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Department of Food Science and Human Nutrition, * Department of Horticulture, Michigan State University, E. Lansing, MI 48824
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENTS
LITERATURE CITED
ABSTRACT 
These studies were undertaken to assess the estrogenic and antiestrogenic effects of dietary genistein. To determine estrogenic effects, genistein was mixed into a modified AIN-76 or AIN-93G semipurified diet at 0 (negative control), 150, 375 or 750 µg/g and 17, 
-estradiol at 1.0 µg/g and fed to ovariectomized 70-d-old Sprague-Dawley rats. Estrogenic potency was determined by analyzing uterine weight, mammary gland development, plasma prolactin and expression of uterine c-fos. Dietary genistein (375 and 750 µg/g) increased uterine wet and dry weights (P < 0.05). Mammary gland regression following ovariectomy was significantly inhibited by dietary genistein at 750 µg/g (P < 0.05). Plasma prolactin was significantly greater in ovariectomized rats fed genistein (750 µg/g) compared with comparable rats not receiving genistein. The relative binding affinity of genistein to the estrogen receptor (ER) was ~0.01 that of estradiol. Genistein (750 µg/g) induced the uterine expression of c-fos. To evaluate potential antiestrogenic effects, genistein and estradiol were mixed into the modified AIN diets at the doses noted above and fed to ovariectomized rats. Dietary genistein (375 or 750 µg/g) did not inhibit the effects of estradiol on uterine weight, mammary gland development or plasma prolactin. Serum concentration of total genistein (conjugated plus free) in rats fed 750 µg/g was 2.2 µmol/L and free genistein was 0.4 µmol/L. Administration of dietary genistein at 750 µg/g can exert estrogenic effects in the uterus, mammary gland and hypothalamic/pituitary axis. Dietary genistein (750 µg/g) did not antagonize the action of estradiol in estradiol-supplemented ovariectomized rats or in intact rats.
INTRODUCTION
Phytoestrogens include the isoflavones, lignans and other nonsteroidal chemicals found in plants and plant products. These compounds can bind to the estrogen receptor and are thought to exert their estrogenic effects through mechanisms similar to those of estradiol.
The consumption of certain plants and plant products can result in impaired reproductive function in livestock. Over five decades ago, clover disease, a syndrome with effects ranging from temporary to permanent infertility, was described in sheep foraging upon subterranean clover in western Australia (Bennets et al. 1946
). Additional studies have documented impaired reproductive function in a number of species (reviewed in Price and Fenwick 1985) including desert quail feeding upon desert brush (Leopold et al. 1976). Furthermore, a decrease in reproductive performance was observed in female rats fed either a soy-based diet or a diet supplemented with genistin at 2 g/kg diet (Carter et al. 1955), and in male mice fed genistin at 36 mg/(mouse·d) (Matrone et al. 1955). All of these effects were attributed to the phytoestrogen content of the diets.
It was later discovered that genistein was responsible for the impaired reproductive performance seen in sheep ingesting subterranean clover (Bradbury and White 1951). Genistein is an isoflavone (4
,5,7-trihydroxyisoflavone), which has estrogenic activity (Folman and Pope 1966), present in various plants including soybeans (Naim et al. 1974). Since the initial discovery of its estrogenic activity, there have been a number of studies in which the effects of soy and genistein on the uterus of mice and rats were evaluated (Carter et al. 1953): all of these studies demonstrated estrogenic effects except the work of Farmakalidis and Murphy (1984). In their study, the potent estrogen agonist diethylstilbestrol also did not promote uterotrophic effects in this strain of mouse (CD-1).
The estrogenic effects of genistein in the uterus are well documented. However, few experiments have been conducted to assess estrogenic effects in other tissues, and to our knowledge, none have examined the effects of dietary genistein on the mammary gland or the hypothalamic/pituitary axis. These studies were undertaken to provide additional insight into the actions of dietary genistein by analyzing its effects on the uterus, pituitary gland and mammary gland. In addition, the plasma concentration of genistein responsible for these estrogenic effects was also determined.
MATERIALS AND METHODS
. In both processes, chemical identity was assessed by nuclear magnetic resonance (NMR)3 and purity assessed at >98%. 3H-17, -Estradiol (3.59 TBq/mmol) and 32P-deoxycytidine 5-triphosphate, tetra(triethylammonium) salt (111 TBq/mmol) were purchased from Dupont New England Nuclear (Boston, MA). All other chemicals, unless otherwise specified, were purchased from Sigma, St. Louis, MO.
Animals.
All rats were maintained according to guidelines in the Guide for the Care and Use of Laboratory Animals (NRC 1985). Intact and ovariectomized 60-d-old Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing from 200 to 225 g were used in Experiments 1 and 2. In Experiment 3, intact 30-d-old rats weighing from 80 to 90 g were used. Upon receipt, rats were weighed and sorted to equalize animal weights within each treatment group. Unless otherwise noted, rats were acclimated to the animal care facility and diet for 7 d prior to initiating the studies. Rats were maintained in the animal care facility with temperature 22 ± 2°C, relative humidity (40-70%), and a 12-h light:dark cycle. Rats were housed in polycarbonate cages (3 rats/cage) with aspen woodchip bedding and were allowed unrestricted access to food and water.
Diets.
Animals were fed the American Institute of Nutrition-93G (AIN-93G) or modified AIN-76 diets prepared in our facilities. For the AIN-76 diet cornstarch was substituted for sucrose to lower the sucrose concentration from 50 g/100 g to 10 g/100 g of the diet. Semipurified diets are required because the potential presence of genistein and other phytoestrogens in the soy portion of commercial nonpurified diets could confound the experimental results. Genistein and 17,-estradiol were mixed into the AIN-76 or AIN-93G diets at the concentrations specified in Experiments 1, 2 and 3.
Experiment 1: estrogenic effects of dietary genistein and estradiol.
Forty-two rats were ovariectomized at 56 d of age and dietary treatments were begun at 70 d of age. The ovariectomized rats (6/treatment) were given free access to food and water for a period of 5 d. Dietary treatments consisted of genistein at 150, 375 and 750 µg/g or estradiol at 0.5, 1.0 and 1.5 µg/g. Eight intact and six ovariectomized 70-d-old rats were fed a modified AIN-76 diet and served as positive and negative controls, respectively. Estrogenic activity was assessed by uterine wet and dry weights.
. The inguinal mammary gland was excised, fixed in a 3:1 (v/v) solution of ethanol:acetic acid for 2 h, transferred to 70% ethanol for 3 d with ethanol changed each day, rinsed in water and stained in alum-carmine for 16 h. The glands were then rinsed in water, placed in 70, 95 and 100% ethanol for 30 min each, transferred to toluene for 24 h for clarification and then stored in absolute methyl salicylate. Lobulo-alveolar and ductal structure (extent of side branching) were assessed using a dissecting microscope and assigned values from 0 to 4, with the higher number representing maximal development. Analyses were performed twice on each gland with the identity of the glands unknown to the analyst.
Northern blot analysis of c-fos.
Total RNA was isolated by the procedure of Chirgwin et al. (1979) as modified by Helferich et al. (1990). Uteri from four rats in each group (randomly selected from the following groups: ovariectomized control, 1 µg/g estradiol and 750 µg/g genistein) were removed from liquid nitrogen, placed in 4.0 mL of 4.0 mol/L guaninidium thiocyanate (GITC) and homogenized on ice with a polytron (Brinkman Instruments, Westbury, NY). N-Lauryl sarcosyl was added (100 µL 10% solution per milliliter GITC), the mixture vortexed and cellular debris removed by centrifugation at 12,000 × g (g max) for 10 min. The supernatant was removed and layered over 1.0 mL of 5.7 mol/L CsCl followed by centrifugation at 110,000 × g (g max) for 16 h. The pellet was resuspended in 7 mol/L guanidine-HCl, 20 mmol/L sodium acetate, 1 mmol/L dithiothreitol, 10 mmol/L iodoacetic acid and 1 mmol/L Na2EDTA and transferred to a 1.5-mL microfuge tube. RNA was precipitated with 2 volumes absolute ethanol and 0.1 volume 300 mmol/L sodium acetate at 
20°C. RNA precipitates were washed once with 3 mol/L sodium acetate and 10 mmol/L iodoacetate, pH 5.0 at 4°C, then with 66% ethanol and 33 mmol/L sodium acetate, pH 5.0, and then with absolute ethanol at 20°C. Ethanol was removed and the RNA pellet dissolved in Tris-EDTA (pH 8.0) and spectrophotometrically quantified at 260/280 nm. Ten micrograms of RNA was loaded onto a 1.2% agarose formaldehyde denaturing gel and electrophoresed for 8 h at 35 V. The RNA was transferred to membrane (Hybond-N, Amersham Life Sciences, Cleveland, OH), and the membrane exposed to UV light at 120 kJ/cm2 for 2 min. Rat c-fos cDNA, 2100 bp, was kindly provided by Tom Curran (Roche Institute of Molecular Biology, Nutley, NJ) and probes made by random priming (Boeringer Mannheim, Indianapolis, IN) with incorporation of 32P-deoxycytidine 5-triphosphate. The membrane was blocked by prehybridizing with herring sperm DNA at 42°C for 4 h followed by hybridization for 12 h and subsequent washing at 65°C with 2X saline sodium citrate buffer (SSC) + 0.1% sodium dodecyl sulfate (SDS) for 30 min each, then 0.3X SSC + 0.1% SDS for 25 min. The hybridized membrane was exposed to Kodak X-OMAT film at 70°C for 3 d and then developed.
Plasma prolactin analysis.
Plasma prolactin concentration was determined in the laboratory of Keith Lookingland at Michigan State University utilizing a double antibody RIA employing reagents and procedures of the National Institute for Diabetes and Digestive and Kidney Disease (NIDDK) assay kit (generously supplied by A. F. Parlow and S. Ratti, National Hormone and Pituitary Program, Rockville, MD). NIDDK rat PRL RP-3 was used as the standard. Using a 100-µL aliquot of plasma, the lower limit of sensitivity was 10.0 µg/L. Samples from each rat were assayed in duplicate at two different dilutions in a single radioimmunoassay.
Competitive binding analysis.
Competitive binding analysis with 3H-estradiol was performed using uterine cytosol prepared from untreated rats consuming a nonpurified diet (22/5 Rodent Diet (w) 8640, Harlan-Teklad, Madison, WI). Rat uteri were placed in ice-cold TEDG (10.0 mmol/L tris-HCl pH 7.4, 1.5 mmol/L EDTA pH 7.4, 1.0 mmol/L dithiothreitol added fresh, and 10% glycerol) and homogenized with a polytron (Brinkman Instruments). The homogenate was centrifuged at 800 × g for 10 min, and the resulting supernatant removed and centrifuged at 110,000 × g (g max) for 1.5 h at 4°C. Protein concentration was 5.0 g/L as determined by the Bradford assay (Bradford 1976). Aliquots were quickly frozen in a isopropanol/dry-ice bath and stored at 70°C. Binding assays were composed of 200 µL uterine cytosol (1.0 mg total protein), TEDG, and genistein or estradiol in ethanol vehicle bringing the total volume to 500 µL. Assays containing 5.0 nmol/L 3H-estradiol and 0-20 nmol/L 17,-estradiol or 0-50 µmol/L genistein were incubated at 4°C for 3 h. After incubation, the contents were removed and placed in a microfuge tube containing the pellet from 250 µL of dextran-coated charcoal (DCC) solution (5% Norit A, 0.5% dextran T-70 in TEDG) to remove the unbound 3H-estradiol. The microfuge tube was vortexed to disperse the DCC pellet and incubated for 3 min at 23°C followed by centrifugation at 13,000 × g to pellet the DCC. Two 200-µL aliquots of the supernatant were collected and counted on a scintillation counter (Beckman Instruments Model LS100, Fullerton, CA). The counts were averaged, divided by the total counts and expressed as a percentage of the total radioactivity.
Serum genistein analysis.
Rats were anesthetized with diethyl ether, and blood (~6 mL/rat) was collected from the tail artery. Blood was placed at 4°C for 16 h to allow clotting. The blood was then centrifuged at 350 × g for 10 min and the serum removed and stored at 70°C. For genistein analysis, 50 µL of serum, from each of four rats fed 750 µg/g genistein in Experiment 2, was sampled in duplicate with one set receiving 5 µL (515 units) of B-glucuronidase Type H-1 (Sigma). All aliquots were incubated in 0.5-mL microfuge tubes at 37°C for 48 h. Following the incubation, 50 µL of absolute methanol was added to each tube, the tubes vortexed and then centrifuged at 15,000 × g for 10 min. Approximately 75 µL was removed and placed at 20°C until analysis. For analysis, the samples were centrifuged at 15,000 × g for 15 min and 20 µL injected onto a C18 column (Microsorb-MV, 5 µm 100A, Rainin Instrument, Woburn, MA) with a flow rate of 1.0 mL/min of 50:50 methanol:water, with 17 mmol/L acetic acid. Recovery was determined by spiking serum from control rats with genistein and then assessing recovery of genistein. Mean recoveries were determined to be 74% (SEM 1.68%). The data presented are corrected for recovery. No genistein was detected in control rats fed the AIN-76A diet.
Statistical analyses.
All statistical tests were performed using a PC-based version of the Statistical Program for the Social Sciences (SPSS) Version SPSS/PC 2.0, Chicago, IL 60611. Uterine weight and plasma prolactin data were analyzed by one-way ANOVA. Variances in uterine weights [Tables 1 and 2] were nonhomogeneous with respect to treatment; thus these data were log transformed prior to ANOVA. Data are means and standard errors before transformation. When a significant (P < 0.05) treatment effect was detected, treatment means were compared using the least significant difference method (Steel and Torrie 1980). Mammary gland data were analyzed by the Kruskal-Wallis nonparametric test (Shavelson 1988). When a significant (P < 0.05) treatment effect was detected, treatment rank means were compared using the least significant difference method (Steel and Torrie 1980). Values in the text are means ± SEM.
RESULTS
-estradiol required to displace 50% of the bound 3H-17,-estradiol in rat uterine cytosol in this study was ~5 nmol/L (Fig. 1). Competitive binding analysis indicated that the relative binding affinity of genistein was ~0.01 that of 17-estradiol.
-estradiol and 3H-estradiol were incubated with rat uterine cytosol for 2 h. Following incubation, free compounds were removed with dextran-coated charcoal and aliquots counted in a Beckman LS-100 scintillation counter. Each point represents the mean of two replicates. Values are expressed as a percentage of the total radioactivity.
Uterotrophic effect of dietary genistein. Genistein administered in the diet to ovariectomized adult rats in Experiment 1 at 150, 375 and 750 µg/g produced a dose-dependent increase in both uterine wet and dry weights (Table 1). In rats fed genistein at 375 and 750 µg/g, significantly greater uterine wet weights and dry weights than those in the control group were measured. Rats fed 750 µg/g genistein had uterine weights similar to those of rats fed 1.0 µg/g estradiol.
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Table 1. Effect of dietary genistein and estradiol on uterine weight in ovariectomized mature rats1 |
Table 2.
Effect of dietary genistein and estradiol on uterine weight and plasma prolactin in adult ovariectomized rats1
-estradiol was evaluated by comparing uterine weights of the 17,-estradiol treated group with those of the groups receiving 17,-estradiol plus genistein. Uterine weights in the base-line group (rats killed at the start of dietary treatment, 14 d after ovariectomy) indicated that substantial regression had occurred (Table 2) when compared with intact rats at the same age used in Experiment 1 (Table 1). Rats receiving genistein and estradiol at all doses had significantly greater uterine weights than those in the control and base-line groups (Table 2). Genistein at 150, 375 or 750 µg/g did not inhibit the uterotrophic effect of concurrently administered 17,-estradiol (Table 2).
Table 3.
Effect of dietary genistein on uterine weight of intact immature rats1
-estradiol and untreated controls were analyzed for the presence of c-fos mRNA (Fig. 2). Gels were stained with ethidium bromide to confirm equal loading of RNA and to assess the integrity of the RNA (data not shown). Both dietary genistein and estradiol induced the expression of c-fos mRNA relative to that of the untreated control rats.
-estradiol. Ten micrograms of total RNA was electrophoresed on a 1.2% denaturing agarose gel, transferred to nylon and hybridized with random primed rat c-fos DNA probe labeled with 32P. The film was exposed for 3 d.
Mammatrophic effect of dietary genistein. In Experiment 2, the mammatrophic effects of dietary genistein were evaluated in ovariectomized rats by analyzing the following two criteria: 1) lobulo-alveolar structure and 2) ductal structure including side branching and infiltration of ducts into the fat pad of the mammary gland. Dietary treatment of ovariectomized rats for 21 d with 750 µg/g genistein prevented mammary gland regression, seen primarily in lobulo-alveolar structure, relative to that of the ovariectomized, untreated control rats (Table 4 and Fig. 3). Average lobulo-alveolar development in the 17,
-estradiol-treated rats did not differ from controls. Average ductal development did not differ for the genistein- or estradiol-treated groups compared with the controls. Lobulo-alveolar development was significantly greater for the groups receiving 750 µg/g genistein or genistein 750 µg/g + 17,-estradiol 1.0 µg/g, compared with the control group. The potential of genistein to antagonize the mammatrophic effect of estradiol was also evaluated. Coadministration of dietary genistein at 150, 375 or 750 µg/g, with 1.0 µg/g 17,-estradiol, did not result in lower mammary scores compared with rats receiving 17,-estradiol alone (Table 4).
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Table 4. Effect of dietary genistein and estradiol on the mammary gland of ovariectomized adult rats1 |
DISCUSSION
), in an estrogen-responsive tissue. Uterine expression of c-fos was induced in ovariectomized rats following the dietary administration of genistein or 17,-estradiol. The variable expression of c-fos may be due to several factors, including the following: 1) the timing of food consumption, 2) variability in food consumption, 3) metabolism of genistein, and 4) the short half-life of c-fos mRNA (Greenburg and Ziff 1984). The induction of c-fos by genistein suggests that genistein is acting through the ER, similar to estradiol, and is capable of forming an active complex with the ER in uterine tissue.
Uterotrophic effects of dietary genistein.
Phytoestrogens have long been recognized for their uterotrophic activity in a variety of animal species. These effects range from temporary to permanent infertility (reviewed by Adams 1989). In the present study, there was a dose-dependent increase in uterine weight with effects seen at a dietary dose of genistein as low as 375 µg/g diet, suggesting that genistein acts in the uterus in a manner similar to that of estradiol. That is, genistein binds to the ER, and the ligand:receptor complex induces the expression of estrogen-responsive genes which ultimately result in increased uterine mass.
inhibited the uterotrophic effect induced by subcutaneous injections in mice of 0.4 µg estrone (total dose) by administering concurrent subcutaneous injections of either 800 or 1600 µg genistein (total dose) twice daily over a 3-d period. In our study, the coadministration of 150, 375 or 750 µg/g genistein with 1.0 µg/g 17,-estradiol did not inhibit the effects of estradiol on uterine weight. In addition, dietary genistein did not affect the development of the uterus, when administered to immature rats, as assessed by uterine weight during maturation of the organ. In the studies reported here, genistein was administered in the diet to rats, whereas in the study by Folman and Pope (1966), genistein was administered subcutaneously to mice. The dose of genistein administered in our studies, relative to the dose of estradiol, was much lower than that of Folman and Pope. Furthermore, variability in species as well as strain in response to compounds with estrogenic activity is well documented (reviewed by Adams 1989, Farmakalidis and Murphy 1984). The amount of genistein absorbed from the gut is currently unknown. All of these variables could account for the different results obtained in this study compared with that of Folman and Pope.
). The model employed in these studies utilized 60-d-old rats and assessed the ability of dietary genistein and/or estradiol to inhibit mammary gland regression postovariectomy.
). Estrogen acts directly at the mammary gland by inducing gene transcription and the subsequent translation of many proteins, including the progesterone receptor required for progesterone action. Estrogen also acts indirectly through the induced synthesis and release of prolactin from the anterior pituitary gland which then exerts its mitogenic effects on the mammary gland. Removal of endogenous estrogen results in regression of the mammary gland, particularly the lobulo-alveolar structures. Mammary gland regression at 35 d postovariectomy in untreated control rats was greater than that in the untreated base-line rats measured at 14 d postovariectomy (the start of dietary treatment). Dietary genistein prevented regression of the mammary gland compared with the untreated controls. Our goal in this set of experiments was to evaluate whether the concurrent administration of genistein at different doses would inhibit the effects of estradiol in preventing regression of the mammary gland. Orally administered estradiol (1 µg/g) was effective in increasing uterine weight and plasma prolactin; however, it was ineffective in the mammary gland. The reason dietary estradiol exerts estrogenic effects in the uterus and not in the mammary gland is unclear. The ability of genistein to inhibit the effects of estradiol in the mammary gland could not be determined with the dose of estradiol used in this study.
). Estrogen is thought to decrease the activity of tyrosine hydroxylase, thereby decreasing the concentration of dopamine in the hypothalamus and pituitary (Jones and Naftolin 1990), resulting in increased plasma prolactin. Genistein or estradiol administered in the diet resulted in increased plasma prolactin compared with the ovariectomized control animals. The increase in plasma prolactin in this study suggests that dietary genistein functions in the hypothalamus and pituitary gland in a manner analogous to that of estradiol, leading to the synthesis and release of prolactin from the anterior pituitary gland.
). Women who consumed soy milk powder daily, which contained 227 mg genistein, had plasma genistein concentrations (conjugated plus free) of up to 6.0 µmol/L (Xu et al. 1995).
showed that soy supplementation produced changes in the uterus similar to those produced by estrogen. However, other studies have failed to show effects different from controls by supplementing soy in the diet of postmenopausal women (Baird et al. 1995).
FOOTNOTES
Manuscript received April 1996. Initial reviews completed 6 June 1996. Revision accepted 25 October 1996.
ACKNOWLEDGMENTS
Sincere appreciation is extended to Keith Lookingland and his laboratory at Michigan State University for their analysis of plasma prolactin. Thanks are also extended to Les Bourquin and Gale Strasburg for their critical review of the manuscript.
LITERATURE CITED
-actin synthesis is increased pretranslationally in pigs fed the phenethanolamine ractopamine.
Endocrinology
1990;
126:3096-3100
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Y. Somekawa, M. Chiguchi, T. Ishibashi, K. Wakana, and T. Aso Efficacy of Ipriflavone in Preventing Adverse Effects of Leuprolide J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3202 - 3206. [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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 C. Munaut, V. Lambert, A. Noel, F. Frankenne, M. Deprez, J.-M. Foidart, and J.-M. Rakic Presence of oestrogen receptor type {beta} in human retina Br. J. Ophthalmol., July 1, 2001; 85(7): 877 - 882. [Abstract] [Full Text] [PDF]
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 M. S. Cotroneo and C. A. Lamartiniere Pharmacologic, but Not Dietary, Genistein Supports Endometriosis in a Rat Model Toxicol. Sci., May 1, 2001; 61(1): 68 - 75. [Abstract] [Full Text] [PDF]
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