand requires further study.

The mechanism through which genisteintreatment increased aromatase activity inthe endometrium remains unknown. Althoughestradiol has been shown to increase aro-matase activity in ESC cultures [38], sev-eral distinct lines of evidence lead us tosuggest that genistein is not acting throughan estrogen receptor mediated pathway in

our cultures to increase aromatase activity.Genistein is a preferential estrogen receptor(ER)-β agonist and has been shown to haveestrogenic actions in a variety of tissues inthe rat [39, 40]. However, ER-α, not ER-βis the dominant ER sub-type expressed inthe endometrium [41]. Furthermore, it isunlikely that genistein causes stimulationof aromatase in the endometrium by actingthrough a functional estrogen receptor path-way because we have shown that the stim-ulation of aromatase in ESC by genistein isnot attenuated by co-treatment of the cellswith ICI 182,780 which is a non-selectiveestrogen receptor antagonist. We thereforepropose that it is unlikely that genisteinstimulates aromatase activity in endome-trial stromal cell cultures by acting directlyvia the ER. Alternatively, we propose thatgenistein can stimulate aromatase activityin the endometrium through inhibition ofphosphodiesterase activity and result inincreased levels of cAMP. Support for thisproposal comes from evidence that genis-tein inhibits cAMP-phosphodiesteraseactivity in a variety of cell types [42–44]. Inaddition, aromatase expression in theendometrium is regulated through cAMP-induced promoter II [45] and cAMP-treat-ment has previously been shown to result ina 26–60 fold increase in endometrial aro-matase activity [46].

Soy products are widely believed by thepublic to provide health benefits. The Foodand Drug Administration has released anapproval for foods that contain at least6.25 g of soy protein/serving to contain acardiovascular health claim (November 10,1999; No. 279) and this has led to a plethoraof soy-based and fortified foods as well assoy supplements to emerge on to the market[47]. Phytoestrogens are efficiently absorbedafter ingestion and their bioavailability ishigh enough to have biological effects [48].Furthermore, contemporary studies revealthat non-Asian women are ingesting increas-ing amounts of phytoestrogens in their dietas part of a trend towards a healthier lifestyle[49, 50]. Despite potential health benefitsfor women of some age groups, we speculate

718 K.M. Edmunds et al.

that genistein consumption by women ofreproductive age may have associated healthrisks. Moreover, epidemiological evidencedemonstrates that Oriental women have ahigher incidence of endometriosis thanCaucasian women suggesting a link betweenendometriosis and dietary phytoestrogens,as Asian diets are high in soy isoflavones[51, 52]. Hence, consumption of soy prod-ucts by women of reproductive age may notbe without consequence for endometrialaromatase activity and potentially endome-triosis.

In summary, the results of this studydemonstrate that dietary compounds, suchas genistein which is present in foodsincluding soy milk and tofu that the generalpublic views as healthy alternatives to tra-ditional foods in the North American diet,can increase the local production of estro-gen in the ESC. Genistein-induced changesin endometrial aromatase activity may havedetrimental effects which could lead toincreased risk for estrogen-dependent dis-eases which involve the dysregulation ofaromatase such as endometriosis, adenom-yosis and uterine leiomyomas.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the tech-nical assistance of Mary Louise Beecroft, RNScott Phillips and Vasko (Bill) Georgievski.This study was supported by funding from theCanadian Network of Toxicology Centres(WGF) and the Canadian Institutes of HealthResearch (MOP 62681; WGF and ACH).

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