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Original Studies |
Epithelial Biology Group (D.F.H., C.S.P., L.E.S.), Biomathematics and Computing Unit (S.A.R.), Paterson Institute for Cancer Research; Department of Medical Oncology, Clinical Research Center (A.H.); and Christie Hospital National Health Service Trust, M20 4BX Manchester, United Kingdom; Tenovus Institute, University of Wales College of Medicine (M.S.M.), CF4 4XN Cardiff, United Kingdom; and the Department of Surgery, University Hospital of South Manchester (C.H., N.J.B.), M20 2LR Manchester, United Kingdom
Address all correspondence and requests for reprints to: Dr. N. J. Bundred, Department of Surgery, University Hospital of South Manchester, M20 2LR Manchester, United Kingdom. E-mail: bundredn{at}fs1.with man.ac.uk.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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0.025). Nipple aspirate (NA)
levels of genistein and daidzein were higher than paired serum levels,
both before (P < 0.001 and P =
0.001, respectively) and after soy supplementation
(P < 0.001 and P = 0.049,
respectively); however, there was no significant increase in NA
isoflavone levels in response to soy. NA levels of apolipoprotein D
were significantly lowered and pS2 levels raised in response to soy
supplementation (P 0.002), indicative of an
estrogenic stimulus. No effect of soy supplementation on breast
epithelial cell proliferation, estrogen and progesterone receptor
status, apoptosis, mitosis, or Bcl-2 expression was detected. In
conclusion, short term dietary soy has a weak estrogenic response on
the breast, as measured by nipple aspirate apolipoprotein D and pS2
expression. No antiestrogenic effect of soy on the breast was detected. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Experimental evidence to prove that dietary soy has a biological effect on mammary tissue is limited to animal models of breast cancer, with several studies demonstrating a reduction in the incidence and size of radiation-induced and chemically induced mammary tumors when rats were fed a soy diet (7, 8, 9). Injection of rodents with isolated isoflavones results in early mammary gland differentiation, protecting against future mammary tumor development (10). Isoflavones also inhibit the proliferation of breast cancer cells in vitro (11, 12, 13). However, there is contradictory evidence to suggest that isoflavones increase rodent mammary gland proliferation (14) and proliferation in breast tumor cell lines (15, 16), which may be a dose-dependent phenomenon (17).
Dietary studies in premenopausal women have shown that isoflavones increase the length of the follicular phase of the menstrual cycle and suppress midcycle LH and FSH peaks, which could reduce breast cancer risk (18, 19). In postmenopausal women, dietary soy results in a slight estrogenic response, including vaginal cell maturation (see Ref. 49) and a reduction in the severity of menopausal hot flushes and hepatic cholesterol production (20, 21). As the possible effects of a soy- and phytoestrogen-rich diet on the human breast remain unknown, the aim of our study was to determine the effects of short term soy supplementation on the rate of proliferation, apoptosis, and steroid hormone receptor status in the premenopausal normal breast. Nipple aspirates (NA) are breast secretions pooled in the lactiferous sinuses and proximal ducts. It has been found that certain proteins, such as apoliprotein D (ApoD or GCDFP-24) and pS2, present in nipple fluid are estrogen regulated and are useful markers of estrogen action upon the breast (22). As part of our study we undertook to examine the effect of dietary soy on these proteins to determine the overall estrogenic effects of phytoestrogens on the breast.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Women attending symptomatic breast clinics at the University Hospital of South Manchester and due to undergo breast biopsy or definitive surgery for breast cancer were selected for this study. This study was approved by the South Manchester ethics committee, and written informed consent was obtained from each of the 84 patients. Details of reproductive status, contraceptive use, history of breast disease, lactation, last menstrual period, normal diet, and recent antibiotic use were recorded. Patients were excluded if they were taking medication likely to alter pituitary function from baseline or consuming soy-rich diets. The day of the menstrual cycle was determined by questioning the patient and asking patients to return details of the first day of their next menstrual cycle. Patients who did not return these details (n = 4) or who had received a hysterectomy but had intact ovaries (n = 3) had menstrual cycle status determined by analysis of serum 17ß-estradiol, progesterone, LH, and FSH levels. As cycle length varied between individuals, for analysis data were fitted to a normalized cycle of 28 days, assuming that the luteal phase was a standard 14 days, with variations in length occurring in the follicular phase. Breast disease was diagnosed from radiographic, echographic, cytological, and histological examination. Women diagnosed with benign breast disease included fibroadenoma (n = 38), reduction mammoplasty (n = 10), fibrocystic masses (n = 9), duct ectasia (n = 6), sclerosing adenosis (n = 3), lipoma (n = 1), and accessory breast removal (n = 1). Thirteen cases of breast cancer were of the invasive ductal type, and 3 were ductal carcinoma in situ. Fourteen patients were confirmed as taking oral contraceptives at the time of surgery, and 61 were parous.
Patients were randomly allocated to either the soy group taking soy protein in addition to a normal diet (n = 28; mean age, 31.6 yr; SD, 7.36 yr) or to the control group consuming their normal unsupplemented diet (n = 23; mean age, 33.74 yr; SD, 8.25). An additional 6 patients randomized to the soy group and 4 to the control group were deemed unsuitable and were removed from the study. Four of these patients were postmenopausal, 2 were receiving either chemotherapy or tamoxifen therapy, 1 was receiving steroidal treatment, 1 had received a depot contraceptive resulting in an interruption of menstruation, 1 failed to take any soy supplement, and 1 stopped soy supplement 7 days before surgery. To strengthen the control data we included tissue from 33 patients, held in a tissue bank at the Paterson Institute for Cancer Research. All control patients gave a dietary history before tissue collection and were added to the control group (entire control group: n = 56; mean age, 34.89; SD, 8.78 yr). The data from the randomized control patients were analyzed both separately and combined with tissue bank patients. The patients were provided with the soy supplement on the day the trial began and requested to consume 4 bread rolls/day [made from a recipe in a previous dietary study (19)] containing a total of 60 g soy in the form of ground textured vegetable protein (which provided 45 mg isoflavones) for 14 days before surgery. Twenty (71.4%) patients completed 13–14 days of soy supplementation, 4 (14.3%) completed 10–12 days, and 4 (14.3%) completed 8–9 days of soy supplementation. However, all patients said they had taken the last soy supplement 24 h before surgery.
Blood was collected before the commencement of soy supplement (day 0) and subsequent to 14 days of soy supplementation (on the day of operation, before any premedication), and serum was stored at -20 C. NAs were obtained by bimanual, four-quadrant compression of the breast. Attempts were made to obtain specimens from both breasts and before any diagnostic study or surgical procedure on the breast. Fluid was collected into capillary tubes, and the volume of neat nipple secretion was calculated by multiplying the length (in millimeters) of nipple fluid in the tube by the cross-sectional area of the capillary tube lumen. The capillary tubes were transferred into 1.5-mL Eppendorf tubes and centrifuged at 6500 rpm for 5 min. PBS was added to the secretion to give a known dilution, and the diluted specimen was stored at -20 C until analyzed.
ApoD and pS2 measurement in nipple aspirates
ApoD was measured using a competitive polyclonal RIA (23). The assay was sensitive to 80 ng/mL, and the intra- and interassay variations at 10,000 ng/mL were less than 9% and 12%, respectively. Purified ApoD and rabbit anti-lipoprotein D were gifts from Dr. D. A. Haagensen (Sacramento, CA). pS2 was measured using a commercially available RIA kit (CIS UK, Ltd., High Wycombe, Bucks, UK). Each specimen, standard, and control was analyzed in duplicate. The intra- and interassay variations were 3.03% and 7.27%, respectively. Total protein was measured by a modification of the Bradford method, and intra- and inter-assay variations were 6.2% and 8.2%, respectively. pS2 and ApoD levels in breast fluid were calculated per mg protein.
Tissue samples
At surgery, a small portion of grossly normal breast tissue
(
1 cm3) was excised at least 1 cm from the
site of the lesion. Half of this specimen was flash-frozen in liquid
nitrogen and stored at -70 C. A portion of the remaining tissue was
fixed in 4% formal saline overnight or in Carnoy’s fixative for
1 h before paraffin embedding and sectioning for the calculation
of apoptotic and mitotic cell counts. Some tissue was immediately
sliced into 1-mm pieces and incubated in DMEM (high glucose, Life Technologies, Inc., Paisley, UK) containing 10 µCi/mL
tritiated thymidine (SA, 6.7 Ci/mmol; NEN Life Science Products, Hounslow, UK) for 1 h in a shaking water bath at
37 C. Tissue was then fixed in 4% formal saline at 4 C overnight
before paraffin embedding and sectioning for autoradiography. Sections
from all biopsies were stained with hematoxylin and eosin and examined
by the same pathologist to confirm that only histologically normal
breast epithelium was studied.
Thymidine and Ki67 dual labeling
Three-micron sections were cut, mounted on APES (3-aminopropyltriethoxysilane, Sigma Chemical Co., Poole, UK)-coated slides, dewaxed, and hydrated before immunohistochemical staining for the Ki67 antigen. Antigen retrieval was achieved by a microwave method (24), and endogenous peroxidase activity was blocked by incubation in 0.3% (vol/vol) H202 in PBS for 15 min. Slides were rinsed in PBS, and endogenous biotin/avidin activity blocked with a blocking kit (Vector Laboratories, Inc., Peterborough, UK) followed by blocking with 0.5% casein in PBS for 1 h at room temperature. Tissue was incubated with rabbit affinity-purified anti-Ki67 polyclonal antibody (DAKO Corp., High Wycombe, UK) at a 1:100 dilution in PBS for 30 min at room temperature. Serial control sections were incubated in rabbit Ig (negative control, DAKO Corp.) diluted to the same protein concentration as the primary antibody. Slides were rinsed, incubated with biotinylated swine F(ab')2 antirabbit Ig (DAKO Corp.) at 1:400 in PBS for 30 min at room temperature, followed by further rinsing in PBS. Slides were incubated in streptavidin biotin-peroxidase complex-horseradish peroxidase (DAKO Corp.) for 30 min, rinsed, and finally visualized in 0.03% (vol/vol) H202, 0.5 mg/mL 3,3-diaminobenzidine 4-HCl (DAB; Sigma Chemical Co.) in Tris-buffered saline (TBS) for 5 min.
Autoradiographs were prepared by leaving the Ki-67-labeled slides in running deionized water overnight before dipping them in Ilford K5 emulsion (Ilford, Knutsford, UK) diluted 1:1 with distilled water. Slides were exposed for 3 days at 4 C, then developed and fixed as described previously (25), and counterstained with Gill’s hematoxylin only.
Progesterone receptor (PR) labeling
Tissue sections were cut and dewaxed, and nonspecific activity was blocked as described above, but the microwave antigen retrieval step was omitted. Sections were labeled for PR using serum from the PR-ICA kit (Abbott Diagnostics, Maidenhead, UK). Briefly, tissue was incubated with the primary rat monoclonal antibody diluted 1:4 in PBS overnight at room temperature, and serial control sections were incubated with control serum at the same dilution. Slides were rinsed in PBS and incubated with biotinylated rabbit antirat Ig (DAKO Corp.) diluted 1:100 in PBS for 30 min at room temperature followed by further rinsing. Tissue was incubated in streptavidin avidin-biotein peroxidase complex-horseradish peroxidase, visualized with DAB tablets from the Abbott kit, and counterstained as described above.
Estrogen receptor (ER) labeling
The protocol for ER immunohistochemical labeling was similar to that described for Ki-67; however, the casein blocking step was substituted by 10% normal rabbit serum for 15 min. Tissue was incubated overnight (4 C) with the primary antibody, a monoclonal mouse IgG1 anti-human ER (DAKO Corp.) diluted 1:100. Serial control sections were incubated with mouse IgG1 negative control serum (DAKO Corp.), and the secondary antibody used was biotinylated rabbit anti-mouse F(ab')2 Ig (DAKO Corp.) diluted 1:350 for 1 h at room temperature.
Bcl-2 labeling
The protocol for Bcl-2 immunohistochemical labeling was similar to that described above, although the casein blocking step was substituted for 2% BSA, 1% normal goat serum in 0.1% Triton x-100 in TBS for 45 min. Tissue was incubated overnight (4 C) with the primary antibody, a monoclonal mouse IgG1 antihuman bcl-2 oncoprotein (DAKO Corp.) diluted 1:100 in PBS. Serial control sections were incubated with mouse IgG1-negative control serum (DAKO Corp.). Slides were then rinsed twice in TBS and 0.5% Tween (TBST), followed by incubation for 45 min at room temperature in biotinylated goat antimouse IgG (Pierce & Warriner, Chester, UK) diluted 1:200 in TBST and 5% normal human serum. Tissue was then rinsed twice in TBST, followed by incubation in ABC Elite (Vector Laboratories, Inc.) for 30 min at room temperature and then rinsed twice in TBST. DAB incubation was performed as previously described, and the tissue was counterstained with methyl green.
Scoring
Lobular epithelium alone was examined, and no attempt was made to distinguish between myoepithelial and luminal epithelial cells. The threshold for 3HTdR labeling was set at 5 or more silver grains over the nucleus, and Ki67-, PR-, and ER-labeled cells were scored as either positive or negative. Apoptotic cells were identified morphologically, by chromatin condensation, condensed and eosinophilic cytoplasm, and the appearance of a halo around the cell. Mitotic cells were distinguished by the appearance of mitotic figures and loss of the nuclear membrane. A minimum of 1000 cells were scored from several areas of the biopsy, randomly selected, and the percentage of labeled cells was expressed as the labeling index.
Bcl-2 immunoreactivity was perinuclear and cytoplasmic, rather that nuclear. For this reason mean optical density (MOD) measurements of bcl-2 immunoreactivity were obtained using an automated image analysis system (Discovery, Becton Dickinson and Co., Leiden, The Netherlands) that has been used previously in our laboratory to measure immunoreactivity in skin tissue (26). The system obtains images at low magnification on a Leitz Autoplan microscope (Rockleigh, NJ), and images are acquired using a CCD camera (Xillix Microlmager, Xillix Technologies, Richmond, Canada). Methyl green-counterstained nuclei were identified using a 620-nm, 10-nm band pass, full-width half-maximum filter. A mask was created by gray level thresholding, defining all nuclei. An area of interest containing the epithelial compartment of the lobular breast unit was defined. Images were segmented into nuclei and artifact. Objects too small (artifact) or too large (overlapping nuclei) were removed from the selected area. Absorption of methyl green and DAB peroxidase staining were then measured using a 460-nm, 10-nm band full-width half-maximum filter. Both filters have maximum absorption for the stains used and are balanced for the same levels of light transmission. The system was programmed to measure 5 pixels around each defined nucleus to obtain a MOD for perinuclear and cytoplasmic immunoreactivity. A slide of tissue cut from the same block was included in each immunohistochemical assay to act as a standard between assays and the image analysis. MOD values from this slide were then used to normalize the data.
Phytoestrogen measurement
Serum and NA phytoestrogen concentrations were measured by isotope dilution gas chromatography mass spectrometry (ID GC-MS), using a Masslab MD800 benchtop quadrupole mass spectrometer (Thermo, Manchester, UK) coupled to a GC8000 gas chromatograph, which houses a 12-m x 0.32-mm SE54 fused silica capillary column. A cocktail of internal standards, containing deuterated analogs of the lignans, enterodiol and enterolactone, and the isoflavanoids, daidzein, genistein, and equol, was added to a weighed aliquot of the biological sample (serum or NA). After a period of equilibration (30 min at room temperature), sulfate and glucuronide conjugates present were hydrolyzed by overnight incubation with a mixed aryl sulfatase/glucuronidase (Helix pomatia) in acetate buffer (pH 5) at 37 C. The aglycones were extracted into fractionated diethyl ether (once, 8 mL), the ether was evaporated under nitrogen, and the residue was chromatographed on 3.5 x 1-cm columns of Sephadex LH-20. The columns were first washed with 5 mL chloroform-heptane-methanol (10:10:1), and then the isoflavones and lignans were eluted with fractionated methanol. The methanol was evaporated under nitrogen, and the eluate was derivatized for gas chromatography-mass spectrometry by forming the trimethylsilyl ethers by reaction with N,O-bis(trimethylsilyl)trifluoroacetamide. Selected ion monitoring was carried out for each analyte and internal standard, and the concentration of the analyte was determined by reference to a standard curve.
For measurement of phytoestrogen levels in breast tissue, tissue was defrosted and weighed into a glass (12 x 75) mm tube. Sodium acetate buffer, pH 5.0 (1 mL; 0.1 mol/L), was added, and the tissue was homogenized. The homogenate was transferred to a B19 test tube, the 12 x 75-mm tube was washed twice with 0.5 mL buffer, and the washings were transferred to the B19 tube. Internal standard (50 µL) containing deuterated analogs of equol, enterodiol, enterolactone, daidzein, and genistein was added, and the homogenate was mixed using a vortex. After an overnight equilibration at room temperature, mixed aryl sulfatase/glucuronidase (H. pomatia; 1000 U) was added, and the mixture was incubated overnight at 37 C. Thereafter, the method was the same as described above.
Statistics
Changes in breast epithelial proliferation rate between dietary groups were compared using hierarchical analysis of covariance, taking week or half of the menstrual cycle, age, current oral contraceptive (OCP) use, and parity into account. Correlations among Ki67, ER, PR, bcl-2 labeling indexes, apoptotic index, mitotic index, and thymidine labeling index (TLI) were assessed by Spearman nonparametric rank correlations. Differences between control/prediet and postdiet serum and NA phytoestrogen levels were compared by the Mann-Whitney U test where the samples were unmatched and by the Wilcoxon signed rank test where the samples were paired (i.e. pre- vs. postdiet and serum vs. NA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Phytoestrogen levels were measured in the serum of 16 control
patients and 27 soy-supplemented patients randomized within the trial
(Fig. 1
). Some 97.6% of control and
presoy patients had serum equol levels that were undetectable or less
than 5 ng/mL, and 1 patient (2.4%) had a level of 23 ng/L; 80.8% of
patients postsoy supplementation had serum equol levels that were
undetectable or less than 5 ng/mL, and 19.2% were considered as equol
producers, having serum levels between 8.1–391 ng/mL. Analysis of
matched serum from patients randomized to the soy diet indicated that
genistein (P = 0.002), daidzein (P =
0.001), and equol (P = 0.025) levels were significantly
higher postsoy supplementation. Serum levels of the lignans
enterolactone and enterodiol were low (not rising above 0.24 and 14.7
ng/L, respectively, in any of the patients studied) and were unaltered
by soy (P 
0.66). Daidzein and genistein serum levels
were significantly higher (P < 0.001) in
soy-supplemented patients compared to unsupplemented control patients.
Equol, enterodiol, and enterolactone were not significantly different
(P 0.16).
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). No
difference in levels between breasts from the same patient was found,
and the phytoestrogen data for each breast were pooled to obtain a
single value for that patient. Higher levels of daidzein and genistein
were present in NAs compared to paired serum (pretherapy:
P < 0.001 and P = 0.001, respectively;
posttherapy: P < 0.001 and P = 0.049,
respectively), but there was no correlation between serum
and NA isoflavone levels. Analysis of matched NA data from patients
randomized to the soy diet indicated no changes in isoflavone or lignan
levels (P 0.18, respectively), although comparisons
with unsupplemented controls demonstrated significantly higher levels
of daidzein (P = 0.001) and genistein
(P = 0.011) in NA of soy-supplemented patients.
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) and at the present
time we are still unable to assay for genistein. Enterolactone,
daidzein, and equol were present in tissue at levels comparable to
those in serum. Daidzein levels were significantly higher in tissue
taken from soy-supplemented patients (P = 0.028).
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ApoD and pS2 levels from randomized patients are shown in Fig. 3. In 35 women who provided NA
bilaterally, there was a good correlation of both ApoD and pS2 between
breasts (r = 0.9; P < 0.001 for both).
Additionally, 18 women gave sequential NAs at least 1 month apart while
not receiving any therapy, and levels did not change between time
points (r = 0.99 for pS2 and r = 0.94 for ApoD). NA pS2
levels were significantly higher in the soy-supplemented patients
compared to those in both presoy and control groups of patients
(P < 0.001; Fig. 3a). Analysis of paired NAs from
patients pre- and postsoy supplementation showed that ApoD levels were
significantly lower subsequent to soy supplementation
(P = 0.002; Fig. 3b). ApoD levels were not
significantly different in the NA of unsupplemented control and
soy-treated patients. Week or half of the menstrual cycle had no
significant effect on either pS2 or ApoD levels.
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Data from patients randomized within the soy study were analyzed
both separately and together with data from the breast bank. As we
found no significant differences between the groups, the data presented
here are for the entire dataset. Menstrual cycle had a significant
effect on the TLI (P < 0.001) and Ki67 labeling index
(P = 0.004) of the normal breast epithelium (Table 2), with the labeling index
increasing in the luteal phase. Age was inversely correlated with TLI
(
= -0.30; P = 0.009) and positively
correlated with ER and Bcl2 expression ( = 0.23;
P < 0.02). Age, menstrual cycle, parity, and
OCP use were considered in a hierarchical ANOVA to determine the effect
of dietary soy supplementation on TLI, Ki67 labeling index, ER labeling
index, PR labeling index, apoptotic index, mitotic index,
and Bcl-2 MOD. However, no differences in any of the parameters
measured were observed between control and soy-supplemented patients
(P = 0.38; Table 2). Duration of soy supplementation
was also considered in the analysis of all of the tissue, serum, and NA
parameters measured, but no correlations were found with any of the
parameters. The origin of normal breast tissue (e.g. from a
breast containing a malignant or a benign tumor) had no significant
effect on any tissue parameter measured.
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Discussion |
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NA phytoestrogen levels were already relatively high compared to serum levels before soy supplementation, and we found that supplementation of patients with soy did not result in significant increases in isoflavone levels. The discrepancy between control and presoy supplementation NA levels may be a chance finding given the small numbers of patients assayed, particularly in the control group. Study women did not receive their supply of soy supplementation until after the first NA sample was taken, ruling out the possibility that supplementation began early. However, accidental consumption of soy products by patients cannot be eliminated. The fact that NA isoflavone levels were significantly higher than serum levels within individuals (in both control and soy-supplemented groups) is suggestive of secretary accumulation of phytoestrogens within the breast ducts. The concentration of various molecules within NA has been noted previously (33), and estrogen and PRL levels in particular are higher in NA compared to serum samples from the same patient (34, 35). The observed concentration of isoflavones within the NA of patients in this study and the lack of any dietary influence may suggest difficulties in the detection of any phytoestrogen supplementation by analysis of NA alone. Serum levels provided a more accurate indicator of recent phytoestrogen consumption, which may be masked in NA fluid due to the effect of concentration. Although this is the first study of isoflavone levels in NA fluid, a similar concentration effect of both isoflavones and lignans has been noted in prostatic fluid (36). Phytoestrogen levels have not previously been measured in tissue of any type. We found that tissue levels are comparable to those found in serum, indicating that concentration of phytoestrogens occurs only in the breast ducts and not within the tissue itself. Daidzein levels were significantly raised in tissue from soy-supplemented patients, confirming that supplementation of the diet with soy leads to an increase in isoflavone levels in the breast tissue itself.
It has been well documented that pS2 and ApoD expression are under hormonal control (37, 38, 39, 40). Although the function of pS2 remains unknown, transcription of the pS2 gene is activated by estradiol (37), and pS2 expression is inhibited by antiestrogens (38) in MCF-7 cells. A recent study of patients receiving antiestrogen therapy demonstrated a significant down-regulation of pS2 expression in NAs and an up-regulation in response to hormone replacement therapy (22). ApoD (or GCDFP-24) is a 24-kDa glycoprotein associated with high density lipoproteins involved in the binding and transport of cholesterol and progesterone in human serum (39, 40). It constitutes 50% of the total protein in NA (41), and expression of ApoD is inhibited in breast cancer cells by estradiol, whereas androgens stimulate its release (42). ApoD levels in NAs are also significantly lower in women receiving hormone replacement therapy, but increased in women receiving the antiestrogen tamoxifen (22).
NA levels of pS2 were raised in patients after soy supplementation. These findings complement a previous study (43) that demonstrated the stimulation of pS2 expression in MCF7 breast cancer cells by the soy isoflavanoids daidzein and equol. Measurement of paired NAs from patients pre- and postsoy supplementation demonstrated a significant reduction in ApoD levels in response to soy. We found no significant difference in levels between unsupplemented control patients and patients postsoy supplementation. This may be due to the power of the study, given the number of patients sampled and variation between patients. A comparison of the paired results provides a more rigorous test of the data and suggests that ApoD levels are affected by dietary soy. The changes in NA pS2 and ApoD levels after soy supplementation are suggestive of an estrogenic stimulus on the breast. Similar estrogenic effects on NAs of premenopausal women in response to dietary soy have been previously reported (44). These researchers noted an increase in NA volume during soy supplementation, a decrease in GCDFP15, and a significant increase in the detection of hyperplastic cells in NAs during or after soy supplementation, both indicative of estrogen stimulation.
In the early stages of this study, our initial analysis of epithelial proliferation and PR expression suggested that both responded to dietary soy supplementation, indicative of an estrogenic response (25). However, with observations of double the number of patients, we are now unable to detect any differences in breast epithelial proliferation, apoptosis, hormone receptor status, and Bcl-2 expression in response to soy supplementation. Breast epithelial proliferation responded to the effects of age and menstrual cycle as expected (45); however, we were unable to detect similar apoptotic responses, possibly due to the much smaller numbers of apoptoses found. It is well documented that age, menstrual cycle, parity, and OCP use have an important influence on premenopausal breast proliferation, apoptosis, and ER expression (45, 46, 47), but there are wide variations between individuals. Recruitment and compliance of patients in the soy arm of the trial proved to be a problem, and many patients found it quite difficult to consume the required amount of soy on a daily basis. We have been unable to find any evidence of an antiestrogenic effect of dietary soy on the breast. In fact, the response of NA proteins is suggestive of an estrogenic stimulus. Failure to detect any significant effect of supplementation on breast epithelial tissue may point to soy exerting only a weak estrogenic effect on the breast, and prolonged supplementation with larger numbers of patients may be required to detect any alterations in epithelial cell proliferation, hormone receptor expression, and apoptosis rates. Recently, several studies have shown the beneficial effects of soy protein on hot flushes in postmenopausal women (21, 48, 49), which is suggestive of an estrogenic action. Genistein also stimulates mouse mammary tissue proliferation (10), although, by doing so, it confers some protection against mammary tumor development in the adult by apparently increasing differentiation of the gland during puberty. Although we have been unable to determine any significant effect of short term soy supplementation on the breast, the safety of longer term soy supplementation with potentially estrogenic effects should be determined. Treatment of neonatal mice with phytoestrogens could be beneficial (10), and indeed, there is some evidence that habitual diets rich in soy products may reduce breast cancer risk (2). Consumption of soy in puberty and early adult life at an age when breast cancers are initiated may protect against carcinogenesis (50). However, sudden dietary intervention in adult women with high doses of phytoestrogens may promote increases in breast proliferation, which could pose a risk in the aging breast or in women with premalignant (already initiated) breast lesions. Estrogenic responses in the breast (e.g. increased PR expression) can occur without a rise in epithelial proliferation at low estrogen doses (51). It is possible that soy phytoestrogens may initiate an estrogenic response in NA proteins, but do not induce a breast epithelial response as the estrogenic activity is too low. Further longer term dietary supplementation studies are required to determine the full effects of phytoestrogens on the breast in a differing range of age groups.
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Acknowledgments |
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Footnotes |
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2 Present address: Bioclinical Services International, St. Mellons,
Cardiff, United Kingdom.
Received February 16, 1999.
Revised July 28, 1999.
Accepted August 9, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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 C. Steiner, W. H.M. Peters, E. P. Gallagher, P. Magee, I. Rowland, and B. L. Pool-Zobel Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system Carcinogenesis, March 1, 2007; 28(3): 738 - 748. [Abstract] [Full Text] [PDF]
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S. Rice and S. A Whitehead Phytoestrogens and breast cancer -promoters or protectors? Endocr. Relat. Cancer, December 1, 2006; 13(4): 995 - 1015. [Abstract] [Full Text] [PDF]
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 M. Messina, W. McCaskill-Stevens, and J. W. Lampe Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings. J Natl Cancer Inst, September 20, 2006; 98(18): 1275 - 1284. [Abstract] [Full Text] [PDF]
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 C. E. Wood, S. E. Appt, T. B. Clarkson, A. A. Franke, C. J. Lees, D. R. Doerge, and J. M. Cline Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys Biol Reprod, September 1, 2006; 75(3): 477 - 486. [Abstract] [Full Text] [PDF]
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 J. A. Ford Jr., S. G. Clark, E. M. Walters, M. B. Wheeler, and W. L. Hurley Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts J Anim Sci, April 1, 2006; 84(4): 834 - 842. [Abstract] [Full Text] [PDF]
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Y. H. Ju, J. Fultz, K. F. Allred, D. R. Doerge, and W. G. Helferich Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice Carcinogenesis, April 1, 2006; 27(4): 856 - 863. [Abstract] [Full Text] [PDF]
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 P. H. Gann, A. S. Geiger, I. B. Helenowski, E. F. Vonesh, and R. T. Chatterton Estrogen and Progesterone Levels in Nipple Aspirate Fluid of Healthy Premenopausal Women: Relationship to Steroid Precursors and Response Proteins Cancer Epidemiol. Biomarkers Prev., January 1, 2006; 15(1): 39 - 44. [Abstract] [Full Text] [PDF]
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 Y. Huang, S. Cao, M. Nagamani, K. E. Anderson, J. J. Grady, and L.-J. W. Lu Decreased Circulating Levels of Tumor Necrosis Factor-{alpha} in Postmenopausal Women during Consumption of Soy-Containing Isoflavones J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 3956 - 3962. [Abstract] [Full Text] [PDF]
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R. T. Chatterton Jr., A. S. Geiger, E. T. Mateo, I. B. Helenowski, and P. H. Gann Comparison of Hormone Levels in Nipple Aspirate Fluid of Pre- and Postmenopausal Women: Effect of Oral Contraceptives and Hormone Replacement J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1686 - 1691. [Abstract] [Full Text] [PDF]
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G. Maskarinec, Y. Takata, A. A. Franke, A. E. Williams, and S. P. Murphy A 2-Year Soy Intervention in Premenopausal Women Does Not Change Mammographic Densities J. Nutr., November 1, 2004; 134(11): 3089 - 3094. [Abstract] [Full Text] [PDF]
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C. D. Allred, K. F. Allred, Y. H. Ju, T. S. Goeppinger, D. R. Doerge, and W. G. Helferich Soy processing influences growth of estrogen-dependent breast cancer tumors Carcinogenesis, September 1, 2004; 25(9): 1649 - 1657. [Abstract] [Full Text] [PDF]
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C. E. Wood, T. C. Register, M. S. Anthony, N. D. Kock, and J. M. Cline Breast and Uterine Effects of Soy Isoflavones and Conjugated Equine Estrogens in Postmenopausal Female Monkeys J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3462 - 3468. [Abstract] [Full Text] [PDF]
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 A. Sparreboom, M. C. Cox, M. R. Acharya, and W. D. Figg Herbal Remedies in the United States: Potential Adverse Interactions With Anticancer Agents J. Clin. Oncol., June 15, 2004; 22(12): 2489 - 2503. [Abstract] [Full Text] [PDF]
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R. T. Chatterton Jr., A. S. Geiger, S. A. Khan, I. B. Helenowski, B. D. Jovanovic, and P. H. Gann Variation in Estradiol, Estradiol Precursors, and Estrogen-related Products in Nipple Aspirate Fluid from Normal Premenopausal Women Cancer Epidemiol. Biomarkers Prev., June 1, 2004; 13(6): 928 - 935. [Abstract] [Full Text] [PDF]
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P. B. Grace, J. I. Taylor, Y.-L. Low, R. N. Luben, A. A. Mulligan, N. P. Botting, M. Dowsett, A. A. Welch, K.-T. Khaw, N. J. Wareham, et al. Phytoestrogen Concentrations in Serum and Spot Urine as Biomarkers for Dietary Phytoestrogen Intake and Their Relation to Breast Cancer Risk in European Prospective Investigation of Cancer and Nutrition-Norfolk Cancer Epidemiol. Biomarkers Prev., May 1, 2004; 13(5): 698 - 708. [Abstract] [Full Text] [PDF]
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 R. L. Smith, S. Pruthi, and L. A. Fitzpatrick Evaluation and Management of Breast Pain Mayo Clin. Proc., March 1, 2004; 79(3): 353 - 372. [Abstract] [PDF]
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E. Nikander, M. Metsa-Heikkila, A. Tiitinen, and O. Ylikorkala Evidence of a Lack of Effect of a Phytoestrogen Regimen on the Levels of C-Reactive Protein, E-Selectin, and Nitrate in Postmenopausal Women J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5180 - 5185. [Abstract] [Full Text] [PDF]
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W. Willett Lessons from dietary studies in Adventists and questions for the future Am. J. Clinical Nutrition, September 1, 2003; 78(3): 539S - 543. [Abstract] [Full Text] [PDF]
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 C. E. Piersen Phytoestrogens in Botanical Dietary Supplements: Implications for Cancer Integr Cancer Ther, June 1, 2003; 2(2): 120 - 138. [Abstract] [PDF]
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M. S. Kurzer Phytoestrogen Supplement Use by Women J. Nutr., June 1, 2003; 133(6): 1983S - 1986. [Abstract] [Full Text] [PDF]
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W. Miltyk, C. N Craciunescu, L. Fischer, R. A Jeffcoat, M. A Koch, W. Lopaczynski, C. Mahoney, R. A Jeffcoat, J. Crowell, J. Paglieri, et al. Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer Am. J. Clinical Nutrition, April 1, 2003; 77(4): 875 - 882. [Abstract] [Full Text] [PDF]
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J. W. Lampe Isoflavonoid and Lignan Phytoestrogens as Dietary Biomarkers J. Nutr., March 1, 2003; 133(3): 956S - 964. [Abstract] [Full Text] [PDF]
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 W. A. Weiger, M. Smith, H. Boon, M. A. Richardson, T. J. Kaptchuk, and D. M. Eisenberg Advising Patients Who Seek Complementary and Alternative Medical Therapies for Cancer Ann Intern Med, December 3, 2002; 137(11): 889 - 903. [Abstract] [Full Text] [PDF]
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L. T Bloedon, A R. Jeffcoat, W. Lopaczynski, M. J Schell, T. M Black, K. J Dix, B. F Thomas, C. Albright, M. G Busby, J. A Crowell, et al. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women Am. J. Clinical Nutrition, November 1, 2002; 76(5): 1126 - 1137. [Abstract] [Full Text] [PDF]
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 M. Maggiolini, A. Vivacqua, A. Carpino, D. Bonofiglio, G. Fasanella, M. Salerno, D. Picard, and S. Ando The Mutant Androgen Receptor T877A Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Genistein and Quercetin on Human LNCaP Prostate Cancer Cells Mol. Pharmacol., November 1, 2002; 62(5): 1027 - 1035. [Abstract] [Full Text] [PDF]
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 M. P. Warren The Effects of Phytoestrogen Supplementation in Postmenopausal Women Reproductive Sciences, July 1, 2002; 9(4): 184 - 185. [PDF]
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K. I. Block, A. Constantinou, L. Hilakivi-Clarke, C. Hughes, D. Tripathy, J. A. Tice, K. Block, and K. Block Point-Counterpoint: Soy Intake for Breast Cancer Patients Integr Cancer Ther, March 1, 2002; 1(1): 90 - 100. [PDF]
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M. Messina, C. Gardner, and S. Barnes Gaining Insight into the Health Effects of Soy but a Long Way Still to Go: Commentary on the Fourth International Symposium on the Role of Soy in Preventing and Treating Chronic Disease J. Nutr., March 1, 2002; 132(3): 547S - 551. [Abstract] [Full Text] [PDF]
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M. S. Kurzer Hormonal Effects of Soy in Premenopausal Women and Men J. Nutr., March 1, 2002; 132(3): 570S - 573. [Abstract] [Full Text] [PDF]
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G. Maskarinec, A. E. Williams, J. S. Inouye, F. Z. Stanczyk, and A. A. Franke A Randomized Isoflavone Intervention among Premenopausal Women Cancer Epidemiol. Biomarkers Prev., February 1, 2002; 11(2): 195 - 201. [Abstract] [Full Text] [PDF]
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 S. E. McNagny, N. K. Wenger, N. Hilmy, S. Banarer, M. Barton, R. K. Dubey, S. R. Davis, J. E. Manson, and K. A. Martin Postmenopausal Hormone-Replacement Therapy N. Engl. J. Med., January 3, 2002; 346(1): 63 - 65. [Full Text] [PDF]
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Y. H. Ju, C. D. Allred, K. F. Allred, K. L. Karko, D. R. Doerge, and W. G. Helferich Physiological Concentrations of Dietary Genistein Dose-Dependently Stimulate Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Athymic Nude Mice J. Nutr., November 1, 2001; 131(11): 2957 - 2962. [Abstract] [Full Text] [PDF]
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M. J. Messina and C. L. Loprinzi Soy for Breast Cancer Survivors: A Critical Review of the Literature J. Nutr., November 1, 2001; 131(11): 3095S - 3108. [Abstract] [Full Text] [PDF]
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C. D. Allred, Y. H. Ju, K. F. Allred, J. Chang, and W. G. Helferich Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein Carcinogenesis, October 1, 2001; 22(10): 1667 - 1673. [Abstract] [Full Text] [PDF]
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M. Maggiolini, D. Bonofiglio, S. Marsico, M. L. Panno, B. Cenni, D. Picard, and S. Ando Estrogen Receptor alpha Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Two Major Phytoestrogens on Human Breast Cancer Cells Mol. Pharmacol., September 1, 2001; 60(3): 595 - 602. [Abstract] [Full Text] [PDF]
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 T. Bayer, T. Colnot, and W. Dekant Disposition and Biotransformation of the Estrogenic Isoflavone Daidzein in Rats Toxicol. Sci., August 1, 2001; 62(2): 205 - 211. [Abstract] [Full Text] [PDF]
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 C. D. Allred, K. F. Allred, Y. H. Ju, S. M. Virant, and W. G. Helferich Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-dependent (MCF-7) Tumors in a Dose-dependent Manner Cancer Res., July 1, 2001; 61(13): 5045 - 5050. [Abstract] [Full Text] [PDF]
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