Disposition of soy isoflavones in normal human breast tissue1–4 by shuifanglj


									Disposition of soy isoflavones in normal human breast tissue1–4
Selin Bolca, Mireia Urpi-Sarda, Phillip Blondeel, Nathalie Roche, Lynn Vanhaecke, Sam Possemiers, Nawaf Al-Maharik,
Nigel Botting, Denis De Keukeleire, Marc Bracke, Arne Heyerick, Claudine Manach, and Herman Depypere

ABSTRACT                                                                      The isoflavones genistein and daidzein, which are present in
Background: Despite decades of research on the relation between            soy-derived foods and dietary supplements, as well as equol,
soy and breast cancer, questions regarding the absorption, metabo-         a microbial metabolite of daidzein (11), are partial agonists of
lism, and distribution of isoflavones in breast tissue largely remain       estrogen receptors (ERs) a and b, with a higher in vitro affinity
unanswered.                                                                to ERb than ERa (12–14). Consequently, depending on the
Objective: We evaluated the potential health effects of isoflavone          endogenous estrogen concentrations and the target tissue, these
consumption on normal breast tissue; isoflavone concentrations,             compounds may interfere with ER signaling. Furthermore,
metabolites, and biodistribution were investigated and compared            several other ER-independent mechanisms of action, such as the
with 17b-estradiol exposure.                                               inhibition of steroidogenic enzyme activities, modulation of

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Design: In this dietary intervention study, healthy women were             growth-factor action, down-regulation of tyrosine and other
randomly allocated to a soy milk (n = 11; 16.98-mg genistein and           protein kinases, and the inhibition of angiogenesis, have been
5.40-mg daidzein aglycone equivalents per dose), soy supplement            suggested for isoflavones on the basis of experiments with ani-
(n = 10; 5.27-mg genistein and 17.56-mg daidzein aglycone equiv-           mal and cellular models (15, 16). However, the physiologic
alents per dose), or control (n = 10) group. After a run-in period !4 d,   relevance of these effects in humans is unclear.
3 doses of soy milk or soy supplements were taken daily for 5 d               As recently reviewed by Messina and Wu (1) and Stubert and
before an esthetic breast reduction. Blood and breast biopsies were        Gerber (2), the relation between soy food, isoflavone intake, and
collected during surgery and analyzed with liquid chromatography–          breast cancer has been investigated for a few decades. Never-
tandem mass spectrometry.                                                  theless, the potential health effects of soy and its isoflavones on
Results: After soy administration, genistein and total daidzein con-
                                                                           breast tissue cannot be completely understood until their bio-
centrations, which were expressed as aglycone equivalents, ranged
                                                                           availability has been fully established (17). In particular, ques-
from 135.1 to 2831 nmol/L and 105.1 to 1397 nmol/L, respectively,
in hydrolyzed serum and from 92.33 to 493.8 pmol/g and 22.15 to               1
770.8 pmol/g, respectively, in hydrolyzed breast tissue. The major              From the Laboratory of Microbial Ecology and Technology, Faculty of
                                                                           Bioscience Engineering (SB and SP), the Laboratory of Chemical Analysis,
metabolites identified in nonhydrolyzed samples were genistein-
                                                                           Faculty of Veterinary Medicine (LV), and the Laboratory of Pharmacognosy
7-O-glucuronide and daidzein-7-O-glucuronide, with an overall glu-
                                                                           and Phytochemistry, Faculty of Pharmaceutical Sciences (SB, DDK, and
curonidation of 98%. Total isoflavones showed a breast adipose/             AH), Ghent University, Ghent, Belgium; the Nutrition and Food Science
glandular tissue distribution of 40:60, and their mean (6SEM) de-          Department, XaRTA, INSA. Pharmacy Faculty, University of Barcelona,
rived 17b-estradiol equivalents toward estrogen receptor b were            Barcelona, Spain (MU-S); the Departments of Plastic and Reconstructive
21 6 4-fold and 40 6 10-fold higher than the 17b-estradiol con-            Surgery (PB and NR) and Uro-Gynaecology (HD) and the Laboratory of
centrations in adipose (0.283 6 0.089 pmol/g, P , 0.001) and               Experimental Cancer Research, Department of Experimental Cancer Re-
glandular (0.246 6 0.091 pmol/g, P = 0.001) fractions, respectively.       search, Radiotherapy and Nuclear Medicine (MB), Ghent University Hos-
Conclusion: After intake of soy milk and soy supplements, isofla-           pital, Ghent, Belgium; the School of Chemistry, University of St Andrews,
vones reach exposure levels in breast tissue at which potential            St Andrews, United Kingdom (NA-M and NB); and Institut National de la
                                                                           Recherche Agronomique, UMR 1019, Unite Nutrition Humaine, Centre
health effects may occur.        Am J Clin Nutr 2010;91:976–84.
                                                                           Clermont-Theix, St Genes Champanelle, France (CM).
                                                                                The study and analyses were conducted independently from the support-
                                                                           ing agencies.
INTRODUCTION                                                                  3
                                                                                Supported by Alpro NV and Frutarom Netherlands BV, a PhD grant
   Estrogens regulate the development and function of many                 from the Institute for the Promotion of Innovation through Science and
tissues in men and women but are also implicated in the etiology           Technology in Flanders (IWT-Vlaanderen; to SB), and an FPI fellowship
of breast cancer. Therefore, estrogen-induced cell proliferation           from the Spanish Ministry of Science and Innovation (to MU-S). Soy milk
has been a major focus in breast cancer research. Despite the              (Alpro Soya Drink Nature, Alpro NV, Wevelgem, Belgium) and soy supple-
substantially lower breast-cancer prevalence in populations with           ments (Frutarom Netherlands BV, Veenendaal, Netherlands) were kindly
                                                                           provided by their manufacturers.
a high soy consumption (1, 2), the findings of Allred et al (3–7)              4
                                                                                Address correspondence to A Heyerick, Laboratory of Pharmacognosy
provoked a heated debate about the safety of dietary phytoes-              and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University,
trogens, such as isoflavones, especially for patients with existing         Harelbekestraat 72, B-9000 Ghent, Belgium. E-mail: arne.heyerick@ugent.be.
estrogen-sensitive tumors and women at a high risk of developing              Received October 23, 2009. Accepted for publication January 6, 2010.
breast cancer (1, 8–10).                                                      First published online February 17, 2010; doi: 10.3945/ajcn.2009.28854.

976                                                          Am J Clin Nutr 2010;91:976–84. Printed in USA. Ó 2010 American Society for Nutrition
                                            ISOFLAVONES IN HUMAN BREAST TISSUE                                                       977
tions regarding the absorption, metabolism, and distribution of       and magnesium stearate, and had a titanium-dioxide and iron-
these compounds in the target tissue need to be clarified because      oxide coating.
these processes highly determine whether isoflavones may elicit
protective or rather adverse responses with respect to breast
carcinogenesis. Two studies (18, 19) quantified isoflavones in
human nipple aspirate fluid and/or breast-tissue homogenates              A total of 31 generally healthy Belgian or Dutch women who
after soy supplementation. However, only total concentrations         were scheduled for an esthetic breast reduction were recruited for
after enzymatic hydrolysis were reported, even though the             this study. The exclusion criteria were breast cancer, an antibiotic
conjugation to glucuronic acid and/or sulfate moieties is known       treatment within the previous month, and a soy allergy.
to alter the pharmacologic profiles of genistein and daidzein (20).       Ethical approval was granted by the Ethics Committee of the
In addition, information is missing concerning the biodistribution    Ghent University Hospital (EC UZG 2005/022; initial re-
of isoflavones within breast tissue, although cell-type-specific        cruitment date: 20 February 2007). The volunteers were fully
responses to estrogen exposure have been reported (21), and           informed about the aims of the study and gave written consent to
aromatase, unlike steroid sulfatase and sulfotransferase, is almost   participate in the study.
exclusively expressed in the adipose part of normal breast tissue
(22). Therefore, we assessed the concentrations, nature of
                                                                      Study design
metabolites (free or conjugated), and biodistribution (adipose or
glandular tissue) of orally administered isoflavones in breast            This study was a randomized dietary intervention trial with
tissue with a randomized dietary intervention trial in generally      a run-in period !4 d and a supplementation phase of 5 d before
healthy women undergoing an esthetic breast reduction. In ad-         breast reduction. After eligibility assessment, volunteers were
dition, the estrogenic potential of the isoflavone exposure was        randomly allocated to the soy milk (n = 11), soy supplement (n =

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compared with the endogenous 17b-estradiol (E2) concentrations        10), or control (n = 10) groups. All participants were counseled
measured in the breast biopsies.                                      not to change their habitual, Western-type dietary patterns but
                                                                      were asked to abstain from soy-based products during the ex-
                                                                      perimental period. A detailed list of isoflavone-containing foods
SUBJECTS AND METHODS                                                  and dietary supplements was distributed to guide the volunteers
                                                                      in this respect. In addition, subjects were instructed to report
Chemicals                                                             every case of doubt or fortuitous consumption and to provide
   Genistein, daidzein, and equol were purchased from                 detailed information on that eating occasion including the type
Extrasynthese (Genay, France), and dihydrodaidzein and O-             and portion size of the food and supplements.
desmethylangolensin were purchased from Plantech UK                      During the supplementation phase, either 250 mL soy milk or
(Reading, United Kingdom). Standards of genistein-7-                  one soy supplement was taken daily with breakfast, lunch, and
O-glucuronide and daidzein-7-O-glucuronide were chemically            dinner. The control group did not receive any supplementation
synthesized according to the method of Al-Maharik and Bot-            before surgery. Compliance was evaluated by a subject inquiry
ting (23). For the hydrolysis of conjugated isoflavones, a 33-g/L      and urinary phytoestrogen excretion.
solution of type H-1 Helix pomatia extract (minimum of 300 U             Subjects delivered 2 spot urine samples: a first one after the
b-glucuronidase/mg and 15.3 U sulfatase/mg; Sigma-Aldrich,            run-in period and a second before anesthesia. During surgery
Bornem, Belgium) in sodium acetate buffer (0.1 mol/L, pH = 5)         (12–18 h after the last soy supplementation), blood and breast
was prepared. 4-Hydroxybenzophenone in methanol was used as           biopsies were collected. Serum was obtained by centrifugation
an internal standard in the quantitative analyses of urine and        [10 min at 600 · g, room temperature (RT)] after coagulation.
serum (400 lmol/L) and breast tissue (40 lmol/L).                     Aliquots of urine and serum samples were stored at 220°C until
                                                                      analysis. The tissue samples were immediately frozen in liquid
                                                                      nitrogen and stored at 280°C until analysis. The timings of the
Isoflavone preparations                                                last soy supplementation and sampling were noted. In addition,
   The isoflavone preparations used in this study were com-            a general questionnaire was used to obtain information on each
mercially available soy milk derived from whole soybeans (Alpro       subject’s history of antibiotic treatments, hormonal therapies,
Soya Drink Nature; Alpro NV, Wevelgem, Belgium) in 250-mL             use of any other medication, food-supplement intakes, and an-
cartons and a soy germ powder (SoyLife EXTRA; Frutarom                thropometric measures, whereas dietary habits were assessed
Netherlands BV, Veenendaal, Netherlands) formulated as tablets.       with a validated food-frequency questionnaire (24). The inves-
One batch of each preparation was analyzed in triplicate at the       tigators were blinded to the treatments when working with the
study onset and closure.                                              samples.
   One portion of soy milk (250 mL) contained a mean (6SEM)
of 16.98 6 0.76-mg genistein and 5.40 6 0.22-mg daidzein              Analytic methods
aglycone equivalents (ratio: 3.14) and 8.25 g protein, 7 g car-
bohydrates, 4.75 g lipids, 1.5 g fiber, 0.375 lg vitamin B-12,         Isoflavones in soy milk and soy supplements
0.6 lg riboflavin, and minerals.                                          Before solid-phase extraction, the soy milk was diluted 10-fold
   Each soy supplement contained 5.27 6 0.04-mg genistein and         in 0.1 mol/L hydrochloric acid. The C18 silica columns (5 mL,
17.56 6 0.28-mg daidzein aglycone equivalents (ratio: 0.30) and       500 mg; Bond Elut, Varian, St-Katelijne-Waver, Belgium) were
was filled with cellulose along with the common processing             preconditioned with 5 mL methanol, 5 mL water, and 5 mL
aids, such as sodium carboxymethylcellulose, silicon dioxide,         of 10 mmol/L hydrochloric acid, consecutively. After sample
978                                                          BOLCA ET AL

application (10 mL), the cartridges were rinsed with 5 mL of          20 lL internal standard, were mixed with sodium acetate buffer
10 mmol/L hydrochloric acid, and the compounds of interest            (0.1 mol/L, pH = 5; 50:50, vol:vol), incubated with 100 lL
were eluted with 5 mL methanol by using a VacMaster 20 sample         b-glucuronidase/sulfatase for 24 h at 37°C, and treated with 800
processing unit (IST, Mid Glamorgan, United Kingdom). Finally,        lL of 200 mmol/L hydrochloric acid in methanol. After cen-
the solvent was evaporated at RT under a gentle stream of ni-         trifugation (5 min at 12,500 · g, 4°C), the supernatant fluid was
trogen gas, and the samples were reconstituted in 1 mL methanol       stored at 220°C until analysis.
per 0.1 mol/L hydrochloric acid (80:20, vol:vol) and analyzed by         To study the biodistribution of isoflavones within the breast,
HPLC-ultraviolet detection. The soy supplements were ground in        tissue samples were first dissected into fractions containing al-
a mortar, and 500 mg powder was extracted with 10 mL ace-             most exclusively either pure fat or glandular tissue on the basis of
tonitrile per 0.2 mol/L hydrochloric acid (50:50, vol:vol) and        gross inspection. Areas of adipose tissue intimately intermixed
diluted 5-fold in methanol before HPLC-ultraviolet analysis.          with fibroglandular tissue were avoided, and connective tissue
   Genistin, acetyl genistin, malonyl genistin, genistein, daidzin,   was removed. Next, ’250 mg adipose or glandular breast tissue,
acetyl daidzin, malonyl daidzin, and daidzein were quantified          with 20 lL internal standard, were homogenized in 2.25 mL ice-
with a Waters 2695 Alliance separations module and 996 pho-           cold 200 mmol/L hydrochloric acid in methanol/water (70:30,
todiode array detector (Waters, Milford, MA) combined with an         vol:vol) and 0.25 mL hexane with a T10 ULTRA-TURRAX
XTerra MS C18 reversed-phase column (5 lm; 250 · 4.6 mm;              homogenizer (Ika, Werk Staufen, Germany). After centrifuga-
Waters) at 35°C and by using a gradient of solvent A (ie,             tion (10 min at 1200 · g, RT), the supernatant fluid was col-
6.6 mmol aqueous formic acid/L) and solvent B (ie, 6.6 mmol           lected, and the pellet was extracted with 1 mL ice-cold 200
formic acid in acetonitrile/L) with the following elution profile      mmol/L hydrochloric acid in methanol/water (70:30, vol:vol).
and a flow rate of 1.5 mL/min: 0–20 min, from 10% to 30%               Pooled supernatant fluids were evaporated to dryness at 37°C
solvent B in solvent A; 20.1–27 min, 30% solvent B in solvent         under a gentle stream of nitrogen gas, reconstituted in 250 lL

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A; and 27.1–30 min, from 30% to 10% solvent B in solvent A.           sodium acetate buffer (0.1 mol/L, pH = 5), and incubated with
The injection volume was 20 lL. Ultraviolet detection was             100 lL b-glucuronidase/sulfatase for 24 h at 37°C. Finally,
conducted at 254 nm for all compounds.                                hydrolyzed samples were treated with 1 mL of 200 mmol/L
                                                                      hydrochloric acid in methanol and centrifuged (5 min at
Isoflavones in hydrolyzed urine                                        12,500 · g, 4°C), and supernatant fluids were stored at 220°C
   Total isoflavones in urine were quantified by enzymatic hy-          until analysis.
drolysis and liquid-liquid extraction by using a method validated        Quantification of genistein, daidzein, dihydrodaidzein, equol,
by Wyns et al (25). Briefly, 2 mL urine, with 20 lL internal           O-desmethylangolensin, and the internal standard in hydrolyzed
standard, was mixed with sodium-acetate buffer (0.1 mol/L,            serum and tissue samples was performed by HPLC-MS/MS with
pH = 5) (50:50, vol:vol), incubated with 30 lL b-glucuronidase/       a Thermo Scientific Accella autosampler and pump system and
sulfatase for 1 h at 37°C, and extracted twice with 5 mL diethyl      a triple quadrupole tandem MS operating in positive and nega-
ether. Pooled extracts were evaporated to dryness at 37°C under       tive electrospray ionization modes (TSQ Vantage; Thermo Sci-
a gentle stream of nitrogen gas, reconstituted in 100 lL of the       entific, San Jose, CA), combined with a Hypersil GOLD C18
mobile phase, and stored at 220°C before quantitative HPLC-           reversed-phase column (3 lm, 2.1 · 50 mm; Thermo Scientific)
ultraviolet/mass spectrometer (MS) analysis.                          at 35°C, and by using a gradient of solvent A (ie, 26.5 mmol
   Genistein, daidzein, dihydrodaidzein, equol, O-desmethyl-          aqueous formic acid/L) and solvent B (ie, 26.5 mmol/L formic
angolensin, and the internal standard in hydrolyzed urine were        acid in acetonitrile) with the following elution profile and a flow
quantified with a reversed-phase HPLC (XBridge C18 column,             rate of 300 lL/min: 0–0.5 min, 10% solvent B in solvent A; 0.5–
3.5 lm, 150 · 3.0 mm; Waters) coupled to a photodiode array           2.5 min, from 10% to 100% solvent B in solvent A; 2.5–4.5 min,
detector and single quadrupole MS operating in the positive           100% solvent B; 4.5–4.6 min, from 100% to 10% solvent B in
atmospheric pressure chemical ionization mode (Hewlett-               solvent A; and 4.6–5.1 min, 10% solvent B in solvent A. The in-
Packard 1200 series; Agilent Technologies, Santa Cara, CA)            jection volume was 10 lL. Ionization and detection variables were
according to Wyns et al (25). The limits of detection (LOD,           optimized during infusion experiments with standards of genis-
signal/noise = 3) and limits of quantification (LOQ, signal/noise =    tein, daidzein, dihydrodaidzein, equol, O-desmethylangolensin,
10) were in the lower nanomoles-per-liter range for all analytes      and 4-hydroxybenzophenone. MS/MS data were collected in a se-
(LOD: 3.00–29.74 nmol/L; LOQ: 9.98–99.11 nmol/L).                     lected reaction monitoring mode by monitoring specific transitions
                                                                      of parent and product ions for each analyte: genistein (m/z 271/91/
Creatinine in nonhydrolyzed urine                                     215), daidzein (m/z 253/208/223), dihydrodaidzein (m/z 257/123/
                                                                      163), equol (m/z 244/123/133), O-desmethylangolensin (m/z 257/
   To allow the standardization of diuresis, the urinary excretion    108/136), and 4-hydroxybenzophenone (m/z 199/77/121). Linear
of creatinine was measured according to the conventional kinetic      (r2 . 0.97) calibration curves were obtained over a range of
Jaffe method as described by Bolca et al (26). On the basis of        0.05–10.00 lmol/L in serum and 0.04–8.00 nmol/g in tissue. The
a creatinine clearance rate of 0.163 mmol/(d Á kg) (27), daily        LOD and LOQ were in the lower nanomoles-per-liter range (LOD:
urinary isoflavone excretions were calculated.                         0.26–44.22 nmol/L; LOQ: 0.66–147.18 nmol/L) and picomoles-
                                                                      per-gram range (LOD: 0.38–52.92 pmol/g; LOQ: 1.08–175.50
Isoflavones in hydrolyzed serum and breast tissue                      pmol/g) for all analytes in hydrolyzed serum and breast tissue, re-
  A sample treatment preceding the quantitative HPLC-MS/MS            spectively, except for equol (LOD: 328.68 nmol/L and 445.50
analysis of isoflavone aglycones in hydrolyzed serum was based         pmol/g; LOQ: 1.10 lmol/L and 1.49 nmol/g). The intra- and in-
on the protocol of Guy et al (28). Serum samples (200 lL), with       terassay CVs were 5% and 15%, respectively.
                                           ISOFLAVONES IN HUMAN BREAST TISSUE                                                     979
Isoflavones in nonhydrolyzed serum and breast tissue                 glandular breast tissue was homogenized in 5 mL ethanol/water
    Semiquantitative analyses of isoflavone metabolites in non-      (70:30, vol:vol) with a T10 ULTRA-TURRAX homogenizer
hydrolyzed serum and tissue samples were based on the protocol      (Ika) and, after precipitation (2 · 24 h at 220°C), extracted with
of Guy et al (28). Briefly, 200 lL serum were mixed with 500 lL      5 mL ethyl acetate/hexane (60:40, vol:vol). The organic phase
200 mmol/L hydrochloric acid in methanol, centrifuged (4 min        was evaporated to dryness at 37°C under a gentle stream of
at 12,500 · g, RT), and analyzed by HPLC-MS/MS. Approxi-            nitrogen gas and reconstituted in 500 lL steroid-free serum
mately 250 mg breast tissue were homogenized in 1 mL ice-cold       (Std0-DRG; DRG Instruments GmbH, Marburg, Germany). Fi-
200 mmol/L hydrochloric acid in methanol/water (70:30, vol:         nally, samples were analyzed for E2 with a commercial quan-
vol) with a System POLYTRON PT2100 homogenizer (Kine-               titative immunoassay (EIA-4499, DRG Instruments GmbH).
matica AG, Luzern, Switzerland). After centrifugation (10 min       According to the manufacturer, this kit had a sensitivity ,5.13
at 1200 · g, 4°C), the supernatant fluid was defatted with 1 mL      pmol/L serum; an intra- and interassay CV of 6.4% and 7.6%,
hexane (centrifugation: 10 min at 12,500 · g, RT), evaporated to    respectively; and a cross-reactivity of 0.2% with estrone, 0.05%
dryness at RT under a gentle stream of nitrogen gas, and recon-     with estriol, and ,0.05% with E2. The cross-reactivity with
stituted in 250 lL methanol. Finally, samples were centrifuged      isoflavones was estimated as 0.01%.
(4 min at 12,500 · g, RT), and analyzed by HPLC-MS/MS.
    As described by Guy et al (28), semiquantitative analyses of
                                                                    Statistical analyses
isoflavone metabolites in nonhydrolyzed serum and tissue
samples were performed by HPLC-MS/MS with a Hewlett-                   A statistical program (SPSS for Windows version 15.0; SPSS,
Packard 1100 HPLC system (Agilent Technologies, Waldbronn,          Chicago, IL) was used for all statistical analyses. Results were
Germany) and an Applied Biosystems API 2000 triple quadru-          considered statistically significant at an a 2-tailed level of 0.05.
pole tandem MS (PE Sciex, Concord, Ontario, Canada) equipped        Means and SEMs of urine, serum, and tissue concentrations

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with a Turbo IonSpray source operating in the positive-ion          were calculated. Tests for normality and equality of the var-
mode. A SymmetryShield C18 reversed-phase column (5 lm, 2.1         iances were performed by using the Kolmogorov-Smirnov and
· 150 mm; Waters) with a linear gradient of solvent A (ie,          Levene tests, respectively. Intrasubject comparisons were eval-
26.5 mmol aqueous formic acid/L) and solvent B (ie, acetoni-        uated with the paired Student’s t test or Wilcoxon’s matched-
trile) was used with the following elution profile at 400 lL/min:    pairs signed-rank test, whereas the Student’s t test, analysis of
0–15 min, from 0% to 100% solvent B; 15–19 min, 100% sol-           variance with Bonferroni correction, or Mann-Whitney U test
vent B, and a 6-min reequilibration. The injection volume was       were used to compare means between groups. Associations were
20 lL. Ionization and detection variables were optimized during     described by using paired Pearson’s correlation coefficients or
infusion and flow injection analysis experiments with standards      nonparametric Spearman’s correlations. With the use of the
of genistein, daidzein, equol, genistein-7-O-glucuronide, and       TwoStep cluster-analysis protocol (SPSS for Windows version
daidzein-7-O-glucuronide (28).                                      15.0; SPSS), subjects were phenotyped as weak, moderate, or
    To determine the nature of isoflavone metabolites in serum and   strong equol producers on the basis of the urinary excretion of
tissue, MS/MS data were collected in a multiple-reaction mon-       daidzein aglycone equivalents as equol (equol/total daidzein;
itoring mode by monitoring specific transitions of parent and        total daidzein = daidzein + dihydrodaidzein + equol + O-
product ions for each analyte as follows: genistein (m/z 271/91),   desmethylangolensin) (30).
dihydrogenistein (m/z 273/255), daidzein (m/z 255/91), dihy-
drodaidzein (m/z 257/95), equol (m/z 244/133), genistein-7-O-
glucuronide (m/z 447/271), dihydrogenistein (m/z 449/273),          RESULTS
daidzein-7-O-glucuronide (m/z 441/255), dihydrodaidzein-            Study population
O-glucuronide (m/z 443/257), equol-O-glucuronide (m/z 419/
244), O-desmethylangolensin-O-glucuronide (m/z 445/259), gen-          A total of 31 generally healthy women undergoing an esthetic
istein-sulfoglucuronide (m/z 527/271), daidzein-sulfoglucuronide    breast reduction, all complying with the study protocol, partic-
(m/z 511/255), genistein-sulfate (m/z 351/271), daidzein-sulfate    ipated in this study. On the basis of self-reported weight and
(m/z 335/255), and equol-sulfate (m/z 323/244). For the quan-       height measurements, their age and BMI (in kg/m2) ranged from
titative analyses, linear (r2 . 0.99) calibration curves were ob-   18 to 62 y and 19 to 36, respectively, and did not differ sig-
tained for genistein, daidzein, genistein-7-O-glucuronide, and      nificantly (P = 0.898 and P = 0.103, respectively) between
daidzein-7-O-glucuronide over a range of 0.01–1.00 lmol/L in        groups. None of the participants reported a change in body
blank serum and tissue homogenate, and the LOQ were esti-           weight after soy supplementation. Eight women (27%; 2 women
mated between 8.3 and 30 nmol/L.                                    in the soy milk group, 3 women in the soy supplement group, 3
                                                                    women in the control group) were in the follicular phase of their
                                                                    menstrual cycle, and 8 women (27%; 3 women in the soy milk
E2 in nonhydrolyzed breast tissue                                   group, 4 women in the soy supplement group, 1 woman in the
   To study the biodistribution of E2 within the breast, tissue     control group) were in the luteal phase, whereas 14 women
samples were dissected into fractions containing, almost ex-        (46%; 6 women in the soy milk group, 3 women in the soy
clusively, either pure fat or glandular tissue on the basis of      supplement group, 5 women in the control group) were meno-
a macroscopic inspection. Areas of adipose tissue intimately        pausal. Seven women followed a therapy with exogenous es-
intermixed with fibroglandular tissue were avoided, and con-         trogens [ie, oral contraceptives (19%; 1 woman in the soy milk
nective tissue was removed. Estrogens were extracted as de-         group, 4 women in the soy supplement group, 1 woman in the
scribed by Chetrite et al (29) as follows: ’200 mg adipose or       control group) or intrauterine contraceptives (3%; 1 woman in
980                                                              BOLCA ET AL
              TABLE 1
              Urinary excretion and serum concentrations of genistein, daidzein, dihydrodaidzein, equol, and O-desmethylangolensin
              algycone equivalents 12–18 h after isoflavone supplementation1
                                                            Soy milk                            Soy supplements

                                                  n             Mean 6 SEM                n            Mean 6 SEM                 P2

              Urine (lmol/d)                      11                                     10
                Genistein                         11          2077.7   6   1165.4        10           267.50   6   88.49        0.138
                Daidzein                          11          148.35   6   72.01         10           214.22   6   73.36        0.533
                Dihydrodaidzein                   11           12.58   6   6.03          10            18.21   6   11.44        0.470
                Equol                             11          154.96   6   79.65         10           287.60   6   148.93       0.419
                O-Desmethylangolensin             11          220.33   6   144.98        10           215.09   6   107.98       0.057
              Serum (nmol/L)                      11                                     10
                Genistein                         11          797.04   6   237.27        10           217.89   6   27.61        0.002
                Daidzein                          11          196.10   6   53.53         10           315.79   6   99.61        0.291
                Dihydrodaidzein                   11           90.08   6   17.60         10           146.34   6   26.56        0.085
                Equol                              4          590.93   6   118.34         3           900.52   6   81.40        0.103
                O-Desmethylangolensin             11          130.25   6   44.35         10           137.68   6   35.76        0.899
                     Isoflavone supplementation: 3 doses of soy milk (ie, 50.94-mg genistein and 16.20-mg daidzein aglycone equiv-
              alents/d) or soy supplements (15.81-mg genistein and 52.68-mg daidzein aglycone equivalents/d) daily for 5 d.
                     Student’s t test was used for all analytes under investigation except genistein, dihydrodaidzein, and O-desmethy-
              langolensin in urine and genistein in serum, for which the Mann-Whitney U test was used.

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the control group)], whereas 2 women were treated with anti-                  conjugated aglycones and deconjugated (sulfo)glucuronides and
estrogens (6%; 1 woman in the soy supplement group, 1 woman                   sulfates, measured in hydrolyzed urine and serum (Table 1) and
in the control group). All individuals had an average fat and fiber            hydrolyzed adipose and glandular tissue (Table 2), and com-
intake (24) and Western-type dietary patterns.                                pared between treatment groups. None of the control samples,
   On the basis of their urinary excretion profiles after soy                  ie, all urine samples collected at the end of the run-in phase and
supplementation, 3 participants (14%; 2 subjects in the soy milk              those of the control group after the intervention phase, contained
group, 1 subject in the soy supplement group) were phenotyped as              detectable amounts of soy-derived phytoestrogens. After iso-
moderate equol producers (45.2 6 5.1% equol), and 6 partic-                   flavone supplementation, estimated daily urinary isoflavone
ipants (29%; 3 subjects in the soy milk group, 3 subjects in the              excretion varied considerably between individuals and was in
soy supplement group) were phenotyped as strong equol pro-                    the micromoles-per-day range (6.56–13,138 lmol genistein/
ducers (76.2 6 4.1% equol).                                                   d and 2.38–2234 lmol total daidzein/d). Genistein and total
                                                                              daidzein serum concentrations ranged from 135.1 to 2831 nmol/
                                                                              L and 105.1 to 1397 nmol/L, respectively, with genistein/total
Total exposure                                                                daidzein ratios (G/D) of 1.56 6 0.44 and 0.28 6 0.04 (P =
   Exposure to genistein, daidzein, and its microbial metabolites             0.015) after soy milk and soy supplement administration, re-
after soy supplementation was assessed as the sum of un-                      spectively. Seven women (33%; 4 women in the soy milk group,

              TABLE 2
              Breast adipose and glandular tissue concentrations of genistein, daidzein, dihydrodaidzein, equol, and O-
              desmethylangolensin algycone equivalents 12–18 h after isoflavone supplementation1
                                                            Soy milk                           Soy supplements

                                                   n           Mean 6 SEM                n             Mean 6 SEM                 P2

              Adipose tissue (pmol/g)             11                                     10
                Genistein                         11           183.59 6 14.84            10          146.79 6 31.27             0.287
                Daidzein                          11            45.35 6 6.58             10           61.96 6 13.99             0.282
                Dihydrodaidzein                    2           116.38 6 44.58             1               234.26                  —
                Equol                             11               ,LOD                  10               ,LOD                    —
                O-Desmethylangolensin             11            15.10 6 3.62                          16.82 6 6.19              0.808
              Glandular tissue (pmol/g)           11                                     10
                Genistein                         11           283.71 6 35.88            10          148.85 6 10.66             0.004
                Daidzein                          11            57.26 6 10.65            10           89.16 6 15.54             0.101
                Dihydrodaidzein                    3           208.40 6 67.82             2          368.52 6 171.71              —
                Equol                              1               559.39                 1               446.25                  —
                O-Desmethylangolensin             11            30.80 6 13.86            10           22.33 6 5.11              0.588
                     Isoflavone supplementation: 3 doses of soy milk (ie, 50.94-mg genistein and 16.20-mg daidzein aglycone equiv-
              alents/d) or soy supplements (15.81-mg genistein and 52.68-mg daidzein aglycone equivalents/d) daily for 5 d. LOD, limit
              of detection.
                     Student’s t test was used for all analytes under investigation.
                                               ISOFLAVONES IN HUMAN BREAST TISSUE                                                          981
3 women in the soy supplement group) had circulating equol                     Breast tissue distribution
concentrations .LOD. In breast adipose and glandular tissue,                      Significantly higher concentrations of total isoflavones were
exposure levels of 92.33–493.8 pmol genistein/g and 22.15–                     measured in glandular breast fractions compared with adipose
770.8 pmol total daidzein/g were measured. The G/D in both                     tissues, with an adipose/glandular tissue distribution of 40:60
tissue fractions (adipose soy milk compared with the soy sup-                  (P = 0.025). Genistein showed an adipose/glandular tissue dis-
plement: 3.00 6 0.44 compared with 1.96 6 0.20, P = 0.046;                     tribution of 41:59 and 47:53 after soy milk consumption (P =
glandular soy milk compared with the soy supplement: 3.64 6                    0.002) and soy supplement intake (P = 0.453), respectively.
0.77 compared with 1.16 6 0.20, P = 0.009) correlated well                     Analogously, total daidzein equivalents were more abundant in
with those shown in serum (adipose-serum r = 0.490, P = 0.024;                 the glandular samples of both treatment groups (soy milk: 42:58,
glandular-serum r = 0.574, P = 0.007). However, no significant                  P = 0.286; soy supplement: 34:66, P = 0.047). However, the
correlations were observed between the serum and tissue con-                   G/D were similar in both tissue fractions of the soy milk group
centrations of genistein, total daidzein, and total isoflavones (ie,            (adipose compared with glandular: 3.00 6 0.44 compared with
genistein + total daidzein). Unlike O-desmethylangolensin, equol               3.64 6 0.77, P = 0.351) and soy supplement group (adipose
was shown in only 2 glandular tissue samples (10%; 1 sample in                 compared with glandular: 1.96 6 0.20 compared with 1.16 6
the soy milk group; 1 sample in the soy supplement group).                     0.20, P = 0.051).

Phase II metabolism
   On isoflavone supplementation, the major metabolites iden-                   Physiologic relevance
tified in nonhydrolyzed serum and breast tissue were genistein-                    The exposure to isoflavones and their microbial metabolites
7-O-glucuronide and daidzein-7-O-glucuronide, whereas neither                  was translated to an overall exposure to E2 equivalents toward
                                                                               ERa and ERb (E2a and E2b equivalents), on the basis of the

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glucuronides nor aglycones of microbial daidzein metabolites
were detected (Table 3). We assume that the other metabolites                  generally accepted dose-addition concept (32) and thereby as-
detected with MS/MS transitions 447/271 and 441/255 were 4#-                   suming relative estrogenic potencies toward ERa of 1/100, 1/
O-glucuronides of genistein and daidzein, respectively (31). In                5000, and 1/200 and toward ERb of 1/2, 1/100, and 1/35 for
serum, similar concentrations of both 7-O-glucuronides were                    genistein, daidzein, and equol, respectively (12–14), and an
shown in nearly all (.90%) samples of the soy milk group,                      overall 98% attenuation because of glucuronidation (33–35)
whereas, after soy supplement intake, daidzein-7-O-glucuronide                 (Table 4). These isoflavone-derived E2a and E2b equivalents
was the most abundant metabolite in all cases with only traces of              showed adipose/glandular tissue distributions of 42:58 (P =
genistein-7-O-glucuronide in 6 (67%) samples. Like serum,                      0.029) and 44:57 (P = 0.037), respectively, whereas no signifi-
breast tissue homogenates collected after soy supplementation                  cant differences were observed between adipose and glandular
contained mostly 7-O-glucuronidated isoflavones, but these                      E2 concentrations (56:44, P = 0.102). On both soy milk and soy
were not detectable in all samples. Only traces of genistein and               supplement administration, breast tissue was exposed to esti-
daidzein aglycones were observed, and an overall total glucur-                 mated concentrations of isoflavone-derived E2b equivalents, which
onidation of 98% was estimated.                                                significantly exceeded the endogenous E2 tissue concentrations

               TABLE 3
               Serum and breast tissue homogenate concentrations of genistein, daidzein, and their phase II metabolites 12–18 h after
               isoflavone supplementation1
                                                               Soy milk                          Soy supplements

                                                       n           Mean 6 SEM               n            Mean 6 SEM                P2

               Serum (nmol/L)                          11                                    9
                 Genistein-7-O-glucuronide             10            122   6   68            6             11 6 4                0.030
                 Genistein-4#-O-glucuronide             6             14   6   5             2              4 6 0.13             0.101
                 Genistein                              2             67   6   19            9              ,LOD                   —
                 Daidzein-7-O-glucuronide              11            151   6   50            9            294 6 119              0.249
                 Daidzein-4#-O-glucuronide              9             16   6   6             7             39 6 19               0.238
                 Daidzein                               3             33   6   7             4             49 6 15               0.445
               Breast tissue (pmol/g)                  11                                   10
                 Genistein-7-O-glucuronide              8            268   6   179           4            191   6   65           0.776
                 Genistein-4#-O-glucuronide             5             86   6   46            4             45   6   7            0.463
                 Genistein                              4             12   6   23            3              8   6   23           0.195
                 Daidzein-7-O-glucuronide               5            145   6   72            7            498   6   226          0.178
                 Daidzein-4#-O-glucuronide              2             97   6   18            3            128   6   27             —
                 Daidzein                               6              8   6   13            6             22   6   43           0.010
                      Isoflavone supplementation: 3 doses of either soy milk (ie, 50.94-mg genistein and 16.20-mg daidzein aglycone
               equivalents/d) or soy supplements (15.81-mg genistein and 52.68-mg daidzein aglycone equivalents/d) daily for 5 d.; LOD,
               limit of detection.
                      Student’s t test was used for all analytes under investigation.
                      Values less than the limits of quantification.
982                                                                BOLCA ET AL

(adipose: P , 0.001 and P = 0.003, respectively; glandular: P =               to genistein and daidzein as occur through a rather high soy food
0.004 and P = 0.005, respectively).                                           or supplement intake.
                                                                                 In agreement with previous reports (28, 31, 39), isoflavone
                                                                              glucuronides were the predominant circulating metabolites, with
DISCUSSION                                                                    preferential conjugation at the C7-position for genistein and
   There is a lot of controversy about the soy–breast cancer                  daidzein. No monosulfates or sulfoglucuronides of genistein and
hypothesis (1, 2). Although soy products contain several bio-                 daidzein were observed, whereas diglucuronides and disulfates
active phytochemicals, most cancer research has focused on                    were not monitored. Like serum, breast tissue contained mostly
                                                                              7-O-glucuronidated isoflavones, but these were not detected in
isoflavones and genistein in particular. However, to properly test
                                                                              all samples. Other than the study of Guy et al (28), we are not
and evaluate the suggested mechanisms of action on breast tissue,
                                                                              aware of additional reports on the disposition of phase II me-
information on the concentrations of orally administered iso-
                                                                              tabolites in human tissue.
flavones that actually reach their target site in a bioactive form is
                                                                                 The question is whether the exposure to isoflavones, as ob-
needed. Our results indicate that, 12–18 h after the last soy milk
                                                                              served in the current study, could result in any protective or
or soy supplement intake, breast adipocytes and mammary gland
                                                                              adverse response related to breast carcinogenesis. Given the
epithelial cells were exposed to up to 20–25 pmol/g total iso-
                                                                              complexity of this interaction, our information on in situ con-
flavone aglycones and 900–1150 pmol/g total isoflavone glu-                     centrations, in addition to the current state of knowledge, only
curonides. Because total isoflavone-derived E2b equivalents                    allows for speculation on the potential activities of orally ad-
were, on average, 21 and 40 times more abundant than the en-                  ministered isoflavones on breast tissue. The prevailing opinion is
dogenous E2 in adipose and glandular breast tissue, respectively,             that treatments that trigger ER antagonistic effects, such as ta-
the clinical implications of these findings require further                    moxifen, and/or reduce E2 and estrone concentrations, such as

                                                                                                                                                  Downloaded from www.ajcn.org by guest on July 12, 2011
investigation.                                                                aromatase inhibitors, are protective against breast cancer and
   Although differences in the time of sampling, dosing regimen,              favorably affect the course of breast cancer once diagnosed.
and formulation (36) often hamper sound comparisons between                   Therefore, we discuss the soy–breast cancer hypothesis in the
dietary intervention trials, the hydrolyzed serum and tissue                  context of the potential of isoflavones to interfere with ER-
isoflavone concentrations observed in this study were in agree-                mediated signaling and estrogen synthesis and deprivation.
ment with previous reports on human breast exposure (18, 19, 37)                 Both ER isoforms, ie, ERa and ERb, are expressed in breast
and prostate exposure (28). Isoflavone tissue concentrations were              tissue. Estrogen-induced cell proliferation and breast carcino-
lower than the corresponding serum concentrations (19, 28).                   genesis have been mainly linked to ERa signaling, whereas ERb
Moreover, the consistent lack of a correlation between serum and              can antagonize ERa-dependent transcription (40). This alleged
tissue concentrations (18, 19) suggests that isoflavone serum                  protective ERb-mediated inhibition of ERa signaling, combined
concentrations do not predict tissue disposition. Nevertheless, the           with the greater affinity of isoflavones for ERb than for ERa
G/D ratios correlated well between the different samples and                  (12–14), fueled the enthusiasm regarding soy consumption as
reflected the composition of the isoflavone preparations (28).                  a possible environmental factor responsible for the striking
Because of the well-known large interindividual variation in                  geographic differences in breast cancer occurrence (1, 2).
phytoestrogen bioavailability (38), the current study was not                 However, ligand-specific conformational changes in ER on
sufficiently powered to detect differences related to the isoflavone            binding result in unique coregulator recruitment and transcrip-
formulation (soy milk compared with soy supplements) (30).                    tional and final biological responses, as illustrated in animal
However, our main objective was to measure the exposure ranges                studies (41, 42). Thus, all ligands and metabolites, and even

               TABLE 4
               Breast adipose and glandular tissue exposure to calculated total isoflavone-derived 17b-estradiol a (E2a) and E2b
               equivalents and unconjugated endogenous E2 12–18 h after isoflavone supplementation1
                                                    Soy milk (n = 11)                     Soy supplements (n = 10)                   P2

               Adipose tissue (pmol/g)
                 Isoflavone-derived E2a                 0.04 6 0.01                               0.03 6 0.01                        0.295
                 Isoflavone-derived E2b                 1.85 6 0.15                               1.48 6 0.32                        0.295
                 Endogenous E2                         0.41 6 0.16                               0.14 6 0.05                        0.133
               Glandular tissue (pmol/g)
                 Isoflavone-derived E2a                 0.06 6 0.01                               0.04 6 0.01                        0.044
                 Isoflavone-derived E2b                 2.88 6 0.38                               1.53 6 0.11                        0.004
                 Endogenous E2                         0.35 6 0.17                               0.13 6 0.15                        0.944
                      All values are means 6 SEMs. Isoflavone-derived E2a and E2b equivalents were estimated from isoflavone tissue
               concentrations (in pmol/g) by using the dose-addition concept and taking the relative estrogenic potencies and glucuroni-
               dation into consideration: isoflavone-derived E2a = 0.02 · (0.01 · genistein + 0.0002 · daidzein + 0.005 · equol) and
               isoflavone-derived E2b = 0.02 · (0.5 · genistein + 0.01 · daidzein + 0.029 · equol). Isoflavone supplementation: 3 doses of
               soy milk (ie, 50.94-mg genistein and 16.20-mg daidzein aglycone equivalents/d) or soy supplements (15.81-mg genistein
               and 52.68-mg daidzein-equivalents/d) daily for 5 d.
                      Student’s t test was used for all values except endogenous E2 in glandular breast tissue, for which the Mann-Whitney
               U test was used.
                                           ISOFLAVONES IN HUMAN BREAST TISSUE                                                                    983
mixtures of ligands and metabolites, should be evaluated. Glu-       several phytochemicals with multiple and perhaps additive or
curonidation may increase [eg, morphine (43)] or decrease [eg,       interfering activities. Finally, the LOD and LOQ hindered the
tamoxifen (44)] the bioactivity of its substrate. The latter seems   accurate quantification of isoflavone aglycones, in particular of
to be the case for the estrogenicity of genistein and daidzein       equol, and an underestimation of isoflavone conjugates in en-
(33–35). The in vitro results of Zhang et al (35) suggest that       zymatically hydrolyzed tissues was reported (54). Moreover,
isoflavone glucuronides need to reach intracellular concen-           theoretical concepts and in vitro data were used to translate
trations exceeding 105-106 times those of endogenous E2 to           aglycones concentrations into an overall exposure to E2 equiv-
compete for ER binding. Therefore, we conclude that the con-         alents.
centrations observed in breast tissue are 100–1000 times too low        Taking these limitations and assumptions into consideration,
to result in direct ER-mediated effects. Although genistein and      we conclude that, after soy milk and soy supplement intakes,
daidzein glucuronides may act as a source of tissue aglycones by     isoflavones can reach exposure levels in breast tissue with po-
means of an in situ glucuronidase activity, intracellular UDP-       tential health effects. This study provides data for a more
glucuronosyltransferases may catalyze the opposite reaction.         comprehensive evaluation of the soy–breast cancer relation on
Therefore, only aglycones were taken into consideration to es-       the basis of physiologically relevant exposure levels and
timate the overall E2a- and E2b-equivalent exposure. The total       metabolites.
isoflavone-derived E2b equivalents significantly exceeded the E2
                                                                        The authors’ responsibilities were as follows—SB: designed the study,
tissue concentrations, which were in agreement with literature       recruited and followed the participants, analyzed all samples, performed
reports (29, 45), suggesting that, in this case, soy consumption     the statistical analyses, interpreted the data, and wrote the initial draft of
could elicit partial ERb agonistic (46) effects in human breast      the manuscript; MU-S, LV, and CM: assisted in the analyses and interpretation
tissue.                                                              of the LC-MS/MS data; PB and NR: recruited participants and collected sam-
   Breast tissue E2 concentrations are maintained by the active      ples; NA-M and NB: provided glucuronide standards; SP, DDK, MB, AH, and

                                                                                                                                                          Downloaded from www.ajcn.org by guest on July 12, 2011
uptake of circulating estrogens and/or local synthesis (the in-      HD: assisted in the study design and data interpretation and provided signif-
tracrine organ concept) (47). The putative attenuation of in situ    icant advice; and all authors: participated in critically revising the manuscript.
                                                                     None of the authors had a personal or financial conflict of interest.
steroidogenesis through the inhibition of aromatase, sulfo-
transferase, and 17b-hydroxysteroid dehydrogenase by iso-
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