Effects of Soya Consumption for One Month on Steroid Hormones … · Effects of Soya Consumption for One Month on Steroid Hormones ... Consumption of rice and tofu (a soy product)

Received 5/23/95: revised 9/25/95: accepted 9/27/95.

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

page charges. This article must therefore be hereby marked ad,’ertise,nent in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.I This study was supported by USPHS Grants CA56273 and CA45181, and NIH

National Center for Research Resources General Clinical Research Center Grant

MOI RROO()73.2 To whom requests for reprints should be addressed. at Department of Preventive

Medicine and Community Health, University of Texas Medical Branch. 2. I 02

Ewing Hall. Rt. I I 10. Galveston. TX 77555-I I 10. Phone: (409) 772-1730: Fax:

(409 ) 772-9 108; E-mail: [email protected].

Vol. 5. 63-70, JWZI4(U%’ 1996 Cancer Epidemiology, Biomarkers & Prevention 63

Effects of Soya Consumption for One Month on Steroid Hormones

in Premenopausal Women: Implications for

Breast Cancer Risk Reduction’

Lee-Jane W. Lu,2 Karl E. Anderson, James J. Grady,and Manubai Nagamani

Departments of Preventive Medicine and Community Health [L-J. W. L..

K. E. A.. J. J. G.j and Obstetrics and Gynecology [M. NI. University of

Texas Medical Branch. Galveston. Texas 77555

Abstract

Soybean consumption is associated with reduced rates ofbreast, prostate, and colon cancer, which is possiblyrelated to the presence of isoflavones that are weaklyestrogenic and anticarcinogenic. We examined the effectsof soya consumption on circulating steroid hormones insix healthy females 22-29 years of age. Starting within6 days after the onset of menses, the subjects ingested a12-oz portion of soymilk with each of three meals dailyfor 1 month on a metabolic unit. Daily isoflavone intakeswere � 100 mg of daidzein (mostly as daidzin) and -100

mg of genistein (mostly as genistin). Serum 17fi-estradiollevels on cycle days 5-7, 12-14, and 20-22 decreased by

31% (P = 0.09), 81% (P = 0.03), and 49% (P 0.02),respectively, during soya feeding. Decreases persisted fortwo to three menstrual cycles after withdrawal from soyafeeding. The luteal phase progesterone levels decreasedby 35% during soya feeding (P 0.002).Dehydroepiandrosterone sulfate levels decreasedprogressively during soya feeding by 14-30% (P 0.03).Menstrual cycle length was 28.3 ± 1.9 days beforesoymilk feeding, increased to 31.8 ± 5.1 days during themonth of soymilk feeding (P = 0.06), remained increasedat 32.7 ± 8.4 days (P 0.1 1) at one cycle aftertermination of soymilk feeding, and returned to pre-soyadiet levels five to six cycles later. These results suggestthat consumption of soya diets containing phytoestrogensmay reduce circulating ovarian steroids and adrenalandrogens and increase menstrual cycle length. Sucheffects may account at least in part for the decreased riskof breast cancer that has been associated with legumeconsumption.

Introduction

Ovarian hormones and other reproductive factors (e.g. , ages of

menarche, menopause, and parity) have important influences

on breast cancer development ( I ). Increased levels of estrogens

in blood and urine are markers for high risk for breast cancer(2-5). For example, in a large prospective, case-control study,

Toniolo et a!. (5) reported a significant relationship betweenserum estrogen levels and the risk for breast cancer in womenin New York. Goldin et a!. (6) reported 44% lower blood levels

of estrogens and androgens in oriental women who emigrated to

the United States from areas of low breast cancer risk, when

compared to Caucasian Americans, who have a higher risk for

breast cancer. Women in rural China have a low risk for breastcancer and 36% lower plasma estrogen levels when comparedto women in Britain where breast cancer is more common (7).

Similarly, postmenopausal women in Japan have lower blood

estrogen levels and a lower risk for breast cancer than do whitewomen in the United States (8). Earlier studies (9) are in

agreement with these recent observations.Dietary factors, such as legumes, may account at least in

part for these differences in hormone levels and breast cancer

risk. Consumption of legumes is greater in countries such asChina, Japan, and Thailand, where breast and prostate cancers

are less common than in Western countries (10-13). Legumesare the major source of protein for vegetarians, another group

that is at low risk for many cancers (14, 15). A cross-sectional

study in Australia found that legume consumption was associ-

ated with lower risk for colon cancer ( I 5). Consumption of riceand tofu (a soy product) was inversely related to prostate cancer

risk among men of Japanese ancestry in Hawaii ( 10). Among

premenopausal women in Singapore, breast cancer risk was

inversely related to soy protein intake (16. 17). These epide-miological observations are supported by results of animal

studies in which soya feeding was protective against experi-mentally induced mammary and other organ cancers ( 13, 18).

Possible mechanisms for the cancer protective effects of

soya in humans are not established. However. soya containssignificant amounts of the isoflavones daidzein and genistein

( 19). These isoflavones are weak estrogens with uterotropic

potencies about I X l0� that ofdiethylstilbestrol and bindingaffinities for mammalian estrogen receptors, including thosefrom MCF-7 cells (20), about 0.1-2 X lO_2 that of 17f3-

estradiol (2 1 ). These isoflavones may act as anti-estrogens by

competing with endogenous estrogens for receptor binding, andthis may reduce estrogen-induced stimulation of breast cell

proliferation (20) and breast tumor formation. Alternatively.

they may reduce breast cancer risk by decreasing endogenous

ovarian steroid levels. For example, genistein stimulates pro-gesterone synthesis by isolated bovine granulosa cells at aconcentration <0.2 ,.LM but inhibits progesterone synthesis at aconcentration >2 �.LM (22) and antagonizes TGF-a-induced

on January 10, 2021. © 1996 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

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554 Soya Diet and Ovarian Steroids

4 L-J. W., Lu, S. N. Lin, J. J. Grady. M. Nagamani, and K. E. Anderson,manuscript in preparation.

estrogen biosynthesis in granulosa and theca cells (23).

Genistein inhibits the enzyme activity ofestrogen-specific 17f3-hydroxysteroid oxidoreductase type I, an enzyme that convertsestrone to l7�-estradiol (24). Unlike some other flavonoids,isoflavones are in general weak inhibitors for aromatase (25)

and inefficient inducers of monooxygenase and transferaseactivities (26). Soya isoflavones may also decrease cancer in-cidence by mechanisms that do not involve alteration of steroidlevels. For example, genistein is a specific inhibitor of tyrosine

kinase (Ref. 27: an important enzyme in transmembrane signaltransduction) induces cell differentiation (28), inhibits the ac-

tivity of topoisomerase II (29), and inhibits angiogenesis (30).l7f3-Estradiol stimulates breast cell proliferation and may

promote breast tumor growth (3 1 ). Progesterone antagonizesthe proliferative effect of l7�3-estradiol on the endometrium butits role in l7�-estradiol-induced breast cell proliferation is not

clear. Human breast epithelial cells proliferate two to threetimes more rapidly during the luteal phase when progesterone

levels are also high (32). This suggests that proliferation ofbreast cells induced by I 7�-estradiol may be further enhancedby high levels of progesterone (3 1). DHEA3 is an adrenalandrogen, an intermediate in the biosynthesis of l7�-estradiol

(33) and has a biphasic dose effect on breast cell proliferationin vitro (34). The role of DHEA as an influence on humanbreast cancer development is still controversial (35). However,a DHEA sulfate-supplemented diet reduced the incidence ofmammary tumors in mice (36). Therefore, l7�3-estradiol, pro-

gesterone, and DHEA sulfate may be useful biomarkers forbreast cancer risk. The purpose of the present study was to

determine if consumption of soya containing isoflavones canalter these three circulating steroid levels in premenopausal

women and, thereby, protect them from developing breast

cancer.

Materials and Methods

Study Design. Six healthy, nonvegetarian women with regularmenstrual cycles (26-3 1 days) were admitted for 33 days to theGeneral Clinical Research Center at The University of TexasMedical Branch. Four subjects were Caucasian, 1 African-American, and 1 Hispanic. Their age range was 22-29 years,

height 1.6-1.7 m, weight 53.8-70.5 kg, and body mass index18.9-26.2 kg/m2. Five subjects had never taken birth controlpills and one had taken an oral contraceptive until 2 monthsbefore the study. History, physical examination, blood cellcounts, blood chemistry profiles, and serum ferritin were nor-mal. The study was approved by the Institutional Review Boardof the University of Texas Medical Branch and written in-formed consent was obtained from each subject.

Subjects consumed a standard hospital diet and three12-oz portions of soymilk (one with each meal) daily for 1month under the supervision of the dietary staff. Soymilk usedfor this study was an homogenized, pasteurized preparationcontaining no preservatives (Banyan Foods Co., Houston, TX).Isoflavone content in soymilk was analyzed as described (37).

Briefly, an 80% methanolic extract was prepared from soymilk,dried and redissolved in water. Portions of the aqueous solutionwith or without f3-glucosidase digestion were purified by

ChemElut chromatography. The dried eluates after silylationwere analyzed by gas chromatography equipped with a flameionization detector. Selected lots used in the study contained

33.49 ± 14.48 mg (mean ± SD; n = 1 1) of daidzin plus

daidzein, and 38.39 ± 14.62 mg of genistin plus genistein/l2-oz lot, mostly as daidzin and genistin, but 15-19% of the

total isoflavones were the aglycones daidzein and genistein.Thus, the subjects ingested - 100 mg daidzin plus daidzein and- 100 mg genistin plus genistein/day. Soymilk ingestion began4.7 ± 1 .0 days (range, 3-6 days) after onset of menses. At

biweekly intervals and for 1-2 days, the subjects ingested allthree l2-oz portions of soymilk within 30 mm for determinationof isoflavone absorption and disposition.4 Blood samples forhormone determinations were obtained before and 1 day after

starting soymilk and, thereafter, at weekly intervals during soyafeeding.

Hormone levels in normal women during a 28-30-daymenstrual cycle are well described (38). 1 7/3-Estradiol levels

are the lowest (<50 pg/ml) during the early follicular phase(days 1-7), peak during days 12-14 (> 200 pg/ml), decreaseprecipitously after the LH surge (to < 100 pg/ml), peak again

during days 20-24 (100-200 pg/ml), and then decrease to

earlier follicular levels before the onset of menses. Progester-one levels are all <0. 1 ng/ml during the follicular phase, beganto rise after the LH surge (near day 14), and peak during themidluteal phase (5-12 ng/ml; days 20-24; Ref. 38). Therefore,blood samples were obtained from our subjects on days 5-7,

12-14, and 20-22 of each menstrual cycle. This was accom-plished for one cycle before soya feeding in three subjects (only

on day 5 for one subject), and for the month of soya feeding andfor at least 3 cycles within the first 6 months after terminationof soya feeding in all subjects. We were able to follow four of

the six subjects as outpatients for up to 10 months by frequentphone contacts; the lengths of their menstrual cycles wererecorded, and blood samples were obtained for hormone

analysis.

Hormone Analysis. 17�-Estradiol and progesterone concen-trations in the serum were measured by specific RIA afterfractionation by microcelite column chromatography as de-

scribed previously (39, 40). Briefly, 1 ml of serum was ex-tracted twice with 3 volumes of ether after adding 3H tracers of17j3-estradiol and progesterone for assessment of recovery. The

extracts were dried under nitrogen and chromatographed onmicrocelite columns with ethylene and propylene glycol as thestationary phase. This chromatographic separation is effective

in removing steroids that can interfere with the assay because ofcross-reactivity. The fractions containing progesterone andl7/3-estradiol were collected and dried, and the residue was

dissolved in the assay buffer. Two aliquots of different volumesof this solution were used in the assay, and 0. 1 ml was used forrecovery calculations. Blank and control sera were run witheach assay. The antibody used in I 7�-estradiol assay washighly specific with <0. 1% cross-reactivity with estrone andestriol. Levels of DHEA sulfate were measured by direct RIA(41). The intra-assay coefficient ofvariation was 4-8% and theinterassay variation was 5-9%. Steroid assays began only afterthe collection of samples from all six subjects was completed.

All of the samples from each subject were analyzed in a singleassay.

Statistical Analysis. To determine if the duration of soymilkingestion had an effect on steroid hormone levels, Friedman’stwo-way ANOVA of the ranked data was used. If Friedman’stest showed that the duration of treatment significantly affected

:‘ The abbreviations used are: DHEA. dehydroepiandrosterone: LH. luteinizing

hormone.



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three I 2-oz portions of soymilk daily for I month.

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Cancer Epidemiology, Biomarkers & Prevention 65

hormone levels, multiple comparisons of the various treatmentswere made using paired t tests on the unranked data. Subjectswith missing data at a particular time point were not includedin the post hoc analysis by paired t tests for only that specific

time point, but their data from other time points were includedin the ANOVA and the calculation of the means shown in thefigures.

Results

Fig. 1 shows the effect of I-month soymilk ingestion on mdi-vidual and mean menstrual cycle lengths. The mean (± SD)cycle length for this group of six women was 28.3 (± 1.9) daysbefore soya feeding; it increased slightly to 31.8 (± 5.1) days

during the month of soymilk feeding (P = 0.06, paired t test)and to 32.7 (± 8.4) days one cycle after termination of soymilkfeeding (P = 0. 1 1 ). Five women experienced increase in cycle

length of 1-12 days during soymilk feeding, whereas onewoman had a 2-day decrease. The subject who was taking an

oral contraceptive 2 months before the study had a 3-dayincrease in cycle length, which was similar to other subjects.

All women returned to their pre-soymilk cycle lengths fivecycles after termination of soymilk feeding (results not shown);this included a subject who had a cycle length of42 days duringsoymilk feeding and 49 days during the first cycle after soymilk

feeding. This subject consumed soymilk for 4 extra days andalso provided one blood sample after the last soymilk dose forhormone measurements.

With only one exception, our subjects had baseline hor-

mone levels within expected normal ranges (see “Materials andMethods;” Ref. 38). Hormone levels from one cycle before and

for five to ten cycles after termination of soymilk feeding fromour study subjects were similar and, therefore, were averagedand regarded as baseline values for each subject. The results ofthe second and third cycle after soymilk feeding were combinedbecause there were no detectable differences in hormone levelsbetween these two cycles.

Fig. 2 shows the effect and persistence of 1 month of

soymilk feeding on individual and mean 17f3-estradiol levels,

when these were averaged over a cycle (Fig. 14 ) and analyzed

separately on specific cycle days (Fig. 2, B-D). One month of

soya feeding reduced 17/3-estradiol levels either when all mea-

surements were averaged during a cycle (Fig. 2A: P = 0.00 15,

Friedman’s test) or when they were analyzed on cycle days 5-7

(Fig. 2B; P = 0.16), 12-14 (Fig. 2C; P = 0.0001), and 20-22

(Fig. 2D; P = 0.08). Fig. 14 shows that compared to the

baseline, the mean cycle levels of 17j3-estradiol decreased by62% during soymilk feeding (P = 0.03, paired t test), by 58%

during the first post-soymilk cycle (P = 0.05), and by 46%during the second to the third cycles after soya (P = 0. 1 1). Fig.2B shows that 17�-estradiol levels in the early follicular phase

(days 5-7) decreased by 31% during chronic soymilk feedingcompared to control periods (P = 0.09). Fig. 2C shows that

compared to the baseline, l7f3-estradiol levels during the latefollicular phases (days 12-14) decreased by 81% during soya

feeding (n = 5; P = 0.03), by 72% during the first cycle

post-soya (n = 5, P = 0.056), and by 68% during the secondto third cycles after soya feeding (n = 5; P = 0.045). The late

follicular phase l7�-estradiol levels during soya feeding were

also 41% lower than those during the second to third cyclesafter soymilk feeding (P = 0.003). Fig. 2D shows that com-pared to the baseline, 17f3-estradiol levels during the luteal

phases (days 20-22) decreased by 49% during soya feeding

(n 5; P = 0.02), by 68% during the first cycle post-soya(n 4; P = 0.04), and by 29% during the second and third

cycles post-soya (n = 4; P = 0.24). Thus, chronic soya inges-tion decreased l7�-estradiol levels that persisted for two cycles

after soya ingestion. During the soymilk feeding, only onewoman (who was previously on an oral contraceptive) had asmall l7�-estradiol peak (57 pg/ml) on day 14. During the

month of soymilk feeding, higher levels of l7f3-estradiol in allsix subjects were detected on cycle days 22-37 (range, 60-135

pg/ml) compared with those on cycle days 12-14 (range, 22-57pg/ml), and this was in contrast to the profiles observed during

baseline periods. If the highest levels (e.g., independent of



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a cycle (A) and for cycle days 5-7 (B). days 12-14 (C). and days 20-22 (D). Baseline values are averages from one menstrual cycle before and/or five to six menstrualcycles after termination of soymilk ingestion. P values (paired I test) refer to comparisons with baseline values.

cycle day) were compared, those during soymilk feeding (84.7± 21 .6 pg/ml; range, 60-135 pg/ml) were also lower than

during baseline study periods (186.9 ± 99.3 pg/ml; range,50-402.5 pg/ml; P = 0.04). The subject who was on an oralcontraceptive previously had a similar but smaller decrease inI 7f3-estradiol levels compared with other subjects.

Follicular phase progesterone levels before, during, andafter soymilk feeding were all <0. 1 ng/ml, as expected (resultsnot shown). Luteal phase progesterone levels during baseline

periods in five subjects were 4-18 ng/ml on cycle days 20-22,which is within the expected range ofwomen with a 28-30 day

cycle (3 1 ). Luteal phase samples could not be obtained in thesixth subject during baseline periods. During soymilk feeding,four subjects had higher progesterone levels on cycle days20-24 than on other cycle days as for baseline periods. Twosubjects who experienced 5- and 12-day increases in cyclelength had progesterone levels <0. 1 ng/ml on cycle days 20-24, and these levels were lower than those on cycle days 27 and37 (9 and 3 ng/ml, respectively). The highest progesteronelevels detected for all subjects during soymilk feeding (7.1 ±

3.0 ng/ml; range, 3.2-12.5 ng/ml) were all lower than their

respective maximal values during baseline periods (9.5 ± 2.9ng/ml; range, 4-17.5 ng/ml; P - 0.06). Fig. 3 shows the effectof 1 month of soymilk feeding on individual and mean proges-

terone levels measured on cycle days 20-22. The progesteronelevels during the soymilk ingestion cycle were 35% lower thanbaseline (P = 0.002) and 28-38% lower than the first threecycles after termination of soya feeding (0. 10 < P < 0.03). Thesubject who used birth control pills before entering the studydid not provide a blood sample on cycle days 20-22 during thebaseline period; therefore, her data was not included in the post

hoc analysis by paired t test.DHEA sulfate levels were measured at least three times!

cycle. Because DHEA sulfate levels do not undergo cyclicchanges, all measurements in a cycle were averaged for eachsubject. Thus, the values during the soymilk feeding repre-

sented means from varying lengths of soymilk ingestion. Base-line intra-individual variability in DHEA sulfate levels within acycle were -15%, and inter-individual variability was -40%.One month of soymilk feeding reduced DHEA sulfate levels



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Fig. 3. The effect in six women of I month (Mon) of

soymilk ingestion on individual and mean serum levels

of progesterone on cycle days 20-22. P value (paired

t test) refers to comparisons with baseline values.

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averaged over a cycle (P = 0.0006. Friedman’s test; Fig. 4A),

and this change increased with the duration soya ingestion (P =

0.06; Fig. 4B). Levels of DHEA sulfate averaged over the cyclewere lowest during soya feeding and were 23% lower than

before soya (P 0.03; Fig. 4A), 20% lower than during thefirst cycle after soya (P = 0.01), and 27% lower than during thesecond to third cycles after soya feeding (P = 0.006). DHEAsulfate levels at two to three cycles post-soya were slightlygreater than pre-soya levels (P 0.08). Fig. 4B shows that

during soymilk ingestion, DHEA sulfate levels decreased pro-gressively as the number of days of soymilk ingestion in-creased. Compared with baseline, the decrease was significantafter >8 days (P values are shown in Fig. 4B).

Discussion

Steroid hormones such as 17�-estradiol, progesterone, andDHEA sulfate can modulate the proliferative rates of mammaryepithelial cells. A change in menstrual cycle length alters therelative duration of mammary epithelial cells in the luteal phase

of a cycle during which time the breast cells are more prolif-erative. Thus, levels of these steroid hormones and menstrual

cycle length are recognized risk factors for breast cancer (re-viewed in Refs. 31, 35). In this study. the effects of consuminga soya diet rich in isoflavones (-200 mg/day) on these riskfactors were studied in six healthy, premenopausal women. Wefound that 1 month of soymilk ingestion effectively reducedlevels of l7�-estradiol, progesterone, and DHEA sulfate andincreased menstrual cycle length.

One month of soya feeding increased menstrual cyclelength in five of the six subjects. The increase was mostpronounced in the two subjects who during soymilk feeding hadvery low levels (<0. 1 ng/ml) of progesterone on cycle days20-24 (Fig. 3), suggesting that there were delays in ovulation.

In general, when cycle length increases, the length of follicularphase increases more than the luteal phase (42). Because breastcells proliferate two to three times more rapidly during the

luteal phase than during the follicular phase (32), an increase in

cycle length, as observed in our subjects with soya feeding. may

shorten the duration of exposure of breast tissues to progester-one in the luteal phase. If this occurs repeatedly over a long

period of time of soya ingestion, the relative amount of timeduring which breast tissue is stimulated to proliferate may

decrease accordingly, and this may decrease the overall risk ofbreast cancer development. Indeed, Henderson et a!. (43) sug-

gested that menstrual cycle length may modulate breast cancer

risk. Differences in cycle lengths between breast cancer cases

and controls (44) and between populations of women in coun-tries with different breast cancer risks are consistent with this

suggestion (45).

During baseline cycles, our subjects had plasma levels of17/3-estradiol and progesterone that were similar to those re-

ported by others in normal women (38). Their levels of l7�-

estradiol were highest on cycle days 12-14, intermediate oncycle days 20-22, and the lowest on cycle days 5-7. 17j3-

Estradiol levels were considerably lower during soymilk feed-ing, when averaged over each cycle (Fig. 14 ), when compared

during certain days of the cycle (Figs. 2, B-D), or when thehighest levels detected were compared. Moreover, during

soymilk feeding in all women, peak levels of 17f3-estradiolwere observed only after cycle day 20. Soya feeding reduced

17�-estradiol levels in all six women, including one who had a2-day decrease in cycle length. These observations suggest thattotal 17f3-estradiol production was decreased during the month

of soymilk ingestion. Progesterone production may also bereduced by soymilk feeding. All six women had lower luteal

progesterone levels during soya consumption than during base-

line cycles. DHEA sulfate levels, which unlike 17�-estradiol

and progesterone do not exhibit cyclicity, were also reduced

during soymilk feeding (Fig. 4). DHEA sulfate levels in thisstudy decreased in a time-dependent manner with longer dura-

tion of soymilk feeding (Fig. 4B). Thus, the effect of soyafeeding on DHEA sulfate levels may result from the inhibition



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ingestion on individual and mean serum levels of

DHEA sulfate averaged over each cycle (A ) and on the

time course of individual and mean serum DHEA sul-

fate levels during soymilk feeding (B). P values (pairedI test) refer to comparisons with baseline values.

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of the synthesis of DHEA. which is an intermediate in the

biosynthesis of 1 7�-estradiol. Reduction of 17j3-estradiol, pro-gesterone, and DHEA sulfate levels during soya feeding mayreduce the extent of breast cell proliferation (3 1 ) and, thus,breast cancer risk.

Reduced 17f3-estradiol and progesterone (but not DHEAsulfate) levels during soymilk ingestion may in part result from

delayed peak formation and subsequently delayed ovulationbecause cycle length increased in five of six women duringsoya ingestion. All six subjects had higher levels of l7�-estradiol during the luteal phase of the cycle when soymilk wasingested in contrast to higher levels on days 12-14 duringbaseline periods (compare Figs. 2, C and D). Because the twowomen who experienced >5-day increases in menstrual cycle

length had progesterone levels > 1 ng!ml and peak l7�-estra-

diol levels on cycle days 27 and 37, reduced levels of ovariansteroids during soymilk feeding may be at least in part aconsequence of delays in peak and ovulation. The delays inovulation during soya feeding would also account for the in-

crease in menstrual cycle lengths in our subjects. This willdecrease the duration of breast cells in the luteal phase and inproliferation and, thus, lower breast cancer risk, as already

discussed.The effectiveness of soya feeding in reducing circulating

ovarian steroid levels in premenopausal women may explain

epidemiological observations that legume consumption is pro-tective against breast cancer development ( 16, 17), and thatChinese and Japanese women from low risk areas have lower




plasma estrogen levels than do women from regions with highrisk (6-8). Among the many chemopreventive components ofsoya (reviewed in Refs. 12, 18, 46-49), only the isoflavones

and their metabolites have been detected in urine and blood ofhumans from populations with low breast and prostate cancerrisks (37, 50-53). Thus, the soya isoflavones may play an

important role in protecting women from developing breastcancer. However, other potentially chemopreventive compo-nents of soya. such as Bowman-Birk protease inhibitor, phyt-

osterols, saponins, and inositols may also contribute to theeffects observed ( 18. 54).

An anti-estrogenic effect may be one of the mechanismsby which the soya phytoestrogens reduce proliferation of breast

epithelial cells and thus breast cancer risk. However, soyaphytoestrogens may also modify breast cell proliferation by

decreasing circulating ovarian steroid levels. Our observationthat the levels of three different steroids, l7f3-estradiol, pro-gesterone, and DHEA sulfate, were reduced suggests that thesynthesis of these steroids were inhibited during soya diets.

Moreover, the effects on I 7�-estradiol levels persisted for twocycles after termination of soymilk feeding, during which timesoya isoflavones were no longer detected in blood and urine

(53). In in vitro assays, genistein inhibits the biosynthesis ofprogesterone by bovine granulosa cells (22), antagonizes trans-forming growth factor a-induced synthesis of estrogen in gran-

ulosa and theca cells (23) and inhibits the enzyme activity of17j3-hydroxysteroid oxidoreductase type I (24), an enzyme that

converts estrone to 17/3-estradiol. The effect of genistein on the

synthesis of these three steroids may result from its effect ontransmembrane signal transduction, a mechanism that may ex-plain the persistence of soya-induced effect on 17j3-estradiollevels. Phytoestrogens are weak estrogens but at high doses

may have sufficient estrogenic activity to down-regulate thehypothalamus and pituitary and, thereby, reduce the ovarian

synthesis of estrogens. However, in vitro studies have shownthat some of biological effects of the genistein are independentof estrogen receptor status (55).

Cassidy et a!. (56) reported that daily consumption of a

soya diet (containing 45 mg of isoflavones) for I month sup-pressed mid-cycle surges of LH and follicle-stimulating hor-

mone in six premenopausal women. We did not measure fol-licle-stimulating hormone and LH in this study, but weobserved increases in menstrual cycle length and delays inprogesterone production during soymilk ingestion, suggestingthat phytoestrogens may delay ovulation. However, Cassidy eta!. (56) reported that soya feeding increased follicular phasel7�-estradiol levels and had no effects on mid-cycle and luteall7�3-estradiol levels, a result different from our observation.Larger amounts of isoflavones (>200 mg) were provided in asoya diet in our study than in the study by Cassidy et a!. (56).

Baird et a!. (57) studied the effects of 1 month of feeding ofsoya containing 165 mg of isoflavones daily in free-livingpostmenopausal women and observed no effect on � 7f3-estra-

diol levels. The reason for these differences in results are notknown but may relate to differences in doses of isoflavones

administered, types of soya products used, and other aspects ofstudy design. The dose responses of genistein in many biolog-ical systems are often biphasic. For example. the effects ofgenistein on progesterone synthesis (22). cell proliferation (24),

and pituitary responsiveness to the stimulation of gonadotropin-releasing hormone (58) differ depending on the doses of

genistein used.In summary, we show that consumption of soymilk con-

taming >200 mg of phytoestrogens daily for 1 month canreduce circulating levels of 17f3-estradiol, progesterone, and

DHEA sulfate and increase cycle length in premenopausalwomen. These endocrine factors are known to influence breastcell proliferation and breast cancer risk (2-5, 43, 44). There-fore, these effects of soya on ovarian steroid levels may provide

a biological basis for epidemiological observations that legumeconsumption is associated with reduced estrogen levels inblood and urine (6-8) and reduced risk for breast cancer (16,17). We also showed that the effects of soya feeding on l7�3-

estradiol levels persisted after termination of soya diets, sug-

gesting that soya consumption may be oncoprotective withoutdaily consumption. However, the persistence of this effect inrelationship to endocrine function also requires more studies.

Acknowledgments

We are grateful to the nursing, dietary, and administrative staff of the General

Clinical Research Center at the University of Texas Medical Branch for theirskillful assistance. We thank Ann Livengood. RD., for diet preparation and

supervision of the subjects and Dr. Lyle D. l3roemeling for statistical advice.

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1996;5:63-70. Cancer Epidemiol Biomarkers Prev L J Lu, K E Anderson, J J Grady, et al. cancer risk reduction.hormones in premenopausal women: implications for breast Effects of soya consumption for one month on steroid

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