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	Isoflavone quantification

TOP Isoflavone quantification Isoflavone absorption and... Osteoporosis Cancer Heart disease and lipids Other topics Isoflavone intake Menopause symptom relief Safety issues REFERENCES

 
Sensitive HPLC Method for Profiling Plasma Phytoestrogens. Tarja Nurmi and Herman Adlercreutz. Institute for Preventive Medicine Nutrition and Cancer, Folkhälsan Research Center, Clinical Chemistry Division, University of Helsinki, Helsinki, Finland.

Phytoestrogens have several beneficial effects on human health, i.e., they lower the risk of hormone-dependent cancers, reduce menopausal symptoms and are known to prevent osteoporosis and coronary heart disease. A Western diet contains low amounts of phytoestrogens; therefore, sensitive analytical methods are required to determine the phytoestrogen profiles in nonsupplemented samples, especially when metabolites are also of interest. Traditional analyses are performed by using gas chromatography–mass spectrometry, which is sensitive and selective but also expensive because of multistage sample pretreatment and a high initial investment. HPLC methods using UV-detection are simpler to use and the devices are usually less expensive; however, the sensitivity is not high enough for reliable analysis of nonsupplemented human plasma samples. Therefore, we developed an HPLC method using coulometric electrode array detection for analyzing plasma phytoestrogens (daidzein, genistein, O-desmethylangolensin, equol, dihydrodaidzein, dihydrogenistein, secoislariciresinol, matairesinol, enterolactone and enterodiol). The method is especially useful for analyzing nonsupplemented plasma samples from Western populations. The method was evaluated by determining intra- and interassay precision, resolution, detection limits, linearities, retention time and detector response repeatability. The simple pretreatment required combined with high sensitivity makes the method a valuable tool in clinical and epidemiologic studies.

Development of Rapid Methods for Measuring Isoflavones in Human Urine. Chunyang Wang and Regina Wixon. South Dakota State University, Department of Nutrition and Food Science, Brookings, SD.

UV-visible spectrophotometry was a very successful technique in measuring isoflavones in soybeans and processed soy protein ingredients. The same approach was used in the development of a rapid method for determining isoflavone concentrations in urine samples. Urine samples (n = 30) with different levels of isoflavones were collected from a dietary intervention study. The original samples were scanned, and mathematical models were developed to predicate isoflavone concentrations on the basis of spectrophotometric properties. The method was found to be ineffective. Various simple sample treatments were conducted before the scanning. They included the following: sample dilution, extraction using tetra-butyl methyl ether and solid-phase extraction. The effectiveness of the UV-visible method was improved, as shown by higher correlation coefficients (R² = 0.3–0.6). Solid-phase extraction was found to be the most effective sample treatment before scanning. Predicating models were developed for genistein, daidzein, glycitein and equol. Other developmental efforts will also be reported. This development effort has great potential to be used in scanning a large number of samples. [Supported by the South Dakota Soybean Research and Promotion Council.]

Liquid Chromatography–Mass Spectrometry Analysis of Isoflavones in Naturally Brewed and Chemically Hydrolyzed Soy Sauce. Amanda Murray, Michelle Smith,* Marion Kirk* and Stephen Barnes.* Department of Pharmacology and Toxicology, Altamont School, Birmingham, AL and *University of Alabama at Birmingham, Birmingham, AL.

Previous reports showed that soy sauce contains very low amounts of isoflavones. However, the method of preparation of soy sauce was not taken into consideration. In this study, we compared the isoflavone composition and concentration of naturally fermented and chemically hydrolyzed soy sauces. Commercially available soy sauces (fermented, Kikkoman, Kikkoman Lite, Kimlan Ponlai; and chemically prepared, Angostura, Chun King) were analyzed by reversed-phase HPLC at 262 nm with a diode array detector and by HPLC–electrospray ionization–mass spectrometry (LC-ES-MS) using a triple quadrupole mass spectrometer. Isoflavones were recovered from the soy sauces either after precipitation with 4 vol of methanol or by C8 Sep-Pak cartridge extraction with and without preliminary treatment with ß-glucosidase. The extracts were taken to dryness, redissolved in 80% methanol, centrifuged and analyzed by HPLC and LC-ES-MS. Initial experiments using HPLC-UV analysis revealed that fermented soy sauce extracts, unlike the chemically prepared soy sauces, had a large number of UV-absorbing compounds. However, the complexity of the profiles prevented unequivocal identification of individual peaks as possible isoflavones and their ß-glucoside conjugates. Analysis of extracts by LC-ES-MS scanning over the m/z range 240–650 allowed the development of reconstructed ion chromatograms for each of the isoflavones in soy (m/z 253 for daidzein, m/z 269 for genistein and m/z 283 for glycitein). Two suspected isoflavone peaks (m/z 285 and m/z 301) corresponding to the addition of two hydroxyl groups to daidzein and genistein were also detected. No evidence was obtained for the presence of isoflavone ß-glucoside conjugates. Confirmation of the identity of each primary isoflavone was made by carrying out MS–MS experiments to obtain daughter ion mass spectra of [M–H]- parent molecular ions. This LC-ES-MS approach clearly established that fermented soy sauces contain each of the primary isoflavones (in the unconjugated form) and in 5–10 times greater concentrations (42–79 µg/mL for total isoflavone concentration) than in the chemically prepared soy sauces. Only minor differences in composition or concentrations of isoflavones were observed between regular and ‘lite’ forms of one of the soy sauces. The results suggest that natural fermentation, unlike chemical hydrolysis, produces soy sauces that contain unconjugated isoflavones, some of which have undergone additional metabolism.

Deuteration of Isoflavone Metabolites, Dihydrodaidzein and Dihydrogenistein. Kristiina Wähälä and Sirpa Rasku.University of Helsinki, Department of Chemistry, Laboratory of Organic Chemistry, University of Helsinki, Helsinki, Finland.

To study the metabolism and biological and physiologic effects of phytoestrogens, a sensitive and specific quantitative method is required. The isotope dilution–gas chromatography–mass spectrometry–selective ion monitoring (ID-GC-MS-SIM) technique using synthesized deuterated internal standards for the correction of losses during the procedure is used to quantitate phytoestrogens from food and biological fluids. An internal standard for quantitative MS must have no unlabeled species, and the isotope labels must remain stable under the analytical conditions used. For polyhydroxy aromatics, it is desirable that the reference compound contain three to five stable deuterium atoms because the unlabeled compound, derivatized with trimethylsilyl for GC, will show fairly intense m+1 and m+2 ions in its mass spectrum. Finally, the standard must be isomerically and isotopically pure. The synthesis of new stable [6,8,3',5'-D₄]-dihydrodaidzein, [3,6,8,3',5'-D₅]-dihydrodaidzein and [3,2',3',5',6'-D₅]-dihydrogenistein involves hydrogen-deuterium exchange of aromatic protons using D₃PO₄·BF₃/D₂O as a deuteration reagent and deprotonation of labile deuteriums from fully deuterated dihydrogenistein. The sites of deuteration were determined from the ¹H and ¹³C nuclear magnetic resonance (NMR) spectra by comparison with those of undeuterated compound. Deuterium-carrying carbon atoms appear as low intensity triplets in the proton noise-decoupled spectra compared with the intensive singlets of undeuterated compounds. The exchange order of hydrogens was determined from different deuteration and dedeuteration experiments by following the progress of the reaction by NMR. The isotopic purity of the product is determined from the mass spectra of trimethylsilylated product to avoid M-1 losses from phenolic hydroxyls. The new deuterium-labeled dihydrodaidzein and dihydrogenistein can be reliably used as reference compounds and introduced at the beginning of the analytical procedure because the deuterium labels are securely bound and will survive the various isolation, purification and derivatization steps. The ID/GC/MS/SIM method has now been used for the quantitation of these isoflavone metabolites in human urine.

Phytoestrogen Content of Various Natural Products. Tarja Nurmi and Herman Adlercreutz. Institute for Preventive Medicine Nutrition and Cancer, Folkhälsan Research Center, Clinical Chemistry Division, University of Helsinki, Helsinki, Finland.

Phytoestrogens, shown to possess several beneficial effects on human health, are of particular interest for the pharmaceutical industry. Phytoestrogens seem to lower the risk of hormone-dependent cancers and reduce menopausal symptoms; they are known to prevent osteoporosis and coronary heart disease in experimental studies. A Western diet contains amounts of phytoestrogens that are too low to allow their levels in plasma to become high enough for biological activity. Instead of changing the traditional diet, it is now possible to supplement it with isoflavone-containing concentrated natural products. Positive health effects may occur with moderate amounts of phytoestrogens and high amounts may not have any effects. There is a difficult dilemma, i.e., what is a moderate amount and what is a sufficient amount? In cancer treatment, on the other hand, very high amounts may be needed. The phytoestrogen content of natural products varies significantly, and the amounts recommended to be consumed are not always specified. Ten commercial products were analyzed by using HPLC with a coulometric electrode array detector. The analytical method was carefully standardized and optimized to separate the following isoflavones: daidzein, genistein, glycitein, formononetin, biochanin A, and daidzein- and genistein-7-O-glucosides and their malonyl-glucosides. The samples were analyzed before and after the hydrolysis to quantify the conjugate forms of the phytoestrogens occurring in the products. Phytoestrogen content of the concentrated natural products was slightly or moderately lower than the amounts claimed by manufacturers. However, in one preparation, the content of phytoestrogens was minimal.

HPLC: Electrochemical Detection of Isoflavones and Lignans in Human Plasma. Paul H. Gamache and Ian N. Acworth. ESA Inc., Chelmsford, MA.

Interest in potential health benefits of phytoestrogens has created the need for simple and reliable techniques for their measurement. A highly sensitive method that uses HPLC with coulometric array detection was developed for the determination of plasma phytoestrogens. Analytes were separated in 25 min by reversed-phase (C18) isocratic elution using a water/methanol/acetonitrile, 68:25:7 (v/v/v) mobile phase containing sodium acetate buffer (0.2 mol/L, pH 4.8) at a flow rate of 0.6 mL/min and column temperature of 42°C. Eight serial electrochemical sensors were used at potentials of 340, 470, 500, 530, 560, 620, 680 and 760 (mV vs. Pd) to generate voltametric response relationships for each analyte. After enzymatic hydrolysis, plasma samples were acidified, washed with hexane and extracted with diethyl ether. Combined extracts were evaporated and the residue was dissolved in 50% (by vol) aqueous methanol before HPLC–electrochemical detection (ECD) analysis. Lower limits of quantification (15% relative standard deviation) estimated from plasma extracts were 0.8, 1.2, 0.8 and 1.4 ng/mL of daidzein, enterolactone, equol and genistein, respectively. By least-squares regression, the response was linear with concentration for all analytes (r 0.99, six levels, 10.0–2500 ng/mL, two replicate extractions at each level on 2 d). Intra- and interassay variability for plasma augmented with 50 ng/mL of standards was <5.5% RSD (n = 10) and <7.7% RSD (4 d), respectively. Plasma levels (mean ± SD) obtained from 15 adult volunteers were 2.7 ± 3.9, 4.2 ± 3.4, <1.2 and 2.7 ± 2.1 ng/mL of daidzein, enterolactone, equol and genistein, respectively. The described method is suitable for measuring plasma phytoestrogen in human subjects at basal levels and after supplementation.

The Synthesis of ¹³ C-Labeled Phytoestrogens. Tara Fryatt, Nigel P. Botting and Mark F. Oldfield. University of St. Andrews, St. Andrews, Scotland.

Accurate analysis of the biological effects of phytoestrogens on human health has been hindered by the lack of stable internal standards for gas chromatography–mass spectrometry analysis and for metabolic studies. The primary objective of this work was to develop efficient syntheses of labeled phytoestrogens for use in studies such as these. The initial targets were ¹³C-labeled daidzein and genistein, compounds that are recognized as protective factors against the development of hormone-dependent cancers. Synthetic procedures for the preparation of the isoflavonoid phytoestrogens formononetin, biochanin A, daidzein and genistein, incorporating a single ¹³C label at the 4-position were developed. These procedures use adaptations of existing methodologies and suitable labeled precursors. Current work is focusing on multiply labeled ¹³C-isoflavonoid derivatives. Work is also being carried out on developing methods for the synthesis of ¹³C-labeled lignans, for example, secoisolariciresinol and matairesinol.

	Isoflavone absorption and metabolism

TOP Isoflavone quantification Isoflavone absorption and... Osteoporosis Cancer Heart disease and lipids Other topics Isoflavone intake Menopause symptom relief Safety issues REFERENCES

 
Transport and Metabolism of Soy Isoflavones (Glycosides vs. Aglycones) in Caco-2 Cells and in Perfused Gut Segments. Aukje Steensma,* Hub P.J.M. Noteborn,* Marcel J. B. Mengelers,* Harry A. Kuiper*. *State Institute for Quality Control of Agricultural Products (RIKILT), Wageningen, The Netherlands and Wageningen Agriculture University, Department of Food Technology and Nutritional Sciences, Sub Department of Toxicology, Wageningen, The Netherlands.

Genistein and daidzein are present in minor amounts in soybeans and soy-derived foods, whereas their sugar-conjugated derivatives are present in relatively high amounts (1–3 mg/g product). Data on the bioavailability of isoflavones are scarce. In this study, Caco-2 cells derived from a human colon adenoma carcinoma were used as a model for intestinal absorption. Information was obtained on the transport, metabolism and mechanism of action of genistein, daidzein and their glycosides by growing the cells on semipermeable filters. These in vitro transport and metabolism data were compared with those from intestinal perfused segments of the rat. In Caco-2 cells, a significant difference in the transport and metabolism between the aglycones and their glycosides was observed. Genistein and daidzein added at the apical side were transported to the basolateral side, whereas their glycosides were hardly transported in this direction. In perfused gut segments, the transport of genistein was also higher than its glycoside. Furthermore, the transport of genistein was highest in the ileal segment, whereas there were no differences in transport of the glycoside in various other segments tested. In both model systems, the glycosides were metabolized to their respective aglycones. Genistein was metabolized mainly to sulfates and glucuronides in the Caco-2 cells and to glucuronides in the perfused gut segments. Our data also indicated that Caco-2 cells and rat segments contained exogenous or endogenous glucosidase activity (or both) because only aglycones could be detected at the basolateral side of the Caco-2 cells and in the resorbate of perfused gut segments.

Absorption of Isoflavone Aglycones and Glycosides in Postmenopausal Women. M. Richelle, S. Pridmore-Merten, S. Bodenstab,* I. Tavazzi, S. Jecklin* and E.A. Offord. Nestlé Research Center, Lausanne, Switzerland and *Nestlé Product Technology Centre, Konolfingen, Konolfingen, Switzerland.

Isoflavones naturally present in soybeans and soy-based foods are mainly in the glucoside form. It is believed that removal of the sugar moiety of the glucoside (by ß-glucosidase activity) is required for its absorption through the intestinal wall. We compared the bioavailability of isoflavone aglycones and glucosides from an isoflavone-rich extract consumed as a drink. The isoflavone-rich extract was hydrolyzed enzymatically by commercial ß-glucosidases to produce aglycones. Drinks were constituted by mixing the isoflavone-rich extracts with water, sugar and orange flavor. The pharmacokinetics of isoflavones in plasma were determined over 34 h in six postmenopausal women. After the ingestion of both soy drinks (aglycone or glucoside), plasma total isoflavone increased rapidly, reaching a maximal concentration of 4 µmol/L between 5 and 7 h. Thereafter, plasma isoflavones decreased slowly, leading to plasma concentrations that were still elevated at 34 h postabsorption. The ratio of plasma daidzein to genistein mimicked the ratio found in the soy drink. A low level of dihydroxydaidzein (0.2 µmol/L), a metabolite of daidzein, appeared in plasma after 4 h. The pharmacokinetics of plasma isoflavones were similar with both products. In conclusion, hydrolysis of glucoside before ingestion does not improve the bioavailability of isoflavones from isoflavone-enriched extracts.

Kinetic Models Describing In Vitro Transport and Metabolism of Isoflavones and Their Glycosides in Human Caco-2 Cells. Aukje Steensma,* Marcel J. B. Mengelers,* Hub P.J.M. Noteborn* and Harry A. Kuiper.* *State Institute for Quality Control of Agriculture Products (RIKILT-DLO), Wageningen, The Netherlands and Wageningen Agriculture University, Department of Food Technology and Nutritional Sciences, Sub Department of Toxicology, Wageningen, The Netherlands.

Kinetic models can be applied to describe the in vitro transport and metabolism of isoflavones by using intestinal epithelial cells (Caco-2). These models may enable a quantitative comparison of in vitro and in vivo bioavailability parameters. An extended kinetic model was developed in Caco-2 cells grown on semipermeable filters that described the transport of isoflavones from the apical to basolateral side and vice versa. The model also included the metabolic activity of the cells. Transport of the isoflavones across the intestinal cells was controlled by diffusion. However, the transport rate of the glycosides across Caco-2 cells was too low to enable a kinetic modeling. Additional metabolic studies were carried out to incorporate metabolic rates into the kinetic model. The metabolic rates obtained from the metabolism studies could be incorporated into the model used for describing the transport experiments without alterations.

Absorption and Deglycosylation of Isoflavone Glycosides in the Small Intestine. A. P. Wilkinson, J. M. Gee, A. J. Day, M. S. DuPont, P. W. Needs, G. W. Plumb, I. T. Johnson, M. R. A. Morgan* and G. Williamson. Institute of Food Research, Norwich Research Park, Norwich, UK and *Proctor Department of Food Science, University of Leeds, Leeds, UK.

The predominant isoflavones in soy are the glycosides, although low levels of the aglycones are also present. Isoflavones appear in plasma within 30 min of ingesting soy, indicating some absorption from the small intestine. The aim of this study was to investigate whether isoflavone aglycones are absorbed preferentially in the small intestine compared with their glycosides. Daidzin and daidzein absorption was studied with the use of an in vitro rat everted-gut model. Everted sacs of rat proximal jejunum were filled with physiologic saline and suspended for 15 min at 37°C in oxygenated (95% CO₂/5% O₂) Krebs buffer (pH, 7.2–7.4) containing either daidzein ( 0, 10, 100 or 1000 µmol/L) or daidzin ( 0, 1, 10 or 100 µmol/L). After the incubation, the serosal fluids were collected and stored. An ELISA for daidzein and HPLC were used to both quantitate amounts of daidzin or daidzein transported across the rat small intestine and investigate the chemical forms present in the serosal fluid. A similar degree of absorption of daidzin and daidzein was observed. Absorbed daidzin and daidzein were rapidly metabolized within the small intestinal enterocytes. The principal metabolite of both daidzin and daidzein found in the serosal fluid was daidzein-O⁷-glucuronide. The mechanisms involved in the absorption and metabolism of daidzein and daidzin may be as follows. Daidzein diffuses passively into the enterocyte where it is conjugated with glucuronic acid, a reaction mediated by UDP-glucuronyl transferase, forming daidzein-O⁷-glucuronide, which is subsequently transferred across the basolateral membrane into the serosal fluid. Two mechanisms may account for daidzin uptake and metabolism. It may enter the enterocyte by interaction with an active sugar transport mechanism. Once inside the cell, daidzin can be hydrolyzed by an intracellular cytosolic ß-glucosidase recently shown to be present in the human small intestine (Day et al. 1998 ). This enzyme can effectively hydrolyze daidzin and genistin. After daidzin hydrolysis, the liberated daidzein is conjugated with glucuronic acid as described above. Alternatively, daidzin may be hydrolyzed at the mucosal brush border membrane by another enzyme (lactase phlorizin hydrolase). Our recent studies have shown that this enzyme can hydrolyze isoflavone glycosides. Thus, at the mucosal brush border, daidzin may be hydrolyzed by lactase phlorizin hydrolase and the daidzein formed then diffuses into the enterocyte to be metabolized as described above. It is frequently inferred that absorption of isoflavone glycosides occurs only after their hydrolysis by ß-glucosidases associated with the large intestinal microflora. The results of this study show that both daidzin and daidzein can be absorbed from the small intestine and that there is little difference between the two in their rate of uptake.

Review of Glycitein Bioavailability and Biological Effects. Suzanne Hendrich, Tong T. Song, Yan Zhang, Gui-Juan Wang and Patricia A. Murphy. Food Science and Human Nutrition, Iowa State University, Ames, IA.

Glycitein (4',7-dihydroxy-6-methoxyisoflavone) constitutes 5–10% of total isoflavones in most soybean foods and ~40% of total isoflavones in soy germ (SoyLife). Glycitein disposition and bioavailability were assessed in humans and its estrogenicity was assessed in mice. Total isoflavones of 4.5 µmol/kg body weight were fed to seven men and seven women from soymilk or SoyLife in a single meal in a randomized crossover design. Urinary excretion and plasma content of isoflavones were analyzed by HPLC-UV. Interindividual variation in isoflavone disposition was controlled by selecting subjects of the moderate fecal isoflavone degradation phenotype (average in vitro fecal genistein half-life of 8.9 ± 4.3 h). Urinary excretion as a percentage of ingested dose differed significantly among isoflavones as follows: glycitein, 55%; daidzein, 46%; and genistein, 29% (P < 0.05). Plasma isoflavone contents after soymilk feeding paralleled soymilk isoflavone contents (genistein daidzein glycitein; P < 0.05) in both sexes. Plasma isoflavones paralleled SoyLife isoflavone contents in men (daidzein glycitein genistein), but at 6 h after dosing, plasma glycitein and genistein did not differ in women. The sex difference may be due to isoflavone biotransformation differences and may require further study. Weanling female B6D2F1 mice were dosed with glycitein or genistein (3.0 mg/d for 4 d) or diethylstilbestrol (0.03 µg/kg for 4 d). Uterine weight was increased by 150% by glycitein, 50% by genistein and 60% by diethylstilbestrol, all significantly greater than the control. Glycitein was threefold more estrogenic than genistein (P < 0.05), suggesting that although glycitein is present in lesser amounts in most soy ingredients and foods, it may be responsible in part for certain biological effects of soy-containing foods.

Metabolites of Dietary Phytoestrogens Daidzein, Genistein and Glycitein. S. Heinonen, K. Wähälä* and H. Adlercreutz. Folkhälsan Institute for Preventive Medicine, Nutrition and Cancer, and Department of Clinical Chemistry, University of Helsinki, Finland and *Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, Finland.

Soy and soy-based foods are rich sources of the isoflavones daidzein, genistein and glycitein. The beneficial properties of these dietary phytoestrogens include prevention of hormone-dependent diseases, such as breast and prostate cancers, osteoporosis and cardiovascular disease. In this investigation, the metabolites of soy isoflavones were studied in humans after soy was consumed. The isolation and the characterization of the urinary metabolites were carried out with absorption chromatography on Sephadex LH-20 and gas chromatography–mass spectrometry (GC–MS). The structures of the isolated isoflavones were confirmed by using authentic reference compounds. We identified dihydrogenistein, 6'-OH-O-desmethylangolensin, and cis-4-OH-equol in addition to the known isoflavonoids, daidzein, genistein and glycitein, and the known metabolites, equol, O-desmethylangolensin and dihydrodaidzein, by comparing the retention times and the spectra of the urinary compounds with those of reference standards. For the first time, the metabolites of glycitein were investigated, and new compounds such as 5'-OH-O-desmethylangolensin and 5'-methoxy-O-desmethylangolensin were isolated and identified tentatively by GC–MS. The metabolic pathways for daidzein, genistein and glycitein are presented on the basis of the isolation and identification of these isoflavonoids from human urine.

P lasma Isoflavone Concentrations in American Men and Women Consuming Different Levels of Isolated Soy Protein for up to 6 Months. S. Teixeira, S. M. Potter* and J. W. Erdman, Jr. Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL and *Protein Technologies International, St. Louis, MO.

Isoflavones are plant compounds with mild estrogenic activity. These compounds are found in high concentrations in soybeans and in many soy foods. It has been known for some time that Asian populations with high intakes of isoflavone-rich products have high concentrations of these compounds in blood and concomitantly a lower incidence of cardiovascular disease, several types of cancer, osteoporosis and menopausal symptoms. As part of two larger studies (a men’s and a women’s study) on the effects of soy protein consumption on blood lipid concentrations, we measured plasma concentrations of several isoflavones by HPLC coulometric array detection (eight-channel detector) and HPLC mass spectrometry. The plasma levels of daidzein, dihydrodaidzein, O-desmethylangolensin, equol, genistein, p-ethyl phenol, glycitein and total isoflavones were analyzed in 81 men and 66 postmenopausal women. In both studies before the soy feeding, the subjects consumed a National Cholesterol Education Program Step I diet for 3 wk (men) or 2 wk (women). In the men’s study, 92 subjects, divided into five groups, were fed 50 g/d of a soy protein–casein mixture in different proportions (0:50, 20:30, 30:20, 40:10 and 50:0 g/d) for 6 wk, with corresponding levels of isoflavones (0, 29.1, 44.8, 62 and 95.1 mg aglycones/d). Blood isoflavones were measured at 0, 3 and 6 wk. In the women’s study, three groups (n = 81 subjects) were fed 40 g/d of soy protein with no isoflavones (casein only) or a moderate (55.6 mg aglycones/d) or high level (90 mg aglycones/d) of isoflavones. The subjects received the study protein for 6 mo. Blood isoflavones were measured at 0 and 24 wk. The effects of chronic consumption of different amounts of isoflavones on blood isoflavone concentrations were analyzed by multiple linear regression. The outcome measured was the isoflavone concentration for each subject, with treatment effects represented as each group contrasted with the control group (casein). In the men’s study, soy feeding resulted in higher plasma concentrations of all isoflavones except for O-desmethylangolensin and glycitein. In the women’s study, soy feeding resulted in higher plasma concentrations of all isoflavones analyzed. Plasma isoflavone concentrations after soy feeding, except for equol, were higher in women than in men. At baseline, plasma isoflavone concentrations were higher in men. [Supported in part by the Illinois Soybean Program Operating Board, the Illinois Council for Food and Agriculture Research, and Protein Technologies International.]

Novel Chlorinated and Nitrated Derivatives of Soy Isoflavones Formed by Proinflammatory Oxidants. Brenda J. Boersma, Rakesh P. Patel, Marion Kirk, Victor M. Darley-Usmar and Stephen Barnes. University of Alabama at Birmingham, Department of Pharmacology and Toxicology, Birmingham, AL.

Several chronic inflammatory diseases including atherosclerosis and many types of cancer are associated with the production of the proinflammatory oxidants hypochlorous acid (HOCl) and peroxynitrite (ONOO^-). Because the soy isoflavones have structural similarities with tyrosine, a biological target of HOCl and ONOO^-, we hypothesized that isoflavones react with HOCl and ONOO^-, thereby modulating the biological responses to these proinflammatory oxidants. In initial studies carried out in vitro using HPLC and liquid chromatography–mass spectrometry (LC–MS), we demonstrated that the isoflavones genistein and biochanin A are converted to their mono- and dichlorinated derivatives by HOCl, whereas daidzein forms a monochlorinated derivative. In the case of ONOO^-, mononitrated products of genistein and daidzein, but not biochanin A, were formed. Treatment of genistein with HOCl and sodium nitrite led to the formation of a doubly substituted chloro-nitro derivative. In biological experiments, we used polymorphonuclear neutrophils (PMN) that were activated with phorbol 12-myristate 13-acetate (PMA) to simulate a respiratory burst that generates several reactive oxygen species, including HOCl. When genistein, daidzein or biochanin A was added to this cellular system, monochlorinated derivatives identical to those found in our in vitro studies were detected by LC–MS. In addition, human leukemia (HL-60) cells, which can be differentiated with dimethylsulfoxide to form PMN-like cells, are activated with PMA to produce reactive oxygen species. Addition of each of the isoflavones to these cells also led to the formation of similar amounts of the monochlorinated products. These results indicate that the chlorinated derivatives of the isoflavones could be formed under pathologic conditions when reactive oxygen species are generated. By using the HL-60 cells, we have a model system in which we can investigate the formation and properties of these novel isoflavone metabolites in a renewable biological system.

Flavones and Isoflavones: Synthesis and Isotopic Labeling. Matthew R. Benton and Nigel P. Botting. School of Chemistry, University of St. Andrews, Andrews, Fife, UK.

At present, we are interested in the synthesis of isoflavones and flavones. Both of these classes of compounds are eliciting considerable interest because of their biological effects. In particular, they show anticancer activity and are being investigated as potential chemopreventive agents. The poster describes our studies on the synthesis of novel isoflavones and the development of improved routes for the synthesis of isoflavone metabolites (e.g., glucosides or glucuronides). Recently, we have also been developing methods for the synthesis of isoflavones containing multiple ¹³C atoms. These derivatives can be used as internal standards for analysis and also in metabolic studies. This work is now being extended to examine the synthesis of ¹³C-labeled flavones. Two of the initial targets are apigenin and naringenin labeled with three ¹³C atoms.

Maternal and Cord Blood Phytoestrogen Levels in Indonesian Women. Fabien S. Dalais, Andreanyta Meliala and Mark L Wahlqvist. International Health and Development Unit, Monash University, Alfred Hospital, Prahran, VIC, Australia.

Epidemiologic, cell, animal and human studies have suggested a potential beneficial link between phytoestrogens from soy and cancer, cardiovascular disease, bone metabolism and menopausal symptoms. Japanese individuals have among the lowest rates of hormone-dependent cancer and low rates of cardiovascular disease and menopausal symptoms. It has been shown that Japanese mothers and their infants have high levels of isoflavones in their plasma and cord blood. Indonesian individuals also consume high levels of soybean in the form of tempeh and tofu. The aim of this study was to determine whether Indonesian mothers and their infants were exposed to levels of isoflavones similar to those of the Japanese individuals and possibly received similar health benefits. Blood and cord blood samples were collected from 30 women giving birth at Bethesda Hospital in Yogyakarta, Indonesia. The maternal levels of the isoflavones genistein and daidzein (mean ± SEM) were 83.1 ± 11.7 and 28.9 ± 6.49 nmol/L, respectively, and the cord blood levels were 91.7 ± 12.5 and 33.9 ± 5.62 nmol/L, respectively. On average, these levels are marginally lower than those published for Japanese individuals. Because these individuals are exposed to isoflavones at an early stage of life, these compounds may modify hormonal metabolism and in turn alter disease profiles later in life.

Absorption of Soy Isoflavone Aglycone in Humans and Its Antiatherosclerotic Effect in Rabbits. Toru Izumi, Jun Yamakoshi, Akio Obata, Koichiro Tobe, Makoto Saito, Shigehiro Kataoka and Mamoru Kikuchi. Kikkoman Corporation, Research and Development Division, Noda City, Japan.

Isoflavone is one of the biologically active compounds in soybeans. There are two types of soy isoflavones, i.e., glucoside forms (IFG) and aglycone forms (IFA). The absorption of IFG has been reported but that of IFA has not yet been reported. We tested the absorption of IFA in humans and their antiatherosclerotic effect in rabbits. We measured the isoflavone concentration in plasma by HPLC after the intake of IFA and IFG by humans. IFA were absorbed faster and in larger amounts than were IFG from both low (0.11 mmol) and high (1.7 mmol) single intakes. The plasma concentration of genistein was higher than that of daidzein after intakes of IFA and IFG. We also tested the antiatherosclerotic effect of IFA and IFG in cholesterol-fed rabbits. IFA significantly inhibited the progression of atherosclerosis in a dose-dependent manner (IFA 1.0%, P < 0.01; IFA 0.33%, P < 0.05). In rabbits fed a diet containing 0.55% IFG (equimolar to 0.33% IFA), the progression was not significantly inhibited. Our data show that IFG are not directly absorbed from the gut but are converted to IFA by intestinal bacteria and then are absorbed. Thus, IFA are more effective than IFG for the prevention of atherosclerosis.

	Osteoporosis

TOP Isoflavone quantification Isoflavone absorption and... Osteoporosis Cancer Heart disease and lipids Other topics Isoflavone intake Menopause symptom relief Safety issues REFERENCES

 
Soy Consumption and Bone Mineral Density in Older Japanese American Women in King County, Washington. Madeline M. Rice,* Andrea Z. LaCroix,* Johanna W. Lampe, Gerald van Belle,* Mark Kestin,*** Barbara L. Drinkwater, Amy B. Graves and Eric B. Larson*. *University of Washington, Seattle, WA; Fred Hutchinson Cancer Research Center, Seattle, WA; **Bastyr University, Seattle, WA; Pacific Medical Center, Seattle, WA; and University of South Florida, Tampa, FL.

The purpose of this study was to examine the relation between soy consumption and bone mineral density (BMD) in 267 older Japanese-American women aged 65–93 y. Soy consumption was measured using a 14-item soy food-frequency questionnaire. Soy isoflavone intake was estimated by using published isoflavone (genistein plus daidzein) content of soy foods. BMD of the hip and spine was measured by using dual-energy X-ray absorptiometry. Least-squares means general linear models were used to estimate mean BMD according to categories of current and lifetime soy consumption. Current soy isoflavone intake was grouped by tertiles (low, moderate, high). Lifetime soy intake was categorized as low (lowest current isoflavone tertile with same or less intake during adolescence, late 20s and late 40s), high (highest two tertiles with same or more intake during adolescence, late 20s and late 40s) and varied (all other women). All analyses were adjusted for age, weight, language spoken at the interview, age at menarche and postmenopausal years without estrogen. Femoral neck BMD in women who consumed high amounts of soy throughout life was 0.680 g/cm² compared with 0.628 g/cm² in women who consumed very little soy throughout their lifetime (P = 0.03). Among women currently using fiber supplements, lumbar spine BMD was 0.968 g/cm² in women in the highest tertile of current isoflavone intake compared with 0.843 g/cm² in women in the lowest tertile (P = 0.01). No association was observed between current isoflavone intake and lumbar spine BMD in women who were not using fiber supplements. Women using postmenopausal estrogen appeared to benefit most from high soy consumption, although this effect modification was not significant. Postmenopausal estrogen users who were high soy consumers had the highest BMD at all sites.

Urinary Isoflavone Levels and Several Factors That Influence Bone Metabolism in Postmenopausal Women. Chung Ja Sung, Sun Hae Choi and Byoung-Seob Ko.* Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea and *Korea Institute of Oriental Medicine, Seoul, Korea.

Several studies showed that isoflavones in soy protein are responsible for its bone-sparing effects. Soy products contain large amounts of isoflavones, which have estrogenic and antiestrogenic properties, and may protect against postmenopausal osteoporosis. The purposes of this study were to investigate the association between urinary isoflavones and a bone resorption biochemical maker, deoxypyridinoline (DPD), and to investigate the correlation of DPD with several factors, including bone mineral density (BMD) of the spine and femoral neck. We interviewed 160 postmenopausal women aged 47–85 y and selected 60 women who had a higher frequency of soy product consumption to answer a questionnaire about soy food consumption. We administered a 24-h dietary recall, made anthropometric measurements and collected spot urine samples. BMD was assessed by dual-energy X-ray absorptionmetry. Urinary DPD and pH values were measured and major isoflavones (genistein and daidzein) were analyzed by using HPLC. The concentration of urinary isoflavones was negatively correlated with urinary DPD (P < 0.01). DPD level was negatively correlated with weight and height. Urinary isoflavone levels were positively correlated with age. With multiple regression analysis, DPD was negatively associated with urinary genistein and femoral BMD; spinal BMD was positively associated with femoral BMD, body mass index and urinary pH; and femoral BMD was negatively associated with DPD. We conclude that urinary isoflavone levels, urinary pH and body mass index affect urinary DPD and BMD. Therefore, consuming soy food can be one way to protect against bone resorption.

Effects of Soy Isoflavones, Daidzein, Genistein and Glycitein, on Bone Loss and Lipid Metabolic Pathway in Ovariectomized Rats. Hitoshi Ishida, Takehiko Uesugi,* Toshiya Toda* and Kuniro Tsuji. School of Pharmaceutical Science, University of Shizioka, Shizuoka Japan and *Fujicco Co. Ltd., Kobe, Japan.

It would be helpful to discover a natural dietary substance that would minimize the risk of bone loss and normalize lipid metabolism in postmenopausal women. Recently, some soy products containing isoflavones (e.g., daidzein, genistein or glycitein) were shown to have positive effects on bone density and to reduce abdominal fat in ovariectomized (ovx) rats. It was found that ipriflavone, which is structurally related to soy isoflavones, shows a similar effect. Therefore, we hypothesized that soy isoflavones might be effective in ameliorating the bone loss and hypercholesterolemia due to ovarian hormone deficiency. To test our hypothesis, we studied the effects of daidzein, genistein and glycitein on bone loss and lipid metabolism in ovx Sprague-Dawley rats (age 11 wk). Each compound was administered orally to ovx rats for 4 wk. The femurs of these rats showed significantly lower density and breaking strength than did those of sham-operated rats. These changes were largely prevented in rats that received daidzein, genistein or glycitein at a dose of 50 mg/(kg · d) and in rats that received subcutaneous estrone [7.5 mg/(kg · d)] as a positive control. Ovariectomy caused atrophy of the uterus and increased the ratio of the urinary excretion of pyridinoline and deoxypyridinoline to endogenous creatinine. This was prevented by administration of daidzein, glycitein or estrone but, interestingly, not genistein. With respect to food intake and body weight, rats in the ovx group had significantly higher final body weights and food intake than did rats in the sham-operated group. Daidzein and glycitein, like estrone, prevented this ovariectomy-induced increase in body weight gain and food intake, whereas genistein did not. Daidzein and glycitein reduced abdominal fat and the level of serum total cholesterol in comparison with the control group as did estrone. The results indicate that daidzein and glycitein seem to be proestrogenic compounds; genistein appears to have a mechanism and site of action different from those of these two compounds.

Relationship Between Urinary Isoflavones and Bone Metabolism in Postmenopausal Japanese Women. Y. Fukui, A. Miura,* Y. Nara,** T. Uesugi, K. Honda and Y. Yamori.* Fujicco Co. Ltd., Kobe, Japan; *WHO Collaborating Center for Research on Primary Prevention of Cardiovascular Diseases, University of Kyoto, Kyoto, Japan; Department of Environmental Preservation and Development, Graduate School of Human and Environmental Studies, University of Kyoto, Kyoto, Japan; and **Graduate School of Integrated Science and Art, University of East Asia, Shimonoseki, Japan.

Soy isoflavones are recognized as having beneficial effects on bone health. This study examines the effect of daily isoflavone intake on bone metabolism. We investigated the association of urinary isoflavones excretion with bone density or urinary bone resorption markers in postmenopausal Japanese women. Subjects included two populations as follows: middle-aged Japanese women (n = 39, 54.8 ± 3.3 y) living in Japan (JJ) and elderly Japanese women (n = 48, 74.4 ± 3.7 y) living in Hawaii (JH). Urine samples were collected for 24 h and were analyzed for the urinary isoflavones daidzein and genistein and bone resorption markers pyridinoline and deoxypyridinoline by HPLC. The bone density, given as the stiffness value, was estimated by an ultrasonic bone densitometer. All subjects were divided by stiffness values into three groups as follows: the higher stiffness, middle stiffness and lower stiffness groups. In both populations, the higher stiffness group had significantly higher urinary isoflavone (daidzein and genistein) excretions than did the lower stiffness group (JJ: 20.0 ± 12.8 and 8.6 ± 7.7 µmol/d; JH: 26.6 ± 24.5 and 9.7 ± 8.9 µmol/d, respectively, for the higher stiffness and lower stiffness groups; P < 0.05). Significant inverse correlations were found between urinary isoflavones excretion and bone resorption markers pyridinoline and deoxypyridinoline in JJ (r = -0.338, P < 0.05 and r = -0.387, P <0.05, respectively, for pyridinoline and deoxypyridinoline). Isoflavones taken from daily Japanese diets were related inversely to bone resorption markers and positively to bone density in menopausal women. Thus, a sufficient daily isoflavone intake is expected to contribute to the prevention of postmenopausal osteoporosis.

Effects of Soy Isoflavones on Blood Lipids, Blood Pressure and Biochemical Markers of Bone Metabolism in Postmenopausal Women. William W. Wong. USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, TX.

Cessation of estrogen production with menopause results in hypercholesterolemia, hypertension and elevated bone loss in postmenopausal women. Estrogen replacement therapy lowers blood lipids, increases arterial compliance and minimizes bone loss in postmenopausal women but increases the risk of cancer. Isoflavones, found in abundance in soybeans, are weak estrogens and were shown to inhibit mammary cancer cell formation and growth. To determine the effects of soy isoflavones on blood lipids, blood pressure and biochemical markers of bone metabolism in postmenopausal women, the fasting blood lipid concentrations, blood pressure and biochemical markers of bone metabolism of six postmenopausal women [ages 55.4 ± 3.5 y, (mean ± SD); body weight, 75.0 ± 10.7 kg; height, 164.7 ± 5.2 cm] ingesting 160 mg/d of soy isoflavones were measured before and after 6 wk of treatment. After isoflavone treatment, no significant changes were observed in body weight (-0.8 ± 2.1%, P = 0.40) or in blood concentrations of total cholesterol (0.5 ± 7.5%, P = 0.88) or LDL cholesterol (-0.5 ± 9.3%, P = 0.90). However, an increase of 10.2 ± 10.1% in apolipoprotein B100, the major lipoprotein in LDL cholesterol, was detected (P = 0.06). Blood concentrations of HDL cholesterol and its major lipoprotein, apolipoprotein A1, were elevated by 5 ± 14% and 3 ± 11%, respectively, although these increases were not significant (P 0.45). No significant change in systolic blood pressure was observed; however, there was a significant reduction in diastolic blood pressure of 12 ± 9% (P <0.03). Isoflavone treatment affected biochemical markers of bone resorption; urinary concentrations of deoxypyridinoline and calcium, serum concentration of parathyroid hormone and urinary calcium excretion were modified by -7 ± 41%, -12 ± 46%, +33 ± 60% and -5 ± 29%, respectively. Biochemical markers of bone formation also were altered by isoflavone treatment; serum concentrations of osteocalcin, bone alkaline phosphatase and insulin-like growth factor I were reduced by 9 ± 15, 16 ± 23 and 8 ± 27%, respectively. However, the changes in biochemical markers of bone metabolism were not significant. Although many of the changes in blood lipids and biochemical markers of bone metabolism induced by the soy isoflavone treatment were not significant because of the small sample size, the size of many of these changes was similar to that reported in postmenopausal women receiving estrogen replacement therapy. Therefore, further studies with a larger number of subjects, a longer treatment period, measurement of lumbar spine bone mineral content and bone density and measurement of calcium kinetics using the dual-tracer technique are warranted. [Funded by the USDA/ARS; the soy isoflavone tablets were provided by Schouten USA Inc., and the Pyrilinks-D kits, for the measurement of urinary Dpd, were kindly provided by Metra Biosystems, Inc.]

	Cancer

TOP Isoflavone quantification Isoflavone absorption and... Osteoporosis Cancer Heart disease and lipids Other topics Isoflavone intake Menopause symptom relief Safety issues REFERENCES

 
Molecular Effects of Soy-Isoflavone Genistein in Prostate Cancer Cells. Joanne N. Davis,* Omer Kucuk* and Fazlul H. Sarkar.** Departments of *Cancer Biology, Internal Medicine, and **Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI.

Prostate cancer is the second leading cause of cancer-related deaths in men in the United States, accounting for 36% of all male cancers and 13% of cancer-related deaths in men. Epidemiologic data provide convincing evidence that dietary factors play an important role in the etiology of cancer. We previously demonstrated that the dietary isoflavone genistein inhibits proliferation, induces apoptosis and modulates important cell cycle regulatory molecules, particularly p21WAF1 and cyclin B, in prostate cancer cells, and therefore may be a potential chemopreventive or therapeutic agent. To further elucidate the molecular mechanism by which genistein elicits its effects, we first investigated the role of a transcription factor NF-B; second, we measured prostate-specific antigen (PSA) levels in prostate cancer cells. NF-B was shown to protect cells against apoptosis by initiating prosurvival mechanisms. We investigated whether genistein modulates NF-B, particularly the inactivation, which may lead to the apoptosis observed in genistein-treated cells. Here we show that genistein decreases NF-B activity in prosate cancer cells in a dose-dependent manner. Prostate cancer cells treated with genistein at 30 and 50 µmol/L for 24 h resulted in reduced NF-B DNA binding. Using confocal microscopy, we showed that genistein blocks the translocation of NF-B p50 and p65 subunits from the cytoplasm to the nucleus, preventing NF-B activation and prohibiting DNA binding. Additionally, we demonstrated that genistein abrogates NF-B activation by two known inducers, H₂O₂ and tumor necrosis factor- (TNF-). Prostate cancer cells pretreated with 50 µmol genistein/L for 48 h inhibited NF-B DNA binding and blocked translocation of NF-B subunits to the nucleus when stimulated with either H₂O₂ or TNF-. These results suggest that the inactivation of NF-B by genistein may lead to the cell growth inhibition and apoptosis observed in genistein-treated cells. The most valuable tumor marker used for the detection and monitoring of prostate cancer is PSA. PSA, a member of the kallidrein family, is a serine protease secreted by prostate epithelial cells. PSA, which is able to cleave the predominant seminal vesicle protein, has been proposed as a candidate growth factor, cytokine or growth factor regulator and has been linked to tumor progression. Therefore, we investigated whether genistein has any effect on PSA expression and secretion in the androgen-sensitive prostate cancer cell line, LNCaP. LNCaP cells were treated with genistein at 0, 30 and 50 µmol/L for 3 d. The medium was collected and assayed for the presence of PSA. We observed that treatment with genistein at 30 µmol/L reduced PSA secretion by 50% and 50 µmol/L reduced PSA by 80% compared with control. Using immunohistochemistry and Western blot analysis, we determined that genistein inhibits PSA protein expression levels but did not affect the protein expression levels of another tumor-associated antigen, prostate-specific membrane antigen. These results indicate that genistein lowers the PSA levels in prostate cancer cells in vitro. In conclusion, the inactivation of NF-B and downregulation of PSA by genistein provide encouraging evidence to support genistein’s role as a chemopreventive and therapeutic agent for prostate cancer. These results also indicate that NF-B may play a pivotal role in genistein-induced apoptosis, providing a mechanism by which genistein promotes cell death.

The Specific Role of Genistein in Estrogen Metabolism. N. B. Kumar, K. Allen, A. Cantor, G. Shaw and C. E. Cox. H. Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL.

Our goal was to evaluate the individual effectiveness of supplementing a group of premenopausal, breast cancer–free women with a dietary supplement of the isoflavone genistein (40 mg/d) in producing a change in sex hormones that are implicated in the initiation and promotion of breast cancer. Consecutively recruited premenopausal omnivorous women (n = 68), of all races and ethnicities, aged 25–55 y, were admitted to the study and randomly assigned to an experimental group supplemented with soy (40 mg genistein/d) or to a control group consuming a placebo for a 12-wk period. Changes in their anthropometric, nutritional and hormonal biomarkers from the early follicular phase were analyzed at baseline and after intervention. Preliminary analysis indicated that hormonal concentrations of free estradiol decreased by 78% in the group consuming genistein compared with 44% in the placebo group. Serum estrone and sex hormone–binding globulin levels were elevated in 47 and 46% of the subjects, respectively, in the experimental group compared with 26 and 40%, respectively, in the placebo group. In the experimental group, the menstrual cycle of 58% of the subjects increased by more than 2 d compared with 36% in the placebo group. These data suggest that increased genistein intake affects estrogen metabolism by altering the sex hormone concentrations that are implicated in breast cancer promotion or inhibition. [Supported by an NIH NCI grant RO3 CA72588-01A1.]

Effect of Soybean Saponins on the Growth and Antioxidant Defense of Human Hepatocarcinoma Cells. M.-K. Sung and M.-Y. Park. Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea.

Carcinogenesis is a multistep process including initiation, promotion and progression. Recent studies indicated that oxygen free radicals, by-products of normal cellular respiratory processes as well as lipid peroxidation processes, induce cellular DNA damage, which is the most plausible mechanism for the initiation of carcinogenesis. Lipid peroxides also were shown to promote tumor cell growth. Saponins are amphiphilic compounds present in a variety of edible and nonedible plants. Recent studies indicated that saponins extracted from soybeans inhibit the formation of lipid peroxides in corn oil samples; this may be due to their ability to scavenge radicals. In this study, effects of soybean saponins on the growth, cellular lipid peroxidation and antioxidative enzyme activities of HepG2 cells were investigated. Effects of saponins were compared with -tocopherol and ascorbic acid. Cells (1–2 x 10⁷) were incubated for 24 h and then treated with tert-butylhydroperoxide (0.5 nmol/L for 45 min) to initiate lipid peroxidation followed by saponin treatment (300 µg/plate for 48 h). Cellular superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione S-transferase (GST) activities were measured. Results showed that tert-butylhydroperoxide treatment significantly increased cellular malondialdehyde content. Cell growth was significantly decreased with saponin, -tocopherol and ascorbic acid treatment. Malondialdehyde content was significantly reduced by saponin (72%) and -tocopherol (40%). Soybean saponins significantly increased cellular SOD, GPX and GST activities. Ascorbic acid significantly decreased GPX activity. However, the activity of GST was not affected by either -tocopherol or ascorbic acid. These results indicate that soybean saponins possess considerable antioxidative capacity, exerting antiproliferative effects on tumor cells.

Soybean Saponins Inhibit the Formation of DNA Adducts in Human Colon and Liver Cells. H.-S. Jeon and M.-K. Sung. Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea.

Numerous chemical carcinogens, activated to form electrophilic agents, react with DNA, which explains the induction of a heritable change in a cell leading to malignant transformation. This may be a main event in the initiation of carcinogenesis. Soybeans contain up to 2% saponins. Soybean saponins were shown to inhibit the growth of human colon carcinoma cells with low toxicity. Also, they were shown to decrease the ornithine decarboxylase activity that is directly related to cancer cell proliferation. These results indicate that soybean saponins are important modulators in the promotion stage of carcinogenesis. This study was performed to examine the effects of soybean saponins on DNA adduct formation, which is the most important reaction of carcinogens with cellular macromolecules initiating carcinogenesis. CCD-18Co and HepG2 cells were used as models for colon and liver cells, respectively. Cells (4–5 x 10⁵) were seeded and allowed to attach. After 18 d (CCD-18Co) and 2 d (HepG2) in culture, soybean saponins at a concentration of 0–50 µg/mL were added and incubated for 1 h. Preincubated tritiated aflatoxin B1 (12 nmol/L; specific activity 25 Ci/mmol) was added to each plate and incubated for a further 48 h. DNA was purified and aliquots of DNA samples were used to measure radioactivity by liquid scintillation counting. Results showed that soybean saponins significantly inhibited the formation of DNA-aflatoxin B1 adducts in CCD-18Co cells by 23.7, 50.7 and 49.4% when 10, 30 and 50 µg saponins/mL, respectively, were used. Amounts of DNA adducts in HepG2 cells were also significantly decreased by 37.3, 50.1 and 49.8%, respectively. These results indicate that soybean saponins may effectively reduce cellular DNA damage by carcinogens and can be regarded as potential chemopreventive agents.

Effects of Genistein on the Activities of Antioxidant Enzymes in Strenuously Exercised Rats. C. Chen and R. M. Bakhit. Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA.

Male Sprague-Dawley rats (n = 48), aged 1 y, were assigned to four groups as follows: normal diet/sedentary, normal diet/exercise, genistein diet/sedentary and genistein diet/exercise. The AIN complete basal diet was supplemented with 500 mg genistein/kg. After 4 wk of consuming the experimental diets, the rats in the exercise groups underwent an acute exercise protocol of 22 m/min at 12° inclination for 1 h. Immediately after the exercise, blood was drawn from each rat and tested for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)]. Genistein concentration was measured in the plasma, liver and muscle. The average feed intakes were not significantly different among the four groups (P 0.05). Plasma, liver and muscle genistein concentrations were significantly higher in the rats undergoing acute exercise than in the nonexercised rats (P < 0.05). In addition, in the exercised rat groups, the genistein-fed rats had significant increases in the activities of two antioxidant enzymes, GPX and CAT, compared with exercised rats fed the normal diet (P < 0.05). GPX and CAT levels in the exercised genistein-fed rats were comparable to those of the sedentary rats. No significant changes were observed for SOD. The results suggest that acute strenuous exercise might lead to the release of relatively more genistein from storage tissue into circulation than would occur with no exercise. There is also an indication that 500 ppm genistein from dietary supplementation might diminish the disadvantages of increased production of free radicals resulting from acute exercise by maintaining the antioxidant defense systems in the acutely exercised rats. [Funded by the Virginia Soybean Association.]

Familial Breast Cancer Dietary Prevention Demonstration Trial. Cristina Bellati and Franco Berrino. Istituto Nazionale Tumori, Milan, Italy.

High serum levels of sex hormones and insulin-like growth factor I (IGF-I) usually precede breast cancer. We recently conducted a randomized dietary trial (the DIANA study) in which a comprehensive modification of Western dietary habits significantly reduced the bioavailability of such hormones (testosterone and estradiol decreased by 18%; sex hormone–binding globulin and IGF binding protein increased). The dietary intervention aimed at reducing insulin resistance and increasing the intake of phytoestrogens, both soy isoflavonoids and lignans from various sources. Insulin inhibits the liver synthesis of sex hormone–binding globulin and IGF-BP and stimulates the ovarian production of sex hormones, whereas phytoestrogens have the opposite effect. The penetrance of genes responsible for hereditary breast cancer is likely to be affected by several environmental factors, including ionizing radiation, tobacco smoking, oral contraceptive use and nutritional habits. The latter refer mainly to fruit and vegetable consumption and the dietary regulation of the availability of sex steroid hormones and IGF and related peptides. Evidence is increasing that these nutritional factors are more strongly related to genetics than to sporadic breast cancer. We are presently carrying out a case-only study (the COS Study) to test this interaction in several thousand European women who were diagnosed with breast cancer before age 40. Seven countries are involved in the study. In Italy, patients who most likely carry high penetrance mutations together with their healthy sisters are being invited to participate in a dietary prevention trial. The dietary modification strategy for the intervention group includes kitchen courses and behavioral and psychological support. Families randomly assigned to the control group receive the usual cancer prevention dietary recommendations, but active intervention will be postponed for several years. The endpoint will be breast cancer in healthy women and contralateral breast cancer in patients. A study including 400 mutation carriers per group has 80% statistical power to detect a 50% reduction in breast cancer incidence.

Urinary Phytoestrogens and Breast Cancer Risk in a Prospective Study in Postmenopausal Dutch Women. P.H.M. Peeters,* I. den Tonkelaar,* P. Van’t Veer,** L. Keinan-Boker,* C.M.J. Arts, H. Adlercreutz and J.H.H. Thijssen. *Julius Center for Patient Oriented Research, University Medical Center, Utrecht, The Netherlands; International Health Foundation, Utrecht, The Netherlands; **Division of Human Nutrition and Epidemiology, Wageningen University, Wageningen, The Netherlands; TNO Nutrition and Food Research Center Institute, Zeist, The Netherlands; Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland; and Department of Endocrinology, UMCU, Utrecht, The Netherlands.

Phytoestrogens, naturally occurring compounds in many foods and especially in soy products, are defined as plant substances that are structurally or functionally similar to estradiol. Two former retrospective studies assessed the relationship between urinary phytoestrogen excretion and breast cancer risk; their results indicated a protective role for phytoestrogens. Urine samples in those studies were collected after breast cancer diagnosis, and urinary phytoestrogen levels (a short-term indicator) might have been affected by the disease state. We chose, therefore, to study the associations of certain phytoestrogens with breast cancer risk by using urinary specimens collected several years before breast cancer was diagnosed. Subjects were 88 postmenopausal women with breast cancer (cases) and 286 postmenopausal women without breast cancer (controls) selected from a cohort of women (n = 14,697) who participated in a breast cancer screening program, the DOM Project, in Utrecht, The Netherlands. Levels of genistein and enterolactone were determined by time-resolved fluorescent immunoassay and expressed in micromoles per mole creatinine. For each subject, the mean value for genistein and enterolactone was computed from two urinary samples collected 1 y apart. Odds ratios of the highest to the lowest tertiles of urinary phytoestrogen per creatinine concentrations were computed. Higher urinary genistein excretion was weakly associated with a reduced breast cancer risk, i.e., the odds ratio for the highest compared with the lowest tertile was 0.83; the 95% confidence interval (CI) was 0.46–1.51. In contrast, higher urinary enterolactone excretion was weakly and nonsignificantly associated with an increased breast cancer risk, i.e., the odds ratio for highest compared with lowest tertile was 1.43, 95% CI, 0.79–2.59. Tests for trends for both phytoestrogens were nonsignificant. We were not able to detect the previously reported firm protective effect of phytoestrogens. Such an effect may be smaller than expected or limited to premenopausal women.

Phytoestrogen and Androgen Levels in Australian Postmenopausal Women Diagnosed with Breast Cancer. A. L. Murkies, F. S. Dalais,* E. M. Briganti, D. L. Heal, H. G. Burge, M. L. Wahlqvist* and S. R. Davis. The Jean Hailes Foundation, Clayton, VIC, Australia; *International Health and Development Unit, Monash University, Clayton, VIC, Australia; Department of Epidemiology and Preventive Medicine, Monash University, The Alfred Hospital, Prahran, VIC, Australia.

Isoflavones are phytoestrogens, plant-derived compounds, that have been implicated as potential anticarcinogenic compounds by in vitro, animal, epidemiologic and human case-control studies. The role of endogenous androgens is still being investigated. The aim of this study was to assess the association between urinary excretion of isoflavones (daidzein and genistein), dietary composition and circulating androgens and their 24-h urinary metabolites in postmenopausal women and the risk of breast cancer. Cases (n = 18) and controls (n = 20) completed a detailed dietary questionnaire; a blood sample and a 24-h urine sample were collected. Analysis and detection of phytoestrogens were carried out by HPLC and UV. Nonparametric analysis was undertaken (Mann-Whitney U test) to compare groups. There were no significant differences in lifestyle and reproductive variables between groups. Urinary daidzein excretion was significantly lower in breast cancer patients [0 nmol/24 h; 0, 1375.5 (median and interquartile ranges)] than in control subjects (1558.2 nmol/24 h; 498, 2773.2; P = 0.02), and there was a trend for decreased genistein (P = 0.08) for patients compared with control subjects. Serum testosterone was elevated in the patients (1.2 nmol/L; 1, 1.6) more than in the control subjects (1 nmol/L; 0.7, 1.25; P = 0.05), whereas no significant differences were observed between the two groups for other hormonal measures or total fat, fiber and vegetable consumption. Women with breast cancer were found to have lower urinary isoflavone levels. The lower urinary phytoestrogens could be attributed to a reduced dietary intake due to future surgery for breast cancer. There was no difference in the usual diet as reported; however, the food intake questionnaire may not have been sufficiently specific for phytoestrogens and may have had limitations in evaluating actual phytoestrogens intake from known sources, such as processed foods. Larger studies are required to examine the association between phytoestrogens and breast cancer more thoroughly while addressing the issues of bias.

Serum Insulin-Like Growth Factor I Is Unaffected by Genistein in Wild-Type or Estrogen Receptor- Knockout Mice. Ruth S. MacDonald, William H. Thornton, Jr., J. Kevin Day and Dennis B. Lubahn. Nutritional Sciences Program, University of Missouri, Columbia, MO.

Consumption of soy foods is ecologically associated with a reduced risk of breast cancer. On the basis of animal experiments, the phytoestrogen genistein is suggested to be one component of soy that provides protection. Recent evidence suggests that the estrogen receptor interacts with growth factor receptors in several cell types to mediate cellular function. In cultured cells, genistein inhibits tyrosine kinase activity, which is an essential component of the insulin-like growth factor-I (IGF-I) signaling pathway. Hence, we examined changes in serum IGF-I in wild-type (WT) and estrogen receptor- knock-out (ERKO) mice fed genistein. WT and ERKO mice were fed a semipurified diet containing 0 (control) or 1 g genistein/kg diet beginning at age 3 wk. The objective of the study was to examine the protective effect of genistein on breast cancer development; hence two medroxyprogesterone pellets (40 mg each) were implanted subcutaneously into each mouse at 6 wk, and DMBA (1 mg/dose) was given orally at 9, 10, 12 and 13 wk. The mice continued to receive their respective dietary treatments for 14 mo. Body weight gain was slightly less in mice fed genistein than in controls throughout the study because of reduced food intake. Tumors developed in all of the WT mice between 29 and 45 wk, but none of the ERKO mice developed tumors. In the WT mice, no protective effect of genistein on breast tumor formation was found. Serum IGF-I concentrations were 199 ± 69, 512 ± 98, 262 ± 60 and 252 ± 85 ng/mL in the WT control, ERKO control, WT genistein and ERKO genistein groups, respectively. When analyzed as a 2 x 2 factorial by ANOVA, there were no significant differences by diet or genotype; however, there was a diet-genotype interaction. The range of serum IGF-I concentration in the WT mice was 13–281 ng/mL and in the ERKO mice was 127–935 ng/mL, including two mice with very high levels. The trend for higher IGF-I in the ERKO mice fed the control diet, which was suppressed by feeding genistein, suggests that genistein influenced serum IGF-I concentrations in these mice. We are continuing to examine the relationship between genistein consumption and the IGF-I axis in both WT and ERKO mice. This animal model provides a useful tool for examining the relationships among estrogen receptors, the IGF-I axis and dietary phytoestrogens. [Funded by Missouri Agricultural Experiment Station Interdisciplinary Regional Research Grant.]

Inositol Hexaphosphate Has an Antioxidant Function That Reduces GST-P⁺ Foci on Hepatocarcinogenesis in Rats. Hae-Jeoung Lee, Hyeon-duck Kim, Sang-A Lee and Haymie Choi. Seoul National University, Department of Food and Nutrition, Seoul, Korea.

Inositol and inositol hexaphosphate (phytate), which are found in plant foodstuffs such as seeds, grains, fruits and vegetables, were demonstrated to have anticancer, anti-cell-proliferation, and antioxidant functions. This study was designed to determine the effects of phytate on rat hepatocarcinogenesis and whether supplementing with inositol would enhance anticarcinogenic and antioxidant effects. Rats received a single intraperitoneal injection of diethylnitrosamine, were subjected to two-thirds hepatectomy 3 wk later and were killed 8 wk after the injection. One week before the partial hepatectomy, the rats were divided into three groups, and inositol or phytate was added to the drinking water (adjusted pH. 7.4); one group received 2% phytate, one received 2% inositol and one received 1% phytate + 1% inositol. In all three groups, the numbers and the areas of glutathione S-transferase–placental positive (GST-P⁺) foci were significantly decreased. Thiobarbituric acid–reactive substances (TBARS) content and catalase and GST activities were significantly reduced in rats fed inositol phosphates. TBARS content and catalase activities were the lowest in the phytate group because phytate potently inhibited the formation of the iron-derived hydroxyl radical and suppressed lipid peroxidation. Catalase and GST activities were not induced likely because of the antioxidant function of inositol phosphates. TBARS content was positively correlated with GST-P⁺ foci. It seemed that inositol phosphates helped the endogenous defense system during carcinogenesis by decreasing TBARS and H₂O₂. Therefore, the preventive effect against hepatocarcinogenesis can be explained in part by the antioxidant function of inositol phosphates, and there were no differences in GST-P⁺ foci, activities of catalase and GST, and TBARS with or without the inositol supplement.

Soy Intake and Colorectal Cancer in Chinese in North America and China. Anna H. Wu, Alice S. Whittemore, Marion Lee, Richard P. Gallagher and Zheng Shu. University of Southern California, Department of Preventive Medicine, Los Angeles CA.

Epidemiologic studies suggest that a high intake of soy foods may reduce the risk of certain types of cancer, including those of the breast, prostate and endometrium. However, the role of soy foods in colorectal cancer is less clear. Our purpose was to examine the relationship of soy foods to risk of colorectal cancer, with a focus on consistency in findings in North America and China, in men and women, or colon and rectal cancer. Cases included persons with histologically confirmed colorectal cancer who were identified through the population-based tumor registries of Los Angeles, San Francisco, Vancouver and Ontario (n = 432 in North America) and in Hangzhou, China (n = 473). Control subjects matched to cases by age, sex and study area were interviewed (n = 1296 in North America, n =1192 in China). Dietary and nondietary (including physical activity and body size characteristics) information was collected during home interviews. Odds ratios were estimated by using conditional logistic regression stratified jointly by age, sex and study area. The intake pattern for soy will be presented by continent, sex, and disease status. Risk of colorectal cancer was 40% lower in association with a high soy intake (at least two times per week compared with fewer than two times per month) for men in North America, but a similar reduction in risk was not observed for women in North America. In China, the data showed no clear patterns of altered risk for colorectal cancer with intake of individual soy foods or all soy foods combined. Further adjustment for other dietary and nondietary factors did not change these findings. Thus, no consistent or strong associations between colorectal cancer risk and consumption of soy foods were found in this study.

Radical-Scavenging Activity in Brown-Colored Soybean Seed Coat. Yashurio Takahata, Shu Furuta, Masakazu Takahashi and Ikuo Suda. Kyushu National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries, Nishigoshi, Kumamoto, Japan.

Soybeans having a brown- or black-colored seed coat have been used in the daily diet traditionally and occasionally in Japan. However, when the effect of soybeans on human health is discussed, most of the background data are derived from a normal soybean that has a yellowish-white–colored seed coat. Here we investigated the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities of a brown-colored soybean seed coat by using indigenous varieties and breeding materials. Among four indigenous varieties, Akita-Zairai showed the highest radical-scavenging activity. We crossed Akita-Zairai and a normal soybean cultivar to obtain progeny seeds as breeding materials. In the F3 seed generation, seeds were classified roughly into the following four phenotypes on the basis of their seed coat phenotype: yellowish-white, lusterless pale brown, lustrous pale brown and dark brown. DPPH radical-scavenging activity in the lustrous pale brown F3 seed coat was higher than that in dark-brown F3 and the original parental Akita-Zairai. The yellowish-white seed coat showed negligible activity as did the lusterless pale brown. The difference in luster seemed to be a key trait in the intensity of radical-scavenging activity. DPPH radical-scavenging activities paralleled the content of polyphenolics. Using an LH-20 column, we fractionated compounds demonstrating radical-scavenging activities and showed that DPPH radical-scavenging activities and proanthocyanidin content were concomitant in each fraction. We could demonstrate that proanthocyanidins are possible candidates for radical-scavenging activities in the brown soybean seed coat. Soymilk made from cotyledons of normal cultivar and seed coats of Akita-Zairai had 2.5-fold higher radical-scavenging activity than did soymilk made from normal soybean alone. This result indicates that the radical-scavenging activity in brown seed coats is retained after processing.

Effect of Estrogen and Estrogen Mimics for the Growth of MCF-7 Cells in Nude Mice. Keiji Sugi, Takahiko Gotoh, Yin Hong and Akihiro Ito. Department of Cancer Research, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.

The estrogen-responsive human breast cancer cell MCF-7 was examined for its growth in nude mice under various doses of 17-ß-estradiol (E₂), bisphenol A and a miso diet. Seven-week-old nude mice were inoculated subcutaneously in six sites each with 5 x10⁶ MCF-7 cells/site. Mice were treated with intramuscular injection of E₂ at 0.001, 0.01 and 0.1 µg/mouse; oral administration of bisphenol A at 10, 100 or 1000 ppm; or a diet containing 10% miso. Twelve weeks later, inoculated tumor sites and weight were scored. Tumor take (incidence) was highest in control mice (47%, 223 mg). For E₂ administration, they were 22%, 194 mg with 0.001 µg; 25%, 393 mg with 0.01 µg; and 39%, 257 mg with 0.1 µg. Similarly, bisphenol A showed a dose-dependent increase by the incidence of 29%, 565 mg with 10 ppm; 40%, 616 mg with 100 ppm; and 42%, 869 mg with 1000 ppm. For the miso group, the values were 17% and 185 mg. Immunohistochemical staining for antibodies to pS2 estrogen-regulated protein and BrdU showed high frequencies in tumor tissues.

Meta-Analysis of Soy Intake and Breast Cancer Risk. Bruce Trock, Leslie White Butler, Robert Clarke* and Leena Hilakivi-Clarke. Departments of Human Oncology, *Physiology and Biophysics, and Psychiatry, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC.

High soy intake in Asian countries was proposed as a factor contributing to the low breast cancer risk for Asian women. However, soy is being marketed and recommended to the public as if a clear protective effect was established when, in fact, the epidemiologic data are rather limited. Because in vivo and in vitro data show estrogenic effects for genistein, the major component of soy, it is important that associations between soy and breast cancer risk be evaluated before recommendations can be made with safety, especially for women who already have breast cancer. Therefore, we performed a meta-analysis of epidemiologic studies examining soy and breast cancer risk. A literature search was based on key words associated with soy or specific isoflavones and phytoestrogens, and breast cancer. An Internet search was also conducted to identify unpublished data. We analyzed data by using the measures of soy intake provided in the studies and also normalized intake to daily grams of soy protein. Odds ratios (OR) were pooled by using Mantel-Haenszel methods, and random effects models were used in the presence of significant heterogeneity of OR across studies. A total of nine studies (eight case-control, one cohort) were included in the analysis. Five studies were done in Asian women living in Asian countries; one was conducted in women with Asian ancestry living in the West; and three were conducted in non-Asian populations. A great deal of variability was observed in the size of the OR and in the level of soy intake that was defined as high intake. A modest significant reduction in risk was associated with high soy intake over all studies [OR = 0.87; 95% confidence interval (CI): 0.80, 0.96]. However, this effect was confined to premenopausal women (OR = 0.80; 95% CI: 0.71, 0.90). There was no protective effect at all in postmenopausal women (OR = 1.01; 95% CI: 0.86, 1.19). There was also no significant effect of soy in women in Asia (OR = 0.95; 95% CI: 0.83, 1.09). Although there is some evidence of a small reduction in premenopausal breast cancer risk associated with soy intake, the number of studies is small, measurement of soy intake is crude and control of confounding factors is inconsistent. Interpretation of these results is further complicated by the similar reductions in risk associated with widely varying soy intakes and the low percentage of subjects consuming soy in studies of non-Asians. There was also a lack of consistency in the effects on hormonal measures observed in soy feeding studies. Coupled with the fact that some studies have suggested potentially adverse effects of soy, these data suggest that recommendations for women to increase their soy intake to prevent breast cancer or prevent its recurrence are premature and that larger, more rigorously controlled studies are required.

Premenopausal Equol Excretors Show Plasma Hormone Profiles Associated with Lowered Risk of Breast Cancer. A. M. Duncan, B. E. Merz-Demlow, X. Xu, W. R. Phipps* and M. S. Kurzer. Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN and *Department of Obstetrics-Gynecology, University of Rochester, Rochester, NY.

Increased urinary excretion of equol, a metabolite of the isoflavone daidzein, has been associated with lowered risk of breast cancer (Ingram et al. 1997 ). This risk reduction has generally been presumed to be a consequence of increased isoflavone consumption. However, only 30–40% of the population excretes more than trace amounts of equol regardless of isoflavone intake. Accordingly, we hypothesized that the observed protective effect of equol may be due to hormonal differences between equol excretors and nonexcretors. To evaluate the effects of equol status per se, we compared plasma hormone and binding globulin concentrations between premenopausal equol excretors (n = 5) and nonexcretors (n = 9) consuming identical isoflavone doses (10, 64 and 128 mg/d) as components of soy protein isolates for 3.5 menstrual cycles each. P < 0.05 was considered significant. Urinary equol for excretors far exceeded that of nonexcretors, even at the lowest dose. At all doses, equol excretors generally had lower concentrations of estrone, estrone sulfate, testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate and cortisol and higher concentrations of sex hormone–binding globulin and midluteal progesterone, a hormonal pattern generally consistent with lowered breast cancer risk. Thus, the association of lowered breast cancer risk with equol excretion may largely reflect the tendency of equol excretors to have more favorable hormonal profiles rather than merely reflecting increased isoflavone intake. Equol may be a marker for the presence of colonic bacterial enzyme activity that increases fecal steroid excretion. Alternatively, equol itself, even with very modest isoflavone intake, may exert beneficial effects on the regulation of endogenous hormones. [Supported by NIH grants CA-66016 and MO1-RR00400, and a gift from Protein Tech. International.]

Phytoestrogen Effect on Human Breast Epithelium. N. J. Bundred, D. F. Hargreaves,* W. R. Miller, M. Morton, I. McFadyen, A. Howell,** S. A. Roberts* and C. S. Potten.* Academic Department of Surgery, South Manchester University Hospital, Manchester, UK; *Paterson Institute of Cancer Research, Manchester, UK; Department of Surgery, Edinburgh Royal Infirmary, Edinburgh, Scotland; **Academic Department of Medical Oncology, Christie Hospital, Manchester, UK; and Tenovus Institute, Cardiff, Wales.

Epidemiologic studies suggest that phytoestrogen consumption prevents breast cancer, but the mechanism is unclear. To determine the effect of dietary soy protein supplementation on estrogenic- and androgenic-induced proteins in breast secretions, we conducted two randomized controlled trials of 14 d and 3 mo of treatment with either placebo or soy protein in premenopausal women. Levels of phytoestrogens rose in both serum (P 0.001 for genistein and daidzein) and nipple aspirate (genistein: 387 ± 433 ng/mL before treatment; 430 ± 430 ng/mL after 14 d of treatment) after supplementation. Nipple aspirate pS₂ (an estrogen-induced protein) rose and apolipoprotein D (apo D) fell after 14 d and 3 mo of supplementation (Table 1 ). Phytoestrogens exert an estrogenic stimulus on human breast as determined by increased secretion of estrogenic proteins.

Epidemiologic evidence suggesting a correlation between diets high in soybean and overall low cancer mortality rates, especially those of colon, breast and prostate, has given impetus to identifying components in soybeans responsible for their anticancer properties. We isolated a soybean cDNA enco