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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0161
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 3956-3962
Copyright © 2005 by The Endocrine Society

Decreased Circulating Levels of Tumor Necrosis Factor-{alpha} in Postmenopausal Women during Consumption of Soy-Containing Isoflavones

Yafei Huang1, Shimin Cao1, Manubai Nagamani, Karl E. Anderson, James J. Grady and Lee-Jane W. Lu

Departments of Preventive Medicine and Community Health (Y.H., S.C., K.E.A., J.J.G., L.-J.W.L.) and Obstetrics and Gynecology (M.N.), the University of Texas Medical Branch, Galveston, Texas 77555-1109

Address all correspondence and requests for reprints to: Lee-Jane W. Lu, Ph.D., the Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, Texas 77555-1109. E-mail: llu{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: TNF-{alpha} is a key mediator of inflammatory responses and may play a pivotal role in the development of cancer and in bone resorption.

Objective: This study determined the effect of soy rich in isoflavones on levels of TNF-{alpha}.

Design: Twelve postmenopausal women ingested a 36-oz portion of soymilk containing isoflavones daily for 16 wk and provided fasting blood samples multiple times before, during, and after soy consumption for the analyses of cytokines and monocyte content.

Results: Compared with prediet levels (36.3 ± 14.0 pg/ml), serum levels of TNF-{alpha} decreased by 25.1% (27.2 ± 10.3 pg/ml; P < 0.01) as early as 2 wk after soy consumption and by 66.7% (11.6 ± 5.3 pg/ml; P < 0.01) 10 wk after soy consumption and recovered to the prediet levels 4 wk after the termination of soy consumption (38.6 ± 19.6 pg/ml; P = 0.66). A similar decrease of up to 56.6 and 14.4% was found for serum IL-1{alpha} and the mean percentage of blood monocytes during soy consumption, respectively, but not for IL-6. In cultures of monocytes or whole blood from postmenopausal women, soy isoflavones (genistein and daidzein, 10–1000 nM), tamoxifen (10–1000 nM), or 17ß-estradiol (0.1–10 nM) inhibited lipopolysaccharide (1 µg/ml)-induced TNF-{alpha} production by up to 55.8%.

Conclusions: Isoflavones may be the active components in soy responsible for the decrease of TNF-{alpha} found in postmenopausal women during a soy diet. This antiinflammatory effect of the isoflavones may be important in immune modulation and the prevention of bone loss and cancer.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
TNF-{alpha}, MAINLY PRODUCED by activated monocytes and macrophages, has a broad spectrum of biological activities (1). TNF-{alpha} is a major mediator of immune and inflammatory responses. TNF-{alpha} promotes acute inflammatory responses, such as occur in sepsis and septic shock (2), and it contributes to the development of several chronic conditions, such as chronic inflammation and autoimmune diseases (3). Recent studies also showed that TNF-{alpha} may be involved in postmenopausal osteoporosis by inhibiting the activities of osteoblasts and stimulating osteoclastogenesis (2).

Several studies have linked chronic inflammation with cancer promotion (4, 5, 6), suggesting a direct involvement of TNF-{alpha} in the development and progression of various types of cancer. For example, tumor promoters that induce precancerous lesions, such as trehalose 6-monomycolate, stimulate TNF-{alpha} gene expression in mouse models (7). In contrast, cancer-preventive compounds, such as epigallocatechin-3-gallate, green tea polyphenols, and tamoxifen, are capable of inhibiting TNF-{alpha} release in both mouse models and cultured cells (7). Moreover, the tumor promoter okadaic acid stimulates TNF-{alpha} release and induces skin tumors in wild-type mice, but not in TNF-{alpha}-deficient mice (8), and anti-TNF-{alpha} treatment in a hepatocellular carcinoma mouse model induces the apoptosis of transformed hepatocytes and results in the failure of cancer formation (9), suggesting that TNF-{alpha} is important in the development of both chemical-induced and spontaneous carcinogenesis. In addition to its production by inflammatory cells, TNF-{alpha} is reported to be constitutively produced by malignant cells, and its expression correlates with tumor progression (10). TNF-{alpha} has been suggested to function as an autocrine and paracrine growth factor for ovarian cancer (11). High levels of TNF-{alpha} in breast tumors correlate positively with tumor growth and are associated with tumor grade and node involvement (12, 13). TNF-{alpha} has been found to induce enzymes that enhance tumor spread, such as collagenases (14), to stimulate angiogenesis (15), and to promote experimental metastasis in mouse models (16).

Gonadal steroid hormones, such as 17ß-estradiol, are known to modulate immune responses (17). 17ß-Estradiol treatment decreases TNF-{alpha} levels in cultures of osteoblast-like cells (18, 19), monocytes/macrophages (20, 21, 22, 23, 24), and whole blood (25) and in animal models (26). Natural or surgical menopause and endotoxin exposure increase circulating levels of TNF-{alpha} in humans (27, 28, 29), and these increases can be reversed by treatment with physiologically relevant doses of estrogen (20). In postmenopausal women diagnosed with osteoporosis, raloxifene, a selective estrogen receptor (ER) modulator, decreases circulating levels of TNF-{alpha} and increases lumbar bone density (30). Potential mechanisms by which 17ß-estradiol affects proinflammatory cytokines include the activation of the ERs (17), binding to the activator protein-1-like site in the promoter region of the TNF-{alpha} gene in monocytic cells (31), down-regulation of the production of reactive oxygen species (32), and rapid nongenomic actions (17). Because soy isoflavones have estrogenic activity (33, 34, 35), this study investigated the effects of soymilk containing isoflavones on circulating cytokines in postmenopausal women and the effects of soy isoflavones on lipopolysaccharide (LPS)-induced TNF-{alpha} release from monocytes in vitro and ex vivo whole-blood cultures.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study design

This was a longitudinal, repeated-measures study to determine the effect of soymilk rich in isoflavones on circulating levels of TNF-{alpha} in healthy postmenopausal women. The study was approved by the Institutional Review Board of the University of Texas Medical Branch (UTMB). Written informed consent was obtained from each subject. Postmenopausal women who were healthy, as determined by history and medical examinations, were recruited by posted advertisements from communities surrounding our institution. To qualify, subjects must have had no menstruation for at least 1 yr and not be on hormone replacement therapy.

Women were observed for 8 wk before the initiation of soymilk consumption to collect baseline prediet data, then consumed soymilk for 16 wk (diet period) and were followed for an additional 8 wk after the termination of soymilk consumption to collect postdiet data. During the baseline prediet observation period, subjects consumed their usual home diets while avoiding soy-containing food products. During the soy diet period, subjects ingested a 36-oz portion of soymilk (Banyang Food Co., Houston, TX) daily in place of one small meal (breakfast or lunch) for 16 wk. Soymilk used in this study contained 112.1 ± 29.7 (mean ± SD) mg isoflavones (expressed as free form, genistein and daidzein equivalent) with 85 mol-% of the isoflavones as glucoside conjugates. The molar ratio of daidzein to genistein was 0.8 ± 0.1. Each 36-oz portion of soymilk provided 400 kcal, 37.9 g protein, 20.3 g fat, and 16.6 g carbohydrates, which did not vary from lot to lot. Lots selected for the study were frozen until the day of ingestion. Several different lots were used for this study, and the isoflavone content of each lot was analyzed as described (36).

Each subject provided fasting blood samples during each study visit. There were four separate visits during the 8 wk of baseline observation period, one visit each at 2, 4, 10, and 16 wk after being placed on the soymilk diet and one visit each at 1, 2, 4, and 8 wk after the termination of soymilk consumption. Women were instructed to ingest their daily portion of soymilk at least 24 h before each scheduled blood draw. Sera were separated and stored immediately at –80 C until analysis. One fasting blood sample from the baseline and from each time point during and after the diet was analyzed for complete blood cell and differential counts by the UTMB clinical laboratory.

Serum levels of cytokines

Serum levels of TNF-{alpha}, IL-1{alpha}, and IL-6 were measured in duplicate using commercial ELISA kits, according to the manufacturers’ instructions. The human TNF-{alpha} kit (Predicta kit) was obtained from Genzyme Diagnostics (Cambridge, MA), and the IL-1{alpha} kit (Quantikine) and the IL-6 kit (Quantikine HS) were from the R&D Systems Inc. (Minneapolis, MN). The Genzyme kit was also used for the monocyte culture study. However, the Genzyme kit was not commercially available, after its acquisition by R&D. Therefore, a TNF-{alpha} kit (OptEIA kit) from BD Biosciences (San Diego, CA) was used for the whole-blood culture study. The detection limits for TNF-{alpha} were 3 pg/ml and 2 pg/ml, respectively, for the Genzyme kit and the BD Biosciences kit; 1.0 pg/ml for the IL-1{alpha} kit, and 0.094 pg/ml for the IL-6 kit.

Serum levels of isoflavones

Serum samples were analyzed for isoflavones by competitive enzyme-linked immunosorbent assays using monoclonal antibodies generated against daidzein and genistein and with horseradish peroxidase conjugates of daidzein and genistein as tracers, as described previously (37). Serum samples (100 µl) were digested with 2 µl ß-glucuronidase (102 U/µl; Sigma, St. Louis, MO) overnight at room temperature, and 10 µl of each sample was used for the analysis. The detection limit of the assay was 0.5 ng/well (0.5 ng/250 µl assay medium or 0.5 ng/10 µl serum) (note: immunosorbent assay was not available for equol, a metabolite of daidzein). Results were expressed as amounts of free forms of the isoflavones.

Human monocyte culture studies

Fasting blood samples were collected in EDTA-coated vaccutainers from five healthy postmenopausal women not on soy or hormone replacement therapy. Mononuclear cells were isolated freshly from the blood by Ficoll-hypaque (Amersham Biosciences, Piscataway, NJ) density gradient separation, washed twice with sterile PBS, and then resuspended in RPMI-1640 medium (Sigma). The cells were plated in 10-ml tissue-culture dishes and incubated at 37 C and 5% CO2 for 1 h to allow the monocytes to attach. The nonadherent cells were removed by washing twice with sterile PBS. The adherent monocytic cells were dislodged by gently scraping with a rubber policeman, washed twice by resuspending in sterile PBS, and collected by centrifugation. The monocytes were resuspended to a final concentration of 5 x 106 cells/ml in RPMI-1640 medium containing 10% charcoal/dextran-treated fetal bovine serum, then plated in 96-well tissue-culture plates with each well containing 1 x 106 cells in 0.2 ml medium, and incubated for 1 h at 37 C and 5% CO2 before any chemical treatment. All culture reagents used had endotoxin levels of less than 0.01 ng/ml. The viability of the monocytes was more than 95%, as determined by trypan blue exclusion. The purity of the monocyte preparation was consistently more than 90%, as assessed by the percentage of cells that stained positive for anti-CD14 during a flow cytometry analysis (FACSCalibur, BD Biosciences).

Stock solutions of soy isoflavones and other estrogens were prepared in dimethylsulfoxide (DMSO) and then diluted with RPMI-1640 to make the final DMSO concentration 0.05%. Monocytes in 96-well plate were pretreated with genistein (10–1000 nM), daidzein (10–1000 nM), 17ß-estradiol (0.1–10 nM), tamoxifen (10–1000 nM), or vehicle control (RPMI-1640 containing 0.05% of DMSO) for 30 min. Each series of experiments used monocytes from one postmenopausal woman’s blood. Each chemical treatment was repeated in two wells. Twenty microliters of LPS (from Salmonella enteritidis, Sigma) were then added to each well to give a final concentration of 1 µg/ml LPS. The plates were incubated at 37 C and 5% CO2. An aliquot (20 µl) of culture media was taken from each well at various time points (4, 8, 24, and/or 72 h after LPS stimulation). Cell-free supernatants obtained after centrifugation (800 x g for 3 min) were stored at –80 C until the analysis of TNF-{alpha} using a commercial ELISA kit (Genzyme Diagnostics). These experiments were performed a total of five times (with the exception of tamoxifen treatment, which was performed three times), using the same experimental design, but each time with blood cells from a different postmenopausal woman. Duplicates from each series of experiments were averaged and expressed as percent of control. More data were obtained for the 24-h time point, and therefore data at this time point from different experiments were pooled for statistical analysis. In view of the natural variability in the data, percent decrease in TNF-{alpha} level was used for statistical analysis. Results were expressed as mean ± SD, unless specified.

Ex vivo whole-blood culture studies

Fasting blood samples were collected, from four healthy postmenopausal women, in EDTA-coated vaccutainers. After a 10-fold dilution with RPMI-1640, 1-ml aliquots were incubated in 24-well plates at 37 C and 5% CO2 for 1 h. The blood cultures were pretreated with genistein (10–1000 nM), daidzein (10–1000 nM), 17ß-estradiol (0.1–10 nM), or vehicle (RPMI-1640 containing 0.05% of DMSO) for 30 min in duplicate, followed by the addition of LPS to a final concentration of 100 ng/ml. Four hours after LPS treatment, cell-free supernatants were obtained after centrifugation (800 x g for 3 min) and were stored at –80 C until the analysis of TNF-{alpha} using a commercial ELISA kit (BD Biosciences). This experiment was performed four times, each time with fresh blood from a different postmenopausal woman.

Statistical analysis

The main outcome measures of interest were the time-dependent effect of soy diet on serum levels of the cytokines and the percentage of monocytes in peripheral blood. Friedman tests were used to examine the overall trends of serum levels of TNF-{alpha}, IL-1{alpha}, IL-6, and the percent composition of monocytes before, during, and after soy consumption. If a statistically significant difference was found using the Friedman test, a paired t test was performed on pair-wise comparisons between various treatment groups. Paired t tests were also used to compare the body mass index of study subjects and serum levels of isoflavones before and during soy consumption. Pearson correlation coefficient was estimated, to examine the association between the percent composition of blood monocytes and circulating levels of TNF-{alpha} at the different time points. To test the effects of estrogens and soy isoflavones on TNF-{alpha} release from in vitro monocyte cultures or ex vivo whole-blood cultures, t tests were used. All statistical analyses were performed using Number Cruncher Statistical Systems (NCSS, Kaysville, UT). A two-tailed {alpha}-level of 0.05 was used to determine statistical significance.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characteristics of the study subjects

A total of 12 women (two Hispanics and 10 Caucasians) participated in the study. The study subjects were 56.8 ± 5.0 (mean ± SD) years old and 5.7 ± 3.9 yr post menopause when entering the study. Body mass index values did not change significantly during the soy diet (26.1 ± 5.4 kg/m2 before and 26.2 ± 5.3 kg/m2 during the diet; P > 0.05). The daily intake of soy isoflavones was 112.1 ± 29.7 mg/d (58.7 ± 21 mg/d for genistein and 53.4 ± 10.7 mg/d for daidzein). Mean serum levels of soy isoflavones increased significantly in all study subjects during the periods of soy consumption, compared with baseline levels. The baseline levels were 47 ± 39 ng/ml for genistein and 46 ± 39 ng/ml for daidzein, which were both at the detection limits of the assay, and the levels during the diet were 193 ± 174 ng/ml for genistein (P < 0.01) and 108 ± 78 ng/ml for daidzein (P < 0.01), indicating compliance with the consumption of soymilk. Peak plasma isoflavone levels are attained 6–9 h after soy consumption (38); and in this study, samples were obtained only at 24 h after soymilk ingestion. Therefore, the average serum levels of isoflavones during soy consumption in this study were expected to be higher than those shown here.

Effects of soy consumption on serum levels of TNF-{alpha}, IL-1{alpha}, and IL-6 and blood levels of monocytes

The levels of cytokines were sampled at four different times during the 8-wk baseline observation period, averaged, and the means compared with values at 2, 4, 10, and 16 wk (during soy consumption) and at 17, 18, and 20 wk (corresponding to 1, 2, and 4 wk after the termination of soy consumption) (Fig. 1Go). The coefficient of variation in the baseline levels of TNF-{alpha} was from 4–9% for each individual. The average baseline value of TNF-{alpha} was 36.3 ± 14.0 pg/ml for the group (Fig. 1AGo). Soy consumption decreased circulating levels of TNF-{alpha} in all twelve study subjects in a time-dependent manner (P < 0.01; Friedman test) (Fig. 1AGo). Paired t tests showed that serum TNF-{alpha} levels decreased significantly as early as 2 wk after soy consumption began (27.2 ± 10.3 pg/ml, a decrease of 25.1%; P < 0.01), and the maximal decrease was observed at 10 wk (11.6 ± 5.3 pg/ml, a decrease of 66.7%; P < 0.01). The decrease persisted at 17 wk, i.e. 1 wk after the termination of soymilk consumption (17.5 ± 8.6 pg/ml; P < 0.01). The effects of the soy diet on circulating levels of TNF-{alpha} were no longer evident at 18 wk, i.e. 2 wk after the termination of soymilk consumption (26.9 ± 16.1 pg/ml; P = 0.10), and TNF-{alpha} levels returned to the prediet levels at 20 wk, i.e. 4 wk after the termination of soy consumption (38.6 ± 19.6 pg/ml; P = 0.66 compared with baseline levels; P < 0.05 compared with levels at 10 wk of soy consumption). The time course of these changes in TNF-{alpha} levels clearly indicated a relationship to soy consumption. Similar results were obtained when samples from these twelve women were analyzed using another commercial TNF-{alpha} kit by BD Biosciences (data not shown). Two of the twelve study subjects were equol producers (results from urine analyses, unpublished observation). However, there is no association between the extent of decreases in TNF-{alpha} and equol metabolic phenotypes (results not shown).



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  		FIG. 1. Circulating levels of TNF-{alpha} (A), IL-1{alpha} (B), and IL-6 (C) and percentage of monocytes (D) before, during, and after soy consumption in 12 healthy postmenopausal women not on hormone replacement therapy. Each study subject ingested a 36-oz portion of soymilk, containing about 112 mg isoflavones, daily for 16 wk during the diet period. Soy diet was initiated at wk 0 and stopped at wk 16. Each symbol in the figure represents an individual subject. The means for all the subjects at each time point are connected by lines. P values refer to two-tailed, paired t tests. WBC, White blood cells.

 
Circulating levels of IL-1{alpha} also decreased during soy consumption (P < 0.01; Friedman test) (Fig. 1BGo). The average concentration of IL-1{alpha} was 6.7 ± 6.0 pg/ml in serum during the baseline observation period, decreased to 2.4 ± 5.8 pg/ml at 10 wk (P < 0.01; paired t test) and 3.0 ± 4.7 pg/ml at 16 wk (P < 0.01) after soy consumption began, and recovered to near baseline at 17 wk, i.e. 1 wk after the termination of soy consumption (5.6 ± 5.9 pg/ml). No significant differences were found in serum levels of IL-6 before, during, or after the soy diet (P = 0.90; Friedman test) (Fig. 1CGo).

Soy consumption did not affect the total white blood cell count (mean baseline value, 5.0 ± 0.8 x 103/ml). However, it did affect the white blood cell differential count. Figure 1DGo shows that the percentage of monocytes decreased during soy consumption (P < 0.05; Friedman test), from 7.6 ± 1.6% at baseline to the lowest value of 6.5 ± 1.3% at the 10th week of soy consumption (a decrease of 14%; P < 0.01) and recovered to prediet levels 2 wk after the termination of soy consumption (7.2 ± 1.8%; P = 0.7). Multiple pair-wise comparisons of data from various time points during the soy diet with baseline data showed a statistically significant decrease in the percentage of monocytes at 10 wk during soy consumption. The percentage of monocytes correlated positively with serum levels of TNF-{alpha} in every study subject before, during, and after soy consumption. The r value between the percent composition of monocytes and circulating TNF-{alpha} levels was 0.81 (P = 0.05).

Effects of genistein, daidzein, tamoxifen, and 17ß-estradiol on LPS-stimulated TNF-{alpha} release from in vitro monocyte cultures and ex vivo whole-blood cultures

Figure 2Go shows the time course of LPS-induced TNF-{alpha} release from monocytes in the presence of low concentrations of test compounds (i.e. 0.1 nM 17ß-estradiol, 10 nM genistein, 10 nM daidzein, or 10 nM tamoxifen) in one experiment. TNF-{alpha} levels from monocytes reached a maximum 8 h after LPS stimulation with or without the presence of test compounds. It has been known that the secretion of TNF-{alpha} and the production of NO in the culture medium from monocytes after LPS stimulation induces apoptosis of monocytes (39). The apoptosis of monocytes, in turn, results in the release of intracellular proteases that degrade TNF-{alpha} in the supernatant. This is consistent with our kinetic observation of TNF-{alpha} levels shown in Fig. 2Go. Genistein, daidzein, 17ß-estradiol, and tamoxifen inhibited LPS-stimulated TNF-{alpha} levels at various time points examined (from 4–72 h after treatment). This pattern was observed for all experiments, and data at the 24-h time point from all experiments were used for statistical analysis.



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  		FIG. 2. Time-dependent effects of genistein, daidzein, 17ß-estradiol, and tamoxifen on LPS (1 µg/ml)-stimulated TNF-{alpha} release from monocyte cultures. Monocytes were isolated from control postmenopausal women. The concentrations of the compounds used were 0.1 nM for 17ß-estradiol and 10 nM for all the other test compounds.

 
Figure 3AGo shows the concentration-dependent effects of various test chemicals on TNF-{alpha} release from monocytes in cultures after 24 h of LPS stimulation. To control for the variability in TNF-{alpha} levels from monocytes of different women, levels are expressed as percent of control. Genistein and daidzein (10–100 nM) and 17ß-estradiol (0.1–10 nM) inhibited LPS-induced TNF-{alpha} release from cultured monocytes by up to 65.7% (P < 0.05) in a biphasic concentration-responsive manner, i.e. with comparatively stronger inhibition at lower than higher concentrations. Decreases observed with tamoxifen (10–1000 nM) may represent a trend but were not statistically significant.



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  		FIG. 3. Concentration-dependent effects of genistein, daidzein, tamoxifen, and 17ß-estradiol on LPS-induced TNF-{alpha} release from human monocytes in cultures (A) and whole-blood cultures ex vivo (B). *, P < 0.05; **, P < 0.01 when compared with controls.

 
The ex vivo whole-blood culture more closely mimics the in vivo environment (40). Figure 3BGo shows the concentration-dependent inhibitory effects of genistein, daidzein, and 17ß-estradiol on LPS-induced TNF-{alpha} release in whole-blood cultures at 4 h of LPS stimulation. The inhibition was up to 74% (P < 0.05) (Fig. 3BGo), with stronger inhibitory effects on TNF-{alpha} at lower than higher concentrations.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Soy consumption has been associated with reduced risk for many human cancers, including breast cancer, but the mechanisms are unknown (41). In this study, we observed that soymilk consumption decreased circulating levels of TNF-{alpha} in all twelve postmenopausal women, and the in vitro data suggest that this effect is most likely attributable to the two phytoestrogens in soy, daidzein and genistein. TNF-{alpha} is an inflammatory cytokine that plays a key role in tumor promotion and progression. Therefore, an effect of soy on TNF-{alpha} may be an important mechanism for reducing cancer risk. These observations are consistent with reports that cancer chemopreventive compounds, such as green tea polyphenol and tamoxifen, also reduce TNF-{alpha} levels (42).

Soymilk consumption also decreased circulating levels of IL-1{alpha} in this study, though the effect was less pronounced than for TNF-{alpha} and its duration shorter. IL-1{alpha} and IL-1ß have similar biological activities. Knowledge of the possible changes in IL-1ß levels should provide more insight into this dietary effect. Due to insufficient amounts of samples available, analysis of IL-1ß was not attempted in our study. Soymilk consumption had no effect on circulating levels of IL-6 (n = 12) or TGF-ß (n = 6, results not shown).

The major source of TNF-{alpha} and IL-1{alpha} production is activated monocytes and/or macrophages. TGF-ß is primarily produced by activated T-lymphocytes (43), and IL-6 originates from many different cell types, including fibroblasts, T-lymphocytes, B-lymphocytes, and monocytes (7, 44). Therefore, the lack of effect of soy on IL-6 and TGF-ß may be due to their origin from cells other than the monocyte/macrophage lineage, which is the major source of TNF-{alpha} and IL-1{alpha} and may be the cells affected by soy isoflavones. Soy consumption decreased the proportion of monocytes in blood without affecting total white blood cell count, suggesting that the differentiation of hematopoietic cells was affected. Consistent with this hypothesis, daidzein has been shown to affect cell differentiation (45). The number of monocytes increases after menopause, when endogenous estrogen levels are low, and this increase can be reversed by hormone replacement therapy (46), suggesting a relationship between estrogens and monocyte production. Because genistein and daidzein are estrogenic, the decrease in monocytes in postmenopausal women in this study may have been due to the estrogenic effects of soy isoflavones.

The proportion of monocytes correlated positively with serum levels of TNF-{alpha} in our study. Although a reduction in monocytes partially explained the decrease in TNF-{alpha} during soymilk consumption, the 14% decrease in the proportion of monocytes (Fig. 1DGo) does not appear to explain the much greater decreases (up to 66.7%) in circulating levels of TNF-{alpha} (Fig. 1AGo). Moreover, the effect of soymilk on monocytes lagged behind its effect on circulating levels of TNF-{alpha}. A significant decrease of monocytes was achieved only at 10 wk after initiating the soy diet, whereas the effect on TNF-{alpha} was detectable as early as 2 wk after the soy diet and was maximal at 10 wk. These data suggest that other mechanisms, such as direct inhibition of TNF-{alpha} production from monocytes by soy isoflavones, may be involved in the early decrease of TNF-{alpha} levels, as is evident from the in vitro and ex vivo effects of daidzein and genistein on LPS-induced TNF-{alpha} release (Fig. 3Go). The in vitro studies using monocyte cultures and the ex vivo studies using whole-blood cultures showed that both natural estrogen (17ß-estradiol), phytoestrogens (daidzein and genistein), and synthetic estrogen (tamoxifen) influence LPS-induced TNF-{alpha} release with similar concentration-response kinetics. The similarity of the relationship between concentration and the TNF-{alpha} responses induced by 17ß-estradiol and soy isoflavones (daidzein and genistein) suggests that the estrogenicity of daidzein and genistein may be directly involved in the inhibition of TNF-{alpha} production. A number of recent studies have also shown that isoflavones have similar inhibitory effects on IL-1 and IL-6 production in stimulated cell cultures (47, 48, 49, 50). Because the effects of the soy diet on IL-1 and IL-6 in humans were weak, the effects of soy isoflavones on these two cytokines in monocyte cultures and ex vivo whole-blood culture studies were not studied.

Tamoxifen is an ER antagonist in breast and an ER agonist in the uterus (51) and, like 17ß-estradiol, preserves bone density (52). Tamoxifen also inhibits activities of cultured osteoclasts (53), which are derivatives of the hematopoietic cells of monocyte/macrophage lineage. In this study, tamoxifen showed an inhibitory effect on TNF-{alpha} release from monocyte cultures. Whether the inhibitory effect of genistein, daidzein, and tamoxifen on TNF-{alpha} release from monocytes is ER-mediated needs to be investigated further.

Soy isoflavones have been reported to have a number of biological effects, including estrogenic/antiestrogenic and antioxidant effects (54), induction of cell differentiation and apoptosis (55, 56), and inhibition of tyrosine kinase (57) and topoisomerases (58). With the exception of estrogenicity, these in vitro effects were observed only at high, nonphysiological concentrations of soy isoflavones (>10–50 µM). Concentrations of circulating isoflavones in humans are usually much less than 10 µM, and only less than 10% of the total (i.e. maximal levels of 1 µM) are present in the circulation as the bioactive aglycones (59). Isoflavones in our study were tested in monocyte cell cultures and in ex vivo whole-blood cultures at physiologically relevant concentrations (0.01–1 µM) and showed inhibitory effects on LPS-induced TNF-{alpha} release. Interestingly, lower concentrations of isoflavones (10 and 100 nM) and 17ß-estradiol (0.1 and 1 nM) were relatively more effective in inhibiting TNF-{alpha} levels than were higher concentrations (1 µM for isoflavones and 100 nM for 17ß-estradiol). Thus, these effects may be relevant to the effects of soy on circulating TNF-{alpha} levels in postmenopausal women, and to soy’s anticancer effects in humans.

In postmenopausal women, endogenous estradiol levels (~0.2 nM) are low, and these isoflavones could act as estrogen agonists. The average plasma levels of isoflavones in our study subjects during the 16-wk of soy consumption were about 0.7 µM (193 ± 174 ng/ml) for genistein and 0.4 µM (108 ± 78 ng/ml) for daidzein at 24 h after soy consumption. Peak levels of isoflavones are 6–9 h after soy consumption. Thus, levels of soy isoflavones (about 1 µM) achievable through soy intake could considerably exceed effective concentrations of endogenous 17ß-estradiol levels.

The estrogenic effect of soy isoflavones may also help maintain bone density in postmenopausal women by decreasing circulating levels of TNF-{alpha}, as well as by reducing detrimental activities of osteoclasts. A study by Walsh et al. (60) showed that the levels of TNF-{alpha} in 47 postmenopausal women after 6-months of raloxifene (60 mg/d) or conjugated equine estrogen (0.625 mg/d) were reduced by an average of 14 and 9%, respectively. Thus, the soy diet containing isoflavones tested in our study led to a greater decrease in TNF-{alpha} levels than those induced by the two synthetic estrogens. However, a strict comparison may not be appropriate because, as shown in Fig. 3Go, the effects of estrogens on TNF-{alpha} levels tended to be biphasic, and similar effects of low doses of raloxifene and conjugated equine estrogen have not been studied in humans. Because TNF-{alpha} plays an important role in bone loss resulting from menopause, the decrease observed in our study may be clinically important. In addition, one of the soy isoflavones, genistein, at physiological relevant concentrations was also reported to decrease the release of IL-6, an osteoclast differentiation marker, in cultured osteoblast cells (47, 49, 50) and increase the mRNA expression of osteoprotegerin, an inhibitor for osteoclast differentiation (49). Therefore, soy isoflavones might be studied for preventing bone loss in women due to natural, surgical, or cancer chemotherapy-induced menopause.

In summary, the decrease in serum levels of TNF-{alpha} in postmenopausal women during soy consumption may be a consequence of both a decrease in monocytes and a diminished release of TNF-{alpha} from monocytes attributable to the estrogenicity of the major soy isoflavones, daidzein and genistein. This antiinflammatory effect of soy isoflavones may also play an important role in reducing cancer risk.


   Acknowledgments
 
We thank the staff of the General Clinical Research Center at UTMB for nursing and dietary research assistance. Special thanks to Ann Livengood for assistance in research diet design and to Dr. Thomas K. Hughes, Dr. Marinel M. Ammenheuser, and Dr. Jeffrey K. Wickliffe for the critical review of this paper.


   Footnotes
 
This work was supported by: United States Public Health Service CA65628, U.S. Army Medical Research and Materiel Command under DAMD17–01-1–0417, American Institute for Cancer Research Grant 01B110, National Institutes of Health (NIH) NCRR General Clinical Research Center M01 RR00073, NIH R01 CA95545, Army MRMC under W81XWH-04–1-0345, and NIH 2 P30 ES06676.

First Published Online April 19, 2005

1 Y.H. and S.C. contributed equally to this project. Back

Abbreviations: DMSO, Dimethylsulfoxide; ER, estrogen receptor; LPS, lipopolysaccharide.

Received January 26, 2005.

Accepted April 13, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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