İsoflavon soya fasulyesinden elde edilen fito östrojendir,vücutta östrojenin etkisini taklit eder ama östrojenin yan etkilerine sebep olmaz.Bu etkisi sayesinde erkek tipi saç dökülmelerinde anti androjenik etkisiyle ve DHT nin saç köküne zararlarını engellemesiyle saç dökülmesini, durdurur.Saç dökülmesi tedavisi yanı sıra aynı nedenlerle ortaya çıkan prostat hastalıklarının tedavisinde de kullanılmaktadır.
Bilimsel yayın 1
Isoflavone supplements stimulated the production of serum equol and decreased the serum dihydrotestosterone levels in healthy male volunteers
M Tanaka, K Fujimoto, Y Chihara, K Torimoto, T Yoneda, N Tanaka, A Hirayama, N Miyanaga, H Akaza and Y Hirao
Department of Urology, Nara Medical University, Kashihara, Japan
Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan
Correspondence: Dr M Tanaka, Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. E-mail: masa-t@naramed-u.ac.jp
Received 28 October 2008; Revised 13 March 2009; Accepted 13 March 2009; Published online 14 July 2009.
The aim of this study was to evaluate the effect of supplementing healthy men with soy isoflavones on the serum levels of sex hormones implicated in prostate cancer development. A total of 28 Japanese healthy volunteers (18 equol producers and 10 equol non-producers) between 30 and 59 years of age were given soy isoflavones (60 mg daily) supplements for 3 months, and the changes in their sex hormone levels were investigated at the baseline and after administration. The serum and urine concentrations of daidzein, genistein, and the levels of equol in the fasting blood samples and 24-h stored urine samples were also measured. All 28 volunteers completed the 3-month supplementation with isoflavone. No changes in the serum levels of estradiol and total testosterone were detected after 3-month supplementation. The serum levels of sex hormone-binding globulin significantly increased, and the serum levels of free testosterone and dihydrotestosterone (DHT) decreased significantly after 3-month supplementation. Among the 10 equol non-producers, equol became detectable in the serum of two healthy volunteers after 3-month supplementation. This study revealed that short-term administration of soy isoflavones stimulated the production of serum equol and decreased the serum DHT level in Japanese healthy volunteers. These results suggest the possibility of converting equol non-producers to producers by prolonged and consistent soy isoflavones consumption.
Bilimsel yayın 2
Isoflavone-Rich Soy Protein Isolate Suppresses Androgen Receptor Expression without Altering Estrogen Receptor-b Expression or Serum Hormonal Profiles in Men at High Risk of Prostate Cancer
Jill M. Hamilton-Reeves,
Salome A. Rebello,
William Thomas,
Joel W. Slaton,
and Mindy S. Kurzer
Department of Food Science and Nutrition;
Division of Biostatistics in the School of Public Health; and
Department of Urologic
Surgery, University of Minnesota, Minneapolis, MN 55455 and
Department of Urology, Veterans Administration Medical Center,
Minneapolis, MN 55417
Abstract
The purpose of this study was to determine the effects of soy protein isolate consumption on circulating hormone profiles
and hormone receptor expression patterns in men at high risk for developing advanced prostate cancer. Fifty-eight men
were randomly assigned to consume 1 of 3 protein isolates containing 40 g/d protein: 1) soy protein isolate (SPI1) (107
mg/d isoflavones); 2) alcohol-washed soy protein isolate (SPI2) (,6 mg/d isoflavones); or 3) milk protein isolate (0 mg/d
isoflavones). For 6 mo, the men consumed the protein isolates in divided doses twice daily as a partial meal replacement.
Serum samples collected at 0, 3, and 6 mo were analyzed for circulating estradiol, estrone, sex hormone-binding globulin,
androstenedione, androstanediol glucuronide, dehydroepiandrosterone sulfate, dihydrotestosterone, testosterone, and
free testosterone concentrations by RIA. Prostate biopsy samples obtained pre- and postintervention were analyzed
for androgen receptor (AR) and estrogen receptor-b expression by immunohistochemistry. At 6 mo, consumption of
SPI1 significantly suppressed AR expression but did not alter estrogen receptor-b expression or circulating hormones.
Consumption of SPI2 significantly increased estradiol and androstenedione concentrations, and tended to suppress AR
expression (P ¼ 0.09). Although the effects of SPI2 consumption on estradiol and androstenedione are difficult to
interpret and the clinical relevance is uncertain, these data show that AR expression in the prostate is suppressed by soy
protein isolate consumption, which may be beneficial in preventing prostate cancer. J. Nutr. 137: 1769–1775, 2007.
Introduction
Steroid hormones modulate growth of the prostate gland, and
elevated levels of androgens have been associated with prostate
cancer risk (1,2). Consumption of soy foods is thought to contribute to prostate cancer prevention as a result of the hormonal
properties of soy isoflavones, either through altered endogenous
circulating hormones or hormone-receptor signaling. Cell culture studies have suggested that the isoflavonoids, genistein and
equol, exert the most noteworthy hormonal effects. Genistein
inhibits the activity of 5a-reductase and 17b-hydroxysteroid dehydrogenase, enzymes required for androgen synthesis (3,4). The
isoflavonoid equol, a bacterially derived metabolite of the iso-
flavone daidzein, sequesters dihydrotestosterone (DHT)from
the androgen receptor (AR) in rat prostate tissue (5). Both isoflavonoids accumulate in the prostate gland (6–9) and may mimic or
modulate endogenous hormones relevant to prostate carcinogenesis.
Despite evidence from in vitro studies, human intervention
studies report inconsistent effects of soy or isoflavone consumption on circulating hormone profiles in men. Although reports
show statistically significant suppression of total testosterone
(10,11), sex hormone binding globulin (SHBG) (12), DHT (13),
dehydroepiandrosterone (14), estrone (15), and free androgen
index (13), and increased concentrations of SHBG (16) and DHT
(17), the majority of the 22 intervention studies to date have not
found significant changes in circulating sex steroid hormones (10–
31). Generally, the studies that report significant changes were
The Soy and Prostate Cancer Prevention (SoyCaP) trial was supported by grant
DAMD 17-02-1-0101 (M.S.K.) and W81XWH-06-1-0075 (J.H.R.) from the United
States Army Department of Defense Prostate Cancer Research Program. The
protein isolates were donated by The Solae Company, St. Louis, MO. Neither
sponsor was involved in writing this report.
Author disclosures: J. M. Hamilton-Reeves, S. A. Rebello, W. Thomas, J. W.
Slaton, and M. S. Kurzer, no conflicts of interest.
Supplemental Tables 1 and 2 are available with the online posting of this paper
at jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: mkurzer@umn.edu.
Abbreviations used: 3a-AG, androstanediol glucuronide; AR, androgen receptor; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; ERb,
estrogen receptor-b; MPI, milk protein isolate; SHBG, sex hormone-binding
globulin; SPI2, alcohol-extracted soy protein isolate; SPI1, isoflavone rich soy
protein isolate.
0022-3166/07 $8.00 ª 2007 American Society for Nutrition. 1769
Manuscript received 15 January 2007. Initial review completed 7 March 2007. Revision accepted 11 April 2007.
by guest on November 14, 2012 jn.nutrition.org Downloaded from
C1.html
http://jn.nutrition.org/content/suppl/2007/06/21/137.7.1769.D
Supplemental Material can be found at:carried out in older men for a relatively long duration. None of the
published studies reported equol-excretor status effects on circulating hormone response to soy isoflavone interventions in men.
Circulating hormone profiles may fail to accurately reflect
prostate tissue exposure, and evaluating hormone receptor expression patterns in the prostate may provide additional evidence concerning the role of soy as a cancer preventive dietary
agent. The AR mediates the action of androgens, and AR expression is a potential marker for prostate cancer prognosis (32).
Dietary genistein has been shown to downregulate AR mRNA
expression in rodents (33,34), and genistein has been shown to
suppress AR activity through an estrogen receptor-b (ERb)-
dependent mechanism in LNCaP cells (35). Despite these data,
to our knowledge, there are no studies published to date that
evaluate the effects of soy protein isolate consumption on AR
and ERb expression in men, although one study reported that an
isoflavone extract derived from red clover failed to alter AR
expression compared with historically matched controls (26).
The objective of this project was to evaluate the effects of
isoflavone-rich soy protein isolate consumption on circulating
concentrations of reproductive hormones and prostate tissue
markers of estrogen and androgen receptor expression in men at
high risk of prostate cancer. The effects of an isoflavone-rich soy
protein isolate were compared with those of an isoflavone-poor
soy protein isolate to determine whether the isoflavones are the
responsible bioactive constituents. The underlying hypothesis
was that isoflavone-rich soy protein isolate consumption would
reduce circulating hormones, downregulate AR expression, and
upregulate ERb expression.
Material and Methods
Subjects. Fifty-eight men, aged 50–85 y, were recruited at the
Minneapolis Veteran’s Administration Medical Center Urology Clinic
from a group of patients that had already undergone a transrectal
ultrasound and biopsy. Patients in this study were either at high risk for
developing prostate cancer (n ¼ 53), or had low-grade prostate cancer
that was being followed by active surveillance (n ¼ 5). Subjects were
considered high risk if they had high-grade prostatic intraepithelial neoplasia (PIN) (n ¼ 50) and/or atypical small acinar proliferation (ASAP)
(n ¼ 14). The subjects with prostate cancer had Gleason scores of ,6 and
were not receiving any other prostate cancer therapy. Subjects were
recruited by urologic physicians, and the research nurse reviewed the
patients’ medical records to determine that eligibility criteria were met.
Exclusionary criteria included BMI .40 kg/m
, prostate cancer that required medical treatment, prostatitis, alcohol consumption .14 drinks/
wk, soy or milk allergy, regular antibiotic use, or renal insufficiency.
Eighty-seven subjects were screened for the study; 21 chose not to
participate after attending the orientation session, and 66 subjects began
the study. Eight subjects withdrew from the study before their 3-mo
appointment [disliked the study treatment powder (n ¼ 3), inconvenienced by study demands (n ¼ 2), gastrointestinal discomfort (n ¼ 1),
chose conventional prostate cancer treatment (n ¼ 1), weight gain (n ¼
1)]. Three subjects completed 3 mo of the study with good compliance
but chose not to finish due to inconvenience of the study demands, and
55 subjects completed the full 6-mo study.
Data from 58 subjects were included in the serum hormone analysis,
and 42 subjects were included in the hormone receptor expression
analysis. Fewer participants were eligible for the hormone expression
analysis because 3 subjects did not undergo the final prostate biopsy
[liver cancer diagnosis (n ¼ 1), heart condition (n ¼ 1), not clinically
indicated (n ¼ 1)], and 13 subjects had insufficient biopsy tissue at either
baseline or postintervention for the analyses. All 58 subjects who completed the study were Caucasian.
Study design. The University of Minnesota Institutional Review Board
Human Subjects Committee, the Minneapolis Veterans Affairs Institutional Review Board, and the U.S. Army Medical Research and Materiel
Command’s Human Subjects Research Review Board approved the
study protocol, and all subjects provided informed consent, attended an
orientation session, and were provided with a study handbook. During
the study orientation, subjects were interviewed and prompted about
incidental exposure to dietary isoflavones (e.g., snack bars, shakes, soy
nuts, canned tuna, legumes, breads) to determine whether they were soy
consumers. Only one participant reported regular soy consumption, but
he did not consume soy-containing products for 1 mo prior to beginning
the study. The 6-mo intervention study used a randomized, singleblinded, placebo-controlled, parallel design. Free-living subjects supplemented their diets with 1 of 3 randomly assigned protein isolates: 1) soy
protein isolate high in isoflavones (SPI1); 2) soy protein isolate that had
most of the isoflavones removed by alcohol extraction (SPI2); or 3) milk
protein isolate (MPI) (The Solae Company). The protein isolates were
consumed in divided doses twice daily and contributed 40 g/d protein
and 200–400 kcal/d (1 kcal ¼ 4.184 kJ). The isoflavone content of the
protein isolates expressed as aglycone equivalents was 107 6 5.0 mg/d
for the SPI1; ,6 6 0.7 mg/d for the SPI2; and 0 mg/d for the MPI
(mean 6 SD). The mean distribution of isoflavones was 53% genistein,
35% daidzein, and 11% glycitein in SPI1, and 57% genistein, 20%
daidzein, and 23% glycitein in SPI2 as analyzed by Dr. Pat Murphy
(Department of Food Science and Human Nutrition, Iowa State University). The packets of protein isolate were numbered and patients were
unaware of the treatment protein isolate they had been assigned until all
subjects completed the intervention. Only the study coordinators who
administered the protein isolates knew the group to which each participant belonged. Compliance was assessed by counting the number of
times the patient consumed the protein isolate, as self-reported in recording calendars given to them, and mean compliance was 94%. Dietary and herbal supplements were allowed, and participants were asked
to avoid changing dosages or adding new supplements to their regimen
during the study. Subjects consumed their habitual diets, and received
detailed instructions to exclude soy products to minimize isoflavone
consumption from other sources.
Serum collection and analysis. Fasting blood was collected in the
morning at 0, 3, and 6 mo. Serum was separated and aliquots were frozen
at –70 C until analysis. All serum samples were analyzed for testosterone, free testosterone, DHT, androstanediol glucuronide (3a-AG),
androstenedione, dehydroepiandrosterone sulfate (DHEAS), SHBG, estradiol, and estrone. Steroid hormones were analyzed in duplicate by
RIA, and SHBG was analyzed by immunoradiometric assay (Diagnostics
Systems Laboratories). Hormone analyses were performed in 3 batches
and all assays required
I-labeled analyte. Intraassay variabilities were
3.7% for testosterone, 4.4% for free testosterone, 6.1% for DHT, 4.5%
for 3a-AG, 4.4% for androstenedione, 2.3% for DHEAS, 4.4% for
SHBG, 3.9% for estradiol, and 4.3% for estrone. An internal control
was utilized to determine variability among batches, and interassay variabilities were between 9 and 30% for all analytes. All 3 serum samples
for each participant were analyzed in the same batch.
Urine collection and analysis. To assess equol-producer status, 24-h
urine was collected in plastic containers containing 1 g/L of ascorbic acid
and separated into aliquots after the addition of sodium azide to a final
concentration of 0.1%. Aliquots were frozen at –20 C until analysis.
Equol was determined by HPLC and MS as previously described (36).
The intraassay CV for equol was 8.2%, and the interassay CV was
12.5%. Subjects were classified as equol excretors if 24-h urine equol
levels exceeded 1000 nmol/d.
Dietary intake and analysis. Food records were completed for 3 d
before each clinic visit. A registered dietitian taught study participants
how to keep accurate food records. Patients were encouraged to use
household scales and volumetric tools and to submit food labels from
unusual foods. Study coordinators reviewed each food record for
completeness and clarified ambiguities with the participant at each clinic
visit. Food records were analyzed with Nutritionist V, version 2.3 (37),
and, for each 3-d food record, mean intakes of energy, macronutrients,
saturated fat, cholesterol, fiber, vitamin D, vitamin E, calcium, selenium,
and zinc were calculated.
1770 Hamilton-Reeves et al.
by guest on November 14, 2012 jn.nutrition.org Downloaded from Tissue collection and analysis. Biopsies were performed before the
initial screening and at the 6-mo clinical visit. Biopsy cores were formalinpreserved for 24 h and paraffin embedded. The histological diagnoses
were determined during a routine pathological evaluation. Immunohistochemistry was performed to assess AR and ERb expression on primarily normal, hyperplastic, or preneoplastic glands collected from
eligible study participants. Antigen retrieval was achieved by pressure
cooking deparaffinized and rehydrated tissue sections at 103 kPa in
citrate buffer. Sections were treated in quenching solution (3% H2O2 in
100% methanol), and then incubated with a protein-blocking solution
(10% milk, 5% serum, and 1% BSA). Samples were incubated overnight
at 4 C with rabbit polyclonal anti-ERb antibody (ab3577; Abcam;
1:1000) for the ERb assay, or for 30 min at room temperature with the
mouse monoclonal anti-AR antibody (AM256–2M; BioGenex; RTU) for
the AR assays. Next, the avidin-biotin peroxidase method was carried
out (Vectastain Elite ABC kit, Vector Laboratories). Color reaction was
developed using diaminobenzidine as the chromagen. Appropriate positive and negative controls were included in all staining runs. Disrupted
glands and glands on the edge of tissue sections were excluded from
analysis to avoid false positives. A technician without prior knowledge of
histological grading scored both the intensity of immunostaining and
the percentage of immunopositive areas at 403 magnification using
the HSCORE system as previously described (38). The range of the
HSCORE is a minimum of 1 and a maximum of 4 (1 indicated absent
staining; 4 indicated intense staining). A mean of 6 intact glands (range:
2–15) per slide for ERb and a mean of 8 intact glands (range: 3–19) per
slide for AR were averaged to derive the HSCORE (Fig. 1).
Excluded from analysis. The following data were excluded from
statistical analysis: 6-mo dietary intake from one participant reporting
unusually low consumption (mean ,500 kcal) (1 kcal ¼ 4.184 kJ)
during the 3-d food diary as a result of illness; 3 mo DHEAS that was
above normal range (16 mmol/L) and inconsistent with the participant’s
baseline and 6-mo measurements; all DHEAS measurements from one
participant with abnormally high 3-mo and 6-mo DHEAS concentrations (9 and 10 mmol/L, respectively) compared with baseline; and all
SHBG measurements from one subject with undetectable SHBG in the
serum (,3 nmol/L). One subject did not consume the treatment powder
for 3 d prior to his 6-mo appointment as a result of illness, so he was
excluded from the 6-mo equol excretion analysis.
Statistical analysis. The data appeared normally distributed and had
similar variance among groups. Demographic comparisons between
groups were performed with 1-way ANOVA for continuous endpoints,
and chi-square for categories of prostate cancer markers. ANCOVA was
used to compare groups adjusted for the baseline value of the final
endpoint. For androstenedione, the model included a treatment by baseline interaction. Preplanned pairwise comparisons of all groups are
reported for each endpoint as dictated by the study hypotheses: each
group’s adjusted mean (least squares mean) was compared with the other
2 groups’ adjusted means. Paired t tests were used to test for significant
within-group changes over time. In addition, these covariates were
screened as adjusters: baseline body weight, equol excretor status, and
energy and nutrient intake. P , 0.05 was considered significant. All
analyses were performed using SAS, version 9.1 (39).
Results
Baseline. Baseline anthropometrics, cancer status, and dietary
intake did not differ among the groups (Table 1), except that the
MPI group had a higher body weight and the SPI2 group consumed significantly more protein, calcium, and zinc at baseline
(Table 2). Baseline prostate steroid receptor expression patterns
(Table 3) and serum hormone and SHBG concentrations (Table
4) did not differ among the groups.
Anthropometrics and dietary intake. Body weight did not
change from baseline to 3 or 6 mo in any group (Table 2), and
the significant differences in body weight among the groups at
baseline were maintained. Protein, calcium, and vitamin D intakes increased in all groups during the study as a result of their
concentrations in the protein isolates, and the differences in
protein, calcium, and zinc intake at baseline were not present at
3 and 6 mo. At 3 mo, total and saturated fat consumption were
reduced in the SPI2 group relative to baseline. During the study,
energy, carbohydrate, cholesterol, fiber, vitamin E, selenium, and
zinc intakes did not change for any group. Dietary and herbal
supplement usage did not differ among groups (data not shown).
Body weight and protein intake differences among groups were
unrelated to altered hormone concentrations or steroid receptor
expression patterns.
Steroid receptors. Baseline-adjusted AR expression was lower
in prostate biopsies after 6 mo in the SPI1 group compared with
the MPI group (P ¼ 0.04) and tended to be lower in the SPI2
group compared with the MPI group (P ¼ 0.09; Table 3). AR
expression significantly increased from baseline in the MPI group,
but not in the other 2 groups. There were no changes from
baseline in ERb expression among the groups (Table 3).
Serum estrogens. The serum estradiol concentration was
significantly increased in the SPI2 group at 3 and 6 mo relative
to baseline, and by 6 mo, baseline-adjusted estradiol concentrations were significantly higher in the SPI2 group compared with
the other 2 groups (Table 4). Serum estrone was also significantly
increased in the SPI2 group at 3 and 6 mo, and was significantly
higher than in the MPI group at 3 mo but not at 6 mo.
Serum androgens and SHBG. The serum androstenedione
concentration was significantly higher in the SPI1 group than in
the MPI group at 3 mo. At 6 mo it was significantly greater than
at baseline in the SPI2 group, resulting in a significantly higher
concentration than in the SPI1 group (Table 4). At both 3 and 6
mo, serum DHEAS was higher in the SPI group than in the other
2 groups, and at 3 mo, 3a-AG was higher in the SPI2 group than
the other 2 groups. At 3 mo, the DHT concentration decreased
from baseline in the SPI group. Serum SHBG concentrations
were decreased significantly from baseline at 3 and 6 mo in all
groups, with no difference among the groups.
Equol-excretor status and hormone profiles. Equol excretor
status was assessed only in the SPI1 group, because only they
consumed sufficient daidzein to excrete equol. At 3 mo, there
were 4 excretors and 15 nonexcretors, but of this group, only
1 excretor remained at 6 mo [dropped out after 3 mo (n ¼ 1),
TABLE 1 Baseline characteristics of subjects
Values are means 6 SD or n (%). Means in a row with superscripts without a
common letter differ, P , 0.05.
Subjects were categorized by most advanced prostate cancer marker.
Soy effects on hormones in men 1771
by guest on November 14, 2012 jn.nutrition.org Downloaded from apparently changed status (n ¼ 1), and excluded data (n ¼ 1)].
Baseline characteristics (Supplemental Table 1) and serum
hormone concentrations at 3 mo (Supplemental Table 2) did
not differ between excretors and nonexcretors.
Discussion
The present study evaluated men at high risk of prostate cancer
to determine the effects of soy protein consumption on serum
hormones and prostate tissue steroid receptor expression levels.
The major finding was lower AR expression levels and no differences in ERb expression or circulating hormones in men
consuming SPI1 compared with those consuming MPI.
AR increased significantly from baseline in the MPI group,
but did not change from baseline in the soy groups. Because AR
expression is expected to increase in this population (40), we
infer that SPI1 apparently prevented or suppressed a rise in AR
expression. Lower tissue AR expression in the SPI1 group is
consistent with research in which dietary phytoestrogens downregulated AR mRNA expression in adult male rats (33,34,41).
Our data differ, however, from those of Jarred et al. (26), who
reported no differences in AR expression patterns between radical prostatectomy patients treated with isoflavones and historically matched controls. The inconsistent results between the
2 studies can be explained by several methodological differences.
In the study by Jarred et al. (26), the subjects, who consumed
160 mg/d of isoflavones in extracts derived from red clover, were
men with advanced prostatic neoplasms treated for short and
varied time periods (7–54 d). The tissue sections studied from
the radical prostatectomies taken from treated subjects represented cancerous glandular acinae and were compared with
sections of cancers from historically matched controls. Our
subjects consumed 107 mg/d of isoflavones in isoflavone-rich
SPI, were earlier in the carcinogenesis continuum, were treated
TABLE 2 Anthropometrics and dietary intake of men at high
risk of prostate cancer that consumed various
protein isolates for 6 mo
All values are means 6 SD. Means in a row with superscripts without a common
letter differ, P , 0.05. *Different from baseline, P , 0.05.
Sample sizes are for all time points except the following: 3 mo, MPI (n ¼ 17), and 6
mo, SPI1 (n ¼ 18), and SPI2 (n ¼ 18).
1 kcal ¼ 4.184 kJ.
FIGURE 1 Representative immunohistochemical staining of AR in
human prostate core biopsies for HSCORE. Arrow indicates stained
acinar cell in MPI control group (enlarged view inset in lower right).
1772 Hamilton-Reeves et al.
by guest on November 14, 2012 jn.nutrition.org Downloaded from for 6 mo each, and all biological samples were evaluated within
the same subject before and after the intervention. Furthermore,
the gland acinae studied presented either benign, hyperplastic, or
preneoplastic tissue.
Consumption of SPI1 did not affect ERb expression or
circulating hormones. The ERb expression results are inconsistent with studies in animals in which prolonged isoflavone exposure decreased ERb expression (33,42), and may be explained
by the variability in commercially available ERb antibodies (43).
Our hormone results, however, are consistent with most published reports from the clinical setting. The testosterone results
are consistent with numerous soy or isoflavone intervention
studies in which no change in total testosterone was observed
(12–31), but differ from 2 studies of short duration (10,11). Our
finding of no effect on directly measured free testosterone is
similar to published soy or isoflavone intervention studies to
date (11,14,15,20,22,24), and our finding of no effect on circulating DHT is consistent with most reports (10,14,16,19–
21,23,30), although it differs from results of 2 studies (13,17),
one of which used red clover extract (17). The lack of effect on
circulating estradiol or estrone is consistent with the literature
(10,11,15,16,19,22,29,30), although there is one report of decreased estrone in men consuming soymilk for 8 wk (15).
Serum SHBG decreased significantly from baseline in all
study groups. The finding that consumption of SPI1 decreased
SHBG is similar to a report by Mackey et al. (12); however, they
did not find a significant decrease in SHBG with an isoflavonepoor protein isolate as we did. In contrast to our findings,
Habito et al. (16) reported increased SHBG in men consuming
35 g of tofu daily for 2 wk, and others have reported no significant changes of SHBG with isoflavone-rich foods or extracts
(13,15,17,20–23,30). Decreased SHBG is a potentially harmful
effect because SHBG-bound hormones are less biologically available to stimulate hormone-sensitive cancers. Because high protein intake has been associated with decreased SHBG (44), it is
likely that the decrease in SHBG from baseline in all groups in
our study resulted from the subjects’ significantly increased
protein intake during the study (45).
The hormonal effects in the SPI2 group were unexpected.
Although AR expression was not significantly lower in the SPI2
group, AR expression appeared to be intermediate between that
of SPI1 and MPI groups. In addition, serum estradiol was increased in the SPI2 group. These results are similar to a study in
young men by Dillingham et al. (20) in which a low-isoflavone
protein isolate containing ,2 mg/d isoflavones significantly increased estradiol and estrone compared with a milk protein isolate after a 8-wk intervention. Our results differ, however, from a
study in older men by Goldin et al. (19) in which a low-isoflavone
soy protein isolate containing ,2 mg/d isoflavones did not change
estradiol or estrone concentrations after a 6-wk intervention.
Interestingly, we found serum estradiol was significantly higher in
the SPI2 group than in the SPI1 group, whereas in Dillingham
et al. (20) found that estradiol in the low-isoflavone group did not
differ from the high-isoflavone group (20).
Serum androstenedione and DHEAS concentrations were
increased in the SPI2 group compared with both SPI1 and MPI
groups. No other soy protein or isoflavone intervention study has
TABLE 4 Serum hormones and SHBG in men at high risk
of prostate cancer that consumed various protein
isolates for 6 mo
Baseline data are unadjusted means 6 SEM. All other data are least-squares means
adjusted for baseline measurement 6 SEM, except androstenedione, which is
additionally adjusted for interaction between treatment and baseline. Means in a row
with superscripts without a common letter differ, P , 0.05. *Different from baseline,
P , 0.05.
Sample sizes are for all time points except: 3 mo MPI (n ¼ 17), and 6 mo SPI1 (n ¼
18), and SPI2 (n ¼ 19).
Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1
(n ¼ 19) and SPI2 (n ¼ 19). At 6 mo, SPI1 (n ¼ 17) and SPI2 (n ¼ 19).
Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1
(n ¼ 19), and at 6 mo, SPI1 (n ¼ 18).
TABLE 3 Steroid receptor expression of men at high risk of
prostate cancer that consumed various protein
isolates for 6 mo
SPI1 SPI2 MPI
Androgen receptor HSCORE
Baseline data are unadjusted means 6 SEM. All other data are least-squares means
adjusted for baseline measurement 6 SEM. Means in a row with superscripts without
a common letter differ, P , 0.05. *Different from baseline, P , 0.05.
Soy effects on hormones in men 1773
by guest on November 14, 2012 jn.nutrition.org Downloaded from reported a change in circulating androstenedione (12,17,19,20,30),
but all other studies to date have intervened for a shorter duration. Higher DHEAS is consistent with other low-isoflavone
soy protein isolate interventions (19,20). Although DHEAS and
androstenedione can be converted by 17b-hydroxysteroid dehydrogenase to testosterone, no significant changes were observed
in circulating testosterone, free testosterone, or DHT. Instead,
our study population had low, but normal, testosterone concentrations throughout the study. Although DHEAS and androstenedione concentrations have been associated with aggressive
prostate cancer (46), our findings of unchanged testosterone and
a trend toward lower AR expression (P ¼ 0.09) suggest neutral effects of SPI2 consumption. In fact, because DHEAS and
androstenedione may be converted to estradiol and estrone in the
prostate gland (47), the increase in DHEAS and androstenedione
may have contributed to the observed increases in circulating
estradiol and estrone. The hormonal effects of SPI2 consumption are likely due to the effects of the alcohol extraction process
on SPI constituents.
In conclusion, we found that consumption of isoflavone-rich
soy protein for 6 mo lowered AR expression levels in the prostate,
but did not change ERb expression or circulating hormones in
men at high risk of prostate cancer. Although consumption of
the alcohol-extracted soy protein did not significantly lower AR
expression, its effect appeared to be intermediate to that of SPI1
and MPI consumption, suggesting that the isoflavones alone may
not be responsible for the AR expression decrease, or, alternatively, that the low level of isoflavones in SPI2 were sufficient to
alter the AR. Unexpectedly, consumption of SPI2, but not SPI1,
significantly increased estradiol and androstenedione concentrations. None of these results were influenced by equol excretion
status. These data suggest that consumption of isoflavone-rich
and isoflavone-poor soy protein isolate exert differing effects on
endogenous hormones and receptor expression, which may
mediate prostate cancer preventive effects.
Acknowledgments
Immunohistochemistry was performed by Kenji Takamura. The
authors thank Kayla Vettling and Ellie Wiener for tissue scoring,
and Lori Sorensen, Nicole Nelson, and Mary McMullen for
assistance with clinic visits and data entry.
Literature Cited
1. Shaneyfelt T. H.R., Bubley G,Mantzoros C. Hormonal predictors of
prostate cancer: A meta-analysis. J Clin Oncol. 2000;18:847–53.
2. Bosland M. The role of steroid hormones in prostate carcinogenesis.
J Natl Cancer Inst Monogr. 2000;27:39–66.
3. Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in
genital skin fibroblasts and prostate tissue by dietary lignans and
isoflavonoids. J Endocrinol. 1995;147:295–302.
4. Makela S, Poutanen M, Kostian ML, Lehtimaki N, Strauss L, Santti R,
Vihko R. Inhibition of 17beta-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells. Proc Soc Exp Biol Med.
1998;217:310–6.
5. Lund TD, Munson DJ, Haldy ME, Setchell KDR, Lephart ED, Handa
RJ. Equol is a novel anti-androgen that inhibits prostate growth and
hormone feedback. Biol Reprod. 2004;70:1188–95.
6. Rowland I, Faughnan M, Hoey L, Wahala K, Williamson G, Cassidy A.
Bioavailability of phyto-oestrogens. Br J Nutr. 2003;89: Suppl 1:S45–58.
7. Morton MS, Chan PS, Cheng C, Blacklock N, Matos-Ferreira A,
Abranches-Monteiro L, Correia R, Lloyd S, Griffiths K. Lignans and
isoflavonoids in plasma and prostatic fluid in men: Samples from
portugal, hong kong, and the united kingdom. Prostate. 1997;32:122–8.
8. Hedlund TE, Maroni PD, Ferucci PG, Dayton R, Barnes S, Jones K,
Moore R, Ogden LG, Wahala K, et al. Long-term dietary habits affect
soy isoflavone metabolism and accumulation in prostatic fluid in
caucasian men. J Nutr. 2005;135:1400–6.
9. Hong SJ, Kim SI, Kwon SM, Lee JR, Chung BC. Comparative study of
concentration of isoflavones and lignans in plasma and prostatic tissues
of normal control and benign prostatic hyperplasia. Yonsei Med J.
2002;43:236–41.
10. Gardner-Thorpe D, O’Hagen C, Young I, Lewis SJ. Dietary supplements
of soya flour lower serum testosterone concentrations and improve
markers of oxidative stress in men. Eur J Clin Nutr. 2003;57:100–6.
11. Spentzos D, Mantzoros C, Regan MM, Morrissey ME, Duggan S,
Flickner-Garvey S, McCormick H, DeWolf W, Balk S, Bubley GJ.
Minimal effect of a low-fat/high soy diet for asymptomatic, hormonally
naive prostate cancer patients. Clin Cancer Res. 2003;9:3282–7.
12. Mackey R, Ekangaki A, Eden JA. The effects of soy protein in women
and men with elevated plasma lipids. Biofactors. 2000;12:251–7.
13. Kranse R, Dagnelie PC, van Kemenade MC, de Jong FH, Blom JH,
Tijburg LB, Weststrate JA, Schroder FH. Dietary intervention in
prostate cancer patients: PSA response in a randomized double-blind
placebo-controlled study. Int J Cancer. 2005;113:835–40.
14. Fischer L, Mahoney C, Jeffcoat AR, Koch MA, Thomas BE, Valentine JL,
Stinchcombe T, Boan J, Crowell JA, Zeisel SH. Clinical characteristics
and pharmacokinetics of purified soy isoflavones: Multiple-dose administration to men with prostate neoplasia. Nutr Cancer. 2004;48:160–70.
15. Nagata C, Takatsuka N, Shimizu H, Hayashi H, Akamatsu T, Murase
K. Effect of soymilk consumption on serum estrogen and androgen
concentrations in japanese men. Cancer Epidemiol Biomarkers Prev.
2001;10:179–84.
16. Habito RC, Montalto J, Leslie E, Ball MJ. Effects of replacing meat with
soyabean in the diet on sex hormone concentrations in healthy adult
males. Br J Nutr. 2000;84:557–63.
17. Lewis JG, Morris JC, Clark BM, Elder PA. The effect of isoflavone
extract ingestion, as trinovin, on plasma steroids in normal men.
Steroids. 2002;67:25–9.
18. Ornish D, Weidner G, Fair WR, Marlin R, Pettengill EB, Raisin CJ,
Dunn-Emke S, Crutchfield L, Jacobs FN, et al. Intensive lifestyle
changes may affect the progression of prostate cancer. J Urol. 2005;
174:1065,9; discussion 1069–70.
19. Goldin BR, Brauner E, Adlercreutz H, Ausman LM, Lichtenstein AH.
Hormonal response to diets high in soy or animal protein without and
with isoflavones in moderately hypercholesterolemic subjects. Nutr
Cancer. 2005;51:1–6.
20. Dillingham BL, McVeigh BL, Lampe JW, Duncan AM. Soy protein
isolates of varying isoflavone content exert minor effects on serum reproductive hormones in healthy young men. J Nutr. 2005;135:584–91.
21. Schroder FH, Roobol MJ, Boeve ER, de Mutsert R, Zuijdgeest-van
Leeuwen SD, Kersten I, Wildhagen MF, van Helvoort A. Randomized,
double-blind, placebo-controlled crossover study in men with prostate
cancer and rising PSA: Effectiveness of a dietary supplement. Eur Urol.
2005;48:922,30; discussion 930–1.
22. Kumar NB, Cantor A, Allen K, Riccardi D, Besterman-Dahan K, Seigne
J, Helal M, Salup R, Pow-Sang J. The specific role of isoflavones in
reducing prostate cancer risk. Prostate. 2004;59:141–7.
23. Dalais FS, Meliala A, Wattanapenpaiboon N, Frydenberg M, Suter DA,
Thomson WK, Wahlqvist ML. Effects of a diet rich in phytoestrogens
on prostate-specific antigen and sex hormones in men diagnosed with
prostate cancer. Urology. 2004;64:510–5.
24. deVere White RW, Hackman RM, Soares SE, Beckett LA, Li Y, Sun B.
Effects of a genistein-rich extract on PSA levels in men with a history of
prostate cancer. Urology. 2004;63:259–63.
25. Hussain M, Banerjee M, Sarkar FH, Djuric Z, Pollak MN, Doerge D,
Fontana J, Chinni S, Davis J, et al. Soy isoflavones in the treatment of
prostate cancer. Nutr Cancer. 2003;47:111–7.
26. Jarred RA, Keikha M, Dowling C, McPherson SJ, Clare AM, Husband
AJ, Pedersen JS, Frydenberg M, Risbridger GP. Induction of apoptosis
in low to moderate-grade human prostate carcinoma by red cloverderived dietary isoflavones. Cancer Epidemiol Biomarkers Prev. 2002;
11:1689–96.
27. Teede HJ, Dalais FS, Kotsopoulos D, Liang YL, Davis S, McGrath BP.
Dietary soy has both beneficial and potentially adverse cardiovascular
effects: A placebo-controlled study in men and postmenopausal women.
J Clin Endocrinol Metab. 2001;86:3053–60.
1774 Hamilton-Reeves et al.
by guest on November 14, 2012 jn.nutrition.org Downloaded from 28. Higashi K, Abata S, Iwamoto N, Ogura M, Yamashita T, Ishikawa O,
Ohslzu F, Nakamura H. Effects of soy protein on levels of remnant-like
particles cholesterol and vitamin E in healthy men. J Nutr Sci Vitaminol
(Tokyo). 2001;47:283–8.
29. Mitchell JH, Cawood E, Kinniburgh D, Provan A, Collins AR, Irvine
DS. Effect of a phytoestrogen food supplement on reproductive health
in normal males. Clin Sci (Lond). 2001;100:613–8.
30. Rannikko A, Petas A, Raivio T, Janne OA, Rannikko S, Adlercreutz H.
The effects of short-term oral phytoestrogen supplementation on the
hypothalamic-pituitary-testicular axis in prostate cancer patients. Prostate. 2006;66:1086–91.
31. Maskarinec G, Morimoto Y, Hebshi S, Sharma S, Franke AA, Stanczyk FZ.
Serum prostate-specific antigen but not testosterone levels decrease in a
randomized soy intervention among men.Eur J Clin Nutr. 2006;60:1423–9.
32. Quinn DI, Henshall SM, Sutherland RL. Molecular markers of prostate
cancer outcome. [review] [415 refs] Eur J Cancer. 2005;41:858–87.
33. Fritz WA, Wang J, Eltoum IE, Lamartiniere CA. Dietary genistein
down-regulates androgen and estrogen receptor expression in the rat
prostate. Mol Cell Endocrinol. 2002;186:89–99.
34. Lamartiniere CA, Cotroneo MS, Fritz WA, Wang J, Mentor-Marcel R,
Elgavish A. Genistein chemoprevention: timing and mechanisms of
action in murine mammary and prostate. J Nutr. 2002;132:552S–8S.
35. Bektic J, Berger AP, Pfeil K, Dobler G, Bartsch G, Klocker H. Androgen
receptor regulation by physiological concentrations of the isoflavonoid
genistein in androgen-dependent LNCaP cells is mediated by estrogen
receptor. [beta] Eur Urol. 2004;45:245–51.
36. Franke AA, Custer LJ, Wilkens LR, Le Marchand LL, Nomura AM,
Goodman MT, Kolonel LN. Liquid chromatographic-photodiode array
mass spectrometric analysis of dietary phytoestrogens from human urine and
blood. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;777:45–59.
37. Nutritionist V Version 2.3. First DataBand Division. San Bruno, CA:
The Hearst Corporation, 2000.
38. Budwit-Novotny DA, McCarty KS, Cox EB, Soper JT, Mutch DG,
Creasman WT, Flowers JL, McCarty KS, Jr. Immunohistochemical
analyses of estrogen receptor in endometrial adenocarcinoma using a
monoclonal antibody. Cancer Res. 1986;46:5419–25.
39. Version SAS 9.1, Cary, NC: SAS Institute, Inc., 2004.
40. Burnstein KL. Regulation of androgen receptor levels: Implications for
prostate cancer progression and therapy. J Cell Biochem. 2005;95:
657–69.
41. Lund TD, Munson DJ, Adlercreutz H, Handa RJ, Lephart ED.
Androgen receptor expression in the rat prostate is down-regulated by
dietary phytoestrogens. Reprod Biol Endocrinol. 2004;2:5.
42. Dalu A, Blaydes BS, Bryant CW, Latendresse JR, Weis CC, Barry
Delclos K. Estrogen receptor expression in the prostate of rats treated
with dietary genistein. J Chromatogr B Analyt Technol Biomed Life Sci.
2002;777:249–60.
43. Skliris GP, Parkes AT, Limer JL, Burdall SE, Carder PJ, Speirs V.
Evaluation of seven oestrogen receptor beta antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast
tissue. J Pathol. 2002;197:155–62.
44. Anderson KE, Kappas A, Conney AH, Bradlow HL, Fishman J. The
influence of dietary protein and carbohydrate on the principal oxidative
biotransformations of estradiol in normal subjects. J Clin Endocrinol
Metab. 1984;59:103–7.
45. Adlercreutz H. Diet and sex hormone metabolism. In: Rowland I, editor. Nutrition, toxicity, and cancer. Boca Raton, FL: CRC Press; 1991.
p. 137–194.
46. Severi G, Morris HA, MacInnis RJ, English DR, Tilley W, Hopper JL,
Boyle P, Giles GG. Circulating steroid hormones and the risk of prostate
cancer. Cancer Epidemiol Biomarkers Prev. 2006;15:86–91.
47. Takase Y, Levesque MH, Luu-The V, El-Alfy M, Labrie F, Pelletier G.
Expression of enzymes involved in estrogen metabolism in human
prostate. J Histochem Cytochem. 2006;54:911–21.
Soybean effect