effects of soy isoflavones on estrogen and phytoestrogen ... · methylation (5, 6). although the...

Received 6/9/98: revised 10/1/98: accepted 10/7/98.

The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I This research was supported by NIH Grant CA-66016 and General Clinical

Research Center Grant MOl-RROO400 from the National Center for Research

Resources. The soy powders were kindly donated by Protein TechnologiesIntemational (St. Louis, MO).2 To whom requests for reprints should be addressed, at the Department of Food

Science and Nutrition. University of Minnesota. I 334 Eckles Avenue. St. Paul.

MN 55108. E-mail: [email protected].

3 The abbreviations used are: E,. estradiol: E1, estrone: I6a-(OH)E�. Iflo-hy-

droxyestrone: E5, estriol: 2-0HE2. 2-hydroxyestradiol: 2-)OH)E1, 2-hy-

droxyestrone: 4-(OH )E2, 4-hydnoxyestradiol: 4-(OH )E, . 4-hydroxyestrone: CYP,

cytochrome P-450: low- and high-iso. low- and high-isotlavone, respectively:

ODMA. O-desmethylangolensin.

Vol. 7. /101-1/08, December 1998 Cancer Epidemiology. Biomarkers & Prevention I/UI

Effects of Soy Isoflavones on Estrogen and Phytoestrogen Metabolism

in Premenopausal Women’

Xia Xu, Alison M. Duncan, Barbara E. Merz, andMindy S. Kurzer2

Department of Food Science and Nutrition. University of Minnesota. St. Paul.

Minnesota 55108

Abstract

Isoflavones and lignans are soy phytoestrogens that havebeen suggested to be anticarcinogenic. The mechanismsby which they exert cancer-preventive effects may involvemodulation of estrogen synthesis and metabolism. Toevaluate this hypothesis, a randomized, cross-over soyisoflavone feeding study was performed in 12 healthypremenopausab women. The study consisted of three dietperiods, each separated by a washout of -3 weeks. Eachdiet period lasted for three menstrual cycles plus 9 days(averaging -100 days), during which subjects consumed

their habitual diets supplemented with soy proteinpowder providing 0.16 (control diet), 1.01, or 2.01 mg oftotal isoflavones per kg of body weight per day (10 ± 1.1,65 ± 9.4, or 129 ± 16 mg/day, respectively). A 72-h urinesample was collected during the midfollicular phase (days7-9) of the fourth menstrual cycle in each diet period.Urine samples were analyzed for 10 phytoestrogens and15 endogenous estrogens and their metabolites by acapillary gas chromatography-mass spectrometry method.Urinary excretion of isoflavonoids and bignanssignificantly increased with increased isoflavoneconsumption. Compared with the control diet, increasedisoflavone consumption decreased urinary excretion ofestradiol, estrone, estriol, and total estrogens, as well asexcretion of the hypothesized genotoxic estrogenmetabolites, 16a-hydroxyestrone, 4-hydroxyestrone, and

4-hydroxyestradiol. Of importance are the observations ofa significant increase in the 2-hydroxyestrone/16a-hydroxyestrone ratio and a decrease in the genotoxic/totalestrogens ratio. These data suggest that soy isoflavoneconsumption may exert cancer-preventive effects bydecreasing estrogen synthesis and altering metabolismaway from genotoxic metabolites toward inactivemetabolites.

Introduction

A role for estrogens in the development of breast cancer has

been known for more than a century, since Beatson demon-strated that oophorectomy induced tumor remissions in humanbreast cancer ( 1 ). This role of estrogen in human breast carci-

nogenesisis is supported by observations of estrogen-relatedrisk factors, such as high serum and urine estrogen bevels (2. 3),

postmenopausal obesity, early onset of menstruation, and latemenopause (4). It has been proposed that the total lifetimeexposure of a woman to estrogen is a determinant of her breast

cancer risk.In addition to the major endogenous estrogens, E23 and

specific estrogen metabobites may also influence breast cancer

risk. E, and E1 are metabolized via three major pathways: the

first pathway involves 16a-hydroxylation to form l6a-(OH)E�

and E3; the second pathway leads to formation of 2-(OH)E2and 2-(OH)E1; and the third pathway forms 4-(OH)E, and

4-(OH)E1 (Fig. I ). Both 2-hydroxybated and 4-hydroxylated E2and E1 are catechol estrogens, which are further metabolized by

methylation (5, 6). Although the mechanisms by which estro-gen increases breast carcinogenesis are not entirely clear, sub-stantiab evidence indicates that the key mechanisms are: (a)mitogenic properties of the parent estrogens and their metabo-lites through classical estrogen receptor-mediated processes;and (b) metabolic activation of E1 and E2 to genotoxic metab-

olites such as l6a-(OH)E1, 4-(OH)E2, and 4-(OH)E1 (7-10).The main estrogen metabolite that has been proposed to be

a risk factor for breast cancer is l6cs-(OH)E1. 16a-(OH)E1 hasbeen shown to exhibit genotoxicity through induction of un-

scheduled DNA synthesis and stimulation of anchorage-inde-

pendent growth of mammary epithelial cells ( 1 1 ). 16a-(OH)E1

also irreversibly binds the estrogen receptor, resulting in long-lasting effects such as persistent hyperpnoliferation and

up-regulated expression of the c-rnvc oncogene, even allenwithdrawal (12). Furthermore, the extent of estrogen b6a-hydroxylation is significantly greater in strains of mice thatexpress mammary tumor virus and show high incidence of

mammary cancer, when compared with those strains that do not

express mammary tumor virus (13). In humans. the relativeextent of estrogen metabolism via the I 6cs-hydroxylation path-way is significantly increased in patients with breast cancer

( I 3-17).

Recent studies suggest that 4-hydroxylated catechol estro-gens may be as harmful as 16a-(OH)E� because their electro-philic quinone products react with DNA to form depuninatingadducts known to generate mutations that initiate cancer both invitro and in vivo (10). In vito treatment with 4-(OH)E2 has been

on March 25, 2021. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

HO

2-MeOE22-(OH)E2

� H

4-(OH)E2HOE2 17-epiE3

16a-(OH)E1 E3

E1

HcV©IZ9�16-ketoE2

�

16-epiE3

2-(OH)E1

HO�99�

HO 4-(OH)E1

2-MeOE1

1102 Soy Phytoestrogens and Estrogen Metabolism

H 4-MeOE2

cH3o

HO

�

cH3o 4-MeOE1

Fig. I. Main pathways of estrogen metabolism.

shown to induce DNA single-strand breaks and other mutagenic

products of oxidative damage in liver and kidney of Syrianhamsters ( 1 8, 19). These effects are of particular significancebecause 4-(OH)E, is known to be a potent long-acting estrogen(20-22). Consistent with these observations are in vitro studies

showing that microsomes prepared from human mammary ad-enocarcinoma and fibroadenoma predominantly catalyze the

4-hydroxylation of E,, although this does not occur in micro-somes prepared from normal tissue (23). In the only human

study to report excretion of these metabolites, Aldercreutz et a!.

(24) reported that the average amount of 24-h urinary 4-(OH)E1in premenopausal Finnish women at high risk of breast cancerwas at least double that in premenopausal Asian women at lowrisk of breast cancer.

Because only -5% of all breast cancer cases can beattributed to genetic predisposition, it has been postulated thatdiet may exert cancer-preventive effects through beneficialeffects on endogenous estrogen concentrations and metabolism(25-28). Vegetarian diets result in anovulation (29), lowered

urinary (30) and plasma (29, 31, 32) estrogen levels and in-creased fecal (3 1 ) estrogens. Consumption of indole-3-carbinol,which is abundant in cruciferous vegetables, significantly de-creases urinary excretion of E,, E�, E3, and 16a-(OH)E1 and

significantly increases urinary excretion of 2-(OH)E, and2-(OH)E1, both of which have been proposed to be benign andweak estrogens in men and women (33). A low-fat, high-fiber

diet decreases plasma E1 sulfate concentrations (34) and urinaryexcretion of I 6a-hydroxylated estrogen metabolites and in-

creases urinary excretion of 2-hydroxylated estrogens inwomen (35).

In addition to the traditional low-fat, high-fiber diet, soy-

bean consumption has been suggested to contribute to the lowincidence and mortality of breast cancer in East and Southeast

Asia, also via effects on endogenous estrogens and estrogenmetabolism (36). Epidemiological studies have shown an in-verse association between soybean consumption and risk of

breast cancer (37-40). Ingram et a!. (41) recently reported astrong inverse association between the risk of both premeno-

pausal and postmenopausal breast cancer and urinary excretionof specific isoflavonoids and lignans, phytoestrogens that are

present in soy. Although numerous nonhormonal properties ofsoy phytoestrogens have been reported, they have also beenshown to inhibit key enzymes of estrogen synthesis (42-45)

and specific CYP isoenzymes responsible for producing geno-toxic estrogen metabolites (46, 47). Data in humans are incon-

sistent, as there have been reports of both reduced (48, 49) and



Cancer Epidemiology. Biomarkers & Prevention I /03

Table I Subject characteristics”

Prestudy Control Low-iso diet High-iso diet

Age (yr)

Body weight (kg)

Body mass index

(kg/rn2)

Body fat (%)

26.0 ± 4.9

63.8 ± 7.6

22.5 ± 1.9

28.2 ± 5.2

65.1 ± 8.4

23.0 ± 2.2

28.4 ± 5.4

64.1 ± 8.1

22.6 ± 2.1

28.1 ± 5.4

64.3 ± 8.2

22.7 ± 2.0

27.2 ± 4.5

‘, Values are means ± SD (ii 12).

increased (50) serum E, concentrations in premenopausal

women and reduced (5 1) serum E2 concentrations in postm-enopausal women after soy consumption. Petrakis et a!. (52)

found that prolonged consumption of soy protein isolate causedbreast cell hyperplasia, suggesting an estrogenic effect of soyon breast cells in premenopausal women. At this time, therehave been no reports of the effect of soy isoflavone consump-tion on estrogen metabolism. For this study, we postulated that

soy isoflavone consumption in women will decrease estrogensynthesis and modulate estrogen metabolism away from for-

mation of potentially carcinogenic metabolites. To evaluate thishypothesis, a randomized, cross-over soy isoflavone feedingstudy was performed in 12 healthy premenopausal women.

Materials and Methods

Subjects. This study was a substudy of a larger project per-formed to evaluate the effects of soy isoflavone consumption onreproductive hormones in women. Fourteen subjects partici-

pated in the larger study. All subjects were recruited from theMinneapolis-St. Paul metropolitan area. Exclusionary criteriaincluded athleticism; vegetarian diet; regular consumption of ahigh-fiber, high-soy, or low-fat diet; cigarette smoking; regular

consumption of vitamin and mineral supplementation greaterthan the Recommended Dietary Allowances; current pregnancyor lactation; regular use of medication including aspirin; use of

hormones or antibiotics within 6 months of the start of thestudy; history of chronic disorders including endocrine or gy-

necological diseases; benign breast disease; irregular menstrual

cycles; <90% or >120% ideal body weight; weight change of> 10 lb within the previous year or weight change of >5 lbwithin the previous 2 months; consumption of more than two

alcoholic beverages per day; and a history of food allergies.Health status of the subjects was verified by health history,

physical exam, and routine blood and urine screening.Data from two of the fourteen subjects recruited into the

larger study were eliminated from this substudy due to anovu-

lation and incorrectly timed urine collections. Thus, 12 subjectsparticipated in this substudy (Table 1 ). Their prestudy averages

for age, body weight, body mass index, and percentage body fatwere 26.0 ± 4.9 years, 63.8 ± 7.6 kg, 22.5 ± 1.9 kg/m2, and

28.2 ± 5.2%, respectively.

Study Design and Diet. Prior to the study, the protocol wasapproved by the University of Minnesota Institutional ReviewBoard Human Subjects Committee. The study was performed

using a randomized, cross-over design following the individualmenstrual cycles of each subject. The study consisted of threediet periods, each separated by a washout period of -3 weeks.Each diet period lasted for three menstrual cycles plus 9 days,during which subjects consumed their habitual diets supple-mented with a soy protein powder (Protein Technologies In-

ternational, St. Louis, MO), providing a daily dose of 0. 16 ±

0.01 (control), 1.01 ± 0.04 (low-iso diet), or 2.01 ± 0.03(high-iso diet) mg of total isoflavones per kg of body weight

(10 ± 1 . 1, 65 ± 9.4, and 129 ± 16 mg of total isoflavones pen

day for control, low-iso, and high-iso diets, respectively). Theconcentrations of all 12 isoflavone isomers (the aglycone, glu-

coside, acetylglucoside, and malonylglucoside forms of daid-zein, genistein, and glycitein) were analyzed in the labonatory

of Professor Pat Murphy at Iowa State University by a reversed-

phase high-performance liquid chromatography method, as de-

scnibed previously (53). On average, the proportions of daid-zein, genistein, and glycitein were 37, 55. and 8%, respectively,

of the total isoflavones in the soy protein powders. Ninety-

seven % of daidzein, 97% of genistein, and 91% of glycitein

were present as their glucoside conjugates, and the remainder

were present as the aglycones. The composition of the soyprotein powders has been described previously (54).

Subjects were free living throughout the entire study. Theywere instructed to minimize phytoestrogen ingestion by avoid-

ing whole grains, oilseeds such as flaxseed (rich in lignans). andlegumes, including soybean foods and foods containing textur-

ized vegetable protein, hydrolyzed vegetable protein, or soy

protein isolate. Food intake was monitored by two 3-day dietrecords kept during each menstrual cycle, one during the fol-

licular phase and one during the luteal phase. Energy, macro-

nutrient, and dietary fiber intakes were analyzed by a comput-

erized nutrient analysis program (Nutritionist IV, Version 4.0:The Hearst Corporation, San Bruno, CA). Body density was

calculated from the sum of the four-skinfold thickness and apredictive equation was used to determine percentage body fat

(55).

Sample Collection and Analysis. Three continuous 24-h

urine samples were collected during the midfolbicular phase(days 7-9) of the fourth menstrual cycle of each diet period.

Twenty-four-h urines were collected in 3-liter containers con-taming 3 g of ascorbic acid. Urinary creatinine was measured to

evaluate the completeness of urine collection using an enzy-matic assay kit (Johnson & Johnson Clinical Diagnostics Inc.,Rochester, NY). After recording the 24-h urine volume, sodium

azide was added to achieve a 0. 1% (w/v) concentration. Urine

samples were stored at -20#{176}C until analysis. Immediatelybefore analysis, the three 24-h urine aliquots from menstrual

cycle days 7-9 were thawed and proportionally combined tocreate a 72-h pooled sample.

Ten-ml aliquots in duplicate from each 72-h pooled urine

sample were extracted and analyzed for 10 phytoestrogens

(equol, ODMA, dihydrodaidzein, daidzein, genistein, glycitein,enterodiol, enterolactone, matairesinol, and coumestnob) and 15

endogenous estrogens and their metabolites [Fig. 1; E1, E2, E3.l6a-(OH)E , 2-(OH)E , 2-(OH)E,, 4-(OH)E1 , 4-(OH)E2,

2-methoxyestrone, 2-methoxyestradiob, 4-methoxyestrone,4-methoxyestradiol, 16-ketoestradiol, 16-epiestriol. and 17-

epiestriol] by an ion-exchange chromatography and capillarygas chromatography-mass spectrometry method originally de-

veboped by Adlercreutz and colleagues (56-58). Briefly, after

ethoximation to protect the carbonyl functions, phytoestnogens

and estrogen metabolites were extracted on Bond Elut C18columns (Chrom Tech, Apple Valley, MN). Seven deuterated

phytoestrogen internal standards (synthesized by Drs. T. Haseand K. W#{228}h#{228}l#{228},Department of Chemistry. University of Hel-

sinki, Helsinki, Finland; Ref. 58) and nine deuterated estrogen

internal standards (Ref. 57; C/D/N Isotopes Inc., Pointe-Claire,Quebec, Canada) were added to each sample for compounds ineach of four analyte fractions described below. The aliquots

were subsequently hydrolyzed with He!ix potnatia enzyme ex-

tract (Sigma Chemical Co., St. Louis, MO). The isoflavonoid

fraction was separated from the nest of the compounds on the



I 104 Soy Phytoestrogens and Estrogen Metabolism

Table 2 Dietary intake”

Prestudy Control Low-iso diet High-iso diet

Length ofdiet periods (day)5 N/A 102 ± 22 99 ± 13 98 ± 12

Isotlavone (mg/day)” N/A 10 ± 1.1 65 ± 9.4 129 ± 16

Isotlavone (mg/kg/day)” N/A 0.16 ± 0.01 1.01 ± 0.04 2.01 ± 0.03

Energy’

kcal/day 2016 ± I29� 2339 ± 82’ 2329 ± 80� 2268 � 79t

Mi/day 8.43 ± 0.54* 9.79 ± OW 9.74 ± 0.34t 949 ± 0.33k

Protein (g/day)’ 71.3 ± 59* 116.0 ± 43t I 5.0 ± 431 I 7.4 ± 4.2k

Fat (5/day) 71.6 t 6.0 79.0 ± 3.3 76.2 ± 3.3 70.6 � 3.2

Carbohydrate (glday)’ 281 ± 17 302 ± 10 305 ± 10 298 ± 10

Dietary fiber (g/day)’ 12.2 ± 0.9* 8.9 ± 0.4k 9.2 ± 0.41 8.3 ± 0.4k

,‘ Prestudy diet data were based on one 3-day food record per subject: Control. low-iso. and high-iso data were based on seven 3-day food records per subject. a 12subjects. N/A. not applicable. Values within rows with different symbols (* or t) were significantly different (P < 0.05).

I, Values are means ± SD.

‘ Least squares mean ± SE.

acetate form of QAE-Sephadex columns (Sigma). Estrogenmetabobites with vicinab cis-hydroxyls (such as catechol estro-

gens) and lignan fractions were separated out on the borate andbicarbonate forms of QAE-Sephadex columns, respectively.

Neutral steroids were removed from the estrogens withoutvicinal cis-hydroxyls [such as E.,, E1, E3, and 16a-(OH)E1] by

the free base form of DEAE-Sephadex columns (Sigma). Tn-methylsilyb derivatives of the samples and standards were an-

alyzed by a Hewlett Packard (Wilmington, DE) 5890 and597 1A quadrupole gas chromatography-mass spectrometry in-

strument operated with a Unix 59940A ChemStation and a HP7673 autosampler in the selective ion-monitoring mode.

All samples from each subject were analyzed in duplicatein the same batch. Duplicate quality control urine samples from

the midfollicular phase were also analyzed with each batch. For

the phytoestrogen analyses, intra-assay coefficients of variationranged from 0.4 to 6.9%, and interassay coefficients of varia-

tion ranged from 2.0 to 10. 1% . For the estrogen metaboliteanalyses. intna-assay coefficients of variation ranged from 1.5

to 6.5%, and interassay coefficients of variation ranged from1.3 to 11.3%.

Statistics. The effects of diet on urinary excretion of phy-toestrogens and estrogen metabolites were determined byANOVA (GLM) using Statistical Analysis System, Version6.12 (SAS Institute, Inc., Cary, NC). Subjects and diet periodswere treated as blocks. Data were examined for homogeneity ofvariance before ANOVA (GLM). If necessary, bog-transforma-tion of the data was performed before analysis. A P value of<0.05 was considered to be significant.

Results

Diet and Body Weight. The diet and bod�’ weight and com-

position results for the entire group of I 4 subjects were reportedseparately (54). Body weight, body mass index, and percentage

body fat of the I 2 subjects in this substudy are presented inTable I . The mean consumption of energy, macronutnients, and

dietary fiber (including the contribution from the daily soypowder) for the 1 2 subjects in this substudy are shown in Table2. There were no significant differences in body weight, bodymass index, percentage body fat, or mean daily consumption of

energy, macnonutnients. or dietary fiber among the three dietperiods, although prestudy energy. protein, and dietary fiber

consumption were significantly different from those during thestudy (Tables I and 2).

Urinary Phytoestrogens. Subjects’ urinary creatinine wasquite consistent, as shown by an average within-subject coef-

ficient of variation of 5-6%. We, therefore, concluded that the24-h urine collections were complete and chose to present our

data as nmol of phytoestrogen per 24 h. When data were

analyzed relative to body weight (nmol of phytoestrogen per kg

of body weight per 24 h), to account for differences in isofla-

vone consumption due to body weight differences, or relative to

urinary creatinine (nmol of phytoestrogen pen mmob of creati-nine), the same results were obtained.

As a result of unequal variance, all phytoestrogen data

were log-transformed before data analysis. Table 3 shows the

geometric means and 95% confidence intervals for the urinaryphytoestrogen data. Compared with the control diet, urinary

excretion of isoflavonoids (genistein, daidzein, dihydrodaid-zein, and glycitein) and lignans (enterodiol and enterolactone)

were significantly increased by isoflavone consumption in a

dose-dependent manner (Table 3). Urinary isoflavone metabo-

bites (ODMA and equol) were also significantly increased byboth the low-iso and high-iso diets, despite the high variability

in excretion (Table 3). Equol variability was particularly high:

4 of the 12 subjects were equol-producers who excreted high

amounts of equol, and the other 8 subjects were predominantODMA-producers who excreted minimal amounts of equol.

During all three diet periods, urinary excretions of coumestrol

and matairesinol were extremely low. Nevertheless, urinary

coumestrol was significantly increased by both the low-iso andhigh-iso diets; urinary matairesinol was significantly increased

by high-iso diet only (Table 3).

Urinary Estrogens and Estrogen Metabobites. Urinary ex-

cretion of E,, E3, 4-(OH)E,, 4-(OH)E1, 2-(OH)E2, 2-me-thoxyestrone, 16-ketoestradiol, and 16-epiestriol were signifi-

cantly decreased by both the low-iso and high-iso diets,

although in most cases, there was no significant difference

between these two diets (Table 4). Urinary excretion of total

estrogens, E1, 16a-(OH)E1, and 4-methoxyestrone were signif-

icantly decreased by the high-iso diet when compared with the

control diet, but they were not decreased by the low-iso diet

(Table 4). Although in most cases, we were unable to statisti-cabby differentiate the low-iso diet from one on both of the other

diets, a trend toward a dose response is apparent. Urinary

excretion of � , 2-methoxyestradiol, 4-methoxyestra-

diob and 17-epiestriol were not significantly affected by diet,although a trend toward decreased excretion with isoflavone

intake is apparent (Table 4).

The ratio of genotoxic estrogen metabolites [l6a-(OH)E,,

4-(OH)E,, and 4-(OH)E�] to total estrogens was significantlydecreased by both the low- and high-iso diets (Table 5). The



Cancer Epidemiology, Biomarkers & Prevention 1105

Table 3 Urinary phytoestrogens (nmol/24 hY’

Control (,i = I 1 )h Low-iso diet (n = 1 1 )‘ High-iso diet (ii = 12)

Isoflavonoids

Genistein 997 (807_l,232)* 6,529 (5.378_7,925)t 14,200 ( I 1.75 I-l7,I59)�

Daidzein 995 (719-l,378)� 4,9f4 (3,634-6.781 )‘ 9,528 (7,1 l()-l2,768)�

Dmhydrodaidzein 364 (249-532)� 1,538 ( l.057_2,236)t 2.799 ( l,986-3.946)�

ODMA 1,155 (805_l,657)* 8,656(6,007_12,474)t 20.292 ( 14.65I_28.l06)t

Equol 1 17 (62-222)� 270 (l42_5I2)t 438 (246-780)’

Glycitemn 262 (205-334)� 1 .4 19 ( 1 , I 06- 1.822)’ 2,5 10 (2.0 I 4-3, I 28)0

Coumestrol 26.9 (22.3_32.6)* 38.6 (3l.9�46.8)t 36.8 (31.1-43.6)’

Lignans

Enterodmol 171 (l43_204)* 251 (211-298)’ 296 (252-347)’

Enterolactone 1.132 (97l_l,320)* 1,874 ( 1,600-2,196)’ 2,782 (2.422_3,l96)0

Matairesinol 8.9 (7.1-1 1 .1 )* 10.9 (8.7-l3.6)�’ I 3.9 ( I I .4-16.9)’

Total 6,438 (5,533_7,49l)* 31,132 (26,696-36,306)� 64,666 (56,418�74.121)�

‘, Due to unequal variance. all data were log-transformed before data analysis. Geometric means (95% confidence intervals) are presented. When data were analyzed relative

to urinary creatinine (nmol of phytoestrogens/mmol of creatinine) or relative to body weight (nmol of phytoestrogens/kg of body weight/24 h). the same results wereobtained. Values within rows with different symbols (8, t, or �) were significantly different. (P < 0.05).b One subject did not collect urine samples at the end of the control diet period.‘. One subject used antibiotics at the end of the low-iso diet period. and her data were not included.

Table 4 Urinary estrogens and estrogen metabolites (nmol/24 h)”

Control Low-iso diet High-iso diet

(n II)” (n 11)’ (n 12)

E, 7.71 ± 0.31* 6.38 ± 0.31’ 5.90 ± 0.28”

E� 17.49 ± 0.65� 16.15 ± 0.64*” 14.95 ± 0.59’

E3 1 1.29 ± 0.39* 9.80 ± o,39t 8.19 ± 0.36�

l6a-(OH)E�” 3.67 � 0.25

2.67 (2.24-3.l9)�

3.36 ± 0.19

2.16 (l.82-2.56)�’

2.84 ± 0.22

1.75 (1.49-2.06)’

4-(OH)E, 1.75 ± 0.I7� 1.04 ± 0.l7t 0.92 ± 0.16’

4-(OH)E, 5.44 ± 0.51

4.81 (4.03_5.74)*

3.87 ± 0.51

3.46 (2.91-4.10)’

3.53 ± 0.46

3.39 (2.89-3.97)’

2-(OH)E, 4.86 ± 0.18* 4.06 ± 0.19t 3.71 ± 0.17t

2-(OH)E1 36.05 ± 1.72 38.27 ± 1.74 34.32 ± 1.57

4-MeOE2 0.14 ± 0.01 0.16 ± 0.02 0.12 ± 0.01

4-MeOE1” 0.47 ± 0.08

0.40 (0.31�0.5l)*

0.30 ± 0.08

0.29 (0.23�0.37)*t

0.28 ± 0.07

0.27 (0.2l�0.33)t

2-MeOE, 2.45 ± 0.1 1 2.34 ± 0.1 1 2.15 ± 0.10

2-MeOE1” 7.77 ± 0.27

7.31 (6.85_7.80)*

6.78 ± 0.27

6.48 (6.06�6.92)t

6.23 ± 0.24

5.94 (5.6O-6.3l)�

l6-ketoE2 2.70 ± 0.1 1* 2.27 ± 0.1 1” 2.00 ± 0.10’

l7-epiE� 0.88 ± 0.08 0.72 ± 0.08 0.79 ± 0.08

16-epiE3 2.59 ± 0.16* 2.01 ± 0.16’ 2.02 ± O.lS�

Total estrogens 105.26 ± 2.60k 97.50 ± 2.67k’ 87.93 ± 2.38”

‘, Least squares means ± SE. When data were analyzed as nmol of estrogenmetabolite/mmol of urinary creatinine, the same results were obtained. 4-MeOE2,

4-methoxyestradiol: 4-MeOE1, 4-methoxyestrone; 2-MeOE2, 2-methoxyestra-

diol; 2-MeOE1, 2-methoxyestrone; l6-ketoE2, 16-ketoestradiol; l7-epiE3, 17-

epiestriol; 16-epiE3. 16-epiestriol. Values within rows with different symbols (*,

t. or �) were significantly different (P < 0.05).

5 One subject did not collect urine samples at the end of the control diet period.

‘- One subject used antibiotics at the end of the low-iso diet period, and her data

were not included.‘I Due to unequal variance, data were log-transformed before ANOVA (GLM).Geometric means (95% confidence intervals) are presented below the least

squares means.

genotoxic estrogen metabolites made up -8% of the totalestrogens in subjects consuming the high- or low-iso diets,

compared with - 10% in subjects consuming the control diet.The ratio of 2-(OH)E1 to 16a-(OH)E1 was significantly in-creased in subjects who consumed the low-iso diet comparedwith the control diet, although the high-iso diet was not statis-

tically distinguishable from the other diets. The ratio of 2E1-total to l6a-total (which reflects the ratio of 2-hydroxylation of

E1 to 16a-hydroxylation of E1; Ref. 59), was significantly

increased by both the low- and high-iso diets (Table 5).

Table 5 Urinary estrogen metabolite ratios”

Control Low-iso diet High-iso diet

(n 11)” (ii = II)’ (‘1 = 12)

Genotoxic/total 0.10 ± 0.01 * 0.08 ± 0.01 � 0.08 ± 0.01’

2-(OH)E1/l6a-(OH)E� 18.00 ± 3.92k 31.37 ± 3.97’ 28.33 ± 3.58*t

2E�-tota1/l6a-totaI” 2.91 ± 0.40 3.92 � 0.41

2.72 (2.34-3.l6)� 335 (2.87-3.91)’

3.76 ± 0.37

3.49 (3.05-4(X))’

2E,-total/4E1-total 8.51 ± 0.79k I 1.91 ± 0.76’ 10.68 ± t).72”

2E,-total/4E,-total 4.42 ± 0.69 5.42 ± 0.7 I 6.44 ± 0.63

2-total/4-total 7.17 ± 0.59* 10.17 ± 0.58’ 9.45 ± 0.54t

“ Least squares means ± SE. Genotoxic/total = (l6a-(OHE1 +4-(OH)E2+4-(OH)E, )/total estrogens: 2E� -totalllfla-total = + 2-MeOE, )/( l6a-

(OH)E1 +E3+ l7-epiE3): 2E1-total/4E1-total = (2-(OH)E, +2-MeOE1 )/(4-(OH)E1+4-MeOE1): 2E�-totaV4E,-total = (2-(OH)E�+ 2-MeOE,)/(4-(OH)E,+4-MeOE2:

2-total/4-total = (2-(OH)E� + 2-(OH)E,+ 2-MeOE1 + 2-MeOE,)/(4-(OH)E1 +4-

(OH)E,+4-MeOE1 +4-MeOE�). 2-MeOE1. 2-methoxyestrone: 2-MeOE2. 2-me-

thoxyestradiol; 4-MeOE� , 4-methoxyestrone: 4-MeOE�. 4-methoxyestradiol. Values

within rows with different symbols (8 or t) were significantly different (P < 0.05).

/‘ One subject did not collect urine samples at the end of the control diet period.‘. One subject used antibiotics at the end of the low-iso diet period. and her datawere not included.

,! Due to unequal variance. data were log-transformed before ANOVA (GLM).

Geometric means (95% confidence intervals) are presented below the least

squares means.

The ratios of 2E1-total to 4E1-total (which reflects the ratio

of 2-hydroxylation to 4-hydroxylation of E1), and 2-total to4-total (which reflects the ratio of 2-hydroxylation to 4-hy-droxybation for both E1 and E2), were significantly increased by

both the low- and high-iso diets (Table 5). There was also atrend toward an increase in the ratio of 2E2-total to 4E,-total

(which reflects the ratio of 2-hydroxylation to 4-hydroxybationof E2) by the low- and high-iso diets, although this was notstatistically significant.

Discussion

As expected, increased urinary excretion of isoflavonoids

(daidzein, genistein, glycitein, dihydrodaidzein. ODMA, andequol) was observed in our subjects after both low and high soyisoflavone consumption for more than three menstrual cycles

(an average of - 100 days), in a clear dose-dependent manner.

These results are consistent with those reported from short-term(1- or 9-day) soy feeding studies (60-62), although the short-



I 106 Soy Phytoestrogens and Estrogen Metabolism

term studies found daidzein to be the major urinary isofla-vonoid, and we observed the daidzein metabobite ODMA to bethe predominant urinary isoflavonoid. This difference suggests

that the length of soy consumption may influence the activitiesof gut microfboral enzymes responsible for ODMA formation.

In addition to providing isoflavones, soy likely contains

small amounts of plant lignans such as secoisobaniciresinol and

matairesinol, which can be converted to the mammalian big-nans. enterodiol, and entenolactone by human gut bacteria (63).Although the quantities are low, they are significant, as shownby significantly increased lignan excretion after soy consump-

tion. It is also possible that consumption of soy isoflavones

alters intestinal microflora toward increased bignan production.The between-subject variability in urinary phytoestrogen

excretion within a diet was great, even after adjusting fordifferences in consumption due to provision of isoflavones

relative to body weight. Within a diet, excretion of genistein,

daidzein, ODMA. and equol varied by 3-. 4-, 7-, and 800-fold.respectively. This high variability in phytoestrogen excretion

after soy consumption is consistent with previous reports (61,62, 64) and is likely due to the dependence of phytoestrogenformation and absorption on the composition of gut microflora

(65), which varies substantially among individuals (66). Thisvariability was particularly high for equol. because only 4 of the

I 2 subjects excreted significant quantities, consistent with pre-vious reports that 30-40% of human subjects produce large

quantities of equol after soy consumption (67).Our data show that overall urinary estrogen excretion was

significantly decreased by soy phytoestrogen consumption, as

reflected by reduced excretion ofE,, E1, E3, and total estrogens.

Because we did not observe significant effects on plasma es-trogen concentrations (54), reduced urinary estrogen excretionis likely due to decreased estrogen synthesis rather than de-creased clearance. This explanation is consistent with in vitro

studies showing that soy phytoestrogens reduce estrogen syn-thesis through inhibition of key steroidogenic enzymes such as

aromatase (42-44), 3/3-hydroxysteroid dehydrogenase, andI 7j3-hydroxysteroid dehydrogenase (45, 68, 69).

Excretion of the genotoxic estrogen metabolites l6a-

(OH)E�, 4-(OH)E,, and 4-(OH)E1 was also significantly re-duced by soy phytoestrogen consumption. Evaluation of theestrogen metabolite ratios suggests that, in addition to the

absolute amounts, the relative amounts of both 16a-hydroxy-lation and 4-hydnoxylation of estrogens (pathways producingthe genotoxic estrogen metabolites) were significantly de-creased after soy phytoestrogen consumption. At the same time,

the proportion of estrogen metabolized by 2-hydroxylation (a

pathway producing benign and weak estrogen metabolites) wassignificantly increased when compared with the proportion

metabolized by 16a-hydroxybation, although the absoluteamount of 2-(OH)E1, the most abundant estrogen metabolitefound in urine, was not significantly changed. These data sug-gest that it is possible to modulate production of estrogens andtheir metabolites through consumption of soy phytoestrogens.In addition, because we did not observe significant effects on

concentrations of plasma estrogens or the menstrual cycle (54),it is likely that urinary estrogen excretion is a more sensitive

indicator of the effects of isoflavones on endogenous estrogen.

Although few data are available. the likely mechanism bywhich soy isoflavones modulate estrogen metabolism is via

effects on the activity of specific CYP isoenzymes responsiblefor estrogen hydroxylations. Genistein has been shown to in-

hibit rat liver CYP1AI (46), an enzyme that catalyzes theconversion of catechol estrogens to their electrophilic quinones,

compounds that may be responsible for the genotoxicity of

4-hydroxybated catechol estrogens (70). It has been shown thata-naphthoflavone, a synthetic flavone and inhibitor ofCYPlAland CYP1 B I , completely suppresses E,-induced tumorigenesis

through inhibition of the formation of 4-(OH)E, and its elec-

trophilic quinone (70). At high concentrations, genistein alsoweakly inhibits CYPIA2 (46), an enzyme that catalyzes the

2-hydroxybation of E, (71) and E2 (72) in human liver. Con-

sumption of the synthetic isoflavone, ipniflavone by rats, resultsin a strong suppression of liver CYP3A4 (47), the enzyme thatcatalyzes the 4- and 16a-hydroxylation of estrogens in humans

(71).This is the first study to report that soy isoflavone con-

sumption in humans lowers urinary estrogen excretion and

leads estrogen metabolism in a favorable direction by prefer-

entialby decreasing production of genotoxic 16a- and 4-hy-droxylated estrogen metabobites. This mechanism may contrib-ute to the observed inverse associations between breast cancer

and soy consumption. Of additional significance is the possi-

bility that, in some studies of dietary effects on endogenousestrogen, urinary estrogen bevels may be more sensitive and

significant end points than plasma concentrations of reproduc-tive hormones or functional end points such as the menstrualcycle.

Acknowledgments

We are grateful to Professor Herman Adlercreutz, Department of Clinical Chem-istry. University of Helsinki, for his advice on our analytical work, and to Dr. Will

Thomas. Division of Biostatistics, School of Public Health, and Dr. Gary Oehlert.

School of Statistics, University of Minnesota, for their advice on our data

analysis. We would also like to thank the subjects and staff at the General Clinical

Research Center, University of Minnesota, for their effort in carrying out this

human feeding study.

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1998;7:1101-1108. Cancer Epidemiol Biomarkers Prev X Xu, A M Duncan, B E Merz, et al. metabolism in premenopausal women.Effects of soy isoflavones on estrogen and phytoestrogen

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effects of soy isoflavones on estrogen and phytoestrogen ... · methylation (5, 6). although the...

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