Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women

¹ From the Department of Nutrition, School of Public Health, School of Medicine, University of North Carolina, Chapel Hill (LTB, WL, MJS, TMB, CA, MGB, and SHZ); the Research Triangle Institute, Research Triangle Park, NC (ARJ, KJD, and BFT); and the Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD (JAC).

² Supported by the National Cancer Institute (NO1-CN-75035 to SZ) and by grants from the National Institutes of Health to the University of North Carolina Clinical Nutrition Research Center (DK56350), the University of North Carolina General Clinical Research Center (RR00046), and the Lineberger Comprehensive Cancer Center (CA16086). Protein Technologies International (PTI), via the National Cancer Institute, provided the isoflavone preparations; WL received some fellowship support from Central Soya, Inc; and SHZ received a grant from Central Soya, Inc.

³ Address reprint requests to SH Zeisel, CB# 7400, McGavran-Greenberg Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7400. E-mail: steven_zeisel{at}unc.edu.

ABSTRACT  
Background: Soy isoflavones are being evaluated as chemopreventive agents for breast and other cancers.

Objective: The objective was to perform safety and pharmacokinetic studies of purified unconjugated isoflavone preparations containing genistein, daidzein, and glycitein in postmenopausal women.

Design: Twenty-four healthy postmenopausal women ingested a single dose of 1 of 2 purified (from soybeans) isoflavone preparations that delivered a genistein dose of 2, 4, 8, or 16 mg/kg body wt. These doses were higher than those previously administered to human females. Toxicity studies were performed 24 h and 3, 6, 14, and 30 d after isoflavone administration. Kinetic studies were performed during the first 24 h.

Results: We observed a 7% decrease in systolic and diastolic blood pressure and a 32% decrease in the neutrophil count 24 h after treatment with formulation A. Isolated episodes of nausea, pedal edema, and breast tenderness were judged to be possibly related to the study treatment. The terminal plasma half-lives for free genistein, daidzein, and glycitein averaged 3.8, 7.7, and 3.4 h, respectively. The terminal pseudo half-lives for total genistein and total daidzein in plasma averaged 10.1 and 10.8 h, respectively. The estimated bioavailabilities of both total genistein and total daidzein from each of the 2 formulations were not significantly different.

Conclusions: A single-dose administration of purified unconjugated isoflavones at amounts that exceed normal dietary intakes had minimal clinical toxicity in healthy postmenopausal women. The pharmacokinetic data suggest that chronic dosing at 12–24-h intervals would not lead to progressive accumulation of these isoflavones.

Key Words: Genistein • daidzein • glycitein • soy isoflavones • cancer • toxicity • pharmacokinetics • postmenopausal women

INTRODUCTION  
Epidemiologic and animal studies suggest that consumption of soybeans and soy-containing foods may lower one’s risk of breast (1–3) and prostate (4–6) cancer. The incidence of breast cancer is significantly lower in Asian countries, where soy isoflavone intake is the highest (7). When Asian populations migrate to the United States, the second—but not the first—generation loses this protection (4). A case-control study in Chinese women residing in Shanghai supports the hypothesis that a high intake of soy foods may reduce the risk of breast cancer (8). The chemopreventive effects of soybeans and soy-containing foods may be related to their isoflavone content (9–13). Dietary intakes of 39.4 and 47.4 mg isoflavones/d in Chinese and Japanese adult populations, respectively, have been reported (14, 15). In the United States, the dietary consumption of soy isoflavones in the general population is < 1 mg/d (16).

The mechanisms responsible for the anticancer effects of soy isoflavones have not been completely characterized. The major soy isoflavones genistein and daidzein exert multiple effects that can result in suppression of tumor growth and diminished tumor survival. These include estrogen receptor activation (17, 18) as well as antiestrogenic effects (19), antioxidant activity (19), inhibition of growth factor receptor signaling via tyrosine kinases (20–23), induction of apoptosis (24–26), induction of cell differentiation (27), and inhibition of angiogenesis (28). Furthermore, isoflavones may help protect against breast cancer because of the ability of these compounds to inhibit enzymes involved in estrogen synthesis (29, 30) and steroid sulfohydrolysis (31), which may reduce the amount of estrogens in the mammary tissue of postmenopausal women (32).

There is no known evidence that the consumption of large amounts of isoflavones in the diet is harmful in humans; however, the multiple and complex actions of genistein and daidzein suggest that the administration of high doses of isoflavones creates a potential risk of adverse effects. Inhibition of growth factor signaling and stimulation of apoptosis (20, 26) could affect rapidly regenerating tissues such as gut mucosa and skin. Estrogen-like effects of soy may increase the growth of estrogen-receptor positive tissues and tumors (33). Genistein, daidzein, and other isoflavones inhibit phenolsulfotransferases, which are important for the metabolism of compounds such as dopamine and some pharmaceuticals (34). Genistein may induce DNA damage (35) via inhibition of the DNA-repair enzyme DNA topoisomerase (EC 5.99.1.2) (36). It is now possible to prepare highly purified soy isoflavones for administration to humans in prospective clinical trials, and we report data on the safety and pharmacokinetics of escalating single doses of 2 such preparations administered to healthy postmenopausal women. In a previous study, we reported data on the safety and pharmacokinetics of a single administration of these purified isoflavone preparations at various doses in healthy men (37).

SUBJECTS AND METHODS  
Subjects
Twenty-four postmenopausal women aged 46–68 y, recruited from the local population in the Research Triangle area of North Carolina, completed the study. Forty-two subjects were screened for the study. Twenty-five subjects were deemed eligible on the basis of inclusion and exclusion criteria and were enrolled in the study. One subject was dropped from the study because of unsuccessful intravenous needle placement that resulted in no available pharmacokinetic data. Demographic data for these subjects is presented in Table 1. Before acceptance into the study, the health status of the volunteers was verified by medical history, physical examination by a licensed physician, screening laboratory tests, and both a chest X-ray and an electrocardiogram. The subjects were required to be in good health and have a body mass index (BMI; in kg/m²) between 18 and 30. The laboratory criteria for eligibility included a white blood cell count 3500 cells/mm³, a platelet count 100 000 cells/mm³, a total lymphocyte count > 1.2 x 10⁹ cells/L, a serum creatinine concentration < 16 mg/L, a serum bilirubin concentration < 16 mg/L, and transaminase concentrations twice the normal limits. The exclusion criteria included premenopausal status (< 12 mo since the last spontaneous menstrual bleeding and a follicle-stimulating hormone concentration < 30 mIU/mL), an age > 70 y, regular use (> 1 dose/wk) of any prescription or over-the-counter medication [except for hormone replacement therapy (HRT)], use of antibiotics within the previous 6 wk, consumption of > 2 alcoholic drinks/d, a history of substance abuse or addiction, and a history of malignancy within the previous 2 y (except for curatively treated nonmelanoma skin cancer). None of the subjects had a calculated isoflavone intake from soy > 10 mg/d on the basis of a diet history, and the subjects were instructed to consume no soy products from the time of screening until after study day 30. The protocol was approved by the Institutional Review Board of the School of Medicine at the University of North Carolina (UNC) at Chapel Hill. The study was fully explained to all volunteers, who gave their written, informed consent.

View this table:
TABLE 1 . Demographic characteristics of subjects¹  
Isoflavone preparations
Two formulations of purified isoflavones were provided by Protein Technologies International via the National Cancer Institute. Both formulations were used under investigational new drug status from the Food and Drug Administration and, therefore, are not available as commercial products. Soy isoflavones sold as dietary supplements may contain one or more compounds used in these formulations. Formulation A contained 100% unconjugated isoflavones (87% genistein,12% daidzein, and 1% glycitein), whereas formulation B contained 70% unconjugated isoflavones (44% genistein, 23% daidzein, and 2% glycitein). The percentages of isoflavones in each formulation were defined by weight. The isoflavone composition and concentration in each formulation were independently analyzed by 2 laboratories (Ralston Analytic Laboratories, St Louis; Sigma Chemical Laboratories, St Louis). The preparations were stable at 40 and 70 °C for 6 mo and at 25 °C for 3 y. (The assays were performed at the University Pharmaceuticals of Maryland, Inc, Baltimore.) The study medication, supplied as a powder, was weighed and packaged into capsules by the Investigational Pharmacy at the UNC hospitals.

Study design
Four doses of genistein were used (2, 4, 8, and 16 mg/kg body wt) for each of the 2 isoflavone preparations. The amounts of genistein and daidzein that were provided by each formulation at each dose are shown in Table 2. The 24 subjects were randomly assigned to the 4 dose groups (n = 6 per dose group). Within each of these 4 groups, 3 subjects were assigned to formulation A and 3 to formulation B. A table of random numbers was used to assign subjects to a formulation within a dose group. Within each dose group, the subjects were stratified in groups of 2 on the basis of whether they were or were not receiving HRT. This blocked randomization ensured a balance between formulations A and B on the basis of HRT status. Both the study personnel and the subjects were blinded to the formulation consumed.

View this table:
TABLE 2 . Doses of genistein, daidzein, and glycitein delivered by formulation A and B  
The study events included a screening visit, inpatient admission, and follow-up visits 3, 6, 14, and 30 d postdose. The subjects were instructed to follow a soy-free diet for 72 h before a 12-h fast before they were admitted to the hospital at 2100 for the inpatient admission. The subjects were confined to the General Clinical Research Center at the UNC hospitals for 24 h after the study drug was provided so that plasma and urine samples could be obtained for pharmacokinetic analysis. Three meals and 2 snacks were served within 24 h after the treatment was administered. All meals were soy-free and isoenergetic with a standardized macronutrient composition of 55% carbohydrate, 30% fat, and 15% protein. Meals and snacks were consumed at specified times (meals: 0.5, 3, and 9 h postdose; snacks: 6 and 12 h postdose) during the study period to standardize any effect of food consumption on drug metabolism.

Clinical measurements
Plasma samples were obtained at the following time points: 0 (immediately before administration of the study drug), 0.5, 1, 1.5, 3, 4.5, 6, 9, 12, 15, 18, and 24 h postdose. Urine was collected over 24 h postdose at the following time points: immediately before administration of the study drug and at intervals ending 3, 6, 12, and 24 h postdose. We analyzed plasma and urine for free genistein, free daidzein, free glycitein, and their respective total (free plus sulfate and glucuronide conjugate) fractions. Blood (14 mL) was collected in vacuum syringes containing sodium heparin. After centrifugation of tubes at 2050 x g for 10 min at 4 °C, plasma was separated and stored at -80 °C until assayed. Urine samples were measured for volume and then a 5-mL sample was stored in a glass vial at -80 °C until assayed.

Measures of clinical toxicity
Toxicity was measured in terms of physical findings on the clinical examination, laboratory variables, vital signs, and any signs or symptoms reported by the subjects. Toxicity was assessed at screening, 24 h postdose, and on days 3, 6, 14, and 30 after administration of the study drug. In addition, lymphocytes from 3 subjects were analyzed within the first 24 h after the study drug was administered to study the rate of apoptosis and to assess tyrosine phosphorylation activity.

The study physician performed a thorough physical examination at baseline and reviewed the major systems at all follow-up visits. Adverse events and abnormalities in behavior were judged and documented by the study physician. Vital signs—including systolic and diastolic blood pressure, respiration, pulse, temperature, and weight—were documented at each visit. In addition, each subject received a chest X-ray and an electrocardiogram at screening and on day 30. Chest X-rays were interpreted in the Radiology Department at the UNC hospitals. Electrocardiograms were performed in the General Clinical Research Center and were interpreted by the study physician. If there were any abnormalities, the electrocardiograms were further evaluated by the Cardiology Department at the UNC hospitals.

The McLendon Clinical Laboratory of the UNC hospitals performed clinical laboratory measurements. This laboratory is both CLIA (Clinical Laboratory Improvement Amendments) and CAP (College of American Pathologists) certified and maintains quality-assurance logs and standard operating procedures. The National Cancer Institute’s Common Toxicity Criteria for the assessment of the toxicity of chemopreventive agents was used to assign a severity grade to all adverse events (version 2, 1998).

ISOFLAVONE MEASUREMENT
The methods used to measure the isoflavones are described in detail elsewhere (37, 38) and are modifications of the HPLC method of Supko and Phillips (39). The current modifications permit the quantitation of the isoflavone glycitein as well as of genistein and daidzein.

Genistein, daidzein, and glycitein standards were obtained from INDOFINE Chemical Company (Somerville, NJ). Dimethylsulfoxide, methanol, acetonitrile, and methyl t-butyl ether (MTBE)—all of which were ultraviolet grade—were obtained from Burdick & Jackson (Muskegon, MI). Reagent-grade ammonium acetate, ammonium formate, formic acid, ß-glucuronidase (EC 3.2.1.31) and aryl-sulfatase (EC 3.1.6.1) from Helix pomatia (Type H-2, catalog number G0876), and the internal standard 4-hydroxybenzophenone (98%) were obtained from Sigma-Aldrich (St Louis). Ascorbic acid and 6 mol hydrochloric acid (reagent) were from JT Baker Inc (Phillipsburg, NJ). Glacial acetic acid (American Chemical Society certified) was purchased from Fisher Scientific (St Louis).

Free isoflavones were measured in 1-mL aliquots of plasma or urine after addition of the internal standard and extraction into MTBE. Total isoflavones are defined as the amount of nonconjugated analyte originally present plus the amount of nonconjugated analyte that is released on treatment of the biological matrix with ß-glucuronidase and aryl-sulfatase. Total isoflavones were determined in 0.25-mL aliquots of plasma or urine after overnight hydrolysis of conjugated isoflavones at 37 °C with ß-glucuronidase and aryl-sulfatase from Helix pomatia, addition of the internal standard, and extraction into MTBE.

HPLC was used for the measurement of all isoflavone (genistein, daidzein, and glycitein). The HPLC instrumentation (Waters, Milford, MA) consisted of a model 600E pumping system, a model 717 automatic injector, a model 2487 ultraviolet detector set at 260 nm, and a Millennium³² Chromatography Information System capable of providing peak retention times, areas, and heights. Injection volumes were typically 100 µL. The plasma samples were analyzed on a Luna phenyl-hexyl column (4.6 x 150 mm, 5 µm; Phenomenex, Torrance, CA), and the urine samples were analyzed on a Zorbax Eclipse XDB-phenyl column (4.6 x 75 mm, 3.5 µm; MacMod, Chadds Ford, PA). A Zorbax Eclipse XDB-phenyl guard column (4.6 x 12.5 mm, 5 µm; MacMod) was used for all analyses. The columns were maintained at 40 °C. The mobile phase (2 mL/min) consisted of various mixtures (by vol) of methanol (plasma samples) or methanol:acetonitrile (1:1; urine samples) in 0.05 mol aqueous ammonium formate/L (pH 4.0).

Concentrations of each isoflavone were calculated from the ratios of the peak heights for these analytes compared with those of the internal standards. Calibration standards and positive controls, each prepared separately in the appropriate matrix, were analyzed along with each set of samples. A linear regression analysis of each set of calibration standards in the appropriate matrix was performed with the use of 1/x weighting to obtain the calibration equation relating the ratios of the peak height to the concentration of the respective analyte for that sample set. Validated ranges for each assay are presented in Table 3.

View this table:
TABLE 3 . Validated ranges of genistein, daidzein, and glycitein by assay¹  
Pharmacokinetic calculations
The dispositions of free and total genistein, daidzein, and glycitein were assessed by standard model-independent pharmacokinetic analyses with the use of the nonlinear least-squares program WINNONLIN (version 1.5; SCI, Morrisville, NC). The terminal elimination rate constant (k_el) and elimination half-life (t_1/2) were estimated by linear least-squares regression of the log-transformed concentrations versus time in the terminal log-linear phase of the disposition profile. Apparent systemic clearance (Cl_P) and apparent volume of distribution (V_d) were estimated for each isoflavone. Because the doses were administered orally, V_d and Cl_P are actually V_d/F and Cl_P/F, where F is bioavailability.

The area under the curve (AUC) for plasma concentration versus time curve, from the time of dosing (time 0) to the time that the last plasma sample was obtained, was calculated by trapezoidal integration. In cases when the analyte concentrations in plasma were less than the limit of quantitation (LOQ) of the assay, the lower (low) and upper (high) limits for the AUC and the maximum plasma concentration (C_max) were determined by setting these concentrations equal to 0 and then equal to the LOQ. To calculate the mean concentrations in plasma, values lower than the LOQ were assigned a value of 0.5 LOQ. At a given dose (mg/kg) of genistein, the relative bioavailability of the 2 formulations was estimated as the ratio of the AUC for plasma concentration versus time curves for the 2 dose forms and as the ratio of the amount excreted in urine over 24 h (40). For daidzein and glycitein, the dose groups receiving similar doses of the particular isoflavone were compared.

Tyrosine phosphorylation and apoptosis in isolated lymphocytes
Protein tyrosine phosphorylation activity was analyzed at 5 time points in purified lymphocytes from 3 subjects. One subject received 8 mg genistein/kg in formulation A, whereas the remaining 2 subjects received 16 mg genistein/kg in formulation B. Lymphocytes were isolated 0, 1, 3, 6, and 24 h postdose from blood samples by centrifugation (1000 x g at 4 °C for 10 min) of peripheral blood over a Ficoll-Hypaque density gradient (41). Two approaches were used for these analyses: dot blot and standard Western analyses (42, 43). The pellet containing 1 million cells was dissolved in 100 µmol Laemmli sodium dodecyl sulfate sample buffer/L containing phosphatase and protease inhibitors (43). For dot blotting, samples (10 µL) were pipetted directly onto the nitrocellulose paper, which was blocked in 2% bovine serum albumin (Sigma Chemical, St Louis) for 1 h. The RC20 (Transduction Laboratories, Lexington, KY) antibody was diluted 1:2500 in tris buffered saline, 0.1% bovine serum albumin, and 0.05% Tween 20 (Sigma Chemical) and incubated at 37 °C for 20 min. The filters were washed 5 times for 5 min with tris buffered saline and 0.05% Tween 20 and incubated with use of the SuperSignal CL-HRP Substrate System (Pierce, Rockford, IL) (mixed 1:1) for 45 s. The blot was wrapped in plastic wrap and exposed to Hyperfilm-ECL (Amersham Pharmacia Biotech, Piscataway, NJ) for 10–60 s. For Western blotting, 20 µL of the lymphocytes suspended in Laemmli sodium dodecyl sulfate sample buffer were added per well and analyzed by polyacrylamide gel electrophoresis (4–15% minigel Bio-Rad system; Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose (0.45 mm). The blot was incubated with antibodies [PhosphoPlus Akt (Ser473) Antibody kit; New England Biolabs, Beverly, MA] followed by incubation with ECL detection reagents (Amersham Pharmacia, Piscataway, NJ) to determine Akt-1 kinase (protein kinase B, EC 2.7.1.37) phosphorylation and activity (44).

In all subjects, lymphocytes were collected 24 h postdose for assessment of apoptosis. The in situ detection of apoptotic cells was performed with the use of free 3'-OH ends of fragmented DNA labeled with digoxigenin-11-deoxyuridine triphosphate (ApoTag-Kit; Intergen, Gaithersburg, MD) and incubated with monoclonal anti-digoxigenin antibody visualized with peroxidase-conjugated secondary antibody and diaminobenzidine (TUNEL assay) (45). In addition, hematoxylin-stained slides were used to assess apoptotic cell morphology, which was quantified with the use of light microscopy.

STATISTICAL ANALYSIS  
Change scores were constructed by subtracting the baseline value (taken at screening visit) from the postdosing values. Wilcoxon signed-rank statistics were used to test whether the change scores of vital signs and laboratory variables deviated significantly from zero. Studies with changes of < 10% in the laboratory variables studied were judged to not be "clinically important." Because we wanted to minimize problems associated with multiple comparisons, only change scores that were statistically significant and were 10% were analyzed with the use of general linear model methods (PROC GLM, SAS version 8.0; SAS Institute Inc, Cary NC). The covariates of interest were the dose, the 2 formulations, and HRT status. Interaction terms of the main effect of dose, formulation, and HRT status were not added to the GLM because of the small sample size. The log (base 2) dose was used to parameterize the 4 doses (2, 4, 8, and 16 mg/kg) in a single covariate. The effects of the covariates were assessed with the use of type III sums of squares. Differences in the urinary excretion of genistein and daidzein between the men and women, with control for the dose, were analyzed by using analysis of variance. The analyses were conducted separately for formulations A and B. All tests were two-sided, and statistical significance was defined as a P value 0.05.

RESULTS  
Twenty-four subjects completed the study; the demographic data for all evaluable subjects are presented in Table 1. Eight subjects were receiving some form of HRT at the time that the study drug was administered, whereas 16 subjects had abstained from the use of any hormones for 30 d before enrollment.

Toxicity
No postmenopausal specific–related symptoms (eg, hot flashes) were reported. There were 4 reported adverse events that were judged to be possibly related to the study drug. Two isolated episodes of trace pedal edema were noted 16 d postdose (2 mg/kg, formulation B) and 5 d postdose (16mg/kg, formulation B). A single episode of nausea was reported in one subject, which lasted for 10 min at 1 h postdose (8 mg/kg, formulation B). This event was judged to be possibly related to treatment because of the close timing of the event to administration of the study drug. Breast tenderness was detected in one subject 2 d postdose (16 mg/kg, formulation A). It was postulated that the reported pedal edema and breast tenderness were caused by the study drug because similar toxic effects were observed in animal studies (46). All 4 events were classified as grade 1 (mild) events.

There were 12 grade 1 adverse events in 7 subjects that were judged unlikely to be related to study drug administration. These events included headaches, abdominal tenderness, fatigue, diarrhea and abdominal discomfort, swelling of the right hand, sinus headache, and mucous or blood in the stool. The adverse events were evenly distributed among formulations and occurred in the 2-, 8-, and 16-mg/kg dose groups. There were 16 reported adverse events (grade 1) in 12 subjects that were judged to be unrelated to treatment. All of the findings could be explained definitively by other known factors. Events were reported across all dose groups and were evenly distributed among formulations.

No significant decrease in blood pressure was observed after treatment with formulation B. On average, in subjects treated with formulation A, the mean (± SD) systolic and diastolic blood pressures decreased from baseline by 24 h after treatment (systolic blood pressure: 132 ± 11 mm Hg at baseline and 116 ± 13 mm Hg at 24 h, P = 0.04; diastolic blood pressure: 74 ± 8 mm Hg at baseline and 61 ± 7 mm Hg at 24 h, P = 0.001). Clinically significant decreases in systolic blood pressure ( 15 mm Hg) were observed 24 h postdose in 5 of these subjects, but only 2 of these subjects continued to show significant decreases 3 d postdose. These changes in systolic blood pressure were seen in the lowest 3 dose groups for formulation A. Systolic blood pressure increased ( 15mm Hg) in 2 subjects 24 h postdose, but neither subject’s blood pressure remained elevated 3 d postdose. Both of these subjects received formulation B (4 and 16 mg/kg). Clinically significant reductions ( 15 mm Hg) in diastolic blood pressure occurred in 8 subjects from various dose groups 24 h postdose. Of these 8 subjects, 6 received formulation A. One subject continued to have a reduced diastolic blood pressure (> 15 mm Hg) on day 3. The diastolic blood pressure increased ( 15 mm Hg) in the same 2 subjects, whose systolic blood pressure increased.

Posttreatment weight change 3 d postdose was statistically (P = 0.001) but not clinically significant (-0.55 kg). This small change may reflect measurement conditions. Blood variables that showed a change of 10% overall and that were statistically significant were examined for differences by dose, formulation, and HRT status. The 7 blood variables that met this criterion were as follows: neutrophil count (decreased on days 1, 6, 14, and 30), white blood cell count (decreased on days 1, 6, 14, and 30), alanine aminotransferase (EC 2.6.1.2; decreased on day 1), aspartate aminotransferase (EC 2.6.1.1; decreased on days 6 and 30), triacylglycerol (decreased on day 6), blood urea nitrogen (increased on day 3), and lactic acid dehydrogenase (EC 1.1.1.27; decreased on day 1). All changes were at most 15%, except for changes in the neutrophil count, which ranged from 20% to 25%. In 4 instances, statistically significant differences were seen in the change scores by formulation: 1) on day 1, the neutrophil count declined from 4.9 to 3.4 x 10⁹ cells/L (32%) in subjects treated with formulation A and from 3.3 to 3.1 x 10⁹ cells/L (5%) in subjects treated with formulation B (change score: P = 0.05); 2) the total white blood cell count was similarly changed on day 1 in subjects treated with formulation A (reflecting the change in neutrophils); 3) from baseline to day 6, triacylglycerol decreased from 140 to 96 mg/dL (31%) in subjects treated with formulation A and increased from 109 to 129 mg/dL (11%) in subjects treated with formulation B (change score: P = 0.003); and 4) on day 1, lactic acid dehydrogenase decreased by 15% (from 516 to 440 IU/L) in subjects treated with formulation A and by 5% (from 478 to 455 IU/L; change score P = 0.01) in subjects treated with formulation B. However, in all cases, the results (at the same time point) did not differ significantly by formulation. No differences by formulation in alanine aminotransferase, aspartate aminotransferase, or blood urea nitrogen were found.

Of the 8 subjects who were currently receiving HRT, the mean change in lipase (EC 3.1.1.3) on day 3 after treatment with isoflavones was a mean (± SD) increase of 30.4 ± 19.23 U/L, whereas lipase among subjects not receiving HRT (n = 16) decreased on day 3 by 5.19 ± 27.37 (P = 0.005). No other statistically significant dose-response effects or effects of HRT status were seen. We observed no clinically significant changes in sodium, potassium, chloride, carbon dioxide, creatinine, alkaline phosphatase (EC 3.1.3.1), total protein, albumin, uric acid, total bilirubin, calcium, red blood cells, hemoglobin, hematocrit, platelet count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocytes, lymphocytes, monocytes, eosinophils, basophils, prothrombin time, partial thromboplastin time, fasting glucose, total cholesterol, HDL, LDL, phosphorous, magnesium, amylase (EC 3.2.1.2), lipase, fibrinogen, luteinizing hormone, follicle-stimulating hormone, urinary pH, urine gravity, or the results of urinalysis (data not shown).

In the 3 subjects in whom protein tyrosine phosphorylation activity was analyzed, activity of Akt-1 kinase increased 3 h postdose. In these same 3 subjects, protein tyrosine phosphorylation decreased 3 and 6 h postdose (Figure 1). No changes in the rate of apoptosis in lymphocytes were noted in any of the 24 subjects 24 h postdose (data not shown).

View larger version (37K):
FIGURE 1. . The effects of isoflavone in lysates isolated from peripheral lymphocytes from a postmenopausal woman after administration of 8 mg isoflavone formulation A/kg body wt. The lysates were subjected to Western blotting with phosphotyrosine (RC20) (pTyr) and phospho-Akt-1 kinase (pAkt1) antibodies. Laemmli sample buffer containing phosphatase and protease inhibitor cocktail was added to samples. Proteins were resolved by 4–15% gradient sodium dodecyl sulfate–mini polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose paper. Dot blotting was performed as described in Subjects and Methods. Samples (10 µL) were pipetted directly onto the nitrocellulose paper. The filter was exposed to film for 1 min. Results are representative of blots obtained from lymphocytes isolated from 3 subjects treated with genistein. C, internal control from epidermal growth factor–stimulated A431 (human lung cancer) cell lysate. The tyrosine phosphorylated epidermal growth factor receptor (EGFR) position (molecular mass: 170 kDa) is marked by an arrow.

 
Plasma and urinary isoflavones
Measurable concentrations of free (unconjugated) genistein were present in plasma from all subjects. Measurable concentrations of free daidzein in plasma were present in all subjects who received daidzein doses of 1 mg/kg. Free glycitein reached measurable concentrations in all subjects receiving glycitein doses of 0.17 mg/kg.

Total genistein was measurable in almost every plasma sample from every subject. Total daidzein was measurable in most samples from subjects who received daidzein doses of 0.55 mg/kg. Measurement of total glycitein in plasma was hampered by the large increase in matrix background from that found for free glycitein. Thus, measurable concentrations of total glycitein were found in only one subject.

The reported average data and pharmacokinetic data are from dose groups in whom measurable concentrations of analytes were present at 5 time points for 2 of the 3 subjects. Plasma concentrations of genistein with time for the subjects in the 8-mg/kg dose groups are shown in Figure 2. Plasma concentrations of daidzein over time for these same subjects are shown in Figure 3. Plasma concentrations of free glycitein over time for the subjects in the 16-mg/kg dose group of formulation B (0.68 mg/kg glycitein) are shown in Figure 4.

View larger version (19K):
FIGURE 2. . Mean (± SD) plasma concentrations of total and free genistein in postmenopausal women after a single dose of 8 mg genistein/kg body wt delivered as formulation A or formulation B. Formulation A consisted of 87% genistein, 12% daidzein, and 1% glycitein; formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. Total genistein, formulation A (); total genistein, formulation B (); free genistein, formulation A (); and free genistein, formulation B (). The curves represent the pattern of pharmacokinetic data for the other treatments administered. The statistical estimates of the pharmacokinetic variables are presented in Table 4. n = 3 per data point.

 

View larger version (21K):
FIGURE 3. . Mean (± SD) plasma concentrations of total and free daidzein in postmenopausal women after a single dose of 8 mg genistein/kg body wt delivered as formulation A or formulation B. Formulation A consisted of 87% genistein, 12% daidzein, and 1% glycitein; formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. Subjects in these groups received either 1.1 (formulation A) or 4.2 (formulation B) mg daidzein/kg body wt. Total daidzein, formulation A (); total daidzein, formulation B (); free daidzein, formulation A (); and free daidzein, formulation B (). The curves represent the pattern of pharmacokinetic data for the other treatments administered. The statistical estimates of the pharmacokinetic variables are presented in Table 4. n = 3 per data point.

 

View larger version (13K):
FIGURE 4. . Mean (± SD) plasma concentrations of glycitein in postmenopausal women after a single dose of 16 mg genistein/kg body wt delivered as formulation B. Formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. Subjects in these groups received 0.68 mg glycitein/kg body wt. Plasma concentrations of glycitein in subjects who received formulation A were too low to measure. The curves represent the pattern of pharmacokinetic data for the other treatments administered. The statistical estimates of the pharmacokinetic variables are presented in Table 4. n = 3 per data point.

 
No significant differences (P > 0.4) between formulations were found in the percentage dose of either genistein or daidzein excreted in urine; however, a somewhat higher percentage (P = 0.03) of glycitein was excreted in subjects treated with formulation B than in subjects treated with formulation A (Figure 5). Averages of 48–74% of the glycitein in formulation B and 34–45% of the glycitein in formulation A were excreted as glycitein conjugates (sulfates and glucuronides) within a day of dosing. Considerable portions of the dose of daidzein were also excreted as sulfate and glucuronide conjugates during the same interval (28–42% of the dose for formulation B and 27–38% of the dose for formulation A). Excretion of genistein conjugates accounted for 10–14% of the dose for each formulation. Some decrement in the percentage of the dose excreted in 24 h as total analytes in urine was seen at the higher doses for each formulation and was probably due to incomplete excretion in this time period. However, excretion rates increased with increasing doses of all analytes and for both formulations (data not shown). In agreement with the results of an earlier study of urinary excretion of dietary isoflavones by Adlercreutz et al (47), conjugation is necessary for the excretion of significant quantities of genistein and daidzein in urine. Less than 0.3% of the dose was excreted as free genistein or free daidzein for any subject, and too little free glycitein was excreted to measure.

View larger version (22K):
FIGURE 5. . Mean (± SD) 24-h urinary excretion of total genistein (), total daidzein (), and total glycitein () in postmenopausal women after a single dose of 2, 4, 8, or 16 mg genistein/kg body wt as formulation A or formulation B. Formulation A consisted of 87% genistein, 12% daidzein, and 1% glycitein; formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. The amount of daidzein was 12% (formulation A) or 52% (formulation B) of the amount of genistein. The amount of glycitein was 0.7% (formulation A) or 4.2% (formulation B) of the amount of genistein. No significant differences were observed after the 2 formulations; however, there was a somewhat higher percentage of glycitein excreted after formulation B than after formulation A. n = 3 per data point.

 
Pharmacokinetics
Similarly to values previously reported for men (37), the estimated terminal plasma half-lives of the 3 isoflavones were all reasonably short (Table 4). The half-life of (free) genistein averaged 3.8 h (range: 1.7–7.3 h), the half- life of (free) daidzein averaged 7.7 h (range: 4.4–21 h), and the half-life of free glycitein (measured in a single dose group) averaged 3.4 h. Half-lives of total genistein and total daidzein are actually pseudo half-lives because each is the combination of metabolism and excretion rates of multiple metabolites. The pseudo half-life of total genistein averaged 10.1 h (range: 6.5–13.4 h). The pseudo half-life of total daidzein averaged 10.8 h (range: 5.7–16.1 h).

View this table:
TABLE 4 . Estimates of noncompartmental pharmacokinetic variables in postmenopausal women after ingestion of formulation A or B¹  
C_max and the times at which C_max were observed (t_max) are shown in Table 4. Mean t_max values for free genistein and free daidzein in the dose-formulation groups ranged from 1.0 to 8.3 h (average: 3.1 h) and for total genistein and total daidzein ranged from 2.5 to 11 h (average: 5.6 h). Mean C_max values for total genistein and total daidzein increased linearly with increasing doses (r² > 0.92) for both formulations. The linearity of mean C_max values with increasing doses could be established for free daidzein in formulation B (r² = 0.97) and for free genistein in formulation A (r² = 0.96). Peak concentrations of free genistein and of free daidzein occurred significantly earlier (P < 0.002) than did the peak concentrations of total genistein and of total daidzein.

Plots of plasma AUCs versus dose are shown in Figure 6 for free isoflavones and in Figure 7 for total isoflavones. Except for free genistein in the 4- and 8-mg/kg dose groups of formulation B, AUC values for free isoflavones increased with increasing doses. Mean AUC values increased linearly with increasing doses for total genistein and total daidzein from both formulations (r² > 0.98) and for free genistein and free daidzein (r² > 0.88). In general, formulation B resulted in higher AUC values for free genistein and free daidzein than did formulation A; however, formulation B yielded lower AUC values for total genistein than did formulation A. The relative bioavailabilities of genistein and daidzein from formulation A were not significantly different from those from formulation B (Table 5).

View larger version (20K):
FIGURE 6. . Mean plasma areas under the curve (AUC) of free isoflavones from 0 to 24 h in postmenopausal women after a single dose of 2, 4, 8, or 16 mg genistein/kg body wt as formulation A or formulation B. Formulation A consisted of 87% genistein, 12% daidzein, and 1% glycitein; formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. Free genistein, formulation B (); free genistein, formulation A (); free daidzein, formulation B (); and free daidzein, formulation A (). Vertical bars indicate the AUC range for each treatment. (If not shown they are smaller than the symbol.) n = 3 per data point.

 

View larger version (22K):
FIGURE 7. . Mean plasma areas under the curve (AUC) of total isoflavones from 0 to 24 h in postmenopausal women after a single dose of 2, 4, 8, or 16 mg genistein/kg body wt as formulation A or formulation B. Formulation A consisted of 87% genistein, 12% daidzein, and 1% glycitein; formulation B consisted of 44% genistein, 23% daidzein, and 2% glycitein. Total genistein, formulation B (); total genistein, formulation A (); total daidzein, formulation B (); and total daidzein, formulation A (•). Vertical bars indicate the AUC range for each treatment. (If not shown, they are smaller than the symbol.) n = 3 per data point.

 

View this table:
TABLE 5 . Relative bioavailability of isoflavones from formulation A compared with that from formulation B in postmenopausal women¹  

DISCUSSION  
Little information is available about the toxicity of high doses of soy isoflavones (> 1 mg/kg body wt) in female humans; we previously published the results of a study in men (37). Our starting genistein dose was 2 mg/kg body wt and provided more than twice the amount of total isoflavones in the average Japanese diet, whereas our highest dose provided > 20 times this amount. Some human infants who consume these isoflavones in soy-based infant formulas could ingest as much as 9 mg total isoflavones, or 3 mg/kg body wt (48). We administered genistein and daidzein at higher doses than had been previously administered to humans and observed little significant toxicity in any subject at any dose. We also described pharmacokinetic variables for these isoflavones after acute high-dose administration.

Toxicity
We observed minimal clinical toxicity with either formulation at all doses provided. The results of physical examination at all points were normal, except as noted. No increase in thyroid size was palpated. Pedal edema was occasionally observed in treated women, and one episode of pedal edema was also noted in our previous study in men (37); however, it occurred 3 wk postdose. The 2 episodes of breast tenderness we observed could have been due to the estrogenic effects of the isoflavones or could have been a symptom of interactions with estrogen replacement therapy; both subjects who experienced breast tenderness were receiving HRT. Most studies investigating hormonal effects in relation to soy used soy-based foods. Studies in postmenopausal women have shown no significant changes in follicle-stimulating hormone (49–51). We too observed no changes in plasma follicle-stimulating hormone concentrations.

In the present study, formulation A was associated with a reduction in blood pressure 24 h postdose. Recent studies have reported that soy-based foods significantly reduce diastolic blood pressure (52, 53), whereas a study of the effects of purified soy isoflavones showed no effect in either systolic or diastolic blood pressure (54). The conditions in our study 24 h postdose may account for the changes in blood pressure. For example, baseline values were measured midway through the 2-h screening visits, whereas postdose values were measured while the patients were in bed or when they may have been more relaxed. However, if this were the case, it is difficult to explain why decreases in blood pressure were observed only after treatment with formulation A. There are reasonable mechanisms whereby genistein could reduce blood pressure. In vitro studies have shown that genistein reduces vascular smooth muscle contractions after angiotensin II administration and that, in resistance arteries of intact rats, an extracellular signal-regulated kinase-1/2 activation pathway using upstream protein kinase C and the tyrosine kinase c-Src are involved in the control of blood pressure (55).

The women treated with formulation A in the present study had significant decreases in neutrophil count (and thus changes in the white blood cell count), but this group started with a higher baseline neutrophil count than did subjects treated with formulation B. The observed decrease in subjects treated with formulation A resulted in mean values that were not significantly different from those of subjects treated with formulation B. We have no explanation for the higher baseline value in the subjects treated with formulation A, but we suggest that this effect of isoflavone treatment was probably not clinically significant. In our previous study, a decrease in neutrophil count was noted in only one man, 24 h after administration of 16 mg formulation B/kg (37). No other statistically significant changes in neutrophil count were noted at any visit in this study in men. There is a plausible mechanism that can be proposed for the observed decrease in neutrophil counts after treatment with genistein. Granulocyte colony-stimulating factor regulates the proliferation, differentiation, and survival of neutrophilic granulocytes and it acts by activating both the Janus kinase (Jak) and Src families of tyrosine kinases, which are inhibited by genistein (56).

In the women who were receiving HRT in the present study and in the men in our previous study (37), we observed elevations in blood lipase activity after isoflavone treatment. The assay for lipase that was used in the clinical laboratories at the UNC hospitals measures the activity of pancreatic lipase, lipases secreted from the gastric and intestinal mucosa, and lipoprotein lipase (EC 3.1.1.34). These elevations in lipase were not associated with increases in amylase, suggesting that they were not due to pancreatic damage. Isoflavones might act by altering the regulation of the activity or distribution of lipoprotein lipase. A protein kinase that is inhibited by genistein mediates the modulation of lipoprotein lipase activity by glucose (57). Also, genistein inhibits the cyclic AMP–mediated release of lipoprotein lipase activity from fat pads (58).

Despite the inhibition of tyrosine kinase activity after women were treated with either isoflavone preparation (Figure 1), we observed no increase in apoptosis in lymphocytes. This may have been because we observed an increased activity of Akt-1 kinase in samples from all 3 subjects after isoflavone administration. Activated (phosphorylated) Akt is implicated in survival signaling in a wide variety of cells (59). Several targets that may underlie the ability of this regulatory cascade to inhibit apoptosis and promote survival have been identified recently. These substrates include 2 components of the intrinsic cell death machinery, BAD (a pro-apoptotic member of the Bcl-2 family) and caspase (EC 3.4.22.36) (60).

Pharmacokinetics
The terminal half-life of free genistein in women (3.8 h) is essentially no different from the terminal half-life we reported earlier (37) in men (3.2 h). The terminal half-life of free daidzein in women (7.7 h) is somewhat longer than that in men (4.2 h). The terminal half-life of free glycitein in women (3.4 h) was not reported previously. We measured the terminal half-life in only one set of 3 subjects. The terminal pseudo half-lives of total genistein and daidzein in women (10.1 and 10.8 h, respectively) were not significantly different from those in men (9.2 h and 8.2 h, respectively). Pseudo half-lives of total genistein and total daidzein in men were reported by King and Bursill (61) to be 5.7 and 4.7 h, respectively, and by Lu and Anderson (62) to be 3.2 and 2.9 h, respectively.

The urinary excretion of total genistein and total daidzein over 24 h in women given single doses of formulation A in the current study were both higher (P < 0.01; with control for the dose) than the urinary excretion in men given equivalent single doses of the same formulation in our previous study (37). Lu and Anderson (62) reported similar differences in urinary excretion between the sexes when soymilk was given to both men and women. After control for the dose, neither the excretion of total daidzein (P = 0.07) nor of total genistein (P = 0.32) was significantly different between men and women who received formulation B. The higher urinary excretion of isoflavones in the women than in the men who received formulation A may have been due to a higher bioavailability of the isoflavones from formulation A by the women. Although we were unable to measure absolute bioavailability in our studies, the relative bioavailability of total genistein in women was not significantly different between the 2 formulations, whereas total genistein was approximately twice as bioavailable from formulation B than from formulation A in men. Similar results were observed for the relative bioavailability of total daidzein.

Summary
In summary, well-defined mixtures of purified unconjugated isoflavones (genistein, daidzein, and glycitein) extracted from soy were administered to women for the first time in the present study. Minimal clinical toxicity was observed, even at single doses that exceeded normal dietary intakes manyfold. We found that genistein and daidzein (free and total) were rapidly cleared from plasma and that doses that are 2 or 3 times the normal daily dietary intake should not result in progressive accumulation of these isoflavones. A large portion of genistein and daidzein is excreted in urine.

ACKNOWLEDGMENTS  
We thank René Mitchell for assistance with the analyses of plasma and urine samples.

REFERENCES  

1. Lee HP, Gourley L, Duffy SW, Esteve J, Lee J, Day NE. Dietary effects on breast-cancer risk in Singapore. Lancet 1991;337:1197–200.
2. Gotoh T, Yamada K, Ito A, Yin H, Kataoka T, Dohi K. Chemoprevention of N-nitroso-N-methylurea-induced rat mammary cancer by miso and tamoxifen, alone and in combination. Jpn J Cancer Res 1998;89:487–95.
3. Fritz WA, Coward L, Wang J, Lamartiniere CA. Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis 1998;19:2151–8.
4. Severson RK, Nomura AM, Grove JS, Stemmermann GN. A prospective analysis of physical activity and cancer. Am J Epidemiol 1989;130:522–9.
5. Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phyto-oestrogens in Japanese men. Lancet 1993;342:1209–10.
6. Jacobsen BK, Knutsen SF, Fraser GE. Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States). Cancer Causes Control 1998;9:553–7.
7. Adlercreutz CH, Goldin BR, Gorbach SL, et al. Soybean phytoestrogen intake and cancer risk. J Nutr 1995;125:757S–70S.
8. Zheng W, Dai Q, Custer LJ, et al. Urinary excretion of isoflavonoids and the risk of breast cancer. Cancer Epidemiol Biomarkers Prev 1999;8:35–40.
9. Messina MJ, Persky V, Setchell KD, Barnes S. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr Cancer 1994;21:113–31.
10. Messina M, Barnes S. The role of soy products in reducing risk of cancer. J Natl Cancer Inst 1991;83:541–6.
11. Hebert JR, Hurley TG, Olendzki BC, Teas J, Ma Y, Hampl JS. Nutritional and socioeconomic factors in relation to prostate cancer mortality: a cross-national study. J Natl Cancer Inst 1998;90:1637–47.
12. Adlercreutz H, Mazur W. Phyto-oestrogens and Western diseases. Ann Med 1997;29:95–120.
13. Messina M, Barnes S, Setchell KD. Phyto-oestrogens and breast cancer. Lancet 1997;350:971–2.
14. Chen Z, Zheng W, Custer LJ, et al. Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai. Nutr Cancer 1999;33:82–7.
15. Arai Y, Uehara M, Sato Y, et al. Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J Epidemiol 2000;10:127–35.
16. Setchell KD, Cassidy A. Dietary isoflavones: biological effects and relevance to human health. J Nutr 1999;129:758S–67S.
17. Willard ST, Frawley LS. Phytoestrogens have agonistic and combinatorial effects on estrogen- responsive gene expression in MCF-7 human breast cancer cells. Endocrine 1998;8:117–21.
18. Hargreaves DF, Potten CS, Harding C, et al. Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast. J Clin Endocrinol Metab 1999;84:4017–24.
19. Gescher A, Pastorino U, Plummer SM, Manson MM. Suppression of tumour development by substances derived from the diet—mechanisms and clinical implications. Br J Clin Pharmacol 1998;45:1–12.
20. Fioravanti L, Cappelletti V, Miodini P, Ronchi E, Brivio M, Di Fronzo G. Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro. Cancer Lett 1998;130:143–52.
21. Uckun FM, Narla RK, Zeren T, et al. In vivo toxicity, pharmacokinetics, and anticancer activity of genistein linked to recombinant human epidermal growth factor. Clin Cancer Res 1998;4:1125–34.
22. Balabhadrapathruni S, Thomas TJ, Yurkow EJ, Amenta PS, Thomas T. Effects of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells. Oncol Rep 2000;7:3–12.
23. Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;262:5592–5.
24. Yoon HS, Moon SC, Kim ND, Park BS, Jeong MH, Yoo YH. Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP. Biochem Biophys Res Commun 2000;276:151–6.
25. Kumi-Diaka J, Butler A. Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells. Biol Cell 2000;92:115–24.
26. Shao ZM, Alpaugh ML, Fontana JA, Barsky SH. Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis. J Cell Biochem 1998;69:44–54.
27. Constantinou AI, Krygier AE, Mehta RR. Genistein induces maturation of cultured human breast cancer cells and prevents tumor growth in nude mice. Am J Clin Nutr 1998;68(suppl):1426S–30S.
28. Fotsis T, Pepper M, Adlercreutz H, et al. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci U S A 1993;90:2690–4.
29. Adlercreutz H, Bannwart C, Wahala K, et al. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 1993;44:147–53.
30. Keung WM. Dietary estrogenic isoflavones are potent inhibitors of beta-hydroxysteroid dehydrogenase of P. testosteronii. Biochem Biophys Res Commun 1995;215:1137–44.
31. Wong CK, Keung WM. Daidzein sulfoconjugates are potent inhibitors of sterol sulfatase (EC 3.1.6.2). Biochem Biophys Res Commun 1997;233:579–83.
32. Xu X, Duncan AM, Wangen KE, Kurzer MS. Soy consumption alters endogenous estrogen metabolism in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2000;9:781–6.
33. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo Cancer Res 1998;58:3833–8. (Published erratum appears in Cancer Res 1999;59:1388.)
34. Ghazali RA, Waring RH. The effects of flavonoids on human phenolsulphotransferases: potential in drug metabolism and chemoprevention. Life Sci 1999;65:1625–32.
35. Kulling SE, Rosenberg B, Jacobs E, Metzler M. The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes. Arch Toxicol 1999;73:50–4.
36. Markovits J, Linassier C, Fosse P, et al. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989;49:5111–7.
37. Busby MG, Jeffcoat AR, Bloedon LT, et al. Clinical characteristics and pharmacokinetics of purified soy isoflavones: single dose administration to healthy men. Am J Clin Nutr 2002;75:126–36.
38. Thomas BF, Zeisel SH, Busby MG, et al. Quantitative analysis of the principal soy isoflavones genistein, daidzein and glycitein, and their primary conjugated metabolites in human plasma and urine using reversed-phase high performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Sci Appl 2001;760:191–205.
39. Supko JG, Phillips LR. High-performance liquid chromatographic assay for genistein in biological fluids. J Chromatogr B Biomed Sci Appl 1995;666:157–67.
40. Rowland M, Tozer TN. Clinical pharmacokinetics: concepts and applications. 3rd ed. Baltimore: Lea and Febiger, 1995.
41. Punt CJ, Rijksen G, Vlug AM, Dekker AW, Staal GE. Tyrosine protein kinase activity in normal and leukaemic human blood cells. Br J Haematol 1989;73:51–6.
42. Lopaczynski W, Terry C. Autophosphorylation of insulin and IGF-1 receptor in vitro. Detection of tyrosine phosphorylation using dot blot. Previews 1996;1:4–6.
43. Lopaczynski W, Harris S, Nissley P. Activation of extracellular signal-regulated kinases 1 and 2 by insulin-like growth factor I in human osteosarcoma MG-63 cells. Horm Metab Res 1999;31:65–9.
44. Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3, 4-bisphosphate. Science 1997;275:665–8.
45. Wijsman JH, Jonker RR, Keijzer R, Vande Velde CJH, Cornelisse CJ, Dierendonck JHV. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 1993;41:7–12.
46. Steele VE, Pereira MA, Sigman CC, Kelloff GJ. Cancer chemoprevention agent development strategies for genistein. J Nutr 1995;125:713S–6S.
47. Adlercreutz H, van der Wildt J, Kinzel J, et al. Human and isoflavonoid conjugates in human urine. J Steroid Biochem Mol Biol 1995;52:97–103.
48. Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE. Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 1997;350:23–7.
49. Duncan AM, Underhill KE, Xu X, Lavalleur J, Phipps WR, Kurzer MS. Modest hormonal effects of soy isoflavones in postmenopausal women. J Clin Endocrinol Metab 1999;84:3479–84.
50. Baird DD, Umbach DM, Lansdell L, et al. Dietary intervention study to assess estrogenicity of dietary soy among postmenopausal women. J Clin Endocrinol Metab 1995;80:1685–90.
51. Murkies AL, Lombard C, Strauss BJ, Wilcox G, Burger HG, Morton MS. Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat. Maturitas 1995;21:189–95.
52. Fujii M, Fukui T, Miyoshi T. Effect of freeze-dried soybean curd (tofu) on various bodily functions. J Med Invest 1999;46:67–74.
53. Washburn S, Burke GL, Morgan T, Anthony M. Effect of soy protein supplementation on serum lipoproteins, blood pressure, and menopausal symptoms in perimenopausal women. Menopause 1999;6:7–13.
54. Hodgson JM, Puddey IB, Beilin LJ, et al. Effects of isoflavonoids on blood pressure in subjects with high-normal ambulatory blood pressure levels: a randomized controlled trial. Am J Hypertens 1999;12:47–53.
55. Matrougui K, Eskildsen-Helmond YE, Fiebeler A, et al. Angiotensin II stimulates extracellular signal-regulated kinase activity in intact pressurized rat mesenteric resistance arteries. Hypertension 2000;36:617–21.
56. Dong F, Larner AC. Activation of Akt kinase by granulocyte colony-stimulating factor (G- CSF): evidence for the role of a tyrosine kinase activity distinct from the Janus kinases. Blood 2000;95:1656–62.
57. Olsen H, Enan E, Matsumura F. 2,3,7,8-Tetrachlorodibenzo-p-dioxin mechanism of action to reduce lipoprotein lipase activity in the 3T3-L1 preadipocyte cell line. J Biochem Mol Toxicol 1998;12:29–39.
58. Motoyashiki T, Miyake M, Yoshida A, Morita T, Ueki H. A vanadyl sulfate-bovine serum albumin complex stimulates the release of lipoprotein lipase activity from isolated rat fat pads through an increase in the cellular content of cAMP and myo-inositol 1,4,5- triphosphate. Biol Pharm Bull 1999;22:780–6.
59. Hemmings BA. Akt signalling: linking membrane events to life and death decisions. Science 1997;275:628–30.
60. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999;13:2905–27.
61. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr 1998;67:867–72.
62. Lu LJ, Anderson KE. Sex and long-term soy diets affect the metabolism and excretion of soy isoflavones in humans. Am J Clin Nutr 1998;68(suppl):1500S–4S.