Phytoestrogens and Health

Phytoestrogens and Health Editors

G. Sarwar Gilani Health Canada Ottawa, Ontario, Canada

John J.B. Anderson University of North Carolina Chapel Hill, North Carolina

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Champaign, Illinois

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AOCS Mission Statement To be a forum for the exchange of ideas, information, and experience among those with a professional interest in the science and technology of fats, oils, and related substances in ways that promote personal excellence and provide high standards of quality. AOCS Books and Special Publications Committee G. Nelson, chairperson, University of California at Davis, WRRC, Davis, California R. Adlof, USDA, ARS, NCAUR, Peoria, Illinois J. Endres, The Endres Group, Fort Wayne, Indiana K. Fitzpatrick, Saskatchewan Nutraceutical Network, Saskatoon, Saskatchewan, Canada T. Foglia, USDA, ARS, ERRC, Wyndmoor, Pennsylvania L. Johnson, Iowa State University, Ames, Iowa H. Knapp, Deaconess Billings Clinic, Billings, Montana M. Mossoba, U.S. Food and Drug Administration, Washington, D.C. A. Sinclair, RMIT University, Melbourne, Victoria, Australia P. White, Iowa State University, Ames, Iowa R. Wilson, USDA, REE, ARS, NPS, CPPVS, Beltsville, Maryland Copyright © 2002 by AOCS Press. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the publisher. The paper used in this book is acid-free and falls within the guidelines established to ensure permanence and durability.

Library of Congress Cataloging-in-Publication Data Gilani, G. Sarwar. Phytoestrogens and health / G. Sarwar Gilani, John J.B. Anderson. p. cm. Includes bibliographical references and index. ISBN 1-893997-32-4 (alk. paper) 1. Phytoestrogens--Physiological effect. 2. Phytoestrogens--Health aspects. I. Anderson, John J. B. (John Joseph Baxter), 1934- II. Title. QP572.P48 G535 2002 615'.321--dc21 Printed in the United States of America with vegetable oil-based inks. 00 99 98 97 5 4 3 2 1

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2002026002 CIP

Preface The major goal of this volume on Phytoestrogens and Health is to provide current reviews on the benefits and disadvantages of these plant molecules for human health. Other subgoals include the showcasing of the diverse approaches being used by investigators who are studying the effects of these plant molecules. The explosion of knowledge about plant crop foods has arisen on two fronts, i.e., many researchers have finally begun to investigate the healthful properties of plant molecules, especially phytoestrogens, in mammalian species, and the food industry has tried to expand its marketing and sales of products based on the potential health benefits of the natural ingredients. This book focuses on the first point, namely, the scientific advancement of our understanding of plant molecules that have estrogenic actions. Although basic information is continually accruing about the phytoestrogens, an incomplete picture remains. This book captures the current status of our knowledge in the early 21st century on the following areas: the relationships of phytoestrogens and diseases, with their general health implications; mechanisms of action of the nonsteroidal molecules; and potential harmful effects. The most significant advances may be occurring at the molecular level, within cells of different tissues and organs, where the mechanisms of action of these molecules, especially soy-derived genistein and daidzein, are exerted. Future investigations will better characterize these cellular mechanisms. Less clear are the effects of soy isoflavones and other phytoestrogens on the prevention of chronic diseases, such as cardiovascular conditions, cancers, and osteoporosis. Ongoing studies, especially prospective randomized controlled trials, should provide new findings that yield clearer answers. Phytoestrogen research over the next few decades should provide increasing understanding of the benefits of the components of plant foods that have estrogen-like effects at doses that can be readily obtained by the consumption of foods in reasonable amounts. The use of phytoestrogen supplements at potentially much higher doses raises the possibilities of potential harmful effects from excessive consumption. The distinction between healthy intakes and excessive intakes must be determined. The risk of deleterious effects of phytoestrogens when ingested in high quantities remains an area of great concern to human investigators, industry, and regulatory agencies. Future research will help illuminate this thorny issue. Thanks are expressed to the authors for their up-to-date contributions, to Gary Nelson, Acquisitions Coordinator, and Chair of the AOCS Books and Special Publications Committee, for the invitation to undertake this worthwhile assignment, and to the publishing staff at AOCS for their gracious assistance in this endeavor.

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Also, gratitude is offered to the anonymous reviewers who helped greatly improve the chapters. Finally, a special thanks is offered to Mary Anthony of Wake Forest University who helped in the conception of this book. G. Sarwar Gilani Ottawa, Ontario, Canada John J.B. Anderson Chapel Hill, North Carolina, USA

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Contents

Chapter 1

Preface

Generic Comments on Isoflavones and Other Phytoestrogens Chapter 1 Brief Historical Overview of Isoflavone Research Mark Messina Chapter 2 Soy Isoflavones as Functional Ingredients in Women’s Health Clare M. Hasler and Susan Kundrat Chapter 3 Isoflavone Supplements: Arguments For and Against Their Use Mark Messina Chapter 4 Industrial Processing and Preparation of Isoflavones Eric T. Gugger

Food Sources and Composition of Phytoestrogens Chapter 5 Human Dietary Sources of Phytoestrogens and Methods of Determination Chung-Ja C. Jackson and H.P. Vasantha Rupasinghe Chapter 6 Tables of Isoflavone, Coumestan, and Lignan Data Chung-Ja C. Jackson and G. Sarwar Gilani

Measurement Methodology of Phytoestrogens in Blood and Tissues Chapter 7 Analysis of Phytoestrogens in Biological Samples by Mass Spectrometry Jeevan K. Prasain, Chao-Cheng Wang, and Stephen Barnes Chapter 8 Measurement Methodology for Phytoestrogens in Blood and Urine Mariko Uehara Chapter 9 Metabolism and Disposition of Genistein, the Principal Soy Isoflavone Daniel R. Doerge, Richard H. Luecke, and John F. Young

Digestion, Absorption and Metabolism of Isoflavones Chapter 10 Digestion, Absorption and Metabolism of Isoflavones Roger A. King

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Cellular Mechanism of Action, Including Estrogen Receptors Chapter 11

Cellular Mechanisms of Action Including Estrogen Receptors: ERα and β Sari Mäkelä and Jan-Åke Gustafsson

Chapter 12 Effects of Phytoestrogens on Bone Cells: Genomic and Nongenomic Mechanisms Xiaowei Chen and John J.B. Anderson

Cardiovascular Effects Chapter 13 Epidemiology of Soy Isoflavones and Cardiovascular Disease M.Z. Vitolins, M.S. Anthony, and G.L. Burke Chapter 14 Soy/Isoflavones and Risk Factors for Cardiovascular Disease Mary S. Anthony Chapter 15 Soy Proteins, Isoflavones, Cardiovascular Risk Factors, and Chronic Disease David J.A. Jenkins, Cyril W.C. Kendall, and Augustine Marchie Chapter 16 Lipoprotein Effects of Soybean Phytoestrogens Sandra R. Teixeira and John W. Erdman, Jr. Chapter 17 Effects of Free (Aglycone) Phytoestrogens and Metabolites on Cardiovascular Functions and Cancer Paul Nestel and Alan Husband

Skeletal Effects Chapter 18 Association Between Soy and/or Isoflavones and Bone: Evidence from Epidemiologic Studies Mary S. Anthony, John J.B. Anderson, and D. Lee Alekel Chapter 19 Skeletal Effects of Phytoestrogens in Humans: Bone Mineral Density and Bone Markers John J.B. Anderson and D. Lee Alekel Chapter 20 Skeletal Effects of Phytoestrogens: Rodent Models: Diet Bahram H. Arjmandi and Brenda J. Smith

Cancer Chapter 21 Phytoestrogens and Cancer: Epidemiologic Evidence Anna H. Wu Chapter 22 Flaxseed Lignans: Health Benefits, Bioavailability, and Safety Lilian U. Thompson and Wendy E. Ward

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Chapter 23 Phytoestrogens, Estrogens and Risk of Colon Cancer Maurice R. Bennink and Elizabeth A. Rondini Chapter 24 Phytoestrogen Actions in the Breast and Uterus Charles E. Wood, Stephen Barnes, and J. Mark Cline Chapter 25 Induction of Apoptosis by Genistein: Potential Applications in Cancer Prevention and Treatment Andreas I. Constantinou

Renal Effects Chapter 26 Phytoestrogens: Diabetic Nephropathy Tammy J. Stephenson and James W. Anderson

Premenopausal Hormone Effects Chapter 27 Hormonal Effects of Phytoestrogens in Premenopausal Women Alison M. Duncan, William R. Phipps, and Mindy S. Kurzer

Postmenopausal, Potential Alternative to Traditional HRT Chapter 28 Phytoestrogens: Effects on Menopausal Symptoms Fabien S. Dalais Chapter 29 Use of Soy Isoflavones as an Alternative to Traditional Hormone Replacement Therapy Mara Z. Vitolins, Mary S. Anthony, and Gregory L. Burke

Safety and Potential Toxicity Chapter 30 Deleterious Effects of Genistein Follow Exposure During Critical Stages of Development Retha R. Newbold, Wendy Jefferson, Elizabeth Padilla-Banks, and Bill Bullock Chapter 31 Evaluation of Phytoestrogen Safety and Toxicity in Rodent Models That Include Developmental Exposure Barry Delclos Chapter 32 The Health Consequences of Soy Infant Formula, Soy Protein Isolate, and Isoflavones Thomas M. Badger, Martin J.J. Ronis, Reza Hakkak, and Sohelia Korourian Chapter 33 Public Health Implications of Dietary Phytoestrogens Joel Rotstein, and G. Sarwar Gilani

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Chapter 1

Brief Historical Overview of Isoflavone Research Mark Messina Loma Linda University, Loma Linda, CA and Nutrition Matters, Incorporated, Seattle, WA

Introduction In 1954, Bradbury and White (1) identified 53 plants, and in 1975, Farnsworth and colleagues (2), 300 plants that possessed constituents with estrogenic activity. Not for another 15 years, however, did the word phytoestrogen emerge as part of nutrition jargon. That certain plants possess hormonal activity is not surprising because they have been used historically to enhance or reduce fertility (2). The dramatic rise in awareness of phytoestrogens can be attributed to several factors. Arguably, it is the intense interest in isoflavones that can be credited with being primarily responsible for bringing the concept of phytoestrogens into mainstream nutrition and even medical thinking. There are currently ~600 papers published on isoflavones annually, compared with just 12 in 1985 (based on Medline search). Those actively investigating the health effects of isoflavones include the U.S. Federal government; in fact, in 1999, the National Institutes of Health convened a 3-d workshop on this subject (3). In that same year, the United States Department of Agriculture (USDA) created an online database of the isoflavone content of foods (http://www.nal.usda.gov/fnic/). That soy foods are the only nutritionally relevant dietary source of these phytoestrogens has certainly heightened interest in isoflavones because soy foods have recently been the subject of considerable investigation (see Table 1.1). Although soybeans do contain numerous biologically active constituents (4), including phytic acid (5–8), phenolic acids (9), saponins (10–14), oligosaccharides (15,16), protease inhibitors (17,18), glyceollins (stressed soybeans only) (19–21), phytosterols (22,23), α-linolenic acid (24), vitamin E (25), and soy protein/peptides (26–34), unquestionably, it is the presence of isoflavones that is overwhelmingly responsible for the interest in soy. Research on the health effects of isoflavones has taken on added importance because soy foods are no longer the only means by which consumers can ingest these phytochemicals. The first isoflavone concentrate, soy germ, which is made from the hypocotyl portion of the soybean, became commercially available in 1996. One year later, the Archer Daniels Midland Company, Decatur, IL, released Novasoy, which is ~40% isoflavones by weight. Several other companies now produce isoflavone concentrates; all of these products are used as food fortificants and/or for the production of supplements.

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TABLE 1.1 Recent Events and Developments Highlighting Interest in Soy and Isoflavones Year

Event or Development

1994

First International Symposium on the Role of Soy in Preventing and Treating Chronic Disease held in Mesa, AZ. (Symposia were also held in 1996, 1999, and 2001.) Food and Drug Administration approves a health claim for the cholesterol-lowering properties of soy protein. United States Department of Agriculture (USDA) in conjunction with Iowa State University creates an online database of the isoflavone content of foods. The National Institutes of Health convenes a 3-d workshop on the health effects of isoflavones. American Heart Association recommends patients with elevated cholesterol include soy protein foods in their diet. USDA issues a ruling allowing soy protein (and other high quality proteins) to completely replace (previous guidelines limited soy to a 30% substitution) animal protein in the National School Lunch Program. USDA for the first time specifically lists calcium-fortified tofu and soy milk in the Dietary Guidelines as good sources of calcium.

1999 1999 1999 2000 2000

2000

The intent of this chapter is to give a brief overview from a conceptual perspective of research developments that have led to the current interest in isoflavones. The approach taken below is to trace the history of isoflavone research for each of the major areas under investigation. Not unexpectedly, research areas specifically related to chronic disease risk have a relatively short history.

Background on Isoflavones Isoflavones are a subclass of a larger and more ubiquitous group of nutraceuticals called flavonoids. In comparison to most flavonoids, isoflavones have a very limited distribution in the plant kingdom. Flavonoids are found in many plant foods such as onions, apples, and grapes, whereas soybeans are the only food to contain nutritionally relevant amounts of isoflavones. The primary isoflavones in soybeans are genistein (4′5,7-trihydroxyisoflavone) and daidzein (4′,7-dihydroxyisoflavone), and their respective β-glycosides, genistin and daidzin. Typically, more genist(e)in exists in soybeans and soy foods than daidz(e)in (35). There are also small amounts of a third isoflavone in soybeans, glycitein (7,4′-dihydroxy-6-methoxyisoflavone) and its glycoside, glycitin. In soybeans and nonfermented soy foods, isoflavones are present primarily as β-glucosides, esterified with malonic or acetic acid (36). In fermented soy products such as tempeh and miso, due to microorganism-induced fermentation and hydrolysis, more of the isoflavones are present in aglycone (unconjugated) form. Isoflavones, like many phytochemicals of interest to nutritionists, are phytoalexins, substances formed by the host tissue in response to physiologic stimuli, infectious agents, or their products, which accumulate to levels that inhibit the

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growth of microorganisms (37). Isoflavones possess properties (e.g., antifungal, antimicrobial, and antioxidant) that enhance the survival of the soybean (37). For this reason, soybean isoflavone concentrations increase greatly in times of stress, such as when moisture is limited, and are influenced by the environmental conditions under which the soybean is grown (38,39). In contrast to many phytoallexins, however, isoflavones are always present in significant quantities in soybeans, because one of their primary functions is to stimulate nodulation genes in soil bacteria called Rhizobium. Rhizobia have the ability to induce the formation of structures called nodules on legume (including soybean) roots (40). The rhizoba within these nodules reduce atmospheric nitrogen to ammonia, which the soybean can then use as a source of nitrogen for growth. Farmers have made extensive use of this property of soybeans in crop rotation to naturally restore nitrogen to their fields.

Identification of Isoflavones Genistein was first isolated in 1899 from Dyer’s Broom (Genista tinctoria) (41) and was chemically synthesized in 1928 (42) (see Table 1.2 for review of key research discoveries). The isoflavone glycosides, genistin and daidzin were isolated from soybeans by Walz in 1931 (43) and then 10 years later by Walter (44). Three decades passed before Naim et al. (45) identified the third isoflavone in soyTABLE 1.2 Important Isoflavone-Related Research Discoveries: 1899–1990s Year

Research

1899 1928 1931/41 1932 1946

Genistein isolated from Dyer’s Broom (Genista tinctoria) Genistein chemically synthesized Genistein and daidzein isolated from soybeans Equol identified in the urine of pregnant mares Breeding problems in sheep in Western Australia grazing on Trifolium subterraneum, leads to isoflavone research especially related to reproductive effects Genistein shown to be estrogenic in young rodents Isoflavones shown to exert antioxidant effects Genistein shown to exert antiestrogenic effects in young rodents Equol established as a bacterial metabolite of daidzein Glycitein identified in soybeans Isoflavones shown to be hypocholesterolemic Equol identified in human urine Daidzein identified in human urine Urinary equol levels increase as much as 1000-fold in humans fed soy Isoflavones hypothesized to account for the hypocholesterolemic effects of soy Genistein inhibits tyrosine protein kinase activity in vitro Isoflavones hypothesized to account for the hypocholesterolemic effects of soy Relative binding affinities of isoflavones greater for estrogen-receptor-α than estrogen receptor-β

1953 1964 1966 1968 1973 1976 1982 1984 1984 1985 1987 1995 1997

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beans, glycitein, in 1973. In 1939, Okano and Beppu reported the presence of other isoflavones in soybeans, which they named tatoin (8-methyl-5,4′-dioxyisoflavon), methylgenistein, isogenistin (5,7,2′-trioxyisoflavon) and methylisogenistin (46). In 1964, György et al. identified 6,7,4′-trihyroxyisoflavone from the fermented soybean product, tempeh (47). In 1932, the isoflavonoid equol, which is a bacterial metabolite of daidzein, was identified in the urine of pregnant mares, hence the basis for the name (48). Approximately three decades later it was found in the urine of goats (49), cows (50), hens (51,52), and sheep (53,54). In 1982, equol was serendipitously found in the urine of rats during the search to better understand the biosynthetic pathways and origins of the lignans, enterolactone and enterodiol (55). That same year equol (56), and 2 years later, daidzein (57), were identified in human urine. Equol was established as a bacterial metabolite of daidzein in 1968 (54). In 1981, Axelson et al. (55) showed that equol was absent in the urine of germ-free rats and also when the typical rat chow (soy-containing) diet was replaced by purified (soyfree) diets, but was present in urine when soy meal alone was added to a purified diet (58). In 1984, two landmark papers showed that the consumption of soy by women resulted in a 100- to 1000-fold increase in urinary levels of equol (58,59). The phytoestrogen, coumestrol, which is a coumestan rather than an isoflavone, was first identified in soy in 1964 by Wada and Yuhara, a finding later confirmed by Knuckles et al. in 1976 (60) and Lockhart et al. 2 years later (61). These last-mentioned investigators also showed that the coumestrol content of soy increases upon germination (62). However, the minute amounts of coumestrol in soybeans are almost certainly physiologically irrelevant (63).

Early Research on the Biological Properties of Isoflavones Reproductive Effects Research initiated during the 1940s on the breeding problems experienced by female sheep in Western Australia led to much investigation of isoflavones (64). Sheep infertility stemmed from the development of cystic endometrium (clover disease); this was attributed to the consumption of Trifolium subterraneum, a type of clover rich in isoflavones, which led to extremely high serum equol levels (1,65). Throughout the 1950s and 1960s, the reproductive effects of isoflavones were the subject of considerable investigation (66-69). Rising concerns over the synthetic estrogen, diethylstilbesterol (DES), helped to fuel interest in this area (70). In 1950, Kendall et al. (71) reported a range of reproductive abnormalities in New Zealand rabbits fed a diet comprised of nearly 50% soybean hay. However, no attempt was made to identify the specific factors responsible for these effects, and the study design did not preclude the possibility that simple nutrient deficiencies were the cause. Nevertheless, the results with soybean hay, in combination with the sheep breeding problems in Western Australia and the known estrogenic effects of isoflavones, led Carter et al. (68) to test the effects of genistin on repro-

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duction in female Swiss albino mice. They found that in mice fed diets to which genistin (0.2%) had been added, the percentage of females dropping litter was decreased relative to the control group. However, no changes were noted in mice fed diets comprised primarily of soybean meal that contained 0.1% genistin. By comparison, the Japanese diet is ~0.005% genistein (72,73). Reproductive issues continued to be cause for concern throughout the 1980s. In 1984, pioneering isoflavone researcher Kenneth D.R. Setchell stated “... repeated soya consumption in man may result in reproductive disorders due to the estrogenic effects of equol or other phytoestrogens, similar to its action in animals...” (59). Three years later, Setchell et al. (74) attributed the inability of the captive cheetah to reproduce to the presence of soy meal in the cheetah’s diet. There is, however, much species variation in response to biologically active compounds; both sheep and cheetahs (the latter because of a reduced hepatic β-glucuronyl transferase activity, an enzyme involved in isoflavone conjugation) are particularly sensitive to the reproductive effects of isoflavones (65,75). The reproductive effects of isoflavones continue to be investigated rigorously (76–79). However, it is now well established that the rodent chow diets used by animal breeders, which clearly lead to normal reproduction, contain large amounts of isoflavone-rich soy meal and produce serum isoflavone levels as high as those found in people who consume soy foods (80). However, although reproduction appears normal, this high level of isoflavones in commercial chow has raised concerns that the resulting estrogenic effect might represent a confounding variable in experimental outcomes (80,81). Drane et al. (82) first raised this issue in 1975 and in 1980, they established that soy was responsible for the estrogenic effects of rat chow (83). However, it was Murphy et al. in 1982 (84) who first quantified the isoflavone content of different soy-based animal diets. Although effects on reproduction dominated much of the research during the early 1950s, the estrogenic effects of isoflavones observed in sheep in the 1940s, in combination with the knowledge that DES stimulated weight gain and feed efficiency in cattle when given during the fattening period, led Cheng and colleagues (85) from the Iowa Agricultural Experiment Station in 1953 to investigate the potential role of isoflavones in livestock feeding because soybean meal was and continues to be a major source of animal nutrition. They demonstrated that genistein stimulated uterine weight in immature mice and concluded that the amount of isoflavones found in soybean meal and certain hays was sufficiently large to exert beneficial influences. These initial findings led numerous groups over the next 10 years to investigate the estrogenic effects of soybean meal, daidzein and genistein, and the methylated isoflavones, biochanin-A and formononetin, which are found in T. subterraneum (86–90). Antiestrogenic Effects By the early 1950s, isoflavones were established as weak estrogens on the basis of their ability to stimulate uterine weight in young rodents (85,87). In 1966, Folman

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and Pope (91) were the first to conduct assays establishing the relative binding affinities of the soybean isoflavones for the estrogen receptor. Ironically, they were also the first to focus on the possible antiestrogenic effects of isoflavones (91). They demonstrated that in female mice, subcutaneously injected genistein inhibited estrone stimulation of uterine growth (91). Three years later, they confirmed their initial findings showing that when genistein was injected along with estradiol, estradiol uptake by the mouse uterus and vagina was substantially decreased (92). They concluded that the importance of genistein “might lie as much in its ability to antagonize the natural steroid estrogens as in its own estrogenic activity” (91). In hindsight, this can be seen as a rather prophetic statement but one that was largely ignored for another 20 years. Certainly, there was no rush to conclude that soy intake might contribute to the low breast cancer incidence in Japan even though estrogen was already suspected at that time of being a causative agent in the etiology of breast cancer and soy was recognized as an important part of the Japanese diet (93). This is not to suggest, however, that the antiestrogenic nature of isoflavones was not investigated. In 1967, Shutt (94) reported that in ovariectomized Sydney White strain mice administered estradiol, subcutaneously injected genistein decreased the amount of assayable estrogenic activity in the reproductive tract compared with estradiol alone. Consistent with these findings, in 1980, Newsome and Kits (95) found that there was a decrease in rat uterine cytoplasmic estradiol receptors 40 h after injection of genistein compared with estradiol injection alone. In that same year, injections of equol and 17β-estradiol in combination induced uterine growth in immature female Wistar rats, which was intermediate between the effects of injections of these two agents administered individually (96). Furthermore, the equol/estrogen receptor complex was shown to compete with the estradiol receptor complex for nuclear binding, but not to initiate the replenishment of estrogen receptors effectively in the cytoplasm (96). In 1978, Martin et al. (97) found that in MCF-7 cells, genistein is processed in the nucleus at about the same rate as the estradiol-bound receptor but is less effective than estradiol in translocating the cytoplasmic estradiol receptor to the nucleus. Interestingly, however, these researchers can be credited with being among the first to suggest that the phytoestrogens might actually stimulate the growth of breast tumors. Unquestionably, of all research areas related to isoflavones, this issue remains as one of the most controversial (98–103). The antiestrogenic effects of isoflavones continue to be widely investigated, especially in regard to breast tissue. Although there are no definitive data, proposed mechanisms for antiestrogenic effects include competitive binding (97,104), downregulation of estrogen receptors (105), an increase in serum levels of sex hormone-binding globulin (SHBG) (106), alteration of the metabolism of estrogen (107,108), a decrease in estrogen synthesis (109), inhibition of estrogen receptor phosphorylation (110), and inhibition of the postreceptor effects of estrogen (111).

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Antioxidants In 1964, György et al. (47) were the first to demonstrate that soybeans possessed antioxidant activity, a finding later confirmed by Ikehata et al. (112) in 1968 and Pratt in 1972 (113). In 1976, Naim et al. (114) systematically studied the antioxidant effects of all three soybean isoflavones as well as the methylated isoflavones found in red clover. Three years later, Pratt and Birac (115) demonstrated that the isoflavones were largely responsible for the antioxidant activity of soybeans. It should be noted, however, that it was recognized early on that there were nonisoflavone antioxidants in soybeans (116). Today, the antioxidant effects of isoflavones are widely studied in a variety of experimental systems (117-125). A notable very recent finding is the reduction in 5-hydroxymethyl-2′-deoxyuridine (5-OHmdU) levels a measure of oxidative damage in DNA from nucleated blood cells in both men and women consuming isoflavone supplements (126).

Isoflavone Databases In 1999, the USDA, in conjunction with Iowa State University, created an online database of the isoflavone content of foods (http://www.nal.usda.gov/fnic/). The lead investigator of this study, Patricia A. Murphy from Iowa State University (35), can be credited in 1982 with publishing the first survey of the isoflavone content of soy foods (127). Of course, numerous groups over the years have made important contributions to understanding of the isoflavone content of soy products (39,73,128–136)

Chronic Disease-Related Research Coronary Heart Disease (CHD) Cholesterol Reduction. Investigation of the hypocholesterolemic effects of soy protein has been underway for >60 years in animals (137,138) and 35 years in humans (139). However, this extensive body of literature was largely ignored until the publication of a meta-analysis in 1995, which summarized the results of nearly 40 clinical trials involving soy (140). The meta-analysis did not directly evaluate the health effects of isoflavones, but helped to draw attention to isoflavones because the authors of the analysis popularized the notion that isoflavones enhanced the cholesterol-lowering effects of soy protein, citing recent work in monkeys in support of this hypothesis (141). However, 10 years earlier, Setchell had suggested that isoflavones, like estrogen, might be responsible for the effects of soy on serum cholesterol levels (142). But it was Indian researchers who first proposed that isoflavones lowered serum cholesterol (143–145). Their initial speculation was based on the cholesterol-lowering effects of Cicer arietinum (Bengal gram or chick pea), which is known to contain isoflavones (146,147). In 1979, Sharma showed that in rats,

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biochanin-A and formononetin, which were first isolated from Bengal gram in 1945 (143), lowered cholesterol (145). Currently, the U.S. Food and Drug Administration does not require that soy protein contain a certain level of isoflavones to qualify for the health claim (148). The notion that isoflavones enhance the effects of soy protein is controversial because there is evidence both for (149–151) and against (152,153) this hypothesis. There is little evidence to suggest that soybean isoflavones independently lower cholesterol in humans although definitive data are yet to be published (154,155). Some data suggest that isoflavones may raise high density lipoprotein (HDL)-cholesterol levels but this is quite speculative (156). Low Density Lipoprotein Cholesterol (LDL-C) Oxidation. The effects of isoflavones on CHD risk factors other than cholesterol reduction have only recently been examined. This is not surprising given that LDL-C oxidation was not recognized as a CHD risk factor (157) until 1989. A year later, estrogen was shown to inhibit LDL-C oxidation in vitro (158). In 1993, Kanazawa et al. (159,160) published the first demonstration that soy reduced LDL-C oxidation in vivo. Tikkanen et al. (161) and Jenkins et al. (162,163) confirmed these findings in 1998 and 2000, respectively. However, these studies did not attempt to identify the antioxidant(s) in soy responsible for this effect. In 1996, isoflavones and their metabolites were first shown to inhibit cholesterol oxidation in vitro (122), a finding confirmed 1 year later (124). Furthermore, studies in animals (164,165) and humans (120) have found that isoflavone-rich soy protein (soy+) inhibits LDL-C oxidation in vivo compared with soy protein low or devoid of isoflavones (soy–). However, three human studies did not find that isolated isoflavones inhibit cholesterol oxidation (139,166,167). Additionally, Djuric et al. (126) did not find that isoflavone supplements affected serum levels of 8-isoprostanes in men or women even though Wiseman et al. (120) found that in human subjects, soy+ significantly reduced isoprostanes levels compared with soy–. However, thus far, the antioxidant effects of isolated isoflavones have not been compared directly with soy in human subjects. Other CHD Risk Factors. Isoflavones are thought to affect a number of biological measures of CHD risk in a favorable manner. Ni et al. (168) found that in apolipoprotein E-deficient rats fed soy+ or casein plus isoflavones, the development of atherosclerosis was reduced compared with rats fed soy– or casein. Also, the atherosclerotic lesion area of the aortic arch was significantly lower in rabbits given isoflavones compared with control rabbits and rabbits given saponins (169). These effects are not surprising given the estrogen-like effects of isoflavones, and the many proposed mechanisms by which estrogen reduces CHD risk (170). The most established effect of isoflavones is the enhancement of systemic arterial compliance (SAC), which was first demonstrated in 1997 (167,171). SAC,

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which is an indicator of vascular elasticity, is considered by some to be an independent measure of CHD risk (172,173). There are, however, conflicting data concerning the effect of isoflavones on endothelium-dependent vascular response, which was first studied in animals in 1997 (174) and then later by several investigators in humans (155,166,167,175–177). Since 1995, as more has been learned about the myriad biological effects of isoflavones and the number of established risk factors for CHD increased, investigation of the relationship between isoflavones and these risk factors increased at an impressive pace. There are at least preliminary data suggesting that isoflavones favorably affect a number of biological processes related to CHD risk, including nitric oxide synthase activity (178), arterial lipid oxidation (165,169), the migration and proliferation of smooth muscle cells (a primary cell type found in arterial plaque) (179–182), platelet aggregation (183–185), platelet serotonin uptake (186), and blood pressure regulation (187,188). Signal Transduction and Cancer Animal research on the anticancer effects of soy has been underway for 20 years, but early studies focused on the protease inhibitors, not isoflavones (189). Pioneering work by Adlercreutz et al. (190) in the early 1980s brought attention to the possible anticancer effects of phytoestrogens, although their focus was more on lignans than isoflavones. Throughout the 1980s, Adlercreutz continued to write about the possible role of both lignans and isoflavones in reducing breast cancer risk, focusing in particular on their antiestrogenic properties (191,192). A historical review of research on the antiestrogenic effects of isoflavones has already been presented; thus no further discussion will be cited here except to again state that Folman and Pope (91) first suggested in 1966 that the antiestrogenic effects of isoflavones may be important. Nearly 20 years later, Setchell began to popularize the notion that isoflavones may be beneficial for breast cancer (59). His collaboration with Stephen Barnes led to the first animal work, published in 1990, suggesting that isoflavones might inhibit mammary tumor development (193,194). The importance of this one animal study cannot be understated because it led to the convening of a workshop by the U.S. National Cancer Institute (NCI) in 1990 on the potential role of soy in reducing cancer risk, which in turn, led to the NCI in 1991 issuing a request for applications (RFA) totaling nearly $3 million for study of the anticancer effects of soy (195). All four grants awarded in response to this RFA focused on isoflavones. The effects of this RFA were far reaching for two reasons; first, it represented acknowledgement by a U.S. federal agency that isoflavones were possible anticarcinogens and second, the availability of funding led to a new pool of investigators interested in conducting research in this area. For cancer, however, it was clearly not the hormonal effects of isoflavones but the nonhormonal effects, especially of genistein, that really first drew attention to the potential anticarcinogenicity of isoflavones. Because the nonhormonal anticancer effects of isoflavones are currently the subject of so much research,

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Akiyama et al. (196) can be credited with having written one of, if not the seminal paper published in this field within the past 15 years. In 1987, they serendipitously discovered that genistein was a specific inhibitor of tyrosine protein kinase (196– 198), an enzyme frequently overexpressed in cancer cells (199). As a result, it immediately became clear that isoflavones could no longer be viewed simply as phytoestrogens. Parenthetically, the nonhormonal effects are also largely responsible for the myriad biological effects of isoflavones, accounting for their possible beneficial roles in diseases as diverse as malaria (200), cystic fibrosis (201), and alcoholism (202). The discovery by Akiyama and colleagues led to the widespread use of genistein as a tool for identifying whether certain cellular processes are under the control of a variety of kinases, which in turn led to research showing that genistein affects a number of enzymes involved in steroid metabolism and influences multiple molecules that modulate cell death and survival (203–208). These effects explain why genistein inhibits the growth of a wide range of both hormone-dependent and -independent cancer cells in vitro (209–211) and why interest in the anticancer effects of soy in general and isoflavones in particular is not limited to breast and prostate cancer, although the focus remains on these two cancers (212,213). In 1994, a comprehensive review of the interrelationships of soy, iso-flavones, and cancer risk published by Messina et al. (210) helped to draw attention to this field. The anticancer effects of isoflavones continue to be the subject of vigorous investigation. Table 1.3 summarizes the major developments and discoveries in this area of research. In 1996, the Chemoprevention Branch of the NCI released its clinical development plan for genistein (214). Two years later, in a comprehensive review of chemopreventive agents, the Chemoprevention Branch in conjunction TABLE 1.3 Isoflavones and Cancer Risk: Important Developments and Research Discoveries Year

Development or Discovery

1966 1982 1984 1987 1990 1990 1991 1993 1993 1994 1994 1995 1998 1999

Isoflavones exert antiestrogenic effects in young rodents Urinary equol levels in healthy women and breast cancer patients examined Isoflavones proposed as possibly preventing breast cancer Genistein inhibits tyrosine protein kinase activity in vitro Soy inhibits chemically-induced mammary tumor development National Cancer Institute (NCI) sponsors workshop on soy NCI allocates funds for isoflavone research Genistein inhibits angiogenesis in vitro Topically applied genistein inhibits mouse skin carcinogenesis Comprehensive review published on soy, isoflavones, and cancer risk Soy extends menstrual cycle length Early genistein exposure reduces mammary tumor development Genistein downregulates epidermal growth factor receptor in rat prostate Isoflavones inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice

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with researchers from the University of Illinois at Chicago concluded that of the 25 agents tested, genistein was one of four chemopreventive agents considered to be superior (215). Wei et al. (119,216) published the first animal study demonstrating the anticancer effects of isolated isoflavones, although in this case genistein was applied topically. Later studies showed isoflavones administered orally and by injection inhibit bladder (217), mammary (218), prostate (219), and lung cancer (220). Although the anticancer effects of isoflavones remain speculative and there are literally hundreds of relevant publications in this area, two papers in particular are worthy of special mention; one deals with breast cancer and the other, with prostate cancer. In 1995, the research group at the University of Alabama headed by Coral Lamartiniere, hypothesized and demonstrated that perinatal exposure to genistein markedly reduces chemically induced mammary cancer in adult animals (221). Numerous publications (222–224) by this group confirm this initial finding and importantly, there is now epidemiologic support for this hypothesis (225). This group can also be credited with being the first to demonstrate that genistein affects signal transduction (downregulation of epidermal growth factor receptor in the rat prostate) in vivo and that the anticancer effects of this isoflavone may be more potent in vivo than in vitro (226). Osteoporosis The role of estrogen in bone health has been definitively established for decades (227) and the potential value of ipriflavone, a synthetic isoflavone, as an osteoporosis drug has been investigated for >15 years (228,229). Thus, there was ample basis for speculating about the benefits of isoflavones in bones. In 1996, the first study demonstrating that isolated isoflavones favorably affect bone mineral density (BMD) in ovariectomized rodents was published (230). That same year, Arjmandi et al. (231) demonstrated that isoflavone-rich soy protein improved BMD in ovariectomized rats. Interestingly, in the former study, Blair et al. (230) attributed the bone protective effects of genistein to the ability of this isoflavone to inhibit tyrosine protein kinase activity, not to its estrogenic properties. Animal studies showing favorable effects of soy on BMD had been published before 1996, but the focus of these studies was on the protein, not isoflavones (232,233). In 1988, Breslau et al. (28), utilizing 12-d dietary periods, found that compared with animal protein, soy protein significantly reduced urinary calcium excretion in humans. Earlier acute human studies had demonstrated similar effects (29,234,235). The decrease in urinary calcium excretion is generally attributed to the lower sulfur amino acid content of soy protein and the resulting reduction in acid ash (236). The year 1998 represents an important year in the short history of isoflavones in bone research. In that year, Ishida et al. (237) established that both genistin and daidzin (the latter in a dose-dependent manner) reduced ovariectomized-induced bone loss in rodents essentially as effectively as estrone. Also, the first human study showing that isoflavones might favorably affect bone health was published. Potter et

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al. (238) found that there was a statistically significant increase in lumbar spine BMD in postmenopausal women consuming 40 g of soy protein/d (90 mg of isoflavones) for 6 mo, whereas there were decreases in BMD in the women fed 40 g of soy protein containing a lesser amount of isoflavones (56 mg) or 40 g of casein-based milk protein. Several human studies have been published since 1998 on the effects of isoflavones on bone health but have produced rather inconsistent results (239–242). Hormonal Effects and Menopausal Symptom Relief It is now commonplace to examine the effects of soy on serum sex hormone levels, although studies in this area have produced very inconsistent findings (107,243– 246). As noted previously, early on, Adlercreutz (192) and Setchell (142) expressed considerable interest in the hormonal effects of phytoestrogens. Adlecreutz (247), on the basis of epidemiologic correlations and then later in vitro data (248), proposed that the phytoestrogens increased serum SHBG levels. The first clinical investigation to look at sex hormone levels was published in 1990 (249); however, it was the finding of Cassidy et al. (250,251) that soy feeding increased the length of the menstrual cycle specifically as a result of an increase in the follicular phase that created considerable excitement about the hormonal and anticancer effects of isoflavones because longer menstrual cycles have been associated with lower breast cancer risk (252,253). Overall however, the effect of soy on menstrual cycle length is unclear (109, 254,255). Generally, clinical studies do not suggest that isoflavones have pronounced hormonal effects (256–258), although one hypothesis is that isoflavones favorably affect estrogen metabolism (107,259). In addition, Kurzer (260) noted that although many studies fail to find significant effects of soy on serum estrogen levels, most studies show decreases. One proposed benefit of isoflavones that has garnered considerable attention from both the public and clinical communities is the alleviation of menopausal symptoms. In 1992, Adlercreutz et al. (261) first suggested that isoflavones were beneficial in this regard although at the time, clinical studies were not available. This suggestion was based largely on the low incidence of hot flashes among women in Japan, information that was popularized by Lock et al. (262,263). The first study to actually test this hypothesis was published by Murkies et al. in 1995 (264). There have been ~15 clinical studies published, but the results are inconsistent and suggest that, at most, soy or isoflavones have very modest benefits (265,266). Cognition Only recently has the effect of diet on cognitive function and risk of developing Alzheimer’s disease begun to be investigated seriously. Not surprisingly, given the interest in estrogen in this regard, isoflavones have also been the subject of speculation. The first animal study to examine this issue was published in 1999 by Pan et al. (267). Since then, several animal and human studies have been published

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(268–272). However, it was an abstract published in 1996 that caused considerable concern about the possible detrimental effects of soy, and isoflavones in particular, on cognitive function (273). This abstract focused on the results of a prospective epidemiologic study, published in full manuscript form in 2000, that found that tofu consumption was associated with impaired cognitive function in Japanese men and women residing in Hawaii (274). This finding contrasts with the findings from animal studies and two short-term human intervention studies that have recently been published (273,275). At this point, the evidence is too preliminary to draw conclusions about the relationship between soy and cognitive function, especially when considering that the effects of estrogen on cognitive function are unclear (276). Alternatives to Conventional Hormone Replacement Therapy Specific discussion of isoflavones as alternatives to conventional hormone replacement (HRT) began around 1995. This is not surprising because only recently has the need to identify alternatives to HRT been recognized. The research group at Wake Forest University headed by Dr. Thomas Clarkson can be credited with highlighting the role of soy as a possible alternative to HRT (277). In the mid 1990s, Dr. Clarkson wrote to the National Heart, Lung, and Blood Institute, of the NIH: “The rationale for choosing soybean estrogens as a potential nutritional alternative to the current standard conjugated equine estrogens therapy is based on the protective effect of those compounds against the development of breast cancer, the likely lack of a harmful effect on the uterus, and an experimental basis for assuming probable favorable effects on coronary artery atherosclerosis and osteoporosis.” Of course, it is now commonplace to compare the health effects of soy, and especially the isoflavones, with HRT. Obviously, if isoflavones duplicated all of the effects of estrogen, they would not be a credible alternative. The search for alternatives to HRT has led the pharmaceutical industry to develop selective estrogen receptor modulators (SERM). SERMs, such as tamoxifen and raloxifene, have estrogenic effects in some tissues but either no effects or antiestrogenic effects in others (278). There is much discussion about whether isoflavones fall into this category (279,280). The identification of a second estrogen receptor in 1996, referred to as estrogen receptor-β (281–283), and the finding that isoflavones bind with much greater affinity to this receptor than estrogen receptor-α (283–285), can be credited with spurring research in this area and with isoflavones increasingly being viewed not only as phytoestrogens but as phyto-SERMs and as phytochemicals important for the health of women, especially postmenopausal women.

Summary Modern research on isoflavones began in the late 1950s. Early work focused on the possible detrimental effects of isoflavones on reproduction because of their weak

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estrogenic properties. In the 1960s, research demonstrated that isoflavones possessed antioxidant activity and were possible antiestrogens, although the latter finding was largely ignored. The number of publications on isoflavones clearly indicates that this area of research did not attract widespread attention until ~1990. Unarguably, more than any other publication, the finding by Akiyama et al. that genistein inhibited tyrosine protein kinase in vitro can be credited with spurring isoflavone research. Funding of isoflavone research by NCI in 1991 also represents a landmark development in this field. Throughout the last decade of the 20th century, isoflavone research was conducted at a phenomenal pace. Aside from the possible anticancer effects, researchers were intrigued by the estrogen-like properties of isoflavones and the role isoflavones might have in promoting bone health, reducing risk of CHD, and alleviating menopausal symptoms. The need for alternatives to conventional HRT further highlighted the potential role of isoflavones in women’s health. The focus on women’s health was accentuated by the finding that isoflavones bind with much greater affinity to estrogen receptor-β than estrogen receptor-α. Naturally, this also led to isoflavones beginning to be viewed as phyto-SERMs. It is hard not to be impressed with the amount of research now being conducted on isoflavones in comparison to just a decade ago. But despite the progress that has been made, it is humbling to recognize the extent to which issues raised 20–40 years ago remain unresolved today. A clear understanding of the antiestrogenic, antioxidant, and hypocholesterolemic effects of isoflavones remains elusive. Fortunately, the pace at which isoflavone research is occurring suggests clarity on these issues will come sooner rather than later. References 1. Bradbury, R.B., and White, D.R. (1954) Estrogen and Related Substances in Plants, in Vitamins and Hormones (Harris, R.S., Marrian, G.F., and Thimann, K.V., eds.) Academic Press, New York. 2. Farnsworth, N.R., Bingel, A.S., Cordell, G.A., Crane, F.A., and Fong, H.S. (1975) Potential Value of Plants as Sources of New Antifertility Agents II, J. Pharm. Sci. 64, 717–754. 3. Lu, L.J., Tice, J.A., and Bellino, F.L. (2001) Phytoestrogens and Healthy Aging: Gaps in Knowledge. A Workshop Report, Menopause 8, 157-170. 4. Messina, M., and Barnes, S. (1991) The Role of Soy Products in Reducing Risk of Cancer, J. Natl. Cancer Inst. 83, 541–546. 5. Henn, R.L., and Netto, F.M. (1998) Biochemical Characterization and Enzymatic Hydrolysis of Different Commercial Soybean Protein Isolates, J. Agric. Food Chem. 46, 3009–3015. 6. Harland, B.F., and Oberleas, D. (1987) Phytate in Foods, World Rev. Nutr. Diet. 52, 235–259. 7. Graf, E., and Eaton, J.W. (1990) Antioxidant Functions of Phytic Acid, Free Radic. Biol. Med. 8, 61–69. 8. Graf, E., and Eaton, J.W. (1993) Suppression of Colonic Cancer by Dietary Phytic Acid, Nutr. Cancer 19, 11–19.

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9. Ramakrishna, M.B.V., Mital, B.K., Gupta, K.C., and Sand, N.K. (1989) Determination of Phenolic Acids in Different Soybean Varieties by Reversed Phase High Performance Liquid Chromatography, J. Food Sci. Technol. 26, 154–155. 10. Tsukamoto, C., Shimada, S., Igita, K., Kudou, S., Kokubun, M., Okubo, K., and Kitamura, K. (1995) Factors Affecting Isoflavone Content in Soybean Seeds: Changes in Isoflavones, Saponins, and Composition of Fatty Acids at Different Temperatures During Seed Development, J. Agric. Food Chem. 43, 1184–1192. 11. Shiraiwa, M., Kudo, S., Shimoyamada, M., Harada, K., and Okubo, K. (1991) Composition and Structure of “Group A Saponin” in Soybean Seed, Agric. Biol. Chem. 55, 315–322. 12. Shiraiwa, M., Harada, K., and Okubo, K. (1991) Composition and Structure of “Group B Saponin” in Soybean Seed, Agric. Biol. Chem. 55, 911–917. 13. Oakenfull, D. (1981) Saponins in Food—A Review, Food Chem. 6, 19–40. 14. Rao, A.V., and Sung, M.K. (1995) Saponins as Anticarcinogens, J. Nutr. 125, 717S– 724S. 15. Hata, Y., Yamamoto, M., and Nakajima, K. (1991) Effects of Soybean Oligosaccharides on Human Digestive Organs: Estimate of Fifty Percent Effective Dose and Maximum Non-Effective Dose Based on Diarrhea, J. Clin. Biochem. Nutr. 10, 135–144. 16. Kuo, T.M., VanMiddlesworth, J.F., and Wolf, W.J. (1988) Content of Raffinose Oligosaccharides and Sucrose in Various Plant Seeds, J. Agric. Food Chem. 36, 32–36. 17. Kennedy, A.R. (1998) Chemopreventive Agents: Protease Inhibitors, Pharmacol. Ther. 78, 167–209. 18. Bierman, B.J., de Banzle, J.S., Handelsman, J., Thompson, J.F., and Madison, J.T. (1998) Methionine and Sulfate Increase a Bowman-Birk Type Protease Inhibitor and Its Messenger RNA in Soybeans, J. Agric. Food Chem. 46, 2858–2662. 19. Burow, M.E., Boue, S.M., Collins-Burow, B.M., Melnik, L.I., Duong, B.N., CarterWientjes, C.H., Li, S., Wiese, T.E., Cleveland, T.E., and McLachlan, J.A. (2001) Phytochemical Glyceollins, Isolated from Soy, Mediate Antihormonal Effects Through Estrogen Receptor Alpha and Beta, J. Clin. Endocrinol. Metab. 86, 1750–1758. 20. Graham, T.L., Kim, J.E., and Graham, M.Y. (1990) Role of Constitutive Isoflavone Conjugates in the Accumulation of Glyceollin in Soybean Infected with Phytophthora megasperma, Mol. Plant-Microbe Interact. 3, 157–166. 21. Graham, T.L., and Graham, M.Y. (1991) Glyceollin Elicitors Induce Major but Distinctly Different Shifts in Isoflavonoid Metabolism in Proximal and Distal Soybean Cell Populations, Mol. Plant-Microbe Interact. 4, 60–68. 22. Rao, A.V., and Janezic, S.A. (1992) The Role of Dietary Phytosterols in Colon Carcinogenesis, Nutr. Cancer 18, 43–52. 23. Weihrauch, J.L., and Gardner, J.M. (1978) Sterol Content of Foods of Plant Origin, J. Am. Diet. Assoc. 73, 39–47. 24. Klein, V., Chajes, V., Germain, E., Schulgen, G., Pinault, M., Malvy, D., Lefrancq, T., Fignon, A., Le Floch, O., Lhuillery, C., and Bougnoux, P. (2000) Low AlphaLinolenic Acid Content of Adipose Breast Tissue Is Associated with an Increased Risk of Breast Cancer, Eur. J. Cancer 36, 335–340. 25. Guzman, G.J., and Murphy, P.A. (1986) Tocopherols of Soybean Seeds and Soybean Curd (Tofu), J. Agric. Food Chem. 34, 791–795. 26. Hawrylewicz, E.J., Huang, H.H., and Blair, W.H. (1991) Dietary Soybean Isolate and Methionine Supplementation Affect Mammary Tumor Progression in Rats, J. Nutr. 121, 1693–1698.

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231. Arjmandi, B.H., Alekel, L., Hollis, B.W., Amin, D., Stacewicz-Sapuntzakis, M., Guo, P., and Kukreja, S.C. (1996) Dietary Soybean Protein Prevents Bone Loss in an Ovariectomized Rat Model of Osteoporosis, J. Nutr. 126, 161, 48167. 232. Kalu, D.N., Masoro, E.J., Yu, B.P., Hardin, R.R., and Hollis, B.W. (1988) Modulation of Age-Related Hyperparathyroidism and Senile Bone Loss in Fischer Rats by Soy Protein and Food Restriction, Endocrinology 122, 1847, 481854. 233. Omi, N., Aoi, S., Murata, K., and Ezawa, I. (1994) Evaluation of the Effect of Soybean Milk and Soybean Milk Peptide on Bone Metabolism in the Rat Model with Ovariectomized Osteoporosis, J. Nutr. Sci. Vitaminol. (Tokyo) 40, 201, 48211. 234. Watkins, T.R., Pandya, K., and Mickelsen, O. (1985) Urinary Acid and Calcium Excretion. Effect of Soy Versus Meat in Human Diets, in Nutritional Bioavailability of Calcium (Kies, C., ed.) American Chemical Society, Washington. 235. Pie, J.-E., and Paik, H.Y. (1986) The Effect of Meat Protein and Soy Protein on Calcium Metabolism in Young Adult Korean Women, Korean J. Nutr. 19, 32, 4840. 236. Barzel, U.S. (1995) The Skeleton as an Ion Exchange System: Implications for the Role of Acid-Base Imbalance in the Genesis of Osteoporosis, J. Bone Miner. Res. 10, 1431, 481–436. 237. Ishida, H., Uesugi, T., Hirai, K., Toda, T., Nukaya, H., Yokotsuka, K., and Tsuji, K. (1998) Preventive Effects of the Plant Isoflavones, Daidzin and Genistin, on Bone Loss in Ovariectomized Rats Fed a Calcium-Deficient Diet, Biol. Pharm. Bull. 21, 62–66. 238. Potter, S.M., Baum, J.A., Teng, H., Stillman, R.J., Shay, N.F., and Erdman, J.W., Jr. (1998) Soy Protein and Isoflavones: Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68, 1375S–1379S. 239. Alekel, D.L., Germain, A.S., Peterson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in the Lumbar Spine of Perimenopausal Women [see comments], Am. J. Clin. Nutr. 72, 844–852. 240. Wangen, K.E., Duncan, A.M., Merz-Demlow, B.E., Xu, X., Marcus, R., Phipps, W.R., and Kurzer, M.S. (2000) Effects of Soy Isoflavones on Markers of Bone Turnover in Premenopausal and Postmenopausal Women, J. Clin. Endocrinol. Metab. 85, 3043– 3048. 241. Alexandersen, P., Toussaint, A., Register, J., Christiansen, C., Devogelaer, J., Gennari, C., Roux, C., and Fechtenbaum, J. (2000) Ipriflavone Has No Effect on Bone Metabolism and Causes Lymphopenia in Osteoporotic Women, J. Bone Miner. Res. 15 (Suppl. 1), 1239. 242. Gallagher, J.C., Rafferty, K., Haynatzka, V., and Wilson, M. (2000) Effect of Soy Protein on Bone Metabolism, J. Nutr. 130, 667S–669S. 243. Duncan, A.M., Merz, B.E., Xu, X., Nagel, T.C., Phipps, W.R., and Kurzer, M.S. (1999) Soy Isoflavones Exert Modest Hormonal Effects in Premenopausal Women, J. Clin. Endocrinol. Metab. 84, 192–197. 244. Duncan, A.M., Underhill, K.E., Xu, X., Lavalleur, J., Phipps, W.R., and Kurzer, M. S. (1999) Modest Hormonal Effects of Soy Isoflavones in Postmenopausal Women, J. Clin. Endocrinol. Metab. 84, 3479–3484. 245. Lu, L.J., Anderson, K.E., Grady, J.J., and Nagamani, M. (1996) Effects of Soya Consumption for One Month on Steroid Hormones in Premenopausal Women: Implications for Breast Cancer Risk Reduction, Cancer Epidemiol. Biomark. Prev. 5, 63–70.

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246. Lu, L.J., Anderson, K.E., Grady, J.J., Kohen, F., and Nagamani, M. (2000) Decreased Ovarian Hormones During a Soya Diet: Implications for Breast Cancer Prevention, Cancer Res. 60, 4112–4121. 247. Adlercreutz, H., Hockerstedt, K., Bannwart, C., Bloigu, S., Hamalainen, E., Fotsis, T., and Ollus, A. (1987) Effect of Dietary Components, Including Lignans and Phytoestrogens, on Enterohepatic Circulation and Liver Metabolism of Estrogens and on Sex Hormone Binding Globulin (SHBG), J. Steroid Biochem. 27, 1135–1144. 248. Loukovaara, M., Carson, M., Palotie, A., and Adlercreutz, H. (1995) Regulation of Sex Hormone-Binding Globulin Production by Isoflavonoids and Patterns of Isoflavonoid Conjugation in HepG2 Cell Cultures, Steroids 60, 656–661. 249. Wilcox, G., Wahlqvist, M.L., Burger, H.G., and Medley, G. (1990) Oestrogenic Effects of Plant Foods in Postmenopausal Women, Br. Med. J. 301, 905–906. 250. Cassidy, A., Bingham, S., and Setchell, K.D. (1994) Biological Effects of a Diet of Soy Protein Rich in Isoflavones on the Menstrual Cycle of Premenopausal Women, Am. J. Clin. Nutr. 60, 333–340. 251. Cassidy, A., Bingham, S., and Setchell, K. (1995) Biological Effects of Isoflavones in Young Women: Importance of the Chemical Composition of Soyabean Products, Br. J. Nutr. 74, 587–601. 252. Whelan, E.A., Sandler, D.P., Root, J.L., Smith, K.R., and Weinberg, C.R. (1994) Menstrual Cycle Patterns and Risk of Breast Cancer, Am. J. Epidemiol. 140, 1081– 1090. 253. Olsson, H., Landin-Olsson, M., and Gullberg, B. (1983) Retrospective Assessment of Menstrual Cycle Length in Patients with Breast Cancer, in Patients with Benign Breast Disease, and in Women Without Breast Disease, J. Natl. Cancer Inst. 70, 17-20. 254. Martini, M.C., Dancisak, B.B., Haggans, C.J., Thomas, W., and Slavin, J.L. (1999) Effects of Soy Intake on Sex Hormone Metabolism in Premenopausal Women, Nutr. Cancer 34, 133–139. 255. Wu, A.H., Stanczyk, F.Z., Hendrich, S., Murphy, P.A., Zhang, C., Wan, P., and Pike, M.C. (2000) Effects of Soy Foods on Ovarian Function in Premenopausal Women, Br. J. Cancer 82, 1879–1886. 256. Kurzer, M.S. (2000) Hormonal Effects of Soy Isoflavones: Studies in Premenopausal and Postmenopausal Women, J. Nutr. 130, 660S–661S. 257. Mitchell, J.H., Cawood, E., Kinniburgh, D., Provan, A., Collins, A.R., and Irvine, D.S. (2001) Effect of a Phytoestrogen Food Supplement on Reproductive Health in Normal Males, Clin. Sci. (Lond.) 100, 613–618. 258. Lu, L.J., Anderson, K.E., Grady, J.J., and Nagamani, M. (2001) Effects of an Isoflavone-Free Soy Diet on Ovarian Hormones in Premenopausal Women, J. Clin. Endocrinol. Metab. 86, 3045–3052. 259. Xu, X., Duncan, A.M., Wangen, K.E., and Kurzer, M.S. (2000) Soy Consumption Alters Endogenous Estrogen Metabolism in Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 9, 781–786. 260. Kurzer, M.S. (2002) Hormonal Effects of Soy in Premenopausal Women and Men, J. Nutr. 132, 570S–573S. 261. Adlercreutz, H., Hamalainen, E., Gorbach, S., and Goldin, B. (1992) Dietary PhytoOestrogens and the Menopause in Japan, Lancet 339, 1233. 262. Lock, M. (1994) Menopause in Cultural Context, Exp. Gerontol. 29, 307–317.

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263. Lock, M. (1992) Contested Meanings of the Menopause, Lancet 337, 1270–1272. 264. Murkies, A.L., Lombard, C., Strauss, B.J., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Post-Menopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 265. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 266. Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L., and Lamia, C.A. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: A Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 267. Pan, Y., Anthony, M., and Clarkson, T.B. (1999) Evidence for Up-Regulation of Brain Derived Neurotrophic Factor mRNA by Soy Phytoestrogens in the Frontal Cortex of Retired Breeder Female Rats, Neurosci. Lett. 261, 17–20. 268. Pan, Y., Anthony, M., and Clarkson, T.B. (1999) Effect of Estradiol and Soy Phytoestrogens on Choline Acetyltransferase and Nerve Growth Factor mRNAs in the Frontal Cortex and Hippocampus of Female Rats, Proc. Soc. Exp. Biol. Med. 221, 118–125. 269. Pan, Y., Anthony, M., Watson, S., and Clarkson, T.B. (2000) Soy Phytoestrogens Improve Radial Arm Maze Performance in Ovariectomized Retired Breeder Rats and Do Not Attenuate Benefits of 17β-Estradiol Treatment, Menopause 7, 230–235. 270. Pan, Y., Anthony, M.S., Binns, M., and Clarkson, T.B. (2001) A Comparison of Oral Micronized Estradiol with Soy Phytoestrogen Effects on Tail Skin Temperatures of Ovariectomized Rats, Menopause 8, 171–174. 271. Kim, H., Xia, H., Li, L., and Gewin, J. (2000) Attenuation of NeurodegenerationRelevant Modifications of Brain Proteins by Dietary Soy, Biofactors 12, 243–250. 272. File, S.E., Jarrett, N., Fluck, E., Duffy, R., Casey, K., and Wiseman, H. (2001) Eating Soya Improves Human Memory, Psychopharmacology (Berl.) 157, 430–436. 273. White, L., Petrovitch, H., Ross, G.W., and Masaki, K. (1996) Association of Mid-Life Consumption of Tofu with Late Life Cognitive Impairment and Dementia: The Honolulu-Asia Aging Study, Neurobiol. Aging 17, S121. 274. White, L.R., Petrovitch, H., Ross, G.W., Masaki, K., Hardman, J., Nelson, J., Davis, D., and Markesbery, W. (2000) Brain Aging and Midlife Tofu Consumption, J. Am. Coll. Nutr. 19, 242–255. 275. File, S.E., Jarrett, N., Fluck, E., Duffy, R., Casey, K., and Wiseman, H. (2001) Eating Soy Improves Memory, Psychopharmacology 156, 430–436. 276. LeBlanc, E.S., Janowsky, J., Chan, B.K., and Nelson, H.D. (2001) Hormone Replacement Therapy and Cognition: Systematic Review and Meta-Analysis, J. Am. Med. Assoc. 285, 1489–1499. 277. Clarkson, T.B., Anthony, M.S., Williams, J.K., Honore, E.K., and Cline, J.M. (1998) The Potential of Soybean Phytoestrogens for Postmenopausal Hormone Replacement Therapy, Proc. Soc. Exp. Biol. Med. 217, 365–368. 278. Jordan, V.C., Gapstur, S., and Morrow, M. (2001) Selective Estrogen Receptor Modulation and Reduction in Risk of Breast Cancer, Osteoporosis, and Coronary Heart Disease, J. Natl. Cancer Inst. 93, 1449–1457. 279. Brzezinski, A., and Debi, A. (1999) Phytoestrogens: The “Natural” Selective Estrogen Receptor Modulators? Eur. J. Obstet. Gynecol. Reprod. Biol. 85, 47–51.

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280. Setchell, K.D. (2001) Soy Isoflavones-Benefits and Risks from Nature’s Selective Estrogen Receptor Modulators (SERMs), J. Am. Coll. Nutr. 20, 354S–362S; discussion 381S–383S. 281. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.A. (1996) Cloning of a Novel Receptor Expressed in Rat Prostate and Ovary, Proc. Natl. Acad. Sci. USA 93, 5925–5930. 282. Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J.A. (1997) Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors Alpha and Beta, Endocrinology 138, 863– 870. 283. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.A. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor Beta, Endocrinology 139, 4252–4263. 284. Nikov, G.N., Hopkins, N.E., Boue, S., and Alworth, W.L. (2000) Interactions of Dietary Estrogens with Human Estrogen Receptors and the Effect on Estrogen Receptor-Estrogen Response Element Complex Formation, Environ. Health Perspect. 108, 867–872. 285. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J., and Nilsson, S. (1998) Differential Response of Estrogen Receptor Alpha and Estrogen Receptor Beta to Partial Estrogen Agonists/Antagonists, Mol. Pharmacol. 54, 105–112.

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Chapter 2

Soy Isoflavones as Functional Ingredients in Women’s Health Clare M. Hasler and Susan Kundrat Department of Food Science and Human Nutrition, and Functional Foods for Health Program, University of Illinois at Urbana-Champaign, Urbana, IL

Introduction Functional foods, “foods that, by virtue of the presence of physiologically active components, provide a health benefit beyond basic nutrition” (1), are one of the leading trends in the food industry today. For the past three years, Food Processing magazine’s Top 100® R&D Survey has identified functional foods/nutraceuticals as one of the hottest categories in which to devote short-term R&D efforts (2–4). In terms of functional food ingredient categories, soy and soy isoflavones clearly lead the industry. In the 2001 Prepared Foods R&D Investment Survey, soy protein was ranked as the leading ingredient of interest for food formulation by 331 marketing and general management respondents (5). This is not surprising, given that more consumers are regularly incorporating soy into their diets. The 7th Annual National Report on consumer attitudes and perceptions on health and nutrition issues from the United Soybean Board (USB) found that the number of consumers eating soy products once a week or more rose from 15% in 1998 to 27% in 2000 (6). Furthermore, the number of consumers who consider soy and soy products to be healthy increased from 59% in 1997 to 76% in 2000. USB’s 2001–2002 survey found that the number of consumers who are aware that soy may lower cholesterol rose from 27% in 1999 to 42% in 2001 (7). Such consumer interest has had a dramatic, positive effect on the market for soy and soy supplements, which has skyrocketed in recent years. Sales of soy foods are expected to reach $6.9 billion in 2005 (8), and sales of soy supplements have been undergoing even more explosive growth, with food, drug, and mass market sales up 686% for the 1-y period ending July 23, 2000 (9). The growing market for soy products and increasing consumer interest in soy is first and foremost stimulated by ongoing soy research in academic and privatesector research centers around the world (10,11). This chapter will briefly summarize some of the most recent findings relevant to the role of soy isoflavones in women’s health, a sector of the food industry of particular interest to food manufacturers today (12), with a specific focus on cardiovascular disease, bone health,

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the alleviation of menopausal symptoms, and breast cancer. The issue of isoflavone safety will also be discussed in relation to breast cancer. Isoflavones Isoflavones are one of the major classes of phytoestrogens that have been the focus of an exponential increase in the number of in vitro, in vivo, epidemiologic, and clinical research studies related to their health effects over the last decade (13). These physiologically active compounds have similarities to 17β-estradiol, the most potent mammalian estrogen. However, the relative binding affinity of isoflavones for the primary estrogen receptor, estrogen receptor-α (ER-α), is only 0.05–1% of the binding affinity of 17β-estradiol (14). The recent discovery of a novel estrogen receptor, ER-β (15), which has a different tissue distribution relative to ER-α, and to which isoflavones bind with an approximately sevenfold greater affinity (16), makes plausible the concept of isoflavones acting as selective estrogen receptor modulators (SERM) (17). In addition, because the concentrations of circulating isoflavones can be several thousand fold greater than that of estradiol (18), isoflavones may exert an overall antiestrogenic effect, particularly when endogenous levels of circulating estrogens are low. This has raised concerns (particularly in postmenopausal women) that isoflavones may act as a double-edged sword in regard to breast cancer. Soy Isoflavones and Cardiovascular Disease Soy Protein and Blood Lipid Reduction. The cardiovascular benefits of soy are the most clinically well-documented health effect associated with consumption of this functional food. Although the cholesterol-lowering effect of soy was initially recognized over 90 years ago (19), awareness of this diet-disease relationship did not become widespread among health professionals and consumers until the mid1990s when a meta-analysis of 38 clinical studies was published in the New England Journal of Medicine (20). This significant review (which involved >700 subjects), demonstrated that, compared with control diets, substitution of soy protein resulted in significant reductions in total cholesterol (TC) (9.3%), low density lipoprotein cholesterol (LDL-C) (12.9%), and triglycerides (10.5%), with a small but insignificant increase (2.4%) in high density lipoprotein cholesterol (HDL-C). Based in large part on data included in the meta-analysis by Anderson et al. (20), as well as their own comprehensive review of 41 clinical studies, the FDA approved a health claim for the relationship between the consumption of soy protein and reduced risk of CHD on October 26, 1999 (21). Thus, the following statement may now be utilized on qualified soy products: Diets low in saturated fat and cholesterol that include 25 grams of soy protein a day may reduce the risk of heart disease. One serving of [name of food] provides [xx] g of soy protein. More recent data indicate that as little as 20 g of soy protein isolate (SPI)/d can lower blood lipids (22). The positive effect of soy on blood lipids prompted the Nutrition

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Committee of the American Heart Association to issue a statement for healthcare professionals in 2000, which recommended including soy protein foods in a diet low in saturated fat and cholesterol to promote heart health (23). Isoflavones and Blood Lipids. Whether isoflavones are the principal physiologically active component responsible for the cardioprotective benefit of soy is a subject of considerable debate (24). Although Anderson et al. (20) concluded that isoflavones were responsible for up to 60% of the hypocholesterolemic activity of soy protein, the FDA stated in the final ruling for the health claim that they were “not persuaded that the isoflavone component of soy protein was a relevant factor to the diet-disease relationship. . . .” This conclusion was based on the observation that clinical studies with isoflavone extracts had been ineffective in improving lipid profiles (25–28). More recent trials have also failed to show that purified isoflavones significantly lower blood lipids (29), including a recent study in 36 postmenopausal women in which 150 mg isoflavones (90 mg as aglycones) was consumed daily for 6 mo (30). In contrast, studies involving increased levels of isoflavones in conjunction with soy protein may be beneficial. A study by Crouse et al. (31) found that isolated soy protein (ISP) containing increasing levels of isoflavones (4, 27, 37, or 62 mg) had a dose-dependent reducing effect on cholesterol. However, the results were modest, with the highest level of isoflavones (62 mg) lowering TC and LDLC levels by only 4 and 6%, respectively. Further, this effect was restricted to a subset of participants with an average LDL-C concentration greater than the median value (166 mg/dL). The effect of a wider range of isoflavone levels (10, 65, or 129 mg isoflavones/d) on blood lipids was examined by Merz-Demlow et al. (32) in 13 healthy, premenopausal women utilizing a crossover design. The high isoflavone diet reduced LDL by 7.6–10%. Similarly, in a separate study by this same group of investigators involving 18 postmenopausal women consuming SPI with ~7.1, 65, or 132 mg isoflavones for three 3-mo treatment periods, the high isoflavone diet reduced LDL cholesterol by 6.5% (33). To date, the evidence does not support the hypothesis that isoflavones per se, separate from soy protein, reduce blood lipids, although this unresolved issue could be related to the fact that different diets may contain different total amounts as well as different ratios of the 12 known isoflavones, i.e., individual isoflavones could differ in biological potency and certain combinations could also have differential physiologic effects (34). Isoflavones and LDL Oxidation. Soy and soy isoflavones are also being investigated for their effects on the oxidation of LDL-C, a pathological event that is now recognized as being critical in the etiology of atherosclerosis (35). Several recent studies have shown a beneficial effect of soy product consumption on the reduction of LDL oxidation in human subjects. In a randomized, crossover design study involving 24 subjects, 56 mg isoflavones significantly increased LDL oxidation lag time by 8% after consumption of 15 g textured soy protein/d (the amount in one

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vegetarian burger) for 17 d (36). When low isoflavone (1.9 mg) burgers were consumed, no significant effect on LDL lag time was noted. Two recent studies by Jenkins and co-workers (37,38) also demonstrate that consumption of soy foods can reduce the oxidation of LDL. In the first study (37), 20 hyperlipidemic men and postmenopausal women consumed 12 ± 2 g soy protein/d from a self-selected menu of soy-based foods as part of a National Cholesterol Education Program (NCEP) step 2 diet. After 8 wk, LDL oxidation was significantly reduced by 8.5 ± 3.3%. In addition, the test diet significantly elevated HDL cholesterol 6.4 ± 2.4%. Similar findings were noted in a second study by these investigators in which 25 hyperlipidemic men and women consumed a soy-based breakfast cereal containing 36 g/d soy protein and 168 mg/d isoflavones for 3 wk (38). Oxidized LDL were significantly reduced by 9.2 ± 4.3%. Collectively, these studies suggest that the consumption of soy protein foods containing isoflavones can increase the resistance of LDL to oxidation. Isoflavones and Arterial Compliance. Another positive effect that soy and/or soy isoflavones may have on heart health is their positive effects on vascular function, including increasing arterial compliance. Reduced arterial compliance, which is an indication of arterial stiffness/peripheral vasodilation, is thought to contribute to cardiovascular disease (39). Because soy isoflavones are plant-derived estrogens, which have been shown to increase brachial artery flow-mediated dilation (40), it is not surprising that they would increase arterial compliance. Isoflavones derived either from soybeans (41) or red clover (42) have been shown to improve systemic arterial compliance in menopausal women by 20–26% even though they failed to lower blood lipids. However, not all studies have shown that isoflavones improve vascular function. A randomized crossover study of 20 postmenopausal women by Simons et al. (43) found that daily consumption of 80 mg isoflavone tablets did not improve endothelium-dependent dilation. More recently, a study that infused increasing doses of genistein or daidzein into the brachial artery of men and premenopausal women found a dose-dependent increase in forearm blood flow with infusion of genistein, but not daidzein (44). Clearly, more research is required to clarify the role of soy isoflavones in vascular function. In summary, there is significant scientific agreement that the consumption of soy protein reduces the major blood lipids associated with cardiovascular disease risk. Although the specific component of soy responsible for its lipid-lowering effect is unknown, isoflavones per se are not likely responsible. However, isoflavones may have other cardiovascular benefits apart from their lipid-lowering effects, including their ability to reduce the oxidation of LDL and enhance arterial compliance. Soy and Menopause Menopause is defined by the Council of Affiliated Menopause Societies (CAMS) of the International Menopause Society as the permanent cessation of menstruation resulting from the loss of ovarian follicular activity (45). In addition, CAMS states that natural menopause is recognized to have occurred after 12 consecutive months

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of amenorrhea (lack of a menstrual cycle for at least three consecutive months), for which there is no other obvious pathologic or physiologic cause. Menopause occurs at a median age of 51.4 y with some women reaching menopause as early as their 30s, and a few in their 60s. Perimenopause includes the period immediately before menopause (when the endocrinologic, biologic, and clinical features of approaching menopause commence) and the first year after menopause. For most women, perimenopause lasts ~4 y. Some of the most common symptoms of the menopausal transition include hot flashes, night sweats, mood changes, loss of libido, and vaginal dryness due to the changing hormonal environment of menopause (46). Hormone replacement therapy (HRT) is presently used to reduce the risk of osteoporosis and heart disease and reduce vasomotor symptoms, including hot flashes and night sweats, which can be quite severe in some women. Although HRT is widely prescribed, some women reject this therapy for a variety of reasons, including the increased risk for breast cancer. A recent study among 705 postmenopausal women with a history of primary invasive breast cancer and 692 controls found that the incidence of breast cancer of all histologic types was increased 60–85% in recent long-term users of HRT. Longer use of HRT [odds ratio (OR), 3.07 for ≥57 mo; 95% confidence interval (CI), 1.55–6.06] and current use of combination therapy (OR, 3.91; 95% CI, 2.05–7.44) were associated with an increased risk of lobular breast cancer, whereas long-term HRT use was associated with a 50% increase in nonlobular cancer (OR, 1.52 for ≥57 mo; 95% CI, 1.01–2.29) (47). According to the American College of Obstetricians and Gynecologists (ACOG), lack of confidence in the espoused benefits of HRT, coupled with a fear of increased cancer risk or other side effects associated with HRT, results in fewer than 1 in 3 women choosing HRT (48). Soy began to be investigated as a candidate alternative therapy for menopause since it was first noted that there are significant differences in how women from various cultures respond to menopause. In a study by Lock et al. (49), which surveyed 1310 Canadian and 1316 Japanese women, 30.9% of the Canadian women reported that they had experienced a hot flash in the preceding 2 wk compared with only 9.7% of the Japanese women. Similarly, only 3.6% of the Asian women had experienced night sweats compared with 19.6% of the Canadian women. Other observations that 85% of women in North America report hot flashes (50), compared with a 15–25% incidence in Asia (51), have led to the hypothesis that soy isoflavones may exert estrogenic effects that alleviate menopausal symptoms. Clinical studies evaluating the effects of soy or soy isoflavones on menopausal symptoms have yielded inconsistent results. This is likely due to the fact that the source and quantity of isoflavones have varied, as have the population group studied (peri- or postmenopausal women), the study design (crossover vs. parallel arm), and the length of the study. More significantly, there is a large placebo effect associated with studies of this nature, which can be >30%. Very few studies have evaluated menopausal symptoms after the consumption of purified isoflavone extracts. The largest study, conducted by Upmalis and co-

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workers (52), examined the effect of an extract of soy isoflavones for relief of menopausal hot flashes in a multicenter study (15 different research sites) in 177 postmenopausal women experiencing ≥5 hot flushes/d. They received either 50 mg unconjugated isoflavones/d or a placebo for 12 wk. Isoflavones reduced hot flash frequency and severity by an additional 15% more than the placebo after 6 wk and the frequency of night sweats by an additional 34%. However, the difference between treatment groups was diminished at 12 wk. Similarly, 50 mg/d conjugated isoflavones was shown to decrease the frequency of hot flashes 20% more than placebo after 6 wk in a study by Scambia et al. (53) involving 39 subjects. Although menopausal symptom reduction was not noted after consumption of 150 mg isoflavones/d in a study by Quella et al. (54), this study had a number of limitations, including the fact that the subjects were breast cancer survivors and the intervention period was only 4 wk. A larger number of clinical trials have investigated the effect of soy products as opposed to purified isoflavones on menopausal symptoms, and the results appear to be more promising. In a study by Murkies and colleagues (55), the incidence of hot flashes was reduced by 40% in those consuming 45 g protein from soy flour daily for 12 wk compared with a 25% reduction in controls. Similarly, a study by Brzezinski and co-workers (56) found that soy foods containing 65 mg isoflavones plus flaxseed reduced hot flash severity at 12 wk by an additional 19% above the reduction seen in the control group. Albertazzi et al. (57) reported a 45% reduction in hot flash frequency (10–12% above the decrease noted in the control group) in a group of women consuming SPI containing 76 mg total isoflavones for 3 mo; symptom severity did not change. Surprisingly, hot flash frequency and severity were not reduced in a relatively long-term study (24 wk) by St. Germain and co-workers (58) involving perimenopausal women consuming 80 mg purified isoflavones compared with soy containing 4 mg isoflavones. One study found that a relatively small dose of isoflavones (34 mg) was effective in reducing hot flash severity by 25% only when divided into two daily doses (59). In summary, consumption of soy isoflavones is associated with small but significant reductions in vasomotor symptoms associated with menopause. At present, however, it does not appear that isoflavones can compete with standard HRT for effectiveness in the relief of vasomotor symptoms associated with menopause. A consensus opinion from the North American Menopause Society states that “the role of isoflavones in the management of short-term menopausal symptoms as well as diseases related to menopause/aging is still uncertain. . .” (60). Thus, although the data at present do not unequivocally support the use of soy as an alternative to HRT, soy isoflavones may provide an attractive addition to the choices available for relief of hot flashes and an option for women who wish to use a dietary approach to relieve hot flushes and associated menopausal symptoms. Osteoporosis Osteoporosis is a reduction in bone tissue resulting in brittle and fragile bones prone to fracture, usually occurring in postmenopausal women and elderly men

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due to hormonal changes or a deficiency of calcium or vitamin D. The World Health Organization has made a distinction in severity of the disease by dividing it into the following two categories: (i) osteopenia, which is a bone density between 1 and 2.5 SD below the average value in young adults, and (ii) osteoporosis, bone density >2.5 SD below the average (61). In the first 3–5 y after menopause, women lose 3–5% of their bone mass per year, whereas after ~10 y, bone loss tapers off, only to increase again after the age of 80 y (62). Estimated cost of health care for osteoporotic fractures in the United States was $13.8 billion in 1995 (80% spent on women, 20% on men) (63); with an aging population, we can expect osteoporosis to become an even greater public health burden. A 2002 report from the National Osteoporosis Foundation documents that there are 44 million women and men ≥50 y old in the United States with either osteoporosis or low bone mass; by the year 2010, this figure will rise to 52 million (64). This report also reiterates the fact that this disease may be largely preventable with lifestyle modification. Epidemiologic data suggest that osteoporosis is about one third as common in Japanese women compared with those consuming a Western diet (65). The extent to which this is genetic is unknown, and it is hypothesized that environmental factors, including diet, and specifically, dietary isoflavones, may play an important role. Isoflavone intake is much higher in Asians than in Westerners, among whom it is negligible. Mean intake of dietary phytoestrogens in postmenopausal women participating in the Framingham study was shown to be 154 µg/d (66). Conversely, isoflavone consumption by certain Asian populations could be in excess of 150 mg/d (17). Because isoflavones are structurally similar to endogenous estrogens, which are known to decrease bone loss in postmenopausal women, this suggests that soy consumption may positively affect bone health (67). Isoflavones have been shown to stimulate osteoblast activity (68) and suppress osteoclast formation (69). Further, ipriflavone, which is a synthetic isoflavone (and also a metabolite of daidzein), has been shown to inhibit the activity of osteoclasts and parathyroid hormone (70) as well as increase alkaline phosphatase activity and collagen formation (71). A beneficial effect of ipriflavone in reducing bone loss (72–74) as well as increasing bone density (75–76) has been demonstrated clinically. Ovariectomized animal models have consistently shown increases in bone mass after treatment with SPI-based diets (77,78), or purified isoflavones (79,80). These studies support epidemiologic investigations showing that a high isoflavone intake is associated with higher bone mineral density (BMD). In a recent longitudinal study involving a cohort of 116 Asian women aged 30–40 y and living in Hong Kong who were followed for an average of 38 months, soy intake had a significant, positive effect on the maintenance of spine BMD (81). Similarly, in a recent study of 650 postmenopausal Chinese women, those with habitually high intake of dietary isoflavone had significantly higher BMD values at both the spine and hip region (82). The bone protective effect of isoflavone was observed at an intake of ~53 mg/d, the mean value of the highest tertile of isoflavone intake. This is lower than the levels of isoflavones that have been shown to be protective in clinical studies.

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Clinical studies examining the effect of soy or soy isoflavones on bone biomarkers have been inconsistent. Although a study by Horiuchi et al. (83) found that postmenopausal women with high soy protein intake had lower levels of bone resorption, a report by Wangen and colleagues (84) did not find that 130 mg isoflavones/d positively changed bone biomarkers. Only two human studies have been published examining the effect of soy consumption on bone health. Both found significant increases in bone mineral content. The first study, conducted by Potter and colleagues (85), involved 66 postmenopausal women who consumed 40 g/d of SPI containing either 56 or 90 mg of isoflavones for a period of 24 wk. Only women in the high isoflavone group had a significant increase (2.2%) in bone mineral density and bone mineral content in the lumbar spine. No significant changes were found in bone mineral density or content in total-body or other skeletal sites. The authors note that the spine, compared with the other measured skeletal sites, is thought to be the most sensitive to estrogen due to its high content of trabecular bone, which is remodeled more rapidly than sites containing a high content of cortical bone, such as the hip. Similar results were noted in a study by Alekel et al. (86) conducted in perimenopausal women. In this 24-wk study, no significant loss in spine BMD occurred in 24 women fed SPI enriched in isoflavones (80 mg) vs. a low isoflavone (4 mg) isolate in which isoflavones had been removed by alcohol extraction. By regression analysis, the SPI with isoflavones had a positive effect on BMD (5.6%; P = 0.023) and bone mineral content (10.1%; P = 0.0032). A loss in BMD did occur in women who received whey protein. In summary, knowledge of the effects of soy or soy isoflavones on bone metabolism is a very new and slowly emerging area of research. Thus, inadequate data exist at present to recommend a level of isoflavone intake to reduce the risk of or prevent osteoporosis. However, clinical data suggest that higher levels of isoflavones (≥80 mg) may be required and longer-term studies (1–2 y) will be necessary to confirm the positive effects seen to date. Breast Cancer Women in the United States who live to be 90 y old have a 1 in 8 chance of being diagnosed with breast cancer. With 205,000 cases anticipated in 2002 (87), breast cancer is expected to be the most frequently diagnosed nonskin malignancy in U.S. women. In that year, breast cancer will kill ~39,600 women, second to lung cancer. According to the American Cancer Society (87), mortality rates declined significantly during 1992–1998, with the largest decreases in younger women, both Caucasian and African-American, most likely the results of both earlier detection and improved treatment. For all ages of women, Caucasian women are more likely to develop breast cancer than African-American women, although among women <50 y old, African-American women have higher incidence rates than Caucasian women (87). Although soy contains a variety of anticancer agents including protease inhibitors, phytate, phytosterols, saponins, and the isoflavones, as well as other

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possible anticarcinogens such as phenolic acids, lecithin, and n-3 fatty acids (88), isoflavones are currently the most intensively researched soy phytochemical with respect to breast cancer. Soy isoflavones have been shown to be anticarcinogenic in several ways. In addition to their weak estrogenic effect discussed earlier, a variety of other nonhormonal anticancer mechanisms have also been proposed. Genistein has been shown to inhibit growth of both human and rodent cancer cell lines in vivo and in vitro; one proposed mechanism is through its inhibition of certain enzymes that stimulate cancer cell growth such as protein tyrosine kinases (89). Genistein also induces differentiation of malignant cells (90) and inhibits angiogenesis (the formation of new blood vessels), thus reducing the blood supply to growing tumors (91). Genistein works as an antioxidant (92) and may also inhibit cancer cell growth by inducing changes in signaling pathways by transforming growth factor (93). The presence of isoflavones in soy may explain why the incidence of breast cancer in Japan and China is one fifth of that in Western women (94). Although soy intake appears to be steadily increasing in the United States, a 1995 study noted that the average intake of soy protein in Southeast Asia ranges from 10–50 g/d in contrast to 1–3 g/d consumed by Americans (89). Asian women have been estimated to consume 20–80 mg isoflavones/d compared with women in the United States whose intake is <5 mg/d (95). Soy has been shown to be protective against breast cancer in women in epidemiologic studies, mammary cancer in rat models, and in human mammary cancer cell lines grown in culture (96). However, no clinical trials have yet been published documenting soy’s ability to reduce breast cancer in women at high risk. There have been no published studies in women regarding soy and tamoxifen taken together. One recent study evaluated the effect of a soy product in combination with tamoxifen against mammary tumorigenesis in female rats (97). Tamoxifen reduced mammary tumors 29%, ISP reduced mammary tumors 37%, and the combination of tamoxifen and ISP led to a 62% reduction in tumors. It appears that genistein can act as both an estrogen and an antiproliferative agent. According to a recent review of genistein and breast cancer (98), these effects may be both dose and tissue dependent. The authors noted that this is in agreement with data from studies with other estrogenic compounds such as tamoxifen and diethylstilbestrol. Diets containing genistein have been shown to decrease tumor numbers and size in animals that were exposed to carcinogens, and the rate at which tumors appear. In a recent review of animal studies published between 1990 and 1997, 16 of 17 studies (94%) showed that soy was protective; five of these were in breast cancer models (99). Santell and colleagues (100) concluded in one study in which genistein was fed to mice at varying doses, that although genistein inhibits cancer cell growth in vitro, it is unlikely that the plasma concentration required to inhibit cancer cell growth in vivo can be achieved from a dietary dose of genistein. Many, but not all of the epidemiologic studies that have specifically examined soy consumption and later incidence of breast cancer have shown that soy intake

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can be protective. In one study done in Singapore among 200 Chinese women with breast cancer and 420 matched controls, a decreased risk of breast cancer was associated with high intakes of soy products in premenopausal but not postmenopausal women (101). A more recent case-control study done at the University of Southern California, Los Angeles, interviewed 597 Asian-American women with previous incidence of breast cancer and 966 controls. Intake of tofu was more than twice as high among Asian-American women born in Asia (62 times/y) compared with those born in the United States (30 times/y). The risk of breast cancer decreased with increasing frequency of tofu consumption in both pre- and postmenopausal women; the adjusted OR associated with each additional serving per week was 0.85 (95% CI, 0.74–0.99) (102). In contrast, however, breast cancer risk was not significantly associated with consumption of soy foods in a recent prospective study by Key et al. (103) involving 34,759 women in Hiroshima and Nagasaki, Japan. Two recent abstracts reviewed studies on soy and cancer. In a meta-analysis of 7 epidemiologic studies (5 case-control studies of Asian women living in Asian countries; 2 cohort studies of AsianAmerican women living in the United States) by Fleischauer et al. (104), a modest reduction of breast cancer was observed among all reports for the highest level of soy consumption compared with the lowest (OR, 0.83; 95% CI, 0.73–0.95). The protective association was in premenopausal women (OR, 0.74; 95% CI, 0.62–0.87) with no apparent benefit among postmenopausal women (OR, 0.94; 95% CI, 0.79–1.12). The authors of this report noted that postmenopausal women who consumed greater amounts of soy did not appear to be at increased risk of breast cancer. In a meta-analysis of eight case-control studies and one cohort study examining soy and breast cancer risk (105), a modest significant reduction in risk was associated with high soy intake over all studies (OR, 0.87; 95% CI, 0.80–0.96). However, this effect was also limited to premenopausal women (OR, 0.80; 95% CI, 0.71–0.90). The authors pointed to the small number of studies, crude measurement of soy intake, and inconsistent control of confounding factors as considerations when interpreting the results. An intake of soy foods early in life may be important for its protective effect. In a case-control study of Chinese women involving 1459 breast cancer cases and 1556 age-matched controls, adolescent soy food intake (ages 13–15 y) was inversely associated with risk for breast cancer after adjustment for a variety of other risk factors (106). In addition, the inverse association was observed for each of the soy foods examined and existed for both pre- and postmenopausal women. This observation was further supported by Lamartiniere et al. (107), who showed that administration of genistein to rats in the perinatal period was sufficient to cause a marked latency in the appearance of mammary tumors after the administration of a mammary cancer causing agent at 50 d of age. This protection was possibly due to enhanced mammary gland differentiation (108), reinforcing the idea that females exposed to soy early in their lives may be protected. Similar results by another group found that both soy and whey reduced chemically induced mammary cancer in the rodent model (109) and parental consumption before conception enhanced benefit for the offspring (110).

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Do Isoflavones Promote Breast Cancer? One of the most controversial topics in the soy and breast cancer research area is whether high concentrations of isoflavones may be contraindicated for postmenopausal women, who have a relatively low level of circulating estrogen. Hsieh et al. (111) found that dietary genistein stimulated the growth of MCF-7 (ER+) cells implanted subcutaneously into the skin of ovariectomized mice. In another study published by this research group, ISP containing increasing concentrations of genistein (15, 150, or 300 µg/g) stimulated growth of estrogen-dependent breast tumors in vivo in a dose-dependent manner (112). In a third study, an assessment of the tumor promoting effects of genistein at doses between 125 and 1000 µg/g in the diet was conducted in mice implanted with MCF-7 cells. Treatment with genistein at physiologic concentrations produced blood levels of genistein sufficient to stimulate breast cancer tumor growth in a dose-dependent manner, increasing cell proliferation at a concentration ≥250 µg/g (113). Few data exist regarding the effects on the normal human breast, but two studies have raised some concerns. The first examined the effect of soy on the proliferation rate of premenopausal normal breast epithelium and found a significant increase after 14 d of soy supplementation of 60 g/d containing 45 mg isoflavones (114). However, this was a very short study and there are other events, such as pregnancy and breastfeeding, that cause mammary cell proliferation and are linked to a reduction, not an increase, in breast cancer risk. Furthermore, in a follow-up study by Hargreaves et al. (115) that included all subjects (n = 84), no effects on cell proliferation were found. A study by Petrakis et al. (116) also suggested that soy has an estrogenic effect on the breast. In that study, 24 pre- and postmenopausal women consumed 38 g SPI/d (containing 38 mg of genistein), an increase in nipple aspirate fluid (NAF) was found in the premenopausal women; a minimal increase or no response was found in postmenopausal women. Also of concern was the appearance of hyperplasic epithelial cells in the NAF of 30% of the women, which is thought to be indicative of a modest increased risk of breast cancer. Although the study concluded that the prolonged daily consumption of SPI appeared to have a stimulatory effect on the premenopausal female breast, this was a poorly designed study with a number of design flaws, including lack of a control group. In summary, epidemiologic, in vitro, and in vivo studies have shown both protective and stimulatory effects of soy and soy isoflavones on breast cancer. Much controversy exists on the topic of whether women with ER+ tumors should avoid consuming soy products, particularly in relation to breast cancer because of the ability of the phytoestrogens to have both estrogenic and antiestrogenic effects. Some researchers suggest that due to the lack of large, rigorously controlled studies, recommendations for women to increase their soy intake to prevent breast cancer or prevent its recurrence are premature (105). Premenopausal and postmenopausal women have vastly different levels of circulating estrogen. It is possible that women with very low circulating levels of estrogen (as would be the case in postmenopausal women) who have an estrogen responsive tumor would respond adversely to very

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high levels of phytoestrogens by having the estrogen-responsive cancer be further stimulated to grow. Perhaps the most prudent recommendation may be to follow the recommendations of Messina and Loprinzi (96), and use Asian soy intake as a general guide for American women.

Summary and Conclusion Isoflavones are one of the major classes of phytoestrogens that have been the focus of an escalating number of in vitro, in vivo, epidemiologic, and clinical research studies related to their health effects over the last decade. The cardiovascular benefits of soy are the most clinically well-documented health effect associated with consumption of this functional food. There is significant scientific agreement that the consumption of soy protein reduces the major blood lipids associated with cardiovascular disease risk. Although the specific component of soy responsible for the lipid-lowering effect of soy is unknown, isoflavones per se are not likely responsible. However, isoflavones may have other cardiovascular benefits apart from their lipid-lowering effects, including their ability to reduce the oxidation of LDL and enhance arterial compliance. Isoflavone consumption causes small but significant reductions of vasomotor symptoms associated with menopause. At present, however, it does not appear that isoflavones can compete with standard estrogen replacement therapy for effectiveness in the relief of vasomotor symptoms associated with menopause. Although the data at present do not unequivocally support the use of soy as an alternative to HRT, soy isoflavones may provide an attractive addition to the choices available for relief of hot flushes and an option for women who wish to use a dietary approach to relieve hot flushes and associated menopausal symptoms. Isoflavones have been shown to have a positive effect on short-term markers of bone health. The limited number of published clinical trials currently precludes a recommended level of isoflavone intake to reduce the risk of osteoporosis. However, higher levels of isoflavones (≥80 mg) may be required and longer term studies (1–2 y) will be necessary to confirm the positive effects seen to date. Soy has been shown to be protective against breast cancer in women in epidemiologic studies, against mammary cancer in rat models, and also in human mammary cancer cell lines grown in culture. However, isoflavones have been shown to promote tumors in ovariectomized, athymic mice. Thus, one of the most controversial topics in the soy and breast cancer research area is whether high concentrations of isoflavones may be contraindicated for postmenopausal women who have a relatively low level of circulating estrogen or women at risk for breast cancer. Although a small number of animal studies have shown a tumor-promoting effect of isoflavones, there is no convincing evidence that soy promotes breast cancer in women. Soy appears to hold a wide range of health-promoting qualities for women if consumed in moderate amounts as part of a balanced diet in the context of an overall healthy lifestyle. There is a wealth of opportunity for further research studies in the area of soy for women’s health. With respect to cardiovascular disease, the leading killer of

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women, future studies should more closely examine nonlipid markers of heart disease risk, including oxidation of LDL, other markers of lipid peroxidation (e.g., F2isoprostanes) and endothelial/vascular function. Clearly, much more extensive work has to be done in investigating the role of soy, and particularly soy isoflavones, in reducing bone loss with special attention to the role this phytoestrogen may play in calcium metabolism. Finally, much further research is required in the area of the anti- vs. proestrogencic effects of soy isoflavones in breast cancer and the interaction of these compounds with breast cancer agents such as tamoxifen. There is a dire need for clinical trials in this area. References 1. International Life Sciences Institute (1999) International Life Sciences Institute North America Food Component Reports, Crit. Rev. Food. Sci. Nutr. 39, 203–316. 2. Dahm, L. (1999) The Top 100® R&D Survey, Food Proc. 60, 45–52. 3. Best, D. (2000) The Top 100® R&D Survey, Food Proc. 61, 18–20. 4. Kindle, L. (2001) The Top 100® R&D Survey, Food Proc. 62, 18–22. 5. Ohr, L. (2000) A Full Plate. Exclusive Annual R&D Survey, Prepared Foods 169, 36–49. 6. United Soybean Board (2001) 7th Annual National Report. Consumer Attitudes About Nutrition, United Soybean Board, Chesterfield, MO. 7. United Soybean Board (2002) 8th Annual National Report. Consumer Attitudes About Nutrition, United Soybean Board, Chesterfield, MO. 8. Industry News. http://www.soyatech.com. Accessed March 10, 2002. 9. Anonymous (2001) Women’s Supplements Show Strong Gains, Nutr. Bus. J. 6, 14–15. 10. Messina, M., and Erdman, J.W., Jr. (1995) First International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, J. Nutr. 125, 567S–808S. 11. Messina, M., and Erdman, J.W., Jr. (1998) The Role of Soy in Preventing and Treating Chronic Disease. Proceedings of a Symposium Held on September 15–18, 1996 and a Satellite Symposium Held on September 19, 1996 in Brussels, Belgium, Am. J. Clin. Nutr. 68, 1329S–1544S. 12. Anonymous (2001) Women’s Health Market Matures, Nutr. Bus. J. 6,13–15. 13. Setchell, K.D.R. (1998) Phytoestrogens: The Biochemistry, Physiology, and Implications for Human Health of Soy Isoflavones, Am. J. Clin. Nutr. 68, 1333S– 1346S. 14. Shutt, D.A., and Cox, R.I. (1972) Steroid and Phyto-Estrogen Binding to Sheep Uterine Receptors In Vitro, J. Endocrinol. 52, 299–310. 15. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.A. (1996) Cloning of a Novel Receptor Expressed in Rat Prostate and Ovary, Proc. Natl. Acad. Sci. USA 93, 5925–5930. 16. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.A. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor β, Endocrinology 139, 4252–4263. 17. Setchell, K.D.R. (2001) Soy Isoflavones—Benefits and Risks from Nature’s Selective Estrogen Receptor Modulators (SERMs), J. Am. Coll. Nutr. 20, 345S–362S. 18. Adlercreutz, H., Markkanen, H., and Watanabe, S. (1998) Plasma Concentrations of Phyto-Oestrogens in Japanese Men, Lancet. 342, 1209–1210. 19. Ignatowsky, M.A. (1908) Influence de la Nourriture Animale sur l’Organisme des Lapins, Arch. Med. Exp. Anal. Pathol., 1–20.

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20. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 21. Department of Health and Human Services, Food and Drug Administration (1999) Food Labeling: Health Claims; Soy Protein and Coronary Heart Disease, 21 CFR, Part 101, Fed. Regist. 64, 57700–57733. 22. Teixeira, S.M., Potter, S.M., Weigel, R., Hannum, S., Erdman, J.W., Jr., and Hasler, C.M. (2000) Effects of Feeding 4 Levels of Soy Protein for 3 and 6 Weeks on Blood Lipids and Apolipoproteins in Mildly Hypercholesterolemic Men, Am. J. Clin. Nutr. 71, 1077–1084. 23. Erdman, J.W., Jr. (2000) Soy Protein and Cardiovascular Disease. A Statement for Healthcare Professionals from the Nutrition Committee of the AHA, Circulation 102, 2555–2559. 24. Tikkanen, M.J., and Adlercreutz, H. (2000) Dietary Soy-Derived Isoflavone Phytoestrogens. Could They Have a Role in Coronary Heart Disease Prevention? Biochem. Pharmacol. 60, 1–5. 25. Nestel, P.J., Yamashita, T., Sasahara, T. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Arterioscler. Thromb. Vasc. Biol. 17, 3392–3398. 26. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., and Croft, K.D. (1998) Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations: A Randomized Controlled Trial in Humans, J. Nutr. 128, 728–732. 27. Nestel, P.J., Pomeroy, S., Kay, S., and Komesaroff, P. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895–898. 28. Samman, S., Lyons-Wall, P.M., Chan, G.S.M., Smith, S.J., and Petocz, P. (1999) The Effect of Supplementation with Isoflavones on Plasma Lipids and Oxidisability of Low Density Lipoprotein in Premenopausal Women, Atherosclerosis 147, 277–283. 29. Howes, J.B., Sullian, D., and Lai, N. (2000) The Effects of Dietary Supplementation with Isoflavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 154, 143–147. 30. Dewell, A., Hollenbeck, C.B., and Bruce, B. (2002) The Effects of Soy-Derived Phytoestrogens on Serum Lipids and Lipoproteins in Moderately Hypercholesterolemic Postmenopausal Women, J. Endocrinol. Metab. 87, 118–121. 31. Crouse, J.R., Morgan, T., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with That of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070–2076. 32. Merz-Demlow, B.E., Duncan, M., Underhill, K.E.W., Xu, X., Carr, T.P., Phipps, L.W.R., and Kurzer, M.S. (2000) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic, Premenopausal Women, Am. J. Clin. Nutr. 71, 1462–1469. 33. Wangen, K.E., Duncan, A.M., Xu, X., and Kurzer, M.S. (2001) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic and Mildly Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 225–231. 34. Friedman, M., and Brandon, D.L. (2001) Nutritional and Health Benefits of Soy Proteins, J. Agric. Food Chem. 49, 1069–1086. 35. Witztum, J.L. (1994) The Oxidation Hypothesis of Atherosclerosis, Lancet 344, 793– 795. 36. Wiseman, W., O’Reilly, J.D., and Adlercreutz, H. (2000) Isoflavone Phytoestrogens Consumed in Soy Decrease F2-Isoprostane Concentrations and Increase Resistance of Low-Density Lipoprotein to Oxidation in Humans, Am. J. Clin. Nutr. 72, 395–400.

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37. Jenkins, D.J.A., Kendall, C.W.C., Vidgen, E., and Mehling, C.C. (2000) The Effect on Serum Lipids and Oxidized Low-Density Lipoprotein of Supplementing Self-Selected Low-Fat Diets with Soluble-Fiber, Soy, and Vegetable Protein Foods, Metabolism 49, 67–72. 38. Jenkins, D.J.A., Kendall, C.W.C., Vidgen, E., and Vuksan, V. (2000) Effect of SoyBased Breakfast Cereal on Blood Lipids and Oxidized Low-Density Lipoprotein, Metabolism 49, 1496–1500. 39. Dart, A., Silagy, C., Dewar, E., Jennings, G., and McNeil, J. (1993) Aortic Distensibility and Left Ventricular Structure and Function in Isolated Systolic Hypertension, Eur. Heart J. 14, 1465–1470. 40. Lieberman, E.H., Gerhard, M.D., and Uehata, A. (1997) Estrogen Improves Endothelium Dependent Flow-Mediated Vasodilation in Postmenopausal Women, Ann. Intern. Med. 121, 936–941. 41. Nestle, P.J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P., Owen, A., and Abbey, M. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Arterioscler. Thromb. Vasc. Biol. 17, 3392–3398. 42. Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J.D., and West, L. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895–898. 43. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2001) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy, Postmenopausal Women, Am. J. Cardiol. 85, 1297–1301. 44. Walker, H.A., Dean, T.S., Sanders, T.A.B., Jackson, G., Ritter, J.M., and Chowienczyk, P.J. (2001) The Phytoestrogen Genistein Produces Acute Nitric OxideDependent Dilation of Human Forearm Vasculature with Similar Potency to 17βEstradiol, Circulation 103, 258–262. 45. International Menopause Society, CAMS Definitions. http://www.imsociety.org/ pages/menuframeset.html, accessed 3/1/02. 46. Love, S.M. (1997) Dr. Susan Love’s Hormone Book. Making Informed Choices About Menopause, Random House, New York. 47. Chen, C., Weiss, N.S., Newcomb, P., Barlow, W., and White, E. (2002) Hormone Replacement Therapy in Relation to Breast Cancer, J. Am. Med. Assoc. 287, 734–741. 48. American College of Obstetricians and Gynecologists (2001) Use of Botanicals for Management of Menopausal Symptoms, ACOG Pract. Bull. 28. (Online at http://www. acog.org/from_home/publications/misc/pb028.htm). 49. Lock, M. (1991) Contested Meaning of the Menopause, Lancet 337, 1270–1272. 50. Notelovitz, M. (1989) Estrogen Replacement Therapy Indications, Contraindications and Agent Selection, Am. J. Obstet. Gynecol. 167, 8–17. 51. Boulet, M.J., Oddens, B.J., Lehert, P., Vemer, H.M., and Visser, A. (1994) Climacteric and Menopause in Seven South-East Asian Countries, Maturitas 19, 157–176. 52. Upmalis, D.H., Lobo, R., and Bradley, L. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: A Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 53. Scambia, G., Mango, D., Signorile, P.G., Angeli, R.A., Palena, C., Gallo, D., Bombardelli, E., Morazzoni, P., Riva, A., and Mancuso, S. (2000) Clinical Effects of a Standardized Soy Extract in Postmenopausal Women: A Pilot Study, Menopause 7, 105–111.

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54. Quella, S.K., Loprinzi, C.L., Barton, D.L., Knost, J.A., Sloan, J.A., LaVasseru, B.I., Swan, D., Kurpp, K.R., Miller, K.D., and Novotny, P.J. (2000) Evaluation of Soy Phytoestrogens for the Treatment of Hot Flashes in Breast Cancer Survivors: A North Central Cancer Treatment Group Trial, J. Clin. Oncol. 18, 1068–1074. 55. Murkies, A.L., Lombard, C., Strauss, B.J.G., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Postmenopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 56. Brzezinski, A., Adlercreutz, H., Shaoul, R., Rosler, A., Shmueli, A., Tanos, V., and Schenker, J.G. (1997) Short-Term Effects of Phytoestrogen-Rich Diet on Postmenopausal Women, Menopause 4, 89–94. 57. Albertazzi, P., Pasini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and Dealoysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 58. St. Germain, A., Peterson, C.T., Robinson, J.G., and Alekel, L. (2001) Isoflavone-Rich or Isoflavone-Poor Soy Protein Does Not Reduce Menopausal Symptoms During 24 Weeks of Treatment, Menopause 8, 17–26. 59. Washburn, S., Burke, G.L., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13. 60. North American Menopause Society (2000) The Role of Isoflavones in Menopausal Health: Consensus Opinion of the North American Menopause Society, Menopause 8, 84–95. 61. Reid, I.R. (1997) Osteoporosis—Emerging Consensus, Aust. N.Z. J. Med. 27, 643–647. 62. Cauley, J.(1997) Hormone Replacement Therapy and Osteoporosis, Soy Connection 5, 2. 63. Fox, R.N., Hcan, J.K., Thamer, M., and Melton, J.L., III (1997) Medical Expenditures for the Treatment of Osteoporotic Fractures in the United States in 1995: Report from the National Osteoporosis Foundation, Journal of Bone and Mineral Research 12, 24– 35. 64. National Osteoporosis Foundation (2002) America’s Bone Health: The State of Osteoporosis and Low Bone Mass in Our Nation, Washington, DC. 65. Cooper, C., Campion, G., and Melton, L.J. (1992) Hip Fractures in the Elderly: A WorldWide Projection, Osteoporos. Int. 2, 285–289. 66. DeKleijn, M.J.S., an de Schouw, Y.T., Wilson, P.W.F., Adlercreutz, H., Mazur, W., Grobbee, D.E., and Jacques, P.F. (2001) Intake of Dietary Phytoestrogens is Low in Postmenopausal Women in the United States: The Framingham Study, J. Nutr. 131, 1826–1832. 67. Anderson, J.J.B., and Garner, S.C. (1998) Phytoestrogens and Bone, in Balliere’s Clinical Endocrinology and Metabolism, (Adlercreutz, H.L., ed.) pp. 1–16, Balliere Tyndall, London. 68. Sugimoto, E., and Yamaguchi, M. (2000) Stimulatory Effect of Diaidzein in Osteoblastic MC3T3-E1 Cells, Biochem. Pharmacol. 35, 471–475. 69. Gao, Y.H., and Yamaguchi, M. (1999) Inhibitory Effect of Genistein on Rat Bone Osteoclasts-Like Cell Formation in Mouse Marrow Cultures, Biochem. Pharmacol. 58, 767–772. 70. Notoya, K., Yoshida, K., Taketomi, S., Yamazaki, I., and Kumegawa, M. (1993) Inhibitory Effect of Ipriflavone on Osteoclast-Mediated Bone Resorption and New Osteoclast Formation in Long-Term Cultures of Mouse Unfractionated Bone Cells, Calcif. Tissue Int. 53, 206–209. 71. Giossi, M., Caruso, P., Civelli, M., and Bongrani, S. (1996) Inhibition of Parathyroid Hormone-Stimulated Resorption in Cultured Fetal Rat Long Bones by the Main Metabolites of Ipriflavone, Calcif. Tissue Int. 58, 419–422.

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72. Gambacciani, M., Ciaponi, M., Cappagli, B., Piaggesi, L., and Genazzani, A.R. (1997) Effects of Combined Low Dose of the Isoflavone Derivative Ipriflavone and Estrogen Replacement on Bone Mineral Density and Metabolism in Postmenopausal Women, Maturitas 28, 75–81. 73. Adami, S., Bufalino, L., Cervetti, R., Dimarco, C., Dimunno, O., Fantasia, L., Isaia, G.C., Serni, U., Vecchiet, L., Passeri, M., Castiglione, G.N., Gardini, F., Letizia, G., Occhipinti, L., Pardini, N., Agamennone, M., Sciolla, A., Matucci, A., Riboldi, R., Costi, D., Dallaglio, E., and Pedrazzoni, M. (1997) Ipriflavone Prevents Radial Bone Loss in Postmenopausal Women with Low Bone Mass over 2 Years, Osteoporosis Int. 7, 119–125. 74. Gennari, C., Adami, S., and Agnusdei, D. (1997) Effect of Chronic Treatment with Ipriflavone in Postmenopausal Women with Low Bone Mass, Calcif. Tissue Int. 61, S19–S22. 75. Kovacs, A.B. (1994) Efficacy of Ipriflavone in the Prevention and Treatment of Postmenopausal Osteoporosis, Agents Actions 41, 86–87. 76. Passeri, M., Biondi, M., and Costi, D. (1992) Effect of Ipriflavone on Bone Mass in Elderly Osteoporotic Women, Bone Min. 19, S57–S62. 77. Arjmandi, B.H., Birnbaum, R., Goyal, N.V., Getlinger, M.J., Juma, S., Alekel, L., Hasler, C.M., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Bone-Sparing Effect of Soy Protein in Ovarian Hormone-Deficient Rats Is Related to Its Isoflavone Content, Am. J. Clin. Nutr. 68, 1364S–1369S. 78. Arjmandi, B.H., Alekel, L., and Hollis, B.W. (1996) Dietary Soybean Protein Prevents Bone Loss in an Ovariectomized Rat Model of Osteoporosis, J. Nutr. 126, 161–167. 79. Fanti, P., Monier-Faugere, M.C., Geng, Z., Schmidt, J., Morris, P.E., Cohen, D., and Malluche, H.H. (1998) The Phytoestrogen Genistein Reduces Bone Loss in ShortTerm Ovariectomized Rats, Osteoporos. Int. 8, 274–281. 80. Picherit, C., Coxam, V., Bennetau-Pelissero, C., Kati-Coulibaly, S., Davicco, M.J., Lebecque, P., and Barlet, J.P. (2000) Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats, J. Nutr. 130, 1675–1681. 81. Ho, S.C., Chan, S.G., Yi, Q.L., Wong, E., and Leung, P.C. (2001) Soy Intake and the Maintenance of Peak Bone Mass in Hong Kong Chinese Women, J. Bone Min. Res. 16, 1363–1369. 82. Mei, J., Yeung, S.S.C., and Kung, A.W.C. (2001) High Dietary Phytoestrogen Intake Is Associated with Higher Bone Mineral Density in Postmenopausal but Not Premenopausal Women, J. Clin. Endocrinol. Metab. 86, 5217–5221. 83. Horiuchi, T., Onouchi, T., Takahashi, M., Ito, H., and Orimo, H. (2000) Effect of Soy Protein on Bone Metabolism in Postmenopausal Japanese Women, Osteoporos. Int. 11, 721–724. 84. Wangen, K.E., Duncan, A.M., Merz-Demlow, B.E., Xu, X., Marcus, R., Phipps, W.R., and Kurzer, M.S. (2000) Effects of Soy Isoflavones on Markers of Bone Turnover of Premenopausal and Postmenopausal Women, J. Clin. Endocrinol. Metab. 85, 3043– 3048. 85. Potter, S.M., Baum, J.A., Teng, H., Stillman, R.J., Shay, N.F., and Erdman, J.W., Jr. (1998) Soy Protein and Isoflavones: Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68, 1375S–1379S. 86. Alekel, L., St. Germain, A., Peterson, C.T., Hanson, K., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in Lumbar Spine of Perimenopausal Women, Am. J. Clin. Nutr. 729, 844–852. 87. American Cancer Society (2002) Cancer Facts & Figures 2002, p. 9, ACS, Atlanta.

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88. Messina, M. and Barnes, S. (1991) The Role of Soy Products in Reducing Risk of Cancer, J. Natl. Cancer Inst. 83, 541–546. 89. Barnes, S., Peterson, T.G., and Coward, L. (1995) Rationale for the Use of GenisteinContaining Soy Matrices in Chemoprevention Trials for Breast and Prostate Cancer, J. Cell Biochem. 22S, 181–187. 90. Constantinou, A.I., Krygier, A.E., and Mehta, R.R. (1998) Genistein Induces Maturation of Cultured Human Breast Cancer Cells and Prevents Tumor Growth in Nude Mice, Am. J. Clin. Nutr. 68, 1426S–1430S. 91. Fotsis, T., Pepper, M., Adlercreutz, H., Fleischann, G., Hase, T., Montesano, R., and Schweigerer, L. (1993) Genistein, a Dietary-Derived Inhibitor of In Vitro Angiogenesis, Proc. Natl. Acad. Sci. U.S.A. 90, 2690–2694. 92. Ruiz-Larrea, M.B. (1997) Antioxidant Activity of Phytoestrogenic Isoflavones, Free Radic. Res. 26, 63–70. 93. Kim, H., Peterson, G.T., and Barnes, S. (1998) Mechanisms of Action of the Soy Isoflavone Genistein: Emerging Role for Its Effects via Transforming Growth Factor Signaling Pathways, Am. J. Clin. Nutr. 68, 1418S–1425S. 94. Stoll, B.A. (1997) Eating to Beat Breast Cancer: Potential Role for Soy Supplements, Ann. Oncol. 8, 223–225. 95. Barnes, S. (1995) Effect of Genistein on In Vitro and In Vivo Models of Cancer, J. Nutr. 125, 777S–783S. 96. Messina, M.J., and Loprimzi, C.L. (2001) Soy for Breast Cancer Survivors: A Critical Review of the Literature, J. Nutr. 131, 3095S–3108S. 97. Constantinou, A.I., Xu, H., Cunningham, E., Lantvit, D., and Pezzuto, J. (2001) Consumption of Soy Products May Enhance the Breast Cancer-Preventive Effects of Tamoxifen, Proc. Am. Assoc. Canc. Res. 42, 826–827. 98. Bouker, K.B., and Hilakivi-Clarke, L. (2000) Genistein: Does It Prevent or Promote Breast Cancer? Environ. Health Perspect. 108, 701–708. 99. Fournier, D.B., Erdman, J.W., Jr., and Gordon, G.B. (1998) Soy, Its Components, and Cancer Prevention: A Review of the In Vitro, Animal, and Human Data, Cancer Epidemiol. Biomark. Prev. 7, 1055–1065. 100. Santell, R.C., Kieu, N., and Helferich, W.G. (2000) Genistein Inhibits Growth of Estrogen-Independent Human Breast Cancer Cells in Culture but Not in Athymic Mice, J. Nutr. 130, 1665–1669. 101. Lee, H.P., Gourley, L., Duffy, S.W., Esteve, J., Lee, J., and Day, N.E. (1991) Dietary Effects on Breast-Cancer Risk in Singapore, Lancet 337, 1197–2000. 102. Wu, A.H., Ziegler, R.G., Horn-Ross, P.L., Nomura, A.M.Y., West, D.W., Kolonel, L.N., Rosenthal, J.F., Hoover, R.N., and Pike, M.C. (1996) Tofu and Risk of Breast Cancer in Asian-Americans, Cancer Epidemiol. Biomark. Prev. 5, 901–906. 103. Key, T.J., Sharp, G.B., Appleby, P.N., Beral, V., Goodman, M.T., Soda, M., and Mabuchi, K. (1999) Soya Foods and Breast Cancer Risk: A Prospective Study in Hiroshima and Nagasaki, Japan, Br. J. Cancer 81, 1248–1256. 104. Fleischauer, A.T., Mead, M.M., Anthony, A.S., Gaudet, M.. and Arab, L. (2001) A Meta- Analysis of Soy Food Consumption and Risk of Breast Cancer, Am. J. Epidemiol. S32 (abstr.). 105. Trock, B., Butler, L.W., Clarke, R., and Hilakivi-Clarke, L. (2000) Meta-Analysis of Soy Intake and Breast Cancer Risk, J. Nutr. 130, 690S–691S (abstr.). 106. Shu, X.O., Jin. F., Dai, Q., Wen, W.Q., Potter, J.D., Kushi, L.H., Ruan, Z.X., Gao, Y.T., and Zheng, W. (2001) Soyfood Intake During Adolescence and Subsequent Risk of Breast Cancer Among Chinese Women, Cancer Epidemiol. Biomark. Prev. 10, 483–488.

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107. Lamartiniere, C.A., Moore, J., Holland, M., and Barnes, S. (1995) Neonatal Genistein Chemoprevents Mammary Cancer, Soc. Exp. Biol. Med. 208, 120–123. 108. Lamartiniere, C.A., Murrill, W.B., Manzolillo, P.A., Zhang, J.-X., Barnes, S., Zhang, X., Wei, H., and Brown, N.M. (1998) Genistein Alters the Ontogeny of Mammary Gland Development and Protects Against Chemically-Induced Mammary Cancer in Rats, Proc. Soc. Exp. Biol. Med. 217, 358–664. 109. Badger, T., Hakkak, R., Korourian, S., Ronis, M., Rowlands, C., and Shelnutt, S. (1999) Differential and Tissue Specific Protective Effects of Diets Formulated with Whey or Soy Proteins on Chemically-Induced Mammary and Colon Cancer in Rats, FASEB J. 13, A583 (abstr.). 110. Hakkak, R., Korourian, S., Ronis, M., Shelnutt, S., Lensing, S., Sweeney, J., Irby, D., Weatherford, C., and Badger, T. (1999) Dietary Whey or Soy Prevents DMBA-Induced Mammary Cancer in Rats: Effects of Diets Fed over Two Generations, FASEB J. 13, A583 (abstr.). 111. Hseih, C.Y., Santell, R.C., Haslam, S.Z. and Helferich, W.G. (2001) Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-Positive Human Breast Cancer (MCF-7) Cells In Vitro and In Vivo, Cancer Res. 58, 3833–3838. 112. Allred, C.D., Allred, K.F., Ju, Y.H., Virant, S.M., and Helferich, W.G. (2001) Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-Dependent (MCF-7) Tumors in a Dose-Dependent Manner, Cancer Res. 61, 5045–5050. 113. Young, H.J., Allred, C.D., Allred, K.F., Karko, K.L., Doerge, D.R., and Helferich, W.G. (2001) Physiological Concentrations of Dietary Genistein Dose-Dependently Stimulate Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Athymic Nude Mice, J. Nutr. 131, 2957–2962. 114. McMichael-Phillips, D.F., Harding, C., Morton, M., Roberts, S.A., Howell, A., Potten, C.S., and Bundred, N.J. (1998) Effects of Soy-Protein Supplementation on Epithelial Proliferation in the Histologically Normal Human Breast, Am. J. Clin. Nutr. 68, 1431S–1435S. 115. Hargreaves, D.F., Potten, C.S., Harding, C., Shaw, L.E., Morton, M.S., Roberts, S.A., Howell, A., and Bundred, N.J. (1999) Two-Week Dietary Soy Supplementation Has an Estrogenic Effect on Normal Premenopausal Breast, J. Clin. Endocrinol. Metab. 84, 4017–4024. 116. Petrakis, N.L., Barnes, S., King, E.B., Lowenstein, J., Wiencke, J., Lee, M.M., Miike, R., Kirk, M., and Coward, L. (1996) Stimulatory Influence of Soy Protein Isolate on Breast Secretion in Pre- and Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 5, 785–794.

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Chapter 3

Isoflavone Supplements: Arguments For and Against Their Use Mark Messina Loma Linda University, Loma Linda, CA and Nutrition Matters, Incorporated, Seattle, WA

Introduction Supplements have become a multibillion-dollar business; reportedly, 60% of Americans use supplements on a regular basis (1). Historically, the established nutrition community has shunned the use of supplements by the general population as a means of meeting nutrient needs and instead, emphasized the importance of consuming a varied diet conforming to government guidelines. However, despite recent disappointing results from clinical trials involving isolated nutrients (2,3), attitudes toward supplements are changing for a multiplicity of reasons. One of these reasons is the recognition that many people do not eat as they should and that as a result, substantial numbers of Americans do not meet the Dietary Reference Intake for a variety of nutrients (4,5). For example, it is readily acknowledged that few postmenopausal women meet calcium requirements and that only through the use of supplements and/or fortified foods is this even possible (6). Similarly, despite the fact that folate is readily available in commonly consumed foods, concerns over inadequate folate intake by women of childbearing age recently led to folate fortification of grains to prevent neural tube defects (7). A second reason for the changing attitude toward supplements is the extent to which there is scientific support for consuming certain nutrients, such as vitamins C (8) and E (9), in amounts beyond what is realistically possible through the consumption of foods alone. Third, many foods that represent the best and in some cases only nutritionally relevant dietary sources of specific phytochemicals, such as flaxseed (lignans) and soy foods (isoflavones), are not currently part of most American diets. Consequently, many consumers view phytochemical supplements as one important means of increasing their phytochemical intake. One phytochemical supplement that is becoming popular is comprised of isoflavones. The increasing interest in isoflavone supplements is undoubtedly linked to the phenomenal rise in attention given to the health effects of soy during the past 10 years. Although there are a number of biologically active compounds in soybeans, the primary focus has been on the isoflavones. At present, ~600 scientific and medical papers on isoflavones are currently published in peer-reviewed journals each year (Fig. 3.1). Consumer interest in isoflavones led to the development of isoflavone supplements derived primarily from soybeans, but also kudzu and red clover. Isoflavones

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700 600 Publications

500 400 300 200 100 0

1985

1991

1993 1995 Years Fig. 3.1. Results of isoflavone MEDLINE search.

1998

are also used as food fortificants and are being added to nonsoy and soy products. One particular type of isoflavone, Novasoy, which is produced by the Archer Daniels Midland Company, Decatur, IL, has achieved generally recognized as safe (GRAS) status through self-affirmation and review by an outside panel of experts. It can be added to adult single meal replacements, and health beverages and bars. Not surprisingly, the availability of isoflavone supplements has prompted considerable discussion within the scientific community. More specifically, many experts have enthusiastically endorsed the consumption of soy foods but have spoken out against the use of isoflavone supplements. The purpose of this chapter is to evaluate the merits of each of the major arguments commonly cited against the use of isoflavone supplements as a guide for health professionals to allow them to give their clients or patients appropriate counsel. For purposes of this discussion, supplements do not include soy protein powders, but only preparations that are primarily isoflavones, or a combination of isoflavones and saponins, and that are derived from soybeans. Also, the comments below apply only to those supplements which have an isoflavone profile that reflects the isoflavone composition of soybeans.

Objection 1. Isoflavone Supplements Do Not Provide the Same Health Benefits as Soy Foods Soybeans contain numerous biologically active constituents (10), including phytic acid (11–14), phenolic acids (15), saponins (16–20), oligosaccharides (21,22), protease inhibitors [Bowman-Birk Inhibitor (BBI)] (23,24), glyceollins (stressed soybeans only) (25–27), phytosterols (28,29), α-linolenic acid (30), vitamin E (31), and soy protein/peptides (32–40). Collectively, these compounds are responsible for the hypothesized health effects of soy, including alleviation of menopausal

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symptoms and protection against breast and prostate cancer, coronary heart disease (CHD), osteoporosis, and declines in cognitive function. Consequently, supplements containing primarily isoflavones cannot duplicate the effects of soy.

Objection 1. Evaluation Cancer Investigation of the anticancer effects of soy, largely because of the low cancer rates in Japan, has focused primarily on breast and prostate cancer (41). There is ample reason to suspect that if soy does reduce the risk of these two cancers, isoflavones are responsible for the protective effects. Multiple mechanisms by which isoflavones reduce breast and prostate cancer risk have been proposed. For example, under some experimental conditions, isoflavones exhibit antiestrogenic (42–48) and possibly antiandrogenic (49) effects. In addition, isoflavones, especially genistein, influence signal transduction in ways that are relevant to the inhibition of both breast and prostate cancer (50–52). This explains why genistein, the primary isoflavone in soybeans, inhibits the growth of hormone-dependent and hormone-independent breast (53,54) and prostate cancer cells (55,56) in vitro. Furthermore, genistein inhibits the invasive potential of cancer cells independent of cell growth (56,57), and inhibits angiogenesis in vitro (58,59) and in vivo in animals (60,61). Although there are several anticarcinogens in soy, as detailed by Messina and Flickenger (62), soy consumption in Japan contributes relatively little to the total intake of most of these anticarcinogens. For example, soy accounts for <5% of the phenolic acid and phytosterol intake, and only 10–20% of the vitamin E, phytic acid, protein, and α-linolenic acid intake (Table 3.1). However, soy contributes ~80, 90, and 100% of the total saponin, protease inhibitor, and isoflavone intake, respectively (62). Thus, logically, if soy does contribute to the low breast and prostate cancer rates in Japan, only the last three constituents are likely responsible for this effect. Although saponins may reduce the risk of colon cancer (63), there is little experimental evidence indicating that saponins reduce breast and prostate cancer risk. Their poor bioavailability limits their potential in this regard (64). Similarly, although the BBI is an exciting chemopreventive agent (23) for certain cancers, there is little evidence to date indicating that the BBI inhibits breast and prostate carcinogenesis especially when considering the relatively low amount of the inhibitor provided by typical soy consumption in Japan. In contrast, although still speculative, the evidence suggests that isoflavones do contribute to the low Japanese breast and prostate cancer rates. Breast Cancer Animal Studies. Findings from animal research are inconsistent, but more studies than not show that the substitution of soy protein for casein modestly (25–50%) reduces tumor incidence or, more commonly, tumor multiplicity (32,65–70). The rel-

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TABLE 3.1 Estimated Amount of Putative Anticarcinogens Provided by Soy Intake and the Relative Contribution of Soy to the Total Japanese Intake of Anticarcinogens Found in Soy Anticarcinogen

Amount in soy

Total Japanese intake

Contribution (%) from soy

30 mg 7.5 mg 34 mg 314 mg 298 mg 8.0 g 690 µg αTEb 15 mg <1 mg

30 mg 8.3 mg 42 mg 1656 mg 2.17 ga 80 g 6.7 mg 370 mg 200 mg

100 90 80 19 14 10 10 4 <1

Isoflavones Bowman-Birk Inhibitor Saponins Phytate α-Linolenic acid Protein Vitamin E Phytosterols Phenolic acids aTotal bαTE,

n-3 fatty acid intake. α-tocopherol equivalents.

evance of these animal data should be questioned, however. These studies are typically designed such that a single protein source provides ~20% (dry weight basis) of the total food intake. In contrast, soy protein accounts for only ~10% of total Japanese protein intake and ~5% of total energy (62). Of course, it is argued that the large amounts of protective agents are required because animals are exposed to such large amounts of carcinogens (sufficient to induce carcinogenesis). Nevertheless, animal studies that focus primarily on protein, reveal only whether soy protein is more or less carcinogenic than casein (the default control protein) when each is provided in amounts far beyond that consumed by the Japanese population. More than 10 years ago, Barnes et al. (67,70) found that alcohol-extracted soy protein was less effective in inhibiting chemically induced mammary tumor development than unextracted protein, suggesting that isoflavones were responsible for the protective effects. More recently, however, Constantiou et al. (71) found that soy protein without isoflavones was slightly more inhibitory against the development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors than isoflavone-rich soy protein. But in contrast to both studies, Cohen et al. (72) did not find that either isoflavone-rich soy protein or isoflavone-depleted soy protein significantly affected N-nitroso-N-methylurea-induced mammary tumorigenesis. Thus, collectively, studies involving soy protein appear to be unable to provide much insight into the possible anticancer effects of isoflavones. The results from studies in which the effects of isolated isoflavones or isoflavone-rich extracts on mammary tumorigenesis have been examined are not particularly impressive; however, they are generally comparable to the findings from studies using soy protein and other soy products (66,69,73–76). Thus, overall, these studies are not inconsistent with the notion that isoflavones account for the anticancer effects of soy, but the data are not especially convincing that either isolated isoflavones or soy protein inhibits the development of mammary cancer.

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Epidemiologic Studies. The epidemiologic data provide little support for the notion that the adult consumption of soy reduces postmenopausal breast cancer risk, and only modest support that it reduces premenopausal breast cancer risk (77– 82). Recently, a large case-control study by Dai et al. (83) found that soy intake among women in Shanghai was associated with reduced risks of both pre- and postmenopausal breast cancer. However, post-hoc subgroup analysis indicated that protective effects were limited to estrogen receptor-positive (ER+) women and women who had a body mass index ≥25 kg/m2. These findings suggest, but by no means prove, that soy was exerting protective effects through an antiestrogenic mechanism, which certainly points to the involvement of isoflavones. Also, these results allow for the possibility that the failure of previous epidemiologic studies to find protective effects of soy against breast cancer may have been because these studies did not stratify women into appropriate subgroups. However, the importance of these findings is seemingly attenuated because no dose-response relationship between soy intake and risk reduction was observed. This suggests that despite controlling for confounding variables, soy consumption may simply be reflective of some lifestyle that is protective against breast cancer, rather than being protective itself. Early Isoflavone Exposure. Epidemiologic data are generally viewed as critical to establishing the validity of any dietary hypothesis. However, arguably, the unimpressive results from the case-control and prospective studies actually strengthen the argument for isoflavones being the primary anticarcinogen in soy. This is because in rats, early exposure to genistein during the perinatal and prepubertal periods for just brief periods of time has repeatedly been shown to markedly reduce chemically induced mammary cancer in adulthood (84,85). Furthermore, Lamartiniere et al. (86) found that when genistein was given to adult animals, it reduced mammary carcinogenesis only when those animals were also first exposed to genistein when young. These findings are consistent with the known protective effects of early pregnancy on breast cancer risk (87), the effects of exposing animals to estrogen early in life (88), and migration data, which highlight the importance of early life experiences on breast cancer risk (89). Most importantly, they are consistent with the results of a large case-control study showing that women who consumed tofu during their teenage years (13–15 y) were ~50% less likely to develop premenopausal and postmenopausal breast cancer as adults compared with women who infrequently consumed soy as teenagers (90). Collectively, these data suggest that if soy contributes to the low breast cancer rates in Japan, it does so because of the effect of isoflavones in girls and young women. Prostate Cancer Animal Studies. The International Health Study Prostate Group recently concluded that isoflavones were responsible for preventing the progression of latent prostate

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cancer to the more advanced stages of this disease (91). In vitro rodent, and human data exist to support this hypothesis. In vitro, genistein inhibits the growth (55,92) and metastatic potential of prostate cancer cells (56). In severe combined immunedeficient mice implanted with LNCaP human prostate cancer cells, Zhou et al. (61) found that isoflavones inhibited tumor growth in a dose-dependent manner. Also, Dalu et al. (93) found that genistein administration downregulated epidermal growth factor receptor levels in the rat prostate despite rather low prostate genistein concentrations. This suggests, as noted by Zhou et al. (61), that genistein may actually be more potent in vivo than in vitro, and therefore, that the rather high genistein concentrations required to inhibit the growth of prostate cancer cells in vitro may be relevant to humans consuming soy. Finally, isoflavone-rich soy protein (soy+) was shown to inhibit both spontaneously-formed and chemically induced prostate cancer in Lobund-Wistar rats compared with soy protein low in, or nearly devoid of isoflavones (soy–) (94–97). Interestingly, on the basis of their work in Lobund-Wistar rats, Pollard et al. (96) concluded that isoflavones inhibit prostate cancer but that components in soy meal may inhibit their anticarcinogenic effects. Human Studies. The epidemiologic data on soy intake and prostate cancer risk are fairly limited, but worth noting are the results from two prospective epidemiologic studies. In one, Japanese men in Hawaii who consumed tofu approximately once per day, were 65% less likely to develop prostate cancer compared with men eating tofu less than once per week (98). In the other study, Seventh-day Adventist men in California who consumed soy milk more than once per day were 70% less likely to develop prostate cancer than men who did not consume soy milk (99). The pronounced protective effects of soy consumption in these studies is striking, but in both studies, the number of men who developed prostate cancer was relatively small. Relatively little clinical work has been conducted, but Morton et al. (100) did find that isoflavone levels in prostatic fluid are higher in men from soy food-consuming countries than in those from countries in which soy is not consumed, and that isoflavones are concentrated in the prostatic fluid by about twofold relative to the serum. Thus, the prostate gland is exposed to high concentrations of isoflavones in men who eat soy foods. However, Urban et al. (101) failed to find that soy consumption lowered prostate specific antigen (PSA) levels in healthy men, but this was a short-term trial (6 wk), involving men with relatively low PSA levels. More importantly, and in contrast to this study, Kucak et al. (102) from the Karmanos Cancer Institute in Detroit, MI, recently reported that in a 6-mo study, more than half of the 41 patients with uncontrolled cancer as determined by a rising PSA level, responded favorably (as judged by PSA levels) to daily supplements of isoflavones (60 mg in aglycone units). Collectively, the evidence overwhelmingly supports the notion that if soy consumption reduces prostate cancer risk, isoflavones alone are responsible for this effect.

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Coronary Heart Disease Cholesterol Reduction. In 1999, the Food and Drug Administration (FDA) approved a health claim for the cholesterol-lowering effects of soy protein (103). Although there are several soybean constituents, e.g., saponins, fiber, and phytic acid, that have been reported to be hypocholesterolemic (104), there is debate in particular about whether isoflavones enhance the cholesterol-lowering effects of soy protein. There is evidence both for (101,105,106) and against this hypothesis (107,108). The FDA does not require that soy protein contain a certain level of isoflavones to qualify for the health claim. Almost certainly, isoflavones alone do not lower low density lipoprotein cholesterol (LDL-C) although there is some evidence that they raise high density lipoprotein cholesterol (HDL-C) levels (109– 111). Thus, isoflavone supplements cannot be used as a means to lower cholesterol; consequently, they are not appropriate substitutes for soy foods for people whose primary purpose for consuming soy is to lower cholesterol. Effects Independent of Cholesterol Reduction. The hypocholesterolemic effects of soy protein are rather modest, and arguably, cholesterol reduction is not the most important means by which soy reduces CHD risk. Independent of cholesterol reduction, isoflavones are thought to favorably affect a number of biological measures of CHD risk. This is not surprising given the estrogen-like effects of isoflavones, and the many proposed mechanisms by which estrogen reduces CHD risk (112). Consistent with this notion, Ni et al. (113) found that in apolipoprotein E-deficient rats fed soy+ or casein plus isoflavones, the development of atherosclerosis was reduced compared with rats fed soy– or casein. Similarly, the atherosclerotic lesion area of the aortic arch was significantly smaller in rabbits given isoflavones compared with control rabbits and rabbits given saponins (114). There has been much study of the effects of soy and isoflavones on the coronary vessels, especially endothelium-independent and -dependent vasodilation. For example, in a 6-mo study in premenopausal monkeys, soy+ inhibited coronary artery vascular constriction in response to acetylcholine (an endothelium-dependent vascular response) by ~12% compared with soy–; intravenously administered genistein in animals fed soy– also produced vasodilation (115). However, no effects on vascular reactivity were noted in ovariectomized monkeys, suggesting that there is an interaction between estrogen and isoflavones (116). Nestel et al. (117) found that isolated isoflavones enhanced systemic arterial compliance (SAC) by 26%, similar to the effect seen with estrogen. SAC is an indicator of vascular elasticity and an independent measure of CHD (118). However, neither in this study nor in one by Simons et al. (109) did isolated isoflavones have an effect on endothelium-dependent flow-mediated dilation. In contrast, Walker et al. (119) found that intravenously administered genistein caused L-arginine/nitric oxide (NO)-dependent vasodilation in forearm vasculature of humans with potency similar to 17β-estradiol and potentiated endotheliumdependent vasodilation to acetylcholine.

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Although the relevance to CHD risk in humans is unclear, there are also studies indicating that isoflavones enhance nitric oxide synthase activity (120), reduce arterial lipid oxidation (114,121), and inhibit the migration and proliferation of smooth muscle cells (a primary cell type found in arterial plaque) (122-125). Additionally, in vitro genistein has been found to inhibit platelet aggregation (126) and platelet serotonin uptake (127). Williams and Clarkson (128) found that in vitro platelet aggregation in response to thrombin and serotonin was reduced in platelets collected from animals fed soy+ compared with soy–. These findings agree with those of Schoene and Guidry (129), who found that platelets from rats fed soy+ had apparent volumes that were significantly smaller than platelets from rats fed soy–, suggesting that these platelets were in a more quiescent state. In contrast, however, Gooderham et al. (130) found no effect of soy protein on platelets in normal men, although this may have been because of the specific assay used to measure platelet aggregation in this study. Finally, some studies suggest that isoflavones (131,132) and soy protein (133,134) have hypotensive effects, although studies are inconsistent in this regard (135–137). There is one area in particular related to CHD in which a seemingly contradictory picture has emerged in regard to the relative effects of soy vs. isolated isoflavones. Several clinical studies have found that soy inhibits LDL-C oxidation (138–141) and in vitro data (142–144) and animal (121,145) and human (146) studies in which the isoflavone content of soy protein has varied suggest that isoflavones are responsible for this effect. Curiously, however, three studies failed to show that isolated isoflavones inhibit LDL-C oxidation (111,117,147). Because none of these studies compared the effects of isoflavones with soy directly, it is not possible to know whether the discrepancy noted above is because isoflavones are not the agents responsible for the antioxidant effects of soy protein, or because the effects of soy+ on oxidation remain to be determined because not all studies show that even soy+ reduces LDL oxidation (141). Interestingly, in healthy men, isoflavone supplements were recently found to reduce 5-hydroxymethyl-2′deoxyuridine (5-OHmdU), a marker for oxidative DNA damage (148). However, in this study there was no effect on isoprostane levels in men or women (149) even though Wiseman et al. (146) found that in human subjects soy+ significantly reduced isoprostanes levels compared with soy–. In conclusion, there is evidence suggesting that isolated isoflavones will have a favorable effect on atherosclerosis; however, this remains speculative, and the magnitude of any protective effects is unknown. However, it is certainly possible that, even independent of cholesterol reduction, isoflavones may not duplicate all of the beneficial effects of soy+. Human studies in which the effects of isolated isoflavones are compared directly with soy+ are required before definitive conclusions can be drawn. Although differences in biological effects between soy+ and soy– point to isoflavones as the critical component, this cannot be assumed to be the case without direct experimental support. Finally, it is important to establish to what extent the effects of soy+ compared with soy– on the atherosclerotic process result from the hypocholesterolemic effects of soy+.

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Osteoporosis Initial speculation about the bone benefits of isoflavones was based on their estrogen-like effects and their similar chemical structure to the synthetic isoflavone, ipriflavone. Ipriflavone has been proven to be a very successful antiosteoporotic agent over the past 20 years (150). Several studies published within the last few years have examined the effect of isolated isoflavones on bone mineral density (BMD) using the ovariectomized rodent model, which is accepted by the U.S. FDA as a model for studying osteoporosis (151). Almost without exception (152), these studies have found beneficial effects (153–159). In addition to the animal data, three clinical studies have found that soy+ improves spinal BMD compared with soy– in perimenopausal (160) and postmenopausal (161,162) women. Also, preliminary results from a randomized, double-blind, placebo-controlled trial indicate that isolated isoflavones improved spinal BMD in premenopausal and perimenopausal women (163). Furthermore, several cross-sectional studies have found that higher soy and isoflavone intake is associated with higher spinal BMD in Asian women, although these studies are not designed to identify the agent in soy responsible for these associations (164–166). Despite the encouraging data, several clinical studies have not found differences in BMD (167,168), markers of bone turnover (108,169), or urinary calcium excretion (170) between women consuming soy protein differing in isoflavone content. Clearly, definitive data regarding both the effects of soy+ and isolated isoflavones have not yet been published. Longer-term studies involving larger numbers of subjects are warranted, a particularly obvious point considering the recent failure of ipriflavone to affect BMD or fracture risk in a recently conducted 3-y randomized controlled study involving 500 postmenopausal women (171). Finally, in addition to the possible direct effects of isoflavones on bone health, soy protein has been shown to decrease urinary calcium excretion when substituted for a similar amount of animal protein, such as whey (35), meat (34,172,173), and a casein-whey mixture (170). The metabolism of the sulfur amino acids (SAA) in protein results in the production of hydrogen ions, which causes bone dissolution so that the buffering agents in bone can be utilized (174). Soy protein has a relatively low SAA content and is more alkaline than animal protein. Nevertheless, because no long-term studies are available, firm conclusions cannot be drawn about the clinical significance of substituting soy protein for animal protein because there could be factors mitigating the apparent beneficial effects of the lower SAA content of soy protein. For example, soy foods could lower serum estrogen levels (175), which might affect BMD unfavorably (176,177). In conclusion, there is strong theoretical and intriguing experimental support for isoflavones promoting bone health, but no firm conclusions can be made at this time. If longer-term studies do show beneficial effects of the protein component of soy foods, this will certainly represent an advantage over isoflavone supplements. To derive this particular benefit, however, it will likely be necessary to substitute soy protein for animal protein.

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Menopausal Symptoms The first published speculation that soy might relieve menopausal symptoms was based on the weak estrogenic effects of isoflavones and the low incidence of hot flashes in Japan (178). Lock (179) found that between 30 and 35% of women in North America reported experiencing hot flashes, whereas only 10% of Japanese women did so. Recent data indicate that women of Chinese and Japanese ancestry residing in the United States are about one third less likely to report hot flashes compared with Caucasian women, whereas African-American women are ~50% more likely (180). Not surprisingly, there is a subjective component to hot flashes. According to Loprinzi et al. (181), placebo will decrease hot flashes by an average of 25% over a 3- to 4-wk period; ~10% of women will experience a 75% reduction in hot flashes with placebo and another 10% will experience a 50–75% reduction. Overall, findings from studies using both soy foods and isoflavones are not impressive. Among the nine studies identified in the literature that used soy foods, six (182–187) found that soy did not significantly affect either hot flash incidence or severity, or both, compared with the control group, whereas two (188,189) found decreases in incidence, and one a very modest reduction in severity (133). However, one of the studies in which incidence was reduced was not blinded; i.e., subjects knew that they were being fed tofu and miso (189). Of the four studies that used isoflavone supplements, two (190,191) found no effects and two (192,193) found modest beneficial effects. However, in one of these, the reduction in the placebo group was only 20% (193), which is lower than all other studies reported. A reduction of even 25% would have almost certainly eliminated any statistical difference between the control and isoflavone groups. The other study in which isoflavones were found to be beneficial was quite small because there were only 20 women in the placebo and treatment groups (192). Parenthetically, the effect of isoflavones derived from red clover on hot flashes are is unimpressive (194). It is worthwhile pointing out however, as noted by Kurzer (195), that although most studies report no statistically significant effects, the direction of these studies is nearly always in favor of soy or isoflavones exerting modest benefits compared with the placebo. Nevertheless, the disappointing results are not totally unexpected because other weak estrogens and selective estrogen receptor modulators, such as tamoxifen (196,197) and raloxifene (198), actually increase hot flash frequency, and the synthetic isoflavone, ipriflavone, is without effect (199). In conclusion, the evidence that soy or isoflavones favorably affect hot flash frequency or severity is relatively unimpressive, although still consistent with modest benefit. But there is no reason, based on either proposed biological mechanisms or the available data, to conclude that soy components other than isoflavones are responsible for any hypothesized beneficial effects. Parenthetically, prospective epidemiologic data suggest that isoflavones may be more effective at preventing

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the onset of hot flashes in women before the menopause than at alleviating hot flashes in women already experiencing them (200). Cognitive Function The possibility that estrogen may prevent declines in cognitive function and reduce risk of Alzheimer’s disease (201) has spurred investigation of the effects of isoflavones in this regard. Studies involving ovariectomized rodents (202-204) and monkeys (205) generally suggest that soy favorably affects cognitive function or biochemical indicators of cognitive function. These studies also suggest that isoflavones are responsible for the favorable effects (203). In support of this contention are results from a recently presented 6-mo trial of postmenopausal women in which subjects given a daily isoflavone supplement (110 mg) experienced an improvement in verbal memory compared with the placebo group (206). These results are consistent with those of File et al. (207) who found that in healthy men and women, the consumption of a high-isoflavone diet for 10 wk was associated with significant improvements in verbal and nonverbal episodic memory and frontal lobe function. In contrast to these rather encouraging data, however, a prospective epidemiologic study by White et al. (208) found that among Japanese men residing in Hawaii, those who consumed the most tofu (2–4 times per week) during their mid40s to mid-60s showed the most signs of mental deterioration in their mid-70s to early 90s. A similar association was seen between subjects’ tofu intake and the cognitive function of their wives (n = 502). At this point, the evidence is too preliminary to draw conclusions about the relationship between soy and cognitive function, especially when considering that the effects of estrogen on cognitive function remain unclear (201). However, there is a strong biological basis and some intriguing experimental support suggesting that if soy does have beneficial effects on cognitive function, they result from the isoflavones in soybeans.

Objection II. Supplements Discourage Dietary Change Independent of any beneficial effect of isoflavones, substituting soy foods for many commonly consumed animal foods will favorably affect overall dietary pattern and nutrient intake. The opportunity to obtain isoflavones via supplements discourages people from making the effort to incorporate soy foods into their diet, causing them to miss out on the full benefits of soy. For example, substituting tofu for red meat will decrease saturated fat and cholesterol intake—changes that should reduce CHD risk. Reducing red meat intake would also decrease the intake of potentially carcinogenic heterocyclic amines (200). Substituting certain soy foods, such as whole soybeans and tempeh, for meat, will increase fiber intake, which could favorably affect the risk of diabetes, CHD, and certain cancers.

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Furthermore, incorporating soy foods into the diet might make the transition to a plant-based diet more likely, thereby resulting in other favorable dietary changes.

Objection II. Evaluation Soy foods do possess a number of desirable nutritional attributes; consequently, they can lead to an improvement in overall dietary intake when substituted for many animal foods. However, many of the newly created soy foods that are routinely endorsed by health professionals are not necessarily intended to replace less healthy foods in the diet. For example, soy nuts are promoted as a substitute for nuts, soy breakfast cereals for other breakfast cereals, and soy milk for cow’s milk. Nuts are associated with a markedly reduced risk of CHD (209), and whole-grain breakfast cereal consumption with reduced risks of CHD (210), diabetes (211), and cancer at several sites (212). Dairy milk (especially lower-fat versions) is generally viewed as a desirable food and recent work shows calcium bioavailability from dairy milk exceeds that from calcium fortified (nonfortified soy milk is very low in calcium) soy milk (213). Similarly, although there is no doubt that soy energy bars are nutritionally superior to traditional candy bars, the foods that they are meant to replace, there is no evidence that these bars are actually used as candy bar substitutes. In addition, soy protein powders, one of the most popular types of soy products, are often added to fruit juice, water, or other types of beverages, rather than replacing less healthy beverages. Overall, it is not at all clear which foods are being displaced when soy energy bars and powders and many other types of soy foods are added to the diet. In some cases, it is certainly possible that other healthy foods are being replaced. If this is the case, one of the potential advantages of using soy foods over supplements is lost. Furthermore, even if one accepts that the nutrient profile and dietary pattern are improved when soy foods are incorporated into the diet, it does not necessarily follow that the availability of isoflavone supplements is undesirable. Arguably, supplements can actually encourage, rather than discourage dietary change. Isoflavone intake recommendations typically range from 30 to 100 mg (aglycone units) per day, the amount of isoflavones provided by 1–3 servings of traditional soy foods. If public perception is that at least two servings of soy per day are needed to derive health benefits, many consumers will opt to consume no soy at all. Conversely, if consumers are told that supplements can be used as a secondary approach when soy intake does not provide the recommended isoflavone intake, attitudes towards soy food consumption may be improved and soy food intake increased. This approach to meeting isoflavone recommendations is not dissimilar to the approach often recommended for meeting the needs of certain nutrients, such as calcium. Nutritionists routinely advise that consumers make calcium-rich foods a part of their diet while at the same time recommending that calcium supplements and calcium-fortified foods be used to ensure that calcium requirements are met.

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Objection III. Safety Soy foods have been consumed for centuries in Asian countries and for decades by subsets of the populations in Western countries. This historical precedent provides a measure of comfort regarding safety that does not exist for supplements. In addition, isoflavone intake via foods is self-limiting. It is unrealistic to think people can ingest >150 mg isoflavones/d via traditional soy foods, whereas some supplements contain this much in one pill or tablet. Furthermore, isoflavones may exert harmful effects when consumed in isolation but not when consumed as part of a biologic mix of nutrients and phytochemicals found in soybeans.

Objection III. Evaluation Historical Precedent There is certainly no historical precedent for consuming isoflavone supplements, but the very popular soy protein powders, which form the basis for so many new soy products, are also a recent development. In fact, although the protein powders are often referred to as supplements, it is common for health professionals to enthusiastically endorse the use of soy powders but not isoflavone supplements. The most commonly consumed powder is soy protein isolate (SPI), a highly processed product. To make SPI requires ridding the bean of the fiber, fat, carbohydrate, and a large portion of the isoflavones and saponins. Arguably, SPI is closer to isoflavone supplements than to traditional soy foods. In any event, historical use alone does not prove safety and this consideration by itself should not be used as a basis for advice regarding supplement use. Excessive Consumption Supplements may make it possible to more easily consume larger amounts of isoflavones than soy foods, but the distinction between pills and foods is becoming blurred, and concerns about overconsumption seem to be exaggerated. The distinction between isoflavone supplements and soy foods is being minimized because isoflavones are being used as food fortificants. As a result, there are several products currently on the market that provide 100 mg of isoflavones (aglycones) per serving, much higher than the ~30 mg that is typical for traditional soy foods. Furthermore, because soy foods are often promoted as safe and pills harmful, isoflavone intake via foods can often exceed isoflavone intake recommendations. It is not uncommon for motivated people to add SPI to their tofu or soy milk smoothie for breakfast, to snack on soy nuts throughout the day, to have a meat substitute for lunch or dinner, and to have a soy dessert for dinner. This level of soy intake would provide well in excess of 100 mg of isoflavones. Because isoflavone content is often not listed on soy products, consumers may not even know their isoflavone intake. This contrasts with supplements, which do list isoflavone content.

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Furthermore, Setchell et al. (214) showed that most isoflavone supplements contain the amount of isoflavones found in one serving of traditional soy products, ~20–35 mg. Of the 32 supplements analyzed, 24 contained ≤26 mg and 8 between 26 and 50 mg. Thus, it would be difficult to consume excessive isoflavones from the use of these supplements. However, a few supplements do contain large amounts of isoflavones, and their use would lead to what most health professionals would consider excessive intake. But this does not justify excluding the use of all isoflavone supplements; instead, consumers should be advised against exceeding isoflavone intake recommendations. Of course, this advice is relevant for all supplement use, not just isoflavone supplements. Because the isoflavone content of pills is indicated, this should not be difficult for consumers. Lack of Safety Data The issue of safety is a complex one and cannot be addressed in its entirety in this chapter. However, several points are worthy of mention in regard to the safety of isoflavone supplements vs. soy foods. First, for those areas for which concerns have been raised, arguably, the most serious findings come from studies involving soy foods, not isolated isoflavones, whether it is breast cancer, cognitive function, reproductive effects, or thyroid function. Furthermore, these studies do not indicate that excessive consumption is the problem. For example, Petrakis et al. (215) and Hargreaves et al. (216) reported that SPI providing 80 mg of isoflavones and textured vegetable protein providing 45 mg of isoflavones, respectively, exerted estrogen-like effects on breast tissue. In addition, Allred et al. (217) showed that SPI and genistein stimulate tumor growth in nude mice implanted with MCF-7 cells to a similar extent, thus indicating no reason to distinguish between pills and foods. In contrast, isoflavone supplements (soy foods were not tested) were shown to decrease breast tissue density in postmenopausal women (218). Concerns about adverse effects of isoflavones on cognitive function are based almost entirely on the results from a prospective epidemiologic study cited previously, which found that tofu consumption was associated with poor cognitive function in older Japanese men and women (208). In regard to the thyroid, isoflavones have been shown to inactivate thyroid peroxidase in vitro (219) and in vivo in animals (220), but other data suggest that nonisoflavone components, such as the protein, are responsible for the goitrogenic effects of soy observed in some studies (221). Furthermore, if isoflavones do have adverse effects on thyroid function in susceptible individuals, there is no reason to think that isoflavone-containing soy foods would not have similar effects and in fact, a few studies have reported adverse effects of soy consumption on thyroid function in infants (222) and adults (223). Finally, there are several components of soy, e.g., phytic acid, lectins, protease inhibitors, oxalate, that have all been labeled as antinutrients and that are not present in significant quantities in isoflavone supplements (224,225). Although soybean oligosaccharides, which may favorably alter the composition of the intestinal microflora (226), are not found in supplements, many soy foods do not contain

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these prebiotics, yet these foods are still recommended. Furthermore, those foods that do contain them may cause flatulence. Therefore, arguably, consuming isoflavones from supplements may actually hold some advantages over soy foods.

Objection IV. Pharmacokinetics When taken in isolation, isoflavones are absorbed and metabolized differently by the body than when taken via soy foods because of the effects of the biological matrix in which they are found. The effects of consuming similar amounts of isoflavones from foods and from supplements may differ in regard to both efficacy and safety.

Objection IV. Evaluation Recent data indicate that isolated isoflavones are absorbed and metabolized similarly to soy foods (214). Consuming identical amounts of isoflavones will lead to similar serum levels whether consumed via isolated isoflavones or soy foods (214). Furthermore, data indicate that isoflavones from supplements are well absorbed (214). There is debate about whether the glycoside isoflavones are absorbed differently than the isoflavone aglycones. Some research suggests aglycones, such as those that exist in fermented soy foods, are absorbed more efficiently (227,228) or more quickly (229) than isoflavone glycosides. However, research using isolated isoflavones indicates that glycosides are actually absorbed more quickly than isolated aglycones (214). Because physiologic effects have been observed in humans in response to the consumption of isolated isoflavones in glycoside form (117) and soy foods in which the isoflavones are present primarily as glycosides (138), it is clear that humans have the ability to absorb isoflavones regardless of isoform. In any event, because soy foods can differ markedly in their percentage of isoflavone glycosides and aglycones, and soy intake recommendations regarding efficacy and safety make no distinction in this regard, this issue would not be directly relevant to the use of supplements. Nevertheless, more research is required to establish whether differences in the absorption and metabolism do exist between isolated isoflavones and isoflavonerich food. Chang et al. (231,232) and Badger (233) noted that differences have been reported in the concentrations of genistein and genistein metabolites in the serum and brain of rats fed isolated genistein or isoflavone-rich soy protein. However, comparisons need to be made within the same study, not across studies before conclusions can be drawn. Furthermore, supplements contain a mixture of isoflavones, not just genistein. Also, results from animal studies may not be applicable to humans because in rodents, equol is the predominant isoflavonoid found in the serum in response to soy consumption. Conceivably, isoflavone absorption from supplements could differ from soy foods because soy foods are generally consumed with other foods, whereas supplements may often be taken on an empty stomach. If this proves to be a legitimate concern, recommendations to take isoflavone supplements with food can be made. This is commonly done for certain supplements and medications.

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Objection V. Label vs. Content Amount The amount of isoflavones in supplements may differ markedly from the amount listed on the product label. Consequently, consumers may be getting much more, or much fewer, isoflavones than thought.

Objection V. Evaluation Quality control concerns plague the supplement industry. Several surveys have identified large discrepancies between the amount of supplement indicated on the product label and the amount in the actual supplement. Recently, Setchell et al. (214) found that of 31 isoflavone supplements analyzed, 15 were within 20% of the stated amount, 9 were within 20–40%, and 6 differed by between 50 and 90%. In nearly all cases, significant discrepancies were due to the supplements containing less, not more, isoflavones than the stated amount. Certainly, in the case of supplements, it is buyer beware, but consumers can try to protect themselves by seeking out the most reputable companies. It should be noted, however, that although the discrepancy between the label and content amount is a legitimate criticism of using isoflavone supplements, there is a large variation in the isoflavone content of different batches of the same brand of soy product, and among different brands of the same type of soy product, which in many cases will not be known to the consumer (234). Thus, using soy foods is also no assurance that one is receiving the expected amount of isoflavones.

Objection VI. Cost Isoflavone supplements are expensive and because the health benefits of isoflavones remain speculative, supplement users may be wasting their money. In contrast, at the very least, consumers who use soy foods are obtaining energy, fiber, and a host of nutrients and phytochemicals.

Objection VI. Evaluation Supplements can be expensive, but because expenditures on supplements are a matter of personal choice, this is not a relevant criticism as long as the consumer understands what is being purchased. Furthermore, many soy products are quite expensive compared with the foods they are intended to replace. Soy milk can easily be two- to fourfold the cost of dairy milk, soy energy bars are two- to fourfold the cost of candy bars, and even soy burgers are two- to threefold more than ground beef. Arguably, it is less expensive to buy the foods soy foods are meant to replace and take an isoflavone supplement as a means of obtaining isoflavones, than it is to actually buy soy foods.

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Conclusions In the opinion of this author, among the objections to the use of isoflavone supplements discussed above, objection V (Label vs. Content Amount) has the most validity. However, as noted previously, this concern is applicable to essentially all supplements. Because health professionals routinely recommend a variety of nutrient supplements, this objection does not qualify as a reason to specifically avoid isoflavone supplements. Nevertheless, the argument presented in this chapter is not that isoflavone supplements should be used in place of soy foods. Soy foods do have some potential advantages over supplements. As discussed, soy protein modestly reduces serum cholesterol levels (235) and when substituted for animal protein reduces urinary calcium excretion (34). Also, soy protein may have a favorable effect on renal function relative to animal proteins (236). Furthermore, in some cases, it is unclear whether isoflavones are responsible for the hypothesized health effects of soy, such as the inhibitory effect of soy on LDL-C oxidation and the reduction in isoprostane levels. Thus, there is little reason to think that isoflavone supplements will exert health benefits that cannot be duplicated by sufficient amounts of soy foods, whereas the opposite may be true. However, the evidence suggests that isoflavones are responsible for most of the key hypothesized benefits of soy, such as protection against breast and prostate cancer, improvements in bone health, alleviation of menopausal symptoms, and certain improvements in the health of the coronary vessels. Therefore, the position of this author is that isoflavone supplements represent a legitimate means of ingesting isoflavones when isoflavone intake via soy foods is inadequate. Most consumers currently do not and probably never will consume soy foods in amounts that provide sufficient (~50 mg/d) isoflavones. Consequently, although health professionals should strongly encourage soy food consumption as the primary and best means of obtaining isoflavones, there appears to be little reason to object to the use of isoflavone supplements as long as total isoflavone intake remains within the recommended range (30-100 mg/d). References 1. Office of Inspector General (2001) Adverse Event Reporting for Dietary Supplements, U.S. Department of Health and Human Services, Washington. 2. Stephens, N.G., Parsons, A., Schofield, P.M., Kelly, F., Cheeseman, K., and Mitchinson, M.J. (1996) Randomised Controlled Trial of Vitamin E in Patients with Coronary Disease: Cambridge Heart Antioxidant Study (CHAOS), Lancet 347, 781–786. 3. Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S., and Hammar, S. (1996) Effects of a Combination of Beta Carotene and Vitamin A on Lung Cancer and Cardiovascular Disease, N. Engl. J. Med. 334, 1150–1155.

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165. Somekawa, Y., Chiguchi, M., Ishibashi, T., and Aso, T. (2001) Soy Intake Related to Menopausal Symptoms, Serum Lipids, and Bone Mineral Density in Postmenopausal Japanese Women, Obstet. Gynecol. 97, 109–115. 166. Ho, S.C., Chan, S.G., Yi, Q., Wong, E., and Leung, P.C. (2001) Soy Intake and the Maintenance of Peak Bone Mass in Hong Kong Chinese Women, J. Bone Miner. Res. 16, 1363–1369. 167. Gallagher, J.C., Rafferty, K., Haynatzka, V., and Wilson, M. (2000) Effect of Soy Protein on Bone Metabolism, J. Nutr. 130, 667S–669S. 168. Vitolins, M.Z., Anthony, M.S., Lenchik, L., Bland, D.R., and Burke, G.L. (2002) Does Soy Protein and Its Isoflavones Prevent Bone Loss in Peri- and Post-Menopausal Women? Results of a Two-Year Randomized Clinical Trial, J. Nutr. 132. 169. Wangen, K.E., Duncan, A.M., Merz-Demlow, B.E., Xu, X., Marcus, R., Phipps, W.R., and Kurzer, M.S. (2000) Effects of Soy Isoflavones on Markers of Bone Turnover in Premenopausal and Postmenopausal Women, J. Clin. Endocrinol. Metab. 85, 3043– 3048. 170. Spence, L.A., Lipscomb, E.R., Cadogan, J., Martin, B.R., Peacock, M., and Weaver, C.M. (2002) Effects of Soy Isoflavones on Calcium Metabolism in Postmenopausal Women, J. Nutr. 132, 581S. 171. Alexandersen, P., Toussaint, A., Christiansen, C., Devogelaer, J.P., Roux, C., Fechtenbaum, J., Gennari, C., and Reginster, J.Y. (2001) Ipriflavone in the Treatment of Postmenopausal Osteoporosis: A Randomized Controlled Trial, J. Am. Med. Assoc. 285, 1482–1488. 172. Kaneko, K., Masaki, U., Aikyo, M., Yabuki, K., Haga, A., Matoba, C., Sasaki, H., and Koike, G. (1990) Urinary Calcium and Calcium Balance in Young Women Affected by High Protein Diet of Soy Protein Isolate and Adding Sulfur-Containing Amino Acids and/or Potassium, J. Nutr. Sci. Vitaminol. (Tokyo) 36, 105–116. 173. Watkins, T.R., Pandya, K., and Mickelsen, O. (1985) Urinary Acid and Calcium Excretion. Effect of Soy Versus Meat in Human Diets, in Nutritional Bioavailability of Calcium (Kies, C., ed.) American Chemical Society, Washington. 174. Barzel, U.S., and Massey, L.K. (1998) Excess Dietary Protein Can Adversely Affect Bone, J. Nutr. 128, 1051–1053. 175. Lu, L.J., Anderson, K.E., Grady, J.J., Kohen, F., and Nagamani, M. (2000) Decreased Ovarian Hormones During a Soya Diet: Implications for Breast Cancer Prevention, Cancer Res. 60, 4112–4121. 176. Nguyen, T.V., Center, J.R., and Eisman, J.A. (2000) Association Between Breast Cancer and Bone Mineral Density: The Dubbo Osteoporosis Epidemiology Study, Maturitas 36, 27–34. 177. Sypniewska, G., and Chodakowska-Akolinska, G. (2000) Bone Turnover Markers and Estradiol Level in Postmenopausal Women, Clin. Chem. Lab. Med. 38, 1115–1119. 178. Adlercreutz, H., Hamalainen, E., Gorbach, S., and Goldin, B. (1992) Dietary Phytooestrogens and the Menopause in Japan, Lancet 339, 1233. 179. Lock, M. (1994) Menopause in Cultural Context, Exp. Gerontol. 29, 307–317. 180. Gold, E.B., Sternfeld, B., Kelsey, J.L., Brown, C., Mouton, C., Reame, N., Salamone, L., and Stellato, R. (2000) Relation of Demographic and Lifestyle Factors to Symptoms in a Multi- Racial/Ethnic Population of Women 40–55 Years of Age, Am. J. Epidemiol. 152, 463–473. 181. Loprinzi, C.L., Barton, D.L., and Rhodes, D. (2001) Management of Hot Flashes in Breast-Cancer Survivors, Lancet Oncol. 2, 199–204.

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182. Murkies, A.L., Lombard, C., Strauss, B.J., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Post-Menopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 183. Dalais, F.S., Rice, G.E., Wahlqvist, M.L., Grehan, M., Murkies, A.L., Medley, G., Ayton, R., and Strauss, B.J.G. (1998) Effects of Dietary Phytoestrogens in Postmenopausal Women, Climacteric 1, 124–129. 184. St. Germain, A., Peterson, C.T., Robinson, J.G., and Alekel, D.L. (2001) IsoflavoneRich or Isoflavone-Poor Soy Protein Does Not Reduce Menopausal Symptoms During 24 Weeks of Treatment, Menopause 8, 17–26. 185. Woods, M.N., Senie, R., and Kronenberg, F. (1998) Effect of a Dietary Soy Bar on Menopausal Symptoms, Am. J. Clin. Nutr. 68, 1553S. 186. Van Patten, C.L., Olivotto, I.A., Chambers, G.K., Gelmon, K.A., Hislop, T.G., Templeton, E., Wattie, A., and Prior, J.C. (2002) Effect of Soy Phytoestrogens on Hot Flashes in Postmenopausal Women with Breast Cancer: A Randomized, Controlled Clinical Trial, J. Clin. Oncol. 20, 1449–1455. 187. Kotsopoulos, D., Dalais, F.S., Liang, Y.-L., McGrath, B.P., and Teede, H.J. (2000) The Effects of Soy Protein Containing Phytoestrogens on Menopausal Symptoms in Postmenopausal Women, Climacteric 3, 161–167. 188. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 189. Brzezinski, A., Adlercreutz, H., Shaoul, R., Rösler, R., Shmueli, A., Tanos, V., and Schenker, J.G. (1997) Short-Term Effect of Phytoestrogen-Rich Diet on Postmenopausal Women, Menopause 4, 89–94. 190. Quella, S.K., Loprinzi, C.L., Barton, D.L., Knost, J.A., Sloan, J.A., LaVasseur, B.I., Swan, D., Krupp, K.R., Miller, K.D., and Novotny, P.J. (2000) Evaluation of Soy Phytoestrogens for the Treatment of Hot Flashes in Breast Cancer Survivors: A North Central Cancer Treatment Group Trial, J. Clin. Oncol. 18, 1068–1074. 191. Baber, R.J., Templeman, C., Morton, T., Kelly, G.E., and West, L. (1999) Randomized Placebo-Controlled Trial of an Isoflavone Supplement and Menopausal Symptoms in Women, Climacteric 2, 85–92. 192. Scambia, G., Mango, D., Signorile, P.G., Anselmi Angeli, R.A., Palena, C., Gallo, D., Bombardelli, E., Morazzoni, P., Riva, A., and Mancuso, S. (2000) Clinical Effects of a Standardized Soy Extract in Postmenopausal Women: A Pilot Study [see comments], Menopause 7, 105–111. 193. Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L., and Lamia, C.A. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: a Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 194. Fugh-Berman, A., and Kronenberg, F. (2001) Red Clover (Trifolium pratense) for Menopausal Women: Current State of Knowledge, Menopause 8, 333–337. 195. Kurzer, M.S. (2002) Hormonal Effects of Premenopausal Women and Men, J. Nutr. 132, 570S–573S. 196. Day, R., Ganz, P.A., Costantino, J.P., Cronin, W.M., Wickerham, D.L., and Fisher, B. (1999) Health-Related Quality of Life and Tamoxifen in Breast Cancer Prevention: A Report from the National Surgical Adjuvant Breast and Bowel Project P-1 Study, J. Clin. Oncol. 17, 2659–2669.

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197. Love, R.R., Cameron, L., Connell, B.L., and Leventhal, H. (1991) Symptoms Associated with Tamoxifen Treatment in Postmenopausal Women, Arch. Intern. Med. 151, 1842–1847. 198. Walsh, B.W., Kuller, L.H., Wild, R.A., Paul, S., Farmer, M., Lawrence, J.B., Shah, A.S., and Anderson, P.W. (1998) Effects of Raloxifene on Serum Lipids and Coagulation Factors in Healthy Postmenopausal Women, J. Am. Med. Assoc. 279, 1445–1451. 199. Agnusdei, D., Gennari, C., and Bufalino, L. (1995) Prevention of Early Postmenopausal Bone Loss Using Low Doses of Conjugated Estrogens and the Non-Hormonal, BoneActive Drug Ipriflavone, Osteoporos. Int. 5, 462–466. 200. Nagata, C., Takatsuka, N., Kawakami, N., and Shimizu, H. (2001) Soy Product Intake and Hot Flashes in Japanese Women: Results from a Community-Based Prospective Study, Am. J. Epidemiol. 153, 790–793. 201. LeBlanc, E.S., Janowsky, J., Chan, B.K., and Nelson, H.D. (2001) Hormone Replacement Therapy and Cognition: Systematic Review and Meta-Analysis, J. Am. Med. Assoc. 285, 1489–1499. 202. Pan, Y., Anthony, M., and Clarkson, T.B. (1999) Evidence for Up-Regulation of Brain-Derived Neurotrophic Factor mRNA by Soy Phytoestrogens in the Frontal Cortex of Retired Breeder Female Rats, Neurosci. Lett. 261, 17–20. 203. Pan, Y., Anthony, M., and Clarkson, T.B. (1999) Effect of Estradiol and Soy Phytoestrogens on Choline Acetyltransferase and Nerve Growth Factor mRNAs in the Frontal Cortex and Hippocampus of Female Rats, Proc. Soc. Exp. Biol. Med. 221, 118–125. 204. Pan, Y., Anthony, M., Watson, S., and Clarkson, T.B. (2000) Soy Phytoestrogens Improve Radial Arm Maze Performance in Ovariectomized Retired Breeder Rats and Do Not Attenuate Benefits of 17β-Estradiol Treatment, Menopause 7, 230–235. 205. Kim, H., Xia, H., Li, L., and Gewin, J. (2000) Attenuation of NeurodegenerationRelevant Modifications of Brain Proteins by Dietary Soy, Biofactors 12, 243–250. 206. Kritz-Silverstein, D., Von Muhlen, D., and Barrett-Connor, E. (2002) The Soy and Postmenopausal Health in Aging (SOPHIA) Study: Overview and Baseline Cognitive Function, J. Nutr. 132, 586–587S. 207. File, S.E., Jarrett, N., Fluck, E., Duffy, R., Casey, K., and Wiseman, H. (2001) Eating Soya Improves Human Memory, Psychopharmacology (Berl) 157, 430–436. 208. White, L.R., Petrovitch, H., Ross, G.W., Masaki, K., Hardman, J., Nelson, J., Davis, D., and Markesbery, W. (2000) Brain Aging and Midlife Tofu Consumption, J. Am. Coll. Nutr. 19, 242–255. 209. Hu, F.B., Stampfer, M.J., Manson, J.E., Rimm, E.B., Colditz, G.A., Rosner, B.A., Speizer, F.E., Hennekens, C.H., and Willett, W.C. (1998) Frequent Nut Consumption and Risk of Coronary Heart Disease in Women: Prospective Cohort Study, Br. Med. J. 317, 1341–1345. 210. Liu, S., Stampfer, M.J., Hu, F.B., Giovannucci, E., Rimm, E., Manson, J.E., Hennekens, C.H., and Willett, W.C. (1999) Whole-Grain Consumption and Risk of Coronary Heart Disease: Results from the Nurses’ Health Study, Am. J. Clin. Nutr. 70, 412–419. 211. Liu, S., Manson, J.E., Stampfer, M.J., Hu, F.B., Giovannucci, E., Colditz, G.A., Hennekens, C.H., and Willett, W.C. (2000) A Prospective Study of Whole-Grain Intake and Risk of Type 2 Diabetes Mellitus in US Women, Am. J. Public Health 90, 1409–1415. 212. Jacobs, D.R., Jr., Marquart, L., Slavin, J., and Kushi, L.H. (1998) Whole-Grain Intake and Cancer: An Expanded Review and Meta-Analysis, Nutr. Cancer 30, 85–96.

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213. Heaney, R.P., Dowell, M.S., Rafferty, K., and Bierman, J. (2000) Bioavailability of the Calcium in Fortified Soy Imitation Milk, with Some Observations on Method, Am. J. Clin. Nutr. 71, 1166–1169. 214. Setchell, K.D., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., and Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131, 1362S–1375S. 215. Petrakis, N.L., Barnes, S., King, E.B., Lowenstein, J., Wiencke, J., Lee, M.M., Miike, R., Kirk, M., and Coward, L. (1996) Stimulatory Influence of Soy Protein Isolate on Breast Secretion in Pre- and Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 5, 785–794. 216. Hargreaves, D.F., Potten, C.S., Harding, C., Shaw, L.E., Morton, M.S., Roberts, S.A., Howell, A., and Bundred, N.J. (1999) Two-Week Dietary Soy Supplementation Has an Estrogenic Effect on Normal Premenopausal Breast, J. Clin. Endocrinol. Metab. 84, 4017–4024. 217. Allred, C.D., Allred, K.F., Ju, Y.H., Virant, S.M., and Helferich, W.G. (2001) Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-Dependent (MCF-7) Tumors in a Dose-Dependent Manner, Cancer Res. 61, 5045–5050. 218. Atkinson, C., Warren, R.M.L., Dowsett, M., Day, N.E., and Bingham, S. (2002) Effects of Isoflavones on Breast Density, Oestradiol, and Gonadotrophins: A Double Blind Randomized Placebo Controlled Trial, J. Nutr. 132, 577S–578S. 219. Divi, R.L., Chang, H.C., and Doerge, D.R. (1997) Anti-Thyroid Isoflavones from Soybean: Isolation, Characterization, and Mechanisms of Action, Biochem. Pharmacol. 54, 1087–1096. 220. Chang, H.C., and Doerge, D.R. (2000) Dietary Genistein Inactivates Rat Thyroid Peroxidase In Vivo Without an Apparent Hypothyroid Effect, Toxicol. Appl. Pharmacol. 168, 244–252. 221. Son, H.Y., Nishikawa, A., Ikeda, T., Imazawa, T., Kimura, S., and Hirose, M. (2001) Lack of Effect of Soy Isoflavone on Thyroid Hyperplasia in Rats Receiving an IodineDeficient Diet, Jpn. J. Cancer Res. 92, 103–108. 222. Tuohy, P.G. (2000) Soy Formulas and the Effects of Isoflavones on the Thyroid [Letter], N.Z. Med. J. 113, 234–235. 223. Ishizuki, Y., Hirooka, Y., Murata, Y., and Togashi, K. (1991) The Effects on the Thyroid Gland of Soybeans Administered Experimentally to Healthy Subjects, Nippon Naibunpi Kai Shi 67, 622–629. 224. Liener, I.E. (1995) Possible Adverse Effects of Soybean Anticarcinogens, J. Nutr. 125, 744S–750S. 225. Liener, I.E. (1994) Implications of Antinutritional Components in Soybean Foods, Crit. Rev. Food Sci. Nutr. 34, 31–67. 226. Koo, M., and Rao, A.V. (1991) Long-Term Effect of Bifidobacteria and Neosugar on Precursor Lesions of Colonic Cancer in CFL Mice, Nutr. Cancer 16, 249–257. 227. Izumi, T., Piskula, M.K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kataoka, S., Kubota, Y., and Kikuchi, M. (2000) Soy Isoflavone Aglycones Are Absorbed Faster and in Higher Amounts than Their Glucosides in Humans, J. Nutr. 130, 1695–1699. 228. Hutchins, A.M., Slavin, J.L., and Lampe, J.W. (1995) Urinary Isoflavonoid Phytoestrogen and Lignan Excretion After Consumption of Fermented and Unfermented Soy Products, J. Am. Diet. Assoc. 95, 545–551.

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229. King, R.A., Broadbent, J.L., and Head, R.J. (1996) Absorption and Excretion of the Soy Isoflavone Genistein in Rats, J. Nutr. 126, 176–182. 230. Holder, C.L., Churchwell, M.I., and Doerge, D.R. (1999) Quantification of Soy Isoflavones, Genistein and Daidzein, and Conjugates in Rat Blood Using LC/ES-MS, J. Agric. Food Chem. 47, 3764–3770. 231. Chang, H.C., Churchwell, M.I., Delclos, K.B., Newbold, R.R., and Doerge, D.R. (2000) Mass Spectrometric Determination of Genistein Tissue Distribution in DietExposed Sprague-Dawley Rats, J. Nutr. 130, 1963–1970. 232. Chang, H.C., Fletcher, T., Ferguson, M., Hale, K., Fang, N., Ronis, M., Prior, R., and Badger, T.M. (2002) Serum and Tissue Profiles of Isoflavones and Conjugates in Rats Fed Diets Containing Soy Protein Isolate (SPI), FASEB J., in press. 233. Badger, T.M., Ronis, M.J., Hakkak, R., Rowlands, J.C., and Korourian, S. (2002) The Health Consequences of Early Soy Consumption, J. Nutr. 132, 559S–565S. 234. Murphy, P.A., Song, T., Buseman, G., Barua, K., Beecher, G.R., Trainer, D., and Holden, J. (1999) Isoflavones in Retail and Institutional Soy Foods, J. Agric. Food Chem. 47, 2697–2704. 235. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 236. Anderson, J.W., Blake, J.E., Turner, J., and Smith, B.M. (1998) Effects of Soy Protein on Renal Function and Proteinuria in Patients with Type 2 Diabetes, Am. J. Clin. Nutr. 68, 1347S–1353S.

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Chapter 4

Industrial Processing and Preparation of Isoflavones Eric T. Gugger General Mills, James Ford Bell Technical Center, Minneapolis, MN

Introduction Although soybeans and soy foods are frequently cited as a source of high-quality protein, fiber, essential fatty acids, phytic acid, saponins, protease inhibitors, and phytosterols, it is without question that the groundswell of recent research and interest in soy has been due to the fact that soy is a rich source of isoflavones, and is the most commonly consumed food source of these compounds. Isoflavones continue to be the focus of research investigating the potential of these compounds to reduce the risk of certain chronic diseases, including heart disease risk factors (1,2), osteoporosis (3), breast cancer, and prostate cancer (4). Industrial preparation of isoflavones has the potential to serve several important needs: (i) Double-blind, placebo-controlled clinical studies that specifically isolate the effect of isoflavones using standardized materials can be conducted. Soy foods are complex and can be variable in their isoflavone content (5), and even isolated soy protein can be challenging to adequately blind in clinical studies (6). Compliance issues with soy foods and soy beverage powders (7) create yet another layer of complexity in the clinical setting, which is more easily overcome with the use of isoflavone tablets and a well-matched placebo. (ii) Individuals who choose to increase dietary isoflavone consumption, but for whom traditional soy foods and powdered beverages are unpalatable or inconvenient, have the option of taking an isoflavone supplement tablet or a more familiar food product to which isoflavones have been added. (iii) Purified isoflavones may eventually serve as a base substrate for the manufacture of novel drugs. Genistein-B43 conjugates have been used experimentally to treat B-cell precursor leukemia in animals (8) and humans (9), and genistein-epidermal growth factor conjugates have been used in a similar fashion to target breast cancer cells (10) and vascular smooth muscle cells (11) in vivo. Continued advances in molecular targeting will allow cell-specific intracellular concentrations of isoflavones to be achieved; this is not possible with soy foods or supplements and will allow us to take advantage of the unique properties of isoflavones, such as protein tyrosine kinase inhibition (12). (iv) In soybeans, the primary function of isoflavones is to induce the expression of nodulation genes of Bradyrhizobium (13), a process ultimately involved in nitrogen fixation. Agricultural applications of isoflavones have been described which utilize this function to enhance nodulation by inoculating soybean seeds with a combination of isoflavones and Bradyrhizobium (14).

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It is the intent of this chapter to give the reader a review of the industrial approaches to isoflavone preparation from natural raw materials, namely, soybeans, as largely described in the patent literature. The predominant applications for these products at this time include dietary supplements and food ingredients. Sources Isoflavones are a subclass of flavonoids common to leguminous plants (15). Soybeans, red clover, and to a lesser extent kudzu root are the most common sources for the industrial production of isoflavones; additional sources (much lower in concentration) include bean varieties such as broad, pinto, navy (5), as well as the American groundnut (16) and lupin (17). As with any industrial naturalproduct extraction, considerations such as isoflavone concentration and profile, availability of raw material, consistency of supply, history of food use and safety, and potential utility of coproducts produced during the extraction process allow the most practical selection of raw material. Soybeans are the most logical and economical choice of raw material, and much of the interest in isoflavones stems from epidemiologic studies relating soy consumption to reduced risk of certain cancers (18). Much of the soy grown in the United States is processed into value-added products including edible oil, animal feed, soy proteins, lecithin, tocopherols, and phytosterols. Existing soy processing procedures common to the soy industry create secondary coproduct streams, such as soy molasses; these contain up to 10 times the isoflavone concentration of raw soybeans, and serve as ideal raw materials for further isolation and purification. The primary focus of this chapter will accordingly center around the production of isoflavones from soy; however, it is anticipated that techniques discussed will apply broadly to a variety of raw materials. Bulk Enrichment The major isoflavone isomers found in soybeans and soy foods are shown in Figure 4.1. In general, the malonyl-glycosides are water and alcohol soluble, and are the predominant glycosides present in soybeans (19,20). These forms are heat labile (>30°C), and are converted to acetyl-glycosides and glycosides depending on processing conditions. Processes such as soybean roasting and texturization (extrusion) of soy flour create large amounts of acetyl-glycosides (20). High temperatures (>100°C) combined with high moisture conditions, such as that observed in the production of soy molasses, favor the conversion of malonyl-glycosides to their respective glycosides. The process of malonyl-glycoside to glycoside conversion can be further aided by employing alkaline conditions (pH > 6) (21,22). The conversion of glycosides to aglycones can be accomplished enzymatically as has been observed with the endogenous β-glucosidases present in soybeans (21,23) and by employing commercially available β-galactosidase, glucoamylase (22), and βglucanase/β-xylanase (24). Traditionally fermented soy foods, such as tempeh,

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genistein

genistin

6′′-O-malonylgenistin

6′′-O-acetylgenistin

daidzein

daidzin

6′′-O-malonyldaidzin

6′′-O-acetyldaidzin

glycitein

glycitin

6′′-O-malonylglycitin

6′′-O-acetylglycitin

Fig. 4.1. Chemical structures of 12 isoflavone isomers found in soy.

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miso, and fermented bean curd, contain much higher levels of aglycones due to the hydrolytic enzyme activity of the microorganisms used in their manufacture (20). Interestingly, malonyl-glycosides are highly resistant to enzymatic hydrolysis by β-galactosidases and must first be converted to their respective glycosides before enzymatic conversion to the aglycone can take place (22). Conversion of glycosides to aglycones can also be achieved with acid hydrolysis (25,26). As summarized in Table 4.1, isoflavones are differentially distributed within the soybean seed. The cotyledon, which constitutes >90% of the soybean weight, accounts for 88% of the total isoflavones. Although the hypocotyl (germ) represents only 2% of soybean weight, it is five- to sixfold more highly concentrated in isoflavones than the cotyledon and is particularly enriched in glycitein and daidzein glycosides compared with genistein glycosides. The elevated isoflavone concentration of soy germ, while quite unique in isoflavone profile compared with whole soybeans and soy foods, has prompted its use as an isoflavone supplement. Commercial soybean dehulling procedures largely separate the soy germ and hull from the cotyledon (Fig. 4.2). The germ can then be further separated from hulls and smaller pieces of broken cotyledon through a combination of sieving and air classification due to the small size and high density of the germ. The germ is then roasted and ground into a flour. During commercial soybean processing, the dehulled cotyledons are flaked and hexane extracted to remove the oil (27). Hexane extraction does not result in any appreciable loss of isoflavones to the oil fraction (28). The resulting defatted flakes serve as the starting material for the production of soy flour, soy protein concentrates, and soy protein isolates (Fig. 4.3). For the production of soy flour, defatted flakes are subjected to various degrees of toasting and grinding, resulting in a product that contains from 2–3 mg total isoflavones/g (5). Most commercial soy protein concentrates are made by subjecting defatted flakes to an aqueous alcohol (~60-80% by volume) extraction at temperature in the range of 44-63°C to remove oligosaccharides (28,29). TABLE 4.1 Isoflavone Distribution Within the Soybeana

Isoflavone Daidzin Genistin Glycitin Daidzein Genistein Glycitein Total isoflavones (mg/g) Total soybean isoflavone content (%) aAdapted

from Reference 49. of soybean total weight. cNot detected. bPercentage

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Hypocotyl (2.2%)b

Seed section Cotyledon (91.5%)

Seed coat (6.3%)

8.38 2.46 10.04 0.35 0.16 0.15 21.54 11.98

1.45 2.10 —c 0.11 0.14 — 3.80 87.94

0.02 0.02 — — 0.01 — 0.05 0.08

Fig. 4.2. Intial stages of soybean processing. Soybeans are cleaned, heated, cracked,

and dehulled. The small pieces of soybean cotyledon, or soy chips, are then flaked to ~0.25–0.3 mm to produce full-fat flakes. These flakes are subsequently extracted with hexane to remove the oil and produce defatted soy flakes, the starting material for commercial soy protein production. Soy germ, which is concentrated (~2%) in isoflavones, particularly glycitin and daidzin, is removed during dehulling and may be further separated from hulls.

This process is also very efficient at removing isoflavones as evidenced by the comparatively low isoflavone content of soy protein concentrates (~0.1-0.2 mg total isoflavones/g) (5). The ethanol extract, or soy solubles, are distilled to recover the ethanol, and may be further concentrated by evaporation to produce soy molasses. Soy molasses contains 1-3% total isoflavones on a dry weight basis, roughly 10-fold higher than raw soybeans, and is an ideal material for further purification of isoflavones. For soy protein isolate manufacture, defatted flakes are subjected to alkaline treatment to solubilize the protein fraction (31). The fiber is then removed by centrifugation, and the protein is precipitated from the supernatant with acid. The resulting aqueous coproduct from this process is known as soy whey; it contains 0.5–2% total isoflavones on a dry weight basis, and is particularly enriched in the highly soluble malonyl-glycosides (22). This process is much less efficient at isoflavone removal compared with aqueous ethanol extraction, with soy protein isolates retaining between 1 and 3 mg/g. Processes have been described that thermally and/or enzymatically

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Fig. 4.3. Processing of defatted soy flakes into the three major commercial soy protein products: soy flour, soy protein concentrate, and soy protein isolate. Soy solubles (molasses) and soy whey, which are coproducts of soy protein concentrate and soy protein isolate manufacturing, respectively, serve as potential starting materials for the industrial preparation of isoflavones.

convert the malonyl glycosides to less soluble glycosides or aglycones during soy isolate processingto retain more isoflavones with the soy protein isolate fraction (32). Similar techniques are described for soy fiber (33) and soy concentrate (34). Several approaches have been described for the bulk solvent extraction of isoflavones from soybeans. For analytical work, HCl-acetonitrile has been described as the best solvent system in terms of maximizing extraction efficiency and minimizing coextractives (35). For practical bulk total isoflavone extraction on a large scale, methanol and ethanol are frequently used as a first step (25,36–38); ethanol is a practical choice from a cost and safety perspective, in addition to its common use in the soy processing industry. Additional solvents have also been investigated; for example, a method has been described whereby soy meal is extracted with acetone, and the extract is then diluted with ice-cold water to precipitate a genistin-enriched fraction, although the composition of this fraction and isoflavone extraction efficiencies were not disclosed (39). Ethyl acetate, which is a poor solvent for glycosides, has also been used as the extraction solvent (24), whereby defatted soy flour, grits, or soy germ are mixed with water and a β-glucanase/β-xylanase (Bio-Feed Beta CT; Novo Nordisk, Denmark) to effect conversion of glycosides to aglycones. Ethyl acetate is then lay-

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ered on top of the aqueous mixture to create a 2-phase system, which is then agitated and recirculated for 18 h. The aglycones are said to partition into the ethyl acetate phase during this process; this is then decanted, evaporated, mixed with hexane to solubilize residual oil, and allowed to stand overnight. The particulate material that settles to the bottom is said to contain ~35% genistein and 28% daidzein on a dry weight basis when using soy flour as raw material, and 18% genistein, 35% daidzein, and 18% glycitein when using soy germ as a raw material. Isoflavone recovery is reported to be between 75 and 80% for this process. Concentration Soy molasses serves as an ideal starting material for further concentration and separation of isoflavones. A typical isoflavone profile of commercial soy molasses is shown in Table 4.2. Modestly enriched isoflavone fractions from soy molasses have been produced by simply diluting high-solids molasses (53% solids, 2.18% total isoflavones, dry basis) to ~18% with water followed by centrifugation to produce an enriched cake (6–8% total isoflavones, dry basis) (37). A similar process is described by Waggle (40) in which concentrated soy molasses (56% solids, 1.2% isoflavones, dry basis) is diluted to 6.6, 13.7, or 28% solids, pH adjusted to 4.5, and centrifuged at 3000 rpm for 30 min at 60 or 0.6°C. Regardless of conditions investigated, total isoflavone concentration of the resulting cake was ~3% (dry basis). To improve recovery, isoflavones were more completely converted to glycosides by adjusting solids content to 6.6 or 13.7%, pH to 11 and holding for 30 min at 35°C, followed by pH adjustment to 4.5, temperature to 4°C, and centrifugation at 10,000 rpm. The resulting cake contained 4.2–4.7% total isoflavones (dry basis). Additional experi-

TABLE 4.2 Isoflavone Content of Soy Molassesa Isoflavone concentration (µg/g, dry basis) Genistin Malonyl-Genistin Acetyl-Genistin Genistein Daidzin Malonyl-Daidzin Acetyl-Daidzin Daidzein Glycitin Malonyl-Glycitin Acetyl-Glycitin Glycitein aSource: bNot

Reference 40. reported.

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4878 1329 0 88 3533 928 210 84 500 105 —b 360

ments were described in which the isoflavones are converted to aglycones followed by centrifugation; however, not enough information was provided to calculate the isoflavone concentration in the resulting cake. From the above examples, it is evident that the isoflavones present in soy molasses are not easily separated by centrifugation. Soy molasses is a brown, creamy, complex mixture containing ~70% sugars, 7-8% protein, 10% ash, and 12% fat (dry basis), which does not readily promote the formation of distinct crystals that could otherwise be separated from the remaining mixture. One simple method (41) for isolating high purity genistin from soy molasses involves subjecting the molasses (~8–12% solids) to high temperature (65–95C°) to increase the solubility of the isoflavones (especially genistin). The molasses is then subjected to ultrafiltration at these elevated temperatures to produce a clear permeate, which contains isoflavones and sugars separated from the large-molecular-weight protein and lipid complexes that otherwise impair proper crytallization. Ultrafiltration under pressure and shear is essential because soy molasses easily blinds most commercial filter papers and similar filter media. As this permeate is slowly allowed to cool to room temperature, fine, white crystals form; they are easily separated by centrifugation at low speeds (900 × g) and contain ~75% genistin. This product can be further purified (>95%) with repeated recrytallization from 80% ethanol as has been described previously (25). Isoflavone enrichment has also been achieved through the use of chromatographic resins. The earliest described use of nonpolar and slightly polar adsorptive resins to purify isoflavones was by Iwamura (42). In this process, hexane-defatted soybeans (100 g) are extracted with 0.4% sodium hydroxide (1.5 L) in a process similar to soy protein isolate manufacture. The insoluble fibrous material is removed by filtration, and the filtrate is either passed directly through a 200-mL bed of absorptive resin (styrene divinylbenzene or acrylic ester based), or the filtrate is acidified with acetic acid to precipitate proteins before being passed through the resin. The resin is then washed with water to remove any residual unbound contaminants and eluted with methanol or aqueous methanol to produce a yellowish-brown powder rich in isoflavones and saponins (analysis not provided in patent). A similar use of styrene divinylbenzene resins was described by Fleury et al. (43), in which an 80% aqueous methanol extract (2.25 L) of defatted soybeans (89 g) was evaporated to 0.38 L, diluted with water to 0.75 L, then slurried with 450 mL of Amberlite XAD-4. The resin was then filtered and washed with water, followed by isoflavone desorption with methanol (750 mL) to produce a product containing 49% isoflavones. Similar processes have also been described using ultrafiltration at elevated temperatures (65–95°C) to aid recovery of isoflavones from soy molasses as well as remove potential fouling agents before passage through the resin bed (38). In addition to the resins mentioned above, the similar use of activated carbons has also been described (21). In general, the use of resins produces a product containing between 30 and 50% isoflavones from the above methods when the eluted product is simply dried; however, additional increases in concentration can be achieved by evaporating the eluted isoflavone product to pro-

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mote crytallization, then filtering or centrifuging the resulting crystals (44). This process can produce a product contaning ~80% isoflavones. Cation-exchange resins with a sulfonic acid functional group have also been utilized as a means to specifically isolate isoflavones, especially 7-glycosides (45). A commercial resin such as Dowex MSC-1 is sodium-charged using sodium hydroxide, rinsed with water, and a filtered soy molasses (pH 9) is passed through the resin. The column is then rinsed with water followed by desorption with 0.1– 1.0 mol/L acetic acid in 86% ethanol or methanol. The resulting product is described as having an isoflavone content of ~50%. The use of anion exchange resins has also been described as a means to reduce the isoflavone content of soy protein isolate by ~90%, and simultaneously produce an isoflavone-rich product (46). In this method, a solution of soy protein isolate (6.5% solids, pH 6.8) is passed though Amberlite IRA-910, a strongly basic quaternary ammonium macroreticular resin, which has been regenerated with 6% NaOH, 1% HCl, and 1.5% NaHCO3. The column is then rinsed with water and eluted with 50% ethanol to produce an isoflavone-rich product (concentration not disclosed). Isoflavone Separation Although nonpolar and slightly polar adsorption resins, as well as cation and anion exchange resins, have all been described for use in concentrating isoflavones, the slightly polar adsorption resins offer the additional advantage that further isoflavone separation is possible by using gradient elution from the resin. As discussed by Zheng et al. (36), a 100% methanol extract (9 L) of defatted soy flakes (909 g) is first diluted with water to 20% methanol, then passed through a 10 × 178 cm column containing polymethacrylate resin (TosoHaas CG-71). The column is then sequentially eluted with five column volumes each of 50, 60, and 75% methanol. Malonyl-glycosides and daidzin are eluted with 50% methanol in the first 5 column volumes, whereas genistin is eluted with 60% methanol between 7 and 10 column volumes. The daidzin and genistin fractions are described as having a purity of 62–63%. Glycitin is found to coelute with daidzin in this process. One can easily see the utility of this resin for both bulk and individual isoflavone separations. If a crude mixture of isoflavones is desired, a simple elution step with 75–80% ethanol or methanol serves to remove all isoflavones from the column. If genistin and daidzin are desired, soy molasses is the ideal starting material, and these two are easily separated as described above using 50 and 60% methanol. Residual genistin can be removed from daidzin with the use of aluminum oxide (47,48), and residual daidzin can be removed from genistin by recrytallization procedures previously discussed (25). If glycitin is desired, a methanol extract of (preferably hexane-defatted) soy germ can be made, diluted to 20% methanol, and passed through the column. Daidzin and glycitin will elute in the 50% methanol fraction, and daidzin can be separated from glycitin by recrystallization of glycitin from absolute ethanol. The use of similar resins to purify malonyl-glycosides has also been described (21). Soy whey (20 L) was passed through a 5 × 21.5 cm (420 mL) column of

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DIAION HP-20 (Mitsubishi Kagaku), washed with distilled water, then eluted with 2 L of 5% aqueous ethanol, followed by 3 L each of 10, 20, 30, and 40% aqueous ethanol, then 2 L of 50% aqueous ethanol. This process is said to allow the separation of malonyl genistin from malonyl daidzin. Further purification of each fraction was performed on ODS resin using 5% ethanol to elute malonyl daidzin and 10% ethanol to elute malonyl genistin from each respective sample.

Summary and Conclusions From an industrial perspective, soy molasses and soy whey offer ideal starting materials for further purification of isoflavones. Both of these starting materials are coproducts from other primary processes, are significantly enriched in isoflavones compared with soybeans, and have historically been considered of low commercial value. Although these products by themselves may seem attractive from the standpoint of isoflavone concentration, they are replete with nondigestible oligosaccharides and are hygroscopic. For the production of isoflavone supplements and food ingredients, a higher concentration can be achieved through the use of a variety of nonpolar and slightly polar adsorptive resins, as well as ion-exchange resins, to produce a product with an isoflavone concentration in the range of 30–50%. This product is satisfactory for tableting and contains the full range of isoflavones present in the starting material. To further improve color, flavor, and increase concentration, the isoflavones eluted from the resin can be concentrated to promote crystallization, then filtered or centrifuged to produce a product of up to 80% purity. If isoflavone separation is desired, a slightly polar resin such as polymethacrylate can be employed, with gradient aqueous alcohol elution required to separate the major isoflavone classes. This review was intended to provide the reader with an industrial perspective on the isolation of isoflavones from natural materials, namely, soy-based raw materials. Such purified isoflavone products are currently used as dietary supplements as well as food ingredients. Although these products are not intended to replace whole soy foods in the diet, they offer an additional choice for the individual. These products have also provided a means to conduct properly blinded placebo-controlled clinical studies, and may eventually serve as substrates for future drug development and special agricultural uses. References 1. Crouse, J.R., 3rd, Morgan, T., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with That of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070–2076. 2. Lichtenstein, A.H. (1999) Soy Protein, Isoflavonoids, and Risk of Developing Coronary Heart Disease, Curr. Atheroscler. Rep. 1, 210–214. 3. Messina, M., Gugger, E.T., and Alekel, D.L. (2001) in Handbook of Nutraceuticals and Functional Foods, pp. 77-98, R.E.C. Wildman, Boca Raton.

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4. Mitchell, J.H. (2001) in Handbook of Nutraceuticals and Functional Foods, pp. 99–112, R.E.C. Wildman, Boca Raton. 5. U.S. Department of Agriculture (1999) USDA-Iowa State University Database on the Isoflavone Content of Foods, http://www.nal.usda.gov/fnic/foodcomp/Data/isoflav/ isfl_tbl.pdf. 6. Klein, B.P., Perry, A.K., and Adair, N. (1995) Incorporating Soy Proteins into Baked Products for Use in Clinical Studies, J. Nutr. 125, 666S–674S. 7. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 8. Uckun, F.M., Evans, W.E., Forsyth, C.J., Waddick, K.G., Ahlgren, L.T., Chelstrom, L.M., Burkhardt, A., Bolen, J., and Myers, D.E. (1995) Biotherapy of B-Cell Precursor Leukemia by Targeting Genistein to CD19-Associated Tyrosine Kinases, Science 267, 886–891. 9. Uckun, F.M., Messinger, Y., Chen, C.L., O’Neill, K., Myers, D.E., Goldman, F., Hurvitz, C., Casper, J.T., and Levine, A. (1999) Treatment of Therapy-Refractory BLineage Acute Lymphoblastic Leukemia with an Apoptosis-Inducing CD19-Directed Tyrosine Kinase Inhibitor, Clin. Cancer Res. 5, 3906–3913. 10. Uckun, F.M., Narla, R.K., Jun, X., Zeren, T., Venkatachalam, T., Waddick, K.G., Rostostev, A., and Myers, D.E. (1998) Cytotoxic Activity of Epidermal Growth FactorGenistein Against Breast Cancer Cells, Clin. Cancer Res. 4, 901–912. 11. Trieu, V.N., Narla, R.K., Myers, D.E., and Uckun, F.M. (2000) EGF-Genistein Inhibits Neointimal Hyperplasia After Vascular Injury in an Experimental Restenosis Model, J. Cardiovasc. Pharmacol. 35, 595–605. 12. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) Genistein, a Specific Inhibitor of Tyrosine-Specific Protein Kinases, J. Biol. Chem. 262, 5592–5595. 13. Kosslak, R., Bookland, R., Barkei, J., Paaren, H., and Appelbaum, E. (1987) Induction of Bradyrhizobium japonicum Common Nod Genes by Isoflavones Isolated from Glycine max, Proc. Natl. Acad. Sci. U S A 84, 7428–7432. 14. Kosslak, R., Bookland, R., and Appelbaum, E., U.S. Patent 5,229,113 (1993) 15 Harborne, J.B., Mabry, T.J., and Mabry, H. (1975) in The Flavonoids, pp. 746–757, Chapman and Hall, London. 16. Mazur, W., and Adlercreutz, H. (1998) Natural and Anthropogenic Environmental Oestrogens: The Scientific Basis for Risk Assessment, Pure and Applied Chemistry 70, 1759–1776. 17. Pantry (1988) in Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medicinal Properties, pp. 52–60, Alan R. Liss, New York. 18. Messina, M.J., Persky, V., Setchell, K.D., and Barnes, S. (1994) Soy Intake and Cancer Risk: A Review of the In Vitro and In Vivo Data, Nutr. Cancer 21, 113–131. 19. Wang, H.-J., and Murphy, P.A. (1994) Isoflavone Composition of American and Japanese Soybeans in Iowa: Effects of Variety, Crop Year, and Location, J. Agric. Food Chem. 42, 1674–1677. 20. Wang, H.-J., and Murphy, P.A. (1994) Isoflavone Content in Commercial Soybean Foods, J. Agric. Food Chem. 42, 1666–1673. 21. Matsuura, M., Obata, A., Tobe, K., and Yamaji, N., U.S. Patent 5,789,581 (1998). 22. Bryan, B.A., Allred, M.C., and Roussey, M.A., U.S. Patent 5,827,682 (1998).

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23. Matsuura, M., and Obata, A. (1993) β-Glucosidases from Soybeans Hydrolyze Daidzin and Genistin, J. Food Sci. 58, 144–147. 24. Kelley, G.E., Huang, J.L., Deacon-Shaw, M.G., and Waring, M.A., U.S. Patent 6,146,668 (2000). 25. Walter, E.D. (1941) Genistin (an Isoflavone Glucoside) and Its Aglucone, Genistein, from Soybeans, J. Am. Chem. Soc. 63, 3273–3276. 26. Wang, K., Kuan, S.S., Francis, O.J., M., W.K., and Carman, A.S. (1990) A Simplified HPLC Method for the Determination of Phyoestrogens in Soybean and Its Processed Products, J. Agric. Food Chem. 38, 185–190. 27. Fulmer, R.W. (1989) The Preparation and Properties of Defatted Soy Flours and Their Products, in Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs (Applewhite, T.H., and Kraft, Inc., eds.) pp. 55– 61, American Oil Chemists’ Society, Champaign, IL. 28. Wang, H.-J., and Murphy, P.A. (1996) Mass Balance Study of Isoflavones During Soybean Processing, J. Agric. Food Chem. 44, 2377–2383. 29. Circle, S.J., and Whitney, R.W., U.S. Patent 3,365,440 (1968). 30. Konwinski, A.H., U.S. Patent 5,097,017 (1992). 31. Lusas, E.W., and Riaz, M.N. (1995) Soy Protein Products: Processing and Use, J. Nutr. 125, 573S–580S. 32. Shen, J.L., and Bryan, B.A., U.S. Patent 6,015,785 (2000). 33. Shen, J.L., U.S. Patent 5,352,384 (1994). 34. Shen, J.L., and Bryan, B.A., U.S. Patent 5,637,562 (1997). 35. Murphy, P.A. (1981) Separation of Genistin, Daidzin and Their Aglycones and Coumesterol by Gradient High-Performance Liquid Chromatography, J. Chromatogr. 211, 166–169. 36. Zheng, B., Yegge, J.A., Bailey, D.T., and Sullivan, J.L., U.S. Patent 5,679,806 (1997). 37. Konwinski, A.H., U.S. Patent 6,228,993 B1 (2001). 38. Gugger, E.T., and Dueppen, D.G., U.S. Patent 5,702,752 (1997). 39. Day, C.E., U.S. Patent 5,932,221 (1999). 40. Waggle, D.H., U.S. Patent 5,919,921 (1999). 41. Gugger, E.T., and Dueppen, D.G., U.S. Patent 5,792,503 (1998). 42. Iwamura, J., U.S. Patent 4,428,876 (1984). 43. Fleury, Y., Welti, D.H., Philippossian, G., and Magnolato, D. (1992) in Phenolic Compounds in Food and Their Effects on Health (Huang, M.-T., Ho, C.-T., and Lee, C.Y., eds.) pp. 98-113, Washington. 44. Gugger, E.T., and Grabiel, R.D., U.S. Patent 6,033,714 (2000). 45. Chaihorsky, A., U.S. Patent 5,670,632 (1997). 46. Johns, P.W., Suh, J.D., Daab-Krzykowski, A., Mazer, T.B., and Mei, F.-I., U.S. Patent 6,020,471 (2000). 47. Wang, L.C. (1971) Separation of Soybean Isoflavones from Their 5-Hydroxy Derivatives by Thin-Layer Chromatography, Anal. Biochem. 42, 296–298. 48. Naim, M., Gestetner, B., Zilkah, S., Birk, Y., and Bondi, A. (1974) Soybean Isoflavones. Characterization, Determination, and Antifungal Activity, J. Agric. Food Chem. 22, 806–810. 49. Kudou, S., Shimoyamada, M., Imura, T., Uchida, T., and Okubo, K. (1991) A New Isoflavone Glycoside in Soybean Seeds (Glycine max Merrill), Glycitein 7-O-β-D-(6”O-Acetyl)-Glucopyranoside, Agric. Biol. Chem. 55, 859–860.

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Chapter 5

Human Dietary Sources of Phytoestrogens and Methods of Determination Chung-Ja C. Jackson and H.P. Vasantha Rupasinghe Guelph Centre for Functional Foods, Laboratory Services, University of Guelph, Guelph, Ontario, Canada

Introduction Phytoestrogens are compounds that occur naturally in plants and have numerous important beneficial effects on human health, primarily by mimicking mammalian estrogens. The classical definition of phytoestrogens is that they are compounds that exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female mammals. There are three major groups of biologically active phytoestrogens: isoflavones, lignans, and coumestans (Fig. 5.1). These phytochemicals belong to a larger class of polyphenols, which are found in all plants; they are characterized by nonsteroidal structures similar to mammalian estrogens, such as estradiol, and have estrogenic properties. With a few exceptions, the presence of a phenolic ring is an essential feature of the chemical structure of phytoestrogens and is a prerequisite for binding to estrogen receptors (ER) (Fig. 5.2). Therefore, phytoestrogens can act either as estrogen agonists or as estrogen antagonists (1). A number of phytoestrogens have been found in fruits, vegetables, and wholegrain foods consumed by humans, but the food sources of phytoestrogens are limited to relatively few of the plant foods commonly consumed (2). Soybeans (Glycine max (L.) Merrill), flaxseeds (Linum usitatissimum L.), and alfalfa (Medicago sativa L.) (consumed as sprouts) are the most significant dietary sources of isoflavones, lignans, and coumestans, respectively. Isoflavones are also found in clover (which is consumed

Fig. 5.1. Classification of the major sources of dietary phytoestrogens.

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Fig. 5.2. Chemical structural similarities between the isoflavone metabolite, equol, and estrogen, 17β-estradiol.

by cattle and sheep) and in chickpeas or garbanzo beans (Cicer arietirum) (3). Other kinds of isoflavonoids occurring in legumes include biochanin A, formononetin, and coumesterol (3,4). In addition, significant quantities of the isoflavone genistein are present in the American groundnut (Apios americana Medikus) (5). Isoflavones and lignans are two major classes of phytoestrogens that have recently attracted the attention of scientists who are interested in the effects of bioactive phytochemicals on human health. The principal dietary sources of isoflavones and lignans are soybeans and flaxseed, respectively. Clinical, epidemiologic, and laboratory studies involving humans, animals, and cell cultures suggest that dietary isoflavones play important roles in the prevention of cancer (1,6–12) and heart disease (13,14), menopausal symptoms, and osteoporosis (8, 15–18). Epidemiologic investigations have also demonstrated that certain lignans may have anticancer effects, whereas others function as antioxidants or as antimitotic, antiviral, antibacterial, or antifungal agents (19). Animal studies have shown, moreover, that flaxseed, the richest source of mammalian lignan precursor, can reduce the incidence of mammary tumors, colon tumors, and metastases of melanoma cells in the lung (20). There are 12 known isoflavone compounds in soybeans (three aglycones, three glucosides, three acetyl-ester glucosides, and three malonyl-ester glucosides). Among them 6”-O-malonylgenistin, genistin, 6”-O-malonyldaidzin, and genistin are the major constituents. In nonfermented soy foods, the isoflavones are mainly

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in the form of glucoside conjugates, whereas in fermented soy foods (e.g., miso and tempeh), the aglycones are common (21,22). The lignans, which are ubiquitous constituents of vascular plants, consist predominantly of dimeric phenylpropanoids. The most common lignans are secoisolariciresinol (SECO) and matairesinol (MAT) in flaxseeds (23). The analytical methods for determining phytoestrogens consist mainly of (i) high-performance liquid chromatography (HPLC) with a photodiode array (PDA), electrochemical, fluorometeric, or mass spectrometric (MS) detector (21,22, 24–32) or (ii) gas chromatography (GC) with an MS detector (6,33,34). Unlike GC-MS systems, the HPLC-MS (generally called LC-MS) systems have the advantages of requiring less preliminary purification of the sample and no derivatization (35,36). A less expensive technique is the combination of HPLC with a coulorimetric array (CoulArray) detector (37). The most recent technological advances in this field have involved the development of methods for performing immunoassays, such as radioimmunoassays (RIA) (38) and fluoroimmunoassays (39). At present, the isotope-dilution GC-MS-selected ion monitoring mode (SIM) technique (ID-GC-MS-SIM) is the only method by which all of the lignans (e.g. SECO, MAT, and anhyroSECO) and isoflavonoids (e.g., genistein and daidzein), together with biochanin A, formonometin, and coumestrol (in short, most of the major phytoestrogens of interest in food samples) can be determined simultaneously (4,23,39). This chapter reviews currently available information on dietary sources of phytoestrogens and analytical methods for determining phytoestrogens in plants and plant-derived foods.

Structural Diversity of Phytoestrogens Isoflavonoids and Isoflavones. Flavonoids represent a large and diverse group of phenylpropanoid-derived natural plant products. Isoflavonoids form a distinct class among flavonoids and have a characteristic chemical structure (Fig. 5.3). The 15carbon (C6-C3-C6) backbone of flavonoids can be arranged as a 1,3-diphenylpropane skeleton (flavonoid nucleus) or as a 1,2-diphenylpropane skeleton (isoflavonoid nucleus) (Fig. 5.3). Isoflavonoids are derivatives of flavone, a heterocyclic compound displaying a wide range of substitution patterns and oxidation states. They include flavonols, flavanols, flavanones, and flavans, or catechins. The hydroxylation and alkoxylation patterns of the A- and B-rings of isoflavones are of great importance in determining the activity of these compounds as antioxidants (Fig. 5.3). In isoflavones, the B-ring is attached to the C-ring at the 3- rather than the 2-position. This precludes the occurrence of a hydrogen-bonded hydroxyl group at the 3-position, diminishing the probability of significant contributions by such a group to the antioxidant activity of an isoflavone (40). There are nearly 900 naturally occurring isoflavonoid aglycones, which can be divided into 9 major classes on the basis of differences in their carbon skeletons (41). The isoflavones and pterocarpans are the most abundant isoflavonoids, with 334 and

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Fig. 5.3. A representative structure of the three major isoflavone aglycones (daidzein, genistein, and glycitein) and the 4-methyl ether derivatives of daidzein and genistein, formononetin and biochanin A, respectively.

152 different structures, respectively. This enormous diversity is due to the wide variety of substituents (e.g., methoxyl, prenyl, and methylenedioxy) that can occur at many different positions of the A- and B-rings. To date, ~90% of all isoflavonoids that have been isolated belong to leguminous plants (41). The principal phytoestrogen isoflavones known are daidzein (4′,7-dihydroxyisoflavone) and genistein (4′,5,7-trihydroxyisoflavone). It is believed that all isoflavonoids are derived from a restricted number of simple isoflavone aglycones, such as daidzein and genistein. In addition, the 4-methyl ether derivatives of genistein and daidzein (biochanin A and formononetin, respectively) (Fig. 5.3) are weakly estrogenic compounds (42). Daidzein and genistein occur in four different forms, namely, the aglycones (daidzein and genistein), the 7-O-β-glucosides (daidzin and genistin), the 6”-O-acetylglucosides (6”-O-acetyldaidzin and 6”-O-acetylgenistin) and the 6”-O-malonylglucosides (6”-O-malonyldaidzin and 6”-O-malonylgenistin) (Fig. 5.4). In addition, a minor component of isoflavones, glycitein (4′,7-dihydroxy-6methoxyisoflavane or 6-methoxydaidzein), has been isolated from plants, but no estrogenic activity has been reported to date for this compound. Similarly, glycitein also exists in four different forms, i.e., the aglycone (glycitein), the 7-O-β-glucosides (glycitin), 6”-O-acetylglycitin, and 6”-O-malonylglycitin (Fig. 5.4). Coumestans are biosynthetically related to the isoflavones. Although a large number of coumestans have been isolated from plants (43), only a few have been shown to possess uterotropic activity. Coumestrol (7,12-dihydroxycoumestan) and 4′-methoxycoumestrol are estrogenic coumestans found in alfalfa, ladino clover (Trifolium repeus L.), and other fodder crops (Fig. 5.5) (44). However, the estrogenic activity of coumestrol is greater than that of the most potent isoflavones with respect to uterine gland development and influence on ER levels (42). Coumestrol and genistein have been shown to be more potent than any other known phytoestrogens when their in vitro estrogenic activity was compared with that of 17βestradiol (45).

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Fig. 5.4. Glucoside forms of the three major isoflavone aglycones (daidzein, genistein,

and glycitein).

Lignans. Lignans are phenolic compounds formed by the union of two monomeric C6C3 moieties (cinnamic acid residues) and thus have a dibenzylbutane skeleton structure. It has been suggested that bacteria in the colon transform plant lignans into the mammalian lignans enterodiol (ED) and enterolactone (EL), which provide protection against cancer. The precursor for ED is the plant lignan SECO, whose precursor, in turn, is SECO diglycoside (SDG). EL, however, is produced from MAT (20,46,47,48) (Fig. 5.6). SECO, MAT, and certain other lignans play important pharmacologic roles (32). These other lignans include pinoresinol from Forsythia intermedia, sesamin and sesamolinol from sesame (Sesamum indicum) seeds, podophyllotoxin from Mayapple (Podophyllum peltatum) rhizomes, gomisin

′

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Fig. 5.5. Structure of coumestrol and 4′-

methoxycoumestrol (coumestan).

Fig. 5.6. The two major plant lignans, secoisolariciresinol (SECO) and matairesinol (MAT) and their metabolites (mammalian lignans), enterodiol and enterolactone.

A isolated from Schizandra chinensis fruits, and nordihydroguaiaretic acid from the creosote bush (Larrea tridendata) (32).

Biosynthesis of Isoflavones The isoflavone biosynthesis pathway and the enzymes involved in it have been studied extensively. Recent molecular biological approaches have been successful in elucidating the mechanism and regulation of isoflavone biosynthesis (49). Isoflavones are synthesized by a branch of the phenylpropanoid pathway of secondary metabolism (Fig. 5.7). The biosynthesis of isoflavone molecules begins with the precursor compound chalcone (C15), which is formed by head-to-tail condensation of 4-coumaroyl CoA and three molecules of malonyl CoA, a reaction catalyzed by the enzyme chalcone synthase (CHS) (Fig. 5.7). CHS catalyzes the addition, condensation, and cyclization reactions leading to the formation of tetrahydroxychalcone (naringenin chalcone). This enzyme has been purified and well characterized, and the genes that produce it have been cloned from many plant

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Fig. 5.7. A partial diagram of the phenylpropanoid pathway showing intermediates and enzymes involved in isoflavone synthesis.

species (50). Chalcone reductase (CHR) activity of legumes is required for production of the second substrate for isoflavone synthesis, trihydroxychalcone. Chalcone isomerase (CHI) catalyzes the conversion of naringenin chalcone and trihydroxychalcone to liquiritigenin and naringenin, respectively. CHI-catalyzed reactions are reported to be the rate-limiting steps in flavonol biosynthesis. Constitutive overexpression of the Petunia gene encoding CHI in tomato resulted in an increase of up to 78-fold of flavonols in the fruit peel (51). The first isoflavonoids are synthesized by aryl migration of the 5-hydroxy- and 5-deoxyflananones, naringenin and liquiritigenin, which are catalyzed by the isoflavone synthase (IFS), yielding genistein

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(5,7,4′-trihydroxyisoflavone) and daidzein (7,4′-dihydroxyisoflavone), respectively. IFS is thought to have evolved independently in the Leguminosae and the other diverse taxa in which isoflavonoids are occasionally found (49). Once the basic isoflavone skeletons (e.g., genistein and daidzein) were formed, all other isoflavonoids are believed to be derived from them as a result of structural modifications and diverse oxygenated side attachments originating from isoprenoid substituents. Genes homologous to the soybean IFS gene have been cloned from several related legume species (red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine) as well as a nonlegume, sugar beet. Two isoforms of IFS have been identified in soybeans, and both of them are evidently able to use both naringenin and liquiritigenin as substrates to produce genistein and daidzein, respectively (52). All of these genes are structurally and functionally similar (52). When the IFS gene was expressed in nonlegume plants such as Arabidopsis thaliana, tobacco, and corn cell lines, production of isoflavones was demonstrated (53), suggesting that the IFS gene could be introduced into other major food crops to produce isoflavones. Yu et al. (53) showed that introduction of IFS1 together with CHR, which provides an additional substrate, liquiritigenin, resulted in the synthesis of daidzein in corn. In summary, one of the significant advances in the biosynthesis of isoflavonoids is the understanding of its regulation at the molecular level. The challenge for the future will be to find the means of manipulating the genes of certain edible plants in such a way as to increase the levels of isoflavones and other phytoestrogens in the plants that produce them, or to induce production of these compounds in plants whose wild types do not produce them. The purpose of this would be, of course, to increase the dietary intake of health-promoting phytoestrogens. In particular, cereal crops such as corn, wheat, and rice, which do not produce isoflavones, are likely to be the target crops for bioengineering in the future because they are major staple foods for both humans and livestock.

Human Dietary Sources of Phytoestrogens Phytoestrogens are found in a wide variety of plants, including cereals, legumes, and grasses. The most important staple plant foods in human diets are those derived from cereals. In addition to their major nutrient components, cereals contain fiber, phytic acid, various phenolic compounds, and phytoestrogens. Irvine et al. (54) found highly variable amounts of phytoestrogens in food products made from cereals, with genistein ranging from 3 to 287 µg/g and daidzein from 2 to 276 µg/g. Legume seeds are rich in complex carbohydrates (both starch and dietary fiber) and contain a broad spectrum of phytochemicals. Legumes are the most important family in terms of isoflavone content. Soybeans, clover, mung bean, alfalfa, peanut, and kudzu (Pueraria lobata) are the major sources of isoflavones (5).

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Isoflavones in Soybeans and Their Products In the plant kingdom most of the flavonoids are ubiquitous, but the isoflavones are restricted primarily to one plant family, the Leguminosae (Fabaceae) and occur only rarely in other families, such as the Apocynaceae, Pinaceae, Compositae, and Moraceae (41). Soybeans and soy foods provide the most abundant dietary sources of isoflavones (1). Soybeans contain ~1.2–3.3 mg of isoflavones/g dry weight (21,22). In Far Eastern populations that consume large amounts of soy products (amounting to 25–100 mg isoflavones/d in Japan), a lower incidence of breast cancer compared with the Western world has been observed (32). This has been attributed to the greater consumption of soy foods in those countries. The phytoestrogen concentrations in soy products depend on plant variety, location, year of harvest, and maturity (21,42,55). Soy products derived from the hypocotyledon are among the most concentrated sources of isoflavones (>20 mg/g). Numerous commercial soy supplements, many of which are made from concentrated extracts of soybean, are now available, obviating the need to consume whole-soy foods to benefit from the health-giving phytoestrogens (isoflavones) of soybeans. Although a high proportion of foods contain soy products, they consist mainly of soy oils and soy lecithins, which are devoid of isoflavones. Isoflavones are associated with the protein fraction of the soybean during its processing, and because soy protein is rarely a normal component of the average Western diet, the average daily dietary intake of isoflavones in Western populations is typically negligible (<1 mg/d) (6,56). Total concentrations of genistein and daidzein (on a dry weight basis) are very different in soy products, such as alcohol-extracted soy protein concentrate and soy protein isolate (SPI), than in whole soybeans. In general, isoflavones are lost not during the defatting of full-fat soybeans, but during the SPI processing steps; 19, 14, and 22% are lost during extraction, precipitation, and washing, respectively (57). During the production of soy foods (e.g., soy milk and tofu), certain percentages of the isoflavones are removed with the by-products (58). It is advisable for the processors to try to retain as much as possible of the isoflavone content in the starting materials. In whole-soybean and nonfermented soy products, isoflavones are present mainly in the form of their 7-O-β-glucoside conjugates, whereas aglycones are the principal isoflavones in fermented soy foods (32). Processes such as toasting, fermentation, and hot extraction can induce decarboxylation and deesterification, resulting in conversion of the isoflavones to 7-O-acetylglucosides, β-glucosides, or even aglycones (21,22,32,58). However, the conjugates (7-O-β-glucosides, 6”-Oacetylglucosides, and 6”-O-malonylglucosides) are transformed into aglycones or “free isoflavones” through hydrolysis in the intestinal tract by β-glucosidase enzymes of gut bacteria (59). Soy food processing appears to affect isoflavone bioavailability. In adults, the glucosides are enzymatically hydrolyzed in the colon by the intestinal flora, whereas aglycones are readily absorbed from the stomach and small intestine (54). In a

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comparative study of isoflavone excretion in urine among 17 men consuming either 112 g of soy tempeh (a fermentation product) or 125 g of unfermented soybean for 9 d, the recovery of daidzein and genistein from urine was higher in the subjects who consumed the tempeh diet than in those who consumed the unfermented soy diet (60). These findings suggest that the isoflavone aglycones in fermented food may be more bioavailable than their glucosides. In contrast, animal studies suggest that the extent of isoflavone absorption is similar for the aglycone and conjugated forms, although the initial absorption rate of aglycone forms is greater than that of the conjugates (1). Wang and Murphy (21,22) characterized the concentrations and distribution of all 12 isoflavone isomers in 29 commercial soybean foods. Unprocessed soybeans contained 1.2–3.3 mg of isoflavones/g dry weight, whereas high-protein soy ingredients such as soy flour and texturized vegetable protein (TVP) contained 1.1–1.4 mg/g dry weight. Soy concentrate, which is produced by a water or alcohol wash of soy flakes to remove soluble carbohydrates and improve functionality, contained an extremely low isoflavone concentration. Processed soy foods, such as tofu yogurt and tempeh burger, contained only 6–20% of the isoflavones found in whole soybeans. Hutabarat et al. (61) quantified daidzein, genistein, formononetin, and biochanin A, in >60 soybeans and soybean products purchased from retail outlets in Australia and Indonesia. They found daidzein and genistein in all soybean products except soy sauce, but Wang et al. (57) and Coward et al. (32), using more sensitive methods, detected them in soy sauce as well. The levels of daidzein and genistein in soybeans and soy foods vary by as much as a factor of six, suggesting that the concentrations of these compounds are much less consistent than the concentrations of nutrients such as protein. The high degree of variability was considered to be the result of genetic factors and environmental (including climatic) variables (21,22,55). Processing is known to affect the forms of isoflavones found in soy foods. In minimally processed soy flour, 6”-O-malonyldaidzin and 6”-O-malonylgenistin are the major isomers. In contrast, TVP contains appreciable amounts of 6”-O-acetyldaidzin and 6”-O-acetylgenistin due to the transformation of the malonyl isoflavones into their acetyl forms by heat treatment during extrusion processing. Nonfermented soy foods (e.g., soy beverages and tofu) contain higher levels of glucosides, whereas fermented soy foods (e.g., miso and tempeh) contain higher levels of aglycones as a result of enzymatic hydrolysis during fermentation (21,22). Jackson et al. (58) determined the concentrations of the 12 major isoflavones in soy products at different stages in the preparation of soy beverage and tofu. The mean recovery of isoflavones in soy beverage and tofu in relation to their initial concentration in the raw soybeans was 54 and 36%, respectively. The estimated percentages of total isoflavones lost in the water used to soak raw soybeans, the okara (waste from heat-treated slurry), and whey were 4, 31, and 18%, respectively. During processing, the detectable levels of aglycones, glucosides, and acetyl glycoside groups increased, whereas the corresponding malonyl glucosides

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decreased. Therefore, it is apparent that as a result of processing, bioavailable forms of isoflavones are increased even though the loss of total isoflavones is considerable. Other Sources of Isoflavones Chickpeas and other legumes, as well as clover, toothed medic (Medicago laciniata L.), and blue grass (Poa Pratenis L.), have also been identified as sources of isoflavones (62). Formononetin and biochanin A, the 4′-methyl esters of daidzein and genistein, respectively, were found in clover (63). Mazur et al. (4) determined the isoflavone concentrations in 68 cultivars of 19 common food legume species and 4 forage legumes. The highest total isoflavone concentration was found in kudzu root (Pueraria lobata and Radix puerariae) (>2 mg/g dry weight); the concentrations in soybeans and chickpeas were 0.37–1.9 and 0.01–0.036 mg/g dry weight, respectively. The same differences in concentration applied to daidzein and its precursor formononetin as well. Interestingly, all soybeans analyzed proved to be the richest source of genistein, the most biologically active of the phytoestrogens reported. Significantly larger quantities of genistein were also found in pigeon pea, groundnuts, pinto beans, and haricot beans. The highest concentration of biochanin A, the precursor of genistein, was found in C. arietinum, another commonly consumed legume species. Daidzein and genistein were detectable in all of the legumes except for the “green split” pea and “Canada” fava bean (4). Moreover, mung bean sprouts contain relatively high concentrations of daidzein (7 µg/g dry weight) and genistein (20 µg/g dry weight). Red clover was found to contain the highest concentrations of formononetin and biochanin A (5). In general, vegetables other than legumes do not contain isoflavones. Nevertheless, cruciferous plants contain low but detectable levels of daidzein and genistein (up to 0.14 µg/g dry weight). Jones et al. (64) analyzed 107 food items other than soy foods on the British market and could not detect isoflavones. Genistein and daidzein have been determined in only a few non-soy foodstuffs, mainly in legumes or cereals. Lignans in Flaxseed and Other Crops Lignans are considered by some researchers to be phytoestrogens because they have certain estrogen-like effects, although they have not been shown to induce estrus (see below). In addition, lignans have antioxidative properties, and they inhibit steroid hormone–metabolizing enzymes such as 5-reductase, 17β-hydroxysteroid dehydrogenase, and aromatase. Consumption of foods containing lignans has been negatively correlated with coronary heart disease (42). Lignans are present in plant foods as well as in human biological fluids. The conversion of plant lignans to mammalian lignans occurs in the gastrointestinal tract as a result of bacterial action. The plant lignans SECO and MAT are the dietary precursors of the

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mammalian lignans enterodiol and enterolactone (Fig. 5.6). These weakly estrogenic lignans also occur as glycosides. Lignans accumulate in various tissues, including roots, stems, leaves, flowers, fruits, and seeds, of higher plants. They are widespread in cereals, fruits, and vegetables. Oilseeds, such as flaxseed, are the richest plant sources of lignans in the plant kingdom. The concentration of SDG in flaxseeds is 75–800 times higher than that in other foods. Intake of flaxseed causes the highest mammalian lignan production (65). Mazur et al. (4) reported that the main lignan component of flaxseed was SECO (>3.6 mg/g dry weight, with minor amounts of MAT). When flaxseed is crushed and defatted, the SECO content rises to 6–7 mg/g dry weight, making it the plant source with the highest concentration of lignans. In general, most legumes contain SECO, but MAT levels seem to be relatively low. Among the most commonly consumed legumes, soybeans and peanuts (Arachis hypogaea) contain the highest levels of SECO (0.13–2.73 and 3.33 µg/g dry weight, respectively), but no MAT, or only a minor amount, has been detected (5). Higher concentrations of SECO (0.3–4.8 µg/g dry weight) have been found in mung (green) bean (Vigna radiata), bocate bean (Vigna sinensis Endl.), and “black pelun” cowpea (Vigna unguiculata) (5). The lignan content of foods has not been fully investigated, and the reported concentrations seem to be dependent upon the nature of the analytical method. When lignans were quantified using ID-GC-MS-SIM, the quantities were 4–5 times greater than those reported by other investigators on the basis of HPLC data (5). Thompson et al. (65) used in vitro fermentation with human fecal flora to determine mammalian lignan production from a variety of plant foods. The concentrations of lignan produced ranged from 0.02 to 67.5 mg/100 g (wet weight) of plant. The highest concentrations were found in oilseeds, including flaxseed and unhulled soybeans, with lesser amounts found in dried seaweeds, whole legumes, cereal brans, legume hulls, whole-grain cereals, vegetables, and fruits (65). Mazur et al. (4) reported lower concentrations of lignans in sesame, clover, sunflower, caraway, and poppy seeds. Lignan is also abundant in most nuts (e.g. cashews, hazelnuts, pistachios, walnuts, and almonds). Lignans are localized mainly in the outer fiber-containing layers of grains, and the highest concentrations occur in the aleurone and pericarp/testa layers (23). Therefore, intake of isolated wheat germ, bran, and white flour does not lead to significant mammalian lignan production. Because of its close association with the outer fiber layers, most milling techniques usually eliminate the aleurone with the pericarp/testa layers; consequently, they are seldom present in commercial products (5). Vegetables contain high levels of the lignan SECO; its concentrations in potato, celery, zucchini, asparagus, and pumpkin are 0.1, 1.14, 8.17, 30.7, and 38.7 µg/g dry weight, respectively. Cruciferous vegetables contain variable concentrations of SECO (e.g., cabbage, 0.33 and broccoli, 4.14 µg/g dry weight) and vegetables of the genus Allium, such as onion, garlic, and chives, contain relatively high levels of SECO (0.83, 3.80, and 12.54 µg/g dry weight, respectively) (5).

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Interestingly, high concentrations of the lignans SECO and MAT have been found in white and red wines (the range for total lignans was 1.53–13.7 µg/L), as well as in both black and green tea (5). Plant Sources of Coumestans Coumestans are produced predominantly during germination of legume seeds but also occur in fodder crops (62). The most significant sources of coumestans in the human diet are legume shoots and sprouts, mainly clover and alfalfa, which have coumestrol concentrations of 5.6 and 0.7 mg/g dry weight, respectively (66). Legumes such as split peas, kala chana seeds, pinto beans, lima beans, and soybean sprouts also contain small amounts of coumestrol (15–80 µg/g dry weight) (66,67). Coumestrol is present only at very low concentrations in most legumes. One of the richest sources of coumestrol found in human food is mung bean sprouts, which contain 20 times as much coumestrol (10 µg/g dry weight) as alfalfa sprouts (0.45 µg/g dry weight) (4). Furthermore, it has been observed that coumestrol concentrations in legumes increase after insect and fungal attack (42). Red clover (Trifolium pratense) contains significant amounts of at least four estrogenic isoflavones, i.e., formononetin, biochanin A, daidzein, and genistein (68). Clover, however (unlike soybeans, which are a common dietary source of phytoestrogens) has never been a dietary staple of humans, although it has been used as a dietary supplement for long-term hormone replacement therapy.

Methods of Determination Isolation and Purification of Isoflavones Isoflavones are present in soybeans as both simple (aglycones) and conjugated (glucoside) forms. It is apparent that the profile of isoflavones could be altered not only during the commercial processing of the soybeans but also during extraction. Therefore, the accuracy of identification and quantification of isoflavones present in soybeans and their products are highly dependent upon the extraction/purification procedures and conditions used in each method. For that reason, it is important to understand that a possible transformation could occur during the analysis designed to determine the isoflavone profile. This is especially critical if soy food products are to be consumed by subjects in clinical studies, in which the quantities and proportions of the various types of isoflavone absorbed into blood must be known (69). Extraction of isoflavones in soy foods using hot aqueous organic solvents (e.g., ethanol or methanol) (62,70) or refluxing in alcohol (71) resulted in complete conversion of 6”-O-malonyl- and 6”-O-acetyl-isoflavone β-glucosides to β-glucosides and aglycone forms. Extraction without heat, however, yielded a different group of isoflavone glucosides, which have been identified as 6”-O-malonyl β-glucoside (6OMalGlc) conjugates (72). Some procedures have employed 80%

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methanol (31), whereas others involved extraction with acidified acetonitrile at room temperature (21,22). However, the acidified acetonitrile procedure was found to be more efficient than any of the other extraction methods currently in use because the recovery was higher and fewer extraneous substances were coextracted (30,73,74). The isoflavone analysis of soy foods using reverse-phase HPLC-MS has shown that most soy foods contain mixtures of the β-glucoside 6OMalGlc and 6”O-acetyl-β-glucoside (6OAcGlc) conjugates (21,22,31). However, the 6OAcGlc conjugates in this case were formed from the 6OMalGlc due to dry-heat treatment during production (75). The isoflavone composition of soybean and soy products could be easily altered adversely by different kinds of treatment, leading to the creation of artifacts. These factors reinforce the importance of using appropriate procedures to extract genuine or intact isoflavones for interpretation of isoflavone content in human diets (69). To determine optimal extraction conditions, an investigation of isoflavone extraction was conducted with 80% methanol at various temperatures for different lengths of time (e.g., 4°C, room temperature, and 80°C for 2–72 h) (32). The results showed that the quantitative and reproducible recovery of the isoflavone glucosides could be achieved after 2 h (at 4°C). The highest concentration of 6OMalGlc conjugates and the lowest concentration of β-glucosides were extracted at 4°C. At room temperature, a slight conversion of the 6OMalGlc conjugates to βglucosides occurred. Extraction at 80ºC caused extensive conversion of the 6OMalGlc conjugates to the β-glucosides, but not to the 6OAcGlc conjugates or to aglycones. With increasing temperature, the concentrations of the individual β-glucosides were greatly altered, although the total quantity of isoflavone extracted was constant. As a result of hot aqueous processing (e.g., the processing of soy milk and tofu), all isoflavones end up in the form of β-glucosides conjugates. Products of fermentation (e.g., miso and tempeh) contain mainly aglycones, and ethanol extraction removes isoflavones from soy flour to produce soy-protein concentrates (32). 6OMalGlc conjugates are converted to β-glucosides by moist heat, but dry heat results in the formation of 6OAcGlc conjugates. The composition of the glucoside conjugates may have significant effects on the bioavailability and pharmacokinetics of the isoflavones. The composition of the isoflavone glucoside conjugates is known to affect the rate of absorption of these compounds, and possibly the extent of their further metabolism. Therefore, these alterations in the chemistry of isoflavones must be considered when interpreting data from clinical trials (32). In general, during isoflavone extraction for HPLC analysis, samples of soy products are extracted with an acidic acetonitrile for 2 h at room temperature (21,22,30,58). The acidified acetonitrile has the following advantages over other solvent systems: (i) more rapid settling of suspending sample particles, (ii) less interference by impurities in the extract, (iii) better recovery, (iv) less coextraction of substances other than isoflavones, and (v) improved efficiency of genistein, daidzein, and coumestrol extraction (74).

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In the isolation and purification of relatively large quantities of isoflavones (60–500 mg), high-speed counter-current chromatography (HSCCC) is a powerful technique (76,77). Isoflavones having a broad range of polarities were separated from a crude soybean extract by HSCCC using a procedure involving three solvent systems: (i) the first solvent system is used to separate less polar isoflavones (glycitein, daidzein, acetyl-genistin, and acetyl-daidzin); (ii) the addition of nbutanol made the second solvent system slightly more polar, resulting in the separation of the more polar isoflavones (genistin, glycitin, and daidzein); and (iii) the more aqueous third system separates the most polar components more effectively than the nonpolar compounds (in the order of decreased polarity, glycitin > daidzin > genistein). Each isolated component showed 98–99% purity as determined by HPLC, and their structures were identified by LC-MS. Due to its great flexibility combined with a high degree of selectivity, HSCCC employing a pair of two-phase solvent systems is an excellent method for large-scale purification of groups of compounds characterized by a wide range of polarities in crude extracts. High-performance size exclusion chromatography (HPSEC) columns allow the rapid characterization of biomolecules and other polymers based on their mass to separate major proteins and isoflavones in soybeans (78). Analytical Methods for Isoflavones in Soybeans and Soy Foods Different laboratories have employed a wide range of analytical techniques for the quantitative determination of isoflavones in soy foods. HPLC and GC are the principal methods, but there are others, including capillary electrophoresis (CE), time-resolved fluoroimmunoassay (TR-FIA) (26,79), luminescent immunoassays (80), enzymebased immunoassays (e.g., enzyme-linked immunosorbent assay; ELISA), and RIA (38). HPLC with different detectors, such as photodiode array/ultraviolet (PDA/UV), electrochemical, fluorometric, and mass spectrometric detectors, is commonly used for the determination of phytoestrogens (6,28–32,35,66,73,74). Thomas et al. (24) developed a method using HPLC-UV with a detection limit of ~2 ng/mL (Table 5.1) for daidzein, genistein, and glycitein. HPLC with PDA/UV detection (using reverse-phase C18 stationary matrices) has been the method of choice for the determination of phytoestrogens in soy foods for many years and is currently the most widely used analytical technique to quantify isoflavones (21,22,30–32,55,58, 61,73,74,81–84) and coumestrol (85). It is particularly suitable for pharmacokinetic studies and has numerous advantages, such as ability to measure (i) both free and nonconjugated molecules, (ii) the combined “free and conjugated molecules,” and (iii) the total free and conjugated molecules in foods, plasma, and urine. A simple, rapid procedure has been developed to determine isoflavonoids and their conjugates in foods by extraction, hydrolysis, and HPLC analysis (66,84). This method is used for the determination of daidzein, genistein, formononetin, biochanin A, and coumestrol. The detection limits obtained from authentic standards range from 1.3 to 4.2 ng/mL (Table 5.1).

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TABLE 5.1 Representative Detection Limits of Phytoestrogens Attainable with Different Analytical Methodsa Analytical methods HPLC/PDA

HPLC-UV

HPLC-fluorescent HPLC-coulorimetric Electrode array detection

HPLC-MS GC-MS-SIM ID-GC-MS-SIM

Analytes (phytoestrogen)

Sensitivity (detection limit)

Daidzein Genistein Coumestrol Formononetin Biochanin A Daidzein Genistein Glycitein Isoflavones Coumestrol Daidzein Genistein Biochanin A Genistein

1.31 ng/mL 2.37 ng/mL 6.89 ng/mL 1.95 ng/mL 3.70 ng/mL 1.76 ng/mL 2.22 ng/mL 2.55 ng/mL 2.0 µg/g 0.5 µg/g 0.90 ng/mL 0.95 ng/mL 1.55 ng/mL 1.8 ng/mL

Isoflavones Daidzein Formononetin Formononetin Biochanin A Daidzein Genistein Coumestrol SECO MAT

1–5 µg/g 2.3 ng/mL 1.0 ng/mL 20–30 ng/g 20–30 ng/g 20–30 ng/g 20–30 ng/g 20–30 ng/g 20–30 ng/g 20–30 ng/g

Reference 66 66 66 66 66 24 24 24 116 116 89 89 89 89 37 31 33 33 4 4 4 4 4 4 4

aAbbreviations:

HPLV/PDA, high-performance liquid chromatography/photodiode array; UV, ultraviolet light; MS, mass spectrometry; GC, gas chromatography; SIM, selected ion monitoring; ID, isotope dilution; SECO, secoisolariciresinol; MAT, matairesinol.

Results of reverse-phase HPLC-PDA analysis of soybeans, soy products, and other common foods, along with data gleaned from the literature, have been used to compile the isoflavone database generated by the USDA/Iowa State University (73). In addition to data for isoflavones, the database includes coumestrol, formononetin, and biochanin A concentrations. This database represents the first systematic, comprehensive documentation of isoflavone concentrations from many different foods (73). Reverse-phase HPLC has also been used to determine isoflavone levels in soy-based infant formulas, which have been used for >30 years and have recently been added to the growing list of isoflavone-containing foods (28,30,83,86,87). In addition, this method is used in research on metabolic excretion of phytoestrogens in urine after consumption of soy-based infant formula (54,87). HPLC analyses have demonstrated that daidzein, genistein, and glycitein are the major “free isoflavones” (aglycones) found in soy foods, although they com-

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prise only a minor component of the isoflavones in soybeans, whereas the isoflavones of raw soybeans are predominantly in the form of glucosides, mainly malonyl glucosides with minor amounts of acetyl glucosides (21,22,28,31,88). The range of average total concentrations of isoflavones in different varieties of soybeans is 1–3 mg/g, but the concentrations of isoflavones vary widely depending on a number of factors, such as environmental variables affecting growth of the plant, the genetic makeup of the plant, time of harvesting, and conditions of processing (21,22,31,32,55). HPLC employing a colourimetric electrode array detector (37,89–91) has proved to be a reliable, sensitive, and eminently suitable technique for routine determination of phytoestrogens. This method has the following advantages: (i) It is convenient and rapid because it requires neither derivatization nor extensive sample preparation; only hydrolysis and extraction of the samples or simple alcohol extraction is required. (ii) The detector, which is highly selective, excludes most contaminating compounds. (iii) The method is suitable for determination of conjugated phytoestrogens. One disadvantage, however, is that the technique is slightly less sensitive than GC-MS, although it has significantly higher sensitivity than HPLC-UV/PDA (91). LC-MS has also been widely used for isoflavone analyses, especially in clinical studies of isoflavone metabolites in humans and animals (25,31,36,37,69,83). HPLC coupled with mass spectrometry (MS), with detection methods such as electrospray ionization (ESI) of heated nebulizer atmospheric pressure chemical ionization (HN-APCI), is a technique that can directly estimate the intact molecular weights of isoflavones, both conjugated and unconjugated without the necessity of hydrolysis or derivatization (31,38). In analyses by HPLC-HN-APCI-MS in the positive ion mode, three different types of conjugates may be identified for each isoflavone. The detection limit for this method is <1–5 µg/g (Table 5.1), which is also the practical limit of detection in HPLC-UV analysis. This technique is reproducible and accurate for the extraction and analysis of individual isoflavone conjugates using reverse-phase HPLC and HPLC-MS. HPLC-triple MS is yet another variant of HPLC which lends itself to isoflavone analysis. It involves an HPLC system equipped with an ion trap mass spectrometer and requires an internal standard (25). Using apigenin (a flavone analog of genistein) as an internal standard with a gradient solvent system, genistein and apigenin are well resolved, whereas they are not separated when an isocratic HPLC solvent system is used. Gas chromatography with mass spectrometry (GC-MS) (12,23) is another common technique for identifying and quantifying isoflavones in soybean and soy foods. The method has relatively high sensitivity, but it requires time-consuming preparative steps, and only the derivatized isoflavones can be determined (23,33,92). A related method, GC with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (GC-MALDI-TOF MS), was first introduced in 1987. It has several advantages over other GC methods including the following: (i) speed of analysis, (ii) high

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sensitivity, (iii) wide applicability, (iv) insensitivity to contaminants, and (v) suitability for analysis of complex mixtures. Wang and Sporns (26) are credited with applying it to isoflavones. Even simple MALDI-TOF MS instruments, however, cannot distinguish between isomers. Isoflavones are predominantly ionized and converted to protonated species, and the relative retention time and peak positions are close to those reported by Murphy et al. (30) and Song et al. (74). Isotope dilution (ID) GC-MS, which provides qualitative and quantitative information, has also been used for the determination of phytoestrogens in various plant foods and food products (23,93). However, the instruments are very expensive; in addition, the requisite isotopic standards are costly and not readily available, and the synthesis of these compounds requires time-consuming derivatization. Consequently, this technique is not suitable for routine analysis, although it is useful for determination of metabolites of phytoestogens in biological fluids or foods containing quantities of phytoestrogens that are too small for detection by the methods used for routine analysis. Extraction and Determination of Lignans Secoisolariciresinol (SECO) and Matairesinol (MAT). Lignans, an important class of phytoestrogens, have attracted keen interest in recent years because of their beneficial effects on human health (94,95). Flaxseeds are the richest source of two aglycone lignans, SECO and matairesinol MAT (23,96). SECO and MAT are currently the most widely known plant lignans because they appear to exert phytoestrogenic effects and are precursors of the mammalian lignans enterolactone and enterodiol (92,97,98), which were first discovered in human urine by Setchell in 1983. The lignans of flaxseed consist mainly of SECO with minor amounts of other compounds, such as MAT, isolariciresinol, and pinoresinol. The plant lignans present a greater challenge to the analytical chemist than do their mammalian counterparts due to the formation of numerous glycoside bonds with monosaccharides and the difficulties posed by the nature of the glycoside linkages (92,99). The SECO of flaxseed is in the form of a precursor, secoisolariciresinol diglucoside (SDG), which is bonded to a polymer containing a number of hydroxyl groups and is involved in the formation of glycosidic bonds to carbohydrates. To release the aglycone lignans from this complex, the carbohydrates must be removed by hydrolysis (57). Enzymatic approaches to hydrolysis of the carbohydrate are complicated by the large variety of carbohydrate-lignan conjugates. The enzymes must not only break glycosidic bonds between the lignan and many different monosaccharides but also overcome steric hindrance of the phenolic linkages of the neighboring methoxy groups and cope with glycosidic bonds linking the lignans to a variety of carbon atoms in the monosaccharide ring (92). Obermeyer et al. (100) developed HPLCUV and HPLC-MS methods for the determination of SECO released from flaxseed by β-glucuronidase hydrolysis. Using this technique, they demonstrated the presence of SECO, but no MAT was detected.

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Two possible strategies for hydrolyzing glycosidic bonds between plant lignans and carbohydrate are described in the literature. Both methods were used to determine the lignan content of a variety of foods (23,65) until they were supplanted by a simple, rapid analytical technique involving alkaline hydrolysis developed for the determination of SDG (27,101). Thompson et al. (65) used cultures of anaerobic fecal bacteria capable of carrying out in vitro fermentation to transform lignans from various foods into the mammalian lignan enterolactone or enterodiol, which was then determined by GC using a flame ionization detector. This method, however, is fraught with difficulties. It is dependent on the survival of a wide variety of anaerobic bacteria from the gut, and setting it up and maintaining it with a uniform capacity for hydrolysis over an extended period of time are technically demanding tasks. Therefore, it is not widely used. A more versatile and sophisticated technique developed by Mazur et al. (23) offered, for the first time, the advantage of being capable of determining several phytoestrogens (SECO, MAT, daidzein, genistein, formononetin, biochanin A, and coumesterol) simultaneously by isotope-dilution GC-MS (ID-GC-MS). The method of Mazur et al. (23) involves multistep procedures employing both enzymes and hot acid to break the glycosidic bonds and remove the carbohydrate component of lignans and isoflavone in foods. This treatment is followed by determination of SECO and MAT using ID-GC-MS. One disadvantage, however, of the acid hydrolysis of natural products is the relative instability of certain natural products. Thus, Mazur et al. (23) found that acid treatment caused one of the major target plant lignans, SECO, to be dehydrated to anhydro-SECO, which was derived solely from SDG. Unfortunately, anhydro-SECO is chemically identical to another naturally occurring lignan called shonanin (3,4-divantillytetrahydrofuran) (23,92). Hence, the method cannot distinguish between naturally occurring shonanin and shonanin produced from SECO during acid hydrolysis. Liggins et al. (92) simplified and improved the procedure of Mazur et al. (23), with the result that it could be used for determination of shonanin as well as SECO and MAT after removal of conjugated carbohydrates by acid hydrolysis. Using another variant of the method of Mazur et al. (23), Mazur and Adlercreutz (34) released the glucose residues by acid hydrolysis and determined the free aglycones by ID-GC-MS. Although this procedure gave reliable results, it was very time-consuming and was highly dependent on the use of isotope-labeled lignans, which are extremely expensive and are not readily available. From the standpoint of human health, SECO and MAT are the most important lignans in flaxseed, but accompanying them are certain minor lignans, including isolariciresinol and pinoresinol, which were first isolated from flaxseed meal and characterized by Meagher et al. (102). The compounds were initially isolated by acid hydrolysis and methanol extraction and then separated and detected by reverse-phase HPLC using a diode array detector. The identity of each lignan was confirmed by GC-MS using retention times and mass spectra of their trimethylsilyl ether (TMS) derivatives (102).

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Secoisolariciresinol Diglucoside (SDG). The most abundant lignans of flaxseed (notably SDG, the precursor of SECO) are the principal precursors of the mammalian lignans enterolactone and enterodiol (46,47,103–108). SDG was first isolated from flaxseed by Bakke and Klosterman (57). Although a number of methods for indirect determination of SDG have been proposed (23,48,100,109,110), no simple, direct method for its determination was available until recently, when Westcott and Muir (101) developed an HPLC technique for the quantitation of SDG in flaxseeds and in baked goods containing flaxseed. They subsequently succeeded in isolating pure SDG from flaxseed and flax meal in significant quantities by extraction with aqueous ethanol followed by hydrolysis and solid-phase extraction (SPE) (111). According to Muir and Westcott (27), SDG with >98% purity was obtained by HPLC using a C-10 radial pack column. In addition, Johnsson et al. (112) developed a method for isolation of SDG from defatted flaxseed by extraction with 1,4-dioxane/95% ethanol. The extract was subjected to alkaline hydrolysis, whereupon the hydrolysate was passed through a reverse-phase column and eluted with methanol. This methanol fraction was concentrated, applied to a column of silica gel 60, and eluted using a mixture of CHCl3/ methanol/water, yielding SDG. The structure of SDG was confirmed by nuclear magnetic resonance (NMR; 400 MHz), and the compound was found to be indistinguishable from a reference sample. Its spectrum was comparable to that described by Qui et al. (113), and the purity was >99% according to NMR analysis. As mentioned before, Westcott and Muir (101,111) succeeded in producing significant quantities of pure SDG using a rapid HPLC technique for determination of SDG (27,101). The presence of SDG in the sample was confirmed by comparison with a standard, by PDA analysis, and also by LC-MS determination of SECO after hydrolysis with β-glucuronidase (Helix pomata) using LC-MS and negative ion electrospray LC-MS/MS (101). SDG does not exist in a free form in flaxseed (even after cooking) but rather as ester-linked polar complexes of carbohydrates (101,114); nevertheless, alkaline hydrolysis results in release of SDG. The yield of SDG increases with decrease in particle size, with a maximum recovery of 99.5 ± 6.5%. Johnsson et al. (112) described an analytical method that is a modification of previous methods. Briefly, it consists of solvent extraction steps, alkaline hydrolysis, and SPE followed by HPLC analysis. Another method involving base hydrolysis followed by SPE was developed by Rickard et al. (115). The recovery of SDG from the SPE column was >99.5%, as shown by analysis of standards. The analyses were done by HPLC employing a UV/DAD detector. A salt produced during the process was removed by SPE for ease of sample handling and for protection of the chromatographic column from salt precipitation. The technique involved a solvent gradient system, which provided good separation of SDG and required less time than other HPLC gradient systems (101). Simultaneous Determination of Isoflavones (Daidzein and Genistein), Coumestrol, and Lignans (SECO and MAT) by ID-GC-MS-SIM. Mazur and co-

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workers (4,5,23) presented, for the first time, a method for simultaneous quantitative determination of phytoestrogens (including the isoflavonoids formononetin, biochanin A, daidzein, and genistein, together with coumesterol and the lignans SECO and MAT) in plant-derived foods. These compounds were determined by ID-GC-MS in the selected ion monitoring mode (ID-GC-MS-SIM) using a synthesized deuterated internal standard for the correction of losses during the procedure. A three-step hydrolysis procedure (rehydration with distilled water followed by enzymatic and acid hydrolysis) was applied to convert diphenolic glycosides to their respective aglycones. The purification and separation steps were followed by derivatization and GC-MS analysis. The detection limit of the analytical method was ~2–3 µg/100g (Table 5.1). This technique has the advantage of being accurate, sensitive enough to require only relatively small amounts of sample (as little as 50 mg of the foodstuff), and capable of performing simultaneous determinations of multiple phytoestrogens in mixtures. On the other hand, it has a serious drawback that limits its usefulness. The synthesis of deuterated internal standards is very time-consuming and expensive, and not many laboratories are equipped to carry out the task. Nevertheless, the method has been applied successfully to the determination of isoflavonoids and lignans in 300 foods.

Concluding Remarks Many different techniques exist for the extraction and determination of phytoestrogens in foods and biological samples. All methods have both advantages and disadvantages; therefore, in any given investigation, it is necessary to select the method that is best suited to the purpose of the analyses. More specifically, the analytical technique must be appropriate for the kind of sample material to be analyzed, the particular analytes to be determined, the concentration range of the phytoestrogens, and so forth. Similarly, the selection of the extraction procedure for phytoestrogens depends on the chemical properties of the analytes, and factors such as the choice of solvents, temperature, and method of hydrolysis must be considered. Methods such as those of Mazur et al. (23) are very useful, because they are highly specific, quantitative, reproducible, and sensitive, and are capable of simultaneous determination of numerous phytoestrogens in mixtures, in addition to being applicable to most of the important phytoestrogens and lignan precursor aglycones. Among other benefits, they can measure the concentrations of seven compounds, including isoflavonoids, lignans, and coumesterol, in urine, plasma, and feces. Such techniques will undoubtedly have many significant applications in future epidemiologic and metabolic investigations. Bear in mind, however, that it is not practical for common use, because it is very costly, requires derivitization, and involves the use of deuterated standards that are not readily available. Accordingly, the most commonly used method for routine analysis of foods for phytoestrogens is HPLC/PDA. It has the advantage of being simple, rapid, and

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inexpensive, and does not require derivitization (21,22,30,73,74); it has the additional advantage of combining well with LC-MS (31), which is highly specific and is useful for confirming the structures of analytes. Furthermore, routine quality control and thorough documentation must be included in the development and validation of analytical techniques for the determination of phytoestogens (73,74,81). According to Song et al. (74), the quality control and documentation should entail the following procedures: (i) performance of quality control measurements at intervals in each series of analyses; (ii) inclusion of standards in every batch of samples during isoflavone analysis; (iii) estimation of accuracy by recovery of external and/or internal standards in soy food matrices; (iv) estimation of precision by calculation of the coefficient of variation for food matrices at different times in the course of a series of analyses; and (v) verification that the HPLC system is operating correctly. Although current knowledge is not sufficient to support dietary recommendations for individual phytoestrogens, available evidence suggests that consumption of plant foods rich in phytoestrogens may greatly benefit human health. More research, however, is required to determine the bioactive components of such foods and the effective doses or daily consumption rates of different plant sources, while considering both possible adverse and beneficial effects. Furthermore, a number of bioengineering techniques have been developed for inducing specific phytoestrogen synthesis by a wide variety of crop plants that do not normally synthesize phytoestrogens. These techniques were made possible by advances in (i) our understanding of the biosynthesis and regulation of phytoestrogen in plants, (ii) our ability to identify human dietary sources of these compounds through quantitative analysis, and (iii) our knowledge of their health benefits. Finally, recommendations regarding the intake of dietary phytoestrogens should be made with caution. In particular, phytoestrogens with relatively high estrogenic potency and known adverse effects (e.g., coumestrol) should be defined as pharmaceuticals and controlled accordingly. In the future, an effort should be made to evaluate the safety and the interactions of phytoestrogens with each other, with other components of the diet, and with prescription drugs. Moreover, the development of improved and standardized analytical techniques for the detection and quantification of phytoestrogens in human foods and natural health products will help to provide a sound basis for recommendations on efficacious and safe daily intake of phytoestrogens. References 1. Kurzer, M.S., and Xu, X. (1997) Dietary Phytoestrogens, Annu. Rev. Nutr. 17, 353–381. 2. Harbone, J.R. (1994) The Flavanoids: Advances in Research Since 1986, p. 676, Chapman and Hall, New York, 3. Reinli, K., and Block, G. (1996) Phytoestrogen Content of Foods—Compendium of Literature Values, Nutr. Cancer 26, 123–148.

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4. Mazur, W.M., Duke, J.A. and Wähälä, K. (1998) Isoflavonoids and Lignans in Legumes: Nutritional and Health Aspects in the Human, J. Nutr. Biochem. 9, 193–200. 5. Mazur, W.M. (1998) Phytoestrogen Content in Foods, Bailliere’s Clin. Endocrinol. Metab. 12, 729–741. 6. Adlercreutz, H. and Mazur, W. (1997) Phytoestrogens and Western Diseases, Ann. Med. 9, 5–120. 7. Peterson, T.G., and Barnes, S. (1996) Genistein Inhibits Both Estrogen and Growth Factor Stimulated Proliferation of Human Breast Cancer Cells, Cell Growth Differ. 7, 1345–1351 8. Messina, M., Persky, V., Setchell, K.D.R., and Barnes, S. (1994) Soy as a Factor in the Lowered Risk of Breast Cancer. A Review of Soy Consumption, Epidemiologic Data and Laboratory Studies, Nutr. Cancer 21, 113–131. 9. Messina, M., and Barns, S. (1991) The Role of Soy Products in Reducing Cancer Risk, J. Natl. Cancer Inst. 83, 541–546. 10. Adlercreutz, H. (1990) Western Diet and Western Diseases: Some Hormonal and Biochemical Mechanisms and Associations, Scand. J. Clin. Lab. Investig. 50 (Suppl. 201), 3–23. 11. Adlercreutz, H., Fotsis, T., Kurzer, M.S., Wähälä, K., Mäkelä, T., and Hase, T. (1995) Isotope Dilution Gas Chromatographic-Mass Spectrometric Method for the Determination of Unconjugated Lignans and Isoflavonoids in Human Feces, with Preliminary Results in Omnivorous and Vegetarian Women, Anal. Biochem. 225, 101–108. 12. Adlercreutz, H., Fotsis, T., Lampe, I.., Wähälä, K., Mäkelä, T., Brunow, G., and Hase, T. (1993) Quantitative Determination of Lignans and Isoflavonoids in Plasma of Omnivorous and Vegetarian Women by Isotope Dilution Gas Chromatography-Mass Spectrometry, Scand J. Clin. Lab. Investig. 53 (Suppl. 215), 5–18. 13. Anthony, M.S., Clarkson, T.B., Bullock, B.C., and Wagner, J.D. (1997) Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet Induced Coronary Artery Atherosclerosis in Male Cynomolgus Monkey, Arterioscler. Thromb. Vasc. Biol. 126, 43–50. 14. Messina, M.J. (1999) Legumes and Soybeans: Overview of Their Nutritional Profiles and Health Effects, Am. J. Clin. Nutr. 70 (Suppl.), 439S–450S. 15. Arjmanci, B.H., Getlinger, M.J., Goyal, N.V., Alekel , L., Hasler, C.M., Jumaa, S., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Role of Soy Protein with Normal or Reduced Isoflavones Content in Reversing Bone Loss Induced by Ovarian Hormone Deficiency in Rats, Am. J. Clin. Nutr. 68 (Suppl.), 1358S–1368S. 16. Arjmanci, B.H., Alekel, L., Hollis, B.W., Amin, D., Stacewicz-Sapuntzakis, M., Guo, P., and Kureja, S.C. (1996) Dietary Soybean Protein Prevents Bone Loss in an Ovariectomized Rat Model of Osteoporosis, J. Nutr. 126, 161–167. 17. Blair, H., Jordan, S.E., Peterson, T.G., and Barnes, S. (1996) Genistein Inhibits Avian Osteoclastic Activity and Reduces Bone Loss in Ovariectomized Rats, J. Cell Biochem. 60, 1761–1769. 18. Potter, S.S., Baum, J.A, Teng, H., Stilman, R.J., Shay, N.E., and Erdman, J.W., Jr. (1998) Soy Protein and Isoflavones: Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68 (Suppl.), 1375S–1379S. 19. Costa, M.A., Xia, Z.-Q., Davin, L.B., and Lewis, N.G. (1999) Towards Engineering the Metabolic Pathways of Cancer-Preventing Lignans in Cereal Grains and Other

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20. 21. 22.

23.

24.

25.

26. 27.

28.

29. 30. 31.

32.

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1

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88. Kudou, S., Shimyamada, M., Imura, T., Uchida, T., and Okubo, K. (1991) A New Isoflavone Glycoside in Soybean Seeds (Glycine max Merrill), Glycitein 7-O-β-D-(6”O-acetyl)-glucopyranoside, Agric. Biol. Chem. 55, 859–860. 89. Müller, C., and Sontal, G. (1999) Determination of Some Phytoestrogens in Soybeans and Their Processed Products with HPLC and Coulorimetric Electrode Array Determination, Presenius J. Anal. Chem. 364, 261–265. 90. Müller, C., and Sontal, G. (2000) HPLC with Coulorimetric Electrode Array Detection Determination of Daidzein and Genistein in Soy Based Infant Food, Soymilk and Soy Based Supplements, Eur. Food Res. Technol. 211, 301–304. 91. Nurmi, T., and Adlercreutz, H. (1999) Sensitive High Performance Liquid Chromatographic Method for Profiling Phytoestrogens Using Coulorimetric Electrode Array Detector: Application to Plasma Analysis, Anal. Biochem. 274, 110–117. 92. Liggins, J., Gimwood, R., and Bingham, S.A. (2000) Extraction and Quantification of Lignan Phytoestrogens in Food and Human Samples, Anal. Biochem. 287, 102–109. 93. Morton, M., Arisaka, O., Miyake, A., and Evans, B. (1999) Analysis of PhytoOestrogens by Gas Chromatography-Mass Spectrometry, Environ. Toxicol. Pharmacol. 7, 221–225. 94. Mousavi, Y., and Adlercreutz, H. (1992) Enterolactone and Estradiol Inhibit Each Other’s Proliferative Effect on MCF-7 Breast Cancer Cells in Culture, J. Steroid Biochem. Mol. Biol. 41, 615–619. 95. Kurzer, M.S., Lampe, J.W., Martinit, M.C., and Adlercreutz, H. (1995) Fecal Lignan and Isoflavonoid Excretion in Premenopausal Women Consuming Flaxseed Powder, Cancer Epidemiol. Biomark. Prev. 4, 358–358. 96. Harris, R.K., and Haggerty, W.J. (1993) Assays for Potentially Anticarinogenic Phytochemicals in Flaxseed, Cereal Foods World 38, 147–151. 97. Borriello, S.P., Setchel, K.D.R., Axelson, M., and Lawson, A.M. (1985) Production and Metabolism of Lignans by Human Faecal Flora, J. Appl. Bacteriol. 58, 37–43. 98. Setchel, K.D.R., Lawson, A.M., Mitchell, F.L., Adlercreutz, H., Kirk, D.N., and Axelson, M. (1980) Lignans in Man and in Animal Species, Nature 287, 740–742. 99. Nagatsu, A., Zhang, H.L., Watanabe, T., Taniguchi, N., Hatano, D., Mizukami, H., and Sakakbar, J. (1998) New Steroid and Matairesinal Glycosides from Safflower (Cathamus tinctorius L.) Oil Cake. Chem. Pharm. Bull. 46, 1044–1047. 100. Obermeyer, W.R., Musser, S.M., Betz, J.M., Casey, R.E., Pohland, A.E., and Page, S.W. (1995) Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chapparal, Proc. Soc. Exp. Biol. Med. 208, 6–12. 101. Westcott, N.D., and Muir, A.D. (1996) Variation in Flaxseed Lignan Concentration with Variety, Location and Year, in Proc. 56th Flax Institute of United States, pp. 77–85, Fargo, ND. 102. Meagher, L.P., Beecher, G.R., Flanagan, V.P., and Li, B.W. (1999) Isolation and Characterization of the Lignans, Isolariciresinol and Pinoresinol, in Flaxseed Meal, J. Agric. Food Chem. 47, 3173–3180. 103. Cunnane, S.C., Hamadeh, M.J., Liede, A.C., Thompson, L.U., Wolever, T.M.S., and Jenkins, D.J.A. (1995) Nutritional Attributes of Traditional Flaxseed in Healthy Young Adults, Am. J. Clin. Nutr. 61, 62–68. 104. Parbtani, A., and Clark, W.F. (1995) Flaxseed and Its Components in Renal Disease, in Flaxseed in Human Nutrition, (Cunnane, S.C, and Thompson, L.U., eds.) pp. 244–260, AOCS Press, Champaign, IL.

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105. Serraino, M.R., and Thompson, L.U. (1991) The Effect of Flaxseed Supplementation or Early Risk Markers for Mammary Carinogenesis, Cancer Lett. 60, 135–142. 106. Serraino, M.R., and Thompson, L.U. (1992) The Effect of Flaxseed Supplementation on the Initiation and Promotional Stages of Mammary Tumorigenesis, Nutr. Cancer 17, 153–159. 107. Serraino, M.R., and Thompson, L.U. (1992) Flaxseed Supplementation and Early Marker of Colon Carcinogenesis, Cancer Lett. 63, 159–165. 108. Axelson, M., Sjövallm, J, Gustaffsson, B.E., and Setchell, K.D.R. (1982) Origin of Lignans in Mammals and Identification of Precursor from Plants, Nature 298, 659–670. 109. Nilsson, M., Aman, P., Harkonen, H., Hallmans, G., Knudsen, K.E.B., Mazur, W., and Adlercreutz, H. (1997) Content of Nutrients and Lignans in Roller Milled Fractions of Rye, J. Sci. Food Agric. 73, 143–148. 110. Nesbitt, P.D., and Thompson, L.U. (1997) Lignans in Homemade and Commercial Products Containing Flaxseed, Nutr. Cancer 83, 297–303. 111. Westcott, N.D., and Muir, A.D. (1998) Process for Extracting Lignans from Flaxseed, U.S. Patent 5,705,618. 112. Johnsson, P., Kamal-Eldin, A., Lundgren, L.N., and Aman, P. (2000) HPLC Method for Analysis of Secoisolariciresinol Diglucoside in Flaxseeds, J. Agric. Food Chem. 48, 5216–5219. 113. Qui, S., Lu, Z., Luyengi, L., Lee, S.K., Pezzuto, J.M., Farnswrorth, N.R., Thompson, L.U., and Fong, H.H.S. (1999) Isolation and Characterization of Flaxseed (Linum usitatissimum) Constituents, Pharm. Biol. 37, 1–7. 114. Bambagiotti-Alberti, M., Coran, S.A., Ghiara, C., Giannellini, V. and Raffaelli, A. (1994) Revealing the Mammalian Lignan Precursor Secoisolariciresinol Diglucoside in Flaxseed by Ion Spray Mass Spectrometry, Rapid Commun. Mass Spectrom. 8, 595–598. 115. Rickard, S.E., Orcheson, L.J., Seid, M.M,. Luyengi, L., Fong, H.H.S., and Thompson, L.U. (1996) Dose-Dependent Production of Mammalian Lignans in Rats and In Vitro from the Purified Precursor Secoisolariciresinol Diglucuroside in Flaxseed, J. Nutr. 126, 2012–2019. 116. Wang, G., Kuan, S.S., Fracis, O.J., Ware, G.M., and Carmen, A.S. (1990) A Simplified PHLC Method for the Determination of Phytoestrogens in Soybeans and Its Processed Products, J. Agric. Food Chem. 38, 185–190.

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Chapter 6

Tables of Isoflavone, Coumestan, and Lignan Data Chung-Ja C. Jacksona, and G. Sarwar Gilanib aGuelph

Centre for Funtional Foods, Laboratory Services, University of Guelph, Guelph, Ontario, N1H 8J7 Canada bNutrition

Research Division, Bureau of Nutritional Sciences, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0L2 Canada

Introduction Phytoestrogens are naturally occurring polyphenolic plant-derived compounds of nonsteroidal structure that are similar to the mammalian estrogen, estradiol (1). Compounds with estrogenic activity have been found in >300 plants, and occur in a number of common edible plants and plant-derived foods and beverages, including beans, vegetables, soybeans, grains, flaxseed, fruits, berries, tea, coffee, and hops (2–4). Although they differ widely in molecular structure, they are collectively classed as “phytoestrogens” (5–7). Hormone-like bisphenolic phytoestrogens, isoflavonoids (daidzein and genistein), coumestans (coumestrol), and lignans [secoisolariciresinol (SECO) and matairesinol (MAT)] are of great interest because of their antiestrogenic, anticarcinogenic, antiviral, antifungal, and antioxidant activities as well as their estrogenic properties (7–14). Most phytoestrogens have one feature in common, i.e., with few exceptions, they possess a phenolic ring, which is a prerequisite for binding to the estrogen receptor (1,14). Therefore, phytoestrogens can function not only as estrogenic but also as antiestrogenic agents (15,16). Moreover, the discovery of a novel second estrogen receptor (ERβ) (17), which has a specific affinity for phytoestrogens and is found in various tissues including bone, brain, vascular endothelia, and bladder, further complicates our understanding of phytoestrogen action (1,18). Dietary Sources of Phytoestrogens Phytoestrogens are found in a variety of food plants, in which their function is to protect the plants against harmful microorganisms, such as pathogenic fungi. After their secretion by the roots of leguminous plants, they also attract specific symbiotic nitrogen-fixing soil bacteria to the roots (19). As noted above, the main classes of phytoestrogens are the isoflavones, coumestrans, and lignans. Isoflavones are a subclass of the more abundant flavonoids of the plant kingdom, and they are found almost exclusively in legumes. The highest concentrations of isoflavones (1–3

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mg/100 g) occur in soybeans in which they are in the form of the glucosides (i.e., mainly malonyl genistin and malonyl daidzein) of the three isoflavone aglycones, daidzein, genistein, and glycitein (2,20–29). Only small quantities exist in their free forms (aglycones) in soybeans. Isoflavones are also abundant in the seeds, sprouts, and leaves of clover (30). An isoflavone called biochanin-A has also been detected in bourbon (31), and genistein and daidzein have been found in beer (32,33). Coumesterol in human food is found in soybean sprouts (24) and is abundant in alfalfa sprouts, clover sprouts (30), and mung bean sprouts (~1 mg/g, which is ~20 times greater than the concentration in alfalfa sprouts). Mung bean sprouts also contain daidzein (~700 µg/g dry weight) and genistein (~2000 µg/g dry weight) (2). Flaxseed (linseed) is the most abundant source of lignans in food (0.8–3.7 mg/g) (34,35). The lignans consist mainly of SECO with minor amounts of MAT (36,37). Other sources of lignans include various grains, seeds, fruits, berries, and vegetables (2,36,37). Tea and coffee, too, have been found to contain lignans along with isoflavones and other flavonoids (4). The richest sources of phytoestrogens in the plant kingdom are the legumes (Leguminosae, or Fabaceae). Human consumption of members of the legume family is generally limited to their seeds and is confined to 20 of the 13,000 species of legumes (3). Edible legumes (dried seeds) are often called “pulses.” Legumes have been major components of traditional diets in many regions throughout the world (e.g., India, the Far East, the Middle East, South America, and Mexico). However, legumes have generally not been important elements in the diets of Western countries (29). The concentrations of phytoestrogens in plants depend on genetic characteristics of plant species or varieties, effects of environmental factors during growth, time of harvesting, and the processing of the crops after harvesting (38,39). The highest levels of phytoestrogens occur in most soy food products, alfalfa, and flaxseed (linseed), with soybean as a major potential source of phytoestrogens for humans (2). Physiological Benefits of Phytoestrogens The beneficial effects of isoflavones on health include prevention of cancer (40–50), heart disease (29,51–54), osteroporosis (55–62), and alleviation of menopausal symptoms (50). Isoflavones also help to control diabetes (63) and improve cognitive function. In addition, the lignans are responsible for many physiologic benefits, such as prevention of cancer and kidney disease (64–69). However, although many epidemiologic studies have suggested that consumption of foods containing phytoestrogens may have beneficial effects, there is no evidence linking such effects directly to phytoestrogens, nor any information concerning the roles that might be played by numerous other biologically active components of soybeans and flaxseed. Accordingly, a pressing need exists for further research in this field.

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Possible Adverse Effects of Phytoestrogens Assuming that consumption of phytoestrogens is always beneficial would be naïve because excessive or inappropriate consumption may be harmful. In addition, the estrogenic effect of phytoestrogens may be unpredictable; e.g., phytoestrogens can have a stimulating (estrogenic) effect instead of an antiestrogenic effect on the premenopausal female breast in the presence of high levels of plasma estradiol (70). Other factors to be considered when assessing these phytoestrogens from the standpoint of safety and efficacy are the rates of absorption and metabolism of these compounds. Phytoestrogens have been shown to induce infertility and developmental toxicity in certain animals, and coumesterol has caused “sheep clover disease,” inhibiting fertility in Australia (71). Furthermore, there have been no long-term studies of possible adverse effects (such as toxicity or allergy resulting from soybean consumption) in humans. Systematic and rigorous investigations of the consequences of long-term consumption of soy foods (especially infant formula) are required, together with research on the relationship between the timing and duration of phytoestrogen consumption and the incidence or severity of disease. One of the goals of such research should be to establish recommended daily intakes.

Tables of Isoflavone and Lignan Concentrations in Foods Analytical Methods High-performance liquid chromatography (HPLC) is generally used to determine isoflavones in foods, notably soybeans and soy foods (22,25,72–77). The USDA-Iowa isoflavone database, the first database of its kind, constitutes an extensive collection of isoflavone data gleaned from the literature or generated by analysis of soybeans and other foods (77). However, there has been a general lack of quantitative data on the isoflavone and lignan content of leguminous plants, and there has been no systematic determination of specific lignans (SECO and MAT) in foods. Moreover, different kinds of phytoestrogens were always determined separately until Mazur et al. (3,4,37) developed a method of isotope dilution/gas chromatography/mass spectrometry in selected monitoring mode (ID-GC-MS-SIM) for simultaneous quantitative determination of isoflavones (daidzein, genistein, formononetin, biochanin-A), coumestrol, and lignans (SECO and MAT) in food samples. The determination of four isoflavonoids (in the range of 0–1.83 mg/g) and lignans (in the range of 0–15.85 mg/kg) was performed for the first time in a single analysis (78). The ID-GC-MS-SIM technique for the determination of phytoestrogens has major advantages over HPLC, such as its higher sensitivity and greater specificity. However, it does have certain disadvantages. It is very expensive and time-consuming (requiring derivatization), and the isotope standards are not readily available (3,4,78). Consequently, it is not suitable for routine analyses. Franke et al. (79) reported HPLC data revealing coumesterol concentrations ranging from 1.48 mg/100 g in lima beans to 561 mg/100 g in clover sprouts. At

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relatively high levels of phytoestrogens in foods, the results of HPLC analysis are comparable to those obtained by ID-GC-MS-SIM (72,78,79). Keep in mind, however, that comparisons among results obtained using different techniques applied are valid only if the same samples are analyzed by these methods. To date, there has been no official procedure for rigorous comparison of different analytical methods and establishment of an optimal standard method (78,80,81). Tables of Isoflavone and Lignan Concentrations There are 12 isoflavone compounds in soybeans: 3 aglycones, or free isoflavones (daidzein, genistein, and glycitein) together with 3 glucosides (daidzin, genistin, and glycitin), 3 actyl glucosides (6”-O-acetyl daidzin, 6”-O-acetyl genistin and acetyl glycitin), and 3 malonyl glucosides (6”-O-malonyl daidzin, 6”-O-malonyl genistin, and 6”-O-malonyl glycitin) formed by the bonding of sugars to the 3 aglycones. There are two ways to perform isoflavone analyses, i.e., determine all 12 forms or determine the 3 aglycones after acid hydrolysis to cleave off the sugar moieties of the glucosides. When all 12 isoflavones are determined, the concentrations of isoflavone glucosides have to be normalized to convert them to concentrations of aglycones. The concentrations of the 3 aglycones, i.e., total daidzein, total genistein, and total glycitein, are calculated by normalizing the analytical data using the molecular weights of the different isoflavones in accordance with the following formulas (38): Total daidzein = 254.23 (daidzin/416.36 + 6”-O-malonyl daidzin/502.41 + 6”-O-acetyl daidzin/458.4 + daidzein/254.23) Total genistein = 270.23(genistin/438.37 + 6”-O-malonyl genistin/518.41 + 6”-O-acetyl genistin/474.4 + genistsein/270.23) Total glycitein = 284 (glycitin/446 + 6”-O-malonyl glycitin/532 + 6”-O-acetyl glycitin/488 + glycitein/284) Representative phytoestrogen data for various foods gleaned from the literature are presented in Tables 6.1–6.9, whose contents are summarized as follows: Table 6.1 shows isoflavone concentrations in soybeans representing different sources and different treatments (e.g., fresh, cooked, mature, and immature soybeans, as well as soybean sprouts and flakes) (4,22,25,72,77–79,82–86). In certain cases, glycitein was not determined, with the result that the total isoflavone values were relatively low. Table 6.2 lists isoflavone levels in soy protein products and processed soy foods, including soy meal (72), defatted and full-fat soy flour (25,80,87), soybean flakes (22,88), soy protein concentrate (25,81,89), SPI, soybean chips, soy fiber, soy noodles, and soy paste (25,26,72,81,82,90).

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TABLE 6.1 Isoflavone Contents of Soybeansa Daidzein Description

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion)

Reference

Soybeans, Brazil, raw Soybeans, Japan, raw Soybeans, Korea, raw Soybeans, Taiwan, raw Soybeans, flakes, full-fat Soybeans, immature, cooked, boiled, drained, without salt Soybeans, immature, seeds, raw Soybeans, green, immature seeds, raw Soybeans, mature cooked, boiled, without salt Soybeans, mature seeds, dry roasted

20.16 34.52 72.68 28.21 48.23 6.85

67.47 64.78 72.31 31.54 79.98 6.94

0.00 13.78 0.00 0.00 1.57 0.00

87.63 118.51 144.99 59.75 128.99 13.79

104 79,82 83 79 22,95 79

9.27 67.79 26.95 52.04

9.84 72.51 27.71 65.88

4.29 10.88 0.00 13.36

20.42 151.17 54.66 128.35

Soybeans, mature seeds, raw (U.S., food quality) Soybeans, mature seeds, raw (U.S., commodity grade) Soybeans, mature seeds, sprouted, raw

46.64 52.20 19.12

73.76 91.71 21.60

10.88 12.07 0.00

128.35 153.40 40.71

85,Unpub. obs.b 26,79,Unpub. obs. 79 25,26,79,85, Unpub. obs. 26,75,79,82,85,86 22,72,79 72,85

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content. bUnpublished observations.

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TABLE 6.2 Isoflavone Contents of Soy Protein Products and Processed Soy Foodsa Daidzein Description

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion)

Reference

Soy meal, defatted, raw Soy flour, full-fat, roasted Soybean flakes defatted Soy flour, full-fat, raw

57.47 99.27 36.97 71.19

68.35 98.75 85.69 96.83

0.00 16.40 14.23 16.18

125.82 198.95 125.82 177.89

Soy flour (textured) Soy protein concentrate, aqueous washed Soy protein concentrate, produced by alcohol extraction Soy protein isolate

59.62 43.04 6.83 33.59

78.90 55.59 5.33 59.62

20.19 5.16 1.57 9.47

148.61 102.07 12.47 97.43

Soybean chips Soy fiber Soy noodles Soy paste

26.71 18.80 0.90 15.03

27.45 21.68 3.70 15.21

0.00 7.90 3.90 7.70

54.16 44.43 8.5 31.52

72 80,87,90 22,76,88,97,98 22,37,75,81, 85,89,99,100 26,62,89,91,101 25,81 25,81,89 25,26,75,85,87, 80,81,87,90,100 25 25,81 26 25,26,72

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content.

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TABLE 6.3 Isoflavone Contents of Tofu Productsa Daidzein Description Tofu, Mori-Nu, silken, firm Tofu, dried-frozen (koyadofu, kori tofu or tung tou-fu) Tofu, Azumaya, extra firm, cooked (steamed) Tofu, Azumaya, extra firm, prepared with nigari Tofu, Azumaya, firm, cooked Tofu, firm, prepared with calcium sulfate and nigari Tofu, fried (aburage) Tofu, okara Tofu, pressed (Tau kwa) raw Tofu, raw, regular, prepared with calcium sulfate Tofu, salted and fermented (fuyu) Tofu, soft, Vitasoy-silken Tofu, soft, prepared with calcium sulfate and nigari Tofu, yoghurt

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion) 11.13 25.34 8.00 8.23 12.80 9.44 17.83 5.39 13.60 9.02 14.29 8.59 11.99 5.70

15.58 42.15 12.75 12.45 16.15 13.35 28.00 6.48 13.90 13.60 16.38 20.65 18.23 9.40

2.40 0.00 1.95 1.95 2.40 2.08 3.37 1.64 2.00 1.98 5.00 0.00 2.03 1.20

Reference 27.91 67.49 22.70 22.63 31.35 24.74 48.35 13.51 29.50 23.61 33.17 29.24 31.10 16.30

25,80 75 80 80 80 73,75,80,91 80,85 75,80 85 26,75,79,91 75,85 91 75,80,85,91 26

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content.

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TABLE 6.4 Isoflavone Contents of Fermented Soy Productsa Daidzein Description Miso soup mix, dry Natto (soybeans, boiled and fermented) Soybean, curd, fermented Tempeh Tempeh burger Tempeh, cooked

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion) 24.93 21.85 14.30 17.59 6.40 19.25

35.46 29.04 22.40 24.85 19.60 31.55

0.00 8.17 2.30 2.10 3.00 2.20

Reference 60.39 58.93 39.00 43.52 29.00 53.00

25 73,80,Unpub. obs.b 26 25,26,80,89,92 26 80

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content. bUnpublished observations.

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TABLE 6.5 Isoflavone Contents of Soy-Based Meat and Cheese Substitutesa Daidzein Description Bacon, meatless Frichick (meatless chicken nuggets), canned, cooked Frichick (meatless chicken nuggets), canned, raw Soy hot dog, frozen, unprepared Soylinks, frozen, cooked, “Morning Star” breakfast Soylinks (raw frozen), “Morning Star” breakfast USDA Commodity, beef patties with VPPb (cooked frozen) USDA Commodity, beef patties with VPP (raw frozen) Worthington Foods (Loma Linda, big franks), meatless franks, canned, prepared Worthington Foods (Loma Linda, big franks), meatless franks, canned Soy cheese, unspecified Soy cheese, cheddar Soybean curd cheese Soy cheese, mozzarella Soy cheese, parmesan

Genistein

Glycitein

Total isoflavones

mg/100 g edible portion) 2.80 4.35 3.45 3.40 0.75 1.18 0.67 0.35 1.35 1.00 11.24 1.80 9.00 1.10 1.50

6.90 9.35 7.90 8.20 2.70 2.45 1.09 0.77 2.00 2.05 20.08 2.25 19.20 3.60 0.80

2.40 0.90 0.85 3.40 0.30 0.30 0.10 0.02 0.40 0.30 0.00 3.10 0.00 3.00 4.10

Reference 12.10 14.60 12.20 15.00 3.75 3.93 1.86 1.14 3.75 3.35 31.32 7.15 28.20 7.70 6.40

26 80 80 26 80 80 80 80 80 80 25,73 26 73 26 26

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content. bVPP, vegetable protein product.

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TABLE 6.6 Isoflavone Contents of Soy-Based Beveragesa Daidzein Description

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion)

Reference

Instant beverage, soy powder, not reconstituted Soy drink Soymilk, fluid

40.07 2.41 4.45

62.18 4.60 6.60

10.90 0.00 0.56

109.51 7.01 9.65

Soymilk, iced Soy milk skin or film (Foo jook or yuba), cooked Soy milk skin or film (Foo jook or yuba), raw

1.90 18.20 79.88

2.81 32.50 104.80

0.00 0.00 18.40

4.71 50.70 193.88

25,26,102 89,91 25,73,74,75,80, 85,86,93,94 25 85 72,85

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content.

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TABLE 6.7 Isoflavone Contents of Soy-Based Formulas for Infants and Adultsa Daidzein Description

Genistein

Glycitein

Total isoflavones

(mg/100 g edible portion)

Reference

Infant formula, Enfamil Nextstep, soy formula, not reconstituted Infant formula, Mead Johnson, Gerber soy, with iron, powder, not reconstituted Infant formula, Mead Johnson, Prosobee with iron, liquid, concentrate, not reconstituted Infant formula, Mead Johnson, Prosobee with iron, powder, not reconstituted, Infant formula, Mead Johnson, Prosobee with iron, ready-to-feed, Infant formula, Ross, Isomil, with iron, Powder not reconstituted, Infant formula, Ross, Isomil, with iron, ready-to-feed Infant formula, Wyethayerst, Nursoy, with iron, liquid concentrate, not reconstituted Infant formula, Wyethayerst, Nursoy, with iron, powder, not reconstituted Infant formula, Wyethayerst, Nursoy, with iron, ready-to-feed

7.23 8.08 1.10

14.75 13.90 2.22

3.00 3.12 0.00

25.00 25.09 6.03

74 74 89,95

7.05 1.71 6.03 1.91 1.02

14.94 2.18 12.23 2.26 2.82

2.95 0.00 2.73 0.00 0.35

24.94 3.89 24.53 4.17 4.02

74 84 74,95 84 74,89

5.70 0.75

13.55 1.60

2.05 0.28

26.00 2.63

Soy-based liquid formula (adults), Ross, Enrich Soy-based liquid formula (adults), Ross, Glucerna Soy-based liquid formula (adults), Ross, Jevity Isotonic

0.14 0.02 0.03

0.40 0.06 0.31

0.00 0.00 0.00

0.54 0.08 0.34

74, 95 Unpub. Obs.b 91 91 91

aValues in the total isoflavones column may not agree with the simple additions of the individual isoflavone values. Several articles did not report glycitein values. Glycitein contributes only ~5–10% of the total content. bUnpublished observations.

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TABLE 6.8 Lignan Contents of Some Common Foods Secoisolariciresinol (Total)a Description Grains and cereals Rye (Secale cereale), whole meal Wheat (Triticum dicoccum), whole meal Barley (Hordeum spp.), whole meal Oats (Avena sativa), whole meal Corn (Zea mays), whole meal Rice (Oryza sediva) Berries and currants Bramble (Rubus fructicosus) Strawberry (Fragaria xananassa) Lingonberry (Vaccinium vitis-ideae) Cranberry (Vaccinium macrocarpum) Red raspberry (Rubus ideaus) Blackcurrant (Ribes nigrum) Redcurrant (Ribes rubrum)

Matairesinol

µg/100 g dry weight)

Reference

47.1 8.1 58.0 13.4 8.0 16.0

65.0 0.0 0.0 trb 0.0 tr

2 2 2 2 Unpub. obs.b Unpub. obs.

3718.0 1500.0 1510.0 1054.0 139.0 388.0 165.3

22.5 78.1 0.0 0.0 0.0 9.5 0.0

Unpub. obs. Unpub. obs. Unpub. obs. 2 Unpub. obs. Unpub. obs. Unpub. obs. (Continued)

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TABLE 6.8 (Cont.) Secoisolariciresinol (Total)a Description Fruits Apple (Pyrus malus) Plum (Prunus domestica) Banana (Musa sapientum) Otaheite gooseberry (Phyllanthus acidus) Avocado (Persea americana) Tomato (Lycopersicum esculentum) Lechee (Litchi chinensis) Papaya (Carica papaya) Guava (Psidium guajava) Cantaloupe (Cucumis melo cantalupensis) Lemon (Citrus limon) Orange (Citrus sinensis) Cruciferous vegetables Cabbage (Brassica oleracea) Broccoli (Brassica oleracea italica) Cauliflower (Brassica oleracea botrytis) Allium vegetables Onion (Allium cepa) Garlic (Allium sativum) Olives (Allium schoenoprasum)

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Matairesinol

µg/100 g dry weight)

Reference

tr.0 5.0 10.0 3040.0 76.7 51.6 53.6 8.2 699.7 183.9 61.3 76.8

0.0 0.0 0.0 5.8 16.0 6.5 tr 0.0 tr 0.0 0.0 0.0

96 96 Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs.

33.0 414.0 97.0

tr 23.0 tr

96 2 96

83.0 379.0 1254.0

8.0 3.6 tr

96 2 Unpub. obs.

Other vegetables Potato (Ipomoea batatas) Carrot (Daucus sativus) Pepper (Capsicum species) Celery (Apium graveolens) Cucumber (Magnolia acuminata) Eggplant (Solanum melongea) Radish (Raphanus sativus radicula)

10.0 192.0 117.0 111.4 25.1 99.7 33.3

6.0 3.0 7 3.5 tr 3.0 3.0

Beverage, nonalcoholic Prince of Wales black tea, brewed China black tea, brewed China green tea, brewed Japanese Seneha green tea, brewed Maxwell coffee Arabica coffee, Nescafé

2420.0 1050.0 2890.0 1890.0 500.0 716.0

305.0 90.0 195.0 277.0 NDb ND

Beverages, wines (origin)c Chardonnay (France), white Chardonnay (Italy), white Cabernet Sauvignon (France), red Chianti, reserve (Italy), red

174.0 135.5 686.0 1280.0

22.0 17.2 74.1 98.0

aSum

96 2 96 Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs.

4 4 4 4 4 4

Unpub. obs. Unpub. obs. Unpub. obs. Unpub. obs.

of anhydroSECO and SECO. tr, present in trace amounts; Unpub. obs., unpublished observation; ND, not determined due to low concentrations and interference by other compounds. for wet weight, µg/L.

bAbbreviations: cGiven

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TABLE 6.9 Isoflavone and Lignan Contents of Legumes Daidzein Description Food legumes, number of cultivars Soybean, 7 (Glycine max)b Kidney bean, 13 (Phaseolus vulgaris) Chickpea, 3 (Cicer arientinum)c Pea, 7 (Pisum sativum) Lentil, 2 (Lens culinaris) Kudzu leaf (Pueraria lobata)d Kudzu root (Pueraria lobata)e Black gram (Vigna mungo) sproutsf Alfalfa (Medicago spp.) sproutsg aSECO, cAlso

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SECOa (Total)

Matairesinol

(µg/100 g dry weight) 10,500–85,000 7.0–40.0 11.0–192.0 0–52.9 3.0–10.0 375.0 185,000 745.0 62.0

secoisolariciresinol. contained formononetin 18.0–121.0. contained formononetin 94.0–215.0, biochanin A 838.0–3080.0. dAlso contained formononetin 87.0, biochanin A 1240, and coumestrol 18.0. eAlso contained formononetin 7090, biochanin A 1400, and coumestrol 1570. fAlso coumestrol 1030. gAlso contained formononetin 4090, biochanin A 124.0, and coumestrol 45.0. bAlso

Genistein

26,800–10,2500 18.0–518.0 69.0–214.0 0–49.7 7.0–19.0 2520 12,600 1900 5.0

13.0–273.0 56.0–153.0 7.0–8.0 3.0–13.0 0–7.0 476.0 31.0 468.0 33.0

Reference Trace Trace 0 0 Trace Trace Trace 0 0

3 3 3 3 3 3 3 2 2

Table 6.3 gives isoflavone data for a variety of tofu products, such as silken, firm, extra firm-steamed, fried, pressed-raw tofu, and tofu prepared with Ca 2SO 4 (25,26,73,75,80,85,91). Table 6.4 presents isoflavone concentrations in fermented soy products, such as miso, natto, and tempeh. The total isoflavone concentrations are much higher in these products than in most other soy foods (25,26,73,80,89,92). Table 6.5 records the isoflavone content of soy-based meat and cheese substitutes (25,26,73,80,85). Note that the meat substitutes have low levels of isoflavones because they contain other constituents besides soy foods. Table 6.6 shows the isoflavone content of soy-based beverages. Instant soy beverage powder and soy milk skin were relatively rich in isoflavones, with concentrations ranging from 100 to 194 mg/g (25,26,72,73,76,85,87,89,91,93,94,102). Table 6.7 lists the isoflavone levels in different forms (powdered, liquid concentrate, and ready-to-feed forms) of soy-based formulas for infants and adults. The powdered, liquid concentrate, and ready-to-feed forms of various soy-based infant formulas contained 24.53–26, 4.02–6.03, and 2.63–4.17 mg/100 g portions of total isoflavones, respectively. The ready-to-use preparations for adults contained 0.08– 0.54 mg/100 g portion of total isoflavones (74,80,84,89,91,95). Table 6.8 contains data for lignans in a selection of common foods, such as grains, cereals, berries, currants, fruits, cruciferous vegetables, and coffee and tea. The foodstuffs were also analyzed for isoflavones; these compounds were not present in detectable concentrations or were present in trace quantities only, with the exception of certain foods that were found to contain significant amounts of isoflavones (clover seed, which had 0.178 mg daidzein and 0.323 mg genistein/100 g, and peanut, which had 0.058 mg daidzein and 0.064 mg genistein/100 g). In most of the berries the dominant lignan was SECO (0.1653–3.718 mg/100 g dry weight), and MAT was not present in detectable amounts or was present in trace quantities only (2). In the most of the coffee and tea samples, the concentrations of SECO fell in the range 0.5–2.89 mg/100 g (3). Table 6.9 records the concentrations of isoflavones and lignans in legumes. The data show that the isoflavone levels in soybeans were high (with ranges of 10.5–85 mg/100 g dry weight for daidzein and 26.8–102.5 mg/100 g dry weight for genistein). The SECO content, however, was only 0.013–0.273 mg/100 g dry weight. Among legumes, the isoflavone concentrations were highest in soybeans (10.5–85.0 mg/100g dry weight for daidzein, and 26.8–102.5 mg/100 g dry weight for genistein); kudzu root (Pueraria lobata) also had remarkably high isoflavone concentrations (185 mg/100 g dry weight for daidzein and 12.6 mg/100 g dry weight for genistein) (2,3,96).

Concluding Remarks Naturally occurring phytoestrogens in foods, such as isoflavones and lignans, appear to be beneficial to human health. Results of epidemiologic studies suggest that phytoestrogen-containing foods may play a significant role in preventing can-

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cer, cardiovascular disease, osteoporosis, and postmenopausal symptoms. However, there is almost no proof that the health-giving effects of phytoestrogen-containing foods are due specifically to phytoestrogens alone. Experiments have demonstrated that many other components of soybean, soy foods, and flaxseed are biologically active and may be responsible, at least in part, for the observed effects in humans and that the combination of these compounds with phytoestrogens might have synergistic effects. More research is required to determine whether phytoestrogens are the main active components, and what mechanisms are involved, and to determine whether consumption of phytoestrogens in particular provides protection against diseases such as cancer and cardiovascular disease. Currently there is insufficient evidence to recommend specific dietary practices for therapeutic purposes. Offsetting the potential health benefits of phytoestrogens are possible risks, especially when large doses of isolated, purified phytoestrogen extracts are taken. Excessive intake of phytoestrogens could exert detrimental, if not toxic, estrogenic effects. This issue is a cause for serious concern because there are many purified isoflavone food supplements on the market, and their use by consumers has increased without the benefit of guidelines for proper dosage and knowledge of possible medicinal effects and side effects, including interactions with prescription drugs. Whole foods, on the other hand, have the advantage of containing a variety of beneficial bioactive components in moderate concentrations, together with valuable nutrients. Their beneficial effects, both medicinal and nutritional, have been demonstrated in clinical and epidemiologic studies, and they are likely to be much safer. For proper evaluation of the health effects of phytoestrogens in foods, accurate, sensitive, and reliable analytical techniques for determining these compounds are absolutely essential. The analytical work must include synthesis of labeled standards and establishment of quality assurance schemes to verify the reliability of the laboratory performing the analyses. Concerning the issue of selecting the best analytical technique for determination of particular phytoestrogens in particular kinds of sample materials, it is necessary to use different analytical methods (e.g., GC-MS, HPLC, and LC-MS) to analyze replicate subsamples of the same sample material and compare the results. The development of suitable analytical methods for determining isoflavones and other phytoestrogens in foods has been difficult due to the large number of phytoestrogens that exist and the range of chemical forms in which they can occur within various biological matrices. Acknowledgement We thank Ms. Estatira Sepehr for assisting in the preparation of the tables.

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40. Messina, M.J., Persky, V., Setchell, K.D.R., and Barnes, S. (1994) Soy Intake and Cancer Risk—A Review of the In Vitro and In Vivo Data, Nutr. Cancer 21, 113–131. 41. Jing, Y., Nakaya, K., and Han, R. (1993) Differentiation of Promyelocytic Leukemia Cells HL-60 Induced by Daidzein In Vitro and In Vivo, Anticancer Res. 13, 1049–1054. 42. Peterson, G., and Barnes, S. (1991) Genistein Inhibition of the Growth of Human Breast Cancer Cells—Independence from Estrogen Receptors and the Multi-Drug Resistance Gene, Biochem. Biophys. Res. Commun. 179, 661–667. 43. Peterson, G., and Barnes, S. (1993) Genistein and Biochanin-A Inhibit the Growth of Human Prostate Cancer Cells but Not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation, Prostate 22, 335–345. 44. Peterson, G., and Barnes, S. (1996) Genistein Inhibits Both Estrogen and Growth FactorStimulated Proliferation of Human Breast Cancer Cells, Cell Growth Differ. 7, 1345– 1351. 45. Zava, D.T., and Duwe, G. (1997) Estrogenic and Antiproliferative Properties of Genistein and Other Flavonoids in Human Breast Cancer Cells In Vitro, Nutr. Cancer 27, 31–40. 46. Naik, H.R., Lehr. J.E., and Pienta, K.J. (1994) An in Vitro and in Vivo Study of Antitumor Effects of Genistein on Hormone Refractory Prostate Cancer, Anticancer Res. 14, 2617–2620. 47. Kuo, S.-M. (1996) Antiproliferative Potency of Structurally Distinct Dietary Flavonoids on Human Colon Cancer Cells, Cancer Lett. 110, 41–48. 48. Kuo, S.-M., Morehouse, H.F., and Lin, C.-P. (1997) Effect of Antiproliferative Flavonoids on Ascorbic Acid Accumulation in Human Colon Adenocarinoma Cell, Cancer Lett. 116, 131–137. 49. Wei, H.C., Bowen, R., Cai, Q.Y., Barnes, S., and Wang, Y. (1995) Antioxidant and Antipromotional Effects of Soybean Isoflavone Genistein, Pro. Soc. Exp. Bio. Med. 208, 124–130. 50. Hirose, M., Hoshiya, T., Akag, K., Takahashi, S., Hara, Y., and Ito, N. (1993) Effects of Green Tea Catechins in a Rat Multiorgan Carcinogenesis Model, Carcinogenesis 14, 1549–1553. 51. Hertog, M.G.L., Hollman, P.C.H., and Vandeputte, B. (1993) Content of Potentially Anticarinogenic Flavonoids of Tea Infusion, Wines, and Fruit Juices, J Agric. Food Chem. 41, 1242–1246. 52. Anderson, J.W., Smith, B.S., and Washnock, C.S. (1999) Cardiovascular and Renal Benefits of Dry Beans and Soybean Intake, Am. J. Clin. Nutr. 70 (Suppl.), 464S–474S. 53. Cassidy, A., Bingham, S., and Setchell, K (1994) Biological Effects of a Diet of Soy Protein Rich in Isoflavones on the Menstrual Cycle of Premenopausal Women, Am. J. Clin. Nutr. 60, 333–340. 54. Cassidy, A., Bingham, S., and Setchell, K. (1995) Biological Effects of Isoflavones in Young Women; Importance of the Chemical Composition of Soybeans Products, Br. J. Nutr. 74, 587–601. 55. Valente, M., Bufalino, L., Castiglinone, G.N., D’Angelo, R., Mancuso, A., Galoppi, P., and Zichella, L. (1994) Effects of 1-Year Treatment with Ipriflavone on Bone in Postmenopausal Women with Low Bone Mass, Calcif. Tissue Int. 54, 377–380. 56. Brandi, M.L. (1992) New Treatment Strategies: Ipriflavone, Strontium, Vitamin D Metabolites, and Analogs, Am. J. Med. 95, 69S–74S 57. Ross, P.D., Fujiwara, S., Huang, C., Davis, J.W., Epstein, R.S., Wasnich, R.D., Kodama, K., and Melton, L.J., III (1995) Vertebral Fracture Prevalence in Women in Hiroshima Compared to Caucasians or Japanese in the US, Int. J. Epidemiol. 24, 1171–1177.

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58. Davis, J. W., Ross, P.D., Nevitt, M.C., and Wasnick, R. D. (1997) Incidence Rates of Falls Among Japanese Men and Women Living in Hawaii, J. Clin. Epidemiol. 50 (Suppl.), 589–594. 59. Schneider, D.L., Barrett-Connor, E.L., and Morton, D.J. (1997) Timing of PostMenopausal Estrogen for Optimal Bone Mineral Density, J. Am. Med. Assoc. 277, 543–547. 60. Potter, S.M., Baum, J.A., Teng, H. Stilman, R.J., Shay, N.F., and Erdman, J.W. (1998) Soy Protein and Isoflavones; Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68 (Suppl.), 1375S–1379S. 61. Dalais, F.S., Rice, G.E., Bell, R.J., Murkies, A.L., Medley, G., Strauss, B.J.G., and Wahlqvist, M.L. (1998) Dietary Soy Supplementation Increases Vaginal Cytology Maturation Index and Bone Mineral Content in Postmenopausal Women, Am. J. Clin. Nutr. 68 (Suppl.), 1518S (Abstr.). 62. Anderson, J.J., Ambrose, W.W., and Garner, S.C. (1995) Orally Dosed Genistein from Soy and Prevention of Cancellous Bone Loss in Two Ovariectomized Rat Models, J. Nutr. 125, 799S (Abstr.) 63. Jenkins, D.J.A., Wolever, T.M.S., Jenkins, A.L., Thorne, M.J., Lee, R., Kalmusky, J., Reicher, R., and Wong, G.S. (1983) The Glycaemic Index of Foods Tested in Diabetic Patients: A New Basis of Carbohydrate Exchange Favouring the Use of Legumes, Diabetologia 24, 257–264. 64. Jenab, M., and Thomson, L.U. (1996) The Influence of Flaxseed and Lignans on Colon Carcinogenesis and Glucuronidase Activity, Carcinogenesis 17, 1343–1348 65. Thompson, L.U., Rickard, S.E., Orcheson, L.J., and Seidl, M.M. (1996) Flaxseed and Its Lignan and Oil Components Reduce Mammary Tumor Growth at a Later Stage of Carcinogenesis, Carcinogenesis 17, 1373–1376 66. Thompson, L.U., Seidl, M.M., Rickard, S.E., Orcheson, L.J., and Fong, H.H.S. (1996) Antitumorigenic Effect of a Mammalian Lignan Precursor from Flaxseed, Nutr. Cancer 26, 159–165. 67. Collin, B.M., Mclachlan, J.A., and Arnold, S. (1997) The Estrogenic and Antiestrogenic Activities of Phytochemicals with the Human Estrogen Receptor Expressed in Yeast, Steroids 62, 365–372. 68. Kurzer, MS., Slavin, J.L., and Adlercreutz, H. (1995) Flaxseed in Lignans and Sex Hormones, in Flaxseed in Human Nutrition, (Cunnane, S., and Thompson, L.U., eds.) pp. 136–144, AOCS Press, Champaign, IL. 69. Mousavi, Y., and Adlercreutz, H. (1982) Enterolactone and Estradiol Inhibit Each Other’s Proliferative Effect on MCF-7 Breast Cancer Cells in Culture, J. Steroid Biochem. Mol. Biol. 41, 615–619. 70. McMichael-Phillips, D. E., McMichael-Phillips, D.F., Harding, C., Morton, M., Roberts, S.A., Howell, A., Potten, C.S., and Bundred, N.J. (1998) Effects of Soy-Protein Supplementation on Epithelial Proliferation in the Histologically Normal Human Breast, Am. J. Clin. Nutr. 68 (Suppl.), 1431S–1436S. 71. Sheehan, D.M., and Medlock, K.L. (1995) The Case for Expanded Phytoestrogen Research, Proc. Soc. Exp. Biol. Med. 208, 3–5. 72. Wang, G., Kuan, S. S., Francis, O. J., Ware, G. M., and Carman, A. S. (1990) A Simplified HPLC Method for the Determination of Phytoestrogens in Soybean and Its Processed Products, J. Agric. Food Chem. 38, 185–190.

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73. Fukutake, M., Takahashi, M., Ishida, K., Kawamura, H., Sugimura, T., and Wakabayashi, K. (1996) Quantification of Genistein and Genistin in Soybeans and Soybean Products, Food Chem. Toxicol. 34, 457–461. 74. Murphy, P. A., Song, T., Buseman, G., and Barua, K. (1997) Isoflavones in Soy-Based Infant Formulas, J. Agric. Food Chem. 45, 4635–4638. 75. Wang, H-J., and Murphy, P. A. (1996) Mass Balance Study of Isoflavones During Soybean Processing, J. Agric. Food Chem. 44, 2377–2383. 76. Jones, A.E., Price, K.R., and Fenwick, G.R. (1989) Development and Application of a High-Performance Liquid Chromatographic Method for the Analysis of Phytoestrogens, J. Sci. Food Agric. 46, 357–364. 77. U.S. Department of Agriculture-Iowa State University (1999) Database on Isoflavone Content of Foods. http://www.nal.usda.gov/fnic/foodcomp/Data/isoflav/isoflav.htm. 78. Mazur, W.M. (1998) Phytoestrogen Content in Foods, Bailliere’s Clin. Endocrinol. Metab. 12, 729–741. 79. Franke, A.A., Custer, L.J., Cerna, C.M., and Narala, K. (1995) Rapid HPLC Analysis of Dietary Phytoestrogens from Legumes and from Human Urine, Proc. Soc. Exp. Biol. Med. 208, 18–26. 80. Murphy, P.A., Song. T., Buseman, G., Barua, K., Beecher, G.R., Trainer, D., and Holden, J. (1999) Isoflavones in Retail and Institutional Soy Foods, J. Agric. Food Chem. 47, 2697–2704. 81. Murphy, P.A., Barua, K., and Song, T. (1998) Soy Isoflavones in Foods: Database Development, in Functional Foods for Disease Prevention, (Shibamoto, T., Terao, J., and Osawa T., eds.) pp. 138–149, American Chemical Society, Washington, DC. 82. Wang, H.-J., and Murphy, P.A. (1994) Isoflavone Composition of American and Japanese Soybeans in Iowa: Effects of Variety, Crop Year, and Location, J. Agric. Food Chem. 42, 1674–1677. 83. Choi, J.-S., C., Kwon,T.-W., and Kim, J.-S. (1996) Isoflavone Contents in Some Varieties of Soybean, Foods Biotechnol. 5, 167–169. 84. Setchell, K.D.R., and Welsh, M.B. (1987) High-Performance Liquid Chromatographic Analysis of Phytoestrogens in Soy Protein Preparations with Ultraviolet, Electrochemical and Thermospray Mass Spectrometric Detection, J. Chromatogr. 386, 315–323. 85. Franke, A.A., Custer, L.J., Wang, W., and Shi, C.Y. (1998) HPLC Analysis of Isoflavonoids and Other Phenolic Agents from Foods and from Human Fluids, Proc. Soc. Exp. Biol. Med. 217, 263–273. 86. Fenner, G.P. (1996) Low-Temperature Treatment of Soybean (Glycine max) Isoflavonoid Aglycon Extracts Improves Gas Chromatgraphic Resolution, J. Agric. Food Chem. 44, 3727–3729. 87. Barnes, S., Kirk, M., and Coward, L. (1994) Isoflavones and Their Conjugates in Soy Foods: Extraction Conditions and Analysis by HPLC Mass Spectrometry, J. Agric. Food Chem. 42, 2466–2474. 88. Farmakalidis, E., and Murphy, P.A. (1985.) Isolation of 6”-O-Acetylgenistin and 6”-OAcetyldaidzin from Toasted Defatted Soyflakes, J. Agric. Food Chem. 33, 385–389. 89. Nguyenle, T., Wang, E., and Cheung, A.P, (1995) An Investigation on the Extraction and Concentration of Isoflavones in Soy-Based Products, J. Pharm. Biomed. Anal. 14, 221–232.

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90. Coward, L., Kirk, M., Albin, N., and Barnes, S. (1996) Analysis of Plasma Isoflavones by Reversed-Phase HPLC-Multiple Reaction Ion Monitoring-Mass Spectrometry, Clin. Chim. Acta 247, 121–142. 91. Dwyer, J.T., Goldin, B.R., Saul, N., Gualtieri, L., Barakat, S., and Adlercreutz, H. (1994) Tofu and Soy Drinks Contain Phytoestrogens, J. Am. Diet. Assoc. 94, 739–743. 92. Hutchins, A.M., Slavin, J.L., and Lampe, J.W. (1995) Urinary Isoflavonoid Phytoestrogen and Lignan Excretion After Consumption of Fermented and Unfermented Soy Products, J. Am. Diet. Assoc. 95, 545–551. 93. Lu, L.W., Broemeling, L.D., Marshall, M.V., and Ramanujam, S. (1995) A Simplified Method to Quantify Isoflavones in Commercial Soybean Diets and Human Urine After Legume Consumption, Cancer Epidemiol. Biomark. Prev. 4, 497–503. 94. Lu, L.W., Grady, J.J., Marshall, M.V., Ramanujam, V.M.S., and Anderson, K.E. (1995) Altered Time Course of Urinary Daidzein and Genistein Excretion During Chronic Soya Diet in Healthy Males, Nutr. Cancer 24, 311–323. 95. Setchell, K.D.R., Zimmer-Nechemias, L., Cai, J., and Heubi, J.E. (1997) Exposure of Infants to Phyto-Oestrogens from Soy-Based Infant Formula, Lancet 350, 23–27. 96. Mazur, W.M., and Adlercreutz, H. (1998) Naturally Occurring Oestrogens in Food, J. Pure Appl. Chem. 70, 1759–1776. 97. Pratt, D.E., and Birac, P.M. (1979) Source of Antioxidant Activity of Soybeans and Soy Products, J. Food Sci. 44, 1720–1722. 98. Seo, A., and Morr, C.V. (1984) Improved High-Performance Liquid Chromatographic Analysis of Phenolic Acids and Isoflavonoids from Soybean Protein Products, J. Agric. Food Chem. 32, 530–533. 99. Naim, M., Gestetner, B., Zilkah, S., Birk, Y., and Bondi, A. (1976) Soybean Isoflavones, Characterization, Determination, and Antifungal Activity, J. Agric. Food Chem. 22, 806–810. 100. Petterson, H., and Kiessling, K.H. (1984) Liquid Chromatographic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed, J. Assoc. Off. Anal. Chem. 67, 503–506. 101. Wang, C., Ma, Q., Pagadala, S., Sherrad, M.S., and Krishnan, P.G. (1998) Changes of Isoflavones During Processing of Soy Protein Isolates, J. Am. Oil Chem. Soc. 75, 337–341. 102. Xu, X., Wang, H.-J., Murphy, P.A., Cook, L., and Hendrich, S. (1994) Daidzein Is a More Bioavailable Soymilk Isoflavone than Is Genistein in Adult Women, J. Nutr. 124, 825–832. 103. Padgette, S.R., Taylor, N.B., Nida, D.L., Bailey, M.R., MacDonald, J., Holden, L.R., and Petterson, H., and Kiessling, K.-H. (1984) Liquid Chromatographic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed, J. Assoc. Off. Anal. Chem. 67, 503–506. 104. Carrao-Panizzi, M., and Kitamura, K. (1995) Isoflavone Content in Brazilian Soybean Cultivars, Breed. Sci. 45, 295–300.

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Chapter 7

Analysis of Phytoestrogens in Biological Samples by Mass Spectrometry Jeevan K. Prasaina,b, Chao-Cheng Wanga,b, and Stephen Barnesa,b,c aDepartment

of Pharmacology and Toxicology, bPurdue-UAB Botanicals Center for Dietary Supplements Research, and cComprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham, Birmingham, AL

Introduction Phenolic compounds comprise one of the largest and most ubiquitous group of phytochemicals and are an important part of the human diet. The compounds most commonly occurring phenolics in foods are flavonoids and phenolic acids. Flavonoids are the largest class of phenolic compounds and are classified mainly into flavones, flavanols (catechins), isoflavones, flavonols, flavanones, and anthocyanins. All are structurally related to the parent compound, flavone (2-phenyl benzopyrone). Isoflavones such as genistein (5,7,4′-trihydroxyisoflavone) and daidzein (7,4′-dihydroxyisoflavone) are commonly regarded as phytoestrogens (Fig. 7.1). Soybeans are a rich source of these compounds. These isoflavones have weak estrogenic activity, but can be estrogen antagonists at higher concentrations (1). Several studies on phytoestrogens have shown that they have an inhibitory effect on mammary tumorigenesis (2,3). Other polyphenols have been the subject of the growing interest in phytoestrogen activity. These include the lignans, the coumestans, and zearalones (the last-mentioned are compounds that arise from microbial contamination of stored food products). The absorption of conjugated and unconjugated phytoestrogens and their biotransformation in humans or in experimental animals is not fully understood. In soybeans, as well as nonfermented soy products, isoflavones occur in conjugated forms as glycosides, malonylglycosides, and acetylglycosides. In contrast, in fermented soy products (for example, miso) the unconjugated aglycones predominate, some of which have undergone further metabolism (4,5). The β-glycosides, genistin and daidzin, are hydrolyzed in the gut by lactose phlorizin hydrolase, an enzyme in the apical membrane of the villi of the small intestine (6), and by intestinal microflora that convert them into aglycone forms (7). The next step involves the reconjugation with glucuronic acid and to a lesser extent with sulfate by the phase II enzymes UDP-glucuronosyltransferase and sulfotransferase in the liver as well as other organs (8,9). In rats, glucuronidation occurs within the intestinal wall, thereby imparting a strong intestinal first-pass effect (8). The phytoestrogen phase II metabolites are taken up by the liver and are excreted in bile,

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Fig. 7.1. Chemical structures of the isoflavones, genistein, daidzein, biochanin A, formononetin, and glycitein (A). Equol (B) and O-desmethylangolensin (C) are common metabolites of isoflavones.

thus transporting them back into the intestines. Intestinal β-glucuronidases (8) and sulfatases release the aglucones; these can be reabsorbed or enter the bacterial-rich large bowel. In the latter, reduction (daidzein to equol) (10), ring opening (daidzein to Odesmethylangolensin) (11), and ring cleavage [to p-ethylphenol and/or 2-(4-hydroxyphenyl)propionic acid] of the heterocyclic ring of the isoflavonoids occur (12). Phytoestrogens of the lignan type are found in various foods. Flaxseed, in particular, has been shown to contain several lignans, among them secoisolariciresinol and matairesinol, which are converted to the mammalian lignans, enterodiol (END) and enterolactone (ENL) (13,14) (Fig. 7.2). END and ENL are excreted in the urine of rats and humans (15,16). Because of their two phenolic groups, both END and ENL are conjugated with glucuronic and sulfuric acid before excretion (17). However, as for the isoflavonoids, the bioavailability and metabolism of these lignans are not well understood. Due to the potential importance of these phytoestrogens to living organisms, the identification of such compounds occurring in biological systems has immense implications for many areas of science. Methods for the qualitative description and quantitative analysis of phytoestrogens and their metabolites have evolved rapidly in the past 10–15 years. These methods include gas chromatography (GC), reversephase high-performance liquid chromatography (HPLC) and capillary electrophoresis in combination with ultraviolet light (UV) absorbance, fluorescence,

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Fig. 7.2. Chemical structures of the lignans. Matairesinol (A) and secoisolariciresinol

(B) are lignans in flax seed. Enterolactone (C) and enterodiol (D) are formed by bacteria in the gut.

electrochemical detection, and mass spectrometry (MS), as well as nonchromatographic techniques such as matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and immunoassay procedures. We recently reviewed the relative merits of these different approaches (18). In this chapter, the focus is on the place of MS in the analysis of phytoestrogens with the goal of providing a practical guide to the use of this powerful technique. In the last few years, MS has become an essential method in the analysis of phytoestrogens. Its high sensitivity and specificity and easy hyphenation with chromatographic techniques are attributes that qualify MS as the most appropriate analytical technique for the study of phytochemicals. It is important to mention here that MS is different from other spectroscopic techniques because of its versatility for applications in detection, identification, and quantification of compounds. The advent of electrospray ionization (ESI), a soft ionization technique, has extended the polarity limit of analysis by MS. Recently, a review appeared on the application of MS for identification of flavonoid glycosides (19). Brief reviews by Barnes et al. (20,21) have described LC-MS analysis of isoflavones, in particular regarding the methods used to prepare samples for analysis, the chromatographic procedures, and types of mass spectrometers that are available for isoflavone analysis. This chapter considers the analysis of phytoestrogens by different MS techniques and compares their usefulness. In

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particular, we present an in-depth overview on the recent use of LC-MS and tandem mass spectrometric (MS-MS) techniques in the analysis of phytoestrogens. Ionization Methods Basically, a mass spectrometer is designed to produce gas phase ions from sample molecules, separate them according to their mass-to-charge (m/z) ratio, and detect and record the separated ions. A large number of different instrumental configurations can be used to perform these functions. There are different methods of ionization, which are described below. Electron Impact Ionization (EI). EI was the first ionization method to be used routinely and is still one of the widely employed methods in MS. The EI source consists of a high-energy electron beam that originates from a heated filament and is then accelerated through a potential of ~70 eV into the source. The gas phase molecules entering the source interact with these electrons. As a result, some of the molecules lose an electron to form a positively charged ion whose mass corresponds to that of the original neutral molecule. This is suitable for the analysis of a large number of synthetic and small molecule natural products. However, EI-MS is limited by the need for sample vaporization before ionization. Thus the technique is unsuitable for the labile, nonvolatile compounds that are encountered in biological samples (Table 7.1). The coupling of EI-MS with capillary gas chromatography (GC/EI-MS) has been widely used in phytoestrogen analysis. However, in the past eight years, LC-MS has emerged as a powerful alternative method for phytoestrogens. Fast-Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS). In FAB, the impact of an energetic particle initiates both the sample vaporization and ionization processes, so that separate thermal volatilization is not required. In the case of LSIMS, a liquid matrix is used and a primary beam of cesium ions instead of fast atoms causes evaporation and ionization. The matrices most often used (each are high boiling solvents) are glycerol, nitrobenzyl alcohol, dithiothreitol/dithioerythritol, 5:1 w/w (magic bullet), and thioglycerol. The choice of matrix has a great effect on the signal-to-noise ratio. Both methods cause a relatively mild ionization process, so that fragment ions are generally of low abundance. The strength of these techniques is the ability to analyze a wide range of thermolabile and ionic compounds. There are several examples of the application of FAB and LSIMS for the analysis of polyphenols, particularly, flavonoid glycosides (see below). Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). Matrix-assisted laser desorption ionization (MALDI) was first introduced by Karas et al. (22) for analysis of nonvolatile compounds. In this technique, samples are co-crystallized with matrices on the target probe, ionized by nitrogen laser pulses (337 nm), and then analyzed with a time-of-flight mass spec-

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TABLE 7.1 Comparison of Mass Spectrometry (MS) Ionization Techniquesa MS ionization technique

Advantages

Disadvantages

Electron impact (EI)

Easy hyphenation with GC Highly sensitive Identification of unknown possible

Derivatization needed, labor intensive Limited mass range Possible thermal decomposition, high fragmentation, often results in no observable molecular ion

Fast Atom Bombardment (FAB)

Extended mass range up to 7 kDa Soft ionization technique Good for analysis of flavonoid glycosides

Low sensitivity Requirement of solubility of sample in matrix High background matrix peaks

Matrix Assisted Laser Desorption/Ionization/ (MALDI)

Practical mass limit up to 300 kDa Tolerant of mmol/L concentration of salts Good for a wide range of mass analysis

Low resolution High matrix background signals little use for Small molecules may be not good for laser-sensitive compounds High-throughput analysis

Atmospheric Pressure Chemical Ionization (APCI)

Practical mass range up to 2 kDa Highly sensitive (fmol) HPLC/MS capable Practical mass limit up to 70 kDa HPLC/MS capable Multiple charge resolution ~2000 Sensitivity fmol–pmol

Sensitivity may be variable with compound type Possibility of thermal decomposition

Electrospray Ionization (ESI)

aAbbreviations:

GC, gas chromatography; HPLC, high-performance liquid chromatography.

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Relatively low salt tolerance Multiple charge can be confused in mixtures Analysis may be difficult for nonionizable compounds No or less tolerance for heterogenous mixture

trometer (TOF-MS) (Fig. 7.3). The use of appropriate matrices, which absorb energy from laser pulses and allow a soft desorption ionization of the sample, is one of the most important aspects of MALDI. There are several compounds that are routinely used as MALDI matrices. Sinapinic acid (3,5-dimethoxy-4-hydroxy cinnnamic acid) is used for large peptides and proteins, and α-cyano-4-hydroxycinnamic acid (CHCA), 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHB) are used for peptides and other small molecules (23). MALDI-TOF-MS has several advantages over other methods, including speed of analysis, sensitivity, good tolerance toward contaminants, and the ability to analyze complex samples (24). Although MALDI-TOF-MS is well known as a powerful tool for the analysis of a wide range of biomolecules such as peptides and proteins, its potential in food analysis has been explored only recently (25). Electrospray Ionization-Mass Spectrometry (ESI-MS)/Atmospheric Pressure Chemical Ionization Mass Spectrometry (APCI-MS). For FAB-MS and LSIMS, electrospray ionization does not require formation of volatile derivatives of the phytoestrogens. ESI is a method of generating highly charged droplets from which ions are ejected by an ion evaporation process (Fig. 7.4). This technique is typical-

Fig. 7.3. Schematic of matrix-assisted laser desorption ionization/time of flight/mass

spectrometric (MALDI-TOF-MS) analysis. The sample for analysis is deposited onto an ultraviolet light (UV)-absorbing crystalline matrix. Once dry, it is inserted into the mass spectrometer. The solutes are volatilized by evaporation of the crystal surface by a short (ns) laser pulse from a N2 laser operating at 337 nm. The resulting ions are focused and then extracted with a 20 kV accelerating pulse. They “drift” through the flight tube and their arrival at the detector is carefully timed, thereby allowing estimation of their molecular weight. In some analyses, the ions are “bounced” off of an electrostatic mirror (reflectron) to obtain a higher resolution mass spectrum.

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ly performed in either the infusion mode, or in combination with HPLC or capillary electrophoresis (see below). In the infusion mode, the sample is introduced into a continuous liquid stream via an injection valve. ESI is most often combined with a quadrupole-based mass spectrometer. A quadrupole is a mass filter, consisting of four rods to which an oscillating electric field is applied, allowing only ions with a certain mass-to-charge (m/z) ratio to pass through. The term APCI denotes those atmospheric pressure ionization processes that involve ion-molecule reactions to create ions in the gas phase (using corona or Ni63 discharge). With the advent of API technology (both ESI and APCI), a wide range of polyphenols can now be analyzed directly. The achievable sensitivity of ESI is at least two orders of magnitude higher than those of FAB or LSIMS. Another advantage of ESI is a better signal-to-noise ratio, due to the reduced number of ions in the spectral range <300 amu originating from the matrix and spraying solvent, a very important region for phytoestrogens such as isoflavones and lignans. These two interfaces (ESI and APCI) are highly sensitive, show greater ionization stability, and have become the methods of choice for isoflavone analysis. It should be noted that both are quenched by the use of common HPLC mobile phase modi-

nebulizing gas sample solution

mass analyzer +HV Atmospheric pressure

Vacuum

[M + nH]n+

1. Solvent evaporation 2. Coulombic repulsion Fig. 7.4. Schematic of the electrospray ionization interface. The sample solution is passed through an electrically charged spraying needle with pneumatic assistance to produce a fine charged droplet spray containing the solute ions. There is rapid evaporation of the droplets forcing the ions into closer contact. This is accelerated by the dry N2 curtain gas. The coulombic pressure exerted by the ions overcomes the surface tension of the droplets causing ejection of a “gas” of wet solute ions. These lose the solvent molecules as the ions are accelerated and pass down the potential gradient leading through the orifice of the mass spectrometer analyzer.

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fiers such as trifluoroacetic acid and sodium or potassium phosphate, thereby requiring modification of existing methods. Alternative modifiers include formic acid in place of trifluoroacetic acid and ammonium acetate or ammonium formate for the phosphate buffers. Both techniques can be used equally well in positive and negative ion modes, although most flavonoids are measured in the negative ion mode. Sample Preparation The analysis of phytoestrogens and their metabolites in biological samples is a particular challenge. First, because of the complexity of the biological matrix, many compounds can interfere in the analysis of the target analytes. Second, some of the phytoestrogens are present at very low concentrations, requiring a high level of sensitivity. Sample preparation is a crucial step in the analysis of biological samples. Proper procedures should be adopted on the basis of the type of analysis being used. A summary of the methods developed to date for the determination of phytoestrogens, the main procedural steps, detection, and recovery is shown in Table 7.2. A detailed discussion of all of these aspects is presented below. Internal Standard. To carry out quantitative measurements of phytoestrogens using chromatographic methods, it has proved necessary to include internal standards (IS) to correct for unknown losses during the procedure used. These standards range from deuterated (2H) or carbon-13 (13C)-labeled stable isotope forms of the phytoestrogen of interest, or compounds with similar chemical structure and properties that are not naturally present in the sample to be studied. GC-MS methods, because of their extensive set of work-up steps, have the greatest requirement for internal standards. However, in most applications, even with 2H- or 13C-labeled phytoestrogen standards, no account is taken of the losses that occur during initial extraction and hydrolysis before addition of the internal standard. For LC-based techniques, glycoside, glucuronide, and sulfate esters of suitable analogous compounds have been used to correct for extraction and incomplete hydrolysis (21,26). Filtration. Because biological samples may contain organic material and suspended particles, filtration is usually the first step of sample preparation. The filtration step is essential when subsequent extraction of the sample involves solid-phase extraction (SPE) because suspended particles could easily clog the adsorbent bed, or when the analysis is performed by immunochemical assay and they cause undesirable adsorption of the antibodies. The filtration step may be performed simultaneously with sample collection and/or extraction, or as a separate step. Centrifugation of the samples can also be used to remove suspended particles. Coldham and Sauer (27) used 1 µm PTFE filters for filtration of the methanolic extract of dietary supplements. Adlercreutz et al. (28) applied a TF 200 membrane filter to the work-up of homogenized samples of human feces in 80% aqueous methanol using a Millipore (Bedford, MA) Swinnex-47 filtration device. In this report, the filtrate (ethanolic extract) was concentrated and further diluted with

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TABLE 7.2 Methods for the Determination of Phytoestrogens in Biological Sample Analytesa Recoveryb (%)

Detection

Ref.

97

C-MS-SIM

28

GC-MS-SIM

32

95.5–105.5

GC-MS-SIM

29

SPE (C18) Liquid/liquid extraction (ChemElut) Adsorption-desorption (Florosil cartridge)

82.2

GC-MS-SIM

31

Preconcentration on a C18 column Cleaned on a ChemElut 1010 column Enzymatic hydrolysis Solvent extraction (dichloromethane/ ethyl acetate 2:1 vol/vol)

63.5–89.2 (free isoflavones) 56.5–77.1 (total isoflavones)

GC-MS

41

Analytes

Sample size

Sample preparation

Lignans and isoflavones in human feces

0.3–0.6 g

IS addition Solvent extraction (ethanol/acetone 9:1 vol/vol) Fats and protein precipitation Cleaned up with (Sep-Pak C18) Purification by ion exchange Chromatography

Lignans and isoflavones plasma and prostatic fluid

Plasma (1 mL) EPS (50–200 µL)

IS addition Enzymatic hydrolysis Ion exchange chromatography

Isoflavonoids, coumestrol and lignans in food

40 mg

Rehydration with water IS addition Enzymatic hydrolysis Ether extraction Acid hydrolysis Ion-exchange chromatography

8-PN in beer

20 mL

Isoflavones in human urine

20 mL

Stationary phase (LC column)

Continued

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TABLE 7.2 (Cont.) Analytes

Sample size

Sample preparation

Flavonoid glycoside Naringin in human urine

1 mL

Isoflavones in Soy foods and nutritional supplements Phytoestrogens in a dietary supplement

1g

IS addition Sep-Pak extraction Enzymatic hydrolysis Ethanol extraction Acetonitrile-water and IS in DMSO addition centrifugation and filtration Aqueous methanol extraction Filtration, acid hydrolysis Centrifugation Ethyl acetate extraction

DS (0.9 g) bread (5 g) Soy flour (40 mg)

Genistein and daidzein in rat serum

100 µL

Genistein, daidzein and conjugates in rat blood

75 µL

Isoflavones in comminuted baby food and soy flour

2g

Isoflavones and coumestrol in soybeans

1g

S addition Enzymatic hydrolysis Filtration Cleaned up with trap cartridge (C8) Acetonitrile addition Protein precipitation Enzymatic hydrolysis IS addition Ethyl acetate extraction Addition of tert-butylhydroquinone in methanol, and hydrochloric acid Spiking, and heating over stem bath Acetonitrile extraction Extracted with acidified ethanol Refluxed, and centrifuged Filtration

Stationary phase (LC column)

Recoveryb (%)

Nova-Pak C18

Detection

Ref.

LC-MS

65

YMC ODS-AM

99–101

LC-MS

38

Hypersil Elite C18

91 (genistein) 97 (8-PN)

LC-MS

27

Ultracarb C18

>80

LC-MS

63

Luna C18

85

LC-MS

62

PrimeSphere5 C18

76–89 (baby food) 84–92 (soy flour) 89–104

LC-MS

40

LC-MS-MS

67

Phenyl NovaPak

aAbbreviations: LC, liquid chromatography; IS, internal standard; GC-MS-SIM, gas chromatography/mass spectrometry/secondary ion mass spectrometry; EPS, expressed prostatic secretion; 8-PN, 8-prenylnaringenin; SPE, solid-phase extraction; DMSO, dimethyl sulfoxide; DS, dietary supplement; MS-MS, tandem mass spectrometry. bAt higher concentration of standards.

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water. The sample was homogenized with a Vortex mixer and kept at –20°C overnight to precipitate fats and proteins. Centrifugation (2000 × g for 10 min at –10°C) of the sample yielded a supernatant fraction. This was evaporated until only water remained. Further extraction and purification of unconjugated lignans and isoflavonoids was carried out using Sep-Pak cartridges (Waters, Milford, MA; see below). Usually, the filtration step is not carried out when liquid-liquid partition is the method of extraction. Extraction and Clean-Up. Extraction of phytoestrogens from biological matrices usually includes SPE followed by liquid-liquid extraction. Octadecyl (C18)-bonded silica has been the most widely used SPE adsorbent. The solvent, volume, and number of steps used for elution depend primarily on the type of adsorbent and the size of the SPE cartridge. Elution is generally performed with pure or aqueous methanol. Mazur et al. (29) used ion exchange chromatography for purification of the ether extract obtained from food samples. Adlercreutz et al. (28) used combined cation-anion exchange column chromatography for extraction and purification of lignans and isoflavonoids in methanolic extracts of human feces. Lu et al. (30) developed a procedure for the extraction of daidzein and genistein from urine samples using a liquid/liquid extraction column (ChemElut column, Varian Sample Preparation Products, Harbor City, CA). These columns are packed with diatomaceous material that absorbs a water matrix. This procedure was also applied to the isolation of the hop-derived phytoestrogen, 8-prenylnaringenin, from beer (31). Morton et al. (32) reported a method for extraction of lignans and isoflavonoids in human plasma and expressed prostatic secretion (EPS). Briefly, the samples were allowed to thaw to room temperature and vortexed to obtain a homogenous sample. After the addition of a deuterated internal standard cocktail, the samples were incubated at 37°C with β-glucuronidase in 0.1 mol/L acetate buffer, pH 5.0, to hydrolyze conjugates, followed by solvent extraction with diethyl ether. A fraction containing diphenolic compounds was then isolated using short columns of diethylaminohydroxypropyl Sephadex LH-20 (LKBPharmacia, Uppsala, Sweden) in the hydroxide form. Sample preparation for analyzing oxidative metabolism of isoflavones and lignans involves incubation of phytoestrogens with liver microsomes and tyrosinase followed by extraction. Microsomes are prepared on the basis of the procedure described by Lake (33). Protein concentrations are estimated using bicinchonic reagent (Pierce Chemical, Rockford, IL) and cytochrome P450 concentrations are measured as described by Omura and Sato (34). Standard incubation mixtures consisted of 2 mg of microsomal protein, 50 nmol/L of isoflavone dissolved in 40 µL of dimethyl sulfoxide (DMSO), and a NADPH-generating system (3 mmol/L MgCl2, 1 mmol/L NADP+, 8 mmol/L DL-isocitrate, and 0.5 U of isocitrate dehydrogenase) in a final volume of 2 mL of 0.05 mol/L potassium phosphate buffer, pH 7.4. After 2 min of preincubation at 37°C, the reaction was initiated by adding the NADPH-generating system and stopped after 60 min followed by extraction with 4 × 2 mL of ice-cold ethyl acetate.

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The enzyme tyrosinase has been also used to generate catechol metabolites. For this assay, the phytoestrogens (500 nmol/L in 50 µL DMSO) were incubated with tyrosinase (500 U) and NADH (5 mg) in 4 mL of 0.1 Tris-HCl buffer, pH 7.4, for 30 min at 37°C. As in the above case, the medium was extracted with ethyl acetate. Sample preparation for GC-MS analysis of lignan metabolites from bile and urine involves a clean-up procedure originally described by Jacobs et al. (35) for human urine and modified for LC-MS by Sfakianos et al. (8). Briefly, equal volumes of bile and ammonium acetate buffer (0.05 mol/L, pH 5.0) are mixed together and applied onto an RP-18 cartridge for SPE. The cartridge is washed with ammonium acetate buffer before the elution of lignans and their metabolites with methanol. Further steps involve dilution of the eluant with water to 70% methanol and application to a DEAESephadex A 25 column. Bile contents are further separated into conjugated and nonconjugated fractions. After evaporation under reduced pressure, the residue is mixed with ammonium acetate and purified using a second RP-18 cartridge. Plant samples are milled before extraction with aqueous ethanol or methanol. Rijke et al. (36) extracted isoflavone glucosides from red clover (Trifolium pratense) with 90% aqueous methanol and aqueous 350 mmol/L Tris buffer (3:1 vol/vol). Borges et al. (37) used a Soxhlet extraction procedure using ethanol (96% vol/vol) to isolate flavonoids and their glycosides from Genista tenera. The extract was concentrated to dryness under vacuum and the residue was dissolved in warm water, filtered, and extracted with diethyl ether. Griffith and Collison’s extraction conditions for soy protein, soy foods, and nutritional supplements involve acetonitrile extraction, followed by the addition of deionized water and internal standard apigenin in DMSO (38). The samples are centrifuged for 2 h and the supernatant is filtered with a polyvinylidene difluoride (PVDF) filter before analysis. In the case of dried, cooked, and canned soybean and tofu powders, the food is extracted with 10 mL of 2 mol/L HCl and aqueous ethanol and refluxed at 100°C. After centrifugation, the supernatant is passed through a microfilter before LC analysis. One of the advantages of LC-MS over GC-MS is that it is often not necessary to use any work-up. For example, urine samples from human subjects consuming soy or rats fed a nonpurified diet can be analyzed directly. The only work-up is centrifugation or filtration of the urine to remove any particles that would clog the HPLC column. In this type of analysis, separation is carried out using gradient elution with acetonitrile or methanol. The electrolyte and other hydrophilic components of urine that would interfere with detection of the phytoestrogens elute before the gradient has begun. For bile samples, the concentration of isoflavones is so high that the bile has to be diluted with the starting HPLC solvent. Again, filtration or centrifugation is used to remove any particulate matter. In the case of serum, the phytoestrogen concentrations are much lower, and it is usually necessary to extract and thereby concentrate the samples. Although several authors have recommended using SPE, this method is not very efficient for conjugated phytoestrogens, particularly the β-glucuronides. To overcome this problem, extraction with the hydrophobic ion pair reagent, trimethylammonium sulfate at pH 7.0 is

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preferred (26). However, this method leads to the recovery of hydrophilic components that are noticeable if the extract is analyzed isocratically. Wang and Murphy (39) utilized acidified acetonitrile at room temperature for extraction of isoflavones from foods and followed this step with concentration under reduced pressure and dissolution in 80% (vol/vol) methanol. Extraction and cleanup of sample from comminuted baby foods and soy flour included mixing of food products with tert-butylhydroquinone in methanol, and hydrochloric acid (40). After gentle stirring, the sample was heated over a steam bath for 2 h. After cooling, it was extracted with acetonitrile and an aliquot was diluted with an equal volume of HPLC solvent and injected into MS. Hydrolysis. Hydrolysis is an important step that converts the conjugated forms into the aglycone forms of phytoestrogens during the sample preparation. This may be necessary or convenient, depending on the nature of the final analysis, e.g., by immunoassay or GC-MS. Enzymatic hydrolysis of the isoflavone β-glucuronides and sulfates was used for the total isoflavone determination in urine (20,21,41). After extraction and cleanup, the sample was dissolved in 0.2 mL of methanol; then 5 mL of a 0.2 mol/L acetate buffer (pH 4.6) and 50 µL of H. pomatia digestive juice were added. The sample was incubated for 90 min at 60°C. After cooling to room temperature, the hydrolyzed urine was cleaned up and extracted with a mixture of dichloromethane and ethyl acetate. An advantage of LC-MS is its ability to determine both free and conjugated forms of phytoestrogens. Derivatization. This step is absolutely required for GC-MS analysis. It usually involves the production of trimethylsilyl ether (TMS) derivatives with N,O-bis (trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane (vol/vol). Pyridine-hexamethyldisilazane-trimethylchlorosilane has also been reported as a silylating reagent (28). In the literature, some authors reported that quantitative derivatization of polyhydroxylated isoflavones such as genistein was difficult (42). TMS derivatives are analyzed using a nonpolar capillary column and a linear temperature gradient. Analysis of Phytoestrogens FAB and LSIMS. There are several reports on the application of FAB and LSIMS for the analysis of flavonoid glycosides, in both positive and negative modes (43–46). The mass spectra of flavonoid glycosides showed ions created after cleavage of bonds between sugars and sugar and aglycone. Collisional activation of [M+H]+, or [M-H]– ions in the MS/MS experiments led to sequential losses of glycoside moieties in a manner that permitted the structure of glycosides to be established. Similarly, characterization and differentiation of diglycosyl flavonoids by positive ion FAB and MS/MS were reported by Li and Claeys (44). According to these authors, O-diglycosyl, O-C-diglycosyl, and di-C-glycosyl flavonoids can be differentiated on the basis of their FAB-MS/MS spectra. Similarly, Ma et al. (47) reported that differentiation of the interglycosidic linkage of isomeric flavonoid glycosides is possi-

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ble with FAB-MS/MS. Recently, two flavones, three isoflavones, and one 7-O-glucosyl isoflavone were isolated from Genista tenera and analyzed by LSIMS in combination with high-energy, collision-induced dissociation, and MS/MS by Borges et al. (37). The MS/MS spectra of 5-O-methylgenistein and 7-O-β-D-glucopyranoside showed the presence of a methoxy group; this is evident because of a m/z 270 ion in the former case. In the latter case, the same loss was observed, not only from the protonated molecules but also from the product ion, giving rise to m/z 432 and 270 ions, respectively. The base peak m/z 285 ion corresponded to the protonated aglycone formed by loss of the sugar unit and a hydrogen transfer. MALDI-TOF-MS. Applications of MALDI-TOF MS to the analyses of anthocyanins and flavonols in red wine and foods have been reported (48–50). Wang and Sporns (51) demonstrated the first example of using MALDI-TOF MS to identify phytoestrogens, isoflavones, in soy products. THAP and DHB were both found to be good MALDI matrices for isoflavones. DHB was used most because it worked well for sample extracts with better spot-to-spot repeatability. In this study, isoflavones exhibited fragmentation corresponding to loss of their carbohydrate residues. As shown in Figure 7.5, daidzin and genistin produced [M-162+H]+ ions at m/z 255 and 271, corresponding to their aglycones, respectively. MALDI-TOF spectra of 6′′-O-malonyl-β-glucoside and 6′′-O-acetyl-β-glucoside conjugates also showed corresponding aglycone fragments due to the glucosidic cleavage. These fragment ions, by glucosidic cleavage of isoflavones, provided characteristic information for structural elucidation. This work demonstrated that MALDI-TOF MS could produce isoflavone profiles and serve as a powerful tool to identify and study the effect of processing on isoflavones in soy products. Because isoflavones feature some UV absorption at 337 nm (the wavelength of the nitrogen laser), it is possible to perform laser desorption of isoflavones without the assistance of other matrices. Figure 7.6 shows the preliminary results of the laser desorption of genistein and daidzein without matrices added at all (Coward et al., unpublished data). Sodium adduct peaks were observed in both spectra, m/z 293 and 315 for genistein m/z 271 and m/z 277 and 293 for daidzein with m/z 255. Further study of laser desorption of phytoestrogens is ongoing in our laboratory. It should be noted that additional fragmentation of the isoflavone molecular and aglucone ions occurs in the drift region after ion acceleration, giving rise to illfocused ions when analysis is performed in the reflector mode. Gas Chromatography-Mass Spectrometry In the preceding chapter, the application of GC-MS for measurement of phytoestrogens in blood and urine was reviewed. As part of our overall review of the application of MS to the analysis of isoflavones, we will instead illustrate analysis by GC-MS on the oxidative metabolism of the major soy isoflavones daidzein and genistein and lignans.

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Fig. 7.5. Matrix-assisted laser desorption ionization/time of flight/mass spectrometry

(MALDI-TOF-MS) positive ion spectra of isoflavones (from soy flour) after high-performance liquid chromatography (HPLC) separation using 2,5-dihydroxybenzoic acid (DHB): (A) peak 1, daidzein; (B) peak 2, 6′′-O-malonyldaidzein; (C) peak 3, genistein, and 6′′-O-malonylglycitin; (D) peak 4, 6′′-O-acetyldaidzin; (E) peak 5, 6′′-O-malonylgenistin. From Reference 51 with permission.

Oxidative Metabolites of Phytoestrogens. As mentioned earlier, phytoestrogen conjugates are excreted through both urine and bile and undergo enterohepatic circulation. However, only a small portion of the ingested amount of these compounds may be recovered in urine and feces. One possible explanation for this low recovery is the formation of different metabolites. Metzler et al. (52–55) investigated the oxidative metabolism of the major soy isoflavones daidzein and genistein and lignans in vitro and in vivo. For GC-MS analysis, the samples were converted to TMS ethers with bovine serum albumin (BSA; 1:10 vol/vol with heptane) and an ion trap mass detector was used.

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Fig. 7.6. Matrix-assisted laser desorption ionization/time of flight/mass spectrometry

(MALDI-TOF-MS) positive ion spectra of isoflavones, A: genistein and B: daidzein,

Roberts-Kirchhoff et al. (56) reported that recombinant human cytochrome P450 1A1, 1A2, 1B1, and 2E1 metabolized genistein to form hydroxylated products. For GC-MS analysis, isoflavones in the dried extracts were converted to their TMS ether derivatives. They were chromatographed on a (12 m × 0.2 mm i.d.) DB1, 0.33-mm methyl silicone film-coated capillary column. GC-MS analysis of the tris-TMS derivative of genistein yielded a molecular weight of 486 although the M+ ion was very weak. In contrast, the M+-15 ion (m/z 471) was strong and could be used to determine the molecular weight. The loss of an O-TMS group resulted in the ion at m/z 399. The ion m/z 559 corresponds to the addition of a fourth O-TMS group because of enolization of the 4-keto group. Klus and Barz (57) investigated the formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from

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tempe (traditional Indonesian food produced from soybeans by fermentation mainly by fungi of the genus Rhizopus). All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavones. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. The GC-MS analyses of the TMS derivatives of the isoflavones provided both the molecular mass of the substrates and the number of hydroxyl groups in the molecule. Total structures of these metabolites were elucidated by GC-MS, HPLC-UV, and chemical degradation. Liquid Chromatography-Mass Spectrometry (LC-MS) and LC-MS/MS Principles. Liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) is a versatile technique for the analysis of phytoestrogens. This methodology combines efficient separation of biological samples and sensitive and specific measurement of the individual components by MS. Although numerous methods for coupling LC to MS have been explored, it is ESI that has transformed LC-MS/MS into a routine laboratory procedure sensitive enough to analyze phytoestrogens and their metabolites contained in biological samples at levels relevant to biochemical research. As described earlier, ESI requires a continuous flow of liquid, and the signal strength is concentration dependent. To achieve maximum sensitivity with limited sample size, efforts have been made to couple nanoscale LC at submicroliter flow rates to the highly sensitive microscale ES interface (usually referred to as nano LC-MS/MS). In a typical LC-MS experiment, the analytes, eluted from a reverse-phase column to separate the polyphenols by hydrophobicity, are ionized and transferred onto the mass spectrometer for analysis. The ion current for each scan can be summed and plotted as a function of time and the display is termed a total ion current (TIC). Postacquisition data can be displayed at a particular m/z value. In the MS/MS experiments, a precursor ion is subjected to fragmentation induced by collision, typically with argon gas. Because the fragmentation achieved using this method is representative of analyte structure, this technique serves as an effective tool for structure identification in complex mixtures. Instrumentation. Earlier, we reported progress in the analysis of isoflavones by LC-MS (20,21). API systems may be coupled to different mass spectrometric analyzers (quadrupole, ion trap, time-of-flight, and certain hybrid configurations), and different designs of API interfaces are available. Mobile phase flow rates in API interfaces range from nL/min (so-called nanoelectrospray) to 2 mL/min. A neutral solvent system is highly suited to reverse-phase HPLC-ESI-MS. The isoflavones and their conjugates readily form negatively or positively charged molecular ions. The temperature control of the APCI desolvation process is far less critical than in thermospray-MS, popular in the 1980s. In ESI, ions are preformed in solution, essential for ion formation in the gas phase. In contrast, APCI relies on the gas phase chemistry in which molecules have to be vaporized into the gas phase before

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ionization through charge or proton transfer. This process involves considerable heating to evaporate the large volume of mobile phase and may lead to compound decomposition. At present, quadrupole-type instruments are widely used because they are simple to operate, rugged, and good for quantitation by LC-MS. They are not very sensitive to impurities. A great advantage of the triple quadrupole instrument is that all tandem scan types can be performed easily. Ion trap instruments are also common and are less expencsive than mass spectrometers. The method has several advantages: (i) it is very sensitive because ions can be accumulated in the ion trap; (ii) the ion trap permits multiple sequential experiments with a given starting molecular ion, MSn; and (iii) the commercially available instruments are about half the price of their triple quadrupole counterparts. However, the fragmentation pathways in a quadrupole ion trap are different from those in a triple quadrupole instrument. MS-MS in ion-trap instruments occurs through a so-called “slow heating” technique that may be disadvantageous compared with other techniques (58,59). The length of the activation period associated with a very slow activation method may constitute a serious weakness because the time required for efficient conversion of parent ions to product ions may not be appropriate for some analytical applications. Neither the ion trap nor the triple quadrupole instruments yield high mass accuracy, particularly in the MS-MS mode. This problem can be overcome by using either a hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer that can deliver mass accuracies of 10–20 µg/g (ppm) in the MS-MS mode, or a Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometer that can provide mass accuracies better than 1 µg/g (ppm) in both the MS and MS-MS modes. The performance of a FT-ICR mass spectrometer is a function of the field strength of its superconducting magnet. Phytoestrogens in Physiologic Samples. Cimino et al. (60) reported a LC-MS method to estimate urinary concentration of genistein and daidzein, and their sulfate and glucuronide conjugates in urine. Human and rat urine samples were extracted with diethyl ether, or predigested with sulfatase and/or β-glucuronidase followed by extraction. The isoflavones were separated using a gradient LCmethod and detected by negative single ion monitoring on an MS system with a heated nebulizer APCI interface. They found 52 ± 4 and 26 ± 4% of genistein in rat urine as aglycone and sulfate conjugates, respectively, compared with 0.36 and 9%, respectively, in human urine. Similarly, Valentin-Blasini and co-workers (61) used APCI-MS/MS for the measurement of seven phytoestrogens in human serum and urine. In this method, enzymatic deconjugation of the phytoestrogen metabolites was carried out followed by SPE and reverse-phase HPLC. The method allows detection of isoflavone and lignans with limits of detection in the low ng/g (ppb) range. LC-MS data on the oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein have been reported (54). Here, the location of the hydroxyl

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groups of the metabolites can be obtained by HPLC/MS with positive API-MS. A base peak [M+H]+, as well as fragment ions derived from the molecular ion by a retro Diels-Alder reaction, appeared. These ions can be used to determine the number of OH groups in the A-ring of the molecules. Fragment ions due to the loss of H2O and CO were also observed in the MS spectrum as an indication of substituent positioning. The ion at m/z 153 is a retro Diel-Alder fragment, indicating that there are two hydroxyl groups on the A-ring. Metzler et al. (52,53) also investigated the mammalian lignans ENL and END by HPLC-APCI-MS and ESI analysis. The development and validation of analytical methods based on LC-ESI-MS for use in determining blood isoflavones in rats was reported by Holder et al. (62). Analysis of rat blood using LC/ES-MS showed that genistein 7-O-β-glucuronide was the major form and that the 4′-isomer was present in small amounts. The method used serum/plasma deproteination, liquid-liquid extraction followed by solvent evaporation and sample dilution. A modified method that obviates the need for protein precipitation, extraction, and solvent removal was developed by Doerge et al. (63) using online SPE for analysis of diluted serum. Total isoflavone content of rat serum was determined by LC-MS with SIM-positive ion mode, after enzymatic deconjugation. The use of a restricted-access/reverse-phase trap cartridge and automated column switching permitted rapid and robust analytical performance with many injections of plasma onto a reverse-phase LC column. The limit of detection for isoflavones in serum samples based on the MS response was 20 nmol/L. Phytoestrogens in Foods. A microbore HPLC/ESI-MS positive ionization method for the determination of total daidzein and genistein in soy flour and baby food was developed (40). Pneumatically assisted EI was used and the limit of detection was 0.2 mg/kg for daidzein and 0.7 mg/kg for genistein in the flour and food samples. Griffith and Collison (38) used reverse-phase HPLC-MS with an ion trap in the positive ion mode for analysis of isoflavones from soy foods. Samples extracted in acetonitrile/water were diluted to 50% acetonitrile and injected directly for gradient HPLC separation onto a C18 reverse-phase column. The ion trap technique takes the most abundant ion found with full scan MS and performs multistage MSMS. The multistage MS-MS experiments can provide unequivocal identification of isoflavones. The report showed that in the case of the malonyl, acetyl, and glucoside forms of isoflavones, MS-MS gives the aglucone core, and MS3 fragments can act as a recognizable fingerprint. The soybean and its products have been considered goitrogenic in humans and animals. LC-APCI/MS analysis has shown that the aglycones genistein and daidzein are the components that inhibit the thyroid peroxidase–catalyzed reaction (64). A hop-based dietary supplement, marketed for natural breast enhancement, was analyzed by LC-MS (31). Results indicated that only hop-associated phytoestrogens [8-prenylnaringenin (8-PN), 6-prenylnaringenin, 6,8-diprenylnaringenin, xanthohumol and isoxanthohumol] were present in the dietary supplements at sig-

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nificant concentrations. Optimal mass spectrometric conditions were investigated for protonated and deprotonated molecular ions of 8-PN, as appropriate, by continuous flow infusion of standards. Negative ion APCI provided the greatest response for 8-PN and for all other analytes except anhydrosecoisolariciresinol, for which ESI (negative ion) was most favorable. Ishii et al. (65) identified a flavonoid glycoside naringin in human urine by LC/ESI-MS-MS technique. Naringin isolation from human urine includes filtration through an activated Sep-Pak cartridge. LC/ESI-MS analysis of the naringin fraction showed an intense peak at m/z 598 (M+NH4)+, which on MS/MS analysis, provided a base peak at m/z 273 (naringenin, M+H+). Recently, LC-ESI-MS was employed in the study of isoflavonoids of soy. ESI-MS and UV data led to the detection of three minor isoflavones (isomers of 6′′-O-malonyl isoflavone glycosides) by Gu and Gu (66). They claimed that these malonyl isoflavone glycosides were new, due to their low concentrations and unstable nature. Although thermally labile, the malonyl glycosides are not decomposed in the ES ion source because of the cooling effects caused by evaporation of solvent. Hutabarat et al. (67) used different stationary phases and a variety of solvents in varying proportion in the quantitative determination of isoflavones and coumestrol in soybean. They claimed that a phenyl reverse-phase column with acetonitrile/water (33:67, vol/vol) provided the best separation. The identity of the individual analytes was confirmed by LC-MS-MS. Phytoestrogens as Plant Secondary Metabolites. APCI-MS analysis of isoflavone glucoside malonates in Trifolium pratense L. (red clover) extract has been reported using ammonium formate at pH 4.0 as an eluent (36). Our group recently analyzed isoflavonoids with greater sensitivity in soy and the American peanut Apios americana using multiple reaction ion monitoring (MRM) during an HPLC analysis (68). LC-MS/MS analysis using a triple quadrupole mass spectrometer has been used for structural information. Examination of the MS/MS spectra of genistein and daidzein indicated that a product ion m/z 133 was diagnostic for these isoflavones, but not for apigenin, the flavonoid isomer of genistein (21,69). Application of LC with UV and MS to monitor changes in profiles of isoflavonoids glycosides and free isoflavonoids in Lupinus albus L. was reported by Bednarek et al. (70). A further attempt to characterize flavonoids in extracts of fresh herbs by negative APCI-MS was made by Justesen (71). His paper explains that negative APCI-MS can provide aglycone fragments by in-source fragmentation of glycosides, and the fragments are further selected for fragmentation by MS-MS. Liquid chromatography with continuous flow fast atom bombardment (CF FAB or CF-LSIMS) interfaces has also been applied for the analysis of plant secondary metabolites (72–74). In this method, only a very small volume of mobile phase eluted from the column enters the ion source of the mass spectrometer with a maximum of 10 µL/min delivered to the probe tip. During the analysis, glycerol

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used as a matrix is added to the mobile phase or delivered postcolumn to the eluate. Application of this method was described for profiling of the acylated isoflavonoid glycosides present in alfalfa (Medicago sativa) and chick pea (Cicer arientium) (75). Capillary Electrophoresis and Mass Spectrometry (CE-MS) CE is a relatively new separation technique, providing different separation mechanisms from other chromatographic methods such as GC and HPLC. The theory of CE has been discussed in detail in many references (76–78). Basically, separation by CE is a result of differences in electrophoretic mobilities of charged species in an electric field in small-diameter capillaries. The use of capillaries, with 50–100 µm i.d. and 150–360 µm o.d., offers advantages of rapid, high-resolution separation (up to 106 theoretical plates) with sample volumes in the nanoliter range, resulting in excellent mass detection limits (femto- to attomole of samples). Since first described in its modern format by Jorgenson and Luckas in 1981 (79,80), CE has been developed into several modes and applied to analyses of various classes of samples, including macromolecules such as proteins, or small molecules such as drug metabolites (81–83). Of the CE techniques, capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) are the most common methods used for analysis of phytoestrogens. CZE is the basic mode of CE techniques. Charged species are separated from each other in the capillary—all neutral species migrate at the same speed. Because most of the phytoestrogens are weak acids, alkaline buffers are used to ensure that the phenolic moiety is charged for electrophoretic separation. Borate buffer, which forms a charged complex with the cis-diol moiety of the sugar rings, is also useful for analyses of β-glycosides of phytoestrogens. The influence of structure and buffer composition on electrophoretic behavior of flavonoids has been discussed in several studies (84–86). MEKC, a modified CE technique, is performed by adding surfactants, such as sodium dodecyl sulfate (SDS), at levels above their critical micellar concentration in the running buffer. The surfactants form charged micelles and migrate in the CE capillary under the electrical field, similar to all charged species. The analytes, both neutral and ionic species, partition between the micelle and running buffer, which contributes additional selectivity to the separation. Therefore, the micelle is referred to as a pseudo-stationary phase, similar to the stationary phase in LC separation. MEKC has been applied extensively to separate various compounds including neutral and hydrophobic species (87,88). The instrumentation format of CE is similar to that of HPLC; therefore, most detection methods used in HPLC can be adapted to monitor CE separations. CE analyses of phytoestrogens using UV detection (89–91), fluorescence detection (92), and electrochemical detection (93,94) have been reported. Mass spectrometry has been adapted as a detection method for CE separation to combine the features of sensitivity, universal detection and capability of providing structural information from MS with the high separation power from CE. In fact, the relatively low flow

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rates of CE (< 1 µL/min) compared with conventional HPLC (1 mL/min) make it much better suited to the electrospray interface because the effluent can be introduced into MS without splitting. However, caution must be taken to maintain the CE separation efficiency and resolution while maintaining the electrical continuity for CE separation and ESI interfacing. The first CE-MS interface, using silver metal deposition onto the capillary terminus as the point for electrical contact, was reported by Smith and co-workers in 1987 (95). Development of other CE-MS interfaces and their applications have been described in many reviews (96,97). Aramendia et al. (98,99) explored the use of on-line CE-MS for separation and characterization of selected isoflavones. A triaxial electrospray probe was used to interface CE with a single-quadrupole MS operating in the negative-ion mode. The triaxial ESI incorporated a sheath tube, allowing additional solvent (the make-up solution) to be transported to the probe tip and mixed coaxially with the sample flow at the end of the CE capillary before spraying. The make-up solution was used to supplement the CE flow by the extent required for ESI and to make electrical contact between CE buffer and the spray tip. CE separation of isoflavones was performed with the MS-compatible ammonium acetate buffer (at pH 9.0), instead of borate buffer used in other detection systems. Fast separation of genistein, daidzein, biochanin A, and isoliquiritigenin was achieved with baseline resolution; however, pseudobatigenin, formononetin, and biochanin A co-migrated in this system. Although those co-migrated isoflavones could not be readily resolved using CE with UV detection, they were readily resolved by CE-MS. The sensitivity of this system relied on many factors. The optimum analytical signal for this system was found when volatile buffers were used at the lowest possible concentrations (10–25 mmol/L); in fact, higher concentrations produced lower ionization efficiencies for the analytes during electrospray. Under optimum conditions and in selected ion recording mode, a limit of detection of ~100 amol, equivalent to 6–7 nmol/L in solution, for almost all the isoflavones was reported. This was 100 times better than working in the scan mode (~10 fmol; 1 µmol/L) (98). Recent Advances in Instrumentation and Strategy Recent improvements in instrument sensitivity, software control, and data analysis tools provide unique high-throughput capabilities for phytoestrogen analysis when using LC-MS-MS. LC-MS analysis using a triple quadrupole mass spectrometer provides both high sensitivity and high specificity. In particular, LC-MS/MS spectral data can be used as a structural fingerprint. Once the diagnostic ions corresponding to substructures are recognized, rapid identification of metabolites is possible. Precursor ion scan, neutral loss scan, and MRM are some of the techniques in MS/MS experiments that allow rapid detection of the metabolites. The last-mentioned technique greatly simplifies the analysis of isoflavonoids, particularly because it largely removes the necessity for gradient chromatography. The effect of HPLC column diameter (and thus mobile-phase flow rate) on the performance of ESI-MS is the sin-

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gle most important consideration for enhancing sensitivity. ESI is commonly viewed as a liquid-phase ionization technique that requires very low flow rates and, thus, very small diameter HPLC column. The reciprocal relationship (to the second power) between column diameter and MS sensitivity implies that the ESI-MS is behaving as a concentration-sensitive detector, much like a UV detector. In an attempt to enhance the sensitivity for phytoestrogen detection in ESI-MS, we developed an interface that utilizes a flow rate of 200 nL/min, enabling us to detect isoflavones in concentrations as low as 2–3 nmol/L and amounts as small as 2–3 fmol (68). The MS-MS-MRM scan (parent ion/daughter ion combination) is a very sensitive technique that detected genistein (m/z 269/133), daidzein (m/z 253/223), and glycetin (m/z 283/240) in commercial soy sauces (68) (Fig. 7.7). Because of the high selectivity of the MRM technique and the use of isocratic conditions, the throughput of sample analysis can be enhanced substantially. The reproducibility of the MRM technique for isoflavones is in the range of 3–8% for concentrations in a 1 mL sample of ≥40 nmol/L. The limit of detection is in the range of 2–10 nmol/L. Thus MRM detects a given reaction occurring in the mass spectrometer. There is no scanning in this case; as a result, the sensitivity is enhanced significantA.

B.

Fig. 7.7. Liquid chromatography-mass spectrometry (LC-MS)/MS-multiple reaction ion

monitoring (MRM) of isoflavonoids in commercial soy sauces. Unconjugated isoflavones in two soy sauces were recovered by solid-phase extraction (SPE) and analyzed by LCMS/MS-MRM under isocratic conditions (30% acetonitrile in 10 mmol/L ammonium acetate) on a 10 cm × 2.1 mm i.d. C8 reverse-phase column. The isoflavones analyzed were daidzein (m/z 253/223), genistein (m/z 269/133), and glycitein (m/z 283/240). The numbers on the top right corner of each chromatogram represent the full scale value of the ion intensity. Ion chromatograms on the left are from a fermented soy sauce, whereas those on the right are from a chemically hydrolyzed soy sauce.

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ly. Furthermore, MRM is particularly well suited to chromatographic applications, to quantitation, and also to detection of a particular compound in mixtures. An improved method of detection of the isoflavone aglycones, genistein, and daidzein was reported using solid phase microextraction-HPLC-ESIMS by Satterfield et al. (100). Their technique of extraction of the isoflavonoids from urine using solid phase microextraction with a Carbowax-templated resin fiber coating allowed rapid preconcentration of the analytes. Limits of detection of daidzein and genistein were 25.4 and 2.70 pg/mL (0.1 and 0.01 nmol/L), respectively. Although LC-MS has enabled investigators to carry out phytoestrogen analyses in physiologic samples without the need for derivatization, a recent innovation (101) has combined LC-APCI-MS with the power of electron capture, a well-known detection method for GC analysis. In the application by Singh et al. (101), the authors prepared pentafluorobenzyl (PFB) derivatives of several compounds (folates, steroids) and analyzed them by LC-APCI. The corona discharge in APCI leads to the formation of electrons that are readily captured by the highly halogenated PFB derivatives. They obtained two orders of magnitude increase in sensitivity, down to the attomole range. Such a technique may well be applicable to the study of phytoestrogens and provide a technology for their detection in small tissue samples.

Concluding Remarks and Future Prospects During the last decade, MS has evolved as an instrumental method of choice for phytoestrogen analysis. Instruments with several different design features are now available in the market, most often interfaced with LC. Further advances of the analytical methodology will be dependent upon the development of both chromatographic separation technology as well as mass spectral detection capabilities. ESI and MALDI represent formidable ionization tools with a new level of sensitivity, accuracy, and mass range. Tandem MS is being used extensively in analyses of phytochemicals and will soon become as widely used in chromatography as massselective detection is at present. Recent development of TOF analyzers for accurate mass and accurate MS/MS has tremendously expanded the possibility of structure characterization. TOF or QTOF detector may be interfaced with LC-ESI systems to provide µg/g (ppm)-level mass accuracy that can enable the determination of the empirical formula of an unknown compound. Acknowledgements Studies on isoflavones, phytoestrogens, and polyphenols are supported by grants-in-aid from the National Cancer Institute (R01 CA-61668) and the National Center for Complementary and Alternative Medicine-sponsored Purdue-UAB Botanicals Center for Dietary Supplements Research (P50 AT-00477). Operation of the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility was supported in part by a NCI Core Research Support Grant to the UAB Comprehensive Cancer (P30 CA-13148). The mass spectrome-

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ters used in our studies were purchased by funds from NIH/NCRR Shared Instrumentation Grants (S10 RR-06487; S10 RR-11329; S10 RR-13795).

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47. Ma, Y.L., Cuyckens, F., van den Heuvel, H., and Claeys, M. (2001) Mass Spectrometric Methods for the Characterization and Differentiation of Isomeric ODiglycosyl Flavonoids, Phytochem. Anal. 12, 159–165. 48. Sugui, J., Bonham, C., Lo, S.C., Wood, K.V., and Nicholson, R.L. (1998) MALDI-TOF Analysis of Mixtures of 3-Deoxyanthocyanidins and Anthocyanins, Phytochemistry 48, 1063–1066. 49. Wang, J., and Sporns, P. (1999) Analysis of Anthocyanins in Red Wine and Fruit Juice Using MALDI-MS, J. Agric. Food Chem. 47, 2009–2015. 50. Wang, J., and Sporns, P. (2000) MALDI-TOF MS Analysis of Food Flavonol Glycosides, J. Agric. Food Chem. 48, 1657–1662. 51. Wang, J., and Sporns, P. (2000) MALDI-TOF Analysis of Isoflavones in Soy Products, J. Agric. Food Chem. 48, 5887–5892. 52. Jacobs, E., and Metzler, M. (1999) Oxidative Metabolism of the Mammalian Lignans Enterolactone and Enterodiol by Rat, Pig, and Human Liver Microsomes, J. Agric. Food. Chem. 47, 1071–1077. 53. Niemeyer, H.B., Honig, D., Bohmer, A.L., Jacobs, E., Kulling, S.E., and Metzler, M. (2000) Oxidative Metabolites of the Mammalian Lignans Enterodiol and Enterolactone in Rat Bile and Urine, J. Agric. Food Chem. 48, 2910–2919. 54. Kulling, S.E., Hoing, Simat, T.J., and Metzler, M. (2000) Oxidative In Vitro Metabolism of the Soy Phytoestrogens Daidzein and Genistein, J. Agric. Food Chem. 48, 4963–4972. 55. Kulling, S.E., Honig, D.M., and Metzler, M. (2001) Oxidative Metabolism of the Soy Isoflavones Daidzein and Genistein in Humans In Vitro and In Vivo, J. Agric. Food Chem. 49, 3024–3033. 56. Roberts-Kirchhoff, E.S., Crowley, J.R., Hollenberg, P.F., and Kim, H. (1999) Metabolism of Genistein by Rat and Human Cytochrome P450s, Chem. Res. Toxicol. 12, 610–616. 57. Klus, K., and Barz, W. (1995) Formation of Polyhydroxylated Isoflavones from the Soybean Seed Isoflavones Daidzein and Glycitein by Bacteria Isolated from Tempe, Arch. Microbial. 164, 428–434. 58. Vekey, K. (2001) Mass Spectrometry and Mass-Selective Detection in Chromatography, J. Chromatogr. A., 921, 227–236. 59. Mcluckey, S.A., and Goeringer, D.E. (1997) Slow Heating Methods in Tandem Mass Spectrometry, J. Mass Spectrom. 32, 461–474. 60. Cimino, C.O., Shelnutt, S.R., Ronis, M.J., and Badger, T.M. (1999) An LC-MS Method to Determine Concentrations of Isoflavones and Their Sulfate and Glucuronide Conjugates in Urine, Clin. Chim. Acta 287, 69–82. 61. Valentin-Blasini, L., Blount, B.C., Rogers, H.S., and Needham, L.L. (2000) HPLCMS/MS Method for the Measurement of Seven Phytoestrogens in Human Serum and Urine, J. Expo. Anal. Environ. Epidemiol. 10, 799–807. 62. Holder, C.L., Churchwell, M.I., and Doerge, D.R. (1999) Quantification of Soy Isoflavones, Genistein and Daidzein, and Conjugates in Rat Blood Using LC/ES-MS, J. Agric. Food Chem. 47, 3764–3770. 63. Doerge, D.R., Churchwell, M.I., and Delclos, K. B. (2000) On-Line Sample Preparation Using Restricted-Access Media in the Analysis of the Soy Isoflavones, Genistein and Daidzein, in Rat Serum Using Liquid Chromatography Electrospray Mass Spectrometry, Rapid Commun. Mass Spectrom. 14, 673–678.

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100. Satterfield, M., Black, D.M., and Brodbelt, J.S. (2001) Detection of the Isoflavones Aglycones Genistein and Daidzein in Urine Using Solid-Phase Microextraction-HighPerformance Liquid Chromatography-Electrospray Ionization Mass Spectrometry, J. Chromatogr. B. Biomed. Sci. Appl. 759, 33–41. 101. Singh, G., Gutierrez, A., Xu, K., and Blair, IA. (2000) Liquid Chromatography/Electron Capture Atmospheric Pressure Chemical Ionization/Mass Spectrometry: Analysis of Pentafluorobenzyl Derivatives of Biomolecules and Drugs in the Attomole Range, Anal. Chem. 72, 3007–3013.

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Chapter 8

Measurement Methodology for Phytoestrogens in Blood and Urine Mariko Uehara Department of Nutritional Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo, Japan

Introduction Since the 1980s, there has been growing interest in the role of the dietary phytoestrogens (isoflavonoids and lignans) in human health (1–5). The precursors of these diphenolic compounds are found in various legumes, pulses, seeds, cereals, unrefined grain products, fruits, and berries. After the intake of various foods containing phytoestrogens, usually in glycosidic form, these compounds are modified by intestinal bacteria in animals and humans, converting them to biologically active substances (6,7). High phytoestrogen consumption and concentrations in plasma and urine are found in subjects who live in countries with low cancer and coronary heart disease incidence; low values have been found in breast cancer patients or in women at high risk for breast cancer. Isoflavones in biological fluids occur mainly as glucuronide and sulfate conjugates (8); they have been analyzed after hydrolysis and extraction by gas chromatography-mass spectrometry (GCMS) (9–12) or by high-performance liquid chromatography (HPLC) (13–15). To date, >20 isoflavonoids, lignans and their metabolites have been identified in human biological fluids by isotope dilution GC-MS in the selective ion-monitoring mode (ID-GC-MS-SIM) (16–18). This methodology is very sensitive but requires many preparative and column chromatographic purification steps. The instrument is expensive and the operator must have considerable experience. Therefore, the development of more rapid and convenient methods has been sought for the measurement of phytoestrogens in biological fluids. This paper will review measurement methodologies of phytoestrogens in blood and urine, and will focus on the development of time-resolved fluoroimmunoassay (TR-FIA) for the purpose of large population screening in epidemiologic studies. GC-MS as Reference Method for Measurement of Phytoestrogens in Blood and Urine In the beginning, GC was used for the determination of lignans (19). GC-MS is used as the reference method for measurement of phytoestrogens in biological fluids.

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The compounds are introduced into the GC-column as trimethylsilyl (TMS) ethers or N,O-bistrimethylsilyl trifluoroacetamides (BSTAF) (10–12, 20–24). Methods using isotopic dilution GC-MS in the selective ion-monitoring mode (ID-GC-MSSIM) for measurements of phytoestrogens in human urine (10), human plasma (22), and human feces (24) have been developed. Ion exchange chromatography is a good method for the purification of all kinds of phenolic compounds and has two purposes: (i) sample clean up; and (ii) group separation of compounds with different acidity. The molecular ions usually used for quantitative work differs in the phytoestrogens from those of steroids, but if low concentrations are quantified extensive purification particularly for urine samples, is necessary. The mammalian lignans were detected for the first time in biological fluids due to their interference with the GC analysis of steroids. The interference is most extensive in the region in which the α-ketolic estrogens are eluted in GC and has in the past resulted in overestimation of some of these compounds. An important step forward in phytoestrogen analysis in biological fluids was the observation that phytoestrogens, compounds with two phenolic hydroxyls, may be separated from estrogens using a combination of three ion exchanges (QAEAC; QAE-Bor, QAE-CO3–). Urine samples were extracted on Sep-Pak cartridge, and conjugated fractions were isolated by chromatography on the acetate form of DEAE-Sephadex. Deuterated internal standards of the compounds were added to the urine samples before hydrolysis. The hydrolysates were extracted on a SepPak cartridge. Two fractions were obtained by chromatography on the acetate form of QAE-Sephadex. Fraction 1 contained equol, enterolactone, enterodiol, matairesinol, and all estrogens, whereas fraction 2 contained O-desmethylangolensin (O-DMA), daidzein, and genistein. The latter fraction was ready for GC-MS, whereas the former was further purified to eliminate a part of the estrogens by chromatography on a QAE column in the borate form followed by chromatography on the carbonate form of QAE-Sephadex to eliminate the rest of the estrogens from the lignans. After silylation, the samples were analyzed by GCMS-SIM. If the pattern of conjugation of the phytoestrogens is to be determined, more complex steps are required (8). At the beginning, free and sulfate fractions of the phytoestrogens (isoflavonoids and lignans) were separated from the glucuronides using ion-exchange chromatography (25). However, later on, the two fractions were combined after hydrolysis (26). Thereafter the plasma extract was further purified by solid phase extraction and ion-exchange chromatography. Losses during the complete procedure are corrected for using 6,7-3H-estradiol17β-glucuronide during the first step and later by adding deuterated internal standards of most compounds measured. The final determination is carried out by ID-GC-MS-SIM (22). The ID-GC-MS-SIM is a highly specific method for measurement of phytoestrogens in blood and urine. This plasma method has now been further simplified by carrying out solvolysis of the sulfates and subsequent enzyme hydrolysis without extraction between the hydrolytic steps (personal communication).

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HPLC a More Convenient, But Less Sensitive and Specific Method for Measurement of Phytoestrogens in Blood and Urine Traditionally, GC-MS was used to determine phytoestrogens and their metabolites in human biological fluids including urine, plasma, and feces. Recently, HPLC was introduced to measure these compounds in human urine, allowing the measurement of a variety of phytoestrogens, including aglycones and conjugates in one run. Compared with GC-MS, the HPLC method requires fewer steps for sample preparation and analysis as well as less time and less expensive instrumentation. Although HPLC was originally applied in previous studies for the determination of isoflavonoid levels in plasma (27,28), glycitein and O-DMA were not included in these assays, and conditions were not evaluated for human specimens. HPLC with a reversed-phase (C18) column can be used directly without a derivatization step for the analysis of isoflavonoids in aglycone and conjugated forms in samples. Franke et al. (13) developed an HPLC technique to determine isoflavonoid levels in human urine and plasma with diode array detection in the ultraviolet (UV) range or electrochemical detection (ECD). Their system was improved by applying a gradient elution system consisting of methanol, acetonitrile, dichloromethane, and 10% aqueous acetic acid. This led to more efficient separation of analytes, especially isoflavones and their metabolites. This method was also applied to separate and quantitate other flavonoids and phenolic acids. The detection limits for daidzein, genistein, equol, and O-DMA using a 20 µL injection volume were found to be 1.09, 0.53, 3.28, and 1.00 pmol, respectively, in their HPLC system with UV detector (13). Coulometric detection at +500 mV (ECD) lowered the detection limits for daidzein, genistein, and equol, compared with diodearray monitoring (Table 8.1). TABLE 8.1 Comparison of High-Performance Liquid Chromatography (HPLC) Detection Limits Between Ultraviolet and Electrochemical Detectiona UVb

ECDb

Detection limitc

Detection limitc

Decrease of detection limitd

15.8 13.9 29.7 85.2 NDb

3.43 1.91 5.53 0.59 ND

(nmol/L) Daidzeine Genisteine Equolf O-Desmethylangolensinf Coumestrole aSource:

54.3 26.6 164.2 50.2 67.4

Reference 13, with modification. diode-array detection in the ultraviolet range; ECD, electrochemical detection coulometrically at (+)500 mV; ND, not determined. cDetermined by peak height with a 20 µL HPLC injection at a signal-to-noise ratio of 5. dCoulometric values relative to UV values. eUV values obtained by absorbance at 260 nm. fUV values obtained by absorbance at 280 nm. bUV,

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An HPLC method for profiling 13 phytoestrogens and their metabolites used eight electrode coulometric array detection; application to plasma analysis was developed by Nurmi and Adlercreutz (29). The sensitivity of the method was slightly less than that of the reference GC-MS method, but significantly higher than the HPLC methods using diode array UV detection. Detection limits varied from 3.4 pg (9.4 fmol) (secoisolariciresinol) to 40.3 pg (93.3 fmol) (genistin) on column, corresponding to concentrations of 0.34 ng/mL (94 pmol/mL) and 4.03 ng/mL (933 pmol/mL), respectively. The sensitivity of coulometric array detection enables the analyses of low-level plasma phytoestrogens. Sensitive and Specific Methods Combining HPLC with MS for Measurement of Phytoestrogens in Blood and Urine Another sensitive HPLC method combined with MS (HPLC-MS) for measurement of isoflavonoids was developed by Barnes et al. (30,31). An important advance in MS was the introduction of effective interfaces between the HPLC and the mass spectrometer, namely, the electrospray ionization (ESI) and the heated nebulizeratmospheric pressure chemical ionization (HN-APCI) interfaces. Because of the isoflavonoid concentrations in fluids such as bile or urine, preliminary extraction, so essential for GC-MS and many other analytical methods, is not necessary. This immediately overcomes the thorny issue of finding an effective solid-phase extraction procedure. Diluted urine, in particular, can be analyzed by HPLC-ESI-MS, without any purification. Using reversed-phase HPLC-ESI-MS, it is possible to obtain mass spectra of all major isoflavonoid metabolites in a single 20-min analysis. Analysis of isoflavonoid conjugates in serum/plasma samples requires initial extraction, but the conjugates can be measured intact either by capillary reversedphase HPLC-ESI-MS/MS or on regular reversed-phase columns by HPLC-HNAPCI-MS. In both cases, specificity is obtained by causing the precursor isoflavonoid molecular ions to undergo collision-induced dissociation to form specific product ions in a triple quadrupole MS instrument. When it is necessary to measure only the total isoflavonoids and their metabolites in blood, hydrolysis can be performed directly in serum/plasma samples and isoflavonoids recovered by ether or ethyl acetate solvent extraction. The isoflavone aglycones can be analyzed by HPLC-MS under isocratic solvent conditions, thereby drastically shortening analysis time and opening up prospects for automation. Therefore, HPLC-MS is a technique that is broadly applicable to the major issues in phytoestrogen research. Holder et al. (32) also developed an LC/electrospray (ES)-MS method for measurement of isoflavones in rat blood. The sensitivity of LC/ES-MS detection in combination with isotopically labeled internal standards (IS) serves to add additional confidence over previous LC-MS in the accuracy and precision of determination by directly providing quality control and assurance information (e.g., retention times and recoveries of IS) in every sample throughout large samples sets. Furthermore, Velentin-Blasini et al. (33) also developed an analytical method for

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seven phytoestrogens in human urine and serum by HPLC-MS/MS. The phytoestrogens are detected using the HN-APCI interface coupled with tandem MS. This method allows the detection of the primary dietary phytoestrogens in human serum and urine with limits of detection in the low parts per billion range. The combination of tandem MS and chromatographic separation of the analytes helps to ensure the selectivity of the method. Stable isotope-labeled internal standards for all seven analytes improve the precision of the assay, resulting in an inter-assay coefficient of variation (CV) of <10% for most compounds studied. The accuracy and precision of the method were monitored over time using quality control samples containing known amounts of phytoestrogens. The majority of phytoestrogens in human sera and urine are present as their glucuronide and sulfate conjugates. Therefore, the thoroughness of deconjugation for each sample was monitored by the addition of a conjugated internal standard and subsequent detection of the deconjugated compounds. Immunoassay, a Rapid and Specific Method for Measurement of Phytoestrogens in Blood and Urine New tools for phytoestrogen assays were developed recently to measure animal and human exposure, which is likely to vary from one diet culture to another. Most of them were immunoassays. Immunoassays may offer a less costly and less timeconsuming alternative to the GC-MS, LC-MS and HPLC methods. The first step developed was radioimmunoassay (RIA). As second and third steps, enzymelinked immunosorbent assay (ELISA) and time-resolved fluoroimmunoassay (TRFIA; also called DELFIA) methods were developed for the purpose of screening large populations. Radioimmunoassay. A radioimmunoassay (RIA) for the analysis of formononetin in blood plasma and rumen fluid of wethers fed red clover utilized antibodies raised against a formononetin-7-O-carboxy-methyl (CME)-bovine serum albumin (BSA) and a 3H-labeled derivative of formononetin as a tracer (34). The RIA for determination of daidzein and genistein in human serum and urine was established with antibodies against daidzein-4′-O-CME-BSA and genistein-4′-OCME-BSA. Both methods used 125I-labeled tracer (33,36). The sensitivity of the assays was 0.4 and 4.4 pg/tube (samples or standards with radioligand and antibody in 400 µL assay buffer) for daidzein and genistein, respectively. The intraand inter-assay coefficients of variation (CV) for the daidzein assay ranged from 4.1 to 11.5% and from 5.6 to 21.7%, respectively, and for genistein it varied from 3.5 to 9.3% and from 6.7 to 19.7%, respectively. Enzyme-Linked Immunosorbent Assay. Enzyme-linked immunosorbent assays (ELISA) for measuring phytoestrogens were developed by Bennetau-Pelissero et al. (37) based on competition between genistein or daidzein and the respective thy-

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roglobulin (Thyr)-hapten conjugates, used for coating of the microtitre wells, for specific antibodies raised in rabbits with genistein or daidzein BSA-conjugates. The specificity of the antisera was relatively good, but no flavonoids were tested. The precision of the assay (intra- and inter-assay CV) was relatively low (13–19%). In their work, seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. Polyclonal antibodies were generated against the BSA conjugates. The 50% inhibitory concentration (IC50) values of the standard curves ranged between 0.8 and 20 ng/mL (0.3 and 9.2 pmol/ well) (38). The specificty of these antibodies, with the exception of the equol antiserum, was relatively low and the authors suggest that a chromatographic step is necessary. However, no definite method for biological fluids was described and validated. Wilkinson et al. also developed ELISA for daidzein and equol (39). However, no suggestions about the best systems are made for the fromononetin, equol and biochanin A assays. Kohen et al. made a monoclonal antibody to genistein generated through the 6-position of genistein (40). Keeping the reactivity against genistein was 100%, the antibody, named 10D8, cross-reacted <5% with genistin, and formonoetin, and did not react with naringenin, quercetin, 7,4′-dihydroxyflavone, equol, daidzein or daidzin. Urine and plasma levels of genistein measured by this ELISA correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. Time-Resolved Fluoroimmunoassay. Time-resolved fluoroimmunoassays (TRFIA) of plasma phytoestrogens combine the advantages of other nonradioisotopic assays with a 10- to 100-fold increase in sensitivity and wide assay range compared with conventional ELISA and other types of FIA methods. Adlercreutz et al. (41) developed the first measurement method of a phytoestrogen, enterolactone, in plasma by TR-FIA. As a second step, measurements of plasma daidzein (42,43) and genistein (42) were developed. Finally, a method with which to measure three urinary phytoestrogens (genistein, daidzein, and enterolactone) was also developed (44,45). In addition, the TR-FIA method for plasma enterolactone was modified in 2000 (46). TR-FIA is a solid phase fluoroimmunoassay, based on competition between a europium (Eu)-labeled phytoestrogen and the sample phytoestrogens in their interaction with polyclonal antiphytoestrogen antibodies derived from rabbit (Fig. 8.1). 4′-Carboxymethylgenistein, 4′-carboxymethyldaidzein, and 5′-carboxymethoxyenterolactone derivatives were synthesized (41,42). The immunogens were prepared according to Yatsimirskaya et al. (47) with minor modifications. Rabbits were immunized, and the antiserum was collected by a standard procedure. For labeling of phytoestrogens with Eu chelate (48), the same derivatives of the aglycones were used. A flow-diagram outlining the TR-FIA method used for measurement of the three phytoestrogens, genistein, daidzein, and enterolactone, in plasma and urine is provid-

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Fig. 8.1. Time-resolved fluoroimmunoassay (TR-FIA) based on competitive immunoassay

for measurement of phytoestrogens. Ig, immunoglobulin.

ed in Figure 8.2. The analytical procedure is described briefly as follows. After addition of 6,7-3H-estradiol-17β-glucuronide (only in the case of plasma for recovery calculation), a hydrolysis reagent (β-glucuronidase and sulfatase in acetate buffer) was added to the samples, which were incubated overnight at 37°C. The volume of hydrolysis reagent added should be the same as the sample volume in the case of plasma. This is quite an important point because a high absolute amount of the sulfatase enzyme per sample released interfering compounds from the matrix. When the concentration of the sulfatase in the hydrolysis reagent is increased from 2 to 50 U/mL, the measured concentration of enterolactone increases linearly by TR-FIA (Fig. 8.3). However, the results were equal throughout the series when the samples were analyzed with HPLC using coulometric electrode array detection (Fig. 8.3). Therefore, incomplete hydrolysis of the enterolactone conjugates was not the cause of the problem. The explanation for the increasing results with higher amounts of enzyme could not be clarified. Some compounds released by sulfatase from the matrix probably exhanced the fluorescence. However, the nature of the compounds remains unknown. β-Glucuronidase, the other enzyme for the hydrolysis, had no such effect. In addition, a similar phenom-

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Fig. 8.2. Flow-diagram of the time-resolved fluoroimmunoassay (TR-FIA) methods for the analysis of phytoestrogens in plasma and urine.

enon occurred in the analysis of the other phytoestrogens, genistein and daidzein, which makes it unlikely that the difference in the results is due to any other lignans present in serum (46). In analyses of urine no such effect was observed; thus, 3–5 times more hydrolysis reagent is required for the hydrolysis of phytoestrogen conjugates in urine. Diethyl ether was used to extract unconjugated phytoestrogens after hydrolysis in the case of plasma, whereas no extraction was performed in the case of urine samples. Before the assay, microstrips coated with goat anti-rabbit immunoglobulin G (IgG) were prewashed with the plate washer. Standard, hydrolyzed urine sample or an amount corresponding to 20 µL of hydrolyzed and extracted plasma, was pipetted into the microstrips; then 100 µL per well of antiserum and 100 µL Eu-labeled phytoestrogen derivatives in the assay buffer were added to each well. The strips were placed on a shaker at room temperature and incubated for 90 min with slow shaking; then the strips were washed with the plate washer. Enhancement solution (200 µL) was added to each well and the strips were shaken slowly for an additional 5 min. The fluorescence was read using a DELFIA Victor 1420 multilabel counter. In the case of plasma samples, another 20 µL of solution was taken for liquid scintillation counting for determination of recovery. On the basis of the results for plasma samples, the final values were corrected for

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Enterolactone, nmol/L)

Sulfatase (U/mL) Fig. 8.3. The effect of increasing concentrations of sulfatase (EC 3.1.6.1; Cat. No. S9626, Sigma Chemical, St. Louis, MO) during the hydrolysis of a serum sample on the result obtained by time-resolved fluoroimmunoassay (TR-FIA) and by high-performance liquid chromatography (HPLC).

losses during hydrolysis and extraction. The final results were calculated using the following formulae: (i) Final value for plasma = Concentration (read) × dilution factor (nmol/L) × 1/recovery (ii) Final value for urine = Concentration (read) × dilution factor (nmol/L) Sensitivity and Accuracy of TR-FIA. Standard curves based on 10 separate assays are shown in Fig. 8.4 (A–C). The results, expressed as means and SD, showed little variation. The working range of the assay for genistein was from 1.7 to 370 nmol/L, that of daidzein was from 1.0 to 216 nmol/L, and that of enterolactone was from 1.5 to 540 nmol/L. The intra- and interassay CV, at three different concentrations varied from 2.9 to 8.0 in the case of plasma samples, and from 1.9 to 9.7 in the case of urine. Specificity of Phytoestrogen Antisera. Cross-reactivity of selected lignans, isoflavonoids, and flavonoids with genistein, daidzein, and enterolactone antisera are shown in Table 8.2. Genistein and daidzein cross-react highly with biochanin A and formononetin. This is seldom a problem because neither of these compounds

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B/BO (%)

B/BO (%) B/BO (%)

Genistein (nmol/L) (Log Scale)

Enterolactone (nmol/L) (Log Scale)

Daidzein (nmol/L) (Log Scale)

Fig. 8.4. (A) Standard curve for genistein assay based on 10 separate standard curves on different days (±SD). (B) Standard curve for daidzein assay based on 10 separate standard curves on different days (±SD). (C) Standard curve for enterolactone assay based on 10 separate standard curves on different days (±SD).

are common in human foods. In addition, biochanin A in food seems to be quantitatively converted to genistein in the gut, and formononetin is converted to daidzein and other metabolites in the gut. No enterolactone antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. Correlation with the GC-MS Method. Comparing the values obtained by the TR-FIA and GC-MS methods in determination of the genistein level in plasma, a highly significant correlation (r = 0.956) and similar mean values were obtained. The urinary excretion levels of total genistein were considerably higher by TR-FIA than by GC-MS. However, the correlation coefficient was relatively good (r = 0.880) (Fig. 8.5A).. Plasma and urinary daidzein levels showed a strong correlation when measured by GC-MS and TR-FIA (plasma: r = 0.951; urine: r = 0.990), and the mean

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TABLE 8.2 Specificity of Genistein, Daidzein, and Enterolactone Antisera Antisera Compound Daidzein Formononetin Biochanin A Daidzin Dihydrodaidzein Genistein Dihydrogenistein Genistin Equol O-Desmethylangolensin Luteolin Quercetin Enterolactone Enteroradiol Matairesinol Anhydrosecoisolarciresinol Secoisolarciresinol aND,

Genistein 2.5 44.4 500.0 1.0 0.1 100.0 11.3 7.6 0.1 0. 0. 0. ND ND ND ND ND

Daidzein

Enterolactone

(% cross section) 100.0 206.00 3.5 6.0 3.1 1.1 0. 0. 0. 0. 0. 0. ND ND ND ND ND

0 NDa ND 0 ND ND ND ND 0 ND ND ND 100.0000 0.28 0 0 0

not determined.

values by TR-FIA were similar to those obtained by GC-MS (Fig. 8.5B). The correlation between the TR-FIA and GC-MS values in the case of enterolactone levels in plasma was also relatively good (r = 0.866). Although the mean value of enterolactone in urine was ~30% higher by TR-FIA than by the GC-MS method, there was a relatively good correlation (r = 0.870) (Fig. 8.5C). It must be remembered that the GC-MS method gives 10–15% lower values than the correct ones due to losses in the beginning of the assay not corrected for by the deuterated internal standards. A highly significant positive correlation between the results obtained by TRFIA and GC-MS was confirmed. No extraction step is necessary in the case of urine samples because of the high concentration and because the protein and the lipid content are usually negligible, although hydrolysis is necessary for the assay by the TR-FIA method. If no hydrolysis is carried out in analysis of phytoestrogens, the values obtained by TR-FIA and GC-MS show large differences. For daidzein, extraction from urine after hydrolysis did not yield different results; thus, the direct assay after hydrolysis was employed. The results in the case of genistein and enterolactone were even better using hydrolysis only, rather than hydrolysis plus the extraction method. The levels of urinary total individual phytoestrogens were higher by TR-FIA than by GC-MS. It is obvious that the TR-FIA method applied to urine samples measures some other metabolites in addition to the target compounds, especially in the case of genistein. There are numerous isoflavone metabolites in urine, and more are

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Genistein (nmol/L) (GC-MS)

Genistein (nmol/L) (GC-MS)

y = 0.930x + 63.44, n = 43 r = 0.956, P < 0.001

Genistein (nmol/L) (TR-FIA)

Daidzein (nmol/L) (GC-MS)

Daidzein (nmol/L) (GC-MS)

Genistein (nmol/L) (TR-FIA)

y = 1.174x + 35.905, n = 45 r = 0.951, P < 0.001

Enterolactone (µmol/L) (GC-MS)

Enterolactone (µmol/L) (GC-MS)

Enterolactone (µmol/L) (TR-FIA)

y = 0.895x – 4.715, n = 25 r = 0.990, P < 0.001

Daidzein (nmol/L) (TR-FIA)

Daidzein (nmol/L) (TR-FIA)

y = 0.942x + 1.685, n = 40 r = 0.866, P < 0.001

y = 0.466x – 0.696, n = 25 r = 0.880, P < 0.001

y = 0.821x – 0.418, n = 25 r = 0.870, P < 0.001

Enterolactone (µmol/L) (TR-FIA)

Fig. 8.5. (A) Linear correlation between plasma and urinary genistein obtained by time-

resolved fluoroimmunoassay (TR-FIA) and by the reference gas chromatography-mass spectrometry (GC-MS) method. (B) Linear correlation between plasma and urinary daidzein obtained by TR-FIA and by the reference GC-MS method. (C) Linear correlation between plasma and urinary enterolactone obtained by TR-FIA and by the reference GCMS method.

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being identified. Recently, Heinonen et al. (17) identified some new isoflavone metabolites. Their cross-reactivities with the daidzein antibody have not been examined. Several new antibodies for genistein coupling the BSA to other side-chains at other positions have been produced, but these antibodies did not improve the method. It is of interest that in analysis of plasma samples using the same antiserum, no overestimation of genistein is observed. However, for screening purposes, the method applied to urine samples for analysis of genistein also seems satisfactory in view of the significant correlation (r = 0.88) between the TR-FIA and GC-MS results. Furthermore, the isoflavone methods applied to plasma or serum is suitable for kinetic studies because the method gives results almost identical to those obtained by the GCMS reference method. It is also possible to use urinary genistein concentration measurements by TR-FIA for kinetic study if the value is adjusted to correspond to the GC-MS value by means of the following formula: y = 0.465x –0.696 (44). In addition, a direct method for plasma isoflavone measurement omitting hydrolysis and extraction, measures only free aglycones, the 4′-monosulfates, and the 4′-monoglucuronides. This method gives underestimated lower values than the values by the normal method including the hydrolysis and extraction step. However, the short direct method can be used for kinetic studies because the difference in isoflavone concentrations between the control and the treatment group after an isoflavone load could be determined (49). A number of reports using these TR-FIA methods have been published with phytoestrogens in various populations and in disease and some kinetic studies have been carried out (45,49–59).

Summary and Conclusions Several excellent methods based on gas chromatography-mass spectrometry (GCMS), liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC) with selective diode array or coulometric electrode array detector and immunoassays have been developed for the measurement of phytoestrogens, including isoflavonoids, lignans, and their metabolites in various biological fluids and in foods. However, some of these methods have not been properly validated. The specificity and sensitivity of the GC-MS methods makes them especially useful as reference methods but they are expensive and time-consuming. This has led to a search for more convenient procedures. The first solution to this problem was the development of the HPLC methods. The introduction of HPLC to measure these analytes has allowed the measurement in a single run of a variety of phytoestrogens, including both aglycones and phytoestrogen glycosides and other conjugates. Compared with GC-MS, HPLC requires fewer steps for sample purification and analysis, and demands less technical time and less expensive instrumentation. However, particularly for urine samples, the specificity of HPLC method is many-times poorer. Neither method is well suited for screening purposes in large populations. Recently, as one solution to this problem, immunoassays were developed for measurement of blood and urinary phytoestrogens. In particular,

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time-resolved fluoroimmunoassay (TR-FIA) applied to plasma could be used for both epidemiologic and kinetic studies because it gives results almost identical to those obtained by the corresponding GC-MS reference method. They are also sensitive and rapid, and can be particularly automated. The methods have recently been adopted to assays in tissue samples, like brain, breast and prostate tissue (personal communication). Acknowledgments The valuable suggestions of Professor Herman Adlercreutz for this review are gratefully acknowledged. Data cited in this presentation represented team efforts of many individuals including Professor Herman Adlercreutz, Dr. Witold Mazur, Dr. Guojie J. Wang, Ms. Katariina Stumpf, Ms. Tarja Nurmi, Ms. Satu Heinonen, Ms. Adile Samaletdin, Ms. Ritva Takkinen, Ms. Anja Koskela, Ms. Inga Wiik, and Ms. Marjatta Valkama in the Institute for Preventive Medicine, Nutrition, and Cancer, the Folkhälsan Research Center, and Division of Clinical Chemistry, University of Helsinki, and Professor Theodore Fotsis, University of Joannina, Greece.

References 1. Setchell, K.D. Lawson, A.M., Mitchell, F.L., Adlercreutz, H., Kirk, D.N., and Axelson, M. (1980) Lignans in Man and in Animal Species, Nature 287, 740–742. 2. Setchell, K.D. Lawson, A.M., Borriello, S.P., Harkness, R., Gordon, H., Morgan, D.M., Kirk, D.N., Adlercreutz, H., Anderson, L.C., and Axelson, M. (1981) Lignan Formation in Man—Microbial Involvement and Possible Roles in Relation to Cancer, Lancet 2, 4–7. 3. Adlercreutz, H., Fotsis, T., Heikkinen, R., Dwyer, J.T., Woods, M., Goldin, B.R., and Gorbach, S.L. (1982) Excretion of the Lignans Enterolactone and Enterodiol and of Equol in Omnivorous and Vegetarian Postmenopausal Women and in Women with Breast Cancer, Lancet 2, 1295–1299. 4. Adlercreutz, H. (1984) Does Fiber-Rich Food Containing Animal Lignan Precursors Protect against Both Colon and Breast Cancer? An Extension of the “Fiber Hypothesis,” Gastroenterology 86, 761–764. 5. Bannwart, C., Fotsis, T., Heikkinen, R., and Adlercreutz, H. (1984) Identification of the Isoflavonic Phytoestrogen Daidzein in Human Urine, Clin. Chim. Acta 136, 165-172. 6. Price, K.R., and Fenwick, G.R. (1985) Naturally Occurring Oestrogens in Food—A Review, Food Addit. Contam. 2, 73–106. 7. Adlercreutz, H., and Mazur, W. (1997) Phyto-Oestrogens and Western Diseases, Ann. Med. 29, 95–120. 8. Adlercreutz, H., van der Wildt, J., Kinzel, J., Attalla, H., Wähälä, K., Mäkelä, T., Hase, T., and Fotsis, T. (1995) Lignan and Isoflavonoid Conjugates in Human Urine, J. Steroid Biochem. Mol. Biol. 52, 97–103. 9. Adlercreutz, H., Fotsis, T., Bannwart, C., Wähälä, K., Mäkelä, T., Brunow, G., and Hase, T. (1986) Determination of Urinary Lignans and Phytoestrogen Metabolites, Potential Antiestrogens and Anticarcinogens, in Urine of Women on Various Habitual Diets, J. Steroid Biochem. 25, 791–797. 10. Adlercreutz, H., Fotsis, T., Bannwart, C., Wahala, K., Brunow, G., and Hase, T. (1991) Isotope Dilution Gas Chromatographic-Mass Spectrometric Method for the Determination

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41. Adlercreutz, H., Wang, G.J., Lapcík, O., Hampl, R., Wähälä, K., Mäkelä, T., Lusa K, Talme, M., and Mikola, H. (1998) Time-Resolved Fluoroimmunoassay for Plasma Enterolactone, Anal. Biochem. 265, 208–215. 42. Wang, G.J., Lapcík, O., Hampl, R., Uehara, M., Al-Maharik, N., Stumpf, K., Mikola, H., Wähälä, K., and Adlercreutz, H. (2000) Time-Resolved Fluoroimmunoassay of Plasma Daidzein and Genistein, Steroids 65, 339–348. 43. Kohen, F., Lichter, S., Gayer, B., DeBoever, J., and Lu, L.J. (1998) the Measurement of the Isoflavone Daidzein by Time Resolved Fluorescent Immunoassay: A Method for Assessment of Dietary Soya Exposure, J. Steroid Biochem. Mol. Biol. 64, 217–222. 44. Uehara, M, Lapcík, O., Hampl, R., Al-Maharik, N., Makelä, T., Wähälä, K., Mikola, H., and Adlercreutz, H. (2000) Rapid Analysis of Phytoestrogens in Human Urine by TimeResolved Fluoroimmunoassay, J. Steroid Biochem. Mol. Biol. 72, 273–282. 45. Uehara, M., Arai, Y., Watanabe, S., and Adlercreutz, H. (2000) Comparison of Plasma and Urinary Phytoestrogens in Japanese and Finnish Women by Time-Resolved Fluoroimmunoassay, Biofactors 12, 217–225. 46. Stumpf, K., Uehara, M., Nutmi, T., and Adlercreutz, H. (2000) Changes in the TimeResolved Fluoroimmunoassay of Plasma Enterolactone, Anal. Biochem. 284, 153–157. 47. Yatsimirskaya, E.A., Gavrilova, E.M. Egorov, A.M., and Levashov, A. (1993) Preparation of Conjugates of Progesterone with Bovine Serum Albumin in the Reversed Micellar Medium, Steroids 58, 547–550. 48. Mukkala, V-M., Mikola,H., and Hemmilä, I. (1989) the Synthesis and Use of Activated N-benzyl Derivatives of Diethylenetriaminetetraacetic Acid: Alternative Reagents for Labeling of Antibodies with Metal Ions, Anal. Biochem. 176, 319–325. 49. Uehara, M., Ohta, A., Sakai, K., Suzuki, K., Watanabe, S., and Adlercreutz, H.(2001) Dietary Fructooligosaccharides Modify Intestinal Bioavailability of a Single Dose of Genistein and Daidzein and Affect Their Urinary Excretion and Kinetics in Blood of Rats, J. Nutr. 131, 787–795. 50. Vanharanta, M., Voutilainen, S., Lakka, T.A., van der Lee, M., Adlercreutz, H., and Salonen, J.T. (1999) Risk of Acute Coronary Events According to Serum Concentrations of Enterolactone: A Prospective Population-Based Case-Control Study, Lancet 354, 2112–2115. 51. Mazur, W.M., Uehara, M., Wahala, K., and Adlercreutz, H. (2000) Phyto-Oestrogen Content of Berries, and Plasma Concentrations and Urinary Excretion of Enterolactone after a single Strawberry-Meal in Human Subjects, Br. J. Nutr. 83, 381–387. 52. Stumpf, K., Pietinen. P., Puska, P., and Adlercreutz H. (2000) Changes in Serum Enterolactone, Genistein, and Daidzein in a Dietary Intervention Study in Finland, Cancer Epidemiol Biomark. Prev. 9, 1369–1372. 53. Arai, Y., Uehara, M., Sato, Y., Kimira, M., Eboshida, A., Adlercreutz, H., and Watanabe, S. (2000) Comparison of Isoflavones Among Dietary Intake, Plasma Concentration and Urinary Excretion for Accurate Estimation of Phytoestrogen Intake, J. Epidemiol. 10, 127–135. 54. Juntunen, K.S., Mazur, W.M., Liukkonen, K.H., Uehara, M., Poutanen, K.S., Adlercreutz, H.C., and Mykkanen, H.M. (2000) Consumption of Wholemeal Rye Bread Increases Serum Concentrations and Urinary Excretion of Enterolactone Compared with Consumption of White Wheat Bread in Healthy Finnish Men and Women, Br. J. Nutr. 84, 839–846. 55. Kilkkinen, A., Stumpf, K., Pietinen, P., Valsta, L.M., Tapanainen, H., and Adlercreutz, H. (2001) Determinants of Serum Enterolactone Concentration, Am. J. Clin. Nutr. 73, 1094– 1100.

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56. Pietinen, P., Stumpf, K., Mannisto, S., Kataja, V., Uusitupa, M., and Adlercreutz, H. (2001) Serum Enterolactone and Risk of Breast Cancer: A Case-Control Study in Eastern Finland, Cancer Epidemiol. Biomark. Prev. 10, 339–344. 57. den Tonkelaar, I., Keinan-Boker, L., Veer, P.V., Arts, C.J., Adlercreutz, H., Thijssen, J.H., and Peeters, P.H. (2001) Urinary Phytoestrogens and Postmenopausal Breast Cancer Risk, Cancer Epidemiol. Biomark. Prev. 10, 223–228. 58. Verkasalo, P.K., Appleby, P.N., Allen, N.E., Davey, G., Adlercreutz, H., and Key, T.J. (2001) Soya Intake and Plasma Concentrations of Daidzein and Genistein: Validity of Dietary Assessment Among Eighty British Women (Oxford Arm of the European Prospective Investigation into Cancer and Nutrition), Br. J. Nutr. 86, 415–421. 59. Yamamoto, S., Sobue, T., Sasaki, S., Kobayashi, M., Arai, Y., Uehara, M., Adlercreutz, H., Watanabe, S., Takahashi, T., Iitoi, Y., Iwase, Y., Akabane, M., and Tsugane, S. (2001) Validity and Reproducibility of a Self-Administered Food-Frequency Questionnaire to Assess Isoflavone Intake in a Japanese Population in Comparison with Dietary Records and Blood and Urine Isoflavones, J. Nutr. 131, 2741–2747.

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Chapter 9

Metabolism and Disposition of Genistein, the Principal Soy Isoflavone Daniel R. Doergea, Richard H. Lueckeb, and John F. Younga aNational

Center for Toxicological Research, Jefferson, AR

bDepartment

of Chemical Engineering, University of Missouri-Columbia, Columbia, MO

Introduction The possible beneficial health effects of soy, which contains significant amounts of the estrogenic isoflavones, genistein and daidzein, are a basis for its popularity as a food constituent and nutritional supplement. Investigations of genistein, the principal and most estrogenic soy isoflavone, in laboratory animals and epidemiologic studies of soy consumption in humans have held out the promise of amelioration of blood lipid profiles associated with cardiovascular disease (1), chemoprevention of breast (2) and prostate cancer (3), and relief of menopausal symptoms (4), including prevention/reversal of osteoporosis (5); however, clinical studies often do not confirm these promises (6). Moreover, the estrogenic mechanisms proposed for such beneficial effects also make possible adverse effects of isoflavones, particularly during fetal and neonatal development (7) or in estrogendependent cancers (8). Additionally, soy has a long-standing association with antithyroid effects in experimental animals (reviewed in Ref. 9), although clinical evidence for such effects in humans is limited (10). Numerous research communications have described various aspects of genistein metabolism and disposition as well as pharmacokinetics in animal models and humans. Such investigations are essential to extending the hypotheses on genistein’s mechanism of action gained through in vitro testing to effects on specific organ systems in animals and to potential soy-based therapies for human diseases. Another underexplored area is the relationship between tissue-specific metabolism of genistein and either terminating or eliciting biological responses. This chapter seeks to describe systematically the body of information related to the metabolism and pharmacokinetics of genistein, its disposition into target tissues of rodents, and the development of a physiologically based pharmacokinetic model that may facilitate extrapolation of this information for use in guiding the design of human clinical trials.

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Extrapolation to Humans from Animal Models for Isoflavone Effects The evaluation of possible human risk-benefit relationships initially requires the use of experimental animal models. The dietary doses selected for use in these animal models are best when they produce circulating isoflavone concentrations similar to those measured in human populations. A recent National Center for Toxicological Research/National Toxicology Program (NCTR/NTP) study of lifetime exposure to dietary genistein exemplifies this connection (11). In that study, rats consuming 5 µg/g and soy-free control diets had very low steady-state serum levels of total genistein (0.01–0.06 µmol/L), similar to those in humans consuming a typical Western diet containing little or no soy; rats consuming a 100 µg/g genistein diet produced serum levels (0.6–0.9 µmol/L) similar to those measured in adults consuming typical Asian diets (12) or soy isoflavone dietary supplements (13); and rats consuming a 500 µg/g genistein diet had serum total genistein levels (6.0–7.9 µmol/L) similar to those measured in infants consuming soy formulas (14). Important components of this study were determination of metabolism, pharmacokinetics, and tissue disposition of genistein in the biologically active aglycone and presumably inactive conjugated forms (11,13,15). Genistein Metabolism and Disposition in Rodents The metabolic biotransformation reactions of genistein are summarized in Figure 9.1. Isoflavones are typically administered either as various glucoside conjugates in soy products (13,16) or as purified aglycones (11,17). Genistein aglycone is liberated in the intestine by the action of microbial β-glucosidases and absorbed into the enterocytes where extensive glucuronidation occurs (18) by the action of microsomal UDP-glucuronosyl transferases (UGT). Therefore, consumption of either genistein or its conjugated forms results in genistein-glucuronides in blood as the predominant circulating form observed. Structural determinations based on liquid chromatography/mass spectrometry (LC/MS) and 1H nuclear magnetic resonance (NMR) showed that both 7- and 4′-glucuronides of genistein were present in rat and human blood (13). Circulating concentrations of genistein aglycone, measured after oral administration, are quite low in adult Sprague-Dawley rats (~2% of total genistein, Ref. 15) and in humans (~1%, Ref. 17). Sulfate conjugates formed through the action of sulfotransferases (SULT) have been observed in vitro (19) in rat and human serum, but the concentrations are very low (13,20). Coldham et al. (20) used radiolabeled genistein and LC-electrospray (ES)/MS to demonstrate the gender-specific formation of additional metabolites resulting from microbial reduction (dihydrogenistein) and ring fission (6′-hydroxy-O-desmethylangolensin, 4hydroxy-phenyl-2-propionic acid) reactions (see Fig. 9.1). Identification of an additional genistein ring-fission product, 4-ethylphenol, was reported (21), and extensive oxidative biotransformation catalyzed by rat cytochromes P450 (Cyt P450) to form multiple catechol-type genistein metabolites has also been reported (22). Reports of biological activities for isoflavone metabolites are few, but include the

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Fig. 9.1. Metabolic biotransformation reactions for genistein.

activation of estrogen receptors (ER) α and β by dihydrogenistein and equol, a reductive metabolite of daidzein (23). Genistein Pharmacokinetics and Tissue Distribution in Rodents Several studies have examined genistein pharmacokinetics in rodents. After an oral dose to CD2F1 mice, plasma genistein aglycone was eliminated with a half-time of 4.8 h, and systemic availability of 21% was observed (24).

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King et al. (25) showed that after a single oral gavage dose of genistein, plasma elimination had a half-time of 8.8 h for total genistein in male Wistar rats, and recovery of total genistein was ~20% in urine and 20% in feces. King et al. (25) also examined the effect on serum pharmacokinetics by administering to these rats an equivalent dose of genistein as glucoside conjugates present in a soy extract. This study showed that although genistein aglycone administration produced serum levels that were initially somewhat higher than those from the glucoside dosing form, the extent of absorption was similar. Uehara et al. (26) performed comprehensive immunochemical analysis of total genistein and total daidzein in portal, central, and peripheral plasma after administration by oral gavage to male Sprague-Dawley rats. Portal vein serum concentrations and areas under the concentration-time curve (AUC) for both isoflavones were approximately double those in peripheral (tail) or central (jugular vein) serum, thus reflecting efficient hepatic uptake at times shortly after dosing. This study also showed that rats consuming a diet rich in complex carbohydrates (fructooligosaccharides) showed altered absorption pharmacokinetics of isoflavones relative to those receiving the control diet. Coldham and Sauer (27) administered a single oral dose of 14C-labeled genistein to male and female Wistar rats, and elimination of total genistein-derived radioactivity from the blood had half-times of 8.5 and 12.4 h in female and male Wistar rats, respectively. Recovery of ~67% of total genistein-derived radioactivity was observed in urine and 33% in feces (27). This study also provided a comprehensive evaluation in many organs of the tissue distribution and elimination of total genistein-derived radioactivity. In addition, several genistein metabolites, including glucuronide and sulfate conjugates, ring fission, and reduction products, were identified at a single time point in selected tissues using LC-ES/MS. Significant gender differences in pharmacokinetics were apparent, including longer elimination half-time, higher maximal plasma concentration (Cmax), higher AUC0-∞, and smaller volume of distribution (Vd) for male rats. Metabolic differences were also apparent, with more extensive sulfation apparent in male liver leading to more rapid elimination from this tissue, and extensive formation of 4-hydroxyphenyl-2propionic acid in prostate, but not uterus. A lifetime dietary exposure study (i.e., in utero, lactational, and dietary exposures) was conducted in male and female Sprague-Dawley rats using a soy-free basal diet and three genistein fortification levels (5, 100, 500 µg/g; see Ref. 11). At postnatal day 140, the rats were denied access to food, serum was obtained sequentially from the tail, and after 12 h, the rats were killed and target endocrine-responsive tissues were removed for analysis. The serum and tissue concentrations of genistein, in both aglycone and conjugated forms, were determined using LCES/MS. Table 9.1 shows the serum levels of total genistein measured immediately after food deprivation for the four dietary groups. As stated above, these “steady state” levels are similar to those measured in several human populations. Pharmacokinetic analysis of serum total genistein showed a significant difference

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TABLE 9.1 Serum Concentrations of Total Genistein in Adult Male and Female Ratsa Dose group Sex

5 µg/g

Control

100 µg/g

500 µg/g

µmol/L Male Female

<0.010 <0.010

0.060 ± 0.006 0.10 ± 0.008

0.59 ± 0.03 0.94 ± 0.21

6.00 ± 0.65 7.94 ± 2.47

aValues

are mean ± SEM determined from individual rats (n = 6) immediately after food deprivation. Aglycone content was in the range of 1–5%. Analysis of the data by two-way ANOVA indicated significant effects of sex and dose as well as a dose × sex interaction (see Ref. 11).

in the elimination half-times and AUC (means ± SEM) between male [2.97 ± 0.14 h and 22.3 ± 1.2 µmol/(L⋅h), respectively] and female rats [4.26 ± 0.29 h and 45.6 ± 3.1 µmol/(L⋅h), respectively]. Tables 9.2 and 9.3 show the tissue levels for both total genistein and aglycone genistein in these male and female rats. Significant TABLE 9.2 Determination of Genistein in Selected Tissues from Male Ratsa Dose group Tissue

Control

5 µg/g

100 µg/g

500 µg/g

0.83 ± 0.16d 0.20 ± 0.04d (24%) 1.09 ± 0.23d 0.49 ± 0.18d (45%) 0.63 ± 0.12d 0.07 ± 0.01d (11%) 0.41 ± 0.08d 0.11 ± 0.03 (25%) 0.67 ± 0.14d 0.23 ± 0.08d (34%) 0.04d 0.04d (100%)

pmol/mg Mammary totalc Mammary aglycone

0.020 ± 0.004b ND

0.020 ± 0.002 ND

Liver totalc Liver aglyconec

<0.02 <0.02

<0.02 <0.02

0.33 ± 0.05d 0.16 ± 0.04d (50%) 0.80 ± 0.23d 0.40 ± 0.13d (50%) 0.42 ± 0.08d 0.040 ± 0.006 (10%) 0.22 ± 0.03d 0.060 ± 0.01 (28%) 0.32 ± 0.10d 0.02

Prostate totalc Prostate aglyconec

0.020 ± 0.003 0.020 ± 0.006

0.030 ± 0.003 ND

Testes totalc Testes aglyconec

0.030 ± 0.01 0.020 ± 0.006

0.040 ± 0.004 ND

Thyroid totalc Thyroid aglycone

0.090 ± 0.01 0.040 ± 0.01

0.10 ± 0.11 0.060 ± 0.07

Brain totalc Brain aglyconec

<0.02 <0.02

<0.02 <0.02

<0.02 <0.02

aValues

are means ± SEM for LC/MS determinations of aglycone and total genistein (pmol genistein/mg tissue, n = 6) in tissues from the four genistein dose groups. The fraction of aglycone as a percentage of total genistein is shown parenthetically for relevant tissues and dose groups (see Ref. 11). ND, not done. bControl total genistein value used for all statistical analyses. cSignificant treatment effect as determined by ANOVA (P < 0.05). dSignificantly different from control as determined by Dunnett’s test (P < 0.05).

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TABLE 9.3 Determination of Genistein in Selected Tissues from Female Ratsa Dose group Tissue

Control

5 µg/g

100 µg/g

500 µg/g

pmol/mg Mammary totalc Mammary aglycone

0.02 ± 0.04b ND

0.030 ± 0.004 ND

Ovary totalc Ovary aglyconec

0.010 ± 0.002 ND

0.059 ± 0.026d ND

Uterus totalc Uterus aglyconec

0.010 ± 0.001 ND

0.037 ± 0.006d ND

Thyroid totalc Thyroid aglyconec

0.047 ± 0.009 0.040 ± 0.014

0.061 ± 0.012 0.043 ± 0.020

Liver totalc Liver aglyconec

0.02 0.01

Brain totalc Brain aglyconec

<0.02 <0.02

0.12 ± 0.01d 0.06 ± 0.01d (50%) ND ND

0.29 ± 0.06d 0.12 ± 0.02d (41%) 0.42 ± 0.05d 0.40 ± 0.04d (95%) 0.78 ± 0.11d 0.64 ± 0.07d (82%) 0.277 ± 0.052d 0.076 ± 0.008 (27%) 1.68 ± 0.39d 1.07 ± 0.21d (64%) ND ND

2.39 ± 0.34d 1.18 ± 0.22d (49%) 1.07 ± 0.11d 0.85 ± 0.09d (80%) 1.42 ± 0.27d 1.43 ± 0.33d (100%) 1.15 ± 0.23d 0.212 ± 0.04c (18%) 7.33 ± 1.62d 5.66 ± 1.31d (77%) 0.06d 0.03 (50%)

aValues

are means ± SEM for LC/MS determinations of aglycone and total genistein (pmol genistein/mg tissue, n = 6) in tissues from the four genistein dose groups. The fraction of aglycone as a percentage of total genistein is shown parenthetically for relevant tissues and dose groups (see Ref. 11). ND, not done. bControl total genistein value used for all statistical analyses. cSignificant treatment effect as determined by ANOVA (P < 0.05). dSignificantly different from control as determined by Dunnett’s test (P < 0.05).

dose-related increases in genistein content were observed in brain, liver, mammary, ovary, prostate, testes, thyroid, and uterus. In all tissues examined, the fraction of total genistein present as aglycone (10–100%) exceeded that in serum (1–5%). Notable were the mammary gland, uterus, ovary, prostate, brain, and the female liver where at least half was present as aglycone. It was remarkable that the maximum tissue levels reached as high as 7 pmol/mg (note: concentration units expressed as pmol/mg are approximately equivalent to micromolar concentrations because the aqueous content of tissues is <100% and varies among the organs). These tissue aglycone levels should be compared with the affinities for competitive binding of genistein to ERα (0.8 µmol/L) and ERβ (0.012 µmol/L; Ref. 28). These results suggested that despite the preponderance of conjugated forms of genistein present in the blood, the small amounts of nonpolar aglycones are sufficient to partition into many lipophilic tissues and accumulate to levels consistent with significant occupancy and activation of ER α and β. The limited accumulation of genistein in brain tissue was striking by comparison with all other tissues examined,

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and may reflect poor penetration of isoflavones into the central nervous system (CNS) of adult rats. In a related study, genistein was administered by oral gavage or through fortified diets to pregnant Sprague-Dawley rats to examine the placental transfer (29). Although the levels of total genistein present in the serum of pups and fetuses were considerably lower (20-fold) than that in the dams’ serum, the level of aglycone genistein was much closer (fivefold) because of a much higher aglycone fraction in offspring (27–53%) relative to dam (2–18%). This higher aglycone fraction in offspring serum probably reflected the diminished capacity to effect conjugation reactions relative to adults. Despite the lower total genistein in offspring serum, the level of genistein in brain (0.2 pmol/mg), essentially exclusively as aglycone (90%), were three- to fivefold higher than that found in adult rats (see Tables 9.2 and 9.3). These findings, and the increased sensitivity of developing brain to toxicological effects of chemicals, are consistent with the transplacental effects of genistein on mammary gland differentiation and promotion of mammary carcinogenesis (30) and effects on the developing CNS (31) in rats treated in utero. Furthermore, the high concentrations of isoflavones measured in cord plasma and amniotic fluid from Japanese infants demonstrated efficient transfer from the maternal circulation in humans (32). Genistein Metabolism and Disposition in Humans The pattern of genistein conjugation in human serum (i.e., predominantly glucuronides with small amounts of sulfate and aglycone) was found to be similar to that in rats (15). An investigation of the enzymology for isoflavone conjugation reactions by human UGT and SULT showed highly efficient glucuronidation activity for genistein, particularly in colon microsomes (13). This efficiency probably resulted from a colon-specific UGT isoform, 1A10, that has high specificity for genistein. The reductive metabolism of genistein to dihydrogenistein by human colonic bacteria under anaerobic conditions (33) was also similar to that observed in rats (20). A recent study by Kulling et al. (34) demonstrated that human liver microsomal metabolism of genistein produced a number of mono- and dihydroxylated catechol-type derivatives and that these metabolites were present in human urine after consumption of soy products (see Fig. 9.1 for structures). The metabolites were similar to those previously reported by this group from incubation of genistein with liver microsomes from male Wistar rats (21). Similar hydroxylation reactions were also observed for daidzein and glycitein. No evidence was found to establish a role for catechol-O-methyltransferase–catalyzed metabolism of genistein and its metabolites in vivo. These metabolites were formed in significant amounts, but at this time, no biological activities of the metabolites have been identified. The many remarkable similarities between genistein metabolism in humans and rats suggests the validity of this interspecies comparison for future assessments of biological activity based on these metabolites.

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Several pharmacokinetic investigations of isoflavone absorption and elimination after human consumption of soy foods have been reported (35,36). These studies showed plasma elimination half-times for genistein of 6–8 h, corresponding to tmax values of ~8 h, and excretion in urine of ~20% of ingested genistein. The most comprehensive determination of isoflavone pharmacokinetics was conducted in women using a single dose of either genistein or an equimolar amount of its 7-glucoside conjugate, genistin (17). Similar studies were also conducted with daidzein and daidzin. Plasma content of total genistein and aglycone genistein was measured using GC/MS, and the fraction present as aglycone was in the range of 1.1–1.7%. The pharmacokinetic parameters determined for genistein and genistin were as follows: the elimination half-times were 6.8 ± 0.8 and 7.0 ± 0.8 h; the Cmax values were 1.3 ± 0.3 and 1.2 0. ± 5 µmol/L; the tmax values were 9.3 ± 1.3 and 9.3 ± 1.3 h; the AUC 0-∞ values were 4.5 ± 1.4 and 5.0 ± 1.0 (µg⋅h)/mL; and the apparent volumes of distribution (Vd/f) values were 161 ± 44 and 112 ± 35 L, respectively. Several conclusions can be made from these data: (i) dosing women with either aglycone or conjugated genistein yielded virtually identical pharmacokinetics; (ii) the elimination half-times were similar to those previously reported for humans consuming genistein in soy foods (35,36) and considerably slower than those from rats (11); (iii) the large values observed for apparent volume of distribution (Vd/f) were consistent with extensive distribution into tissues; and (iv) the very small proportion of aglycone genistein present in plasma was similar to that observed in rats. Enterohepatic Cycling of Genistein Extensive evidence has been published to support a significant role for enterohepatic recycling of genistein in rodents and humans. Yasuda et al. (37) isolated and identified several glucuronide and sulfate conjugates of genistein in the bile of rats receiving an oral dose of genistein and determined that ~16% of the administered dose was excreted in bile within 36 h. Sfakianos et al. (18) examined the intestinal absorption, biliary excretion, and metabolism of genistein by fitting ratswith in-dwelling biliary cannulas and using everted intestinal sac preparations. Infusion of 14C-labeled genistein into the duodenum resulted in efficient absorption and excretion into the bile as the 7-glucuronide conjugate. The presence of primarily genistein glucuronide in portal blood after duodenal infusion of genistein was consistent with extensive conjugation in the intestine as opposed to the liver. Infusion of genistein glucuronide into the duodenum also led to its appearance in bile, an observation best explained by an efficient recirculation process. This study also predicted that such an efficient process for enterohepatic recirculation would have important effects on the pharmacokinetics of genistein. Supko and Malspeis (24) administered genistein intravenously to mice and observed an initial rapid elimination of aglycone genistein from plasma (half-time,

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3–7 min) followed by a secondary phase during which genistein concentration increased and subsequently declined (half-time, 40 min). This kinetic behavior was attributed to enterohepatic cycling and the cumulative amount of genistein reabsorbed was ~6% of the administered dose. Several other pharmacokinetic studies have reported responses in rodents and humans for kinetic behavior that are best explained by efficient enterohepatic recirculation of genistein (17,26,27,36). These responses are typically observed as fluctuations in the plots of blood concentrations vs. time and are important because enterohepatic recirculation can extend the elimination process and increase AUC. Physiologically Based Pharmacokinetic (PBPK) Model of Genistein in Rats The ultimate goal of performing rodent metabolism and disposition studies is to extrapolate such information to humans. An important technique for achieving this goal is the physiologically based pharmacokinetic model (PBPK). In the first stage of model development, the Wistar rat data produced by Coldham and Sauer (27) were selected because this data set represents the most extensive compilation of plasma and tissue pharmacokinetics for genistein. In an attempt to model the enterohepatic recirculation and pharmacokinetics of genistein, the genistein levels in plasma and liver reported by Coldham and Sauer (27) were simulated using a multipurpose PBPK model of Luecke et al. (38). Male plasma total genistein data were obtained from the second figure of Coldham and Sauer (27) by scanning and importing the image into UN-SCAN-IT (Silk Scientific, Orem, UT) where the x,y coordinates of the data points were estimated. The concentration-time data were then used as input values for the PBPK program. The third figure from this same paper (27) allowed estimates of the aglycone and sum of conjugates for plasma and liver at the time of killing. Figure 9.2 depicts the PBPK simulation results. The input parameters to the PBPK program were iteratively adjusted to achieve optimal fit of data points from total plasma genistein and the four individual values at 420 min (i.e., plasma aglycone and conjugates, liver aglycone/conjugates) to the simulated curves. It is interesting to observe that the liver tissue simulation curve parallels the effect of enterohepatic recirculation apparent in the total plasma data. Taking this model one step further to evaluate genistein in additional tissues, the male Sprague-Dawley rat data from the genistein feeding study of Chang et al. (11) were used. The PBPK values for recirculation and metabolism from the simulation in Figure 9.2 were held intact for this second simulation. Chang et al. (11) reported only four plasma levels of total genistein from the time when food was removed until killing 12 h later; at the time of killing, aglycone and conjugate measurements were made for mammary, ovary, prostate, testes, thyroid, liver, and brain tissues (see Tables 9.2 and 9.3). An average value of 1.1% genistein aglycone, which was determined previously in male Sprague-Dawley rat serum (14), was used in the simulation. Figure 9.3 illustrates the simulation of these data. A multidosing effect was used as input to depict this continuous feeding study and is

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Fig. 9.2. Physiologically based pharmacokinetic (PBPK) simulation of Coldham and

Sauer (27) plasma and liver levels of genistein aglycone and conjugates. Serial data points for total plasma genistein and single determinations at 420 min of conjugated and aglycone genistein in liver and plasma from male rats were used to produce optimized simulated curves using the multipurpose PBPK model of Leucke et al. (38).

shown for only the last 3 d. Only the curves for liver and plasma conjugates are shown because the simulated conjugate levels in all tissues were similar in magnitude, although the aglycone curves from several tissues were clearly different. This simulation also illustrates the lower uptake of the aglycone into male brain tissue.

Time (d)

Fig. 9.3. Physiologically based pharmacokinetic (PBPK) simulation of male rat serum and tissue levels of conjugated and aglycone forms of genistein. The PBPK model from Figure 9.2 was applied to the serial data for serum and terminal determinations of tissue genistein aglycone and conjugates from Chang et al. (11) to produce simulated curves for uptake from repeated oral dosing and elimination.

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The results from this PBPK modeling provide additional information beyond the simple half-time or AUC calculations. Not only are the measured tissue data from Chang et al. (11) simulated, but values both before and after the time of killing can be estimated. From examination of Figure 9.3, the maximal steady-state tissue levels achieved through repeated dosing are predicted to be much higher (40- to 80-fold) than the values shown in Table 9.2, which were determined after 12 h of elimination. Alterations of dose amount and route of administration can easily be made, as was done between Figures 9.2 and 9.3. Extrapolation to other species, including humans, can be accomplished through proper use of scaling and will be the subject of future investigations.

Conclusions The studies reported here present a reasonable understanding of genistein metabolism and distribution in animal models. Genistein has similar bioavailability when ingested as either aglycone or as the glucoside conjugates present in soy. The extensive conjugation of genistein that occurs in the intestine and liver leads to enterohepatic recirculation, which significantly prolongs the time of elimination and helps maintain serum steady-state levels. Despite the extensive conjugation of circulating genistein, the small remaining fraction of aglycone (1–10%) is sufficient to drive accumulation in lipophilic tissues from repeated dosing to levels consistent with significant occupancy and activation of ER α and β. Such estrogenic activity is presumably responsible for many of genistein’s biological activities (see other chapters in this volume). In addition, clear evidence exists for significant biotransformation of genistein from analyses of in vitro metabolism, of urinary and fecal excretions, and tissue levels. The challenges ahead are to determine the biological activities, if any, associated with these metabolites of genistein and to produce valid extrapolations to humans of the important biological effects observed from genistein in rodent models. References 1. Anderson, J.J.B., Anthony, M.S., Cline, J.M., Washburn, S.A., and Garner, S.C. (1999) Health Potential of Soy Isoflavones for Postmenopausal Women, Public Health Nutr. 2, 489–504. 2. Lamartiniere, C.A., Zhao, Y.X., and Fritz, W.A. (2000) Genistein: Mammary Cancer Chemoprevention, In Vivo Mechanisms of Action, Potential for Toxicity, and Bioavailability in Rats, J. Women’s Cancer 2, 11–19. 3. Pollard, M., and Wolter, W. (2000) Prevention of Spontaneous Prostate-Related Cancer in Lobund-Wistar Rats by a Soy Protein Isolate/Isoflavone Diet, Prostate 45, 101–105. 4. Duncan, A.M., Underhill, K.E., Xu, X., Lavalleur, J., Phipps, W.R., and Kurzer, M.S. (1999) Soy Isoflavones Exert Modest Hormonal Effects in Postmenopausal Women, J. Clin. Endocrinol. Metab. 84, 3479–3484. 5. Arjmandi, B.H., Birnbaum, R., Goyal, N.V., Getlinger, M.J., Juma, S., Alekel, L., Hasler, C.M., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Bone-Sparing Effect of Soy Protein in Ovarian Hormone-Deficient Rats Is Related to Its Isoflavone Content, Am. J. Clin. Nutr. 68, 1364S–1368S.

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6. Eden, J.A. (2001) Managing the Menopause: Phytoestrogens or Hormone Replacement Therapy? Ann. Med. 33, 4–6. 7. Newbold, R.R., Banks, E.P., Bullock, B., and Jefferson, W.N. (2001) Uterine Adenocarcinoma in Mice Treated Neonatally with Genistein, Cancer Res. 61, 4325–4328. 8. Ju, Y.H., Alred, C.D., Alred, K.F., Karko, K.L., Doerge, D.R., and Helferich, W.G. (2001) Physiological Concentrations of Dietary Genistein Dose-Dependently Stimulate Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Athymic Nude Mice, J. Nutr. 131, 2957–2962. 9. Doerge, D.R., and Chang, H.C. (2002) Inactivation of Thyroid Peroxidase by Soy Isoflavones, In Vitro and In Vivo, J. Chromatogr. B., in press.(AQ2) 10. Ishizuki, Y., Hirooka, Y., Murata, Y., and Togashi, K. (1991) The Effects on the Thyroid Gland of Soybeans Administered Experimentally in Healthy Subjects, Nippon Naibunpi Gakkai Zasshi 67, 622–629. 11. Chang, H.C., Churchwell, M.I., Delclos, K.B., Newbold, R.R., and Doerge, D.R. (2000) Mass Spectrometric Determination of Genistein Tissue Distribution in Sprague-Dawley Rats from Dietary Exposure, J. Nutr. 130, 1963–1970. 12. Adlercreutz, H., Fotsis, T., Watanabe, S., Lampe, J., Wähälä, K., Mäkelä, T., and Hase, T. (1994) Determination of Lignans and Isoflavonoids in Plasma by Isotope Dilution GC/MS, Cancer Detect. Prev. 18, 259–271. 13. Doerge, D.R., Chang, H.C., Holder, C.L., and Churchwell, M.I. (2000) Enzymatic Conjugation of the Soy Isoflavones, Genistein and Daidzein, and Analysis in Human Blood Using Liquid Chromatography and Mass Spectrometry, Drug Metab. Disp. 28, 298–307. 14. Setchell, K.D.R., Zimmer-Nechimias, L., Cai, J., and Heubi, J.E. (1997) Exposure of Infants to Phyto-Estrogens from Soy-Based Formula, Lancet 350, 23–27. 15. Holder, C.L., Churchwell, M.I., and Doerge, D.R. (1999) Quantification of Genistein and Metabolites in Rat Blood Using LC-ES/MS, J. Agric. Food Chem. 47, 3764–3770. 16. Barnes, S., Coward, L., Kirk, M., and Sfakianos, J. (1996) HPLC-Mass Spectrometry Analysis of Isoflavones, Proc. Soc. Exp. Biol. Med. 217, 254–262. 17. Setchell, K.D.R., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131, 1362S–1375S. 18. Sfakianos, J., Coward, L., Kirk, M., and Barnes, S. (1997) Intestinal Uptake and Biliary Excretion of the Isoflavone Genistein in Rats, J. Nutr. 127, 1260–1268. 19. Peterson, T.G., Coward, L., Kirk, M., Falany, C.N., and Barnes, S. (1996) The Role of Metabolism in Mammary Epithelial Cell Growth Inhibition by the Isolflavones Genistein and Biochanin A, Carcinogenesis 17, 1861–1869. 20. Coldham, N.G., Howells, L.C., Santi, A., Montesissa, C., Langlais, C., King, L.J., Macpherson, D.D., and Sauer, M.J. (1999) Biotransformation of Genistein in the Rat: Elucidation of Metabolite Structure by Product Ion Mass Fragmentology, J. Steroid Biochem. Mol. Biol. 70, 169–184. 21. Batterham, T.J., Hart, N.K., and Lamberton, J.A. (1965) Metabolism of Oestrogenic Isoflavones in Sheep, Nature 206, 509. 22. Kulling, S.E., Honig, D.M., Simat, T.J., and Metzler, M. (2000) Oxidative In Vitro Metabolism of the Soy Phytoestrogens Daidzein and Genistein, J. Agric. Food Chem. 48, 4963–4972. 23. Morito, K., Hirose, T., Kinjo, J., Hirakawa, T., Okawa, M., Nohara, T., Ogawa, S., Inoue, S., Muramatsu, M., and Masamune, Y. (2001) Interaction of Phytoestrogens with Estrogen Receptors α and β, Biol. Pharm. Bull. 24, 351–356. 24. Supko, J.G., and Malspeis, L. (1995) Plasma Pharmacokinetics of Genistein in Mice, Int. J. Oncol. 7, 847–854. 25. King, R.A., Broadbent, J.L., and Head, R.J. (1996) Absorption and Excretion of the Soy Isoflavone Genistein in Rats, J. Nutr. 126, 176–182.

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26. Uehara, M., Ohta, A., Sakai, K., Suzuki, K., Watanabe, S., and Adlercreutz, H. (2001) Dietary Fructooligosaccharides Modify Intestinal Bioavailability of a Single Dose of Genistein and Daidzein and Affect Their Urinary Excretion and Kinetics in Blood of Rats, J. Nutr. 131, 787–795. 27. Coldham, N.G., and Sauer, M.J. (2000) Pharmacokinetics of [14C]Genistein in the Rat: Gender-Related Differences, Potential Mechanisms of Biological Action, and Implications for Human Health, Toxicol. Appl. Pharmacol. 164, 206–215. 28. Nikov, G.N., Hopkins, N.E., Boue, S., and Alworth, W.L. (2001) Interactions of Dietary Estrogens with Human Estrogen Receptors and the Effect on Estrogen Receptor Response Element Complex Formation, Environ. Health Perspect. 108, 867–872. 29. Doerge, D.R., Churchwell, M.I., Chang, H.C., Newbold, R.R., and Delclos, K.B. (2001) Placental Transfer of the Soy Isoflavone, Genistein, Following Oral Administration to Sprague-Dawley Rats, Reprod. Toxicol. 15, 105–110. 30. Hilakivi-Clarke, L., Cho, E., and Clarke, R. (1998) Maternal Genistein Exposure Mimics the Effects of Estrogen on Mammary Gland Development in Female Mouse Offspring, Oncol. Rep. 5, 609–616. 31. Faber, K.A., and Hughes, C.L., Jr. (1993) Dose-Response Characteristics of Neonatal Exposure to Genistein on Pituitary Responsiveness to Gonadotropin Releasing Hormone and Volume of the Sexually Dimorphic Nucleus of the Preoptic Area (SDNPOA) in Postpubertal Castrated Female Rats, Reprod. Toxicol. 7, 35–39. 32. Adlercreutz, H., Yamada, T., Wähälä, K., and Watanabe, S. (1999) Maternal and Neonatal Phytoestrogens in Japanese Women During Birth, Am. J. Obstet. Gynecol. 180, 737–743. 33. Hur, H.G., Lay, J.O., Jr., Beger, R.D., and Rafii, F. (2000) Isolation of Human Intestinal Bacteria Metabolizing the Natural Isoflavone Glycosides Daidzin and Genistin, Arch. Microbiol. 174, 422–428. 34. Kulling, S.E., Honig, D.M., and Metzler, M. (2001) Oxidative Metabolism of the Soy Isoflavones Daidzein and Genistein in Humans In Vitro and In Vivo, J. Agric. Food Chem. 49, 3024–3033. 35. King, R.A., and Bursill, D.B. (1998) Plasma and Urinary Kinetics of the Isoflavones Daidzein and Genistein After a Single Soy Meal in Humans, Am. J. Clin. Nutr. 67, 867–872. 36. Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., Mazur, W., Wähälä, K., and Adlercreutz, H. (1998) Pharmacokinetics of Soybean Isoflavones in Plasma, Urine, and Feces of Men After Ingestion of 60 g Baked Soybean Powder (Kinako), J. Nutr. 128, 1710–1715. 37. Yasuda, T., Mizonuma, S., Kano, Y., Saito, K., and Ohsawa, K. (1996) Urinary and Biliary Metabolites of Genistein in Rats, Biol. Pharm. Bull. 19, 413–417. 38. Luecke, R.H., Wosilait, W.D., Pearce, B.A., and Young, J.F. (1994) A Physiologically Based Pharmacokinetic Computer Model for Human Pregnancy, Teratology 49, 90–103.

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Chapter 10

Digestion, Absorption and Metabolism of Isoflavones Roger A. King CSIRO Health Sciences and Nutrition, Adelaide BC, South Australia

Introduction Understanding the role of the isoflavones in human health depends very heavily upon an understanding of their digestion, absorption, and metabolism. Over the last few years, substantial advances have been made in these areas, although many questions remain. Similarly, there has been a significant increase in our understanding of the bioavailability and the pharmacokinetics of the isoflavones, which represents an integration of these processes. There are three main potential sources of isoflavones for humans, i.e., soy extracts, soy foods (and to a lesser extent other legumes), which contain genistein, daidzein, and glycitein; dietary supplements extracted from red clover, which contain mainly formononetin and biochanin A; and the pharmaceutical, ipriflavone. In this chapter, I will review current knowledge regarding the digestion, absorption, metabolism, bioavailability, and pharmacokinetics of the isoflavones, with particular reference to the soy isoflavones, although the clover isoflavones will also be discussed briefly. The reader is directed to the extensive literature for studies involving ipriflavone [see for example (1–7)]. Intestinal Transport of Isoflavones The intestinal transport of the broad flavonoid group, including the isoflavones, has been an area of strong research interest over the last few years, and our understanding of the mechanisms and sites of absorption has increased substantially. The flavonols, and in particular quercetin and its glycosides (found in high concentrations in onions), have been most thoroughly investigated (8–19), although a substantial number of studies have involved the isoflavones (20–31). One of the major questions has been whether, as originally proposed (32), the glycosidic forms of the isoflavones and other flavonoids must first be deconjugated to the respective aglycones by bacterial enzymes in the large bowel before absorption can occur, or whether they can be transported across the intestinal wall either intact or after deglycosylation by mammalian enzymes. Consideration of these possibilities was largely prompted by indirect evidence from studies of quercetin absorption in ileostomists, which suggested that quercetin glucosides were more extensively absorbed than the aglycone (8). Subsequent studies have

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generally supported this hypothesis. However, there appears to be a complex interplay between glycoside structure and rate of transport (10,12,33), and in contrast to the flavonols, the evidence suggests that the isoflavone aglycones are more rapidly transported than their glucosides (22,27–31). Other studies have focused on identification of the mechanisms and enzymic processes that may be involved in intestinal transport of flavonoids, and evidence has been obtained for the involvement of the sodium-dependent glucose co-transporter (SGLT1) (12,13,15,16), the multidrug resistance protein-2 (MRP-2) (34,35), lactase phorizin hydrolase (LPH) (18), and other intestinal β-glucosidases (19,23); however, relatively few of these studies have specifically involved the isoflavones. None of the studies have involved the clover isoflavones. In a series of studies, Andlauer et al. (29–31) examined the transport of isoflavones and their glucosides using isolated rat intestine preparations. In the first of these studies (30), the entire small intestine and its associated mesenteric vascular bed were suspended in an organ bath and the lumen perfused with medium containing genistein for 60 min. In a single pass through the intestine, ~40% of the applied dose of isoflavone was transported to the vascular side, with ~23% of this as aglycone and ~77% as glucuronides, whereas ~54% of the dose passed unabsorbed in the luminal effluent, of which ~75% was aglycone and ~25% glucuronide conjugates. Thus, there was an efficient extraction of genistein from the luminal perfusate, followed by glucuronidation in the intestinal wall and transport to the vascular side, with return of a significant fraction of the glucuronides back into the lumen. In the second study (31), the same protocol was used for genistin, the glucoside of genistein. In this case, at an equivalent dose, only ~13% of the luminal genistin was recovered on the vascular side, of which ~72% was glucuronides, ~15% was genistein and ~13% genistin. Thus, in this model, genistein was much more efficiently extracted and transported from the lumen than genistin. Importantly, only ~2% of the glucoside dose passed to the vascular side intact. In a third series of experiments (29), the perfusate consisted of a pepsin-pancreatin digest of tofu to resemble more closely the situation after ingestion of soy foods. The isoflavones in the digest were predominantly the glucosides, daidzin, genistin, malonyl daidzin, and malonyl genistin, with small proportions of daidzein and genistein. Although direct comparisons with the other studies are difficult because of the complex isoflavone mix provided by the tofu digest, the results suggested that the efficiency of extraction from this more complex milieu was significantly reduced compared with the pure compounds, with <2% of the total isoflavones recovered on the vascular side. Consistent with these results with intestinal preparations, genistein and daidzein were transported much more efficiently than their respective glucosides across monolayers of human intestinal Caco-2 cells cultured on semipermeable membranes (27,28). Studies with rats and humans have also provided evidence that the isoflavone aglycones are absorbed more rapidly, and in addition, that they can be absorbed from more proximal regions of the gastrointestinal (GI) tract compared with gluco-

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sides. Piskula et al. (22) investigated the absorption of genistein, daidzein, genistin, and daidzin in anesthetized rats over a period of 30 min after their oral administration. Genistein and daidzein (measured after enzymic hydrolysis of glucuronides, sulfates, and glucosides) were detectable in tail vein blood 3 min after administration of the aglycones; however, when the respective glucosides were administered, no genistein or daidzein was detectable until after 10 min. In a second series of experiments using the same dosing procedure, the pylorus was ligated so that absorption could occur only from the stomach. These showed that the aglycones were readily absorbed within 3 min, but no absorption of glucosides was detectable even after 30 min (22). Consistent with these studies, peak plasma genistein concentration occurred earlier and was substantially higher after an oral dose of genistein compared with a soy glucoside extract in rats (20); in humans, peak concentrations of daidzein and genistein were reached earlier after consumption of aglycones compared with glucosides (24,26). In another human study, isoflavones were detectable in plasma as early as 30 min after consumption of a soy meal (which contained a small proportion of aglycones) and reached ~40% of peak concentrations by 1 h (25), when the undigested portion of the meal would be expected to be only just commencing to enter the large bowel (36). Taken together, these studies suggest that the isoflavone aglycones are more efficiently transported across the wall of the GI tract than their respective glucosides, and that they can be absorbed from more proximal regions. A large fraction of ingested glucosides are absorbed in the distal small intestine and in the colon after hydrolysis to the aglycones. Whether flavonoid glycosides are transported across the wall of the GI tract and into the circulation intact has been an area of some debate, with some studies supporting significant transport in this form (14,37–40) and others suggesting little or no such transport (12,31,33,41–44). Only one of these studies involved isoflavone glucosides. This suggests that intact transport is quantitatively unimportant (31), and the evidence from the most recent of these studies (44) suggests that the use of analytical methods with insufficient specificity resulting in the misidentification of glucuronides as glycosides may explain much of this disagreement (44). Mechanisms of Intestinal Transport of Isoflavones. As indicated earlier, studies investigating possible transport mechanisms of the broad flavonoid group have focused on SGLT1, MRP-2 (a membrane-associated export pump of glucuronate and sulfate conjugates), and intestinal β-glucosidases including LPH. Studies with rat jejunal everted sacs (15,45) and intestinal segments (13) provided evidence for the interaction of a number of quercetin glucosides with SGLT1, although transport by this mechanism was not directly demonstrated and isoflavones were not studied. More direct evidence that quercetin glucosides could be transported by SGLT1 was obtained from cell culture studies (16), but again the isoflavones were not studied. In these studies, apical uptake of quercetin 4′-glucoside (Q4′G), one of

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the main onion flavonols, into Caco-2 cells was inhibited by phloridzin, an inhibitor of SGLT1, and uptake into Chinese hamster ovary cells occurred only after transfection of parent cells with SGLT1. Apical uptake of Q4′G into Caco-2 cells increased (16), and basolateral-apical efflux of Q4′G and genistin (27) was inhibited in the presence of the drug MK-571, an inhibitor of MRP-2. Because MRP-2 has been localized to the apical surface of Caco-2 cells (35), this suggests that there was an MRP-2–mediated efflux from the apical surface. In the last few years, there have been a number of studies on the role of mammalian intestinal enzymes in the transport of flavone and isoflavone glycosides. In 1998, Day et al. reported the ability of a cytosolic preparation from human small intestine to hydrolyze genistin, daidzin, and a number of flavonol glycosides (23). Evidence was obtained for the involvement of a cytosolic β-glucosidase, and the authors suggested that hydrolysis by this enzyme may be an important early step in glucoside uptake. They also noted the existence of lactase phlorizin hydrolase as the second of three β-glucosidases present in human tissue. Subsequently, the same group demonstrated the ability of highly purified LPH from lamb small intestine to hydrolyze daidzin and genistin as well as quercetin-3-glucoside and quercetin-4′glucoside (18). Using separate inhibitors of the lactase site and of the phlorizin hydrolase site of LPH, they obtained evidence that the former was primarily responsible for hydrolysis of glycosides. They noted that LPH is located in the brush border membrane in close proximity to SGLT1 and is therefore strategically positioned to be accessible by glycosides present in the gut lumen. They hypothesized that (iso)flavonoid glycosides would be hydrolyzed by LPH, with the cleaved sugar being transported into the enterocyte by the adjacently positioned SGLT1 and the aglycone entering the cell by passive diffusion (18). The postulated cellular metabolic and transport processes involving SGLT1, LPH, and MRP-2, are summarized in Figure 10.1. Bacterial Metabolism of Isoflavones As with any food, the process of digestion of soy begins in the mouth, continues along the remainder of the GI tract and is terminated with the excretion of feces. The release of isoflavones from the food matrix is facilitated by the various secretions that are present, as well as by the action of the bacterial populations present throughout the GI tract. It is the GI bacteria, however, that play a pivotal role in the further metabolism of the released isoflavones. The human stomach is essentially sterile, but bacterial numbers increase by several log units from the upper small intestine to the large bowel (46,47). Certainly, significant numbers are present in the distal small intestine, and the large bowel contains a vast array of bacterial families (47). The role of bacteria in the metabolism of isoflavones is essentially threefold. First, they can hydrolyze the βglucosidic bonds of glucosidic forms (e.g., genistin) to produce the aglycones (e.g., genistein). This may possibly occur to some extent as proximally as the distal

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Fig. 10.1. Two postulated transport and metabolic processes for isoflavones in enterocytes. According to the sequence summarized in the upper half of the cell, the isoflavone glucoside (e.g., genistin) is hydrolyzed to glucose and the aglycone genistein by the lactase site of lactase phlorizin hydrolase (LPH) in the brush border of the intestinal cells. Glucose is released in the unstirred boundary layer and is transported into the cell by the adjacent sodium-dependent glucose-transporter 1 (SGLT1), whereas the released aglycone is initially retained in the unstirred layer and then diffuses into the cell. According to the scheme at the lower half of the cell, glucoside is transported into the cell by SGLT1. Once in the cell it can be hydrolyzed by β-glucosidase or can return to the lumen via the multidrug-resistance protein-2 (MRP-2). Intracellular genistein can be glucuronidated by uridine diphosphate glucuronosyl transferase (UDPGT) or leave the cell via the basolateral surface and enter the circulation; a proportion may exit the cell via the apical surface and return to the intestinal lumen. Genistein can enter via passive diffusion, with small losses back into the lumen. Adapted from and based on data of Day et al. (18,23), Walgren et al. (16,35), and Andlauer (30,31).

small intestine, based on the presence of significant numbers of bacteria with βglucosidase activity in this region (46) and on pharmacokinetic data (25,26). Certainly, extensive hydrolysis occurs in the large intestine (48,49). Second, the bacteria in the large bowel can, in addition to hydrolysis of the β-glucosidic bond, structurally modify and degrade the resulting aglycones to a series of metabolites (49–53). Third, a number of bacterial species in the bowel express glucuronidase

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(46,49,54) and/or sulfatase activities (54), and can therefore hydrolyze the isoflavone glucuronides and sulfates that are excreted in the bile to regenerate the aglycones, which then become available for reabsorption or further bacterial metabolism (the enterohepatic cycle). The first metabolite of the soy isoflavones to be identified in human urine was equol, a major metabolite of daidzein (55–57); since that time, a number of others have been identified (Table 10.1). The role of bacteria in the generation of many of these metabolites is supported by the following evidence. First, equol was detected in the urine of normal rats but not germ-free rats when fed soy (55,60). Second, young infants who were fed soy formula to 4 mo of age did not excrete equol, which has been attributed to the absence of appropriate bacterial populations (61). Third, upon incubation with human fecal extracts, isoflavones were converted to a number of products (49–53,62), including those detected in urine. Fourth, individual fecal bacterial species have been shown to metabolize isoflavones to a range of products that have been identified in vivo (49,53,63,64). Most studies of the bacterial metabolism of the clover isoflavones have been in animals (65–67), especially sheep, because of the devastating effects they can have on their reproductive performance. However, one study has examined the metabolism of formononetin and biochanin A to daidzein and genistein by human TABLE 10.1 Isoflavone Metabolites Identified in Human Urine After Consumption of Soy, or After Incubation of Soy or Isoflavones with Fecal Extracts or Fecal Bacteria Reference Metabolite Dihydrodaidzein Tetrahydrodaidzein 2-Dehydro-O-desmethylangolensin O-Desmethylangolensin Equol cis-4-OH-Equol 3′-Methoxy-7,4′-dihydroxyisoflavone 3′,7-Dihydroxyisoflavane Benzopyran-4,7-diol-,3-(4-hydroxyphenyl) 4-Hydroxybenzoic acid 2,4-Dihydroxybenzoic acid 2′,4′-Dihydroxyacetophenone 4-Hydroxyphenylacetic acid Dihydrogenistein Tetrahydrogenistein 6′-Hydroxy-O-desmethylangolensin 4-Ethylphenol 6,7,4′-Trihydroxyisoflavone aDetected

Urine 59,69,97 59,69 59,69 58,59,69,97,98 57–59,69,97 97

Fecal extract or fecal bacteria 51

51,52 53

99a

59,69,97 59 56,69,97 100

51 49 49 49 49 51

63

in urine without a soy challenge; origin from other foods (e.g., milk) cannot be excluded.

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fecal bacteria (63). In another human study, the daily consumption of a red clover extract tablet containing mostly biochanin A and formononetin, with little daidzein or genistein, resulted in the urinary excretion of mainly daidzein, genistein, and Odesmethylangolensin (O-DMA, a bacterial metabolite of daidzein), with very small amounts of formononetin and biochanin A (68). These patterns of excretion are consistent with the bacterial conversion of the clover isoflavones as noted above, with subsequent conversion of genistein and daidzein to other metabolites. The pathways of bacterial metabolism of the clover and soy isoflavones are illustrated in Figure 10.2. Metabolism of Isoflavones by Specific Fecal Bacterial Species. A number of recent studies have focused on the metabolism of isoflavones by individual bacterial species, and these promise a much better understanding of the role of microbial populations in isoflavone metabolism. In the first of these studies, Bacteriodes J37, Eubacterium A-44 and Fusobacterium K-60 hydrolyzed the β-glucosidic bond of daidzin to produce daidzein when incubated under anaerobic conditions (49). The same study reported the further degradation of the aglycone to a series of phenolic acids by Bacteroides JY-6. However, identification of the products was based only upon thin-layer chromatographic (TLC) separation and selective colorimetric sprays. In a later study, however (53), the same group used more specific mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods to demonstrate the conversion of daidzin to daidzein and calycosin (3′-methoxy-7,4′-dihydroxyisoflavone) by fecal extracts and by Bifidobacterium spp. designated as K111. In two recent extensive studies, Hur et al. (63,64) tested the ability of a number of individual fecal bacterial species to metabolize glycitein, biochanin A, and formononetin (63), and daidzin and genistin (64). In the first study (63) Eubacterium limosum (ATCC 8486) demethylated biochanin A, formononetin, and glycitein to genistein, daidzein, and 6,7,4′-trihydroxyisoflavone, respectively, but was not capable of further metabolism of these products, even after 26 d incubation. In the second study (64), a large number of bacterial strains isolated from human feces were screened. Two strains, a gram-positive organism (ATCC BAA96), designated HGH6, and an Escherichia coli (ATCC BAA-97), designated HGH 21 were shown to be capable of hydrolyzing daidzin and genistin to daidzein and genistein, respectively. However, only HGH6 was capable of reduction of the double bond between atoms 2 and 3 of the central C-ring of daidzein and genistein to produce dihydrodaidzein and dihydrogenistein as end products, respectively. On the other hand, the total colonic flora from which HGH6 and HGH21 were isolated were capable of production of equol from daidzein, and 6′-OH-O-DMA from genistein, indicating that other species are responsible for these transformations. Interestingly, HGH6 was incapable of metabolizing apigenin and chrysin, the flavone analogs of genistein and daidzein, respectively, in which the B-ring is joined to the C-ring at the 2-position, rather than the 3-position as in the isoflavones, indicating considerable bacterial specificity. Although a large number

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2-Dehydro-O-desmethylangolensin

O-desmethylangolensin

6′-Hydroxy-O-desmethylangolensin

Fig. 10.2. Bacterial metabolism of formononetin and daidzein (top), and biochanin A and genistein (bottom)

according to Joannou et al. (59) and Batterham et al. (67). Dashed arrows indicate pathways for which there is limited evidence. Note that the isoflavones are present as glucosides in clover and most soy foods and are first hydrolyzed to the respective aglycones by bacteria expressing β-glucosidase (not shown). Copyright 2002 by AOCS Press. All rights reserved.

of bacterial species are capable of hydrolyzing isoflavone glucosides to produce the aglycones, there appear to be a smaller number that are capable of metabolism of the isoflavonoid ring structures. Thus, it appears that the complete metabolism of the isoflavones requires the sequential action of a number of different bacterial species. Interindividual Variation in Metabolite Excretion. The reasons for the large interindividual variation in isoflavone metabolite excretion and the possible implications for bioavailability and biological effects of the isoflavones have intrigued researchers since the phenomenon was first reported (62). Unpublished data cited by Kelly et al. (69) suggest that excretion by an individual is relatively stable over time, and it is highly likely that differences in bacterial populations in the gut underlie much of this variation. Certainly, colonization of germ-free rats with human fecal flora obtained from “equol excreters” led to excretion of equol by these rats after soy consumption, whereas those colonized with flora from “nonexcreters” did not excrete equol (60), suggesting that the ability to excrete equol is an inherent characteristic of the bacterial population of the gut and not due to some other influence. In that first study referred to above (62), four subjects increased urinary equol excretion by 50- to 1000-fold after soy consumption, whereas the other two were essentially incapable of production of equol. Subsequent studies in both men and women (58,69–75) have repeatedly confirmed these large interindividual variations in metabolite excretion, with ranges of 500- to 1000-fold in equol excretion when consuming a soy diet, irrespective of gender. Substantial variations were also seen for ODMA, daidzein, and genistein excretion (58,70–73). Although the criteria for selection as a “good excreter” or a “poor excreter” have varied from study to study, it is generally accepted that approximately one-third of the individuals in a population have the capacity to excrete significant amounts of equol after soy consumption (73). On the basis of the possible effects on colonic microflora, a number of studies have examined the influence of diet composition on the excretion of equol and other bacterial metabolites. In a study of nine men and ten women in Japan, Adlercreutz et al. (70) reported a large interindividual variation in urinary equol excretion, from 0–10.95 µmol/d, and a positive correlation with estimated intake of total fat (P < 0.01), meat (P < 0.05), and fat:fiber ratio (P < 0.05). There were no significant correlations of daidzein or O-DMA excretion with these intakes. In a larger study involving 30 men and 30 women consuming a Western-type diet (73), women who were classified as equol excreters (2.2–20.3 µmol/d) consumed significantly more dietary fiber (P < 0.05), plant protein (P < 0.05), carbohydrate (P < 0.05), and percentage of energy as carbohydrate (P < 0.05) than nonexcreters (0.02–0.23 µmol/d). In contrast to the study of Adlercreutz et al. (70), the estimated fat:fiber ratio was significantly lower (P < 0.01) and fat and animal protein intakes tended to be lower in the female excreters (73). Excretion of daidzein, genistein, and O-DMA did not differ between excreters and nonexcreters for either

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sex. There were no significant differences in nutrient intake between excreters and nonexcreters in men. The authors suggested that sex-dependent interactions between diet and gut physiology, and possible differences in the accuracy of diet record keeping between the sexes could explain the observed differences between men and women. Consistent with this report, a study involving 19 women and 5 men found that “good excreters” consumed less fat as a percentage of energy (26 ± 2.3% compared with 35 ± 1.6%, P < 0.01) and more carbohydrate as a percentage of energy (55 ± 2.9% compared with 47 ± 1.7%, P < 0.05) (58). The positive correlation between carbohydrate intake and equol excretion seen in these last-mentioned two studies is consistent with in vitro studies that have shown greatly increased production of equol from daidzein by fecal flora incubated in high carbohydrate media compared with low carbohydrate media (76). It has been suggested (73) that the differences between the studies involving Western subjects (58,73) and that involving Japanese subjects (70) may relate to differences in basal diet, such as a higher general contribution of carbohydrate to energy intake in the Japanese study. In summary, although some studies have suggested that dietary composition may influence metabolite excretion, this does not appear to be a major factor. The bacterial origin of equol indicates that differences in colonic flora must largely underlie these substantial interindividual differences, and, as discussed earlier, progress is now being made in the identification of individual fecal bacterial species that are capable of specific transformations of isoflavones. However, with the massive number of bacterial species present in the human colon, the challenge now is to characterize these particular metabolic capacities in a greater range of enteric bacteria, and to correlate the presence of these populations with the capacity to produce equol and other metabolites. Metabolism of Isoflavones by Mammalian Tissues The major role of mammalian tissues in the metabolism of the isoflavones and their metabolites is the formation of glucuronide, sulfate, and sulfoglucuronide conjugates. Glucuronides predominate, with lesser proportions of sulfates and sulfoglucuronides (77,78). Generally, <10% of the total isoflavones remain unconjugated in plasma and urine (78), although this fraction may be higher in some tissues (79). Glucuronidation is catalyzed by UDP-glucuronosyltransferases, and current evidence suggests that a major site of glucuronidation is the wall of the GI tract (11,14,21,30,78,80), with other organs, notably the liver, having the capacity to conjugate that which escapes conjugation in enterocytes (78,81). Recombinant microsomal UDP-glucuronosyltrasferases and microsomes from human colon, liver, and kidney have been shown to produce both 7- and 4′-glucuronides of genistein and daidzein, with the ratio of the two isomers depending on the tissue (78). The contribution of the intestine to sulfation of isoflavones is less clear. Sulfotransferases are present in GI tissue and other organs, such as the liver, kidney, and lung (82), and evidence has been obtained for sulfation of quercetin and

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rutin by rat small intestine (11,39) and of chrysin by Caco-2 cells (13); however, studies with isoflavones have not detected sulfates after perfusion of isolated intestinal tissue (21,29–31), suggesting that their sulfation occurs in other organs. Although limited demethylation of biochanin A to genistein by rat liver slices (83), and of formononetin to daidzein by sheep and bovine liver microsomes (84) were reported some time ago, it has generally been thought that metabolism of soy isoflavones by mammalian tissues was essentially limited to glucuronidation and sulfation as discussed above. However, recent studies have now provided evidence of hydroxylation and methylation of isoflavones by rat (85–87) and human tissues (85,87). Incubation of daidzein with human liver microsomes resulted in the formation of three monohydroxylated and three dihydroxylated metabolites, whereas incubation with genistein led to the formation of up to six hydroxylated metabolites (85,87). Importantly, a number of these metabolites were identified (after deconjugation with glucuronidase/sulfatase) in the urine of humans who had consumed soy, and it was calculated that these may account for up to ~10% of the excreted isoflavones and metabolites (87). A number of these metabolites contained orthohydroxy (catechol) groups and were substrates for catechol-O-methyltransferase in vitro; however, only very small amounts of the methylated metabolites were detected in the post-soy urine samples (87). The highly hydroxylated nature of these newly identified metabolites suggests that they may possess more powerful antioxidant properties than the parent isoflavones, making it important to investigate their potential biological significance. The bacterial and mammalian metabolism of the isoflavones is summarized in Figure 10.3. Bioavailability and Pharmacokinetics of Isoflavones in Humans Most studies on the bioavailability of the isoflavones in humans have used urinary excretion data, with only a few studies employing theoretically more rigorous pharmacokinetic methods. All studies that have measured urinary excretion of isoflavones after consumption of soy have shown substantially greater recoveries of ingested daidzein than genistein (25,50,88–92) (Table 10.2). However, the fraction of the respective doses excreted has varied widely among studies, ranging from 16 to 62% for daidzein and from 9 to 37% for genistein. In the single study in which it was measured (89), 47% of ingested glycitein was excreted. The reasons for these large interstudy differences remain to be explained (25), but do not appear to relate to differences in the soy food consumed, dose of isoflavones, or urine collection period. Generally <5% of ingested isoflavones were excreted in feces (50,88,91,92), due to significant absorption from more proximal regions of the GI tract, degradation by the resident colonic bacteria, and reabsorption from the colon after excretion in the bile. There have been no studies that have determined the urinary excretion of biochanin A and formononetin after a single dose of clover extract. However, as discussed earlier, daily excretions were determined in a study in which subjects consumed daily for 4 wk a clover extract tablet containing either 44 mg or 87 mg total isoflavones of which 60% was biochanin A, 37% formononetin, and the

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Isoflavone Glucosides

Aglycones + glucuronides + sulfates (?)

Small intestine Bile

Aglycones + glucuronidees + sulfates + hydroxylated metabolites + methylated metabolites

Aglycones Liver Glucuronides + sulfates Isoflavone Glucuronides Large Bowel

Kidney

Bladder Aglycones

Bacterial metabolites Urine

Feces

Aglycones + glucuronides + sulfates + bacterial metabolites

Aglycones + glucuronides + sulfates + hydroxylated metabolites + methylated metabolites + bacterial metabolites

Fig. 10.3. Bacterial and mammalian metabolism of isoflavones. The diagram shows a

stylized small intestine (upper) and large bowel (lower). Solid arrows signify movement of isoflavones, dashed arrows signify pathways of bacterial conversions. Isoflavones from nonfermented soy foods enter the small intestine as glucosides from the stomach. A proportion of these may be glucuronidated and possibly sulfated in the enterocytes of the small intestine (see Fig. 10.1) and enter the portal circulation. The remainder is either hydrolyzed to aglycones by bacteria in the distal small intestine, or passes into the large intestine. In the bowel, bacteria can also hydrolyze the glucosides to aglycones. The aglycones produced in this way, as well as those entering from the small intestine, can be absorbed or converted to a series of metabolites (e.g., equol or dihydrogenistein) by certain fecal bacteria, and these metabolites can also be absorbed. Absorbed isoflavones and bacterial metabolites that are not glucuronidated or sulfated in the intestine wall pass to the liver via the portal vein, where the majority are conjugated, but a fraction may also be hydroxylated and then methylated to a number of metabolites. The conjugates are partitioned between urine and bile for excretion. Those that enter the bowel with the bile are deconjugated by fecal bacteria to release the aglycones, which may again be either metabolized or absorbed (the enterohepatic cycle). Isoflavones and metabolites that are not finally absorbed and glucuronides and sulfates that are not hydrolyzed in the colon are excreted in the feces.

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TABLE 10.2 Recovery of Isoflavones in Urine and Feces After a Soy Meal in Humansa Recovery of ingested dose (%) Urine

Collection period (h)

Daidzein

Genistein

Glycitein

Fecesb

n

Soy food

Dose (µmol)

7M 7F 7F 12F 6M 7F 7M

Soy milk TVP/tofu Soy milk Soy flour Soy milk Baked soy powder Soybeans TVP Tofu Tempeh

4.5 total isoflavones/kg body 83–102 Daid; 111–126 Gen 98–279 Daid; 70–207 Gen 2.7 Daid/kg; 3.6 Gen/kg ~91–276 Daid; 119–352 Gen 102 Daid; 111 Gen

48 24c 24c 35 48c 72

52 49d 21 62 16 36

37 15–20d 9 22 10 18

47 ND ND ND ND ND

ND ND 1–2 ND 0.4–8 3–4

79 Daid; 89 Gen 110 Daid; 119 Gen 146 Daid; 159 Gen 87 Daid; 111 Gen

24c 24c 24c 24c

45 51 50 38

13 13 16 9

ND ND ND ND

2 2 1 2

10F 10F 5F 4F

aAbbreviations:

Reference 89,101 90 91 25 50 92 88 88 88 88

n, number of subjects; M, male; F, female; TVP, textured vegetable protein; Daid, daidzein aglycone equivalents; Gen, genistein aglycone equivalents; ND, not determined. bRecovery of total isoflavones. cPlus first void on the following day. dRecoveries not separately reported for daidzein; 15% genistein for TVP, 20% for tofu.

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remainder genistein and daidzein (68). Only ~1–5% of the ingested doses were excreted intact in urine over 24 h, suggesting that the bioavailabilities of biochanin A and formononetin were extremely low. On the basis of the differences in urinary excretion discussed above, it was concluded that the bioavailability of daidzein was greater than that of genistein (91). However, more recent pharmacokinetic studies in which areas under plasma concentration vs. time curves (AUC) were measured have come to different conclusions. These are summarized in Table 10.3. In the first of these, King and Bursill (25) concluded that the bioavailabilities of daidzein and genistein were similar in humans because the ratio of AUC for the two isoflavones was close to the ratio of their concentrations in the soy meal consumed. The lesser excretion of genistein compared with daidzein in urine was attributed to a greater fractional excretion in bile. This was supported by subsequent studies in cannulated rats, which showed significantly greater excretion of genistein compared with daidzein in bile (21) and correspondingly less excretion in urine (21,93). Watanabe et al. (92) measured plasma and urinary daidzein and genistein for 72 h after a single meal of a baked soybean powder product in which the two isoflavones were present at almost the same concentration (1.7 µmol daidzein/g and 1.8 µmol genistein/g); 36% of the daidzein dose and 18% of the genistein dose were recovered in urine. Unfortunately the AUC were not reported, but the mean peak plasma concentration of daidzein was less than that for genistein (1.56 ± 0.34 µmol/L compared with 2.44 ± 0.65 µmol/L), and it remained lower for the 72-h study period, which is suggestive of a greater, not lesser, bioavailability of genistein, as implied from the urinary excretion ratio. Setchell et al. (26) compared the pharmacokinetics of daidzein and genistein after the separate consumption of these isoflavones, or their respective glycosides, daidzin or genistin, as pure substances in pill form in female volunteers. In agreement with King and Bursill (25), plasma AUC for daidzein and genistein after consumption of 50 mg genistin (n = 3) or daidzin (n = 4) (the forms found in unfermented soy foods) were similar [4.95 ± 1.03 and 4.52 ± 0.49 µg/(mL⋅h)], respectively. The mean AUC after consumption of 50 mg daidzein by six subjects was 2.94 0.22 µg/(mL⋅h) compared with 4.54 ± 1.41 µg/(mL⋅h) after consumption of an equal amount of genistein by six other subjects. On the basis of the latter differences the authors suggested that genistein was more bioavailable than daidzein. However, no statistical analysis was presented to establish the significance of this difference. The same study examined the pharmacokinetics of glycitein after consumption of 25 mg of its glucoside, glycitin, by a single male subject (26). On an equivalent dose basis, the AUC for glycitein was somewhat less than the values for daidzein and genistein, suggesting lower bioavailability; however, a confounding effect of dose cannot be excluded, and it is clear that further studies are required. There appears to have been only one published study of the pharmacokinetics of the clover isoflavones in humans. Plasma concentrations of formononetin, biochanin A, daidzein, and genistein were measured in a single subject for 48 h after consumption of a clover extract tablet that contained mainly formononetin and

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TABLE 10.3 Plasma Pharmacokinetic Parameters of Isoflavones in Humansa

n 6F 4F 6F 3F 1M 7M 6M 4F 4M 4F 4M 4F 4M + 4F 4M

Test isoflavone or food Daidzein Daidzin Genistein Genistin Glycitin Baked soy powder Soy flour Daidzin + Genistin Daidzin + Genistin Daidzein + Genistein Daidzein + Genistein

Daidzein Dose (µmol) 197 118 185 114 55 103 Daid 112 Gen 2.7 Daid 3.6 Gen/kg 60 Daid 50 Gen 900 Daid 800 Gen 62 Daid 48 Gen 920 Daid 780 Gen

Cmax (µmol/L) 0.76 ± 0.12 1.55 ± 0.24

tmax (h) 6.6 ± 1.36 9.0 ± 1.0

Genistein t1/2 (h)

Cmax (µmol/L)

tmax (h)

Glycitein t1/2 (h)

Cmax (µmol/L)

9.33 ± 1.33 9.3 ± 1.3

3.14 ± 0.36

7.42 ± 0.74

Ref.

4

8.9

26 26 26 26 26

6.78 ± 0.84 ND ~0.7b

6

t1/2 (h)

ND ND 1.26 ± 0.27 1.22 ± 0.47

1.56 ± 0.34

tmax (h)

5.79

2.44 ± 0.65

6

8.36

ND

ND

92

4.7 ± 1.1

4.09 ± 0.94

8.42 ± 0.69

5.7 ± 1.3

ND

ND

25

0.2 ± 0.1b

4

0.4 ± 0.2b

4

24

2 ± 1b

4

4 ± 1b

6

24

0.8 ± 0.3b

2

1.0 ± 0.4b

2

24

17 ± 5b

4

21 ± 6b

4

24

aAbbreviations:

n, number of subjects; F, female; M, male; Daid, daidzein aglycone equivalents; Gen, genistein aglycone equivalents; Cmax, maximum plasma concentration; tmax, time to reach maximum plasma concentration; t1/2, elimination half life; ND, not determined. bValues estimated from graphical presentation of data.

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biochanin A in aglycone forms (26). Consistent with the urinary excretion pattern discussed earlier (68), the major isoflavones in plasma were genistein and daidzein, with much lower levels of formononetin and biochanin A, again suggesting very low bioavailability of the last-mentioned two compared with genistein and daidzein. In summary, it is clear from pharmacokinetic studies that the bioavailability of genistein is approximately equal to (25) or greater than that of daidzein (26,92) in humans, not significantly less as urinary excretion values would suggest (91), and it emphasizes the need for caution in interpreting urinary excretion data. The pharmacokinetic studies that have been conducted in humans to date have lacked the statistical power to fully quantitate differences in bioavailability of the soy isoflavones and it is important therefore that more studies with appropriate statistical power be conducted. In addition, with the recent marketing of soy hypocotyl products, which are high in glycitein, it is also important that further studies be conducted to evaluate the pharmacokinetics and bioavailability of this isoflavone. Limited urinary excretion and pharmacokinetic data for formononetin and biochanin A suggest that the bioavailabilities of the major clover isoflavones are low in humans; however, more studies with clover extracts are also required. Influence of Background Diet on Isoflavone Bioavailability. There are a number of possible reasons why background diet is postulated to influence the bioavailability of isoflavones. First, dietary components may sequester isoflavones and hinder their absorption from the intestinal tract. Second, dietary fiber, certain oligosaccharides, and resistant starch, which escape digestion in the small intestine, could alter the total microfloral population of the bowel, as well as the proportions of individual bacterial species. This may lead to changes in β-glucosidase and/or glucuronidase activity, which may influence the extent of hydrolysis both of the isoflavone glycosides entering the bowel from the small intestine, and of the glucuronides entering with the bile, thus modulating the levels of the more efficiently absorbed aglycones. Third, effects on gut microflora populations could modulate the ability of the bowel contents to degrade the isoflavones. Finally, bioavailability could be altered by dietary components such as fiber which influence transit time, i.e., a shorter transit time may lead to a shorter exposure of the isoflavones to the absorptive surface of the bowel and hence to lesser uptake. As discussed above, the use of urinary excretion data to compare the bioavailabilities of different isoflavones may lead to erroneous conclusions; however, this is a valid and convenient method with which to assess effects on the bioavailability of a single isoflavone. Based on the limited number of studies to date (88,90,94), background diet appears to have little influence on urinary excretion of isoflavones and presumed bioavailability. Xu et al. (88) compared urinary excretion after ingestion of soymilk in subjects who consumed a “basic food” diet, a “self-selected” diet, or an “ad libitum” diet, which differed in intakes of fat (range 44–63 g/d), protein (range 58–85 g/d), and cholesterol (range 54–118 g/d), but not carbohydrate or total dietary fiber. Urinary and fecal excretions of isoflavones were not

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different among the diets. Tew et al. (90) compared urinary excretion of isoflavones after consumption of a single meal of textured vegetable protein (TVP) or tofu by subjects consuming a high wheat fiber diet (~40 g dietary fiber/d) or a control diet (~15 g dietary fiber/d). On the basis of the combined tofu and TVP data (there were no significant differences between the two foods), urinary excretion of genistein was 19% lower (P < 0.03) in those consuming the high fiber diet compared with the control diet, suggesting that fiber did reduce the bioavailability of genistein; however, excretion of daidzein was not affected. Lampe et al. (94) compared urinary daidzein and genistein excretion in female volunteers who consumed for 1 mo either a regular diet that provided ~16 g fiber/d or a diet that provided ~32 g fiber/d, of which 16 g was provided by the same wheat bran breakfast cereal used by Tew et al. (90). In different arms of the study, soy protein was consumed for either 4 d or 1 mo and urine was collected for 2 d at the end of the intervention period. Contrary to the study of Tew et al. (90), there was no significant effect of fiber on urinary excretion of either daidzein or genistein in either arm of the study. In a study in rats, a diet containing 5% dietary fructooligosaccharide increased cecal content weight by 240% and total short-chain fatty acid concentration by 34% and decreased cecal pH from 6.75 to 5.21. These changes would be expected, and suggest significant change in bacterial numbers and activities in the cecum. As assessed by plasma AUC in tail blood, the bioavailability of daidzein was increased by 37% (P < 0.05) and that of genistein by 60% (P < 0.01) after a single dose of a proprietary isoflavone glucoside preparation (93); however, there was no significant effect on urinary excretion of genistein or daidzein over 48 h. In summary, dietary wheat fiber appears to have little or no effect on isoflavone bioavailability in humans. On the basis of the data from rats (93), the effects of other dietary fibers, and in particular nondigestible oligosaccharides and resistant starch, deserve further study in humans. Influence of Soy Food Type on Isoflavone Bioavailability. There are 12 isoflavone chemical entities that can be present in soy foods, three aglycones and nine glucosides, and the proportions of these differ among the various soy foods (95). The most obvious structural differences occur between the aglycones (genistein, daidzein, and glycitein) and their respective glucosides, but there are also more subtle chemical differences within each of the glucoside groups, e.g., among genistin, acetyl genistin and malonyl genistin. Therefore, there is potential for differences in the bioavailabilities of these various chemical forms, both in terms of the possible influence of structure on cellular transport as discussed earlier, as well as on resistance to bacterial metabolism. In addition to the differences in the chemical forms of isoflavones in the different foods, other factors such as the physical form and consistency of the food (e.g., liquid, solid, or pill) may also potentially influence availability of isoflavones for absorption. Because of the large between-study differences in urinary excretion values, no clear conclusions can be drawn regarding the relative bioavailabilities of the

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isoflavones from different foods by comparisons of the data obtained from different studies. In addition, because large individual-to-individual differences exist, particular care must be taken with study design, especially the use of adequate subject numbers, and ideally the use of the same individuals for testing the different soy foods. This has not always been the case, and there is a need for more robust studies to be undertaken. Three studies have compared the bioavailabilities of isoflavones from different soy foods on the basis of urinary excretion (75,88,90), and two have used plasma pharmacokinetic data to compare glucosides with aglycones (24,26). Some data are also available from an animal study in which the bioavailability of genistein from a glucoside-rich extract was compared with pure aglycone (20). In the study of Tew et al. discussed earlier (90), urinary excretion of isoflavones over 24 h was compared in seven women after consumption of a meal containing tofu or TVP against a background high-fiber or control diet. Tofu and TVP both provided 0.9 mg total isoflavones/kg body, although the ratio of total daidzein species to total genistein species in the two foods differed slightly, i.e., 62.0% genistein species in tofu and 53.6% in TVP. There was no significant difference between the two diet groups in urinary excretion of either isoflavone. The same authors compared urinary excretion of isoflavones after consumption of cooked soybeans, TVP, tofu, or tempeh in a randomized crossover study (88). Although analyses of individual conjugates in the foods were not reported in either study, substantial differences would be expected on the basis of other analyses of the respective food types (75,95,96). For example, cooked soybeans and TVP would be expected to contain the highest proportions of simple glucosides (daidzin and genistin) and acetyl glucosides, and the lowest proportions of malonyl glucosides; tofu would be expected to have low proportions of acetyl forms and high proportions of simple glucosides; tempeh, as a fermented food, would be expected to contain the highest proportions of aglycones. The percentage of recovery of daidzein over 24 h after consumption of each food ranged from 38 ± 19% for tempeh to 51 ± 10% for TVP and of genistein from 9 ± 4% for tempeh to 16 ± 5% for tofu, but there were no significant differences among food types. However, although group numbers were 10 for soybeans and TVP, they were only 5 for tofu and 4 for tempeh due to technical problems; thus the statistical power of the study was limited. In a randomized crossover study with 17 men, urinary excretions of isoflavones were compared on d 7–9 after consumption of cooked soybean pieces or tempeh (75). In contrast to the study of Xu et al. (88), urinary excretion of daidzein was 70% higher and genistein excretion 46% higher (both P < 0.05) after tempeh consumption. The tempeh meal contained 59% of the total daidzein and 34% of the total genistein as aglycones, compared with only 16 and 12%, respectively, for the soybean meal. This suggests that, as might be predicted from differences in GI transport discussed earlier, the bioavailabilities of the aglycone forms of the isoflavones were higher than those of the conjugates, although possible effects of other factors such as the differing physical consistencies of the foods, as

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discussed by the authors (88), must also be considered. In addition, the amount of isoflavones provided by the two diets was quite different, i.e., 18.4 mg total daidzein and 35.7 mg total genistein from soybeans compared with 10.8 mg total daidzein and 27.0 mg total genistein from tempeh; thus, confounding effects of dose on bioavailability also cannot be ruled out. Two studies in humans (24,26) and one in rats (20) have avoided possible effects of differing food matrices by using isoflavone extracts or pure substances, but the conclusions from these studies are also conflicting. In the study in rats (20), plasma pharmacokinetics and urinary excretion profiles of genistein were compared over 48 h after a single oral dose of the aglycone, or an equivalent dose of a soy extract that contained >99% glucosides. Over the 48-h collection period, the recovery of genistein in urine was slightly higher from aglycone-treated rats compared with those receiving the soy extract (19.9 ± 2.4% vs. 17.5 ± 1.1%) but these differences were not significant, suggesting that the bioavailabilities of the two forms of genistein were similar in this species. In the first of the human studies (24), plasma genistein and daidzein concentrations were measured in four men and four women at 2, 4, 6, and 24 h after consumption of two proprietary products (one containing aglycones and one containing glucosides, but both containing similar ratios of daidzein to genistein) at doses equivalent to 30 and 450 mg total aglycones. No AUC or urinary excretion data were reported, but plasma genistein and daidzein concentrations at 2, 4, and 6 h after aglycone consumption at both doses were up to five times higher (P < 0.05) than after glycoside consumption. After daily consumption of 80 mg aglycones for 4 wk, plasma levels of daidzein and genistein were also substantially higher (P < 0.005) compared with an equivalent dose of glucosides. Although the absence of AUC data means it is not possible to draw firm conclusions, these results are consistent with the transport studies discussed earlier (22,27–31) and are suggestive of a greater bioavailability of isoflavone aglycones compared with glycosides. In contrast, in the study outlined earlier, Setchell et al. concluded that isoflavone glucosides were more bioavailable than their corresponding aglycones. In this study, AUC were compared in women after single doses of pure daidzein or genistein or their respective glucosides, daidzin, or genistin (26). Unfortunately, on an aglycone-equivalent basis, the dose of the glucosides was only ~60% that of the aglycones; thus, the authors used a dose-normalized AUC to compare bioavailabilities. On this basis, the bioavailability of genistin was 1.8 times higher and daidzin 2.5 times higher compared with their respective aglycones. However, no statistical analysis was conducted to determine the significance of this difference. In summary, there is no clear consensus from studies that have compared the bioavailabilities of isoflavone aglycones and glucosides, either from foods or from purified extracts, with some studies suggesting little difference (20,88,90), some suggesting that aglycones may be more bioavailable than glucosides (24,88), and others suggesting the reverse (26). It is clear that more and better-designed studies are required to resolve this question.

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Summary and Conclusions In the last few years, there has been substantial progress in our understanding of the digestion, transport, and metabolism of the isoflavones, and the related areas of bioavailability and pharmacokinetics. For example, contrary to earlier belief, it is now recognized that the hydrolysis of glucosides by bacterial hydrolases is not mandatory for intestinal transport, and the weight of evidence suggests that little or no isoflavone glucosides enter the blood stream intact. Although mechanisms for the transport of flavonols have been investigated extensively, there have been few studies of the isoflavones and this should be a focus for future research. Although a large number of bacterial and mammalian metabolites have now been identified, we have yet to account for all of the ingested isoflavones. The recent synthesis of 14C- and 13C-labeled isoflavones should enable substantial progress to be made in the near future. The determinants of equol-producer status of individuals remain of intense interest, and planned studies signaled at the recent 4th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease should lead to a major step forward in our understanding of these determinants. The increasing availability of molecular techniques for the identification of specific gut microflora should also contribute in this area. Pharmacokinetic studies have made it clear that urinary excretion of isoflavones is an unsatisfactory index of their relative bioavailabilities, and much is now known about the time course and routes of absorption and excretion of isoflavones in humans. However, further pharmacokinetic studies with appropriate statistical power, particularly with glycitein and the clover isoflavones, are required. Although major progress has been made in the last 5–6 years, the exciting challenge researchers in this area face in the next few years is to work toward answering the many questions that remain. References 1. Reginster, J.-Y.L. (1993) Ipriflavone: Pharmacological Properties and Usefulness in Postmenopausal Osteoporosis, Bone Miner. 23, 223–232. 2. Yoshida, K., Tsukamoto, T., Torii, H., Doi, T., Naeshiro, I., Uemura, I., and Tanayama, S. (1985) Metabolism of Ipriflavone (TC-80) in Rats, Radioisotopes 34, 612–617. 3. Yoshida, K., Tsukamoto, T., Torii, H., Doi, T., Naeshiro, I., Shibata, K., Uemura, I., and Tanayama, S. (1985) Disposition of Ipriflavone (TC-80) in Rats and Dogs, Radioisotopes 34, 618–623. 4. Kim, S.H., Lee, J.S., and Lee, M.G. (2000) Pharmacokinetics and Tissue Distribution of Ipriflavone, an Isoflavone Derivative, After Intravenous Administration to Rabbits, Biopharm. Drug Dispos. 21, 147–156. 5. Rondelli, I., Acerbi, D., and Ventura, P. (1991) Steady-State Pharmacokinetics of Ipriflavone and Its Metabolites in Patients with Renal Failure, Int. J. Clin. Pharmacol. Res. 11, 183–192. 6. Saito, A.M. (1985) Pharmacokinetic Study of Ipriflavone (TC-80) by Oral Administration in Healthy Male Volunteers, Jpn. Pharm. Ther. J. 13, 7223–7233.

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7. Sato, T., Koite, T., Hamada, A., Oshashi, M., and Shimazu, A. (1986) Pharmacokinetics of Ipriflavone Tablets in Long-Term Administration to Patients with Osteoporosis, Jpn. Pharm. Ther. J. 14, 873–883. 8. Hollman, P.C.H., de Vries, J.H.M., van Leeuwen, S.D., and Mengelers, M.J.B. (1995) Absorption of Dietary Quercetin Glycosides and Quercetin in Healthy Ileostomy Volunteers, Am. J. Clin. Nutr. 62, 1276–1282. 9. Walle, T., Otake, Y., Walle, U.K., and Wilson, F.A. (2000) Quercetin Glucosides Are Completely Hydrolyzed in Ileostomy Patients Before Absorption, J. Nutr. 130, 2658–2661. 10. Hollman, P.C.H., Bijsman, M.N.C.P., van Gameren, Y., Cnossen, E.P.J., de Vries, J.H.M., and Katan,M.B. (1999) The Sugar Moiety Is a Major Determinant of the Absorption of Dietary Flavonoid Glycosides in Man, Free Radic.Res. 31, 569–573. 11. Crespy, V., Morand, C., Manach, C., Besson, C., Demigné, C., and Rémésy, C. (1999) Part of Quercetin Absorbed in the Small Intestine Is Conjugated and Further Secreted in the Intestinal Lumen, Am. J. Physiol. 40, G120–G126. 12. Gee, J.M., DuPont, M.S., Day, A.J., Plumb, G.W., Williamson, G., and Johnson, I.T. (2000) Intestinal Transport of Quercetin Glycosides in Rats Involves Both Deglycosylation and Interaction with the Hexose Transport Pathway, J. Nutr. 130, 2765–2771. 13. Ader, P., Block, M., Pietzsch, S., and Wolffram, S. (2001) Interaction of Quercetin Glucosides with the Intestinal Sodium/Glucose Co-Transporter (SGLT-1), Cancer Lett. 162, 175–180. 14. Spencer, J.P.E., Chowrimootoo, G., Choudhury, R., Debnam, E.S., Srai, S.K., and Rice-Evans, C. (1999) The Small Intestine Can Both Absorb and Glucuronidate Luminal Flavonoids, FEBS Lett. 458, 224–230. 15. Gee, J.M., DuPont, M.S., Rhodes, M.J.C., and Johnson, I.T. (1998) Quercetin Glucosides Interact with the Intestinal Glucose Transport Pathway, Free Radic. Biol. Med. 25, 19–25. 16. Walgren, R.A., Lin, J.T., Kinne, R.K.H., and Walle, T. (2000) Cellular Uptake of Dietary Flavonoid Quercetin 4′-β-Glucoside by Sodium-Dependent Glucose Transporter SGLT1, J. Pharmacol. Exp. Ther. 294, 837–843. 17. Walgren, R.A., Walle, K., and Walle, T. (1998) Transport of Quercetin and Its Glucoside Across Human Intestinal Epithelial Caco-2 Cells, Biochem. Pharmacol. 55, 1721–1727. 18. Day, A.J., Canada, F.J., Diaz, J.C., Kroon, P.A., Mclauchlan, R., Faulds, C.B., Plumb, G.W., Morgan, M.R., and Williamson, G. (2000) Dietary Flavonoid and Isoflavone Glycosides Are Hydrolysed by the Lactase Site of Lactase Phlorizin Hydrolase, FEBS Lett. 468, 166–170. 19. Ioku, K., Pongpiriyadacha, Y., Konishi, Y., Takei, Y., Nakatani, N., and Terao, J. (1998) β-Glucosidase Activity in the Rat Small Intestine Toward Quercetin Monoglucosides, Biosci. Biotechnol. Biochem. 62, 1428–1431. 20. King, R.A., Broadbent, J.L., and Head, R.J. (1996) Absorption and Excretion of the Soy Isoflavone Genistein in Rats, J. Nutr. 126, 176–182. 21. Sfakianos, J., Coward, L., Kirk, M., and Barnes, S. (1997) Intestinal Uptake and Biliary Excretion of the Isoflavone Genistein in Rats, J. Nutr. 127, 1260–1268. 22. Piskula, M.K., Yamakoshi, J., and Iwai, Y. (1999) Daidzein and Genistein but Not Their Glucosides Are Absorbed from the Rat Stomach, FEBS Lett. 447, 287–291. 23. Day, A.J., DuPont, M.S., Ridley, S., Rhodes, M., Rhodes, M.J.C., Morgan, M.R.A.,

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and Williamson, G. (1998) Deglycosylation of Flavonoid and Isoflavonoid Glycosides by Human Small Intestine and Liver Beta-Glucosidase Activity, FEBS Lett. 436, 71–75. Izumi, T., Piskula, M.K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kataoka, S., Kubota, Y., and Kikuchi, M. (2000) Soy Isoflavone Aglycones Are Absorbed Faster and in Higher Amounts than Their Glucosides in Humans, J. Nutr. 130, 1695–1699. King, R.A., and Bursill, D.B. (1998) Plasma and Urinary Kinetics of the Isoflavones Daidzein and Genistein After a Single Soy Meal in Humans, Am. J. Clin. Nutr. 67, 867–872. Setchell, K.D.R., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., and Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131, 1362S–1375S. Walle, U.K., French, K.L., Walgren, R.A., and Walle, T. (1999) Transport of Genistein-7-Glucoside by Human Intestinal Caco-2 Cells: Potential Role for MRP2, Res. Commun. Mol. Pathol. Pharmacol. 103, 45–56. Steensma, A., Noteborn, H.P.J.M., vanderjagt, R.C.M., Polman, T.H.G., Mengelers, M.J.B., and Kuiper, H.A. (1999) Bioavailability of Genistein, Daidzein, and Their Glycosides in Intestinal Epithelial Caco-2 Cells, Environ. Toxicol. Pharmacol. 7, 209–211. Andlauer, W., Kolb, J., and Furst, P. (2000) Isoflavones from Tofu Are Absorbed and Metabolized in the Isolated Rat Small Intestine, J. Nutr. 130, 3021–3027. Andlauer, W., Kolb, J., Stehle, P., and Furst, P. (2000) Absorption and Metabolism of Genistein in Isolated Rat Small Intestine, J. Nutr. 130, 843–846. Andlauer, W., Kolb, J., and Furst, P. (2000) Absorption and Metabolism of Genistin in the Isolated Rat Small Intestine, FEBS Lett. 475, 127–130. Kuhnau, J. (1976) The Flavonoids. A Class of Semi-Essential Food Components: Their Role in Human Nutrition, in World Review of Nutrition and Dietetics 24 (Bourne, G.H., ed.,) pp. 117–191, Karger AG, Basel. Morand, C., Manach, C., Crespy, V., and Rémésy, C. (2000) Quercetin 3-O-βGlucoside Is Better Absorbed than Other Quercetin Forms and Is Not Present in Rat Plasma, Free Radic. Res. 33, 667–676. Walle, U.K., Galijatovic, A., and Walle, T. (1999) Transport of the Flavonoid Chrysin and Its Conjugated Metabolites by the Human Intestinal Cell Line Caco-2, Biochem. Pharmacol. 58, 431–438. Walgren, R.A., Karnaky, K.J., Lindenmayer, G.E., and Walle, T. (2000) Efflux of Dietary Flavonoid Quercetin 4′-β-Glucoside Across Human Intestinal Caco-2 Cell Monolayers by Apical Multidrug Resistance-Associated Protein-2, J. Pharmacol. Exp. Ther. 294, 830–836. Malagelada, J.-R., Robertson, J.S., Brown, M.L., Remington, M., Duenes, J.A., Thomforde, G.M., and Carryer, P.W. (1984) Intestinal Transit of Solid and Liquid Components of a Meal in Health, Gastroenterology 87, 1255–1263. Paganga, G., and Rice-Evans, C.A. (1997) The Identification of Flavonoids as Glycosides in Human Plasma, FEBS Lett. 401, 78–82. Aziz, A.A., Edwards, C.A., Lean, M.E.J., and Crozier, A. (1998) Absorption and Excretion of Conjugated Flavonols, Including Quercetin-4′-O-β-Glucoside and Isorhamnetin-4′-O-β-Glucoside by Human Volunteers After the Consumption of Onions, Free Radic. Res. 29, 257–269. Andlauer, W., Stumpf, C., and Furst, P. (2001) Intestinal Absorption of Rutin in Free and Conjugated Forms, Biochem. Pharmacol. 62, 369–374.

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40. Mauri, P.L., Iemoli, L., Gardana, C., Riso, P., Simonetti, P., Porrini, M., and Pietta, P.G. (1999) Liquid Chromatography/Electrospray Ionization Mass Spectrometric Characterization of Flavonol Glycosides in Tomato Extracts and Human Plasma, Rapid Commun. Mass Spectrom. 13, 924–931. 41. Crespy, V., Morand, C., Besson, C., Manach, C., Demigné, C., and Rémésy, C. (2001) Comparison of the Intestinal Absorption of Quercetin, Phloretin and Their Glucosides in Rats, J. Nutr. 131, 2109–2114. 42. Moon, J.H., Nakata, R., Oshima, S., Inakuma, T., and Terao, J. (2000) Accumulation of Quercetin Conjugates in Blood Plasma After the Short-Term Ingestion of Onion by Women, Am. J. Physiol. 279, R461–R467. 43. Shimoi, K., Okada, H., Furugori, M., Goda, T., Takase, S., Suzuki, M., Hara, Y., Yamamoto, H., and Kinae, N. (1998) Intestinal Absorption of Luteolin and Luteolin 7O-β-Glucoside in Rats and Humans, FEBS Lett. 438, 220–224. 44. Sesink, A.L.A., O’Leary, K.A., and Hollman, P.C.H. (2001) Quercetin Glucuronides but Not Glucosides Are Present in Human Plasma After Consumption of Quercetin-3Glucoside or Quercetin-4′-Glucoside, J. Nutr. 131, 1938–1941. 45. Lamon-Fava, S. (2000) Genistein Activates Apolipoprotein A-I Gene Expression in the Human Hepatoma Cell Line Hep G2, J. Nutr. 130, 2489–2492. 46. Hawkesworth, G., Drasar, B.S., and Hill, M.J. (1971) Intestinal Bacteria and the Hydrolysis of the Glycosidic Bonds, J. Med. Microbiol. 4, 451–459. 47. Drasar, B.S. (1988) The Bacterial Flora of the Intestine, in Role of the Gut Flora in Toxicity and Cancer (Rowland, I.R., ed.) pp. 23–38, Academic Press, London. 48. Bokkenheuser, V.D., Shackleton, C.H.L., and Winter, J. (2001) Hydrolysis of Dietary Flavonoid Glycosides by Strains of Intestinal Bacteroides from Humans, Biochem. J. 248, 953–956. 49. Kim, D.-H., Jung, E.-A., Sohng, I.-S., Han, J.-A., Kim, T.-H., and Han, M.J. (1998) Intestinal Bacterial Metabolism of Flavonoids and Its Relation to Some Biological Activities, Arch. Pharm. Res. 21, 17–23. 50. Xu, X., Harris, K.S., Wang, H.-J., Murphy, P.A., and Hendrich, S. (1995) Bioavailability of Soybean Isoflavones Depends upon Gut Microflora in Women, J. Nutr. 125, 2307–2315. 51. Chang, Y.C. and Nair, M.G. (1995) Metabolism of Daidzein and Genistein by Intestinal Bacteria, J. Nat. Prod. 58, 1892–1896. 52. Setchell, K.D.R., Borriello, S.P., Kirk, D.N., and Axelson, M. (1984) Nonsteroidal Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Disease, Am. J. Clin. Nutr. 40, 569–578. 53. Kim, D.-H., Yu, K.-U., Bae, E.-A., and Han, M.J. (1998) Metabolism of Puerarin and Daidzin by Human Intestinal Bacteria and Their Relation to In Vitro Cytotoxicity, Biol. Pharm. Bull. 21, 628–630. 54. Laube, B., Winkler, S., Ladstetter, B., Scheller, T., and Schwarz, L.R. (2000) Establishment of a Novel In Vitro System for Studying the Interaction of Xenobiotic Metabolism of Liver and Intestinal Microflora, Arch. Toxicol. 74, 379–387. 55. Axelson, M., Kirk, D.N., Farrant, R.D., Cooley, G., Lawson, A.M., and Setchell, K.D. (1982) The Identification of the Weak Oestrogen Equol [7-Hydroxy-3-(4′-hydroxyphenyl)chroman] in Human Urine, Biochem. J. 201, 353–357. 56. Adlercreutz, H., Fotsis, T., Heikkinen, R., Dwyer, J.T., Woods, M., and Goldin, B.R. (1982) Excretion of the Lignans Enterolactone and Enterodiol and of Equol in Omnivorous and Vegetarian Postmenopausal Women and in Women with Breast

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Cancer, Lancet 2, 1295–1299. 57. Axelson, M., Sjovall, J., Gustafsson, B.E., and Setchell, K.D. (1984) Soya—A Dietary Source of the Non-Steroidal Oestrogen Equol in Man and Animals, J. Endocrinol. 102, 49–56. 58. Rowland, I.R., Wiseman, H., Sanders, T.A.B., Adlercreutz, H., and Bowey, E.A. (2000) Interindividual Variation in Metabolism of Soy Isoflavones and Lignans: Influence of Habitual Diet on Equol Production by the Gut Microflora, Nutr. Cancer 36, 27–32. 59. Joannou, G.E., Kelly, G.E., Reeder, A.Y., Waring, M., and Nelson, C. (1995) A Urinary Profile Study of Dietary Phytoestrogens. The Identification and Mode of Metabolism of New Isoflavonoids, J. Steroid Biochem. Mol. Biol. 54, 167–184. 60. Rowland, I., Wiseman, H., Sanders, T., Adlercreutz, H., and Bowey, E. (1999) Metabolism of Oestrogens and Phytoestrogens: Role of the Gut Microflora, Biochem. Soc. Trans. 27, 304–308. 61. Cruz, M.L.A., Wong, W.W., Mimouni, F., Hachey, D.L., Setchell, K.D.R., Klein, P.D., and Tsang, R.C. (1994) Effects of Infant Nutrition on Cholesterol Synthesis Rates, Pediatr. Res. 35, 135–140. 62. Setchell, K.D., Borriello, S.P., Hulme, P., Kirk, D.N., and Axelson, M. (1984) Nonsteroidal Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Disease, Am. J. Clin. Nutr. 40, 569–578. 63. Hur, H.G., and Rafii, F. (2000) Biotransformation of the Isoflavonoids Biochanin A, Formononetin, and Glycitein by Eubacterium limosum, FEMS Microbiol. Lett. 192, 21–25. 64. Hur, H.G., Lay, J.O., Beger, R.D., Freeman, J.P., and Rafii, F. (2000) Isolation of Human Intestinal Bacteria Metabolizing the Natural Isoflavone Glycosides Daidzin and Genistin, Arch. Microbiol. 174, 422–428. 65. Dickinson, J.M., Smith, G.R., Randel, R.D., and Pemberton, J. (1988) In Vitro Metabolism of Formononetin and Biochanin A in Bovine Rumen Fluid, J. Anim. Sci. 66, 1969–1973. 66. Lundh, T.J.-O. (1995) Metabolism of Estrogenic Isoflavones in Domestic Animals, Proc. Soc. Exp. Biol. Med. 208, 33–39. 67. Batterham, T.J., Shutt, D.A., Hart, N.K., Braden, A.W.H., and Tweeddale, H.J. (1971) Metabolism of Intraruminally Administered [4- 14C] Formononetin and [4- 14C] Biochanin A in Sheep, Aust. J. Agric. Res. 22, 131–138. 68. Howes, J.B., Sullivan, D., Lai, N., Nestel, P., Pomeroy, S., West, L., Eden, J.A., and Howes, L.G. (2000) The Effects of Dietary Supplementation with Isoflavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 152, 143–147. 69. Kelly, G.E., Nelson, C., Waring, M.A., Joannou, G.E., and Reeder, A.Y. (1993) Metabolites of Dietary (Soya) Isoflavones in Human Urine, Clin. Chim. Acta 223, 9–22. 70. Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T., Hamalainen, E., Hasegawa, T., and Okada, H. (1991) Urinary Excretion of Lignans and Isoflavonoid Phytoestrogens in Japanese Men and Women Consuming a Traditional Japanese Diet, Am. J. Clin. Nutr. 54, 1093–1100. 71. Karr, S.C., Lampe, J.W., Hutchins, A.M., and Slavin, J.L. (1997) Urinary Isoflavonoid Excretion in Humans Is Dose Dependent at Low to Moderate Levels of Soy-Protein Consumption, Am. J. Clin. Nutr. 66, 46–51. 72. Kelly, G.E., Joannou, G.E., Reeder, A.Y., Nelson, C., and Waring, M.A. (1995) The

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Variable Metabolic Response to Dietary Isoflavones in Humans, Proc. Soc. Exp. Biol. Med. 208, 40–43. Lampe, J.W., Karr, S.C., Hutchins, A.M., and Slavin, J.L. (1998) Urinary Equol Excretion with a Soy Challenge: Influence of Habitual Diet, Proc. Soc. Exp. Biol. Med. 217, 335–339. Shelnutt, S.R., Cimino, C.O., Wiggins, P.A., and Badger, T.M. (2000) Urinary Pharmacokinetics of the Glucuronide and Sulfate Conjugates of Genistein and Daidzein, Cancer Epidemiol. Biomark. Prev. 9, 413–419. Hutchins, A.M., Slavin, J.L., and Lampe, J.W. (1995) Urinary Isoflavonoid Phytoestrogen and Lignan Excretion After Consumption of Fermented and Unfermented Soy Products, J. Am. Diet. Assoc. 95, 545–551. Setchell, K.D.R., and Cassidy, A. (1999) Dietary Isoflavones: Biological Effects and Relevance to Human Health, J. Nutr. 129, 758S–767S. Adlercreutz, H., Fotsis, T., Watanabe, S., Lampe, J.W., Wähälä, K., Mäkelä, T., and Hase, T. (1994) Determination of Lignans and Isoflavonoids in Plasma by Isotope Dilution Gas Chromatography-Mass Spectrometry, Cancer Detect. Prev. 18, 259–271. Doerge, D.R., Chang, H.C., Churchwell, M.I., and Holder, C.L. (2000) Analysis of Soy Isoflavone Conjugation In Vitro and in Human Blood Using Liquid Chromatography-Mass Spectrometry, Drug Metab. Dispos. 28, 298–307. Chang, H.C., Churchwell, M.I., Delclos, K.B., Newbold, R.R., and Doerge, D.R. (2000) Mass Spectrometric Determination of Genistein Tissue Distribution in DietExposed Sprague-Dawley Rats, J. Nutr. 130, 1963–1970. Lundh, T.J.-O. (1990) Conjugation of the Plant Estrogens Formononetin and Daidzein and Their Metabolite Equol by Gastrointestinal Epithelium from Cattle and Sheep, J. Agric. Food Chem. 38, 1012–1016. Mulder, G.J., Coughtrie, M.W.H., and Burchell, B. (1990) Glucuronidation, in Conjugation Reactions in Drug Metabolism: An Integrated Approach (Mulder, G.J., ed.) pp. 51–105, Taylor & Francis, London. Mulder, G.J., and Jakoby, W.B. (1990) Sulphation, in Conjugation Reactions in Drug Metabolism: An Integrated Approach (Mulder, G.J., ed.) pp. 107–161, Taylor & Francis, London. Nilsson, A. (1961) Demethylation of the Plant Estrogen Biochanin A in the Rat, Nature 192, 358. Lundh, T.J.-O., Pettersson, H., and Kiessling, K.H. (1988) Demethylation and Conjugation of Formononetin and Daidzein in Sheep and Cow Liver Microsomes, J. Agric. Food Chem. 36, 22–25. Roberts-Kirchhoff, E.S., Crowley, J.R., Hollenberg, P.F., and Kim, H. (1999) Metabolism of Genistein by Rat and Human Cytochrome P450s, Chem. Res. Toxicol. 12, 610–616. Kulling, S.E., Honig, D.M., Simat, T.J., and Metzler, M. (2000) Oxidative In Vitro Metabolism of the Soy Phytoestrogens Daidzein and Genistein, J. Agric. Food Chem. 48, 4963–4972. Kulling, S.E., Honig, D.M., and Metzler, M. (2001) Oxidative Metabolism of the Soy Isoflavones Daidzein and Genistein in Humans In Vitro and In Vivo, J. Agric. Food Chem. 49, 3024–3033. Xu, X., Wang, H.J., Murphy, P.A., and Hendrich, S. (2000) Neither Background Diet Nor Type of Soy Food Affects Short-Term Isoflavone Bioavailability in Women, J. Nutr. 130, 798–801.

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89. Zhang, Y., Wang, G.J., Song, T.T., Murphy, P.A., and Hendrich, S. (1999) Urinary Disposition of the Soybean Isoflavones Daidzein, Genistein and Glycitein Differs Among Humans with Moderate Fecal Isoflavone Degradation Activity, J. Nutr. 129, 957–962. 90. Tew, B.-Y., Xu, X., Wang, H.-J., Murphy, P.A., and Hendrich, S. (1996) A Diet High in Wheat Fiber Decreases the Bioavailability of Soybean Isoflavones in a Single Meal Fed to Women, J. Nutr. 126, 871–877. 91. Xu, X., Wang, H.-J., Murphy, P.A., Cook, L., and Hendrich, S. (1994) Daidzein Is a More Bioavailable Soymilk Isoflavone than Is Genistein in Adult Women, J. Nutr. 124, 825–832. 92. Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., Mazur, W., Wähälä , K., and Adlercreutz, H. (1998) Pharmacokinetics of Soybean Isoflavones in Plasma, Urine and Feces of Men After Ingestion of 60 g Baked Soybean Powder (Kinako), J. Nutr. 128, 1710–1715. 93. Uehara, M., Ohta, A., Sakai, K., Suzuki, K., Watanabe, S., and Adlercreutz, H. (2001) Dietary Fructooligosaccharides Modify Intestinal Bioavailability of a Single Dose of Genistein and Daidzein and Affect Their Urinary Excretion and Kinetics in Blood of Rats, J. Nutr. 131, 787–795. 94. Lampe, J.W., Skor, H.E., Li, S., Wähälä , K., Howald, W.N., and Chen, C. (2001) Wheat Bran and Soy Protein Feeding Do Not Alter Urinary Excretion of the Isoflavan Equol in Premenopausal Women, J. Nutr. 131, 740–744. 95. King, R.A. and Bignell, C.M. (2000) Concentrations of Isoflavone Phytoestrgens and Their Glucosides in Australian Soya Beans and Soya Foods, Aust. J. Nutr. Diet. 57, 70–78. 96. Murphy, P.A., Song, T.T., Buseman, G., Barua, K., Beecher, G.R., Trainer, D., and Holden, J. (1999) Isoflavones in Retail and Institutional Soy Foods, J. Agric. Food Chem. 47, 2697–2704. 97. Heinonen, S., Wähälä , K., and Adlercreutz, H. (1999) Identification of Isoflavone Metabolites Dihydrodaidzein, Dihydrogenistein, 6′-OH-O-DMA, and cis-4-OH-Equol in Human Urine by Gas Chromatography-Mass Spectroscopy Using Authentic Reference Compounds, Anal. Biochem. 274, 211–219. 98. Bannwart, C., Adlercreutz, H., Fotsis, T., Wähälä, K., Hase, T., and Brunow, G. (1984) Identification of O-Desmethylangolensin, a Metabolite of Daidzein, and of Matairesinol, One Likely Plant Preursor of the Animal Lignan Enterolactone, in Human Urine, Finn. Chem. Lett. 4–5, 120–125. 99. Bannwart, C., Adlercreutz, H., Wähälä , K., Kotiaho, T., Hesso, A., and Hase, T. (1988) Identification of the Phyto-Estrogen 3′,7-Dihydroxyisoflavan, an Isomer of Equol, in Human Urine and Cow’s Milk, Biomed. Environ. Mass. Spectrom. 17, 1–16. 100. Setchell, K.D.R. (1998) Phytoestrogens: The Biochemistry, Physiology, and Implications for Human Health of Soy Isoflavones, Am. J. Clin. Nutr. 68, 1333S–1346S. 101. Zhang, Y., Wang, G.J., Song, T.T., Murphy, P.A., and Hendrich, S. (2001) Differences in Disposition of the Soybean Isoflavones, Glycitein, Daidzein and Genistein in Humans with Moderate Fecal Isoflavone Degradation Activity, J. Nutr. 131, 147–148.

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Chapter 11

Cellular Mechanisms of Action Including Estrogen Receptors: ER α and β Sari Mäkeläa,b and Jan-Åke Gustafssona aDepartment

of Medical Nutrition, Karolinska Institute, S-14186 Huddinge, Sweden

bInstitute

of Biomedicine and Functional Foods Forum, University of Turku, FIN-20014 Turku, Finland

Introduction Phytoestrogens, also called plant estrogens, constitute a heterogeneous group of plant-derived compounds that interact with estrogen receptors (ER) and/or induce estrogen-like effects in vitro or in vivo. Over 300 plant species, including edible and medicinal plants, with estrogen-like activity have been identified. The concentrations and combinations of different phytoestrogens vary enormously among individual plant-species. In the human diet, soybeans constitute the most abundant source of phytoestrogens. On the basis of their chemical structures, phytoestrogens can be divided into four subgroups as follows: isoflavonoids, flavonoids, coumestans, and lignans. Phytoestrogens are nonsteroidal polyphenolic compounds, and are structurally different from the endogenous mammalian steroid hormones (Fig. 11.1). Two forms of ER proteins are expressed in mammalian tissues. ERα was identified >30 years ago by Jensen and Jacobsen (1). Until recently, ERα was thought to be solely responsible for mediating all of the physiologic and pharmacologic effects of endogenous and exogenous estrogens. In 1995, a second ER, named ERβ, was cloned from a rat prostate cDNA library (2), and later from several other mammalian species, including humans (3). This discovery has initiated a thorough reevaluation of mechanisms and targets of estrogen action. Although the two ER subtypes share a high degree of homology in some parts of the receptor protein, they differ markedly from each other in multiple aspects, such as ligand binding properties, regulation of gene expression, and organ distribution; their biological functions are thus clearly different (4,5). There is increasing interest in the possible effect of dietary phytoestrogens on human health because phytoestrogens are suggested to play a role in the development of hormone-dependent diseases. Epidemiologic studies indicate that life-long exposure to phytoestrogen-rich diets may be associated with a low risk of breast and prostate cancer, postmenopausal osteoporosis, and cardiovascular diseases. On the other hand, it has been suggested that phytoestrogens, similar to any other hormonally active agents in the environment, may act as endocrine disruptors and cause adverse effects in the reproductive system. Clearly, if a compound is an estrogen

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Fig. 11.1. Chemical structures of phytoestrogens.

(i.e., interacts with ER), it may have the potential for beneficial or adverse effects, depending on the target cell or tissue, age and time of exposure, the hormonal milieu, and the dose or concentration in the target tissue. Furthermore, in addition to estrogen-like activity, phytoestrogens exert multiple other biological actions that occur independently of ER, and it is possible that the non-ER–mediated effects may also be of importance in vivo. However, they are not in the scope of this review and will not be discussed further. Very little, if anything, is known about the ER-mediated effects of phytoestrogens in humans. Most of the information was obtained from studies using complex phytoestrogen-rich diets rather than purified compounds. It is thus not possible to conclude whether the effects are due to phytoestrogens because multiple other biologically active compounds are present in the same plants. The major questions remain open: (i) Do dietary phytoestrogens exert ER-mediated actions in vivo in humans? (ii) Do phytoestrogens act as "typical" estrogen agonists? (iii) Do phytoestrogens act as antiestrogens (i.e., inhibit the actions of more potent endogenous estrogens)? And (iv) Do phytoestrogens act as selective estrogen receptor modulators (SERM)?

Effects In Vitro Phytoestrogens Bind to Estrogen Receptors α and β It has been known for decades that phytoestrogens bind to estrogen receptors isolated from uterine tissue. The relative binding affinities were reported to be much lower than those of steroidal estrogens, and phytoestrogens were concluded to be

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very weak estrogens (6–8). Binding to uterine ER is now known to reflect mainly binding to ERα, and the earlier conclusions must be reevaluated. The studies by Kuiper and co-workers in 1996 and 1998 (9,10) created an enormous interest in phytoestrogens because they were the first to indicate that some phytoestrogens, such as genistein, may act in a unique manner and bind at higher affinity to ERβ than to ERα (Table 11.1). These results were later confirmed in other laboratories (11,12). Such a phenomenon had not been observed for any other group of natural compounds, and this generated the hypothesis of phytoestrogens as "natural ERβselective agents" or "natural SERM." However, this may be an overstatement because it has been demonstrated that phytoestrogens bind relatively well also to ERα. Furthermore, the receptor binding studies demonstrate that there are marked differences among individual compounds, and indicate that only a minor difference in the chemical structure, such as the lack of one hydroxyl group in the molecule, may result in a significant change in the biological activity, as for genistein (4′,5,7trihydroxyisoflavone) vs. daidzein (4′,7-dihydroxyisoflavone). Phytoestrogens Regulate the Expression of Estrogen-Responsive Genes and Cell Proliferation via Estrogen Receptors At a high concentration (1 µmol/L), several phytoestrogens induce the expression of the estrogen-sensitive reporter gene via both ER, some even beyond the maximal activation by 17β-estradiol (E2) (Table 11.2) (10). As expected on the basis of receptor binding affinities, marked differences exist in the transcriptional activity of the individual compounds, although the differences in the chemical structures TABLE 11.1 Relative Binding Affinities of Phytoestrogens to Human Estrogen Receptor (hER)α and hERβa hERα 17β-Estradiol

100

100

Coumestans Coumestrol

20

140

Isoflavonoids Genistein Daidzein Formononetin Biochanin A Flavonoids Apigenin Naringenin aSource:

hERβ

Reference 10.

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4 0.1 <0.01 <0.01

87 0.5 <0.01 <0.01

0.3 0.01

6 0.11

TABLE 11.2 Transcriptional Activity of Phytoestrogens via Human Estrogen Receptor (hER)α and hERβa hERα

hERβ

17β-Estradiol (1 nmol/L)

100

100

Coumestans Coumestrol (1 µmol/L)

102

98

Isoflavonoids Genistein (1 µmol/L) Daidzein (1 µmol/L) Formononetin (1 µmol/L) Biochanin A (1 µmol/L)

198 97 6 36

182 80 2 53

50 36

49 45

Flavonoids Apigenin (1 µmol/L) Naringenin (1 µmol/L) aSource:

Reference 10. indicate the expression of the reporter gene, relative to the maximal activation induced by 10 nmol/L 17β-estradiol (=100).

bValues

are relatively small. Interestingly, the "ER-subtype specificity" of genistein, or its preference to ERβ over ERα observed in the receptor binding studies at lower ligand concentrations (10–100 nmol/L), cannot be demonstrated at 1 µmol/L, and genistein induces the reporter gene expression as well via both ER (10). Similar results have been obtained by other investigators using different reporter gene constructs (11,13). Several earlier studies demonstrated that genistein acts as an estrogen agonist and stimulates cell proliferation and expression of estrogen-responsive marker genes in human breast cancer cells (11,12,14–19). However, it is not known whether the effects of phytoestrogens are similar in all aspects to those of E2 or other known estrogen agonists. Recently, Diel and co-workers (20) investigated the effects of genistein and coumestrol in MCF-7 breast cancer cells in vitro, using the expression of progesterone receptor and ERα as markers of estrogen action. They reported that coumestrol acts similarly to E2, whereas the effect of genistein is comparable to that of the SERM compounds raloxifen and ICI 182,780. In these studies, cell lines expressing predominantly ERα were used, and the results are likely to reflect ERα-mediated actions. It is not yet known how genistein, or other phytoestrogens, regulate the expression of intrinsic ("natural") estrogen-responsive genes and/or cell proliferation via ERβ, or whether the effect is similar to those of other known estrogen agonists. Kuiper and co-workers (10) demonstrated that genistein exerts more pronounced activity via ERβ, but only at a relatively low concentration range (<100–200 nmol/L). At higher concentrations, no difference exists, and genistein acts as a superagonist via both ER subtypes (10,13). In contrast, Barkhem and coworkers (21) showed that genistein is only a partial agonist via ERβ, but a full ago-

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nist via ERα. Different reporter gene constructs and treatment times were used in these studies, which may explain the different responses. Coumestrol, albeit showing higher binding affinity to ERβ, does not differentiate between the two ER with regard to transcriptional activity at any concentration (10). There is no obvious explanation for this discrepancy. The ER subtype expression pattern (ERα only, ERβ only, ERα and ERβ) of the target cell is also likely to modulate the response to phytoestrogens. Recently, it was shown that coexpression of ERα with ERβ enhances the agonistic effect of genistein at a low concentration (20 nmol/L), and in the presence of both ER subtypes, the enhanced response to genistein involves the N-terminal parts of the receptors (13). These results indicate that ERα/ERβ-heterodimers are likely to act in a different manner compared with ERβ homodimers. Interaction of Genistein with ERb Ligand Binding Domain It is not yet fully understood why genistein may act differently from the other known estrogens with regard to its interaction with ERβ. Two recent studies indicate that this is determined by the ERβ ligand binding domain (LBD). Using X-ray crystallography, Pike and co-workers (22) investigated the interaction of genistein and two other ER ligands, E2 (natural steroidal estrogen) and raloxifen (a synthetic SERM compound), with ERβ LBD. It was shown that genistein binds to the hormone-binding cavity in a manner similar to E2, whereas the conformation of the LBD (especially the orientation of the AF-2 helix) adopted after ligand binding is different from that elicited by E2, and resembles that seen in the ERβ LBD-raloxifen complex. In another study utilizing chimeric proteins, the selective response to a low concentration of genistein was shown to be determined by ERβ-LBD (13). Taken together, these results suggest that genistein may interact with ER in a unique manner, different from other known estrogens. Whether this indicates SERM-like activity in vivo, is a very intriguing question. Are Phytoestrogens Antiestrogens? Genistein and other phytoestrogens have also been suggested to act as antiestrogens. This is a logical theory because phytoestrogens are weaker estrogens than endogenous steroidal estrogens and, in theory, could compete with the more potent estrogens for binding to ER, thus inhibiting their action. However, no convincing evidence exists for ER-mediated antiestrogenic activity of genistein or other isoflavonoid phytoestrogens. In transfected cells with an ERE-driven reporter gene construct, genistein did not block the action of E2, via either ERα or ERβ (10). Furthermore, in earlier studies with ERα-expressing breast cancer cells, genistein did not inhibit E2-induced cell proliferation, again indicating that genistein does not act as an antiestrogen (20). However, the possibility remains, that antiestrogenicity may occur via other mechanisms or in other estrogen-mediated processes. The assay systems used to date give information about the effects on only selected

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or artificial target genes under the control of classical estrogen response elements (ERE). In addition, there are other estrogen-responsive genes that are under the control of other response elements (such as AP-1). It has been shown before that the hormone response element, together with the ER subtype, may determine whether the very same ligand acts as an agonist or an antagonist (23). It is not yet known if such a phenomenon may occur in the presence of phytoestrogens. Moreover, effects of single compounds are likely to be different from the effects of phytoestrogen mixtures present in the diet. The four major soy-derived isoflavonoids (genistein, daidzein, biochanin A, and formonetin) all appear to act as ER agonists, and no clear evidence exists for antiestrogenic activity. In addition to isoflavonoids, there are multiple other phytochemicals that have been shown to interact with ER and act as antiestrogens in vitro (24–27). On the basis of current knowledge, it is not yet possible to predict the effects of complex phytoestrogen mixtures. Selective Recruitment of Steroid Receptor Coregulators by Isoflavonoid Phytoestrogens Interaction of nuclear receptors (including the ER) with their target genes is modulated by multiple proteins or protein complexes collectively called nuclear receptor coregulators, which may either enhance or repress transcription (coactivators and corepressors, respectively) (5). Which coregulators are involved in the process, is determined by many factors, including the ligand itself, i.e., different ligands may recruit different coregulators. Recent studies indicate that isoflavonoid phytoestrogens may recruit coregulators in a selective manner that is different from that of E2, and modulate both ERα- and ERβ-mediated transcription. An and co-workers (28) showed that genistein, daidzein, and biochanin A are ERβ-selective agonists causing transcriptional repression or activation via two different hormone response elements [tumor necrosis factor response element and ERE, respectively], and their ERβ-selectivity is associated with enhanced recruitment of the coregulator GRIP1. Wong and co-workers (29) reported that genistein promotes ERα interaction with some coactivators (SRC1 and SRC3), but exerts a minimal effect on interaction with others (DRIP205 and CBP), unlike E2, which enchances ERα interaction with all four coactivators.

Effects In Vivo Genistein Acts as an Estrogen In Vivo Several in vivo studies indicate that genistein exerts the typical estrogenic effects in many target organs of male and female rodents (30–32). The dose is critical, and in most cases, compared with E2, a hundred- or thousand-fold dose of genistein is required to elicit similar effects. These studies clearly indicate that genistein acts similarly to E2 in many target organs, but do not necessarily indicate that this is always the case because only a limited number of estrogen targets have been investigated.

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Furthermore, it is not yet known which ER subtype mediates the phytoestrogen effects in the different target organs. Studies with ER knockout mouse models (ERαKO, ERβKO) will help to clarify this question. Using ERαKO model, Klotz and co-workers (33) recently demonstrated that ERα is responsible for the activation of the uterine insulin-like growth factor 1 signaling pathway, an important regulator of uterine growth, by different estrogens, including genistein. ER-subtype–specific effects in other target organs remain to be investigated. Perhaps one of the most interesting questions in regard to the putative ERmediated adverse effects of phytoestrogens is the following: Is it possible that genistein, or any other phytoestrogen, would stimulate the growth of estrogenresponsive breast cancer cells in vivo, and/or antagonize the effect of antiestrogen therapy? Clearly, growth stimulation occurs in the rodent models with transplanted MCF-7 human breast cancer cells (34–37). The proliferative effect of genistein is likely to be mediated via ERα, as has been shown in vitro (11). As a further indication of genistein's ability to stimulate mammary cancer growth via ERα, genistein has been shown to enhance the growth of carcinogen-induced mammary tumors in normal mice, but not in ERαKO mice (38). It remains to be clarified whether the tumor growth stimulation could occur in humans consuming diets or natural products rich in phytoestrogens. Furthermore, it is not yet known how ERβ-expressing cells, which have been found in human breast tumors, would respond to genistein in vivo. This question is intriguing in the light of recent findings that indicate that expression of ERβ, and not only ERα, is a prognostic factor in breast cancer (39–43) and may play a role in the development of tamoxifen resistance (44). Is Genistein A Tissue-Selective Estrogen? On the basis of in vitro results it might be expected that genistein would act in a tissueor ER-subtype–selective manner. At least three studies indicate that such effects may occur in vivo. Very recently, Diel and coworkers (45) reported that orally administered genistein may induce selective effects in rat uterine cancer. In normal uterus, genistein acted in a manner similar to ethinyl estradiol (EE2), although a higher dose of genistein was required, and its effect was more moderate for most end points. However, the endometrial tumors responded differently to the two estrogens. As expected, EE2 stimulated the growth and induced the expression of estrogen-responsive genes of the tumors, known to be estrogen-responsive. Interestingly, genistein did not affect tumor growth, although it upregulated the expression of the marker genes. Whether the selective effect of genistein is related to ER-subtype selectivity is not yet known. Another interesting putative target for genistein is the vascular wall. Estrogens regulate the proliferation and migration of vascular smooth muscle cells (VSMC), and protect the vascular wall from trauma-induced intimal growth (i.e., formation of neointima) (46), which is thought to be one of the mechanisms by which estrogens may protect against cardiovascular diseases. Interestingly, after vascular trauma, the proliferating and migrating smooth muscle cells express high levels of

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ERβ, whereas the expression of ERα remains unaltered or low (47,48). Therefore, VSMC could be a likely site for ERβ-mediated actions. In ovariectomized rats, genistein was found to be active in the vascular wall at doses comparable to E2, whereas the uterotrophic responses were observed only in E2-treated animals (48). The vasculoprotective effect of genistein was also demonstrated in male and female rabbits (49). These results (48) indicate that proliferating and migrating VSMC, which express a high level of ERβ, may indeed be more sensitive to genistein, and responses in VSMC may occur at lower doses than the uterotrophic response, which is mediated primarily by ERα (4). Later, Ishimi and co-workers (50) reported that a similar phenomenon could be observed in rodent bone, i.e., the effect of genistein in bone occurs at lower doses compared with uterus.

Are Lignans Phytoestrogens? Plant lignans, such as secoisolariciresinol and matairesinol, are polyphenolic compounds present in fiber-rich plants. Plant lignans are converted to mammalian lignans, enterolactone (Fig. 11.1) and enterodiol, by gut microflora, and mammalian lignans are thought to be the bioactive forms of lignans (51). For many years, lignans have been classified as a subgroup of phytoestrogens, and have been claimed to act via ER. However, very little evidence exists for ER-mediated activity in vitro or in vivo. In two in vitro studies, mammalian lignans were shown to stimulate the proliferation of estrogen-sensitive breast cancer cells, or to induce the expression of the estrogen-sensitive pS2 gene (18,52). However, high concentrations (≥1 µmol/L) are required for these effects, and it is not known whether they are truly ER mediated. More recently, the transcriptional activities of enterolactone and enterodiol were investigated in a reporter gene assay, and no induction via either ERα or ERβ was observed at lignan concentrations <10 µmol/L (53). When the test compounds were added together with E2, no effect was seen at any concentration. Furthermore, no convincing evidence exists for ER-mediated activity of purified lignans in vivo. The mammalian lignan enterolactone was reported to be inactive in a mouse uterus bioassay (54), and a 4-wk oral exposure to hydroxymatairesinol, a plant lignan, did not cause any estrogen-like effects in male rats (53). Ward and co-workers (55) reported that lactational exposure to secoisolariciresinoldiglycoside (SDG), a plant lignan isolated from flaxseed, modulates mammary gland development in female rats, but in a more recent study, they did not find any alterations in the reproductive indices of female or male rats after oral life-long exposure to SDG (56).

Summary and Conclusions Several in vitro studies indicate that isoflavonoid, flavonoid, and coumestan phytoestrogens interact with both ER subtypes, and regulate the expression of estrogenresponsive genes via ERα and ERβ. Among individual compounds, marked differences exist in the biological activities, i.e., both agonistic and antagonistic effects have

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been observed, and some compounds that are currently classified as phytoestrogens (i.e., lignans) appear not to interact with ER. The binding affinity to ER and the capability to induce ER-mediated transcriptional activity vary greatly among the subgroups, in the following order: coumestans ≥ isoflavonoids >> flavonoids (1,2). Furthermore, marked differences exist among the individual compounds in the same subgroup, indicating that only minor differences in the chemical structure may result in significant changes in estrogenic activity. Interestingly, some phytoestrogens, (e.g., genistein), show higher binding affinity to ERβ than ERα, and, at low concentrations, may induce more pronounced transcriptional activity via ERβ. It has been suggested that genistein acts as a "natural SERM," on the basis of observations on its unique interaction with ERβ ligand-binding domain and selective recruitment of nuclear receptor coregulators in vitro (5–8). Only a limited number of compounds have been tested in vivo in experimental animals. These studies indicate that isoflavonoids and coumestans can induce typical estrogen-like effects in many estrogen target organs. At present, very little is known about the ER-subtype–specific actions of phytoestrogen, but it is likely that effects may occur via both ER subtypes. At a low dose range, isoflavonoids may induce tissueselective effects, but it is not yet known whether this is related to ERβ-mediated effects. Currently, no information about the effects of purified phytoestrogens exists for humans. On the basis of the experimental findings, both beneficial and adverse effects are possible, but there is no firm evidence for either. Epidemiologic studies suggest that life-long consumption of phytoestrogen-rich diets may protect against hormone-dependent diseases, such as breast cancer, but the causal relationship between dietary phytoestrogens and reduced disease risks remains to be established. On the other hand, experimental studies clearly indicate that isoflavonoids stimulate the growth of estrogen-responsive breast cancer cells, and that this effect is likely to occur via ERα. Phytoestrogen-rich foods and/or natural products are increasingly consumed by adult Western women, and it is of critical importance to know whether phytoestrogens stimulate the growth of human breast cancer and/or interfere with antihormone therapy. It should be noted, however, that human breast cancers also commonly express ERβ, not only ERα, and therefore multiple effects are possible, depending on the ER expression pattern of the tumor. More profound knowledge on the mechanisms of estrogen action in general, and the specific roles of ER subtypes in particular, is required to improve our understanding of the actions of phytoestrogens in different estrogen target tissues. Whether the ER-mediated actions of phytoestrogens demonstrated under experimental conditions occur in humans consuming phytoestrogen-rich diets and play a role in the development of hormone-related diseases remain to be determined. References 1. Jensen, E.V., Jacobsen, H.I., (1962) Basic Guides to the Mechanism of Estrogen Action, Rec. Prog. Horm. Res. 18, 387–414.

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2. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.-Å. (1996) Cloning of a Novel Receptor Expressed in Rat Prostate and Ovary, Proc. Natl. Acad. Sci. U S A 93, 5925–5930. 3. Mosselman, S., Polman, J., and Dijkema, R. (1996) ER Beta: Identification and Characterization of a Novel Human Estrogen Receptor, FEBS Lett. 392, 49–53. 4. Couse, J.F., Curtis, S.W. Hewitt, S., and Korach, K.S. (2000) Receptor Null Mice Reveal Contrasting Roles for Estrogen Receptor Alpha and Beta in Reproductive Tissues, J. Steroid Biochem. Mol. Biol. 74, 287–296. 5. Nilsson, S., Mäkelä, S., Treuter, E., Tujague, M., Thomsen, J., Andersson, G., Enmark, E., Pettersson, K., Warner, M., and Gustafsson, J.-Å. (2001) Mechanisms of Estrogen Action, Physiol. Rev. 81, 1535–1565. 6. Martin, P.M., Horwitz, K.B., Ryan, D.S., and McGuire, W.L. (1978) Phytoestrogen Interaction with Estrogen Receptors in Human Breast Cancer Cells, Endocrinology 103, 1860–1867. 7. Shutt, D.A., and Cox, R.I. (1972) Steroid and Phyto-Oestrogen Binding to Sheep Uterine Receptors In Vitro, J. Endocrinol. 52, 299–310. 8. Verdeal, K., Brown, R.R., Richardson, T., and Ryan, D.S. (1980) Affinity of Phytoestrogens for Estradiol-Binding Proteins and Effect of Coumestrol on Growth of 7,12-Dimethylbenz[a]anthracene-Induced Rat Mammary Tumors, J. Natl. Cancer Inst. 64, 285–290. 9. Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., and Gustafsson, J.-Å. (1997) Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors Alpha and Beta, Endocrinology 138, 863–870. 10. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.-Å. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor Beta, Endocrinology 139, 4252–4263. 11. Maggiolini, M., Bonofiglio, D., Marsico, S., Panno, M.L., Cenni, B., Picard, D., and Ando, S. (2001) Estrogen Receptor Alpha Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Two Major Phytoestrogens on Human Breast Cancer Cells, Mol. Pharmacol. 60, 595–602. 12. Morito, K., Hirose, T., Kinjo, J., Hirakawa, T., Okawa, M., Nohara, T., Ogawa, S., Inoue, S., Muramatsu, M., and Masamune, Y. (2001) Interaction of Phytoestrogens with Estrogen Receptors Alpha and Beta, Biol. Pharm. Bull. 24, 351–356. 13. Pettersson, K., Delaunay, F., and Gustafsson, J.-Å.(2000) Estrogen Receptor Beta Acts as a Dominant Regulator of Estrogen Signaling, Oncogene 19, 4970–4978. 14. Mäkelä, S., Davis, V.L., Tally, W.C., Korkman, J., Salo, L., Vihko, R., Santti, R., and Korach, K.S. (1994) Dietary Estrogens Act Through Estrogen Receptor-Mediated Processes and Show No Antiestrogenicity in Cultured Breast Cancer Cells, Environ. Health Perspect. 102, 572–578. 15. Mayr, U., Butsch, A., and Schneider, S. (1992) Validation of Two In Vitro Test Systems for Estrogenic Activities with Zearalenone, Phytoestrogens and Cereal Extracts, Toxicology 74, 135–149. 16. Miksicek, R.J. (1993) Commonly Occurring Plant Flavonoids Have Estrogenic Activity, Mol. Pharmacol. 44, 37–43. 17. Miksicek, R.J. (1994) Interaction of Naturally Occurring Nonsteroidal Estrogens with Expressed Recombinant Human Estrogen Receptor, J. Steroid Biochem. Mol. Biol. 49, 153–160.

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34. Allred, C.D., Allred, K.F., Ju, Y.H., Virant, S.M., and Helferich, W.G. (2001) Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-Dependent (MCF-7) Tumors in a Dose-Dependent Manner, Cancer Res. 61, 5045–5050. 35. Allred, C.D., Ju, Y.H., Allred, K.F., Chang, J., and Helferich, W.G. (2001) Dietary Genistin Stimulates Growth of Estrogen-Dependent Breast Cancer Tumors Similar to That Observed with Genistein, Carcinogenesis 22, 1667–1673. 36. Hsieh, C.Y., Santell, R.C., Haslam, S.Z., and Helferich, W.G. (1998) Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-Positive Human Breast Cancer (MCF-7) Cells In Vitro and In Vivo, Cancer Res. 58, 3833–3838. 37. Ju, Y.H., Allred, C.D., Allred, K.F., Karko, K.L., Doerge, D.R., and Helferich, W.G. (2001) Physiological Concentrations of Dietary Genistein Dose-Dependently Stimulate Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Athymic Nude Mice, J. Nutr. 131, 2957–2962. 38. Day, J.K., Besch-Williford, C., McMann, T.R., Hufford, M.G., Lubahn, D.B., and MacDonald, R.S. (2001) Dietary Genistein Increased DMBA-Induced Mammary Adenocarcinoma in Wild-Type, but Not ER α KO Mice, Nutr. Cancer 39, 226–232. 39. Järvinen, T.A., Pelto-Huikko, M., Holli, K., and Isola, J. (2000) Estrogen Receptor β Is Coexpressed with ERa and PR and Associated with Nodal Status, Grade, and Proliferation Rate in Breast Cancer, Am. J. Pathol. 156, 29–35. 40. Mann, S., Laucirica, R., Carlson, N., Younes, P.S., Ali, N., Younes, A., Li, Y., and Younes, M. (2001) Estrogen Receptor Beta Expression in Invasive Breast Cancer, Hum. Pathol. 32, 113–118. 41. Omoto, Y., Inoue, S., Ogawa, S., Toyama, T., Yamashita, H., Muramatsu, M., Kobayashi, S., and Iwase, H. (2001) Clinical Value of the Wild-Type Estrogen Receptor Beta Expression in Breast Cancer, Cancer Lett. 163, 207–212. 42. Roger, P., Sahla, M.E., Mäkelä, S., Gustafsson, J.-Å., Baldet, P., and Rochefort, H. (2001) Decreased Expression of Estrogen Receptor Beta Protein in Proliferative Preinvasive Mammary Tumors, Cancer Res. 61, 2537–2541. 43. Speirs, V., Parkes, A.T., Kerin, M.J., Walton, D.S., Carleton, P.J., Fox, J.N., and Atkin, S.L. (1999) Coexpression of Estrogen Receptor Alpha and Beta: Poor Prognostic Factors in Human Breast Cancer? Cancer Res. 59, 525–528. 44. Speirs, V., Malone, C., Walton, D.S., Kerin, M.J., and Atkin, S.L. (1999) Increased Expression of Estrogen Receptor Beta mRNA in Tamoxifen-Resistant Breast Cancer Patients, Cancer Res. 59, 5421–5424. 45. Diel, P., Smolnikar, K., Schulz, T., Laudenbach-Leschowski, U., Michna, H., and Vollmer, G. (2001) Phytoestrogens and Carcinogenesis-Differential Effects of Genistein in Experimental Models of Normal and Malignant Rat Endometrium, Hum. Reprod. 16, 997–1006. 46. Mendelsohn, M.E., and Karas, R.H. (1999) The Protective Effects of Estrogen on the Cardiovascular System, N. Engl. J. Med. 340, 1801–1811. 47. Lindner, V., Kim, S.K., Karas, R.H., Kuiper, G.G., Gustafsson, J.-Å., and Mendelsohn, M.E. (1998) Increased Expression of Estrogen Receptor-Beta mRNA in Male Blood Vessels After Vascular Injury, Circ. Res. 83, 224–229. 48. Mäkelä, S., Savolainen, H., Aavik, E., Myllärniemi, M., Strauss, L., Taskinen, E., Gustafsson, J.-Å., and Häyry, P. (1999) Differentiation Between Vasculoprotective and Uterotrophic Effects of Ligands with Different Binding Affinities to Estrogen Receptors Alpha and Beta, Proc. Natl. Acad. Sci. U S A 96, 7077–7082.

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49. Finking, G., Wohlfrom, M., Lenz, C., Wolkenhauer, M., Eberle, C., and Hanke, H. (1999) The Phytoestrogens Genistein and Daidzein, and 17 β-Estradiol Inhibit Development of Neointima in Aortas from Male and Female Rabbits In Vitro After Injury, Coron. Artery Dis. 10, 607–615. 50. Ishimi, Y., Arai, N., Wang, X., Wu, J., Umegaki, K., Miyaura, C., Takeda, A., and Ikegami, S. (2000) Difference in Effective Dosage of Genistein on Bone and Uterus in Ovariectomized Mice, Biochem. Biophys. Res. Commun. 274, 697–701. 51. Adlercreutz, H., and Mazur, W. (1997) Phyto-Oestrogens and Western Diseases, Ann. Med. 29, 95–120. 52. Welshons, W.V., Murphy, C.S., Koch, R., Calaf, G., and Jordan, V.C. (1987) Stimulation of Breast Cancer Cells In Vitro by the Environmental Estrogen Enterolactone and the Phytoestrogen Equol, Breast Cancer Res. Treat. 10, 169–175. 53. Saarinen, N.M., Wärri, A., Mäkelä, S.I., Eckerman, C., Reunanen, M., Ahotupa, M., Salmi, S.M., Franke, A.A., Kangas, L., and Santti, R. (2000) Hydroxymatairesinol, a Novel Enterolactone Precursor with Antitumor Properties from Coniferous Tree (Picea abies), Nutr. Cancer 36, 207–216. 54. Setchell, K.D., Lawson, A.M., Borriello, S.P., Harkness, R., Gordon, H., Morgan, D.M., Kirk, D.N., Adlercreutz, H., Anderson, L.C., and Axelson, M. (1981) Lignan Formation in Man—Microbial Involvement and Possible Roles in Relation to Cancer, Lancet 2(8236), 4–7. 55. Ward, W.E., Jiang, F.O., and Thompson, L.U. (2000) Exposure to Flaxseed or Purified Lignan During Lactation Influences Rat Mammary Gland Structures, Nutr. Cancer 37, 1871–92. 56. Ward, W.E., Chen, J., and Thompson, L.U. (2001) Exposure to Flaxseed or Its Purified Lignan During Suckling Only or Continuously Does Not Alter Reproductive Indices in Male and Female Offspring, J. Toxicol. Environ. Health A 64, 567–577.

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Chapter 12

Effects of Phytoestrogens on Bone Cells: Genomic and Nongenomic Mechanisms Xiaowei Chen and John J.B. Anderson Department of Nutrition, Schools of Public Health and Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

Introduction Postmenopausal osteoporosis, one of the major health problems in women, results in part from endogenous estrogen deficiency (1), but the roles of estrogens and estrogen-like molecules, including phytoestrogens, in regulating bone cell activities require clarification. Then, the etiology, prevention, and treatment of this disease can be based on more solid evidence (2). Although estrogen replacement therapy (ERT) has been a major approach used in treating or preventing postmenopausal osteoporosis, ERT may increase the risk of endometrial and breast cancer and has other unwanted side effects (3). Therefore, the naturally occurring estrogen-like molecules, i.e., phytoestrogens, have been investigated for their potential roles in the prevention and treatment of osteoporosis and other chronic diseases in recent years (4). According to their structures and health effects, phytoestrogens in food can be divided into three major groups, i.e., isoflavones, lignans, and coumestans. The major dietary sources of phytoestrogens in Asian populations are isoflavones from soybeans and soy products. For example, the dietary intake of phytoestrogens for a Japanese adult is between 20 and 80 mg/d (5). Compared with Asians, Western populations have much lower intakes of dietary phytoestrogens (<1 mg/d), lignans and isoflavones (6). This review is focused more on the biological effects of isoflavones on bone cells rather than the other phytoestrogens because reports on lignans and coumestans remain limited. Data from young ovariectomized animal models have consistently demonstrated an improvement in bone mass after isoflavone treatments (7–17), and a few reports of human studies also show that dietary isoflavones at sufficiently high doses may improve bone mass in both periand postmenopausal women (18,19) (see chapters on bone in this monograph). The cell mechanisms by which these molecules maintain or improve bone retention, however, remain uncertain. The purpose of this review is to provide current evidence for the direct actions of phytoestrogens on osteoblasts and osteoclasts. Three reviews on the relationships between phytoestrogens and bone loss were published previously (2,20,21). The major topics covered in this chapter are the mechanisms of action of isoflavones and other phytoestrogens at the cellular and molecular levels.

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Actions at the Cellular Level Many bone cell lines have been used in culture to test the effects of isoflavones on bone functions that relate to remodeling in animal or human studies. Results from in vitro studies have established that osteoblast/osteoclast-like cells respond directly to isoflavones. Depending on the concentrations used, isoflavones modulate cell proliferation and cell differentiation. Also, isoflavones have been shown to display antioxidant effects against reactive oxygen species (ROS), which adversely affect osteoblast-like cells. Effects of Isoflavones on Osteoblasts. Investigations of the effects of isoflavones, at dietarily related concentrations, on osteoblastic phenotype development remain limited. One group of researchers (22–24) reported that genistein and daidzein had an anabolic effect on MC3T3-E1 cells, an osteoblast-like cell line derived from newborn mouse calvariae (25). In their studies, osteoblastic MC3T3-E1 cells were cultured for 48 h in the presence of genistein and daidzein (10–7–10–5 mol/L). Genistein and daidzein caused a significant elevation of protein content, alkaline phosphatase (ALP) activity, and DNA content in the cells. In addition, 3H-leucyltRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein or daidzein in the enzyme reaction mixture. Genistein and daidzein increased protein content, and ALP activity in the cells was clearly abolished by the presence of an antiestrogen, tamoxifen (10–6 mol/L). Elevations of protein and ALP activity by 17β-estradiol (10–9 mol/L) were also found in these cell lines. The investigators suggested that the effects of isoflavones (both genistein and daidzein) on osteoblastic cells might, at least in part, involve estrogen receptor (ER)-dependent activities. Chen et al. (26,27) examined the effects of genistein, at dietarily achievable concentrations (10–10 to 10–8 mol/L), on biomarkers synthesized by MC3T3-E1 osteoblast-like cells. Genistein had little effect on ALP and osteocalcin production at d 12 of culture. Genistein treatment, however, significantly decreased interleukin-6 (IL-6) production by ~40–70% in both a dose- and time-dependent manner over a 12-d period; it also increased the ratio of osteoprotegerin (OPG) to the receptor activator of nuclear factor (NF)-κB ligand (RANKL) mRNA more than twofold (26,27). These findings suggest that part of the beneficial effects of genistein on bone mass retention may be mediated by inhibition of osteoclastogenesis through altering the expression of IL-6 and the ratio of OPG to RANKL. Dang et al. (28) reported that genistein at concentrations of 10–6 or 10–7 mol/L stimulated osteoblastogenesis, but inhibited adipogenesis, in preosteoblastic KS483 cells via ER-dependent pathways. In that study, genistein at 10–6 mol/L led to a twofold increase in DNA content and a threefold increase in ALP activity, nodule formation, and calcium content. These stimulatory effects by genistein were completely blocked by the antiestrogen, ICI 164,384. In addition, Choi et al. (29) investigated the effect of a soybean ethanol extract on MC3T3-E1 cells and found that soy extract increased MC3T3-E1 cell viabilities and

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DNA synthesis in a dose-dependent manner. Soy extract was also found to increase ALP activity and collagen synthesis. Additionally, tamoxifen, an antiestrogen, abolished the effects of soy extract on the MC3T3-E1 cell proliferation, ALP activity, and collagen synthesis. Soy extract has been suggested to have direct effects on bone formation in cultured osteoblast-like cells, and these effects may be ER-dependent. For the research on antioxidant effects of isoflavones, a recent study reported that genistein protects against oxidative cellular damage of free radicals in osteoblast-like cells (30). In this research, MC3T3-E1 cells were treated with oxidative catalysts, H2O2 and FeSO4, in combination with genistein. The damage, i.e., decrease in cell viability, caused by free radicals was significantly reduced in cells treated with genistein at physiologic concentrations. Effects of Isoflavones on Osteoclasts. Direct inhibitory effects of isoflavones have been demonstrated on osteoclasts in vitro, and these actions are believed not to be ER-dependent because mammalian osteoclasts are generally considered not to express ER. The suppressive effect of genistein (10–7–10–5 mol/L) on osteoclastlike multinucleated cells from rat femoral tissues has been reported by Gao and coworkers (31–33). Also, genistein was found to modulate the expression of mRNAs for osteoclast differentiation factor and OPG in osteogenic stromal cells in osteoclastogenesis; this regulation was shown to involve the topoisomerase II pathway (34). These studies suggest that the suppressive effect of genistein on bone osteoclasts results in part from its interference with specific enzymatic systems, such as the tyrosine kinase activity of epidermal growth factor receptor protein, i.e., a nongenomic mechanism. In contrast to these effects of genistein, at least one study, using a pit formation assay, found that daidzein stimulated osteoclastic resorption at a concentration of 10–8–10–10 mol/L (35). In some studies, genistein, the isoflavone most commonly tested, has been used as a tyrosine kinase inhibitor to investigate the signal transduction related to tyrosine phosphorylation during osteoblastic cell proliferation, but the concentrations of genistein in those investigations were in the pharmacologic range (~10–5–10–3 mol/L). For example, one study reported that genistein inhibited the influence of type I collagen on gene expression in osteoblast-like cells. Treatment of osteoblastic UMR106-06 cells with genistein abolished, in a dose-dependent manner, the effects of collagen on the expression of parathyroid hormone/parathyroid hormone–related protein receptor, ALP, and OP mRNA. The authors concluded that a type I collagen substrate influences the expression of osteoblast-associated genes in a cell model of mature osteoblasts and suggested that this action involves, at least in part, changes in intracellular tyrosine phosphorylation (36). Another study also showed that genistein exerted its inhibiting effects on resorbing activities in rat osteoclasts through a tyrosine kinase inhibitor mechanism (37). Effects of Coumestans and Lignans on Bone Cells. One study of oophorectomized rats by Draper et al. (38) showed that coumestrol (1.5 µmol, intramuscularly injected twice per week) significantly reduced bone loss at all measured bone sites, and also

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reduced urinary calcium excretion and bone resorption markers after 1 wk of treatment, compared with the control group. Dodge et al. (39) also found that oral coumestrol spared bone loss that was caused by estrogen deficiency in ovariectomized rats. In an in vitro study, KCA-098, an analog of coumestrol, was found to increase, in a dose-dependent manner, ALP activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvariae (40). In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells (40). KCA-098 was also found to stimulate the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats (41–43), but it had no effect on the basal synthesis of osteocalcin. In addition, KCA-098 inhibited the formation of osteoclasts in cultures of mouse bone marrow cells, previously treated with 1α,25(OH)2D3, parathyroid hormone, and prostaglandin E2 (40). Coumestrol had no effect on the proliferation and ALP activity of ROS 17/2.8 cells (40). However, coumestrol increased the calcium content of 9-d-old chick embryonic femurs in organ culture, and dose dependently inhibited osteoclast-like cell formation in a manner similar to KCA-098 (40,44). Colonic bacteria can convert plant lignan secoisolariciresinol diglycoside to two major mammalian lignans, enterodiol (ED) and enterolactone (EL) (45). Both ED and EL can produce biological effects similar to estrogen, and exposure to lignans during the early stages (birth through postnatal d 21) reduces bone strength in young but not in older male rats (46). No in vitro study, however, has been performed to explain this finding. Summary of Actions at the Cellular Level. At high concentrations (10–5–10–3 mol/L), genistein, the most common isoflavone studied, acts mainly as a tyrosine kinase or topoisomerase II inhibitor, thereby inhibiting cell growth, arresting cell cycle progression at G2-M, and inducing apoptosis. At lower “more physiologic” concentrations (10–8–10–6 mol/L), which are closer to dietarily achievable levels, genistein appears to function more as a weak estrogenic agonist via ER (47,48). These speculations are supported by the important finding of a biphasic response of ALP synthesis by osteoblast-like cells over a wide range of concentrations of isoflavones (Fig. 12.1) (7,49). Actions at the Molecular Level The basic structure of isoflavones consists of two benzene rings, to which one or more hydroxyl groups (–OH) may be found attached. A heterocyclic pyrane ring is located between these two benzene rings. The chemical structures of genistein and other isoflavones are compared with the structure of 17β-estradiol, one of the most potent estrogens, in Fig. 12.2. Generally, the structures of isoflavones and estradiol share two characteristics, i.e., both have an aromatic ring with a hydroxyl group, and both have a nearly identical distance between two hydroxyl groups (50). Therefore, isoflavones are thought to bind to the ER (α and β) because of their structural similarity to estradiol. Compared with true estrogens, however, the isoflavones have much lower binding

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Fig. 1. Effects of genistein (Gen) and 17β-estradiol (E2) on the alkaline phosphatase activity of ROS.SMER-14 osteoblast-like cells in culture. (Adapted from Ref. 2.)

affinities to ER (Table 12.1) (51–53). In addition, the binding affinities of isoflavones to ER are ER subtype–dependent. For example, genistein has a much higher binding affinity to ERβ than ERα (54). These findings help explain the selective effects of isoflavones in different estrogen-responsive tissues, a topic discussed in the next section. Although the interaction of isoflavones with ER may explain many effects of these compounds on bone cell, isoflavones also can function via non-ER–dependent pathways. These mechanisms are reviewed in the following sections. Estrogen Receptor–Mediated Mechanism. ER respond to both true estrogens and isoflavones, as well as to other environmental estrogen-like molecules. The isoflavones have higher binding affinities for the ER in mammalian cells than most other estrogen-like molecules but lower than for 17β-estradiol (see Table 12.1).

Fig. 2. Chemical structures of 17β-estradiol and major isoflavones. (Adapted from Ref.

50.)

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TABLE 12.1 Relative Binding Affinity to Estrogen Receptor (ER)α and ERβ and Relative Potencies in Inducing Alkaline Phosphatase Between 17β-Estradiol and Isoflavones Relative binding affinity Molecule 17β-estradiol Genistein Daidzein

ERαb

ERβc

Relative potencya

1 0.0025 0.0010

1 0.15 NAd

1.0 0.0008 0.00013

aSource:

Reference 53. Reference 51. cSource: Reference 52. dNot available. bSource:

When isoflavones reach the target tissue, isoflavones are believed to cross the cell membrane by passive diffusion; they then bind to the ER in the cytosol and form an isoflavone-ER complex; this complex then translocates into the nucleus for activation of the estrogen response element (ERE), which is involved in the regulation of DNA-directed mRNA synthesis and the production of new proteins. This sequence represents the classical steroid hormone action at the cellular and molecular levels. It was demonstrated recently that estrogen can act through the so-called “nonclassical” mechanism (55), but no study has yet been done to test this mechanism in the action of isoflavone. A good example is the regulation of IL-6 synthesis in the fibroblasts and hematopoietic cell lines (56,57). The inhibition of IL-6 expression by estradiol results from an indirect action via nuclear transcription factors, which involves the ER but not its direct interaction with the ERE on the DNA (56,57). ER of both subtypes are found in osteoblasts (54,58,59), but their presence in mammalian osteoclasts remains controversial. In addition to secreting bone formation–related proteins, such as ALP and osteocalcin, osteoblasts are also capable of synthesizing many other cytokines. These cytokine products (e.g., IL-6 and OPG) have been demonstrated to have critical roles in the regulation of osteoclast differentiation and activities (60,61). Isoflavones may therefore exert indirect effects on osteoclasts by mediating cytokine production in osteoblasts. The net effect of these actions of isoflavones results in inhibition of osteoclast differentiation and bone resorption activities, including reduction of their numbers. The discovery of a new subtype of ER, ERβ (58), has raised a number of questions regarding the respective physiologic roles that these two receptors, ERα and ERβ, may play in an estrogen-responsive tissue. This new receptor may help explain the selective effects of estrogen-like molecules in different tissues. A recent report indicates that genistein may differentially bind to ERα and ERβ receptors, acting as a partial agonist with ERβ receptors (62). At this point, understanding of the relationship between genistein stimulation and ER expression is based on limited data, and the roles of ER in the activation of gene transcription in

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osteoblasts may be cell type- and promoter-dependent, as suggested previously (63). Because of the different binding affinity to ERα and β, isoflavones may act differently on the two ER. In addition, not all isoflavones, such as genistein compared with daidzein, are equally effective. A recent report, for example, suggested that daidzein is more effective in conserving bone than genistein in rodent studies (16). Non-ER-Mediated Mechanism. In addition to the ER-dependent pathway, some isoflavones, such as genistein, can also bind with membrane receptors and function as tyrosine kinase inhibitors (64) and topoisomerase II inhibitors (34). In this way, isoflavones influence cell cycle and metabolism through second messengers in the cytoplasm (Fig. 12.3). In addition, isoflavones also act as antioxidants by protecting unsaturated fatty acids, nucleic acids, and proteins against oxidant damages (30). Isoflavones likely have multiple roles in osteoblastic cells. Summary of Actions at the Molecular Level. Cellular and molecular investigations of the effects of isoflavones, at dietarily or supplementally achievable concentrations, on phenotypic development of osteoblasts remain limited. Most stud-

Protein Products (e.g., ALP, IL-6, or OPG)

Fig. 3. The mechanisms of action of isoflavones on osteoblastic cells using genistein as the model. (1) Estrogen receptor (ER)-dependent pathway: genistein is taken up by passive diffusion across the cell membrane and acts as a weak agonist via the classical estrogen mechanism. Typical protein products synthesized and secreted by osteoblasts, such as IL-6, are inhibited by the action of genistein. (2) Non-ER-dependent pathway: isoflavones interact with membrane receptors and function as tyrosine kinase inhibitors or topoisomerase II inhibitors: then, isoflavones influence cell cycle and metabolism through second messengers in the cytoplasm. In addition, isoflavones also acts as antioxidants by protecting unsaturated fatty acids, nucleic acids, and proteins against oxidant damages (not shown). Abbreviations: ALP, alkaline phosphatase; IL-6, interleukin-6; OPG, osteoprotegerin.

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ies of osteoblasts used genistein as a tyrosine kinase inhibitor to investigate the roles of second messengers in osteoblastic cell mitogenesis; the concentrations of genistein used in these investigations were in the pharmacologic range (~10–4– 10–3 mol/L). At lower, more physiologic concentrations (10–10–10–6 mol/L), which are closer to dietarily achievable levels, genistein appears to function more as a weak estrogen agonist via ER rather than as a tyrosine kinase or topoisomerase II inhibitor.

Summary and Conclusions The positive effects of isoflavone-rich supplements and soy foods enriched with these molecules on bone tissue in young ovariectomized rodent models indicate that these isoflavones may have similar beneficial effects in humans. In humans, the beneficial effects on bone have been observed only in woman with low circulating estrogen concentrations. Too few data are available for lignans and coumestans to reach conclusions on their effects on bone. Cellular mechanisms for the functions of isoflavones remain incompletely understood. The discovery of a new isoform of the ER, ERβ, suggests that the molecular regulation of bone remodeling by estrogens, or estrogen-like molecules, is more complicated than previously thought. Such work may help stimulate scientists to uncover the roles played by isoflavones in bone tissue, especially those involved in maintaining a healthy balance between the activities of osteoblasts and osteoclasts. Because of the structural similarities between isoflavones and true estrogens, isoflavones are able to bind to the ER even though they have much lower binding affinities. In addition, the small structural differences between estrogens and isoflavones may help explain the selective effects of isoflavones in different estrogen-responsive tissues. Isoflavones may benefit bone tissues with no adverse effects on other organs, such as breast. In the past, most cell studies have focused on the effects of isoflavones on bone formation markers, but the actions of isoflavones in stimulating the synthesis and secretion of other important osteoclastogenesis-related proteins, including IL6, OPG, and RANKL, have been largely ignored. Results from future experimental work should provide improved understanding of the roles of isoflavones on osteoblasts at the molecular and cellular levels and help advance knowledge of the process of bone remodeling. References 1. Albright, F., Bloomberg, F., and Smith, P.H. (1940) Postmenopausal Osteoporosis, Trans. Assoc. Am. Physicians 55, 298–305. 2. Anderson, J.J.B., and Garner, S.C. (1998) Phytoestrogens and Bone, Baillieres Clin. Endocrinol. Metabol. 12, 543–557. 3. Vihtamaki, T., Savilahti, R., and Tuimala, R. (1999) Why Do Postmenopausal Women Discontinue Hormone Replacement Therapy? Maturitas 33, 99–105.

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4. Anderson, J.J.B., Anthony, M., Messina, M., and Garner, S.C. (1999) Effects of PhytoOestrogen on Tissues, Nutr. Res. Rev. 12, 75–116. 5. Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T., Hamalainen, E., Hasegawa, T., and Okada, H. (1991) Urinary Excretion of Lignans and Isoflavonoid Phytoestrogens in Japanese Men and Women Consuming a Traditional Japanese Diet, Am. J. Clin. Nutr. 54, 1093–1100. 6. de Kleijn, M.J., van der Schouw, Y.T., Wilson, P.W., Adlercreutz, H., Mazur, W., Grobbee, D.E., and Jacques, P.F. (2001) Intake of Dietary Phytoestrogens Is Low in Postmenopausal Women in the United States: The Framingham Study (1–4), J. Nutr. 131, 1826–1832. 7. Anderson, J.J., Ambrose, W.W., and Garner, S.C. (1998) Biphasic Effects of Genistein on Bone Tissue in the Ovariectomized, Lactating Rat Model, Proc. Soc. Exp. Biol. Med. 217, 345–350. 8. Anderson, J.J.B., Ambrose, W.W., and Garner, S.C. (1995) Orally Dosed Genistein from Soy and Prevention of Cancellous Bone Loss in Two Ovariectomized Rat Models, J. Nutr. 125, 799S (abstr.). 9. Arjmandi, B.H., Birnbaum, R., Goyal, N.V., Getlinger, M.J., Juma, S., Alekel, L., Hasler, C.M., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Bone-Sparing Effect of Soy Protein in Ovarian Hormone-Deficient Rats Is Related to Its Isoflavone Content, Am. J. Clin. Nutr. 68, 1364S–1368S. 10. Arjmandi, B.H., Getlinger, M.J., Goyal, N.V., Alekel, L., Hasler, C.M., Juma, S., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Role of Soy Protein with Normal or Reduced Isoflavone Content in Reversing Bone Loss Induced by Ovarian Hormone Deficiency in Rats, Am. J. Clin. Nutr. 68, 1358S–1363S. 11. Blair, H.C., Jordan, S.E., Peterson, T.G., and Barnes, S. (1996) Variable Effects of Tyrosine Kinase Inhibitors on Avian Osteoclastic Activity and Reduction of Bone Loss in Ovariectomized Rats, J. Cell. Biochem. 61, 629–637. 12. Fanti, P., Monier-Faugere, M.C., Geng, Z., Schmidt, J., Morris, P.E., Cohen, D., and Malluche, H.H. (1998) The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats, Osteoporos. Int. 8, 274–281. 13. Ishida, H., Uesugi, T., Hirai, K., Toda, T., Nukaya, H., Yokotsuka, K., and Tsuji, K. (1998) Preventive Effects of the Plant Isoflavones, Daidzin and Genistin, on Bone Loss in Ovariectomized Rats Fed a Calcium-Deficient Diet, Biol. Pharm. Bull. 21, 62–66. 14. Ishimi, Y., Arai, N., Wang, X., Wu, J., Umegaki, K., Miyaura, C., Takeda, A., and Ikegami, S. (2000) Difference in Effective Dosage of Genistein on Bone and Uterus in Ovariectomized Mice, Biochem. Biophys. Res. Commun. 274, 697–701. 15. Ishimi, Y., Miyaura, C., Ohmura, M., Onoe, Y., Sato, T., Uchiyama, Y., Ito, M., Wang, X., Suda, T., and Ikegami, S. (1999) Selective Effects of Genistein, a Soybean Isoflavone, on B-Lymphopoiesis and Bone Loss Caused by Estrogen Deficiency, Endocrinology 140, 1893–1900. 16. Picherit, C., Coxam, V., Bennetau-Pelissero, C., Kati-Coulibaly, S., Davicco, M.J., Lebecque, P., and Barlet, J.P. (2000) Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats, J. Nutr. 130, 1675–1681. 17. Picherit, C., Chanteranne, B., Bennetau-Pelissero, C., Davicco, M.J., Lebecque, P., Barlet, J.P., and Coxam, V. (2001) Dose-Dependent Bone-Sparing Effects of Dietary Isoflavones in the Ovariectomised Rat, Br. J. Nutr. 85, 307–316. 18. Alekel, D.L., Germain, A.S., Peterson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T.

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35. Tobe, H., Komiyama, O., Komiyama, Y., and Maruyama, H.B. (1997) Daidzein Stimulation of Bone Resorption in Pit Formation Assay, Biosci. Biotechnol. Biochem. 61, 370–371. 36. Celic, S., Katayama, Y., Chilco, P.J., Martin, T.J., and Findlay, D.M. (1998) Type I Collagen Influence on Gene Expression in UMR106-06 Osteoblast-Like Cells Is Inhibited by Genistein, J. Endocrinol. 158, 377–388. 37. Kajiya, H., Okabe, K., Okamoto, F., Tsuzuki, T., and Soeda, H. (2000) Protein Tyrosine Kinase Inhibitors Increase Cytosolic Calcium and Inhibit Actin Organization as Resorbing Activity in Rat Osteoclasts, J. Cell. Physiol. 183, 83–90. 38. Draper, C.R., Edel, M.J., Dick, I.M., Randall, A.G., Martin, G.B., and Prince, R.L. (1997) Phytoestrogens Reduce Bone Loss and Bone Resorption in Oophorectomized Rats, J. Nutr. 127, 1795–1799. 39. Dodge, J.A., Glasebrook, A.L., Magee, D.E., Phillips, D.L., Sato, M., Short, L.L., and Bryant, H.U. (1996) Environmental Estrogens: Effects on Cholesterol Lowering and Bone in the Ovariectomized Rat, J. Steroid Biochem. Mol. Biol. 59, 155–161. 40. Kawashima, K., Inoue, T., Tsutsumi, N., and Endo, H. (1996) Effect of KCA-098 on the Function of Osteoblast-Like Cells and the Formation of TRAP-Positive Multinucleated Cells in a Mouse Bone Marrow Cell Population, Biochem. Pharmacol. 51, 133–139. 41. Tsutsumi, N., Kawashima, K., Arai, N., Nagata, H., Kojima, M., Ujiie, A., and Endo, H. (1994) In Vitro Effect of KCA-098, a Derivative of Coumestrol, on Bone Resorption of Fetal Rat Femurs, Bone. Miner. 24, 201–209. 42. Kojima, M., Tsutsumi, N., Nagata, H., Itoh, F., Ujiie, A., Kawashima, K., Endo, H., and Okazaki, M. (1994) Effect of KCA-098, a New Benzofuroquinoline Derivative, on Bone Mineral Metabolism, Biol. Pharm. Bull. 17, 504–508. 43. Tsutsumi, N., Kawashima, K., Nagata, H., Ujiie, A., and Endo, H. (1995) Effects of KCA-012 on Bone Metabolism in Organ Culture, Jpn. J. Pharmacol. 67, 169–171. 44. Tsutsumi, N. (1995) Effect of Coumestrol on Bone Metabolism in Organ Culture, Biol. Pharm. Bull. 18, 1012–1015. 45. Wang, L.Q., Meselhy, M.R., Li, Y., Qin, G.W., and Hattori, M. (2000) Human Intestinal Bacteria Capable of Transforming Secoisolariciresinol Diglucoside to Mammalian Lignans, Enterodiol and Enterolactone, Chem. Pharm. Bull. 48, 1606–1610. 46. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Flaxseed and Its Purified Lignan Reduces Bone Strength in Young but Not Older Male Rats, J. Toxicol. Environ. Health 63, 53–65. 47. Anderson, J.J.B. (1999) Plant-Based Diets and Bone Health: Nutritional Implications, Am. J. Clin. Nutr. 70, 539S–542S. 48. Hsieh, C.Y., Santell, R.C., Haslam, S.Z., and Helferich, W.G. (1998) Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-Positive Human Breast Cancer (MCF-7) Cells In Vitro and In Vivo, Cancer Res. 58, 3833–3838. 49. Yoon, H.K., Chen, K., Baylink, D.J., and Lau, K.H. (1998) Differential Effects of Two Protein Tyrosine Kinase Inhibitors, Tyrphostin and Genistein, on Human Bone Cell Proliferation as Compared with Differentiation, Calcif. Tissue Int. 63, 243–249. 50. Setchell, K.D., and Cassidy, A. (1999) Dietary Isoflavones: Biological Effects and Relevance to Human Health, J. Nutr. 129, 758S–767S. 51. Miksicek, R.J. (1994) Interaction of Naturally Occurring Nonsteroidal Estrogens with Expressed Recombinant Human Estrogen Receptor, J. Steroid Biochem. Mol. Biol. 49, 153–160.

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52. Routledge, E.J., White, R., Parker, M.G., and Sumpter, J.P. (2000) Differential Effects of Xenoestrogens on Coactivator Recruitment by Estrogen Receptor (ER)α and ERβ, J. Biol. Chem. 275, 35986–35993. 53. Markiewicz, L., Garey, J., Adlercreutz, H., and Gurpide, E. (1993) In Vitro Bioassays of Non-Steroidal Phytoestrogens, J. Steroid Biochem. Mol. Biol. 45, 399–405. 54. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.A. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor β, Endocrinology 139, 4252–4263. 55. Brann, D.W., Hendry, L.B., and Mahesh, V.B. (1995) Emerging Diversities in the Mechanism of Action of Steroid Hormones, J. Steroid Biochem. Mol. Biol. 52, 113–133. 56. Ray, A., Prefontaine, K.E., and Ray, P. (1994) Down-Modulation of Interleukin-6 Gene Expression by 17β-Estradiol in the Absence of High Affinity DNA Binding by the Estrogen Receptor, J. Biol. Chem. 269, 12940–12946. 57. Pottratz, S.T., Bellido, T., Mocharla, H., Crabb, D., and Manolagas, S.C. (1994) 17βEstradiol Inhibits Expression of Human Interleukin-6 Promoter-Reporter Constructs by a Receptor-Dependent Mechanism, J. Clin. Investig. 93, 944–950. 58. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.A. (1996) Cloning of a Novel Receptor Expressed in Rat Prostate and Ovary, Proc. Natl. Acad. Sci. USA 93, 5925–5930. 59. Onoe, Y., Miyaura, C., Ohta, H., Nozawa, S., and Suda, T. (1997) Expression of Estrogen Receptor Beta in Rat Bone, Endocrinology 138, 4509–4512. 60. Jilka, R.L., Hangoc, G., Girasole, G., Passeri, G., Williams, D.C., Abrams, J.S., Boyce, B., Broxmeyer, H., and Manolagas, S.C. (1992) Increased Osteoclast Development After Estrogen Loss: Mediation by Interleukin-6, Science 257, 88–91. 61. Simonet, W.S., Lacey, D.L., Dunstan, C.R., Kelley, M., Chang, M.S., Luthy, R., Nguyen, H.Q., Wooden, S., Bennett, L., Boone, T., Shimamoto, G., DeRose, M., Elliott, R., Colombero, A., Tan, H.L., Trail, G., Sullivan, J., Davy, E., Bucay, N., Renshaw-Gegg, L., Hughes, T.M., Hill, D., Pattison, W., Campbell, P., and Boyle, W.J. (1997) Osteoprotegerin: A Novel Secreted Protein Involved in the Regulation of Bone Density, Cell 89, 309–319. 62. Pike, A.C., Brzozowski, A.M., Hubbard, R.E., Bonn, T., Thorsell, A.G., Engstrom, O., Ljunggren, J., Gustafsson, J.A., and Carlquist, M. (1999) Structure of the LigandBinding Domain of Oestrogen Receptor Beta in the Presence of a Partial Agonist and a Full Antagonist, EMBO J. 18, 4608–4618. 63. Jones, P.S., Parrott, E., and White, I.N. (1999) Activation of Transcription by Estrogen Receptor Alpha and Beta Is Cell Type- and Promoter-Dependent, J. Biol. Chem. 274, 32008–32014. 64. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) Genistein, a Specific Inhibitor of Tyrosine-Specific Protein Kinases, J. Biol. Chem. 262, 5592–5595.

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Chapter 13

Epidemiology of Soy Isoflavones and Cardiovascular Disease M.Z. Vitolinsa, M.S. Anthonya,b, and G.L. Burkea aDepartments of Public Health Science and bPathology, Wake Forest University School of Medicine, Winston-Salem, NC

Introduction Mortality rates from coronary heart disease (CHD) in Asian countries are markedly lower than in Western countries. Although there are many potential explanations for these differences, at least a portion of the CHD protection is thought to be due to lifestyle factors, including dietary composition. These data on chronic disease differences are reinforced by the fact that when Asians emigrate to Western countries and begin to acculturate (e.g., consume more Western-type diets), rates of chronic diseases become similar to those observed in the host country. Recent evidence has suggested that soy might be one of the components in the Asian diet responsible for the lower rates of CHD. Pertinent cross-cultural, migrant, and observational epidemiologic studies that have evaluated the association between soy consumption and cardiovascular disease (CVD) risk are reviewed in this chapter. Overview of the Epidemiology of Cardiovascular Disease Diseases of the heart and blood vessels, collectively known as cardiovascular disease (CVD), comprise the major cause of death in most Western countries. A decrease of >50% in the age-specific mortality rates has been noted since the late 1960s in the United States (1,2). Factors contributing to the dramatic decline in U.S. CVD mortality rates include better identification and treatment of traditional risk factors in the population and also improvements in medical care delivery for treatment of coronary artery disease and CHD events. Changes in dietary intake are an important part of primary prevention efforts to reduce the burden from CHD in Western countries. However, it is important to note that despite these marked improvements, CVD continues to be the leading cause of death in the United States (3), and recent projections suggest that these diseases will be the leading causes of death in both developed and developing regions of the world by the year 2020 (4). In fact, 958,775 deaths in the United States were attributed to CVD in 1999.

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A number of factors have been shown to be associated with the risk of developing CHD. Demographic factors such as age and gender are associated with CVD risk. Worldwide, men have higher CHD rates, and these rates are approximately equal to those observed in women 10 y older (3). Older adults have higher CHD rates compared with younger people, and this rate rises at a nearly exponential rate beyond the age of 75 y in the United States (3). A number of modifiable risk factors for CHD have been identified (2,3). A risk factor that is highly related to dietary intake and hence pertinent for a chapter focused on soy and isoflavone intake is dyslipidemia. Elevated cholesterol and abnormal lipid/lipoprotein profiles greatly increase the risk of heart disease with clinical trial data suggesting that each 1% reduction in low density lipoprotein cholesterol (LDL-C) is associated with a 2% lower incidence of CHD (3,5). In the United States, ~19% of the population aged 20–74 y have high serum cholesterol with a mean serum cholesterol value of ~203 mg/dL. An important fact is that blood cholesterol levels early in adulthood correlate strongly with risk of developing heart disease later in life (6). International studies suggest that there is a linear association between total blood cholesterol and long-term mortality from CHD in different countries (7,8). Although less linked to the issue of soy and isoflavone intake, other major modifiable risk factors for heart disease include elevated blood pressure, smoking and diabetes. Data linking these risk factors to CHD incidence and the efficacy of preventive efforts focused on these and other risk factors have been described in detail elsewhere (9). Behavioral risk factors such as physical inactivity, tobacco use, and dietary intake are important etiologic factors in the development of chronic diseases, including CVD (10). Dietary modifications have been shown to modify heart disease risk factors in a favorable direction by reducing elevated cholesterol and triglyceride levels, and lowering blood pressure; thus, they have the potential to have a major influence on CHD burden. CHD in younger individuals greatly affects health care costs, productivity and work years, and quality of life. Therefore, making modifications in diet and lifestyle will delay the onset of CHD and have a notable effect on longevity, productive years, and health care costs. Observational epidemiologic studies have played a critical role in identifying lifestyle and diet-disease associations. Cross-Cultural and Ecologic Comparisons Asian populations with high soy consumption have CVD rates that are characteristically lower than those of Western populations with low soy intakes. The striking differences in coronary heart disease mortality between men and women living in Japan and the United States can be seen in Figure 13.1 (11). The age-adjusted CHD mortality rates are ~8 times higher for U.S. men and women relative to Japanese men and women. More recently, Nagata (12) conducted an ecologic study in Japan evaluating the relationship between soy and isoflavone intake (at a population level) with

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Rate per 100,000

Men

Women

300

Fig. 13.1. Age-adjusted

200 100 0 USA

Japan

USA

Japan

coronary heart disease mortality rates in United States Caucasian men and women and Japanese men and women, 40–69 y of age. Data adapted from Reference12.

mortality from heart disease and cancer. Soy product and major nutrient intake was derived from the National Nutritional Survey in which dietary habits were surveyed annually from 1980 to 1985 by 3-d diet records in 6000 randomly selected households in 12 geographical districts covering 47 prefectures. The survey included the following four soy products: miso, tofu, fried tofu, and soybeans, as well as other soy products (e.g., yuba, soy milk). Soy protein intake was estimated from food tables and isoflavone intake was derived from data published for Japanese foods. The mean values for soy and isoflavone intake were assigned to the prefectures forming the district. Nagata reported a significant inverse correlation between heart disease mortality rate and soy protein consumption in women and a modest correlation in men. The author emphasized the importance of utilizing various study designs that include a full range of soy products to better assess the health effects of soy product consumption. Migrant Studies Both genetic and lifestyle differences may contribute to the differences in CHD rates in the United States compared with Japan. Studies of individuals who migrate from areas of low CVD prevalence to areas of higher CVD prevalence provide valuable corroborating evidence to the observed ecological comparisons between countries. Therefore, migrant studies are often done to determine whether these differences may be attributable to many factors, including country/regional differences in genotypes, gene-environment interactions, differences in health behaviors, and also differences in the awareness and diagnosis of CVD. Robertson et al. (13) reported the incidence of myocardial infarction and death from CHD in Japanese men, 45–68 y of age, living in Japan, Hawaii, and California. As can be seen in Figure 2, the lowest incidence rate was seen in Japanese men living in Japan, which was reported to be half that of the Japanese men living in Hawaii (P < 0.01). For Japanese men in California, the incidence rate was ~50% higher than that of the Japanese men in Hawaii (P < 0.05). Thus, Japanese men who have emigrated from Japan to Hawaii and to California have increased CHD risk with increased Westernization. Similar patterns have also been observed in women. This information suggests that environmental factors likely play a key role in mediating some of

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Rate per 1000 person (y)

5

P < 0.05

4.3

4 P < 0.01 3

3

2.8

2 1.4 1 0 Japan

Hawaii

Hawaii

California

Fig. 13.2. Myocardial infarction incidence in men of Japanese ancestry in Japan, Hawaii,

and California (13). Age-adjusted rate for men 45–64 y of age.

the large differences observed between countries. It is unlikely that individuals genetically predisposed toward a more abnormal CVD risk profile and higher rates of CVD morbidity and mortality are more likely to migrate. However, the specific diet and lifestyle changes that contribute to the observed differences in CHD rates are less clear. Cross-Sectional Studies Nagata et al. (14) examined the relationship between soy intake and total cholesterol levels of 3596 women and 1242 men who were participants in a prospective cohort study, the Takayama Study, conducted in Takayama, Japan. Data on soy intake was collected via a food-frequency questionnaire (FFQ). Men and women were categorized into quartiles on the basis of soy consumption. A significant trend (P for trend = 0.0001) was found for decreased total cholesterol concentrations with a higher intake of soy foods for both men and women. As soy intake increased, total cholesterol decreased. This study supports the notion that soy protein may be beneficial in lowering serum total cholesterol concentrations. Somekawa et al. (15) evaluated the association between consumption of isoflavones (nonsteroidal phytoestrogens that are naturally occurring in soy) and plasma lipid profiles in 478 postmenopausal Japanese women. A standardized questionnaire to assess soy intake was not utilized; rather, participants were asked to report weekly, monthly, and/or yearly consumption of soy products for both present and past consumption (i.e., when they were 40 y old). Participants were assigned to two groups (early or late menopause) depending on the length of time in menopause, and then each group was categorized into quartiles on the basis of dietary isoflavone consumption. The mean estimated isoflavone intake was 54.3 mg/d. Although intake of isoflavones was slightly higher in older women, differences in isoflavone intake between the early and late menopause groups were not

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significant. Although a favorable association between soy/isoflavone consumption and bone density was noted, there was no significant association between soy/isoflavone intake and total cholesterol, serum triglyceride, LDL-C, high density lipoprotein cholesterol (HDL-C), apolipoprotein (apo) AI, apo B, or apo E concentrations in either the early or late postmenopausal group. Arai et al. (16) calculated intake of flavonols, flavones, and isoflavones consumed by 115 Japanese women between the ages of 27 and 78 y. Dietary intake was assessed by 3-d food record. The mean intake of isoflavones was found to be 47.2 mg/d, and the major source of isoflavones reported was tofu. Total intake of flavonoids was inversely correlated with total cholesterol and LDL-C concentrations, but associations between total cholesterol or LDL-C and isoflavones by themselves were not significant. The authors proposed that the intake of flavonoids and isoflavones was potentially one of the contributors to the low incidence of CHD in Japanese women compared with women in other countries. Ho et al. (17) studied patterns of soy intake and its association with blood lipid concentrations of 500 men and 510 women between the ages of 24 and 74 y in Hong Kong. Dietary intake of soy was assessed by a dietary practice questionnaire that was similar to the Singapore Ministry of Health Food Consumption survey and a FFQ. Mean intake of soy protein was ~46 g/wk for men and ~34 g/wk for women. The isoflavone intake was estimated to be ~120 mg/wk for men and 77 mg/wk for women. Older women (>50 y old) consumed lower amounts of soy protein and isoflavones than younger women. The most frequently consumed soy products were tofu and soy milk. Soy products were consumed ~1-2 times/wk in this population. In men, there were significant associations between soy protein intake and total cholesterol concentrations (r = –0.09, P = 0.04) and LDL-C concentrations (r = –0.11, P = 0.02). Similarly, in women <50 y old, there were significant associations between soy protein intake and total cholesterol concentrations (r = –0.11, P = 0.04) and LDL-C concentrations (r = –0.11, P = 0.05). The authors hypothesized that older women may not have consumed enough soy products to affect lipid and lipoprotein levels. Additionally, they commented that this population consumed only moderate amounts of soy and they would have to increase their soy intake two- to threefold to meet the China Nutrition Association guidelines for soybean consumption. A study conducted in the United States examined the CVD benefit of dietary intake of isoflavones (18). Associations were evaluated between usual dietary isoflavone intake and CVD risk factors, including lipids, body mass index, blood pressure, and several other factors. Postmenopausal women (n = 208) participated in the study. Usual daily intake of isoflavones, assessed by FFQ, was found to be inversely associated with fasting insulin concentrations, 2 h postglucose challenge insulin levels, and obesity, and positively associated with HDL-C levels. No significant associations were found between usual isoflavone intake and blood pressure, total cholesterol, LDL-C, or triglycerides. The authors proposed two reasons for the difference in their findings from those of Nagata et al. (14). One was the differ-

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ence in average isoflavone intake between the populations (much lower in the U.S. population); the second possibility was that no adjustment for estrogen replacement use was made in the Nagata study. It is notable that although the usual isoflavone consumption of their study participants was very low, significant associations were still found, suggesting the possibility that even very modest soy consumption can have beneficial effects on CVD risk factors. Alternatively, it is possible that women who ate soy, even infrequently, also had other healthy lifestyle characteristics that were not considered in the analysis. This study suggests, however, that usual dietary intake of isoflavones may be associated with a beneficial cardiovascular profile for a number of factors including obesity, HDL-C, and insulin levels in postmenopausal women. Recently de Kleijn et al. (19) studied 939 postmenopausal women in the United States who were Framingham Offspring Study participants and found significant inverse association between consumption of isoflavones and lignans and plasma triglyceride levels. Dietary data for that study were collected by a FFQ, and careful scoring of phytoestrogen intake (lignans and isoflavones) was conducted. Women were categorized into quartiles on the basis of isoflavone or lignan intake. In the highest quartile of intake of isoflavones, plasma triglyceride levels were lower by 0.16 mmol/L [95% confidence interval (CI), –0.30 to –0.02] compared with the lowest quartile of isoflavone intake. For lignan intake, the highest quartile had plasma triglyceride concentrations that were lower by 0.23 mmol/L (95% CI, –0.37 to –0.09) compared with the lowest intake quartile. In the highest quartile of isoflavone consumption, the mean cardiovascular risk factor metabolic score (based on an aggregate score using multiple cardiovascular risk factors) was 0.43 points lower (95% CI, –0.70 to –0.16) than the lowest quartile of consumption. For lignans the difference in this score was 0.55 points, with the highest lignan intake quartile lower than lowest quartile (95% CI, –0.82 to –0.28). This study supports the premise that phytoestrogen intake may be associated with improvement in CVD risk profiles in menopausal women. Limitations of Epidemiologic Data on Soy and Isoflavones Several methodological issues pertaining to epidemiologic and cross-cultural studies warrant discussion. Ecological studies are useful if there are large differences in exposure (such as soy consumption) in the different regions studied, but the conclusions that can be made are limited. Because exposure and outcome are not measured at the individual level, it is possible that associations are spurious in that population-wide associations may not be seen in individuals (ecologic fallacy). Much stronger conclusions about associations between soy and CHD could be made from other study designs, in which intake and outcome are measured in individuals. Thus, evidence from both observational studies and clinical trials is essential to a fuller understanding of the important role of dietary soy and isoflavone intake in chronic disease risk. Another of the issues present in all of these studies is the measurement of dietary intake of isoflavones. Different studies used different

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methods for quantifying soy intake and used different databases for calculating isoflavone intake. This makes comparisons of isoflavone intake across studies questionable. Because isoflavone content of soybeans can vary depending on growing conditions and type of soybean, precise estimates of individual isoflavone intake are difficult to estimate in epidemiologic studies. Databases for isoflavone content of foods available in different countries are generally accurate for estimating isoflavone content of foods that are eaten by populations. A second issue is that FFQ, dietary recalls, and most methods of diet measurement are prone to error, both systematic and random. Random error will typically result in an underestimation of the true association between the food and outcome, suggesting that the true associations might actually be stronger than those reported (20).

Conclusions CVD is a major cause of morbidity and mortality in Western countries and is rapidly increasing worldwide. Behavioral factors, including dietary intake, play an important role in the etiology of CVD risk. The associations between dietary intake and CVD risk factors in these observational studies suggest that soy/isoflavone intake is associated with decreased CVD risk. CVD prevention efforts including those focused on modifying dietary patterns will play an important role in helping to offset the projected worldwide epidemic of CVD. Data from both observational studies and clinical trials have shown that soy can have beneficial effects on CVD risk factors. However, fewer data are available concerning the role of soy or isoflavone consumption in reducing CHD morbidity and mortality. It may be possible to evaluate this question in an existing observational epidemiologic study to determine whether the beneficial relationship between soy or isoflavone intake and CVD risk factors is also reflected in a reduction in CVD events. References 1. Metropolitan Insurance Companies (1993) Heart Disease Mortality: International Comparisons, Stat. Bull. Metrop. Life Insur. Co. 74, 19–26. 2. National Heart, Lung, and Blood Institute (2000) 2000 Chartbook on Cardiovascular, Lung, and Blood Diseases, National Institutes of Health, Washington. 3. American Heart Association (2001) 2002 Heart and Stroke Statistical Update, American Heart Association, Dallas. 4. Murray, J.L., and Lopez, A.D. (1996) The Global Burden of Disease: A Comprehensive Assessment of Global Mortality and Disability from Diseases, Injuries and Risk Factors in 1990 and Projected to 2020, World Health Organization, Geneva. 5. Lipid Research Clinics Program (1984) The Lipid Research Clinics Coronary Primary Prevention Trial results: II. The Relationship of Reduction in Incidence of Coronary Heart Disease to Cholesterol Lowering, J. Am. Med. Assoc. 251, 365–374. 6. Klag, M.J., Ford, D.E., Mead, L.A., Jiang, H., Whelton, P.K., Liang, K.Y., and Levine, D.M. (1993) Serum Cholesterol in Young Men and Subsequent Cardiovascular Disease, N. Engl. J. Med. 328, 313–318.

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7. Keys, A., Aravanis, C., Blackburn, H.W., Van Buchem, F.S.P., Buzina, R., Djordjevic, B.S., Dontas, A.S., Fidanza, F., Karvonen, M.J., Kimura, N., Lekos, D., Monti, M., Puddu, V., and Taylor, H.L. (1967) Epidemiologic Studies Related to Coronary Heart Disease: Characteristics of Men Aged 40–59 in Seven Countries, Acta. Med. Scand. (Suppl.) 460, 1–392. 8. Vershuren, W.M.M., Jacobs, D.R., Bloemberg, B.P.M., Kromhout, D., Menotti, A., Aravanis, C., Blackburn, H., Buzina, R., Dontas, A.S., Fidanza, F., Karvonen, M.J., Nedelijkovic, S., Nisinen, A., and Toshima, H. (1995) Serum Total Cholesterol and Long-Term Coronary Heart Disease Mortality in Different Cultures: Twenty-Five Year Follow-Up of the Seven Countries Study, J. Am. Med. Assoc. 274, 131–136. 9. U.S. Department of Health and Human Services (1994) Report of the Task Force on Research in Epidemiology and Prevention of Cardiovascular Disease, National Heart, Lung, and Blood Institute, Washington. 10. U.S. Department of Health and Human Services (1982) Diet, Nutrition and Cancer, National Academy of Sciences, National Academy Press, Washington. 11. Beaglehole, R. (1990) International Trends in Coronary Heart Disease Mortality, Morbidity, and Risk Factors, Epidemiol. Rev. 12, 1–15. 12. Nagata, C. (2000) Ecological Study of the Association Between Soy Product Intake and Mortality from Cancer and Heart Disease in Japan, Int. J. Epidemiol. 29, 832–836. 13. Robertson, T.L., Kato, H., Rhoades, A., Kagan, M., Marmot, M., Syme, S.L., Gordon, T., Worth, R.M., Belsky, J.L., Dock, D.S., Miyanishi, M., and Kawamoto, S. (1977) Epidemiologic Studies of Coronary Heart Disease and Stroke in Japanese Men Living in Japan, Hawaii and California: Incidence of Myocardial Infarction and Death from Coronary Heart Disease, Am. J. Cardiol. 39, 239–243. 14. Nagata, C., Takatsuka, N., Kurisu, Y., and Shimizu, H. (1998) Decreased Serum Cholesterol Concentration Is Associated with High Intake of Soy Products in Japanese Men and Women, J. Nutr. 128, 209–213. 15. Somekawa, Y., Chiguchi, M., Ishibashi, T., and Aso, T. (2001) Soy Intake Related to Menopausal Symptoms, Serum Lipids, and Bone Mineral Density in Postmenopausal Japanese Women, Obstet. Gynecol. 97, 109–115. 16. Arai, Y., Watanabe, S., Kimira, M., Shimoi, K., Mochizuki, R., and Kinae, N. (2000) Dietary Intakes of Flavonols, Flavones and Isoflavones by Japanese Women and the Inverse Correlations Between Quercetin Intake and Plasma LDL Cholesterol Concentration, J. Nutr. 130, 2243–2250. 17. Ho, S.C., Woo, J.L.F., Leung, S.S.F., Sham, A.L.K., Lam, T.H., and Janus, E.D. (2000) Intake of Soy Products Is Associated with Better Plasma Lipid Profiles in the Hong Kong Chinese Population, J. Nutr. 130, 2590–2593. 18. Goodman-Gruen, D., and Kritz-Silverstein, D. (2001) Usual Dietary Isoflavone Intake Is Associated with Cardiovascular Disease Risk Factors in Postmenopausal Women, J. Nutr. 131, 1202–1206. 19. De Kleijn, M.J.J., van der Schouw, Y.T., Wilson, P.W.F., Grobbee, D.E., and Jacques, P.F. (2001) Dietary Intake of Phytoestrogens Is Associated with Favorable Metabolic Cardiovascular Risk Profile in Postmenopausal U.S. Women: The Framingham Study, J. Nutr. 132, 276–282. 20. Willett, W. (1998) Nutritional Epidemiology, 2nd edn., p. 288, Oxford University Press, New York.

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Chapter 14

Soy/Isoflavones and Risk Factors for Cardiovascular Disease Mary S. Anthony Departments of Pathology and Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC

Introduction Cardiovascular disease (CVD) is the leading cause of death for both men and women in Western countries. In the United States, coronary heart disease (CHD) accounts for ~1 in every 5 deaths, and stroke is the cause of ~1 in every 14 deaths (1). CHD is the leading cause of premature, permanent disability, accounting for ~19% of disability allowances. Currently, an estimated 6.2 million men and 6.4 million women have a history of CHD. Atherosclerosis is an underlying process that manifests in clinical events of heart attack and stroke. In addition to the accumulation of lesions that can occlude the lumen of arteries, the atherosclerotic process is associated with endothelial and vascular dysfunction that can result in vasospasm, and plaques that can become unstable and rupture, resulting in emboli or thrombi. Some of the primary modifiable risk factors for atherosclerosis and cardiovascular disease include dyslipidemia [e.g., elevated low density lipoprotein (LDL) cholesterol, elevated plasma triglycerides, reduced high density lipoprotein (HDL) cholesterol concentrations] and elevated blood pressure. Approximately 40–50% of Americans ≥20 y old have LDL cholesterol concentrations >130 mg/dL, a level that is associated with higher CHD risk (1). In addition, ~25% of adults in the United States have high blood pressure (1). Therefore, there is a large population that could benefit from therapies to improve these risk factors and thereby possibly reduce CHD risk. Because the relationship between nutrition and CHD risk is well established, there seems to be great potential to reduce the chronic disease burden by nutritional education and dietary modifications. These have been proven strategies for reducing total fat, and particularly saturated fat intake in the United States (2). Increasing the consumption of soy foods is another dietary change that could have an important effect on improving cardiovascular health and has been included in the recommendations made in the recent report of the Nutrition Committee of the American Heart Association (3). Substantial data exist concerning the effects of soy consumption on CVD risk factors to support this recommendation. Clinical trials and some studies in animal models that have investigated the effects of soy protein or its components on cardiovascular disease risk factors will be reviewed.

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Components of Soy That Might Affect Cardiovascular Disease Although the atheroprotective effects of soy protein are well accepted, the components responsible for the effects have been the subject of much research (4–6). Beginning in the 1970s, the amino acid composition of soy protein was evaluated for its effect on plasma lipid/lipoprotein metabolism and its role in atherogenesis. More recently, other protein components, including specific protein fractions and globulins have been studied. Nonnutritive components of soy protein isolate (SPI) have also been investigated, including the saponins, phytic acid, trypsin inhibitors, and isoflavones. A study by Huff and colleagues (7) suggested that there was a component of SPI, other than the amino acids, that contributed to the hypocholesterolemic properties of soy. In that experiment, groups of rabbits (n = 6–10/group) were fed diets that contained casein, SPI, or a mixture of amino acids that duplicated the amino acid composition of either casein or SPI. The total plasma cholesterol concentrations of the groups fed casein or the casein amino acid mixture were essentially the same (casein: 213 ± 53 mg/dL, casein amino acids: 213 ± 42 mg/dL). In contrast, the soy protein amino acid mixture was not as hypocholesterolemic as the intact protein (soy: 69 ± 12 mg/dL, soy amino acids: 124 ± 30 mg/dL), suggesting that components of soy protein other than the amino acids are important for plasma cholesterol modulation. Other soy protein components, including the 7S globulin (β-conglycinin) (8) and the high-molecular-weight fraction of soy (9), have been investigated for their effects on plasma lipoprotein metabolism. Lovati and colleagues (10) reported that the 7S soy globulin (β-conglycinin) and in particular the α′ subunit can increase LDL receptor expression in a human hepatoma cell line (Hep G2 cells). More recently, these investigators reported that specific low-molecular-weight peptides in soy appear to be responsible for the regulation of LDL receptor expression and LDL receptor–mediated uptake of LDL in vitro (8). Because it is unlikely that large peptides of soy reach the hepatocytes, the applicability of these observations must await in vivo confirmation. Sugano and co-workers (11) described the effects of a high-molecular-weight fraction of soy protein on cholesterol metabolism. Compared with whole soy protein, the high-molecular-weight fraction increased neutral and acidic sterol excretion in rats (11) and lowered LDL cholesterol concentrations in women (9). Evidence has been accumulating that the components of soy protein responsible for a large part of its hypocholesterolemic and atheroprotective effects are alcohol extractable, or at least affected by alcohol washing (12–25). The isoflavones (nonsteroidal phytoestrogens) are largely removed from SPI when it is alcoholwashed. These molecules are being studied intensively for their role in cardiovascular health, cancer prevention, bone health, and postmenopausal therapy. Other alcohol-extractable components that might affect plasma lipid concentrations are

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the saponins and phytosterols. The bulk of evidence regarding saponins is that they exert no hypocholesterolemic effect in the presence of soy protein (26). Most SPIs have low amounts of phytosterols because of delipidation and extensive processing in making SPI (27). Therefore, on the basis of current knowledge, the isoflavones and protein seem the most likely components responsible for the cardiovascular benefits of SPI.

Effects of Soy Protein with Isoflavones on Cardiovascular Disease Risk Factors Studies citing the protective effects of soy for cardiovascular disease, risk factors, and inhibition of atherosclerosis have been ongoing for many years. Many of the studies have focused on the effects of soy protein and its components on plasma lipid and lipoprotein concentrations. However, there is also evidence that intact soy protein (i.e., not alcohol-washed) might modify cardiovascular disease independently of effects on plasma lipoprotein concentrations (28). Some of these potential mechanisms that are independent of plasma lipid concentrations are as follows: blood pressure, vascular function, platelet function, carbohydrate metabolism, and LDL oxidation. Plasma Lipids and Lipoproteins The atheroprotective effect of soy seems to be mediated in part by effects on plasma lipoprotein concentrations. Because other chapters in this book deal specifically with effects on lipoproteins, this section will simply summarize this large body of data. The plasma cholesterol-lowering properties of soy protein have been studied since the 1940s. A meta-analysis that included 38 clinical trials in humans reported that soy consumption, compared with a control diet, resulted in 13% lower LDL cholesterol concentrations, 10% lower plasma triglycerides, and slightly, but not significantly higher HDL cholesterol (~2% higher) (29). There was also evidence that those with higher baseline plasma cholesterol concentrations had a larger decrease in LDL cholesterol with soy consumption than those with normal cholesterol concentrations at baseline. These beneficial effects of soy protein on plasma lipoprotein concentrations culminated in the U.S. Food and Drug Administration’s approval of a health claim in October 1999, stating that “25 grams of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease.” Several studies in animal models have found that alcohol washing of soy protein (a process that largely removes the isoflavones) results in a soy protein that is less effective than the isoflavone-intact soy proteins for improving plasma lipoprotein concentrations (i.e., lowering LDL cholesterol, increasing HDL cholesterol) (13,14,16,19). Several recent studies in humans have suggested that soy protein with higher levels of isoflavones might have more robust effects on lowering LDL

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cholesterol concentrations than soy protein with lower isoflavone content (17, 18,20,24). In the study by Crouse and colleagues (17), there was an increasing reduction in LDL cholesterol concentrations with increasing isoflavone content (from 3 to 62 mg) in 25 g of soy protein and slightly higher (not significant) HDL cholesterol concentrations. In a study in normocholesterolemic premenopausal women, soy protein (53 g/d) with the highest isoflavone content (129 mg) had a more robust effect than the same amount of soy protein with about half the isoflavone content (65 mg) and was significantly more effective than alcoholwashed soy protein in lowering LDL cholesterol concentrations (20). In postmenopausal women, consumption of soy protein (63 g/d) with 132 mg isoflavones lowered LDL cholesterol more than the same amount of soy protein with ~65 mg isoflavones and was also significantly better than alcohol-washed soy (24). In the study by Gardner and colleagues (18), postmenopausal women who consumed soy protein (42 g/d) with 80 mg isoflavones (n = 31) had greater reductions in LDL cholesterol than a group that received alcohol-washed soy protein (n = 33, P = 0.005). In all of these studies, consumption of the SPI with the highest isoflavone content resulted in 7–10% lower LDL cholesterol concentrations compared with the alcohol-washed SPI. However, significant effects of soy protein with isoflavones are not always found (30). Baseline LDL cholesterol concentrations and the hormonal milieu are likely important determinants of effects on plasma lipoprotein concentrations. Other Cardiovascular Disease Risk Factors Vascular Function. There is evidence that soy protein with isoflavones can affect vascular function. However, this effect might be different in males than in females and might differ by estrogen status. In addition, the benefits of soy on vascular reactivity might be evident only in those with impaired vascular function. In a study by Honoré et al. (31) young male (n = 11) and premenopausal female (n = 11) nonhuman primates were fed soy protein with isoflavones [SPI(+), isoflavone dose approximately equivalent to a human dose of 150 mg/d] or alcohol-washed SPI [SPI(–)] for 6 mo. Among the females, the SPI(+) group had an increase of ~6.4% in coronary artery lumen diameter in response to acetylcholine (an endothelium-dependent vascular response), whereas the SPI(–) group had a 6.2% constriction after acetylcholine [SPI(+) vs. SPI(–), P = 0.01]. However, in the males, both groups showed vasoconstriction after acetylcholine infusion [SPI(–), 1.9% constriction; SPI(+), 7.8% constriction; SPI(+) vs. SPI(–), P = 0.13]. Another study with surgically postmenopausal cynomolgus monkeys (32) evaluated the separate and combined effects of estrogen replacement therapy (ERT) and SPI(+). The ovariectomized (estrogen-devoid) casein-fed group (Control, n = 12) showed vasoconstriction in response to acetylcholine and there was no significant improvement in the SPI(+) group (n = 11) compared with Control (P = 0.45). However the ERT group (n = 12) showed vasodilation that was significantly different from that of the

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Control group (P = 0.01), and the combined ERT + SPI(+) group had vasodilation that was significantly better than the Control group (P = 0.001), the SPI(+) group (P = 0.01), and the ERT group (P = 0.04). In a study presented in abstract form (33), postmenopausal women (n = 18) with impaired vascular function (i.e., abnormal endothelium dependent flow-mediated dilation), were given a beverage with 40 g of soy protein containing 80 mg of isoflavones daily for 1 mo and then reassessed. Flow-mediated dilation was significantly improved by 5.3% (P < 0.0001) with soy consumption and the response returned to baseline after a 1-mo washout. More recently, Teede and colleagues (34) evaluated the effects of consumption of soy with isoflavones (40 g of soy containing 118 mg isoflavones daily), compared with a group given casein, on CVD risk factors. Treatment was for 3 mo and the study included men (n = 96) and postmenopausal women (n = 83), 50–75 y of age, who were not selected for impaired vascular function. In women, there was a slight, nonsignificant improvement in endothelial-dependent vascular function with soy treatment, and in men there was an adverse effect (i.e., greater vascular constriction in the soy group) (34). In that study, there were no significant improvements in LDL cholesterol concentrations; thus, it would be of interest to evaluate vascular function in men who demonstrated LDL cholesterol lowering in response to soy. Data for the clinical trials in humans are shown in Table 14.1. In summary, there appear to be beneficial effects of soy with isoflavones for women, particularly those with impaired vascular function and with some circulating estradiol. However, there seems to be no benefit, and possibly an adverse effect, of soy with isoflavones on vascular function in men. Platelet Function. Another mechanism by which soy with isoflavones might improve cardiovascular disease is by exerting effects on platelets. In a study with female nonhuman primates (n = 12), a group fed SPI(–) had a 26% greater reduction (P = 0.02) in blood flow after collagen-induced platelet activation compared with a group fed SPI(+) (35). Although the precise mechanism for this protection against reduction in blood flow by the isoflavones could not be determined in that study, there are several possible explanations. When platelets are activated they release their vasoactive substances, including serotonin, which is a potent vasoconstrictor. In the study by Peluso et al. (21) described below, thrombin-stimulated platelets from rats fed SPI(+) released less serotonin than when rats were fed casein or SPI(–). Williams and Clarkson (35) also found that in vitro platelet aggregation in response to thrombin and serotonin was reduced in platelets collected from the SPI(+) group compared with platelets from the SPI(–) group. Schoene and Guidry (36) found that platelets from rats fed SPI(+) had apparent volumes that were significantly smaller than platelets from rats fed SPI(–), suggesting that these smaller platelets were in a more quiescent state. In that study, other indicators of platelet activation were also improved (i.e., reduced platelet activation) by SPI(+) compared with SPI(–). In a study with rats, Peluso and colleagues (21) found that both SPI(–) and SPI(+) resulted in a 36% lower serotonin release from

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TABLE 14.1 Clinical Trials in Humans Evaluating the Effects of Soy Protein with Isoflavones or Isoflavone Pills on Vascular Functiona Ref.

Study population (n)

Study design

Tx length

Active Tx

Isoflavones (mg/d)

Flow-mediated dilation (absolute difference between treatments for % change from baseline): (33) Postmenopausal women Sequential 1 mo 40 g SPI 80 mg with impaired FMD (18) (34) Postmenopausal women (83) Parallel arm 3 mo 40 g SPI 118 mg and men (96), age 50–75 y (53) Postmenopausal women Crossover 8 wk Soy iso pills 80 mg with impaired FMD (20) Systemic arterial compliance (% difference between treatments): (34) Postmenopausal women (83) Parallel arm 3 mo 40 g SPI 118 mg and men (96), age 50–75 y (52) Peri- and postmenopausal Sequential 5 wk Soy iso pills 80 mg women (21) (58) Postmenopausal women (17) Sequential 5 wk Red clover pills 40 mg 80 mg aAbbreviations:

iso, isoflavones; NS, not significant; SPI, soy protein isolate, Tx, treatment.

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Outcome

P-value

Comparison

↑5.3%

P < 0.0001

Baseline

Women: ↑1.8% Men: ↓3.7%

NS P < 0.02

40 g casein

↑0.8%

NS

Placebo

↑5.7%

NS

40 g casein

↑22.8%

P = 0.01

Placebo

↑20.3% ↑23.9%

P = 0.04 P = 0.02

Placebo

unstimulated platelets, compared with a group fed casein (P ≤ 0.002). In that same study, the group fed isoflavone-intact soy protein had a 13% lower thrombin-stimulated serotonin release from platelets, compared with the casein-fed group (P = 0.003); the group fed isoflavone-depleted soy protein had an intermediate response, ~6% lower (P = 0.1) (21). Thus soy with the isoflavones might inhibit platelet activation and aggregation and reduce the amount of serotonin in the platelets, all of which could contribute to a reduction in coronary vasospasm and thrombosis. Lipoprotein Oxidation. Oxidized LDL particles are thought to play an important role in exacerbating atherogenesis. Several recent studies have evaluated the effects of soy protein with isoflavones on LDL oxidation in human beings (37–40). Data for these clinical trials are summarized in Table 14.2. In a randomized crossover study by Jenkins et al. (37), consumption of soy foods containing 86 mg isoflavones resulted in significantly lower concentrations of conjugated dienes in the LDL fraction (–10.8%, P < 0.001) and significantly lower conjugated diene/LDL cholesterol ratio (–4.5%, P = 0.03), compared with a lacto-ovovegetarian control diet period in 19 men and 12 postmenopausal women. In a second crossover study by Jenkins and colleagues (38), hyperlipidemic men and women (n = 25) ate breakfast cereals containing soy protein (36 g/d with 168 mg isoflavones) or an isocaloric control cereal for 3-wk periods. Although the soy cereal had no significant effects on plasma lipids, there was a 9.2% reduction in total dienes in the LDL fraction (P = 0.04) and an 8.7% reduction of the conjugated diene/LDL cholesterol ratio (P = 0.05). Wiseman et al. (40) evaluated both F2-isoprostanes and LDL resistance to copper-induced conjugated diene formation in 19 premenopausal women and 5 men who consumed textured soy protein that was either high (56 mg/d) or low (1.9 mg/d) in isoflavones for 17-d periods. They reported that the high-isoflavone soy resulted in 19.5% lower concentrations (P = 0.03) of 8-epi-prostaglandin F2α, a biomarker of in vivo lipid peroxidation, and longer lag time for copper-induced LDL oxidation (~9% longer, P = 0.02). Tikkanen et al. (39) found that consumption of soy protein containing 60 mg isoflavones prolonged copper-induced LDL oxidation lag time by ~15% (P < 0.02) after 2 wk of soy consumption compared with measures done at baseline and after a 2-wk washout in a group of healthy volunteers (n = 6). These studies are remarkably consistent in finding lower LDL oxidation potential with consumption of isoflavoneintact soy protein. Blood Pressure. In four trials in humans (17,34,41,42), SPI with isoflavones was shown to lower diastolic, and in some cases systolic blood pressure (Table 14.3). The study by Washburn et al. (42) was a crossover study in perimenopausal women (n = 51). In that study, 20 g of SPI (containing 34 mg isoflavones) given in a split dose, significantly reduced diastolic blood pressure by 5 mm Hg compared with an isocaloric carbohydrate placebo (P < 0.01) (42). In a study by Crouse et al.

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TABLE 14.2 Clinical Trials in Humans Evaluating the Effects of Soy Protein with Isoflavones or Isoflavone Pills on LDL Oxidationa Ref.

Study population (n)

Study design

Tx length

Active Tx

(37)

Hyperlipidemic postmenopausal women (12) and men (19)

Crossover

1 mo

Soy foods with 33 g SPI

86 mg

Hyperlipidemic post -menopausal women (10) and men (15)

Crossover

Soy cereal with 36 g SPI

168 mg

Premenopausal women (3) and men (3) Premenopausal women (19) and men (5)

Sequential

Bars with 21 g SPI Soy protein

57 mg

(38)

(39) (40)

(62) (52)

(59)

Postmenopausal women (13) and men (46) Peri- and postmenopausal women (21)

Premenopausal women (14)

aAbbreviations:

Crossover

Parallel arm Sequential

Crossover

3 wk

2 wk 17 d

8 wk 5 wk

~2 mo

ST clover pills Soy iso pills

Red clover pills

Isoflavones (mg/d)

56 mg

55 mg 80 mg

86 mg

Outcome Conj diene: ↓10.8% Conj diene/LDL-C: ↓4.5% Conj diene: ↓9.2% Conj diene/LDL-C: ↓8.7% Lag time to Cu ox ↑15.2% F2-isoprostanes: ↓19.5% Lag time to Cu ox: ↑9.1% F2-isoprostane excr: ↑~6.6% Cu ox conj diene: ↓0.7% Lag time to Cu ox: ↓0.6% TBARS: ↑2.9% Cu ox conj diene: ↓6.5% Lag time to Cu ox: ↓7.6%

P-value

Comparison Control diet

P <0.001 P = 0.03 Control cereal P = 0.04 P = 0.05 Baseline: P < 0.02 P = 0.03

Soy protein with 1.9 mg iso

P = 0.02 Placebo NS Placebo NS NS NS Placebo NS NS

Conj diene, conjugated diene concentration in LDL fraction; Cu ox, copper-induced oxidation; excr, urinary excretion; iso, isoflavones; LDL-C, low density lipoprotein cholesterol; NS = not significant; SPI, soy protein isolate; ST, subterranean; TBARS, thiobarbituric acid reactive substances, method that measures malondialdehyde generated in oxidized LDL; Tx, treatment.

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TABLE 14.3 Clinical Trials in Humans Evaluating the Effects of Soy Protein with Isoflavones or Isoflavone Pills on Blood Pressurea Ref.

Study population (n)

Study design

Tx length

Active Tx

Isoflavones (mg/d)

(41)

Tx HPT men (18) and women (18)

Parallel arm

8 wk

66 g SPI

23 mg

(17)

Mod HC men (94) and women (62)

Parallel arm

9 wk

25 g SPI

3, 27, 37, 62 mg

(34)

Parallel arm

3 mo

40 g SPI

118 mg

(42)

Postmenopausal women (83) and men (96), age 50–75 y Perimenopausal women (51)

Crossover

6 wk

20 g SPI

34 mg-one 34 mg-split

(60)

Postmenopausal women (80)

Parallel arm

4 mo

Soy iso pills

(63)

Postmenopausal women (13) and men (46) with high normal BP

Parallel arm

8 wk

ST clover pills

100 mg+ 150 mg SPI 55 mg

Peri- and postmenopausal women (21) Postmenopausal women (17)

Sequential

5 wk

Soy iso pills

80 mg

Sequential

5 wk

Crossover

8 wk

Red clover pills Soy iso pills

40 mg 80 mg 80 mg

(52) (58) (53)

Postmenopausal women with impaired FMD (20)

Outcome 24-h SBP: ↓5.9 mm Hg 24-h DBP: ↓2.6 mm Hg Women: ↑iso dose, ↓DBP Men: No differences SBP: ↓3.9 mm Hg DBP: ↓2.4 mm Hg SBP: ↓2.4 mm Hg DBP: ↓2.3 mm Hg SBP: ↓1.3 mm Hg DBP: ↓4.9 mm Hg MinBP: ↑1 mm Hg MaxBP: 0 mm Hg 24-h SBP: ↓1.4 mm Hg 24-h DBP: ↓0.8 mm Hg MBP: 0 mm Hg MBP: ↓1 mm Hg MBP: ↑5 mm Hg SBP: ↓1 mm Hg DBP: 0 mm Hg

P-value

Comparison

P = 0.001

66 g maltodextrin

P = 0.006 P for trend = 0.04 NS P < 0.05 P < 0.05 NS NS NS P < 0.01 NS NS

40 g casein 20 g complex carbohydrate

Placebo Placebo

NS NS NS NS NS NS NS

Placebo Placebo Placebo

aAbbreviations: DBP, diastolic blood pressure; FMD, flow-mediated dilation; iso = isoflavones, MBP; mean arterial blood pressure; mod HC, moderately hypercholesterolemic; NS, not significant; SBP, systolic blood pressure; SPI, soy protein isolate; ST, subterranean; Tx HPT, treated hypertensives, Tx, treatment.

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(17) in which moderately hypercholesterolemic men (n = 94) and women (n = 62) were given supplements containing 25 g casein or 25 g SPI with differing concentrations of isoflavones (3–62 mg isoflavones/25 g protein), there was a significant trend for lower diastolic blood pressure with increasing isoflavone dose in the women (P for trend = 0.04). There was no effect on blood pressure in men. More recently, Burke and colleagues (41) found that supplementation with soy protein (66 g/d containing 23 mg isoflavones) compared with an isocaloric carbohydrate supplement, resulted in a 5.9 mm Hg lower 24-h systolic blood pressure (P = 0.001) and 2.6 mm Hg lower diastolic blood pressure (P = 0.006) in treated hypertensive subjects (n = 36). There were no apparent gender differences. In a larger study (34), 83 women and 96 men, ages 50–75 y, were given 40 g of soy protein containing 118 mg isoflavones each day or a casein placebo for 3 mo. Systolic and diastolic blood pressure decreased in both groups; however, the decrease was significantly greater for the soy group relative to the casein group for both systolic blood pressure (3.9 mm Hg lower, P < 0.05) and diastolic (2.4 mm Hg lower, P < 0.05). Again, there were no differences in the effects of soy between men and women. One study (38) reported no effect on blood pressure of soy cereal (36 g SPI/d with 168 mg isoflavones) consumed for 3 wk compared with control cereal in 25 hypercholesterolemic men and women. In summary, four of of the five clinical trials that tested soy with isoflavones and reported blood pressure found reductions in diastolic, and in some cases systolic, blood pressure. The reductions in diastolic blood pressure were ~2–5 mm Hg in the four studies that reported significant effects and in the two that reported effects on systolic blood pressure, soy with isoflavone consumption resulted in reductions of 4–6 mm Hg. Carbohydrate Metabolism and Obesity. Insulin resistance, impaired carbohydrate metabolism, and obesity are known risk factors for CHD. Two studies have investigated the effects of soy protein with isoflavones on measures of carbohydrate metabolism. In a study in 48 nonhuman primates (43), the separate and combined effects of soy protein containing isoflavones and estrogen replacement therapy were evaluated. Soy protein, compared with casein protein resulted in improved insulin sensitivity (P = 0.03) and improved glucose effectiveness (P = 0.04), as determined by minimal-model analyses. However, there were no significant effects of soy protein on fasting insulin concentrations, insulin:glucose ratios, fructosamine concentrations, or body mass index. More recently, in an observational study with 208 postmenopausal women, 45–74 y of age and living in California, associations were done between usual intake of isoflavones and cardiovascular disease risk factors (44). Usual soy intake during the previous year was determined from food-frequency questionnaires and then isoflavone intake was calculated on the basis of published isoflavone content of the different foods. Women were categorized into tertiles by isoflavone intake, and then associations with cardiovascular disease risk factors were determined. Higher usual isoflavone intake was associated with lower body mass index (P for trend = 0.05) and a tendency toward lower

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fasting insulin concentrations (P for trend = 0.07). After adjustment for body mass index, ethnicity, total energy intake, and fiber intake, higher isoflavone consumption was associated with lower insulin concentrations 2 h after glucose challenge (P = 0.05). Most clinical trials have not reported changes in body weight. Many may have been too short in duration for any changes to be detected. On the other hand, most studies counseled participants to maintain a consistent body weight during the trial. Because the study by Goodman-Gruen and Kritz-Silverstein (44) was an observational study, one cannot conclude a causal relationship between isoflavone intake and body mass index or carbohydrate metabolism. Therefore, further data are required to verify these effects of soy and isoflavones on carbohydrate metabolism; nevertheless, these findings are intriguing. Lipoprotein(a) Concentrations. Lipoprotein(a) [Lp(a)] is a risk factor for cardiovascular disease, with higher concentrations associated with higher coronary heart disease and cerebrovascular disease. Although it is uncertain whether it plays a role in the development of atherosclerosis, its structural similarity to plasminogen suggests that it might have a role in the thrombosis/thrombolysis pathway. Three studies have reported higher Lp(a) concentrations in response to consumption of soy protein with isoflavones (34,45,46). Data for these studies are summarized in Table 14.4. In the first study (46), normolipidemic men (n = 9) were given two liquid formula diets containing either casein or soy as the protein source for 45 d (n = 7) or 33 d (n = 2) in a crossover design. Daily protein intake was ~154 g and constituted ~20% of total energy. After 30 d, there was a significant decline in Lp(a) concentrations with casein consumption relative to the baseline self-selected diet and no difference from baseline for the soy protein diet. Men consuming the soy protein diet had Lp(a) concentrations approximately twice as high as those conbsuming the casein diet (soy protein, 93.5 ± 57.2 mg/L; casein, 44.7 ± 32.5 mg/L; P < 0.001). In a second study by this same group (45), soy protein with isoflavones [SPI(+)] was compared with alcohol-washed soy protein devoid of isoflavones [SPI(–)] and with casein. Twelve healthy volunteers (6 men and 6 premenopausal women) were studied in a crossover design with diet periods that lasted 32 d and were separated by wash-out periods. The casein, SPI(–), and SPI(+) diets were liquid diets providing ~150 g protein/d, which constituted ~20% of total energy. In the casein and SPI(–) groups, Lp(a) concentrations decreased from baseline levels by ~67.7% (P < 0.001 vs. baseline) and 61.6% (P < 0.01 vs. baseline) respectively. Lp(a) concentrations with SPI(+) treatment were ~12.3% lower than baseline, but this change was not significant. Casein and SPI(–) diets resulted in significantly lower Lp(a) concentrations compared with SPI(+) (P < 0.001). In the randomized, placebo-controlled trial by Teede et al. (34), men (n = 96) and postmenopausal women (n = 83) received either 40 g casein or 40 g soy/d with 118 mg isoflavones for 3 mo. Lp(a) concentrations increased by 42 mg/L compared with baseline for both men and women in the soy group, whereas in the casein group, the increase was only 4 mg/L (soy vs. casein, P < 0.05). The results

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TABLE 14.4 Clinical Trials in Humans Evaluating the Effects of Soy Protein with Isoflavones or Isoflavone Pills on Lipoproteina Ref.

Study population (n)

Study design

Tx length

Active Tx

Isoflavones (mg/d)

(17)

Mod HC men (94) and women (62) Perimenopausal women (69)

Parallel arm

9 wk

25 g SPI

Parallel arm

6 mo

40 g SPI 40 g SPI 150 g SPI 150 g SPI 53 g SPI 53 g SPI 154 g SPI

3, 27, 37, 62 mg 4 mg 80 mg 16 mg 315 mg 65 mg 129 mg ~260 mg

40 g SPI 10 g SPI + 40 g C 20 g SPI + 30 g C 30 g SPI + 20 g C 50 g SPI 63 g SPI 63 g SPI ST clover pills Soy iso pills

(30) (45)

Crossover

32 d

(20)

Premenopausal women (6) and men (6) Premenopausal women (13)

Crossover

~3 mo

(46)

Normolipidemic men (9)

Crossover

(34)

Parallel arm

(47)

Postmenopausal women (83) and men (96), age 50–75 y Mod HC men (81)

33– 45 d 3 mo

Parallel arm

6 wk

(24)

Postmenopausal women (18)

Crossover

3 mo

(56)

Postmenopausal women (13) and men (46) Postmenopausal women with impaired FMD (20)

Parallel arm

8 wk

Crossover

8 wk

(53)

Outcome

P-value

Comparison

No difference

NS

25 g casein

No difference No difference ↑6.1% ↑55.4% ↑3.8% 0% ↑109.2%

NS NS NS P < 0.001 NS NS P < 0.001

40 g whey protein 150 g casein 53 g SPI with 10 mg iso 154 g casein

118 mg

↑13.5%

P < 0.05

40 g casein

19 mg 38 mg 57 mg 95 mg 65 mg 132 mg 55 mg

↓7.9% ↑15.4% ↓6.3% ↓10.7% ↑1.5% ↑2.2% ↓5.7%

NS NS NS NS NS NS NS

50 g casein

63 g SPI with 7 mg iso Placebo

80 mg

↓7.3%

NS

Placebo

aAbbreviations: C, casein; FMD, flow-mediated dilation; iso, isoflavones; mod HC, moderately hypercholesterolemic; NS, not significant; SPI, soy protein isolate; ST, subterranean; Tx, treatment.

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of these three studies are in contrast with five other studies that have compared soy protein containing isoflavones to alcohol-washed soy protein and/or milk proteins (17,20,24,30,47). In these other studies, moderately hypercholesterolemic men (47), normocholesterolemic premenopausal women (20), perimenopausal women (30), postmenopausal women (24), and a moderately hypercholesterolemic group of men and women (17) were evaluated. The soy treatment periods ranged from 6 wk up to 24 wk and study designs were either crossover (20,24) or parallel-arm (17,30,47). The number of individuals in each treatment group ranged from 13 to 30. The amounts of soy protein were between 10 and 63 g/d and contained between 3 and 132 mg isoflavones. None of these five studies reported higher Lp(a) concentrations with SPI(+). Although the two studies in Denmark (45,46) used 2–15 times more soy and isoflavones than did these other five studies (17,20,24,30,47), the trial by Teede et al. (34) used amounts of soy protein and isoflavones similar to these five studies. Therefore, more studies should be done to clarify which populations/individuals might be susceptible to Lp(a) increases and at what intake level of soy and/or isoflavones. Direct Effects on the Artery Wall. There are no studies that directly evaluate the effects of soy protein with isoflavones on cells of the artery wall. However, two studies in animal models (12,48) suggest that the inhibition of atherosclerosis by treatment with soy with isoflavones can be independent of effects on plasma lipid and lipoprotein concentrations. Other CVD Risk Factors. There is one other observational study that evaluated the association between intake of isoflavones or lignans (another type of phytoestrogen found in whole grains and some seeds) and cardiovascular disease risk factors (49). This study was done with 939 postmenopausal women in the Framingham Offspring Study. A metabolic syndrome score was calculated for each woman based on World Health Organization criteria. This score can range from 0 to 6 and is calculated by assigning one point for each of the following: systolic blood pressure ≥160 mm Hg or use of antihypertensive medications, diastolic blood pressure ≥90 mm Hg, plasma triglycerides ≥150 mg/dL or use of cholesterol-lowering medications, plasma HDL cholesterol <40 mg/dL, waist:hip ratio >0.85, body mass index >30 kg/m2. After adjustment for age, use of hormone replacement therapy, smoking, and dietary fiber intake, the authors reported a significant trend for lower metabolic syndrome score with increasing isoflavone intake (P for trend = 0.01). There was a significantly lower score in the highest isoflavone intake quartile compared with the lowest quartile (–0.43, 95% confidence interval: –0.70 to –0.16). Although the only significant difference between high and low isoflavone intake groups among the components of this score was for a lower triglyceride concentration with higher isoflavone intake (P < 0.05), there appeared to be small improvements in each of the other components, which contributed to a more favorable metabolic syndrome score, a global indicator of CVD risk.

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Effects of Isolated Isoflavones on Cardiovascular Disease and Risk Factors Although there is less information about the effects of isolated isoflavones (i.e., without soy protein) on CVD risk factors, the data are accumulating because isoflavone supplements are readily available. These results are an interesting contrast to the effects of soy protein containing isoflavones. The data are summarized below. Plasma Lipids and Lipoproteins There is little evidence that isoflavones, isolated from soy (such as found in isoflavone pills), can improve plasma lipid concentrations, either in humans (50–53) or nonhuman primates (54). In the studies in humans, isoflavone doses ranged from 80–150 mg/d. The study populations have been peri- and postmenopausal women (52) or postmenopausal women (50,51,53) and in one study, a moderately hypercholesterolemic group was selected (50). Isoflavone supplements made from red clover have also been tested and have similarly had no significant effect on plasma lipid concentrations (55–59). There is one study that did report an effect of isoflavone supplements on plasma lipid concentrations in postmenopausal women in Brazil (60). In that study, the authors examined the effects of 100 mg isoflavones with 150 mg soy protein, given in three doses, and found significantly lower LDL cholesterol (P < 0.01) concentrations in the active treatment group (n = 40) compared with placebo (n = 40). Whether the small amount of soy protein could make the difference in this study is unlikely; however, this finding remains to be confirmed. Thus, although isoflavone-devoid (alcohol-washed) soy protein appears to be less effective at lowering LDL cholesterol concentrations, the isolated isoflavones do not appear to modulate plasma lipid concentrations at all, suggesting either that the isoflavones are not an active compound in isoflavone-intact soy protein, or that alcohol extraction alters their effectiveness. Therefore, the current data suggest that soy protein that has not been alcohol-washed and contains naturally occurring isoflavones provides the maximum benefit for improving plasma lipoprotein concentrations. Other Cardiovascular Disease Risk Factors Vascular Function. The data regarding the effects of isoflavone pills on vascular function are summarized in Table 14.1. In a placebo-controlled, randomized, crossover study with peri- and postmenopausal women (n = 21), treatment for 5 wk with 80 mg/d of soy isoflavone pills (i.e., no soy protein) improved systemic arterial compliance, an indicator of vascular elasticity, by 23% (52); however, there was no effect of the isoflavone pills on vascular reactivity measured as a percentage decrease in resistance in the microcirculation of the forearm after ischemia, or acetylcholine or nitroprusside administration. In a second study by this group (58),

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isoflavones from red clover given at a dose of 40 or 80 mg/d increased systemic arterial compliance by 20% in the 40-mg group (P = 0.04) or 24% in the 80-mg group (P = 0.02). The lack of effect of soy isoflavone pills on vascular function as measured by flow-mediated dilation was recently confirmed in a group of 20 postmenopausal women in a crossover study (53). In contrast, in a study by Walker et al. (61), infusion of increasing concentrations of genistein resulted in dose-dependent improvements in forearm blood flow in men (n = 9) and premenopausal women (n = 6). Daidzein did not affect forearm blood flow. These authors also determined that this effect of genistein on vasodilation was nitric oxide dependent. Whether the differences found in this study compared with previous studies with isolated isoflavones (52,53) are the result of different techniques with which to measure vascular function, or the result of higher genistein doses is uncertain. Isoflavone pills do appear to affect systemic arterial compliance, a measure of arterial elasticity, but not endothelium-dependent vascular reactivity, an indicator of endothelial function. Platelet Function. There are no studies of isoflavone pills that have measured platelet function. Lipoprotein Oxidation. Three studies have evaluated the effects of isoflavone pills on LDL oxidation (Table 14.2), all with negative results (52,59,62). Hodgson et al. (62) found no effect on urinary F2-isoprostane concentrations after 8 wk of treatment with 55 mg of isoflavones in 46 men and 13 postmenopausal women. In the study by Nestel et al. (52) in peri- and postmenopausal women, there were no effects of 80 mg isoflavones on LDL oxidizability as indicated by malondialdehyde measured by the thiobarbituric acid-reactive substances method, or lag time to copper-induced oxidation, maximum diene formation, or oxidation rate. Samman et al. (59) reported that in 14 premenopausal women, 86 mg of isoflavones given for 2 menstrual cycles did not affect lag time for copper-induced LDL oxidation, oxidation rate, or maximum oxidation. Thus, these results are very different from the studies evaluating the effects of soy with isoflavones. Blood Pressure. In five studies (Table 14.3) that have evaluated the effect of isoflavone pills on blood pressure (52,53,58,60,63), there were no significant effects of isoflavones. Isoflavone dose in these studies ranged from 40–100 mg, similar to the doses at which soy with isoflavones had effects. Although four of the studies included normotensive peri- and postmenopausal women (52,53,58,60), the study by Hodgson et al. (63) was with men and postmenopausal women with highnormal blood pressure. Again, these results are different than the studies using soy with isoflavones. Lipoprotein(a) Concentrations. Only two studies (Table 14.4) have tested the effect of isoflavone pills on Lp(a) concentrations (53,56). Hodgson et al. (56)

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reported no effect of isoflavone pills on Lp(a) concentrations in men (n = 46) and postmenopausal women (n = 13) randomly assigned to receive 55 mg isoflavone or placebo pills. Similarly, Simons and colleagues (53) found no effect of 80 mg isoflavones in 20 postmenopausal women. Direct Effects on the Artery Wall. There are several studies that suggest that isoflavones might have direct effects on the artery wall, including inhibition of the migration and proliferation of smooth muscle cells, which might inhibit the promotion and progression of atherosclerosis (64–67). In a study of arterial injury–induced atherogenesis in rats, Mäkelä and colleagues (65) found that the injured arteries markedly increased expression of estrogen receptor β (ERβ) and that genistein (which binds with high affinity to ERβ) inhibited neointima formation (i.e., reduced the proliferation of cells in the artery wall that form atherosclerotic lesions). These studies suggest that isoflavones might have direct effects on the artery wall to inhibit atherosclerosis formation and maintain cardiovascular health.

Effects of Soy Protein with Endogenous Isoflavones and Isolated Isoflavones on Atherosclerosis There are studies in animal models suggesting that soy protein with the isoflavones compared with isoflavone-devoid soy protein, can reduce atherosclerosis. In two studies in nonhuman primates (14,16), soy protein with isoflavones [SPI(+)] inhibited atherosclerosis formation relative to alcohol-washed (isoflavone-devoid) soy protein [SPI(–)]. In the first study, young males fed casein/lactalbumin (C/L) had the most atherosclerosis, those fed SPI(+) had the least (90% smaller lesions than the C/L group) and those fed SPI(–) had an intermediate amount (50% smaller lesions than the C/L group) (14). In a study in postmenopausal females (16), compared with the SPI(–) group, the SPI(+) group had significantly less atherosclerosis in the arteries of the head and neck (common carotid, 34% smaller; internal carotid, 61% smaller). In the coronary arteries, the SPI(+) group had 25% smaller atherosclerotic lesions than the SPI(–) group; however this difference was not significant (P = 0.12). Kirk et al. (19) reported an inhibition of atherosclerosis in LDL receptor–intact mice fed SPI(+) relative to a group fed SPI(–). However, there was no difference in atherosclerosis between these diet groups in a strain of mouse without LDL receptors (LDL receptor null), which they suggested meant that at least a portion of the atheroprotective effect of SPI(+) is mediated by LDL receptors. Adams and colleagues (12) compared the effects of SPI(+) and SPI(–) to casein on atherogenesis in male and female LDL receptor–null mice and apolipoprotein E (apo E)–null mice. They found that SPI(+) resulted in significantly less atherosclerosis in both the LDL receptor–null and apo E–null mice compared with both SPI(–) and casein. They also found that reductions in atherosclerosis were not necessarily related to improvements in plasma lipoprotein concentrations. Ni et al. (48) also used the apo

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E–deficient mouse model and found that soy protein feeding, compared with casein, reduced atherosclerosis in the absence of effects on total plasma cholesterol concentrations. There is only one published study that has evaluated the effect of an isoflavone extract on atherosclerosis. In rabbits (25), two doses of an isoflavone aglycone–rich extract, without soy protein, were tested. The isoflavone extract treatment resulted in significantly lower LDL oxidation, attenuated atherosclerosis, and less malondialdehyde (oxidation product) in the artery wall. However, because isolated isoflavones appear to have minimal effects on many mediators of atherosclerosis, it seems reasonable to speculate that isolated isoflavones will not have as potent an effect on inhibition of atherosclerosis as soy protein with the isoflavones. Thus, the beneficial effects of soy on CVD risk factors appear to translate into reductions in the extent of atherosclerosis. Alcohol-washed soy protein does not have the same benefits for inhibiting atherosclerosis that are seen with intact soy protein. Although at least a portion of the inhibition of atherosclerosis is likely mediated by effects on plasma lipid concentrations, there are also data to suggest that the effects on LDL oxidation and other nonlipid mechanisms might be involved.

Summary There are many mechanisms by which soy protein and/or the isoflavones might decrease atherosclerosis and cardiovascular disease. There are the well-recognized improvements in plasma lipid and lipoprotein concentrations, i.e., lower LDL cholesterol, lower triglycerides, and possibly higher HDL cholesterol; these are addressed more fully in other chapters. There is also evidence that soy protein with isoflavones can have beneficial effects on blood pressure, vascular function in women, platelet function, LDL oxidation, and possibly carbohydrate metabolism. Fewer data exist for isoflavone pills, but the data seem convincing about the lack of effect on plasma lipid concentrations. Further research is required before a conclusion can be made about potential effects of isoflavone pills on blood pressure, LDL oxidation, vascular function, and platelet function. The possible adverse effects of soy with isoflavones on vascular function in men and Lp(a) concentrations should be examined further. Data exist that show inhibition of atherosclerosis with soy containing isoflavones in established animal models, but only one study thus far has tested an isoflavone extract. To date, no studies have been published that evaluated the effects of soy protein and/or isoflavones on atherosclerosis in humans. Because of the effects on CVD risk factors, soy foods containing isoflavones could have an important effect on reduction of CVD burden. However, neither isoflavone-devoid soy protein nor isoflavone pills appear to have the same benefits for improving cardiovascular health. Epidemiologic studies to evaluate the associations between soy and/or isoflavone intake and CHD morbidity and mortality are necessary to determine whether these effects of soy/isoflavones on CVD risk factors translate into

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effects on CHD risk. At this time, including foods containing intact soy with isoflavones in a “heart healthy” diet seems the best recommendation for cardiovascular health. References 1. American Heart Association (2002) 2002 Heart and Stroke Statistical Update, American Heart Association, Dallas. 2. Krauss, R.M., Eckel, R.H., Howard, B., Appel, L.J., Daniels, S.R., Deckelbaum, R.J., Erdman, J.W., Kris-Etherton, P., Goldberg, I.J., Kotchen, T.A., Lichtenstein, A.H., Mitch, W.E., Mullis, R., Robinson, K., Wylie-Rosett, J., St. Jeor, S., Suttie, J., Tribble, D.L., and Bazzarre, T.L. (2000) AHA Dietary Guidelines Revision 2000: A Statement for Healthcare Professionals from the Nutrition Committee of the American Heart Association, Circulation. 102, 2284–2299. 3. Erdman, J.W. (2000) Soy Protein and Cardiovascular Disease: A Statement for Healthcare Professionals from the Nutrition Committee of the AHA, Circulation 102, 2555–2559. 4. Erdman, J.W., and Fordyce, E.J. (1989) Soy Products and the Human Diet, Am. J. Clin. Nutr. 49, 725–737. 5. Potter, S.M. (1995) Overview of Proposed Mechanisms for the Hypocholesterolemic Effect of Soy, J. Nutr. 125, 606S–611S. 6. Potter, S.M. (1998) Soy Protein and Cardiovascular Disease: The Impact of Bioactive Components in Soy, Nutr. Rev. 56, 231–235. 7. Huff, M.W., Hamilton, R.M.G., and Carroll, K.K. (1977) Plasma Cholesterol Levels in Rabbits Fed Low Fat, Cholesterol-Free, Semipurified Diets: Effects of Dietary Proteins, Protein Hydrolysates and Amino Acid Mixtures, Atherosclerosis 28, 187–195. 8. Lovati, M.R., Manzoni, C., Gianazza, E., Arnoldi, A., Kurowska, E., Carroll, K.K., and Sirtori, C.R. (2000) Soy Protein Peptides Regulate Cholesterol Homeostasis in Hep G2 Cells, J. Nutr. 130, 2543–2549. 9. Wang, M.-F., Yamamoto, S., Chung, H.-M., Chung, S.-Y., Miyatani, S., Mori, M., Okita, T., and Sugano, M. (1995) Antihypercholesterolemic Effect of Undigested Fraction of Soybean Protein in Young Female Volunteers, J. Nutr. Sci. Vitaminol. 41, 187–195. 10. Lovati, M.R., Manzoni, C., Gianazza, E., and Sirtori, C.R. (1998) Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. I. Identification of Active β-Conglycinin Subunits, J. Agric. Food. Chem. 46, 2474–2480. 11. Sugano, M., Goto, S., Yamada, Y., Yoshida, K., Hashimoto, Y., Matsuo, T., and Kimoto, M. (1990) Cholesterol-Lowering Activity of Various Undigested Fractions of Soybean Protein in Rats, J. Nutr. 120, 977–985. 12. Adams, M.R., Golden, D.L., Anthony, M.S., Register, T.C., and Williams, J.K. (2002) The Inhibitory Effect of Soy Protein Isolate on Atherosclerosis in Mice Does Not Require the Presence of LDL Receptors or Alteration of Plasma Lipoproteins, J. Nutr. 132, 43–49. 13. Anthony, M.S., Clarkson, T.B., Hughes, C.L., Morgan, T.M., and Burke, G.L. (1996) Soybean Isoflavones Improve Cardiovascular Risk Factors Without Affecting the Reproductive System of Peripubertal Rhesus Monkeys, J. Nutr. 126, 43–50. 14. Anthony, M.S., Clarkson, T.B., Bullock, B.C., and Wagner, J.D. (1997) Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet-Induced Coronary Artery Atherosclerosis of Male Cynomolgus Monkeys, Arterioscler. Thromb. Vasc. Biol. 17, 2524–2531.

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15. Balmir, F., Staack, R., Jeffrey, E., Berber-Jimenez, M.D., Wang, L., and Potter, S.M. (1996) An Extract of Soy Flour Influences Serum Cholesterol and Thyroid Hormones in Rats and Hamsters, J. Nutr. 126, 3046–3053. 16. Clarkson, T.B., Anthony, M.S., and Morgan, T.M. (2001) Inhibition of Postmenopausal Atherosclerosis Progression: A Comparison of the Effects of Conjugated Equine Estrogens and Soy Phytoestrogens, J. Clin. Endocrinol. Metab. 86, 41–47. 17. Crouse, J.R., Morgan, T.M., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with that of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070–2076. 18. Gardner, C.D., Newell, K.A., Cherin, R., and Haskell, W.L. (2001) The Effect of Soy Protein with or Without Isoflavones Relative to Milk Protein on Plasma Lipids in Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 728–735. 19. Kirk, E..A., Sutherland, P., Wang, S.A., Chait, A., and LeBoeuf, R.C. (1998) Dietary Isoflavones Reduce Plasma Cholesterol and Atherosclerosis in C57BL/6 Mice but Not LDL Receptor-deficient Mice, J. Nutr. 128, 954–959. 20. Merz-Demlow, B.E., Duncan, A.M., Wangen, K.E., Xu, X., Carr, T.P., Phipps, W.R., and Kurzer, M.S. (2000) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic, Premenopausal Women, Am. J. Clin. Nutr. 71, 1462–1469. 21. Peluso, M.R., Winters, T.A., Shanahan, M.F., and Banz ,W.J. (2000) A Cooperative Interaction Between Soy Protein and Its Isoflavone-Enriched Fraction Lowers Hepatic Lipids in Male Obese Zucker Rats and Reduces Blood Platelet Sensitivity in Male Sprague-Dawley Rats, J. Nutr. 130, 2333–2342. 22. Sugano,, M., and Koba, K. (1993) Dietary Protein and Lipid Metabolism: A Multifunctional Effect, Ann. N.Y. Acad. Sci. 676, 215–222. 23. Tovar-Palacio, C., Potter, S.M., Hafermann, J.C., and Shay, N.F. (1998) Intake of Soy Protein and Soy Protein Extracts Influences Lipid Metabolism and Hepatic Gene Expression in Gerbils, J. Nutr. 128, 839–842. 24. Wangen, K.E., Duncan, A.M., Xu, X., and Kurzer, M.S. (2001) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic and Mildly Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 225–231. 25. Yamakoshi, J., Piskula, M.K., Izumi, T., Tobe, K., Saito, M., Kataoka, S., Obata, A., and Kikuchi, M. (2000) Isoflavone Aglycone-rich Extract Without Soy Protein Attenuates Atherosclerosis Development in Cholesterol-fed Rabbits, J. Nutr. 130, 1887–1893. 26. Potter, S.M., Jimenez-Flores, R., Pollack, J.-A., Lone, T.A., and Berber-Jiminez, M.D. (1993) Protein-Saponin Interaction and Its Influence on Blood Lipids, J. Agric. Food. Chem. 41, 1287–1291. 27. Anderson, R.L., and Wolf, W.J. (1995) Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflavones Related to Soybean Processing, J. Nutr. 125, 581S–588S. 28. Anthony, M.S. (2000) Soy and Cardiovascular Disease: Cholesterol Lowering and Beyond, J. Nutr. 130, 662S–663S. 29. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 30. Dent, S.B., Peterson, C.T., Brace, L.D., Swain, J.H., Reddy, M.B., Hanson, K.B., Robinson, J.G., and Alekel, D.L. (2001) Soy Protein Intake by Perimenopausal Women Does Not Affect Circulating Lipids and Lipoproteins or Coagulation and Fibrinolytic Factors, J. Nutr. 131, 2280–2287.

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31. Honoré, E.K., Williams, J.K., Anthony, M.S., and Clarkson, T.B. (1997) Soy Isoflavones Enhance Vascular Reactivity in Atherosclerotic Female Macaques, Fertil. Steril. 67, 148–154. 32. Williams, J.K., Anthony, M.S., and Herrington, D.M. (2001) Interactive Effects of Soy Protein and Estradiol on Coronary Artery Reactivity in Atherosclerotic, Ovariectomized Monkeys, Menopause 8, 307–313. 33. DuBroff, R., and Decker, P. (1999) Soy Phytoestrogens Improve Endothelial Dysfunction in Postmenopausal Women, North American Menopause Society Meeting Abstracts, p. 53, Abstract # 99.085. 34. Teede, H.J., Dalais, F.S., Kotsopoulos, D., Liang, Y.-L., Davis, S., and McGrath, B.P. (2001) Dietary Soy Has Both Beneficial and Potentially Adverse Cardiovascular Effects: A Placebo-Controlled Study in Men and Postmenopausal Women, J. Clin. Endocrinol. Metab. 86, 3053–3060. 35. Williams, K., andClarkson, T.B. (1998) Dietary Soy Isoflavones Inhibit In Vivo Constrictor Responses of Coronary Arteries to Collagen-Induced Platelet Activation, Coron. Artery Dis. 9, 759–764. 36. Schoene, N.W., and Guidry, C.A. (1999) Dietary Soy Isoflavones Inhibit Activation of Rat Platelets, J. Nutr. Biochem. 10, 421–426. 37. Jenkins, D.J.A., Kendall, C.W.C., Garsetti, M., Rosenberg-Zand, R.S., Jackson, C.-J., Agarwal, S., Rao, A.V., Diamandis, E.P., Parker, T., Faulkner, D., Vuksan,V., and Vidgen, E. (2000) Effect of Soy Protein Foods on Low-Density Lipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity—A Controlled Crossover Trial, Metabolism 49, 537–543. 38. Jenkins, D.J.A., Kendall, C.W.C., Vidgen, E., Vuksan, V., Jackson, C.-J., Augustin, L.S.A., Lee, B., Garsetti, M., Agarwal, S., Rao, A.V., Cagampang, G.B., and Fulgoni, V. (2000) Effect of Soy-Based Breakfast Cereal on Blood Lipids and Oxidized LowDensity Lipoprotein, Metabolism 49, 1496–1500. 39. Tikkanen, M.J., Wähälä, K., Ojala, S., Vihma, V., Adlercreutz, H. (1998) Effect of Soybean Phytoestrogen Intake on Low Density Lipoprotein Oxidation Resistance, Proc. Natl. Acad. Sci. 95, 3106–3110. 40. Wiseman, H., O’Reilly, J.D., Adlercreutz, H., Mallet, A.I., Bowey, E.A., Rowland, I.R., and Sanders, T.A.B. (2000) Isoflavone Phytoestrogens Consumed in Soy Decrease F2Isoprostane Concentrations and Increase Resistance of Low-Density Lipoprotein to Oxidation in Humans, Am J. Clin. Nutr. 72, 395–400. 41. Burke, V., Hodgson, J.M., Beilin, L.J., Giangiulioi, N., Rogers, P., and Puddey, I.B. (2001) Dietary Protein and Soluble Fiber Reduce Ambulatory Blood Pressure in Treated Hypertensives, Hypertension 38, 821–826. 42. Washburn, S., Burke, G.L., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13. 43. Wagner, J.D., Cefalu, W.T., Anthony, M.S., Litwak, K.N., Zhang, L., and Clarkson, T.B. (1997) Dietary Soy Protein and Estrogen Replacement Therapy Improve Cardiovascular Risk Factors and Decrease Aortic Cholesteryl Ester Content in Ovariectomized Cynomolgus Monkeys, Metabolism 46, 698–705. 44. Goodman-Gruen, D., and Kritz-Silverstein, D. (2001) Usual Dietary Isoflavone Intake Is Associated with Cardiovascular Disease Risk Factors in Postmenopausal Women, J. Nutr. 131, 1202–1206.

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45. Meinertz, H., Nilausen, K., and Hilden, J. (2002) Alcohol-Extracted, but Not Intact Dietary Soy Protein Lowers Lipoprotein(a) Markedly, Arterioscler. Thromb. Vasc. Biol. 22, 312–316. 46. Nilausen, K., and Meinertz, H. (1999) Lipoprotein(a) and Dietary Proteins: Casein Lowers Lipoprotein(a) Concentrations as Compared with Soy Protein, Am. J. Clin. Nutr. 69, 419–425. 47. Teixeira, S.R., Potter, S.M., Weigel, R., Hannum, S., Erdman, J.W., and Hasler, C.M. (2000) Effects of Feeding 4 Levels of Soy Protein for 3 and 6 wk on Blood Lipids and Apolipoproteins in Moderately Hypercholesterolemic Men, Am. J. Clin. Nutr. 71, 1077–1084. 48. Ni, W., Tsuda, Y., Sakono, M., Imaizumi, K. (1998) Dietary Soy Protein Isolate, Compared with Casein, Reduces Atherosclerotic Lesion Area in Apolipoprotein EDeficient Mice, J. Nutr. 128, 1884–1889. 49. De Kleijn,, M.J.J., van der Schouw, Y.T., Wilson, P.W.F., Grobbee, D.E., and Jacques, P.F. (2002) Dietary Intake of Phytoestrogens Is Associated with a Favorable Metabolic Cardiovascular Risk Profile in Postmenopausal U.S. Women: The Framingham Study, J. Nutr. 132, 276–282. 50. Dewell, A., Hollenbeck, C.B., and Bruce, B. (2002) The Effects of Soy-derived Phytoestrogens on Serum Lipids and Lipoproteins in Moderately Hypercholesterolemic Postmenopausal Women, J. Clin. Endocrinol. Metab. 87, 118–121. 51. Hsu, C.-S., Shen, W.W., Hsueh, Y.-M., and Yeh, S.-L. (2001) Soy Isoflavone Supplementation in Postmenopausal Women. Effects on Plasma Lipids, Antioxidant Enzyme Activities and Bone Density, J. Reprod. Med. 46, 221–226. 52. Nestel, P.J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P., Owen, A., and Abbey, M. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Arterioscler. Thromb. Vasc. Biol. 17, 3392–3398. 53. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2000) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy, Postmenopausal Women, Am. J. Cardiol. 85, 1297–1301. 54. Greaves, K.A., Parks, J.S., Williams, J.K., and Wagner, J.D. (1999) Intact Dietary Soy Protein, but Not Adding an Isoflavone-Rich Soy Extract to Casein, Improves Plasma Lipids in Ovariectomized Cynomolgus Monkeys, J. Nutr. 129, 1585–1592. 55. Hale, G.E., Hughes, C.L., Robboy, S.J., Agarwal, S.K., and Bievre, M. (2001) A Double-Blind Randomized Study on the Effects of Red Clover Isoflavones on the Endometrium, Menopause 8, 338–346. 56. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., and Croft, K.D. (1998) Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations: A Randomized Controlled Trial in Humans, J. Nutr. 128, 728–732. 57. Howes, J.B., Sullivan, D., Lai, N., Nestel, P., Pomeroy, S., West, L., Eden, J.A., and Howes, L.G. (2000) The Effects of Dietary Supplementation with Isoflavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 52, 143–147. 58. Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J.D., and West, L. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895–898.

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59. Samman, S., Wall, P.M.L., Chan, G.S.M., Smith, S.J., and Petocz, P. (1999) The Effect of Supplementation with Isoflavones on Plasma Lipids and Oxidisability of Low Density Lipoprotein in Premenopausal Women, Atherosclerosis 147, 277–283. 60. Han, K.K., Soares, J.M., Haidar, M.A., de Lima, G.R., and Baracat, E.C. (2002) Benefits of Soy Isoflavone Therapeutic Regimen on Menopausal Symptoms, Obstet. Gynecol. 99, 389–394. 61. Walker, H.A., Dean, T.S., Sanders, T.A.B., Jackson, G., Ritter, J.M., and Chowienczyk, P.J. (2001) The Phytoestrogen Genistein Produces Acute Nitric Oxide-Dependent Dilation of Human Forearm Vasculature with Similar Potency to 17β-Estradiol, Circulation 103, 258–262. 62. Hodgson, J.M., Puddey, I.B., Croft, K.D., Mori, T.A., Rivera, J., and Beilin, L.J. (1999) Isoflavonoids Do Not Inhibit In Vivo Lipid Peroxidation in Subjects with High-Normal Blood Pressure, Atherosclerosis 145, 167–172. 63. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., Burke, V., Croft, K.D., and Rogers, P.B. (1999) Effects of Isoflavonoids on Blood Pressure in Subjects with HighNormal Ambulatory Blood Pressure Levels. A Randomized Controlled Trial, Am. J. Hypertens. 12, 47–53. 64. Fujio, Y., Fumiko, Y., Takahashi, K., and Shibata, N. (1993) Responses of Smooth Muscle Cells to Platelet-Derived Growth Factor Are Inhibited by Herbimycin-A Tyrosine Kinase Inhibitor+, Biochem. Biophys. Res. Commun. 195, 79–83. 65. Mäkelä, S., Savolainen, H., Aavik, E., Myllärniemi, M., Strauss, L., Taskinen, E., Gustafsson, J.Å., and Häyry, P. (1999) Differentiation Between Vasculoprotective and Uterotrophic Effects of Ligands with Different Binding Affinities to Estrogen Receptors α and β, Proc. Natl. Acad. Sci. USA 96, 7077–7082. 66. Shimokado, K., Yokota, T., Umezawa, K., Sasaguri, T., and Ogata, J. (1994) Protein Tyrosine Kinase Inhibitors Inhibit Chemotaxis of Vascular Smooth Muscle Cells, Arterioscler. Thromb. 14, 973–981. 67. Shimokado, K., Umezawa, K., and Ogata, J. (1995) Tyrosine Kinase Inhibitors Inhibit Multiple Steps of the Cell Cycle of Vascular Smooth Muscle Cells, Exp. Cell. Res. 220, 266–273.

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Chapter 15

Soy Proteins, Isoflavones, Cardiovascular Risk Factors, and Chronic Disease David J.A. Jenkinsa,b,c, Cyril W.C. Kendalla,c, and Augustine Marchiea,c aClinical Nutrition and Risk Factor Modification Center; bDepartment of Medicine, St. Michael’s Hospital; and cDepartment of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada

Introduction Soy has attracted much interest as a dietary component that may reduce a range of cardiovascular risk factors. In recent years, much emphasis has been placed on the protein-associated isoflavones as the component of soy responsible for the benefits. There is evidence, however, that the soy protein per se appears to be responsible, at least in part, for many of the effects. Other soy components such as saponins, which are extracted from the soy protein on alcohol washing along with the isoflavones, may also confer some of the advantages of soy ascribed to the isoflavones. Part of the reason for the uncertainty over the function of the isoflavones also relates to the many effects ascribed to soy. These include lipid reduction (1–6), antioxidant- (7–11) and homocysteine-lowering ability (12), together with improved arterial compliance (13) and possibly heightened immune responsiveness (14) in those consuming soy foods. There is even a suggestion that soy may be more satiating (15) and may aid in weight reduction. Possibly it is because of this broad spectrum of soy effects that there have been divergent views on some of the details. This paper will review some of the health issues surrounding soy consumption and will focus especially on soy and cardiovascular disease risk factors (blood lipids, antioxidant activity, vascular reactivity, thrombosis, homocysteine, immune response, and blood pressure), body weight, isoflavone bioavailability, osteoporosis, and cancer. Blood Lipids Early studies in the 1970s from the laboratories of Sirtori (16), Carroll (17) and Kritchevsky (18,19) demonstrated the effectiveness of soy protein in reducing serum lipids, notably low density lipoprotein (LDL) cholesterol (LDL-C). By the mid 1990s, Anderson et al. (3) were able to report a meta-analysis of 39 studies, which concluded that for an average intake of 47 g soy protein daily, a reduction of 13% in LDL-C might be expected. They concluded that the isoflavones might be the effective agent and cited abstracts of the work of Clarkson and colleagues

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that systematically explored this phenomenon in monkeys; the report was published subsequently in The Journal of Nutrition (1). Later studies by Potter et al. (20) also demonstrated effects of soy protein in raising high density lipoprotein (HDL) cholesterol (HDL-C) and lowering LDL and very low density lipoprotein (VLDL) cholesterol in 6-mo studies. However, the effects were greatest with the lower-isoflavone soy (56 mg/d) than with the higher-isoflavone soy (90 mg/d). They also demonstrated increased mononuclear cell LDL receptor mRNA on soy vs. dairy control (20). Studies by Crouse et al. (4) went on to demonstrate a clear dose response to isoflavones taken with a constant 25 g of soy protein. However, even in these studies, soy protein was found to have a cholesterol-lowering effect in the absence of isoflavones. Furthermore, the effect was seen only in hyperlipidemic subjects (4). Additional studies by Kurzer et al. (5) demonstrated the cholesterol-lowering effect of soy but failed to detect a clear difference in LDL-C between high and low isoflavone treatments. Most recently the studies of Gardner et al. (6), although noting a LDL difference between high and low isoflavone treatments favoring lower LDL-C levels on high isoflavone, did not find the dairy protein control to be significantly different from either isoflavone treatment. Unlike the animal studies, the studies of soy isoflavone in humans have been less clear in demonstrating a cholesterol-lowering effect. The question arose, therefore, concerning whether the differences related to the use of supplements, taken mainly as beverages rather than in foods, as an integral part of the diet. However, studies using foods delivering either ~10 or 70 mg of isoflavones daily also failed to show differences between high and low treatments, although both soy treatments reduced LDL-C compared with the dairy control treatment (21,22). The role of isoflavones in LDL reduction is not clear from current human studies; although some effect seems probable, there seems to be a requirement for the presence of soy proteins because studies in which isoflavones have been given in isolation have not reduced serum cholesterol levels (13,23). Furthermore, despite differences in isoflavone level, soy proteins per se appear to lower serum cholesterol levels. Antioxidant Activity Antioxidant activity appears to be more consistently associated with the isoflavone component of soy protein. Genistein has been shown to reduce LDL oxidation in vitro and attenuate inducible nitric oxide synthase expression (7,24). Studies of soy consumption have shown reduced urinary isoprostane excretion as a marker of lipid oxidative damage (8), and soy feeding has been associated also with reduced levels of conjugated dienes in the LDL fraction as a marker of LDL oxidation (9–11). Furthermore, the antioxidant effect is still seen clearly even when the effect on LDL reduction is not significant as with soy in cereal form in which raised temperatures in processing may have denatured the protein and prevented the lipidlowering effect (10). These antioxidant effects are likely to contribute to the cardiovascular protective effects of soy foods.

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Vascular Reactivity It has been suggested that soy phytoestrogens may increase NO synthesis, the endothelial relaxation factor, because estrogens increase the activity of nitric oxide synthesis. However, the ability of genistein to attenuate inducible NO synthase expression suggest that in vivo studies are required to determine the relative importance of these potentially conflicting effects. Furthermore, plant proteins have higher arginine to lysine ratios than animal proteins and this might also favor increased nitric oxide synthesis. Original studies by Clarkson and colleagues (25,26) on female rhesus monkeys fed soy containing isoflavones or alcohol-washed soy from which the isoflavones had been removed indicated that acetylcholine injection caused vasodilatation in the presence of isoflavones and vasoconstriction with the isoflavone-depleted soy diet. The intravenous injection of genistein reversed the vasoconstriction. Other studies have shown that genistein acts like estrogen to improve endothelial dysfunction induced by ovariectomy in rats (27). Furthermore, purified isoflavones from both soy and clover have been shown to improve systemic arterial compliance in menopausal women. This parameter is an index of the elasticity of large arteries such as the thoracic aorta. Compliance diminishes with age and menopause, and the improvement in this derived assessment by isoflavone ingestion is considered to be beneficial for cardiovascular health (13,28). Unfortunately, the one study to measure flow-mediated vasodilatation in the brachial arteries of 50- to 70-y-old postmenopausal women after consuming an isoflavone tablet (80 mg/d) or placebo for 8 wk showed no effect (29). The hypothesized similarity between the isoflavones and estrogens with respect to vascular function, therefore, remains to be demonstrated. The effect on arterial compliance is difficult to explain. It occurs in a relatively short time (a matter of weeks) and has been suggested to be due to endothelial events influencing arterial smooth muscle relaxation. However, the lack of an effect of isoflavones on flow-mediated vasodilatation makes this explanation less satisfactory. The effect of soy on the human vascular system requires much further study before any of the hypothesized benefits can be clearly defined. Thrombosis Risk Although estrogens may negatively affect thrombosis risk, it has been suggested that the effect of isoflavones may be beneficial. Production of plasminogen activator inhibitor (PAI-1), an important risk factor for thrombosis that has been shown to be modifiable by dietary means (30), is stimulated by inflammatory mediators, tumor necrosis factor (TNF)-α, interleukin (IL)-1, and bacterial lipopolysaccharide. In vitro genistein has been shown to block synthesis of PAI-1 by human endothelial cells stimulated with TNF-α. The effects of genistein were accompanied by a decrease in PAI-1 mRNA and suppression of the PAI-1 transcription rate (31). More recently, two studies were reported (12,32) in which soy protein with

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isoflavones failed to alter PAI-1 levels and other indices of thrombosis risk. In perimenopausal women, 40 g/d of soy protein fed for 24 wk did not alter blood lipids or PAI-1 or serum fibrinogen vs. the control group irrespective of whether the soy was high or low in isoflavones (32). In another study (12) of type 2 diabetic subjects fed 50 g soy protein/d, containing a minimum of 165 mg of isoflavone, despite beneficial effects on the blood lipid profile (LDL-C and LDL-C:HDL-C ratio) no beneficial effects were seen over 6 wk on PAI-1 or factor VIIc, von Willebrand factor, or fibrinogen. Thus, despite in vitro data, in vivo data are still required to demonstrate a beneficial effect of soy on clotting factors and thrombosis risk. Serum Homocysteine A number of reasons suggest that another risk factor for cardiovascular disease, serum homocysteine, may be reduced when soy replaces animal proteins in the diet. First, soy is a relatively poor source of methionine and thus, the amino acid precursors may be reduced. Second, if soy isoflavones possess estrogen activity, they may reduce homocysteine levels, as do mammalian estrogens. In premenopausal women, homocysteine levels are lower than in men, but rise after the menopause (33–35). Few studies have assessed homocysteine levels after soy consumption, but measured homocysteine levels appear to be reduced in both type 2 diabetes (12) and hyperlipidemic subjects (21,22). However in these hyperlipidemic subjects, the isoflavone content did not influence the effect of the soy protein (21,22). Immune Response Few data are available in this area. Estrogens are known to modulate the immune response and enhance cytokine production. Feeding high isoflavone soy has been shown to increase IL-6 production in postmenopausal women but not men, and low isoflavone soy protein was without effect in either sex (14). Hypercholesterolemic men and postmenopausal women were fed three 1-mo diets consisting of a low-fat dairy food control phase and high- and low-isoflavone soy food test phases (containing 50 and 52 g/d soy protein, respectively, and 73 and 10 mg/d isoflavone, respectively). For the group as a whole, no treatment differences were observed for acute phase proteins or proinflammatory cytokines. However, assessing the results of men and women separately, women showed significantly higher IL-6 values after the high-isoflavone soy diet compared with control values and a significantly higher IL-6 value than the low-isoflavone IL-6 values, on the unadjusted data (14). No significant effects were seen for men or women in Creactive protein, serum amyloid A, or TNF-α (14). The relation of this action to other immune phenomena is not known but a heightened inflammatory response and increased immunosurveillance may play a role in tumor suppression and perhaps relate to the reduced incidence of certain cancers in the parts of the world that consume soy.

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Blood Pressure Reasons exist why soy isoflavones may increase vascular reactivity and possibly prevent rises in blood pressure. A number of studies have confirmed blood pressure reductions from soy in both men and women (36,37). The reduction in blood pressure recorded is modest and the level of isoflavone in the soy did not appear to be a factor (21,22). Body Weight Few data have been generated on the relationship between soy and weight reduction. Preliminary studies indicate that soy has a more prolonged and greater satiating effect than egg and casein (15). In one study of obese women, the body weight reduction (4 kg in 4 wk) was twice that of the dairy control but the difference was not significant despite a very significant difference in LDL-C favoring the soy treatment (38). Isoflavone Bioavailability The amount of isoflavone absorbed, its rate of absorption, and the length of time serum levels remain elevated may all influence the action of the isoflavone. Isoflavones are present largely in the conjugated form in food. They are absorbed as aglycones after deconjugation, likely by colonic bacteria, but the absorption of conjugated isoflavones may also occur after deconjugation by small intestinal brush border glucosidases (39). The absorption for soy foods is slower, more prolonged, and reaches lower serum concentrations (39). Although it would appear that physiologic processes such as increased fermentation of fermentable fiber may enhance deconjugation and facilitate isoflavone absorption, it is also possible that enhanced fermentation may result in bacterial metabolism of the aglycone rendering it unavailable for absorption, and less rather than more isoflavone may be absorbed. Studies to assess these effects are required. Furthermore, on absorption, the aglycone is conjugated in the gastrointestinal tract, circulates in the blood in the conjugated form, and may be hydrolyzed in the target tissues as occurs with other bioactive compounds such as estrogens. The aglycone is presumed to be the active form of the isoflavone; however, recent data suggest that the conjugated form, although it may bind to the ER receptor more weakly, may stimulate growth of MCF-6 cells more than the aglycone isoflavone (40). Osteoporosis and Cancer There is growing evidence that soy isoflavones may protect from the development of osteoporosis through the action on the estrogen receptor in bone. Studies have demonstrated reduced urinary N-telopeptide excretion in the urine and reduced bone loss assessed by dual-energy X-ray absorptiometry (DXA) scans in women

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fed soy (41,42). The effect may be dose dependent with benefits in the normal range of soy consumption but possibly with negative side effects at isoflavone levels above the normal consumption range (43). The area shows promise and further studies are required. The reduced incidence of prostate and breast cancer in Asian countries in which soy is consumed on a regular basis, has prompted speculation that soy consumption may reduce the incidence of these diseases. It has been reasoned that estrogens have been shown to inhibit prostate cancer growth in men and that in women, the high affinity of the isoflavone for the receptor coupled with the weak estrogenic activity effectively blocks endogenous estrogen activity, thus also reducing the risk of breast cancer. However, few human data exist. One study of Seventh Day Adventist men indicated that with greater daily intakes of soy milk, there was a reduced risk of prostate cancer (44). Recently, the Shanghai Breast Cancer Study demonstrated that breast cancer incidence was negatively related to the consumption of soy by the women during their adolescence and even more strongly negatively correlated with the soy intakes of the women’s mothers (45). These data are encouraging but many more studies in this area are required. Studies of soy consumption and serum prostate-specific antigen (PSA) levels in men have indicated no effect even at levels of soy consumption and over a time period in which serum LDL-C levels have been reduced (46). Of additional importance, young adults fed soy as infants show no developmental problems in young adult life apart from some increase incidence of discomfort at the time of menstruation (47). The only negative effect of soy is the suggestion that isoflavones may cause a rare form of childhood leukemia (48) and brain atrophy in old age (49). However, the rareness of this form of childhood leukemia renders this concern difficult to assess and the problem remains only a theoretical possibility. The greater brain atrophy after soy consumption observed in the Hawaiian study could be accounted for by the older age at death of the high vs. low soy consumers (49). Furthermore, increased estrogenic activity has been associated with preservation of mental function and negative effects of isoflavones in men would therefore be more difficult to explain.

Conclusions Overall, soy food consumption appears to have valuable cardioprotective effects in terms of cholesterol lowering, antioxidant activity, and homocysteine reduction. The extent to which these effects are the result of the isoflavone component or the soy protein remains to be defined, although a role for isoflavones has clearly been demonstrated in some studies. Isoflavones may have independently promising roles in improving cardiac function and vascular compliance, preventing osteoporosis, stimulating the immune response, and possibly reducing the risk of certain cancers. It is also possi-

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ble that other soy components, e.g., saponins, are responsible for some of the benefits. The many useful facets of soy continue to support its increased use in human nutrition. References 1. Anthony, M.S., Clarkson, T.B., Hughes, C.L., Jr., Morgan, T.M., and Burke, G.L. (1996) Soybean Isoflavones Improve Cardiovascular Risk Factors Without Affecting the Reproductive System of Peripubertal Rhesus Monkeys, J. Nutr. 126, 43–50. 2. Potter, S.M., Bakhit, R.M., Essex-Sorlie, D.L., Weingartner, K.E., Chapman, K.M., Nelson, R.A., Prabhudesai, M., Savage, W.D., Nelson, A.I., Winter, L.W., and Erdman, J.W., Jr. (1993) Depression of Plasma Cholesterol in Men by Consumption of Baked Products Containing Soy Protein, Am. J. Clin. Nutr. 58, 501–506. 3. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 4. Crouse, J.R., 3rd, Morgan, T., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with That of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070–2076. 5. Wangen, K.E., Duncan, A.M., Xu, X., and Kurzer, M.S. (2001) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic and Mildly Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 225–231. 6. Gardner, C.D., Newell, K.A., Cherin, R., and Haskell, W.L. (2001) The Effect of Soy Protein with or Without Isoflavones Relative to Milk Protein on Plasma Lipids in Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 728–735. 7. Patel, R.P., Boersma, B.J., Crawford, J.H., Hogg, N., Kirk, M., Kalyanaraman, B., Parks, D.A., Barnes, S., and Darley-Usmar, V. (2001) Antioxidant Mechanisms of Isoflavones in Lipid Systems: Paradoxical Effects of Peroxyl Radical Scavenging, Free Radic. Biol. Med. 31, 1570–1581. 8. Wiseman, H., O’Reilly, J.D., Adlercreutz, H., Mallet, A.I., Bowey, E.A., Rowland, I.R., and Sanders, T.A. (2000) Isoflavone Phytoestrogens Consumed in Soy Decrease F(2)Isoprostane Concentrations and Increase Resistance of Low-Density Lipoprotein to Oxidation in Humans, Am. J. Clin. Nutr. 72, 395–400. 9. Jenkins, D.J., Kendall, C.W., Vidgen, E., Mehling, C.C., Parker, T., Seyler, H., Faulkner, D., Garsetti, M., Griffin, L.C., Agarwal, S., Rao, A.V., Cunnane, S.C., Ryan, M.A., Connelly, P.W., Leiter, L.A., Vuksan, V., and Josse, R. (2000) The Effect on Serum Lipids and Oxidized Low-Density Lipoprotein of Supplementing Self-Selected Low-Fat Diets with Soluble-Fiber, Soy, and Vegetable Protein Foods, Metabolism 49, 67–72. 10. Jenkins, D.J., Kendall, C.W., Vidgen, E., Vuksan, V., Jackson, C.J., Augustin, L.S., Lee, B., Garsetti, M., Agarwal, S., Rao, A.V., Cagampang, G.B., and Fulgoni, V., 3rd. (2000) Effect of Soy-Based Breakfast Cereal on Blood Lipids and Oxidized LowDensity Lipoprotein, Metabolism 49, 1496–1500. 11. Jenkins, D.J., Kendall, C.W., Garsetti, M., Rosenberg-Zand, R.S., Jackson, C.J., Agarwal, S., Rao, A.V., Diamandis, E.P., Parker, T., Faulkner, D., Vuksan, V., and Vidgen, E. (2000) Effect of Soy Protein Foods on Low-Density Lipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity—A Controlled Crossover Trial, Metabolism 49, 537–543.

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12. Hermansen, K., Sondergaard, M., Hoie, L., Carstensen, M., and Brock, B. (2001) Beneficial Effects of a Soy-Based Dietary Supplement on Lipid Levels and Cardiovascular Risk Markers in Type 2 Diabetic Subjects, Diabetes Care 24, 228–233. 13. Nestel, P.J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P., Owen, A., and Abbey, M. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Arterioscler. Thromb. Vasc. Biol. 17, 3392–3398. 14. Jenkins, D.J., Kendall, C.W., Connelly, P.W., Jackson, C.J., Parker, T., Faulkner, D., and Vidgen, E. (2002) Effects of High and Low (Phytoestrogen) Soy Foods on Inflammatory Biomarkers and Proinflammatory Cytokines in Hyperlipidemic Men and Women, Metabolism, in press. 15. Tecimer, S.N., and Anderson, G.H. (2001) Effect of Protein Source in Suppression of Food Intake in Young Men, Can. Fed. Biol. Soc. Proc. 44, 63 (Abstr.). 16. Sirtori, C.R., Agradi, E., Conti, F., Mantero, O., and Gatti, E. (1977) Soybean-Protein Diet in the Treatment of Type-Ii Hyperlipoproteinaemia, Lancet 1, 275–277. 17. Carroll, K.K., Giovannetti, P.M., Huff, M.W., Moase, O., Roberts, D.C., and Wolfe, B.M. (1978) Hypocholesterolemic Effect of Substituting Soybean Protein for Animal Protein in the Diet of Healthy Young Women, Am. J. Clin. Nutr. 31, 1312–1321. 18. Vahouny, G.V., Adamson, I., Chalcarz, W., Satchithanandam, S., Muesing, R., Klurfeld, D.M., Tepper, S.A., Sanghvi, A., and Kritchevsky, D. (1985) Effects of Casein and Soy Protein on Hepatic and Serum Lipids and Lipoprotein Lipid Distributions in the Rat, Atherosclerosis 56, 127–137. 19. Kritchevsky, D., Tepper, S.A., Williams, D.E., and Story, J.A. (1977) Experimental Atherosclerosis in Rabbits Fed Cholesterol-Free Diets. Part 7. Interaction of Animal or Vegetable Protein with Fiber, Atherosclerosis 26, 397–403. 20. Potter, S.M., Baum, J.A., Teng, H., Stillman, R.J., Shay, N.F., and Erdman, J.W., Jr. (1998) Soy Protein and Isoflavones: Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68 (Suppl.), 1375S–1379S. 21. Kendall, C.W., Jenkins, D.J., Connelly, P.W., Parker, T., Faulkner, D., and Josse, R.G. (2002) Effects of High and Low Isoflavone Soy Foods on Blood Lipids, Oxidized LDL, Homocysteine and Blood Pressure in Hyperlipidemic Men and Women, Am. J. Clin Nutr. 75 (Suppl.), 385S (Abstr.). 22. Jenkins, D.J., Kendall, C.W., Jackson, C.-J., Faulkner, D., Parker, P., Vidgen, E., Cunnane, S.C., Connelly, P.W., Leiter, L.A., and Josse, R.G. (2002) Effects of High an Low Isoflavone Soy Foods on Blood Lipids, Oxidized LDL, Homocysteine and Blood Pressure in Hyperlipidemic Men and Women, Am. J. Clin. Nutr., in press. 23. Gooderham, M.H., Adlercreutz, H., Ojala, S.T., Wähälä, K., and Holub, B.J. (1996) A Soy Protein Isolate Rich in Genistein and Daidzein and Its Effects on Plasma Isoflavone Concentrations, Platelet Aggregation, Blood Lipids and Fatty Acid Composition of Plasma Phospholipid in Normal Men, J. Nutr. 126, 2000–2006. 24. Kim, H., Peterson, T.G., and Barnes, S. (1998) Mechanisms of Action of the Soy Isoflavone Genistein: Emerging Role for Its Effects Via Transforming Growth Factor Beta Signaling Pathways, Am. J. Clin. Nutr. 68 (Suppl.), 1418S–1425S. 25. Honore, E.K., Williams, J.K., Anthony, M.S., and Clarkson, T.B. (1997) Soy Isoflavones Enhance Coronary Vascular Reactivity in Atherosclerotic Female Macaques, Fertil. Steril. 67, 148–154.

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26. Williams, J.K., Honore, E.K., Washburn, S.A., and Clarkson, T.B. (1994) Effects of Hormone Replacement Therapy on Reactivity of Atherosclerotic Coronary Arteries in Cynomolgus Monkeys, J. Am. Coll. Cardiol. 24, 1757–1761. 27. Squadrito, F., Altavilla, D., Squadrito, G., Saitta, A., Cucinotta, D., Minutoli, L., Deodato, B., Ferlito, M., Campo, G.M., Bova, A., and Caputi, A.P. (2000) Genistein Supplementation and Estrogen Replacement Therapy Improve Endothelial Dysfunction Induced by Ovariectomy in Rats, Cardiovasc. Res. 45, 454–462. 28. Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J.D., and West, L. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895– 898. 29. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2000) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy, Postmenopausal Women, Am. J. Cardiol. 85, 1297–1301. 30. Jarvi, A.E., Karlstrom, B.E., Granfeldt, Y.E., Bjorck, I.E., Asp, N.G., and Vessby, B.O. (1999) Improved Glycemic Control and Lipid Profile and Normalized Fibrinolytic Activity on a Low-Glycemic Index Diet in Type 2 Diabetic Patients, Diabetes Care 22, 10–18. 31. van Hinsbergh, V.W., Vermeer, M., Koolwijk, P., Grimbergen, J., and Kooistra, T. (1994) Genistein Reduces Tumor Necrosis Factor Alpha-Induced Plasminogen Activator Inhibitor-1 Transcription but Not Urokinase Expression in Human Endothelial Cells, Blood 84, 2984–2991. 32. Dent, S.B., Peterson, C.T., Brace, L.D., Swain, J.H., Reddy, M.B., Hanson, K.B., Robinson, J.G., and Alekel, D.L. (2001) Soy Protein Intake by Perimenopausal Women Does Not Affect Circulating Lipids and Lipoproteins or Coagulation and Fibrinolytic Factors, J. Nutr. 131, 2280–2287. 33. Verhoef, P. (2000) Hyperhomocycteinemia and Risk of Vascular Disease in Women, Semin. Thromb. Hemost. 26, 325–334. 34. Hak, A.E., Polderman, K.H., Westendorp, I.C., Jakobs, C., Hofman, A., Witteman, J.C., and Stehouwer, C.D. (2000) Increased Plasma Homocysteine After Menopause, Atherosclerosis 149, 163–168. 35. Wouters, M.G., Moorrees, M.T., van der Mooren, M.J., Blom, H.J., Boers, G.H., Schellekens, L.A., Thomas, C.M., and Eskes, T.K. (1995) Plasma Homocysteine and Menopausal Status, Eur. J. Clin. Investig. 25, 801–805. 36. Washburn, S., Burke, G.L., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13. 37. Duncan, A.M., Underhill, K.E., Xu, X., Lavalleur, J., Phipps, W.R., and Kurzer, M.S. (1999) Modest Hormonal Effects of Soy Isoflavones in Postmenopausal Women, J. Clin. Endocrinol. Metab. 84, 3479–3484. 38. Jenkins, D.J., Wolever, T.M., Spiller, G., Buckley, G., Lam, Y., Jenkins, A.L., and Josse, R.G. (1989) Hypocholesterolemic Effect of Vegetable Protein in a Hypocaloric Diet, Atherosclerosis 78, 99–107. 39. Setchell, K.D., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., and Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131 (Suppl.), 1362S–1375S.

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40. Morito, K., Hirose, T., Kinjo, J., Hirakawa, T., Okawa, M., Nohara, T., Ogawa, S., Inoue, S., Muramatsu, M., and Masamune, Y. (2001) Interaction of Phytoestrogens with Estrogen Receptors Alpha and Beta, Biol. Pharm. Bull. 24, 351–356. 41. Scheiber, M.D., Liu, J.H., Subbiah, M.T., Rebar, R.W., and Setchell, K.D. (2001) Dietary Inclusion of Whole Soy Foods Results in Significant Reductions in Clinical Risk Factors for Osteoporosis and Cardiovascular Disease in Normal Postmenopausal Women, Menopause 8, 384–392. 42. Alekel, D.L., Germain. A.S., Peterson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in the Lumbar Spine of Perimenopausal Women, Am. J. Clin. Nutr. 72, 844–852. 43. Anderson, J.J., Ambrose, W.W., and Garner, S.C. (1998) Biphasic Effects of Genistein on Bone Tissue in the Ovariectomized, Lactating Rat Model, Proc. Soc. Exp. Biol. Med. 217, 345–350. 44. Jacobsen, B.K., Knutsen, S.F., and Fraser, G.E. (1998) Does High Soy Milk Intake Reduce Prostate Cancer Incidence? The Adventist Health Study (United States), Cancer Causes Control 9, 553–557. 45. Shu, X.O., Jin, F., Dai, Q., Wen, W., Potter, J.D., Kushi, L.H., Ruan, Z., Gao, Y.T., and Zheng, W. (2001) Soyfood Intake During Adolescence and Subsequent Risk of Breast Cancer Among Chinese Women, Cancer Epidemiol. Biomark. Prev. 10, 483–488. 46. Urban, D., Irwin, W., Kirk, M., Markiewicz, M.A., Myers, R., Smith, M., Weiss, H., Grizzle, W.E., and Barnes, S. (2001) The Effect of Isolated Soy Protein on Plasma Biomarkers in Elderly Men with Elevated Serum Prostate Specific Antigen, J. Urol. 165, 294–300. 47. Strom, B.L., Schinnar, R., Ziegler, E.E., Barnhart, K.T., Sammel, M.D., Macones, G.A., Stallings, V.A., Drulis, J.M., Nelson, S.E., and Hanson, S.A. (2001) Exposure to SoyBased Formula in Infancy and Endocrinological and Reproductive Outcomes in Young Adulthood, J. Am. Med. Assoc. 286, 807–814. 48. Ross, J.A. (2000) Dietary Flavonoids and the MLL Gene: A Pathway to Infant Leukemia? Proc. Natl. Acad. Sci. USA 97, 4411–4413. 49. White, L.R., Petrovitch, H., Ross, G.W., Masaki, K., Hardman, J., Nelson, J., Davis, D., and Markesbery, W. (2000) Brain Aging and Midlife Tofu Consumption, J. Am. Coll. Nutr. 19, 242–255.

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Chapter 16

Lipoprotein Effects of Soybean Phytoestrogens Sandra R. Teixeiraa and John W. Erdman, Jr.b aVitamins-Human

Nutrition and Health, Roche Vitamins Limited, CH-4070 Basel,

Switzerland bDepartment

of Food Science and Human Nutrition and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL

Introduction Phytoestrogens, and in particular isoflavones, have been postulated to have a positive effect on blood lipoproteins. Therefore, the consumption of these compounds has been advocated by some as a means to help fight against coronary heart disease (CHD). Because most of the attention in this area has been directed toward soybeans and isoflavones, this chapter will focus on the effect of isoflavones on blood lipoprotein profiles. Nevertheless, other phytoestrogens, such as lignans, may also exert similar physiologic effects.

Phytoestrogens: Isoflavones Phytoestrogens are plant compounds with estrogenic and/or antiestrogenic biological activities (1). There are three main groups of phytoestrogens, i.e., the isoflavones, lignans, and coumestans. These compounds are found in many products of plant origin, but soybeans and flaxseed have received most of the attention due to their high natural concentrations of isoflavones and lignans, respectively (2). Isoflavones are also found in relatively high amounts in clover; thus, isoflavone extracts from red and subterranean clovers have been used by some researchers as well (3,4). Soybeans contain three main isoflavones, each found in the following four chemical forms: aglycone (genistein, daidzein, glycitein), glycoside (genistin, daidzin, glycitin), acetylglycoside (6′-O-acetylgenistin, 6′-O-acetyldaidzin, 6′-O-acetylglycitin), and malonylglycosides (6′-O-malonylgenistin, 6′-O-malonyldaidzin, 6′-O-malonylglycitin) (2). In clover, biochanin-A and formononetin are also found; these are the 4′-methy ethers of genistein and daidzein, respectively (2). The isoflavone content in soy foods can be reduced substantially by ethanol extraction (5); thus, ethanol-extracted concentrates and isolates typically have very low levels of isoflavones. Other than ethanol extraction, processing generally does not remove isoflavones from soy protein products. However, the chemical forms of the isoflavones can be altered as a result of heat treatment or enzyme reactions during fermentation (5).

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Isoflavone absorption is facilitated by hydrolysis of the sugar moiety by the βglucosidases in foods and in the human gut (2), and by gastric hydrochloric acid. Isoflavones in the aglycone form are further metabolized by gut bacteria (2). Both the aglycone and several of its metabolites are found in human plasma (6) after consumption of soy protein. Most of the circulating isoflavones are found conjugated with glucuronic acid and sulfate (2). Like estrogens, isoflavones are excreted in urine and bile, and they undergo enterohepatic circulation. Considerable intersubject variation is found in the absorption, metabolism, and excretion of isoflavones (2). This variability results from intersubject variations in several important steps, including the removal of the sugar moiety and the series of conjugation and deconjugation steps involved in metabolism. Additionally, the effects of isoflavone consumption may depend on the isoflavone composition of the product being studied. Together, these variations may contribute to the inconsistent results found in the literature, and they may account in part for the controversy regarding the biological effects of isoflavones on blood lipids.

Isoflavones and Blood Lipoprotein Concentrations Soy protein consumption has been shown to reduce total cholesterol (TC) and low density lipoprotein (LDL) cholesterol, while increasing high density lipoprotein (HDL) cholesterol, in a variety of human studies (7–11). These effects of soy protein on blood lipoprotein cholesterol concentrations led to a Food and Drug Administration (FDA) approved health claim for the consumption of 25 g soy protein/d as part of a diet that is low in saturated fat and cholesterol for the reduction of CHD risk (12). Although the effects of soy protein consumption on lipoprotein cholesterol concentrations in hypercholesterolemic individuals are generally accepted, the mechanisms by which soy protein produces these effects remain controversial. In particular, the role of isoflavones alone or in combination with soy protein on blood lipoprotein cholesterol has been the topic of debate. This issue must be addressed promptly for two main reasons. First, the FDA-approved health claim is not specific about the isoflavone content in the soy protein. Many soy protein–containing products have little or no isoflavones; thus, it is important to know whether these products affect blood lipoproteins in the same way as soy products that are naturally rich in isoflavones. Second, isoflavone supplements are becoming common, and there is increasing evidence to suggest that pure isoflavones supplements may be harmful to some individuals (13,14). Thus, the public should be informed about the products that can reduce their risk of CHD and those that have either no beneficial effects or potentially harmful ones. Studies Supporting an Involvement of Isoflavones on Blood Lipoprotein Concentration Changes Several studies in rodents, monkeys, and humans suggest a possible role of isoflavones in the beneficial effects of soy protein consumption on blood lipoproteins

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(Table 16.1). However, a closer inspection of these studies reveals that most of them did not examine the role of isoflavones per se, but rather the role of intact soy protein. This is an important distinction because other compounds (e.g. saponins), in addition to isoflavones, are also removed during ethanol extraction. In fact, only the study by Clifton-Bligh et al. (4) directly examined the role of isoflavones on blood lipoprotein cholesterol concentrations in the absence of soy protein. In the following sections, these studies are grouped according to the species examined. Rodents. Balmir et al. (15) compared the effects of soy protein naturally rich in isoflavones (intact soy protein) with the effects of isoflavone extract from soy flour (ethanol/acetone extract) added to soy protein and/or casein. This comparison was studied in two different animal models, male Sprague-Dawley rats and male Golden Syrian hamsters (15). The authors found that in both rodent species, intact or isoflavone-added soy protein reduced TC and LDL cholesterol concentrations. Furthermore, adding the ethanol/acetone extract to casein at 0.36 mg/g protein also resulted in the reduction of LDL cholesterol compared with animals fed casein alone. However, addition of the ethanol/acetone extract at 0.72 mg/g protein to either soy protein or casein did not further improve the blood lipoprotein profile in these animals. Although this study did not conclusively show that isoflavones in soy protein were required for the observed improvements in blood lipoproteins in rodents, it suggested that a component in the ethanol/acetone extract had an effect on lipoprotein cholesterol, especially on LDL cholesterol concentrations in rodents. Moreover, increasing the dose of this component may not lead to a concomitant increase in the beneficial effect. Kirk et al. (16) evaluated the role of intact soy protein vs. soy protein from which isoflavones were removed by ethanol extraction in C57BL/6 LDL receptor–deficient mice (LDLr-null) and wild-type mice. Although there was no apparent effect of isoflavones on the LDLr-null mice, intact soy protein led to a 30% reduction in TC and non-HDL cholesterol concentrations in wild-type mice. Because this study did not include a control group without soy protein, the results could not conclusively show that the ethanol-extracted soy protein had no effect on cholesterol levels. However, it showed that compared with ethanol-extracted soy protein, which was virtually free of isoflavones, intact soy protein led to a reduction in cholesterol concentrations by a LDLr-mediated mechanism. Recently, Adams et al. (17) evaluated the effect of intact soy protein, ethanolextracted soy protein, and casein diets on atherosclerosis in two types of mice, i.e., mice that are devoid of LDL receptors but overproduce apolipoprotein (apo) B (LDLr-/-), and apo E-deficient mice (apoE-/-). The authors found that although both soy protein diets reduced atherosclerosis in the two mice strains compared with casein, the effects were stronger with intact soy protein and in LDLr-/- mice. Furthermore, the effects of soy protein were independent of plasma lipoprotein concentrations. These results suggested the existence of a pathway independent of the LDL receptor and plasma lipoproteins for the antiatherogenic effects of soy

Copyright 2002 by AOCS Press. All rights reserved.

TABLE 16.1 Human or Animal Studies Supporting the Involvement of Isoflavones on Blood Lipoprotein Concentration Changesa Subjects

Duration

Treatment

Results

Male SpragueDawley rats

4 wk

• Lower TC in SP and SP- compared with CAS • Lower LDL cholesterol in SP, SP-, and CAS+ compared with CAS

Male golden Syrian hamsters

8 wk

Peripubertal male and female rhesus monkeys

6 mo

Male cynomolgus macaques

14 mo

Soy protein with intact isoflavones (SP) Soy protein with isoflavones extracted (SP–) Casein with isoflavone extract added at 0 mg/g protein (CAS) 0.36 mg/g protein (CAS+) Soy protein with intact isoflavones (SP) Soy protein with intact isoflavones + isoflavone extract added (0.36 mg/g protein) (SP+) Casein with isoflavone extract added at 0 mg/g protein (CAS) 0.36 mg/g protein (CAS+) 0.72 mg/g protein (CAS++) Soy protein with intact isoflavones (1.27 mg genistein and 0.42 mg daidzein per g protein; SP) Soy protein with isoflavones extracted (0.121 mg genistein and 0.052 mg daidzein per g protein; SP–) Casein/lactalbumin (CAS) Soy protein with intact isoflavones (SP) Soy protein with isoflavones extracted (SP–)

C57BL/6 and LDLr-null mice

6 or 10 wk

Soy protein with intact isoflavones (27 mg genistein and 9 mg daidzein per 100 g protein) Soy protein with isoflavones extracted (2 mg genistein and 1 mg daidzein per 100 g protein)

LDLr-/- and apoE-/- mice

16 wk

Casein/lactalbumin (CAS) Alcohol-extracted soy protein (total isoflavones 0.04 mg/g protein; SP–) Intact soy protein (total isoflavones 1.72 mg/g protein)

156 Moderately hypercholesterol-

9 wk

25 g/d casein 25 g/d soy protein with aglycone isoflavones at

Copyright 2002 by AOCS Press. All rights reserved.

Reference (15) (15)

• Lower TC and non-HDL-cholesterol in SP and CAS+ compared with CAS • No effects on SP+ or CAS++

• SP males and females had lower LDL + VLDL cholesterol, and lower TC-to-HDL-cholesterol ratio • SP females had higher HDL cholesterol • Lower TC and LDL + VLDL cholesterol for SP • No effect of isoflavones on plasma lipoprotein cholesterol in LDLr-null mice • 30% decrease in C57BL/6 mice fed SP with isoflavones • Atherosclerosis inhibited for SP and SP- in both LDLr-/- and apoE-/- mice • Stronger effects for SP and in LDLr-/- mice • No significant effect of 3 mg isoflavones on TC and LDL cholesterol

(19)

(18) (16)

(17)

(21) Continued

TABLE 16.1 (Cont.) Subjects

Duration

emic men and women

Treatment 3 mg/d 27 mg/d 37 mg/d 62 mg/d

Results

Reference

• Dose-response effect with increasing amounts of isoflavones

13 Normocholesterolemic premenopausal women

3 menstrual cycles

Soy protein with total isoflavones at 10.0 mg/d 64.7 mg/d 128.7 mg/d

• No significant effects with 64.7 mg/d isoflavones • 7.6–10.0% decrease in LDL cholesterol, 10.2% decrease in TC-to-HDL-cholesterol ratio, and 13.8% decrease in LDL-to-HDL cholesterol ratio with 128.7 mg/d isoflavones

(22)

94 Moderately hypercholesterolemic postmenopausal women

12 wk

42 g/d milk protein 42 g/d soy protein with aglycone isoflavones at trace amounts 80 mg/d

• Larger decrease in LDL cholesterol for 80 mg/d group than for trace amount group • However, changes were not different from those in milk group

(24)

46 Postmenopausal women

6 mo

Red clover isoflavone tablets at 28.5 mg/d 57.0 mg/d 85.5 mg/d

• 15.7-28.6 % increase in HDL cholesterol and 11.5-17.0% decrease in apo B • However, the response magnitude was independent of the isoflavone dose

(4)

18 Postmenopausal women with borderline high cholesterol concentrations

93 d

Soy protein with intact isoflavones at 65 mg/d total isoflavones (SP65) 132 mg/d total isoflavones (SP132) Soy protein with isoflavones extracted (7.1 mg/d isoflavones remaining)

• 6.5% decrease in LDL cholesterol for SP132 • 8.5 and 7.7% decrease in LDL-to-HDLcholesterol ratio for SP65 and SP132, respectively

(23)

Female cynomolgus macaques

36 mo

Soy protein with intact isoflavones (SP) Soy protein with isoflavones extracted (SP–) Soy protein with isoflavones extracted + equine estrogens added (SPEE)

• Lower TC and LDL + VLDL cholesterol in SP and SPEE compared with SP• Higher HDL cholesterol and apo A-1 in SP compared with SP-

(20)

aAbbreviations:

TC, total cholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; VLDL, very low density lipoprotein; LDLr, LDL receptor; apo, apolipoprotein.

Copyright 2002 by AOCS Press. All rights reserved.

protein. Also, intact soy protein seemed to be more effective than soy protein depleted of isoflavones. Monkeys. Anthony et al. (18,19) showed that in male and female rhesus monkeys (19) and in male cynomolgus macaques (18), only intact soy protein led to a beneficial effect on blood lipoprotein concentrations. In two separate studies, the authors found that only monkeys fed the intact soy protein diet had significant reductions in non-HDL cholesterol concentrations. Additionally, the authors found an increase in HDL cholesterol concentrations only in female monkeys when intact soy protein was fed. More recently, Clarkson et al. (20) studied female cynomolgus macaques for 36 mo. In that study, the effect of intact soy protein was compared with ethanolextracted soy protein (with vestigial levels of isoflavones) and ethanol-extracted soy protein with equine estrogens added. The authors found that intact soy protein was required to improve the lipoprotein profile in the study subjects and that the effects of intact soy protein were similar to the effects resulting from the addition of equine estrogens. Thus, these results suggest that the compound(s) removed from soy protein by ethanol extraction may have a mechanism of action similar to estrogens and that the active compound(s) are inactivated by ethanol extraction. Humans. Crouse et al. (21) studied the effect of 25 g/d of casein or soy protein with increasing amounts of isoflavones on the blood lipoprotein profile of hypercholesterolemic men and women. The authors found that although soy protein with vestigial amounts (3 mg/d) of isoflavones (ethanol extracted) slightly reduced TC and LDL cholesterol concentrations compared with casein, the differences were not statistically significant. However, soy protein with higher levels of isoflavones resulted in significant reductions. Additionally, a dose-response effect was found with increasing amounts of isoflavones. The results from that study clearly showed that intact soy protein had a greater effect on blood lipoprotein cholesterol concentrations than ethanol-extracted soy protein in moderately hypercholesterolemic individuals. Merz-Demlow et al. (22) studied the effects of soy protein with different levels of isoflavones in normocholesterolemic premenopausal women. In that study, significant reductions in lipoprotein cholesterol concentrations were found only for soy protein with the highest level of isoflavones (128.7 mg/d), suggesting that high levels of isoflavones were required for the beneficial effects of soy protein on blood lipoproteins. Similar results were found by Wangen et al. (23) in postmenopausal women with borderline high cholesterol concentrations. In Wangen's study, only intact soy protein at 132 mg/d of isoflavones led to a significant reduction in lipoprotein cholesterol concentrations. However, both of these studies lacked a control group without soy protein, which somewhat compromised their conclusions. More complex results were found by Gardner et al. (24) in moderately hypercholesterolemic postmenopausal women. In that study, intact soy protein appeared to have a stronger effect than ethanol-extracted soy protein, suggesting a possible

Copyright 2002 by AOCS Press. All rights reserved.

role of isoflavones. However, the two groups were not statistically significantly different from the control group consuming milk. These results led the authors to conclude that perhaps other factors may have contributed to their findings. Recently, Clifton-Bligh et al. (4) reported the only study to date that found a significant effect of isoflavones provided in the form of isoflavone tablets. In that study, the authors examined the effects of red clover isoflavone tablets at three concentrations (28.5, 57.0, and 85.5 mg/d) on blood lipoproteins and apolipoproteins in mildly hypercholesterolemic postmenopausal women. The authors found a significant increase in HDL cholesterol concentrations and a significant reduction in apo B with time, but no significant change in either TC or LDL cholesterol. No effect of dose was found. Although these results suggest that isoflavones do play a role in lipoprotein concentration changes, they also suggest that levels as low as 28.5 mg/d are sufficient for the observed effects on lipoprotein. However, the results from that study were compromised by the lack of a placebo group. Studies Opposing an Involvement of Isoflavones on Blood Lipoprotein Concentration Changes Table 16.2 lists a number of animal and human studies in which the results did not support an involvement of isoflavones in blood lipoprotein concentration changes. Approximately half of these studies examined the addition of soybean ethanolextracts to either soy protein or casein. The other half examined the effect of isoflavone supplements provided in the form of tablets or powder. Thus, most of the studies that argued against the involvement of isoflavones on blood lipoprotein profile changes did not evaluate the effects of intact soy protein with naturally occurring isoflavones, but only the effects of isoflavone-rich extracts. Rodents and Rabbits. Tovar-Palacio et al. (25) examined the effects of ethanolextracted soy protein alone or with the addition of an isoflavone-rich soy protein ethanol extract at different levels vs. casein in Mongolian gerbils. The authors found that all soy protein groups had a reduction in TC and non-HDL cholesterol concentrations compared with casein, but no differences were found among the soy protein groups. At first glance, this study seems to suggest that the presence of isoflavones is not necessary for the hypocholesterolemic effects of soy protein in gerbils, but that something else in soy protein may be responsible for the improvement in the lipoprotein profile. However, this study did not use any form of intact soy protein. Thus, it did not examine the effects of intact soy protein and of isoflavones that are part of intact soy protein. As mentioned previously, the isoflavone-rich ethanol extract seems to lose its activity compared with the naturally occurring isoflavones. Yamakoshi et al. (26) examined the effects of a cholesterol-rich diet with and without the addition of soybean extracts in male rabbits. The authors compared the addition of an isoflavone-rich extract at two levels (also containing saponins) and a

Copyright 2002 by AOCS Press. All rights reserved.

TABLE 16.2 Human or Animal Studies Opposing the Involvement of Isoflavones on Blood Lipoprotein Concentration Changesa Subjects

Duration

Treatment

Results

Refernce

21 Mildly hypercholesterolemic peri- and postmenopausal women

5–10 wk

Red clover isoflavones tablets with 80 mg/d (45 mg/d genistein) Placebo tablet

• No effect on plasma lipoproteins

(31)

Mongolian gerbils

21 d

Casein (CAS) Soy protein with isoflavones extracted (SP–) Soy protein with isoflavones extracted + isoflavone extract added at 2.1 mg/g protein (SP+) 3.6 mg/g protein (SP++) 6.2 mg/g protein (SP+++)

• Lower TC, LDL + VLDL cholesterol, and apolipoprotein B in SP-, SP+, SP++, and SP+++ compared with CAS • No differences among SP-, SP+, SP++, and SP+++ in lipoprotein cholesterol concentrations

(25)

46 Postmenopausal women with normal-toborderline high cholesterol concentrations

8 wk

Subterranean clover isoflavones tablets with 55 mg/d isoflavones (biochanin A 16 mg/d, genistein 30 mg/d, formononetin 8 mg/d, and daidzein 1 mg/d) Placebo tablets

• No effects on plasma lipoprotein cholesterol concentrations

(3)

66 Hypercholesterolemic postmenopausal women

6 mo

Casein/nonfat dry milk (CAS) Soy protein with intact isoflavones at 56 mg/d total aglycone isoflavones (SP56) 90 mg/d total aglycone isoflavones (SP90)

• Lower non-HDL cholesterol and TCto-HDL-cholesterol ratio in SP56 and SP90 compared with CAS • Higher HDL cholesterol in SP56 and SP90 compared to CAS

(9)

Continued Copyright 2002 by AOCS Press. All rights reserved.

TABLE 16.2 (Cont.) Subjects

Duration

Treatment

Results

14 Healthy premenopausal women

2 menstrual cycles

Red clover isoflavones tablets with 86 mg/d isoflavones (biochanin A 51.4 mg/d, formononetin 18.6 mg/d, genistein 8.6 mg/d, and daidzein 7.4 mg/d) Placebo tablets

• No effect on TC or LDL cholesterol concentrations

(32)

60 Female cynomolgus macaques

12 wk

Casein/lactalbumin (CAS) CAS with isoflavone extract added (CAS+) Soy protein with intact isoflavones (SP)

• Lower TC, VLDL + IDL, and LDL cholesterol for SP • Lower HDL cholesterol for SP compared with CAS • Lower cholesteryl ester in LDL particles for SP than CAS.

(28)

66 Normo- to hypercholesterolemic postmenopausal women

4 wk

Red clover isoflavones tablets with 43.5 mg/d isoflavones (biochanin A 26 mg/d, formononetin 16 mg/d, daidzein 0.5 mg/d, and genistein 1 mg/d), or double the dose (87 mg/d total isoflavones) Placebo tablets

• No effects on plasma lipoprotein cholesterol concentrations

(30)

20 Moderately hypercholesterolemic postmenopausal women

8 wk

Soybean isoflavones tablets with 80 mg/d total isoflavones Placebo tablets

• No effect on plasma lipoprotein cholesterol c ncentrations

(33)

Copyright 2002 by AOCS Press. All rights reserved.

Reference

40 Female cynomolgus macaques

20 wk

Casein/lactalbumin (CAS) Soy protein with intact isoflavones (SP) CAS with isoflavone extract added (CAS+;

• Lower TC and VLDL + IDL cholesterol in SP group compared with all other groups

similar isoflavones amount to SP) CAS with equine estrogen added (CASEE)

• Higher HDL cholesterol in SP group compared with CAS and CASEE

(27)

54 Hypercholesterolemic postmenopausal women

12 wk

Soy protein with intact isoflavones Soy protein with isoflavones extracted

• Lower TC and LDL cholesterol in both groups over time • No differences between treatments.

(29)

27 Moderately hypercholesterolemic men

12 wk

Soy protein with intact isoflavones

• Increase in HDL cholesterol

(29)

New Zealand white male rabbits

8 wk

Control diet (no isoflavones, saponins, or cholesterol) 1% cholesterol-rich diet 1% cholesterol-rich diet with 0.33 g total aglycone isoflavones/100 g 1.00 g total aglycone isoflavones/100 g 1.09 g soy saponin-rich extract/100 g

• No effect on serum lipoprotein cholesterol

(26)

37 Postmenopausal women

6 mo

Isoflavone supplement with 150 mg/d total isoflavones

• No effects on plasma lipoprotein cholesterol concentrations

(34)

Abbreviations: See Table 16.1.

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saponin-rich extract (virtually free of isoflavones). No effects of blood lipoprotein cholesterol concentrations were found with the addition of the soybean extracts, suggesting that neither isoflavones nor saponins promote changes in lipoprotein cholesterol in this animal model. However, the study lacked an intact soy protein group; thus the findings could be accounted for by several explanations including the following: (i) the extracts were ineffective; (ii) the specific animal model did not respond to soy protein; or (iii) the active components were inactivated in the extracts. In that study, the authors found a reduction in copper ion–induced LDL oxidation with both isoflavone diets, but not with the saponin-rich extract. Monkeys. Two studies by Greaves et al. (27,28) showed no effect of adding an isoflavone-rich soybean ethanol extract to casein on blood lipoprotein cholesterol in female cynomolgus macaques. In both studies, intact soy protein had positive effects on the blood lipoprotein profile, but adding the extract to casein failed to promote a similar effect. Due to the lack of an ethanol-extracted soy protein group, a definitive conclusion could not be drawn concerning whether intact soy protein was necessary for the positive changes seen in blood lipoproteins, or whether the ethanol-extracted soy protein alone could exert an hypocholesterolemic effect. This study adds to the mounting evidence that the isoflavone-rich ethanol extract may be inactive, but it did not oppose the role of isoflavones in intact soy protein. Humans. Our group compared the effects of soy protein at two levels of isoflavone aglycones (56 and 90 mg/d) with casein in hypercholesterolemic postmenopausal women (9). In this study, Baum et al. (9) found an improvement in blood lipoprotein profile with soy protein consumption, but no differences were seen between the two isoflavone groups. However, it is likely that both levels of isoflavones were sufficiently high to promote an effect on lipoproteins and that there was a plateau in the dose response. This idea is supported by recent work from our group in which 25 g/d of soy protein (with 29.1 mg/d isoflavone aglycones) was found to be sufficient for promoting an improvement in the lipoprotein profile of mildly hypercholesterolemic men after 6 wk consumption (8). Recently, Mackey et al. (29) provided evidence that intact soy was not necessary for the hypocholesterolemic effects of soy protein, but that it might be required for the increase in HDL cholesterol. However, Mackey's results were compromised by the lack of appropriate control groups. Six other independent studies (3,30–34) have evaluated the effects of isoflavone supplementation in the form of tablets or powder in humans and found no effects on the blood lipoprotein profile. In those studies, the isoflavones were derived from various sources (soybeans, red or subterranean clovers) and the isoflavone amounts and profiles varied across studies. Among the six studies, Nestel et al. (31) found that supplementation with 80 mg/d of isoflavones extracted from red clover had no significant effect on the blood lipoprotein profile in mildly hypercholesterolemic peri- and postmenopausal women. According to the authors, the

Copyright 2002 by AOCS Press. All rights reserved.

tablets used had an isoflavone profile similar to that of soy protein with a ratio of genistein/daidzein/glycitein of 1.3:1.0:0.1. Although no effect was found on lipoprotein concentrations, the authors found an improvement in systemic arterial compliance in individuals taking the isoflavone tablets. Hodgson et al. (3) found no effect on plasma lipoprotein cholesterol concentrations when supplementing with 55 mg/d isoflavones from subterranean clover in men and postmenopausal women with normal-to-borderline-high cholesterol concentrations. Isoflavone profile of the tablets used by Hodgson et al. (3) was 16, 30, 8, and 1 mg/d for biochanin A, genistein, formononetin, and daidzein, respectively. In healthy premenopausal women, Samman et al. (32) also found no significant effect on TC and LDL cholesterol concentrations using a supplement with 86 mg/d of isoflavones extracted from red clover. The isoflavone profile (51.4, 18.6, 8.6, and 7.4 mg/d for biochanin A, formononetin, genistein, and daidzein, respectively) of the product used in that study was considerably different from the profile usually found in soy protein. Howes et al. (30) used an isoflavone supplement derived from red clover in normo- and hypercholesterolemic postmenopausal women. The product used in that study also had an isoflavone profile (26, 16, 1.0, and 0.5 mg/d for biochanin A, formononetin, genistein, and daidzein, respectively) considerably different from the typical profile found in soy protein. The supplement was provided at two levels, 43.5 or 87 mg/d. Once again, no effects were found on plasma lipoprotein cholesterol concentrations. Simons et al. (33) examined the effects of soybean isoflavone tablets at 80 mg/d on moderately hypercholesterolemic postmenopausal women and found no effect on lipoproteins. In that study, the isoflavone profile of the product used was not described. More recently, Hsu et al. (34) reported their results from an intervention trial on mildly hypercholesterolemic postmenopausal women. The subjects in that study were provided with a daily supplement of 150 mg/d of total isoflavones, and they were studied over time. No effect on plasma lipoprotein isoflavones was observed. Unfortunately, this study did not include a placebo group, which undermined the final conclusions. Additionally, the origin and isoflavone profile of the supplement used were not described by the authors. In the studies reviewed above, the general lack of consensus regarding the effects of isoflavones may be attributed to a number of confounding factors. The first concerns the type of subjects studied (i.e. healthy normocholesterolemic vs. mildly to moderately hypercholesterolemic, premenopausal vs. postmenopausal women, or women vs. men). For example, the effect of soy protein and perhaps of isoflavones on blood lipoproteins may depend on the initial concentration of lipoprotein cholesterol as suggested by a meta-analysis (7). Also, the effects may be specific to or strongly dependent upon gender. For example, increases in HDL cholesterol are more often found in women than in men. Second, the isoflavone amounts and profiles varied across studies, making comparisons difficult to interpret. For example, some studies used isoflavone extracts derived from red or sub-

Copyright 2002 by AOCS Press. All rights reserved.

terranean clover; others used extracts from soybeans. As mentioned above, clover is rich in biochanin A and formononetin, whereas these two forms of isoflavones are absent from soybeans. Additionally, the processing of soy protein including extraction, purification, and readdition of the extracted fractions was inconsistent among studies, and inactivation of some bioactive components during processing may be responsible for the conflicting results. Third, extrapolation of results across species is difficult especially in the area of lipid metabolism. Thus, ultimately, only the results from human studies will have direct clinical relevance. Fourth, studies have addressed slightly different questions, as indicated previously. Some studies evaluated whether intact soy protein, which is naturally rich in isoflavones, is strictly essential for the improvement in lipoprotein profile. Others evaluated the effects of adding isoflavone-rich ethanol extracts to a diet virtually depleted of isoflavones and/or soy protein. Still others investigated the role of isoflavone supplements taken in the form of pills. Finally, questions concerning the possible presence of other compounds in the soybean isoflavone extracts and how those compounds might compare with compounds found in clover extracts have not been fully investigated. Together, these confounding factors make it difficult to evaluate the possible effect of isoflavones on blood lipoproteins in a definitive manner. At this point, no direct evidence exists to show that the isoflavones in soy protein are indeed the main compounds responsible for the changes in lipoproteins. Evidence is accumulating though, suggesting that isoflavones alone in the form of tablets do not affect lipoprotein concentrations. However, naturally occurring isoflavones in intact soy protein appear to be required for the full positive effect of soy protein consumption. Alternatively, other ethanol-soluble compounds may be necessary for the full effects of soy protein on lipoprotein profiles. Furthermore, adding isoflavone-rich ethanol extracts to soy protein that was previously depleted of isoflavones by ethanol extraction does not seem to affect blood lipoproteins. Therefore, some active compounds may become inactivated during the extraction procedure; consequently, the extract loses its activity.

Isoflavones and Low Density Lipoprotein Oxidation The role of isoflavones in LDL oxidation has also been the focus of some controversy. Fewer studies have been conducted in this area (Table 16.3), but the results are similar to those for lipoprotein concentrations. In a study on healthy premenopausal women, isoflavones alone in the form of tablets (86 mg isoflavones/d, isoflavones extracted from red clover) were found not to affect lag time for copperinduced LDL oxidation, oxidation rate, or maximum oxidation (32). On the other hand, Jenkins et al. (35) found significantly lower concentrations of conjugated dienes in the LDL fraction of men and postmenopausal women after consumption of soy foods with 86 mg isoflavones/d, compared with a control diet. Wiseman et al. (36) specifically compared the effects of soy protein with high (56 mg/d) and low (1.9 mg/d) isoflavones on several biomarkers of oxidation in 5 men and 19

Copyright 2002 by AOCS Press. All rights reserved.

TABLE 16.3 Human or Animal Studies Investigating the Role of Isoflavones and Low Density Lipoprotein (LDL) Oxidation Subjects

Duration

Treatment

Results

14 Healthy premenopausal women

2 menstrual cycles

Red clover isoflavones tablets with 86 mg/d total isoflavones Placebo tablets

• No effects on LDL oxidation

(32)

24 Healthy men and women

17 d

Textured soy protein with intact isoflavones (21.2 mg/d daidzein and 34.8 mg/d genistein) (SP) Textured soy protein with isoflavones extracted (0.9 mg/d daidzein and 1.0 mg/d genistein) (SP–)

• Lower 8-epi-prostaglandin F2α for SP • Longer lag time for copper-ion-induced LDL oxidation for SP

(36)

New Zealand white male rabbits

8 wk

Control diet (no isoflavones, saponins, or cholesterol) 1% cholesterol-rich diet 1% cholesterol-rich diet with 0.33 g total aglycone isoflavones/100 g 1.00 g total aglycone isoflavones/100 g 1.09 g soy saponin-rich extract/100 g

• Lower copper-ion-induced LDL oxidation for both aglycone isoflavone diets

(26)

Abbreviations: LDL, low-density lipoprotein.

Copyright 2002 by AOCS Press. All rights reserved.

Reference

premenopausal women. The authors found significantly lower concentrations of 8epi-prostaglandin F2α and a longer lag time for copper-induced LDL oxidation when the high isoflavone diet was consumed. As described earlier, Yamakoshi et al. (26) found a reduction in copper ion–induced LDL oxidation when soy protein isoflavones were provided in the diet of male rabbits. As with lipoprotein concentrations, the presence of naturally occurring isoflavones in soy protein seems to be necessary for the reduction of LDL oxidation. However, this area warrants further investigation.

Conclusions The work discussed above suggests that in humans, supplementation with isoflavones alone does not lead to an improvement in the blood lipoprotein profile. A pertinent question that must be addressed is whether soy protein consumption alone is sufficient to bring about the positive effects on the lipoprotein profile, or whether isoflavone-rich intact soy protein is required. At this point, only a handful of human studies have investigated the effect of intact soy protein on lipoprotein concentrations (9,21,23) or LDL oxidation (36), and only Crouse et al. (21) compared different levels of naturally occurring isoflavone concentrations with a constant level of dietary soy protein. Results from Crouse et al. (21), Wangen et al. (23), and Wiseman et al. (36) all suggest the need for intact soy protein with intact isoflavones to bring about an improvement in the blood lipoprotein profile. However, these studies were unable to attribute the bioactive effect definitively to isoflavones, and rule out other potentially active compounds that may be present. As recently reviewed by Erdman (37), “There is apparently synergy among the components of intact soy protein, which provides the maximum hypocholesterolemic effect.” There is now emerging evidence that isoflavones may have other beneficial effects in CHD prevention, such as improved arterial compliance (31), in addition to its possible effect on blood lipoprotein concentrations. Therefore, at the moment, the consumption of soy protein that is naturally rich in isoflavones offers a better prospect in the fight against CHD. In fact, the North American Menopause Society recently stated that “. . . clinicians may wish to recommend that menopausal women consume whole foods that contain isoflavones, especially for the cardiovascular benefits of these foods. . .” (38). Nevertheless, further work in this area is required and the molecular mechanisms for the effects of soy protein and isoflavones on the risk factors of CHD remain to be identified. References 1. Humfrey, C.D. (1998) Phytoestrogens and Human Health Effects: Weighing Up the Current Evidence, Nat. Toxins 6, 51–59. 2. Kurzer, M., and Xu, X. (1997) Dietary Phytoestrogens, Annu. Rev. Nutr. 17, 353–381. 3. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., and Croft, K.D. (1998) Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations: A Randomized Controlled Trial in Humans, J. Nutr. 128, 728–732.

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4. Clifton-Bligh, P.B., Baber, R.J., Fulcher, G.R., Nery, M.L., and Moreton, T. (2001) The Effect of Isoflavones Extracted from Red Clover (Rimostil) on Lipid and Bone Metabolism, Menopause 8, 259–265. 5. Anderson, R. L., and Wolf, W.J. (1995) Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflavones Related to Soybean Processing, J. Nutr. 125, 581S–588S. 6. Teixeira, S.R., Potter, S.M., and Erdman, J.W., Jr. (1999) Plasma Isoflavone Concentrations in American Men and Women Consuming Different Levels of Isolated Soy Protein (ISP) Up to 6 Months, in Third International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, pp. 683S–685S, Washington, D.C. 7. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 8. Teixeira, S.R., Potter, S.M., Weigel, R., Hannum, S., Erdman, J.W., Jr., and Hasler, C.M. (2000) Effects of Feeding 4 Levels of Soy Protein for 3 and 6 Wk on Blood Lipids and Apolipoproteins in Moderately Hypercholesterolemic Men, Am. J. Clin. Nutr. 71, 1077–1084. 9. Baum, J.A., Teng, H., Erdman, J.W., Jr., Weigel, R.M., Klein, B.P., Persky, V.W., Surya, P., Bakhit, R.M., Shay, N.F., and Potter, S.M. (1998) Long-Term Intake of Soy Protein Improves Blood Lipid Profiles and Increases Mononuclear Cell LDL Receptor mRNA in Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 68, 545– 551. 10. Bakhit, R.M., Klein, B.P., Essex-Sorlie, D., Ham, J.O., Erdman, J.W., Jr., and Potter, S.M. (1994) Intake of 25 G of Soybean Protein with or Without Soybean Fiber Alters Plasma Lipids in Men with Elevated Cholesterol Concentrations, J. Nutr. 124, 213–222. 11. Potter, S.M., Bakhit, R.M., Essex-Sorlie, D.L., Weingartner, K.E., Chapman, K.M., Nelson, R.A., Prabhudesa, M., Savage, W.D., Nelson, A.I., Winter, L.W., and Erdman, J.W., Jr., (1993) Depression of Plasma Cholesterol in Men by Consumption of Baked Products Containing Soy Protein, Am. J. Clin. Nutr. 58, 501–506. 12. Food and Drug Administration (1999) Food Labeling: Health Claims; Soy Protein and Coronary Heart Disease, Fed. Reg. 64, 57699-57733. 13. Sirtori, C.R. (2000) Dubious Benefits and Potential Risk of Soy Phyto-Oestrogens, Lancet 355, 849. 14. Busby, M.G., Jeffcoat, A.R., Bloedon, L.T., Koch, M.A., Black, T., Dix, K.J., Heizer, D.W., Thomas, B.F., Hill, J.M., Crowell, J.M., and Zeisel, S.H. (2002) Clinical Characteristics and Pharmacokinetics of Purified Soy Isoflavones: Single-Dose Administration to Healthy Men, Am. J. Clin. Nutr. 75, 126–136. 15. Balmir, F., Staack, R., Jeffrey, E., Jimenez, M.D., Wang, L., and Potter, S.M. (1996) An Extract of Soy Flour Influences Serum Cholesterol and Thyroid Hormones in Rats and Hamsters, J. Nutr. 126, 3046–3053. 16. Kirk, E.A., Sutherland, P., Wang, S.A., Chait, A., and LeBoeuf, R.C. (1998) Dietary Isoflavones Reduce Plasma Cholesterol and Atherosclerosis in C57BL/6 Mice but Not LDL Receptor-Deficient Mice, J. Nutr. 128, 954–959. 17. Adams, M.R., Golden, D.L., Anthony, M.S., Register, T.C., and Williams, J.K. (2002) The Inhibitory Effect of Soy Protein Isolate on Atherosclerosis in Mice Does Not Require the Presence of LDL Receptors or Alteration of Plasma Lipoproteins, J. Nutr. 132, 43–49. 18. Anthony, M.S., Clarkson, T.B., Bullock, B.C., and Wagner, J.D. (1997) Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet-Induced Coronary Artery

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19.

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Atherosclerosis of Male Cynomolgus Monkeys, Arterioscler. Thromb. Vasc. Biol. 17, 2524–2531. Anthony, M.S., Clarkson, T.B., Hughes, C.L., Jr., Morgan, T.M., and Burke, G.L. (1996) Soybean Isoflavones Improve Cardiovascular Risk Factors Without Affecting the Reproductive System of Peripubertal Rhesus Monkeys, J. Nutr. 126, 43–50. Clarkson, T.B., Anthony, M.S., and Morgan, T.M. (2001) Inhibition of Postmenopausal Atherosclerosis Progression: A Comparison of the Effects of Conjugated Equine Estrogens and Soy Phytoestrogens, J. Clin. Endocrinol. Metab. 86, 41–47. Crouse, J.R., III, Morgan, T., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with That of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070–2076. Merz-Demlow, B.E., Duncan, A.M., Underhill, K.E.W., Xu, X., Carr, T.S., Phipps, W.R., and Kurzer, M.S. (2000) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic, Premenopausal Women, Am. J. Clin. Nutr. 71, 1462–1469. Wangen, K.E., Duncan, A.M., Xu, X., and Kurzer, M.S. (2001) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic and Mildly Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 225–231. Gardner, C.D., Newell, K.A., Cherin, R., and Haskell, W.L. (2001) The Effect of Soy Protein with or Without Isoflavones Relative to Milk Protein on Plasma Lipids in Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 728–735. Tovar-Palacio, C., Potter, S.M., Hafermann, J.C., and Shay, N.F. (1998) Intake of Soy Protein and Soy Protein Extracts Influences Lipid Metabolism and Hepatic Gene Expression in Gerbils, J. Nutr. 128, 839–842. Yamakoshi, J., Piskula, M.K., Izumi, T., Tobe, K., Saito, M., Kataoka, S., Obata, A., and Kikuchi, M. (2000) Isoflavone Aglycone-Rich Extract Without Soy Protein Attenuates Atherosclerosis Development in Cholesterol-Fed Rabbits, J. Nutr. 130, 1887–1893. Greaves, K.A., Wilson, M.D., Rudel, L.L., Williams, J.K., and Wagner, J.D. (2000) Consumption of Soy Protein Reduces Cholesterol Absorption Compared to Casein Protein Alone or Supplemented with an Isoflavone Extract or Conjugated Equine Estrogen in Ovariectomized Cynomolgus Monkeys, J. Nutr. 130, 820–826. Greaves, K.A., Parks, J.S., Williams, J.K., and Wagner, J.D. (1999) Intact Dietary Soy Protein, but Not Adding an Isoflavone-Rich Soy Extract to Casein, Improves Plasma Lipids in Ovariectomized Cynomolgus Monkeys, J. Nutr. 129, 1585–1592. Mackey, R., Ekangaki, A., and Eden, J.A. (2000) The Effects of Soy Protein in Women and Men with Elevated Plasma Lipids, Biofactors 12, 251–257. Howes, J.B., Sullivan, D., Lai N., Nestel, P., Pomeroy, S., West, L., Eden, J.A., and Howes, L.G. (2000) The Effects of Dietary Supplementation with Isoflavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 152, 143–147. Nestel, P.J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P., Owen, A., and Abbey, M. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Arterioscler. Thromb. Vasc. Biol. 17, 3392–3398. Samman, S., Lyons, Wall, P.M., Chan, G.S., Smith, S.J., and Petocz, P. (1999) The Effect of Supplementation with Isoflavones on Plasma Lipids and Oxidisability of Low Density Lipoprotein in Premenopausal Women, Atherosclerosis 147, 277–283.

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33. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2000) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy, Postmenopausal Women, Am. J. Cardiol. 85, 1297–1301. 34. Hsu, C.S., Shen, W.W., Hsueh, Y.M., and Yeh, S.L. (2001) Soy Isoflavone Supplementation in Postmenopausal Women. Effects on Plasma Lipids, Antioxidant Enzyme Activities and Bone Density, J. Reprod. Med. 46, 221–226. 35. Jenkins, D.J., Kendall, C.W., Garsetti, M., Rosenberg-Zand, R.S., Jackson, C.-J., Agarwall, S., Rao, A.V., Diamandis, E.P., Parker, T., Faulkner, D., Vuksan, V., and Vidgen, E. (2000) Effect of Soy Protein Foods on Low-Density Lipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity—A Controlled Crossover Trial, Metabolism 49, 537–543. 36. Wiseman, H., O'Reilly, J.D., Adlercreutz, H., Mallet, A.I., Bowey, E.A., and Rowland, I.R. (2000) Isoflavone Phytoestrogens Consumed in Soy Decrease F(2)-Isoprostane Concentrations and Increase Resistance of Low-Density Lipoprotein to Oxidation in Humans, Am. J. Clin. Nutr. 72, 395–400. 37. Erdman, J.W., Jr. (2000) AHA Science Advisory: Soy Protein and Cardiovascular Disease: A Statement for Healthcare Professionals from the Nutrition Committee of the AHA, Circulation 102, 2555–2559. 38. Anonymous (2000) The Role of Isoflavones in Menopausal Health: Consensus Opinion of the North American Menopause Society, Menopause 7, 215–229.

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Chapter 17

Effects of Free (Aglycone) Phytoestrogens and Metabolites on Cardiovascular Functions and Cancer Paul Nestela and Alan Husbandb aBaker

Medical Research Institute, Melbourne, Australia Ltd., North Ryde NSW, Australia

bNovogen

Introduction Free refers to the aglycone forms of isoflavones that are formed through the hydrolysis of the naturally occurring isoflavone glycosides, a process that takes place in the intestine during the digestion and absorption of phytoestrogens. Most of the studies relating to isoflavones relate to soy glycosides that are extracted from the beans with the protein, whereas less is known about the physiologic effects of consuming the purified aglycones. This is the area that will be primarily reviewed here. Additionally, some of the characteristics of the highly active metabolites that are formed through the metabolism of the primary isoflavones will also be discussed. Much less is known about these, but the availability of pure, synthesized compounds has allowed us to investigate this very interesting subgroup of isoflavones. The focus is primarily on cardiovascular effects and on the potential of the metabolites in the treatment of cancer.

Estrogen as Benchmark The view of estrogens as protective of women against premature cardiovascular disease (CVD) has taken an unexpected turn for the worse. Although it had been recognized that rational prescribing of estrogen supplements to women after the menopause would require confirmation in randomized, controlled trials, the negative outcomes of recent and current trials have been surprising (1). The concept of protection had been based on excellent criteria, i.e., the infrequent occurrence of CVD events in women before menopause, the strong biological credentials of estrogens on vascular functions, and the strong inference of protection from large prospective cohort surveys. The list of physiologic cardiovascular functions that are beneficially modified by estrogens in vivo or in vitro is lengthy and robust: (i) improvement in lipoprotein profile; (ii) restoration of normal endothelial function; (iii) reduction in circulating inflammatory cytokines; and (iv) inhibition of experimental atherosclerosis. Particularly noteworthy are the direct effects on the arterial wall and vasculature, on the mediators of vasodilation, and in reversing the biomarkers of vascular

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dysfunction. Vascular responsiveness to vasodilators declines with the menopause (2). Vasodilation in the microcirculation, flow-mediated dilation of muscular arteries such as the brachial, and distensibility of large arteries such as the carotid and thoracic aorta all become impaired. In the majority of reports, these functions are restored with estrogen replacement (3). The plasma cholesterol-lowering effect of estrogens, especially on low density lipoprotein (LDL) cholesterol [because high density lipoprotein (HDL) cholesterol generally rises] has been known for some time. These are potent antiatherogenic effects, but they may be partially countered by the increase in plasma triglyceride. Biomarkers of endothelial dysfunction, other than impaired vascular dilation, that decline with the menopause include circulating adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and other inflammatory markers such as E-selectin and C-reactive protein (CRP). These molecules appear to be proatherogenic, as are prothrombotic molecules that lead to platelet aggregation. Estrogens have been reported to oppose these inflammatory and atherogenic processes (4). It is against these effects that phytoestrogens have to be measured.

Effects of Free Phytoestrogens on Cardiovascular Functions in Humans Absorption Isoflavones occur in plants almost solely as glucosides. Their absorption and bioavailability require several metabolic steps that depend in part on intestinal microflora (5). The several glucosides with which the isoflavones are conjugated are hydrolyzed by intestinal glucosidases. The released aglycones are absorbable without further change; however, the methylated precursors of genistein and daidzein, biochanin and formononetin, respectively, may be further demethylated in the gut. Additional degradation of daidzein to active metabolites such as equol may occur, especially in the context of highly fermentable nutrients. The considerable interindividual variability in the apparent absorption of isoflavones (6) results from the complex nature of their absorption, which involves microbial activity and fermentation. Absorption has been estimated from the excretion of isoflavones in the urine. However, isoflavones are excreted in feces as well; not all metabolites are measured in the urine, and metabolism within the body is incompletely understood. Hence urinary values almost certainly underestimate true absorption. Absorption occurs over a period of hours with peak plasma concentrations 6–9 h after ingestion (faster for aglycones than for glycosides) (7) and halflives of the order of 6–9 h; the faster excretion of daidzein explains in part the greater systemic bioavailability of genistein (7). Given the complexity of the process of absorption, we have examined in unpublished observations whether the absorption of isoflavone aglycones might be more rapid than that of the corresponding glucosides, as has been suggested previ-

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ously. Free isoflavones from red clover and glucosides of soybean were separately incorporated into similar foods and consumed in random order over 2 wk each. Mean excretion at the end of each phase was not significantly different for aglycones and glucosides, averaging ~25% for the major isoflavones measured. Of additional interest were the following observations: interindividual variability was high; intraindividual variability was less in that the excretion (absorption) during the two phases of the study correlated significantly (R = 0.69); the differing isoflavone compositions derived from soy and red clover did not influence absorption (although much of the biochanin and formononetin in red clover was converted to genistein and daidzein, respectively). Although no firm conclusion can be drawn from the few studies carried out to date, the absorption of aglycones and glucosides appears not to be dissimilar. Effects on Plasma Lipids This is a controversial and as yet, inconclusive area of free phytoestrogen research. Published data that support a plasma cholesterol (LDL cholesterol; LDL-C)-lowering effect of isoflavones have been derived almost entirely from trials of soy protein (8). As with other nutritional interventions, the responsiveness of individuals has varied substantially, although the totality of the observations has been accepted by the U.S. Food and Drug Administration as adequate for a health claim. Although nonisoflavone effects of soy protein have not been excluded, several reported studies have shown a relationship between the isoflavone content of the soy flour and the reduction in LDL-C. In one large trial among 156 men and women, the consumption of 62 mg soy isoflavone in a single serving reduced LDL-C by an average of 6% and by 9% in those with an initially raised LDL-C concentration (9). A 37-mg dose was less effective. A similarly suggestive dose-related lowering of LDL-C was reported in a group of 81 mildly hypercholesterolemic men (10). Neither plasma triglycerides nor HDL cholesterol were affected. Several trials with isoflavone aglycones have been disappointing. Isolated and purified aglycones from soy or red clover have failed to lower LDL-C (11–15) although at least one published study demonstrated a significant rise in HDL cholesterol (HDL-C) (16). One large double-blind, randomized, placebo-controlled trial in Australia failed to show an effect on plasma lipids and lipoproteins of an aglycone mix of red clover isoflavones (14). That study was carried out in hypercholesterolemic postmenopausal women and the result was consistent with that in a similar trial conducted in a smaller group of postmenopausal women (13). Three studies of soy aglycone isoflavones in postmenopausal women also failed to show a change in plasma lipids (11,12,15). On the other hand, one study reported only in abstract form, conducted in women enrolled in the U.S. Women’s Ischemia Syndrome Evaluation (WISE) Study, found an inverse correlation between the plasma concentration of daidzein and plasma lipoprotein concentrations (17). Among 239 predominantly postmenopausal women, the highest tertile of the plasma daidzein concentration was

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associated with the lowest levels of LDL-C, plasma total triglyceride, and cholesterol. The concentrations of other isoflavones were not related to improved lipid profiles. This finding raises the possibility that the variability in the absorption of isoflavones may be a factor in the failure to demonstrate lipid-lowering effects. We therefore examined, in a recent 5-wk trial of red clover isoflavone aglycones in hypercholesterolemic postmenopausal women, whether the amounts absorbed (estimated from steady-state excretion values) might correlate with the changes in LDL-C. In this double-blind, randomized, placebo-controlled trial, the percentage of change in LDL-C between placebo and active intervention values, correlated significantly with the amounts of isoflavones excreted (absorbed), R = 0 0.67. Future trials of the effects of isoflavones, whether tested as glucosides or aglycones, should take into account a measure of bioavailability. It would therefore be premature to draw conclusions about the capacity of free isoflavones to influence plasma lipids. Effects on Vascular Functions Isoflavones resemble estradiol structurally, allowing occupation of estrogen receptors (ER). Although binding with much lower affinity to these receptors than do estrogens, their higher concentration in plasma that is achievable with supplementation leads to phytoestrogens exerting both estrogen-like activity and competitive inhibition against estradiol. Because the isoflavones bind preferentially to ER-β than to ER-α, they exert particularly strong effects on the vasculature that is enriched with ER-β (5). Blood Pressure. One report found no change in blood pressure in 59 subjects with systolic pressures in the high-normal range (18). In a randomized, placebo-controlled, parallel-design trial, 55 mg of isoflavone aglycones were consumed daily; 24-h ambulatory blood pressure measurements did not differ significantly between the placebo and active intervention groups over 8 wk, nor between the pre- and postintervention periods. Endothelial Function. In the U.S. WISE study of women with suspected myocardial ischemia, 71% of whom were postmenopausal, flow-mediated dilation of the brachial artery was correlated with the concentrations of each of several isoflavones in plasma (19). Only plasma genistein correlated positively with brachial artery reactivity (postischemic dilation). This is in contrast to the inverse relationship between the plasma daidzen concentration and LDL-C, reported in this same group of women (17). However, in a direct intervention with 80 mg/d soy isoflavone aglycone, Simons et al. (15) failed to find a significant difference in flow-mediated dilation between the active and placebo phases of a double-blind, randomized trial in 20 postmenopausal women. In a recent important paper, endothelial function was evaluated by plethysmography in the microcirculation of the forearm in 15 subjects (20). Pure genistein and daidzein were infused on separate days into the brachial artery to produce increasing concentrations in the microcirculation. The dilatory response to genistein was similar to that induced by 17β-estradiol; daidzein was

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ineffective. [This response resembled that observed in the WISE study (19), i.e., responsiveness to raising the concentration of genistein but not that of daidzein.] Genistein also potentiated acetylcholine-induced dilation and was inhibited when nitric oxide activity was suppressed. Systemic Arterial Compliance (SAC). We have reported two studies with two different sources of free isoflavones, one from soy (13) and the other from red clover (11). In both trials, SAC increased significantly with the active preparation compared with placebo. SAC reflects the distensibility of the large elastic arteries in contrast to the smaller muscular arteries such as the brachial. The components of SAC are more complex and probably include several constituents of the artery (elastic tissue, smooth muscle cells, glycoproteins, endothelium) and as measured by our method represent mainly upper aorta. The clinical significance of reduced SAC includes systolic hypertension, increased left ventricular load, and possibly coronary insufficiency due to diminished diastolic flow. SAC decreases with age and the menopause and can be restored to average values with estrogen therapy. Our finding of ~25% increments in SAC with isoflavone aglycones is therefore clinically important. In the first study (13), 80 mg/d soy isoflavones was taken in a placebo-controlled cross-over trial of 21 perimenopausal and postmenopausal women. Five weeks of active intervention raised SAC by 26%, which was independent of changes in blood pressure, plasma lipids, or LDL oxidizability. In the second study (11), 14 postmenopausal women were given in turn placebo, 40 mg, and 80 mg red clover isoflavones in a single blind study, leading to a 23% rise in SAC that was not significantly different between the two active doses (Fig. 17.1). The effects of free isoflavones on vascular function suggest that they resemble estrogen in this important role of improving cardiovascular health. A later section describes supporting evidence from in vitro experiments. In Vivo Antioxidant Functions The number of reports is inadequate to provide useful conclusions. By contrast, the isoflavones and their metabolites have been shown to be powerful antioxidants, albeit at concentrations higher than achievable with isoflavone consumption at current dietary levels. We found no significant changes in several parameters of antioxidant capacity such as lag time, rate of oxidation, and accumulation of thiobarbituric acid reactive substances (TBARS) after 5 wk of 80 mg/d treatment with soy isoflavone aglycones (13). Excretion of F2-isoprostane, an index of oxidant burden superior to ex vivo LDL oxidizability, was found to be reduced when 55 mg/d soy isoflavone was consumed (21). Effects in Experimental Animals and In Vitro Preparations Although less extensive than the data utilizing isoflavone glycosides within soy protein, the reported effects of aglycones are highly promising, strongly suggesting

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Arterial compliance units

Run-in

Placebo

40 mg

80 mg

Fig. 17.1. Effect of red clover isoflavones on systemic arterial compliance (SAC) in

menopausal women. Source: Reference 10.

that the active components in soy are the isoflavones. A recent report showed significant reduction in aortic arch atherosclerosis in cholesterol-fed rabbits, which also were fed aglycone isoflavones derived from soy (22). Of additional interest was the reduction in the aorta of lipid hydroperoxides and malondialdehyde, markers of oxidation. A 37% suppression in atherosclerosis was independent of plasma lipid concentration. Rabbits fed a low-isoflavone but saponin-rich supplement showed no improvement. In nonhuman primates, genistein appears to be responsible for the vasodilatory effect observed with consumption of soy protein. Genistein, injected intravenously enhanced acetylcholine-induced coronary dilation in female monkeys with coronary atherosclerosis (23). This is particularly interesting because genistein, but not daidzein, was vasodilatory in the human forearm microcirculation (20). Aortic rings, prepared from ovariectomized rats, showed restored vascular function when the rats were pretreated with daily injections of genistein (24). Similar vasodilatory effects have been observed with rat mesenteric arteries perfused in vitro with genistein and daidzein (25). Genistein has also been shown to have antioxidant effects in vitro. The addition of genistein prevents excessive oxidation of LDL (26). Effects of Isoflavone Metabolites The recent demonstration that several metabolites, formed in vivo after the consumption of isoflavones, showed powerful biological activity has focused attention on these compounds for their therapeutic potential. In this review, we will summa-

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rize mainly unpublished observations of the effects on cardiovascular parameters derived from in vitro experiments . Pure compounds have been synthesized for this purpose by Novogen Ltd. (North Ryde, NSW, Australia). Vascular Reactivity, Anti-inflammatory and Antioxidant Effects. Several metabolites have been studied by Chin-Dusting et al. (27) in classic pharmacologic studies using the rat aortic ring. The models investigated included reversal of norepinephrineinduced contraction, direct vasodilation, protection from the vasoconstrictive effects of oxidized LDL, and endothelial denudation. Four metabolites derived from daidzein, i.e., dehydroequol, dihydrodiadzein, and cis- and trans-tetrahydrodiadzein, were compared with 17β-estradiol. All four metabolites inhibited preconstricted arteries with dehydroequol equipotent with estradiol. Trans-tetrahydrodiadzein was much more potent than estradiol in blocking the damaging effect of oxidized LDL. The mechanisms were not uniform; although all metabolites were endothelium dependent; they became ineffective when the endothelium was denuded, and some remained effective in the presence of nitric oxide inhibition. The inhibition of endothelial activation by cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 has also been impressive. In endothelial cell preparations, the expression of the adhesion molecules VCAM-1 and E-selectin was found to be suppressed by both metabolites tested, in comparison with the up-regulation seen in control cells incubated with the cytokines alone (J. Gamble and M. Vadas, unpublished data). The metabolites have also been found to have powerful antioxidant effects, with dehydroequol substantially exceeding the potency of ascorbate in several models of oxidation (R. Stocker, unpublished data). Tetrahydrodiadzein has been found to be a potent inhibitor of smooth muscle cell synthesis and proliferation; its activity is dependent on estrogen receptor ligation (K. Sudhir et al., unpublished data). Thus, several key processes in the putative prevention of atherosclerosis appear to be potentiated by these metabolites, at least in vitro, raising the strong possibility of future therapeutic benefits.

Anticarcinogenic Effects In addition to their cardiovascular effects, compounds based on a flavonoid ring structure are emerging as a potentially important new class of pharmaceutical compounds with a possible role as anticancer agents. Interest in the potential role of plant flavonoids in cancer was stimulated by epidemiologic evidence of an inverse relationship between the incidence of both breast and prostate cancer and the dietary intake of foods rich in plant flavonoids (28). For example, the incidence of metastatic prostate cancer is ~30 times higher in Western men than in age-matched men in China and eight times higher than in Japanese men (29). The essential points of distinction between the typical Western diet vs. the Asian or vegetarian diet are a greater reliance on meat for protein requirements and greater use of refined, fiber-free carbohydrates for energy requirements. Although early interest

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in the link between diet and increased rates of cancer in Western populations focused on dietary factors such as animal fats, the general lack of success in that area has led in recent years to an alternative view, i.e., it is the high isoflavone intake in the Asian (or vegetarian) diet that may prevent induction and restrain the progression of many common cancers. The flavonoid quercetin, a flavone found in vegetables such as onions and apples and a common component of most human diets, was one of the first to be tested for anticartcinogenic potential (30). However, it did not progress beyond a Phase 1 study in cancer patients (30). Considerable attention has also been focused on the isoflavone genistein for cancer treatment. Genistein is a considerably more potent anticancer agent than quercetin and has been described as having anticancer activity in vitro against various types of human and animal cancers. Genistein was considered by the U.S. National Cancer Institute (NCI) in 1998 as an oral chemotherapeutic for prostate cancer in particular; its high safety index and potent and novel anticancer mechanisms made it an attractive potential anticancer agent. However, genistein failed to meet the NCI activity criteria when tested in xenograft animal models bearing human tumors and Phase 1 studies did not proceed. Another flavonoid to receive attention as a potential anticancer agent has been flavopiridol. This semisynthetic compound, based on a naturally occurring plant flavonoid, is a potent cyclin-dependent kinase inhibitor capable of mitotic arrest in either G1 or G2 phase (31). Flavopiridol exhibits antiproliferative activity in vitro against a broad range of human cancer cells including non-small cell lung cancer cells and prostate cancer and melanoma cells (32). Flavopiridol underwent early stage clinical studies in 1998–1999; although those early studies revealed promising anticancer activity, flavopiridol was found also to have dose-limiting disadvantages. The potential for isoflavones in the treatment of cancer has been rekindled by observations that although these compounds are moderately effective in native form, they form the substrate for a range of human metabolites, and a variety of metabolites of the isoflavones daidzein, genistein, formononetin and biochanin have been described. The potential for each of these isoflavonoid metabolites to express anticancer properties and their potency relative to the parent isoflavones have not been described previously. However, a number of reports have noted considerable variation among individuals in their profile of isoflavonoid metabolites, leading to speculation on a possible link between metabolite profiles and differences in individual susceptibility to various diseases including cancer (33,34). For instance, the lower rates of excretion of equol (35) and O-demethylangolensin (36) in the urine of women with breast cancer compared with women without breast cancer have been noted. However, the relative contributions of the parent isoflavones and their individual metabolites to the overall anticancer effect have not been established. In pursuit of the pharmaceutical opportunities represented by these compounds, Novogen research has focused on the synthesis of a panel of compounds based on the structure of selected metabolites for screening with regard to anticancer effect. This has produced a lead compound, phenoxodiol, which is a propri-

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etary synthetic derivative of one of these compounds which, in preclinical screening, produced potent anticancer activity against a range of cancer targets, including prostate cancer. In view of its potential to inhibit proliferation of prostate cancer cells, it has been further tested for anticancer properties as a potential prostate cancer therapeutic. Phenoxodiol inhibits the proliferation of a variety of human cancer cell lines in vitro in a dose-dependent fashion and is between 5- and 20-fold more potent than genistein in its antiproliferative effects (Fig. 17.2). Flow cytometric analysis (Fig. 17.3) shows that phenoxodiol arrests cells in G1 phase. The effects of phenoxodiol on LNCaP and PC3 prostate cancer cell line colonies growing in soft agar were tested at a range of concentrations and compared with the control (vehicle only) and positive control, etoposide. Phenoxodiol was highly effective at inhibiting both LNCaP and PC3 colony formation. Human leukemia and human prostate cancer cells undergo apoptosis when exposed to phenoxodiol. Cancer cells progress from viable cells to an early apoptotic stage in which phosphatidylserine (PS) is exposed externally from the inner to the outer leaflet of the plasma membrane, and then to a late apoptotic stage characterized by nuclear fragmentation. The degree of sensitivity appears to correlate with the functional state of the apoptotic regulatory gene p53. Phenoxodiol is a potent inhibitor of topoisomerase II, sphingosine kinase, and protein tyrosine kinases, all recognized as highly relevant targets for cancer treatment. The potential anticancer therapeutic benefits of phenoxodiol have been reflected in animal studies in which a significant inhibitory effect on the growth of human prostate cancer cells (PC3 cell line) implanted subcutaneously in male or female athymic mice was observed. Extensive toxicology studies have also demon-

Cell Proliferation

Genistein

HT29 Colon LNCaP Prostate Du145 Prostate MDA-MB-468 Breast MCF7 Breast

Phenoxodiol

K562 Leukemia

Concentration (mg/mL) Fig. 17.2. Inhibition of proliferation of several human cancer lines by genistein and

phenoxodiol. Note greater potency of the latter.

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0

50

100

G0G1 S G2M

50% 25% 25%

150

200

Channel

G0G1 S G2M

250 0

50

100

70% 8% 22%

150

200

250

Channel

Fig. 17.3. Flow cytometric analysis shows that phenoxodiol arrests prostate cancer cells in G0/G1 phase.

strated that phenoxodiol is nonclastogenic and nonmutagenic, with no drug-related toxicities in either acute or chronic dosing regimens in rodents. The evidence for efficacy of isoflavones compounds, and in particular, the synthetic flavonoid phenoxodiol, is sufficiently encouraging now to prompt the initiation of human clinical trials in cancer patients. Phase I/II trials are currently in progress for phenoxodiol, and it will be of considerable interest to determine the tolerability as well as efficacy of this compound, which may herald a new era of preventative and therapeutic options in the management of a range of cancers.

Summary and Conclusions Free aglycone isoflavones have been studied less extensively than the naturally occurring glycosides. Their effects on cardiovascular function include mainly studies of plasma lipoproteins, arterial function, and lipid oxidation. Most impressive are improvements in arterial function although these are not invariable. In humans, systemic arterial compliance has been improved with both soybean and red clover isoflavones. Intraarterial infusion of genistein is a potent vasodilator in humans and macacque monkeys, and brachial dilation in humans correlates with plasma genistein concentration. Of particular interest is the potency of metabolites formed in vivo after isoflavone consumption. These are powerful vasodilators and antioxidants and suppress atherogenic processes such as expression of adhesion molecules. In addition, these metabolites are potentially powerful anticarcinogens and are now in PhaseI/II trials. References 1. Hulley, S., Grady, D., Bush, T.L., Furberg, C., Herrington, D., Riggs, B., and Vittinghof, E. (1998) Randomized Trial of Estrogen Plus Progestin for Secondary Prevention of Coronary Heart Disease in Postmenopausal Women, J. Am. Med. Assoc. 280, 605–613.

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2. Westendorp, I.C.D., Bots, M.L., Grobbee, D.E., Reneman, R.S., Hoeks, A.P.G., Van Popele, N.M., Hofman, A., and Witteman, J., C.M. (1999) Menopausal Status and Distensibility of the Common Carotid Artery, Arterioscler. Thromb. Vasc. Biol. 19, 713–717. 3. Koh, K.K., Blum, A., Hathaway, L., Mincemoyer, R., Csako, G., Waclawiw, M.A., Panza, J.A., and Cannon, R.O. (1999) Vascular Effects and Vitamin E Therapies in Postmenopausal Women, Circulation 100, 1851–1857. 4. Cushman, M., Meilahn, B.M., Kuller, L.H., Dobs, A.S., and Tracy, R.P. (1999) Hormone Replacement Therapy, Inflammation and Hemostasis in Elderly Women, Arterioscler. Thromb. Vasc. Biol. 19, 893–899. 5. Setchell, K.D.R., and Cassidy, A. (1999) Dietary Isoflavones: Biological Effects and Relevance to Human Health, J. Nutr. 129, 758S–767S. 6. Zhang, Y., Wang, G.J., Song, T.T., Murphy, P.A., and Hendrichs, S. (1999) Urinary Disposition of the Soybean Isoflavones Daidzein, Genistein and Glycetin Differs Among Humans with Moderate Fecal Isoflavone Degradation Activity, J. Nutr. 129, 957–962. 7. Setchell, K.D., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Brashear, W.T., Kirschner, A.S., and Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131, 1362S–1375S. 8. Anderson, J.W., and Johnstone, B. M.(1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 9. Crouse, J.R., Morgan, T., Terry, J.G., Ellis, J., Vitolins, M., and Burke, G.L. (1999) A Randomized Trial Comparing the Effect of Casein with That of Soy Protein Containing Varying Amounts of Isoflavones on Plasma Concentrations of Lipids and Lipoproteins, Arch. Intern. Med. 159, 2070– 2076. 10. Teixeira, S.R., Potter, S.M., Weigel, R., Hannum, S., Erdman, J.W., and Hasler, C.M. (2000) Effects of Feeding 4 Levels of Soy Protein for 3 and 6 Weeks on Blood Lipids and Apolipoproteins in Moderately Hypercholesterolemic Men, Am. J. Clin. Nutr. 71, 1077–1084. 11. Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J.D., and West, L. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895–898. 12. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., and Croft, K.D. (1998) Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations, J. Nutr. 128, 728–732. 13. Nestel, P.J., Yamashita,T., Sashara, T., Pomeroy, S.T., Dart, A., Komesaroff, P., Owen, A., and Abbey, M. (1997) Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women, Atrerioscler. Thromb. Vasc. Biol. 17, 3392–3398. 14. Howes, J.B., Sullivan, D., Lai, N., Nestel, P., Pomeroy, S., West, L., Eden, J.A., and Howes, L.G. (2000) The Effects of Dietary Supplementation with Isolavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 152, 143–147. 15. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2000) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy Postmenopausal Women, Am. J. Cvardiol. 85, 1297–1301.

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16. Samman, S., Lyons Wall, P.M., Chan, S.M., Smith, S.J., and Petocz, P. (1999) The Effect of Supplementation with Isoflavones on Plasma Lipids and Oxidizability of Low Density Lipoprotein in Premenopausal Women, Atherosclerosis 147, 277–283. 17. Merz, C.N.B., Johnson, D.B., Braunstein, G., Pepine, C.J., Jordon, R.L., Reis, S.E., Paul-Labrador, M., Sopko, G., and Kelsey, S.F. (1999) Phytoestrogen Levels Mediate Lipoproteins in Women Independent of Estrogen Level: The NHLBI-Sponsored WISE Study, Circulation 100 (Suppl. 1), I-29. 18. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., Burke, V., Croft, K.D., and Rogers, P.B. (1999) Effects of Isoflavonoids on Blood Pressure in Subjects with HighNormal Ambulatory Blood Pressure Levels, Am. J. Hypertens. 12, 47–53. 19. Merz, C.N.B., Johnson, D.B., and Kelsey, S.F. (1999) Phytoestrogen Levels Mediate Brachial Artery Reactivity in Women Independent of Estrogen Level: The NHLBISponsored WISE Study, Circulation 100 (Suppl. 1), I-221. 20. Walker, H.A., Dean, T.S., Sanders, T.A.B., Jackson, G., Ritter, J.M., and Chowienczyk, P.J. (2001) The Phytoestrogen Genistein Produces Acute Nitric Oxide-Dependent Dilation of Human Forearm Vasculature with Similar Potency to 17β-Estradiol, Circulation 103, 258–262. 21. Wiseman, H., O’Reilly, J.D., Adlercreutz, H., Mallet, A.J., Bowey, E.A., Rowland, I.R., and Sanders, T.A.B. (2000) Isoflavone Phytoestrogens Consumed in Soy Decrease F2isoprostane Concentrations and Increase Resistance to Low-Density Lipoprotein to Oxidation in Humans, Am. J. Clin. Nutr. 72, 395-400. 22. Yamakoshi, J., Piskula, M.K., Izumi, I.T., Tobe, K., Saito, M., Kataoka, S., Obata, A., and Kikuchi, M. (2000) Isoflavone Aglycone-Rich Extract Without Soy Protein Attenuates Atherosclerosis in Cholesterol-Fed Rabbits, J. Nutr. 130, 1887–1893. 23. Honore, E.K., Williams, J.K., Anthony, M.S., and Clarkson, T.B. (1997) Soy Isoflavones Enhance Coronary Vascular Reactivity in Atherosclerotic Female Macaques, Fertil. Steril. 67, 148–154. 24. Squadrito, F., Altavilla, D., Squadrito, G., Saitta, A., Cucinotta, D., Minutoli, L., Deodato, B., Ferlito, M., Campo, G.M., Bova, A., and Caputi, A.P. (2000) Genistein Supplementation and Estrogen Replacement Therapy Improve Endothelial Dysfunction Induced by Ovariectomy in Rats, Cardiovasc. Res. 45, 454–462. 25. Nevala, R., Korpela, R., and Vapaatalo, H. (1998) Plant Derived Estrogens Relax Rat Mesenteric Artery In Vitro, Life Sci. 63. 26. Kerry, N., and Abbey, M. (1998) The Isoflavone Genistein Inhibits Copper and Peroxyl Radical Mediated Low Density Lipoprotein Oxidation In Vitro, Atherosclerosis 140, 341–347. 27. Chin-Dusting, J.P.F., Fisher, L.J., Lewis, T.V., Piekarska, A., Nestel, P.J., and Husband, A.H. (2001) The Vascular Activity of Some Isoflavone Metabolites: Implications for a Cardioprotective Role, Br. J. Pharmacol. 133, 595–605. 28. Adlercreutz, H., Fotsis, T., Heikkinen, R., Dwyer, J.T., Woods, M., Goldin, B.R., and Gorbach, S.L. (1982) Excretion of the Lignans Enterolactone and Enterodiol and of Equol in Omnivorous and Vegetarian Postmenopausal Women and in Women with Breast Cancer, Lancet 2, 1295–1299. 29. Dhom, G. (1991) Epidemiology of Hormone-Depending Tumors, in Endocrine Dependent Tumors (Voigt, K.D., and Knabbe, C., eds.) pp. 1–42, Raven Press, New York. 30. Wei, Y.Q., Zhao, X., Kariya, Y., Fukata, H., Teshigawara, K., and Uchida, A. (1994) Induction of Apoptosis by Quercetin: Involvement of Heat Shock Protein, Cancer Res. 54, 4952–4957.

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31. Carlson, B.A., Dubay, M.M., Sausville, E.A., Brizuela, L., and Worland, P. (1996) Flavopiridol Induces G1 Arrest with Inhibition of Cyclin-Dependent Kinase (CDK)2 and CDK4 in Human Breast Carcinoma Cells, Cancer Res. 56, 2973–2978. 32. Drees, M., Dengler, W.A., Roth, T., Labonte, H., Mayo, J., Malspeis, L., Grever, M., Sausville, E.A., and Fiebig, H.H. (1997) Flavopiridol (L86-8275): Selective Antitumor Activity In Vitro and Activity In Vivo for Prostate Carcinoma Cells, Clin. Cancer Res. 3, 273–279. 33. Setchell, K.D., Borriello, S.P., Hulme, P., Kirk, D.N., and Axelson, M. (1984) Nonsteroidal Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Disease. Am. J. Clin. Nutr. 40, 569-578. 34. Kelly, G.E., Nelson, C., Waring, M.A., Joannou, G.E., and Reeder, A.Y. (1993) Metabolites of Dietary (Soya) Isoflavones in Human Urine, Clin. Chim. Acta 223, 9–22. 35. Ingram, D., Sander, K., Kolybaba, M., and Lopez, D. (1998) Case-Control Study of Phyto-Oestrogens and Breast Cancer, Lancet 350, 990–994. 36. Adlercreutz, H., Goldin, B.R., Gorbach, S.L., Hockerstedt, K.A., Watanabe, S., Hamalainen, E.K., Markkanen, M.H., Mäkelä, T.H., and Wähälä, K.T. (1995) Soybean Phytoestrogen Intake and Cancer Risk, J. Nutr. 125, 757S-770S.

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Chapter 18

Association Between Soy and/or Isoflavones and Bone: Evidence from Epidemiologic Studies Mary S. Anthonya, John J.B. Andersonb, and D. Lee Alekelc aDepartments

of Pathology and Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC bDepartment

of Nutrition, Schools of Public Health and Medicine, University of North Carolina, Chapel Hill, NC

cDepartment

of Food Science and Human Nutrition, Human Metabolic Unit, Iowa State University, Ames, IA

Introduction A recent upsurge has occurred in research exploring whether soy and/or the isoflavones in soy, termed phytoestrogens, might improve bone health. Much of this interest has focused on soy and isoflavones as potential alternatives to pharmacologic hormone replacement therapy (HRT). However, soy and/or the isoflavones could also affect bone health in young women and men. In this chapter, the magnitude of the problem of osteoporosis will be described briefly and the evidence from observational epidemiologic studies regarding the association between soy and/or isoflavone intake and bone will be reviewed. Overview of Osteoporosis. Osteoporosis is a silent epidemic, afflicting 10 million people and accounting for 1.5 million new fractures each year in the United States alone (National Osteoporosis Foundation Fact Sheet 2002). Women are ~4 times more likely to be affected by osteoporosis than men, but both men and women are at increasing risk as they age. The longer life expectancy of women amplifies their disease burden. Although only ~15% of women aged 50–59 y have osteoporosis, ~70% of women 80 y old and older are affected (1). Many areas of the world are experiencing increases in hip fracture incidence, in part because of an increase in longevity. In 1990, ~1.66 million hip fractures occurred worldwide, and it has been estimated that the number could rise to 6.26 million by 2050 (2). At present, the majority of hip fractures occur in Europe and North America, but enormous increases in the number of elderly in South America, Africa, and Asia will shift this burden of disease from the developed to the developing world (3). Effective prevention strategies will have to be designed and disseminated in these parts of the world to prevent the expected increase in hip fractures, and a nutritional strategy might be more easily implemented than pharmacologic ones.

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Osteoporosis is defined as a “skeletal disorder characterized by compromised bone strength and predisposing a person to an increased risk of fracture” (4). Both bone density and quality of the bone are important aspects of bone strength. The World Health Organization (WHO) has developed an operational definition of osteoporosis based on bone mineral density (BMD) of young adult Caucasian women (5). Unfortunately, because of insufficient data on the relationship between BMD and fracture risk in men or non-Caucasian women, the WHO did not offer a definition of osteoporosis for groups other than Caucasian women. The WHO defines osteoporosis as a BMD measurement <2.5 SD below the mean for young women and osteopenia as a BMD between 1 and 2.5 SD below the mean for young women; a BMD that is at the mean or higher indicates normal bone mass. On the basis of these cut-off values, it has been estimated that 30% of postmenopausal Caucasian women in the United States and ~23% of European women >50 y old have osteoporosis, i.e., BMD 2.5 or more SD below the mean (6). However, more recently, The National Osteoporosis Risk Assessment (NORA) study (7) estimated that, using WHO criteria, 39.6% of postmenopausal women in the United States have osteopenia (T score of –1 to –2.49) and 7.2% have osteoporosis (T score at or below –2.5). Lower BMD is associated with higher facture risk; for each 1 SD below the mean BMD at the hip, a woman’s risk of fracture is 2.6 times higher (8). Estrogens and Osteoporosis. Bone is continually undergoing remodeling, with osteoclast cells resorbing bone and osteoblasts forming new bone. Estrogen deficiency during perimenopause increases the rate of bone remodeling, resulting in a relative imbalance between bone resorption and formation, i.e., resorption rates are higher than formation rates, leading to net bone loss. Bone loss during the perimenopausal period may result in a 20–30% loss in cancellous (trabecular) bone and a 5–10% loss in cortical bone (9); these high rates of loss may continue for the next decade. HRT prevents bone loss at the spine and hip (10) and has been estimated to reduce nonvertebral fracture rates by ~27%, based on a meta-analysis of 22 randomized trials (11). HRT decreases bone loss by slowing bone turnover, reducing both formation and resorption. HRT reduces bone loss regardless of when therapy is initiated, but when therapy is discontinued, bone loss ensues at a rate similar to that immediately after menopause (12). Because of the fear of cancer and adverse side effects in some women, only ~20% of naturally menopausal women continue HRT for >5 y (13). Hence, it is important to find therapies that are effective for preventing bone loss and will be continued long term. Cross-Cultural Comparisons of Caucasian vs. Asian Populations: Bone Density and Fractures Ethnic and genetic differences may make some groups more susceptible than others to osteoporotic fractures (14). However, lifestyle and dietary differences are also likely to contribute to cross-cultural differences in osteoporosis and fracture

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rates. Hip fracture rates for Japanese living in Okinawa are approximately half that seen in American Caucasian populations (Fig. 18.1), for both men and women (15). Similar differences in hip fractures rates have been reported between Hong Kong and the U.S. (16) and between Chinese, Japanese, and Koreans living in the U.S. compared with U.S. Caucasians (17). Because hip fractures almost always require hospitalization, there are fewer concerns about complete case ascertainment than for other types of fractures. However, although there are lower rates of hip fractures in Japanese compared with U.S. populations, there do not appear to be differences in the rates of osteoporosis as determined by the percentage of the populations with low BMD (18). Thus, the lower hip fracture rates in Japanese living in Japan compared with U.S. Caucasian populations cannot be explained by differences in BMD or calcium intake, which is lower in Japan. Some of the factors that have been postulated to account for lower hip fracture rates in Asian populations include: higher physical activity and more developed hip musculature (18), fewer falls (19,20), and shorter hip axis length (21,22). However, lower osteoporotic fracture risk for the hip in Asian populations might also be related to dietary factors, such as lower animal protein intake (23) or higher soy and isoflavone intake. Despite lower hip fracture rates, Taiwanese have comparable vertebral fracture incidence (24) and, hence, dietary factors may not be the primary factors in determining ethnic differences in spine fracture risk.

Observational Studies of Soy Intake, Bone Density, and Fractures Several reports from observational studies on the relationship between soy and/or isoflavones and bone density have been published over the last few years. These Rate/100,000

Rate/100,000

2000

Men 1500 1000

US Japan

500 0

2000

Women

1500 1000

US Japan

500

50–54 55–59 60–64 65–69 70–74 75–79 80–84 Age group

0

50–54 55–59 60–64 65–69 70–74 75–79 80–84 Age group

Fig. 18.1. Age-specific incidence rates of hip fractures in Japanese (Niigata Prefecture)

and U.S. Caucasian men and women. Rates are per 100,000 persons. Adapted from Reference 15.

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Change in spinal BMD (%)

reports are largely in Asian populations, in which soy consumption is more common; one study, however, was published in Dutch postmenopausal women (25). In the Asian population studies, two studies evaluated premenopausal women (26,27), three studies were in postmenopausal women (27–29), and two studies did not specify menopausal status (30,31). Ho and colleagues (26) evaluated the association between soy isoflavone intake and change in bone density over a 3-y follow-up period in a cohort of premenopausal Hong Kong Chinese women (n = 132), ages 30–40 y. They assessed soy food intake and other dietary factors by a semi-quantitative food-frequency questionnaire (FFQ) for the12 mo before the baseline BMD measurement. Spinal BMD was measured by dual-energy X-ray absorptiometry (DXA) at baseline and at 3 y of treatment; 116 women also had an intermediate measurement (at 1 or 2 y of follow-up), and it was these women with three measurements who were included in the analyses. Isoflavone intake was calculated on the basis of published data on the isoflavone content of various soy foods. Tofu, bean curd pudding, and soy milk were the soy foods included in the questionnaire and account for ~70% of the total soy intake in Hong Kong Chinese. Women were categorized into quartiles on the basis of soy isoflavone intake, and the change in spinal BMD was compared among quartiles. Isoflavone intake in the lowest quartile was estimated to be 0–2.96 mg/d and in the highest quartile, 7.43–48.30 mg/d. The percentage of change in spinal BMD over 38 mo of follow-up (unadjusted) is shown in Figure 18.2. A significant difference was found in spinal BMD change in the highest vs. lowest isoflavone quartiles (P < 0.01) when adjusting for lean body mass, physical activity, and follow-up time; the high-isoflavone group lost ~2.4% less bone than the low-isoflavone group. These variables together accounted for ~20% of the variability in spinal BMD loss in this population. 0.0 –0.5 –1.0 –1.5 –2.0 –2.5 –3.0 –3.5 –4.0 –4.5 –5.0 0–2.96

2.98–4.45 4.47–7.38 7.43–48.30 Isoflavone intake (mg/d) Fig. 18.2. The percentage of change in spinal bone mineral density (BMD) by isoflavone consumption quartile in premenopausal women (n = 116) followed for 38 mo. Data are unadjusted means ± SEM. Source: Reference 26.

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In a cross-sectional study of premenopausal (n = 293) and postmenopausal (n = 357) Hong Kong women, soy food intake was assessed by a semi-quantitative FFQ that included nine soy food items (27). As in the study by Ho et al. (26), isoflavone intake was estimated on the basis of published isoflavone content of various foods. Women were categorized into tertiles according to that intake. Average isoflavone intake in the highest tertile was estimated at 53 mg/d. BMD was measured by DXA for lumbar spine, Ward’s triangle, femoral neck, trochanter, and total hip. No association was found between isoflavone intake and BMD at any of these sites in the premenopausal women. However, in postmenopausal women, the highest isoflavone tertile had significantly higher BMD in the spine and Ward’s triangle (both areas rich in trabecular bone) compared with the low isoflavone tertile (spine, 6.3% higher in tertile 3 vs. tertile 1, P = 0.02; Ward’s triangle, 8.4% higher in tertile 3 vs. tertile 1, P = 0.05). In a study by Horiuchi et al. (28) in postmenopausal Japanese women (n = 85), diet was assessed by 3-d diet records and included weighed portion sizes. Intake of soy protein was estimated from these records by a dietitian. Lumbar spine BMD was measured by DXA. Soy protein intake was positively associated with lumbar spine BMD, expressed as a Z-score (r = 0.23, P = 0.04), when controlling for energy, protein, and calcium intake. The women in this study may represent a selected population because they were referred to an osteoporosis unit. About 60% of the women were osteopenic or osteoporotic on the basis of the measurements of their lumbar BMD. The measures of association in this study were not adjusted for other potential confounders, such as body weight, although the Z-scores used for the BMD measures were derived from age-matched healthy Japanese women. Somekawa and colleagues (29) examined the relationship between soy isoflavone intake and spinal BMD measured by DXA in 478 postmenopausal Japanese women. Women were divided into two postmenopausal groups (PMG) based on time since menopause, early PMG: <5 years since menopause and late PMG: >5 y since menopause. Consumption of soy foods was asked for current intake and past intake (i.e., when participants were 40 y old) and isoflavone intake was estimated from a Japanese database. Individuals were divided into quartiles on the basis of isoflavone intake (Quartile 1, <35 mg/d; Quartile 2, 35–50 mg/d; Quartile 3, 50–65 mg/d; Quartile 4, >65 mg/d. A linear association was found between isoflavone intake and spinal BMD for both early (P for trend ≤0.001) and late (P > for trend = 0.01) postmenopausal groups, after adjusting for weight and years since menopause. Higher isoflavone consumption in women was associated with higher BMD (Fig. 18.3). In the early PMG, lumbar spine BMD was 7.9% higher in quartile 4 vs. quartile 1 and in the late PMG, BMD was 8.8% higher in quartile 4 compared with quartile 1. A study in middle-aged (40–49 y) Japanese women (n = 995) examined the relationship of various dietary factors (including soybean intake) to metacarpal BMD as measured by computed X-ray densitometry (31). Dietary intake was assessed by FFQ. Women who consumed soybeans 2–5 times/wk had 4.6% higher

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Adjusted lumbar BMD (g/cm2)

Early PMG

Late PMG

P for trend ≤ 0.001

P for trend = 0.01

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 <35 35–50 50-65 >65 <35 35–50 50–65 >65 Isoflavone intake (mg/d)

Fig. 18.3. Lumbar spine bone mineral density (BMD, g/cm2) in postmenopausal women by isoflavone intake quartile. Women were stratified by time since menopause: the early postmenopausal group (early PMG, n = 269) included those <5 y postmenopause and the late postmenopausal group (late PMG, n = 209) included those >5 y postmenopause. Data are adjusted means (adjusted for weight and years since menopause) ± SEM. Source: Reference 29.

BMD than those who had soybeans once or 0–1 times/wk (P = 0.02) and those who consumed soybean 6–7 times/wk had 5.0% higher BMD than the lowest intake group (P = 0.004). This trend remained significant (P for trend = 0.03) after controlling for age, height, weight, and weekly calcium intake. In a small cross-sectional study of women (n = 50) 32–68 y of age in Japan, isoflavone intake was estimated from 3-d dietary records (30). Bone density was measured by ultrasound. Daidzein intake ranged from 3.2 to 35.6 mg/d and genistein intake from 4.6 to 52.1 mg/d. Isoflavone intake in this population of volunteer housewives was not correlated with bone density. However, age and menopausal status were not taken into account in the analysis, nor were any other covariates. In the Netherlands, Kardinaal and colleagues (25) tested the hypothesis that the rate of postmenopausal radial bone loss measured by single photon absorptiometry is inversely related to urinary excretion of phytoestrogens. Women with a high rate of bone loss (≥2.5%/y, n = 35) and a low rate of bone loss (≤0.5%/y, n = 32) were selected from a cohort study with 10 y of follow-up. Urine was collected annually and for each individual, an aggregate urine sample was made from the annually collected urine specimens. Urinary concentrations of genistein, daidzein, and equol did not differ between the two bone loss groups. The Dutch typically consume very low amounts of dietary phytoestrogens, and this group of women had similarly low intakes. Interestingly, urinary equol excretion was positively

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associated with bone loss during the first 5 y of follow-up. Because equol has been measured in infants consuming exclusively cow’s milk formula (32), and dairy products are a dietary staple of the Dutch, it is possible that this unexpected relationship between higher bone loss and higher equol excretion is due to an association with higher animal (milk) protein rather than soy consumption.

Conclusions These published studies differ with respect to the type and site of bone measured, the amount of dietary soy and/or isoflavones habitually consumed, and study design. Nonetheless, a positive association appears to exist between soy and/or isoflavone intake and bone health. Of the seven studies in Asian populations, five (26–29,31) found a positive association between soy protein or isoflavone intake and higher bone density (or reduced bone loss). Two studies (27,30) found no significant association between isoflavone intake and bone density, although one of these studies (30) included only 41 women, used ultrasound rather than DXA to assess BMD, and did not account for many important determinants of bone density in the analyses (e.g., age, menopausal status, smoking, or body weight). The study by Kardinaal et al. (25) in postmenopausal women in the Netherlands found no significant association between isoflavone excretion and bone loss. Although this was a well-designed study, isoflavone intake in this population was quite low and thus unlikely to reveal a relationship. There are no definitive differences in the effects of soy and isoflavones on bone by menopausal status in these studies. Positive associations between soy intake and bone density were found in premenopausal (26), pre-/perimenopausal (31), and postmenopausal women (27–29). However, the association might be more consistently found in postmenopausal women because all three of the studies in postmenopausal women (27–29) found a significant association between soy and/or isoflavone intake and bone density; however, only one (26) of the two studies in premenopausal Asian women (26,27) found a significant association. The lack of difference between pre- and postmenopausal women in the effect of soy and its isoflavones might suggest a nonhormonal (or non-estrogen receptor-mediated) mechanism of action. Although these observational studies suggest an association between soy and/or isoflavone intake and bone health, a causal association cannot be determined. If there is a critical time for soy and/or isoflavones to affect bone, it cannot be determined from these studies in Asian populations in which life-long soy consumption occurs. Further, these observational studies cannot determine whether it is the soy protein or the isoflavones that are responsible for the relationship because most of the soy consumed in Asia contains isoflavones. Therefore, evidence of a causal relationship between soy protein or the isoflavones and bone must be evaluated further in animal studies and followed up by carefully controlled clinical trials. These studies are reviewed in other chapters.

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Data on soy intake with respect to osteoporotic fractures are lacking because prospective observational studies and clinical trials require very large samples of subjects that must be followed long term and are thus very costly. Other observational study designs, however, might be useful in evaluating fracture outcomes. Cross-sectional and retrospective case-control studies in soy-consuming Asian populations could be done relatively inexpensively, particularly if nested within ongoing cohort studies. A long-term prospective observational study in soy-consuming postmenopausal women living in Asia, however, could provide much more convincing evidence regarding soy and/or isoflavones and fracture incidence. References 1. Melton, L.J., III. (1996) Global Aspects of Osteoporosis Epidemiology, in Osteoporosis 1996 (Papapoulos, S.E., ed.) pp. 79–86, Elsevier Science BV, Amsterdam. 2. Cooper, C., Campion, G., and Melton, L.J., III. (1992) Hip Fractures in the Elderly: A Worldwide Projection, Osteoporos. Int. 2, 285–289. 3. Genant, H.K., Cooper, C., Poor, G., Reid, I., Ehrlich, G., Kanis, J., Nordin, B.E.C., Barrett-Connor, E., Black, D., Bonjour, J-P., Dawson-Hughes, B., Delmas, P.D., Dequeker, J., RagiEis, S., Gennari, C., Johnell, O., Johnston, C.C., Lau, E.M.C., Liberman, U.A., Lindsay, R., Martin, T.J., Masri, B., Mautalen, C.A., Meunier, P.J., Miller, P.D., Mithal, A., Morii, H., Papapoulos, S., Woolf, A., Yu, W., and Khaltaev, N. (1999) Interim Report and Recommendations of the World Health Organization TaskForce for Osteoporosis, Osteoporos. Int. 10, 259–264. 4. NIH Consensus Development Panel (2001) Osteoporosis Prevention, Diagnosis, and Therapy, J. Am. Med. Assoc. 285, 785–795. 5. World Health Organization (1994) Assessment of Fracture Risk and Its Application to Screening for Postmenopausal Osteoporosis, WHO Technical Report Series no. 843, World Health Organization, Geneva. 6. Cooper, C., and Barrett-Connor, E. (1996) Epidemiology of Osteoporosis, in Osteoporosis 1996 (Papapoulos, S.E., ed.) pp.75–78, Elsevier Science BV, Amsterdam. 7. Siris, E.S., Miller, P.D., Barrett-Connor, E., Faulkner, K.G., Wehren, L.E., Abbott, T.A., Berger, M.L., Santora, A.C., and Sherwood, L.M. (2001) Identification and Fracture Outcomes of Undiagnosed Low Bone Mineral Density in Postmenopausal Women: Results from the National Osteoporosis Risk Assessment, J. Am. Med. Assoc. 286, 2815–2822. 8. Cummings, S.R., Black, D.M., Nevitt, M.C., Browner, W., Cauley, J., Ensrud, K., Genant, H.K., Palermo, L., Scott, J., and Vogt, T.M. (1993) Bone Density at Various Sites for Prediction of Hip Fractures, Lancet 341, 72–75. 9. Riggs, B.L., Khosla, S., and Melton, L.J. III. (1998) A Unitary Model for Involutional Osteoporosis: Estrogen Deficiency Causes Both Type I and Type II Osteoporosis in Postmenopausal Women and Contributes to Bone Loss in Aging Men, J. Bone Miner. Res. 13, 763–773. 10. Komulainen, M., Kroger, H., Tuppurainen, M.T., Heikkinen, A.M., Alhava, E., Honkanen, R., Jurvelin, J., and Saarikoski, S. (1999) Prevention of Femoral and Lumbar Bone Loss with Hormone Replacement Therapy and Vitamin D3 in Early Postmenopausal Women: A Population-Based 5-Year Randomized Trial, J. Clin. Endocrinol. Metab. 84, 546–52.

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11. Torgerson, D.J., and Bell-Syer, S.E.M. (2001) Hormone Replacement Therapy and Prevention of Nonvertebral Fractures: A Meta-Analysis of Randomized Trials, J. Am. Med. Assoc. 285, 2891–2897. 12. Schneider, D.L., Barrett-Connor, E.L., and Morton, D.J. (1997) Timing of Postmenopausal Estrogen for Optimal Bone Mineral Density. The Rancho Bernardo Study, J. Am. Med. Assoc. 277, 543–547. 13. Brett, K.M., and Madans, J.H. (1997) Use of Postmenopausal Hormone Replacement Therapy: Estimates from a Nationally Representative Cohort Study, Am. J. Epidemiol. 145, 536–545. 14. Pollitzer, W.S., and Anderson, J.J.B. (1989) Ethnic and Genetic Differences in Bone Mass: a Review with a Hereditary vs Environmental Perspective, Am. J. Clin. Nutr. 50, 1244–1259. 15. Ross, P.D., Norimatsu, H., Davis, J.W., Yano, K., Wasnich, R.D., Fujiwara, S., Hosoda, Y., and Melton, L.J. III. (1991) A Comparison of Hip Fracture Incidence Among Native Japanese, Japanese Americans, and American Caucasians, Am. J. Epidemiol. 133, 801–809. 16. Ho, S.C., Bacon, E., Harris, T., Looker, A., and Maggi, S. (1993) Hip Fracture Rates in Hong Kong and the United States, 1988 Through 1989, Am. J. Public Health. 83, 694–697. 17. Lauderdale, D.S., Jacobsen, S.J., Furner, S.E., Levy, P.S., Brody, J.A., and Goldberg, J. (1997) Hip Fracture Incidence Among Elderly Asian-American Populations, Am. J. Epidemiol. 146, 502–509. 18. Fujita, T., and Fukase, M. (1992) Comparison of Osteoporosis and Calcium Intake Between Japan and the United States, Proc. Soc. Exp. Biol. Med. 200, 149–152. 19. Aoyagi, K., Ross, P.D., Davis, J.W., Wasnich, R.D., Hayashi, T., and Takemoto, T-I. (1998) Falls Among Community-Dwelling Elderly in Japan, J. Bone Miner. Res. 13, 1468–1474. 20. Davis, J.W., Ross, P.D., Nevitt, M.C., and Wasnich, R.D. (1997) Incidence Rates of Falls Among Japanese Men and Women Living in Hawaii, J. Clin. Epidemiol. 50, 589–594. 21. Cummings, S.R., Cauley, J.A., Palermo, L., Ross, P.D., Wasnich, R.D., Black, D., and Faulkner, K.G. (1994) Racial Differences in Hip Axis Lengths Might Explain Racial Differences in Rates of Hip Fracture. Study of Osteoporotic Fractures Research Group, Osteoporos. Int. 4, 226–229. 22. Nakamura, T., Turner, C.H., Yoshikawa, T., Slemenda, C.W., Peacock, M., Burr, D.B., Mizuno, Y., Orimo, H., Ouchi, Y., and Johnston, C.C. (1994) Do Variations in Hip Geometry Explain Differences in Hip Fracture Risk Between Japanese and White Americans? J. Bone Miner. Res. 9, 1071–1076. 23. Abelow, B.J., Holford, T.R., and Insogna, K.L. (1992) Cross-Cultural Association Between Animal Protein and Fracture: A Hypothesis, Calcif. Tissue. Int. 50, 14–18. 24. Tsai, K.S. (1997) Osteoporotic Fracture Rate, Bone Mineral Density, and Bone Metabolism in Taiwan, J. Formos. Med. Assoc. 96, 802–805. 25. Kardinaal, A.F.M., Morton, M.S., Brggermann-Rotgans, I.E.M., and van Beresteijn, E.C.H. (1998) Phyto-Oestrogen Excretion and Rate of Bone Loss in Postmenopausal Women, Eur. J. Clin. Nutr. 52, 850–855. 26. Ho, S.C., Chan, S.G., Yi, Q., Wong, E., and Leung, P.C. (2001) Soy Intake and the Maintenance of Peak Bone Mass in Hong Kong Chinese Women, J. Bone Miner. Res. 16, 1363–1369.

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27. Mei, J., Yeung, S.S.C., and Kung, A.W.C. (2001) High Dietary Phytoestrogen Intake Is Associated with Higher Bone Mineral Density in Postmenopausal but Not Premenopausal Women, J. Clin. Endocrinol. Metab. 86, 5217–5221. 28. Horiuchi, T., Onouchi, T., Takahashi, M., Ito, H., and Orimo, H. (2000) Effect of Soy Protein on Bone Metabolism in Postmenopausal Japanese Women, Osteoporos. Int. 11, 721–724. 29. Somekawa, Y., Chiguchi, M., Ishibashi, T., and Aso, T. (2001) Soy Intake Related to Menopausal Symptoms, Serum Lipids, and Bone Mineral Density in Postmenopausal Japanese Women, Obstet. Gynecol. 97, 109–115. 30. Kimira, M., Arai, Y., Shimoi, K., and Watanabe, S. (1998) Japanese Intake of Flavonoids and Isoflavonoids from Foods, J. Epidemiol. 8, 168–175. 31. Tsuchida, K., Mizushima, S., Toba, M., and Soda, K. (1999) Dietary Soybeans Intake and Bone Mineral Density Among 995 Middle-Aged Women in Yokohama, J. Epidemiol. 9, 14–19. 32. Setchell, K.D.R., Zimmer-Nechemias, L., Cai, J., and Heubi, J.E. (1997) Exposure of Infants to Phyto-Oestrogens from Soy-Base Infant Formula, Lancet 350, 23–27.

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Chapter 19

Skeletal Effects of Phytoestrogens in Humans: Bone Mineral Density and Bone Markers John J.B. Andersona and D. Lee Alekelb aDepartment

of Nutrition, Schools of Public Health and Medicine, University of North Carolina, Chapel Hill, NC bDepartment

of Food Science and Human Nutrition, Human Metabolic Unit, Iowa State University, Ames, IA

Introduction This review highlights research findings in humans on the skeletal effects of phytoestrogens. Phytoestrogens broadly encompass several classes of plant molecules, such as isoflavones, lignans, and coumestans, that have some estrogenic activity in animal and human tissues. Because most of the reported studies have been conducted on the naturally occurring nonsteroidal isoflavones obtained from soy foods, this review focuses on isoflavones. Supplements of soy- and clover-derived molecules have recently been introduced into the marketplace, but published data on these extracted phytoestrogens are limited. This review covers reports on subjects participating in prospective investigations; cross-sectional observational studies in the context of an epidemiologic perspective were reviewed in Chapter 18. Although relatively few data are available from prospective studies on the effect of phytoestrogens on bone mineral density (BMD), these reports as well as studies on biochemical markers of bone in humans are reviewed. Animal and in vitro cell studies were reviewed in Chapter 20. Readers are referred to previous reviews published on the skeletal effects of soy isoflavones (1–5). Two types of end points have been used to assess changes in skeletal tissue with treatment, i.e., measurements of BMD by dual-energy X-ray absorptiometry (DXA) and of biochemical markers of bone in blood or urine. Therefore, this chapter covers the potential skeletal benefits of phytoestrogens from soy and other sources in human subjects. At this time, the only clear maintenance of BMD has been found in women, whereas no data from studies of men have been reported. Isoflavones act on cells in several different ways, but one way the bone-conserving action of these molecules is thought to work is through the estrogen receptor (ER)-mediated pathway (see Chapter 12). Isoflavones are weak agonists of 17βestradiol in bone cells, primarily in osteoblasts, but they act as estrogen antagonists in reproductive tissues (6). These differential tissue responses relate to the relative number of α and β ER subtypes in various cells. The stronger binding affinity of isoflavones for ER-β may be particularly important because this receptor has been identified in bone tissue (7). Dietary isoflavones are weakly estrogenic, particular-

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ly when there is a lack of endogenous estrogen, but preferentially bind to ER-β (8), implying that their actions are distinct from those of classical steroidal estrogens that bind to both ER-α and ER-β. Isoflavones indeed may exert tissue-selective effects because some tissues contain predominately ER-α or ER-β. Thus, isoflavones behave much like drugs known as selective estrogen receptor modulators (SERM). Although further study is required to determine which isoflavones in vivo inhibit bone resorption and/or stimulate formation, in vitro studies demonstrate that the responses of osteoblasts and osteoclasts to isoflavones depend on the dose used. With achievable dietary doses, isoflavones suppress osteoclastic and enhance osteoblastic function in cell lines. Whether isoflavones will be used to prevent or treat osteoporosis requires more extensive human investigation. The emphasis of this review is on the effects of isoflavones on BMD and bone biomarkers in human subjects. Phytoestrogens and Bone Mineral Density: Prospective Studies Seven prospective studies that include a measurement of bone mass as an outcome in response to isoflavone intake have been published (9–15). Three of these studies (11,14,15) were designed specifically to examine bone as the primary outcome. Three studies used soy protein isolate (SPI) (10,11,15), one used soy foods (9), and one examined usual soy food intake among Asians (14) as the source of isoflavones; two used extracted phytoestrogens (12,13). Dalais and colleagues (9) provided either 45 g soy grits (flour) containing 53 mg isoflavones, 45 g linseed (flaxseed with mammalian lignan precursors), or 45 g wheat kibble (control) daily to 44 postmenopausal women for 12 wk using a cross-over design. They reported that total body bone mineral content (BMC) increased 5.2% (P = 0.03) in the soy, 5.2% [not significant (NS)] in the linseed, and 3.8% (NS) in the wheat groups, but there was no change in BMD. It is rather surprising that there were such substantial increases in BMC but not in BMD in the soy, linseed, and wheat control (albeit NS) groups, and hence these results must be interpreted with caution. The next published study (10), designed to examine the lipid-related effects of soy protein, randomly assigned 66 postmenopausal women to one of three treatments for 6 mo: (i) casein + nonfat dry milk protein, (ii) SPI (40 g/d) with 56 mg/d of isoflavones, or (iii) SPI with 90 mg/d of isoflavones. The time since menopause and age (49–83 y) varied widely in these hypercholesterolemic women. Also, some of the women gained weight, but this key factor was not taken into account. At the end of treatment, women in the high-isoflavone group experienced an increase (~2%; P < 0.05) in lumbar spine BMD and BMC, whereas those consuming the casein + milk–based protein had slight decreases. Although baseline values were not taken into account, women in the high-isoflavone group began the study with lower BMD and BMC than did the other two groups. Those with lower bone mass typically have a greater response to treatment (16) and, thus, baseline BMD should be considered. Another published study (11) in 69 perimenopausal women is in general agreement with the previously described work. Women were randomized (double-

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blind) to treatment, with dose expressed as aglycone units: isoflavone-rich soy (+SPI, 80.4 mg/d; n = 24), isoflavone-poor soy (–SPI, 4.4 mg/d; n = 24), or whey (control; n = 21) protein. No change was reported in the +SPI (-0.2%, P = 0.7; +0.6%, P = 0.5) or –SPI (–0.7%, P = 0.1; –0.6%, P = 0.3) groups, but loss (P = 0.004) occurred in controls (–1.3%, –1.7%) in the lumbar spine BMD and BMC (Fig. 19.1), respectively, values. Because baseline BMD and BMC (P ≤ 0.0001) affected (negatively) the percentage of change in these outcomes, baseline values were taken into account in the analysis of covariance (ANCOVA) and regression analysis. Treatment had a significant effect on the percentage of change in BMC (P = 0.021), but not on the percentage of change in BMD (P = 0.25) using ANCOVA. Contrast coding in ANCOVA revealed that isoflavones, not soy protein, exerted a positive effect on BMD and BMC. Using multiple regression analysis, +SPI had a significant positive treatment effect on the percentage of change in both BMD (5.6%, P = 0.023) and BMC (10.1%, P = 0.0032), while accounting for various contributing factors. Body weight at baseline rather than final weight or weight gain was related to the percentage of change in BMD, suggesting that weight gain did not confound the effect of +SPI on bone. Contrary to their hypothesis, the authors did not find an effect of endogenous reproductive hormones or estrogen status on bone loss. Soy (–SPI) or whey protein had no effect on the spine, and treatment in general had no effect on bone sites other than the spine. These two studies (10,11) provide support for the contention that isoflavones are the bioactive component of soy with respect to bone. A recently published study was designed to examine habitual soy intake and bone mass in premenopausal Chinese women 30–40 y of age living in Hong Kong

Fig. 19.1. Mean (± SEM) percentage (%) change in lumbar spine bone mineral density (BMD; left panel) and bone mineral content (BMC; right panel) from baseline to post-treatment in three treatment groups of perimenopausal women: isoflavone-rich soy (+SPI = X; n = 24), isoflavone-poor soy (–SPI = ■; n = 24), and whey (control = ◆; n = 21) protein. Lumbar spine bone mass was determined using dual-energy X-ray absorptiometry. *Significantly different from baseline (paired t tests with Bonferroni adjustment; P-values noted). Reproduced with permission by the American Journal of Clinical Nutrition (11).

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(14). After adjusting for age and body size (height, weight, and bone area), researchers reported a positive effect of soy isoflavones on spinal BMD after an average follow-up time of 38.1 mo. The mean percentage of decline in spinal BMD in 116 women was greater (P < 0.05) in the lowest (–3.5%) vs. highest (–1.1%) quartile of soy isoflavone intake. Multiple regression analysis revealed that soy isoflavone intake (along with lean body mass, physical activity, energyadjusted calcium intake, and follow-up time) accounted for 24% of the variance in spinal BMD in these women. This 3-y study indicated that soy isoflavone intake had a positive effect on maintaining spinal BMD in premenopausal women 30–40 y of age. In contrast, after a year-long study in premenopausal women (15), neither the isoflavone-containing nor the isoflavone-deficient soy had any affect on bone. Young adult eumenorrheic women (21–25 y of age) were randomly assigned to either 90 mg of isoflavones (60% in aglycone form) contained in soy protein (n = 15) or to an isoflavone-deficient SPI (n = 13) for 12 mo (15). These investigators reported no change in BMC, BMD, or body composition with treatment. A study in 37 postmenopausal women supplemented with soy isoflavones (150 mg/d, but undefined) for 6 mo also did not produce a significant change in calcaneous BMD (12). That study is difficult to interpret because there was no control group. Alternatively, because the calcaneous (heel) is weight-bearing and has greater trabecular content (17) than vertebral bone, it may respond differently to isoflavone treatment than the lumbar spine. Another human study (13) also used extracted isoflavones, but from red clover containing genistein, daidzein, formononetin, and biochanin that was administered (double-blind) to 46 postmenopausal women for 6 mo. Subjects were randomized to one of three treatments: 28.5, 57, or 85.5 mg/d of isoflavones. Women who received the middle dose experienced a 4.1% (P = 0.002) and those at the high dose a 3.0% (P = 0.023) increase in the proximal radius and ulna (predominantly cortical bone) BMD, whereas those at the low dose did not have a significant (P = 0.12) increase (2.9%). The distal radius and ulna (predominantly trabecular bone) BMD did not change in relation to isoflavone dose, nor did endometrial thickness increase with treatment. The interpretation of this study is difficult, given the lack of a control group. Yet, results of this study suggest that isoflavones may have a significant effect on cortical bone, unlike the previously presented trials, or that appendicular bone responds differently than the axial (i.e., spine) skeleton. Alternatively, formononetin and biochanin may exert effects on cortical bone, whereas other soy isoflavones may have greater effects on trabecular bone. In summary, results of these human studies suggest that isoflavones may attenuate bone loss from the lumbar spine in estrogen-deficient women, who may otherwise be expected to lose 2 to 3%/y (Table 19.1). Such attenuation of loss, particularly if continued throughout the postmenopausal period, could translate into a decreased risk of osteoporosis. Because a bone-remodeling cycle ranges from 30 to 80 wk (18), such short-term preliminary studies cannot answer the question whether these bone-sparing effects would be sustained over a longer period. From these results, we cannot determine whether the reported bone-sparing effect is due to treat-

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TABLE 19.1 Phytoestrogens and Bone Mineral Density (BMD), Bone Mineral Content (BMC): Prospective Studies in Humansa Study cited Subject characteristics No. of subjects

Treatment Design

Duration Dose (mg/d)

Outcome Reported

Result Comments

Reference 9 Postmenopausal Caucasian(?) 45–65 y 44

45 g Soy grits Wheat kibble Linseed Double-blind

12 wk 53 mg isoflavones

Total body BMC, BMD

No change in BMD, but substantial ↑ BMC: Soy grits: ↑ 5.2% (P = 0.03) Wheat: ↑ 3.8% (NS) Linseed: ↑ 5.2% (NS)

Reference 10 Postmenopausal Caucasian(?) 49–83 y 66

SPI (40 g/d): high vs. low dose vs. control (casein + non-fat dry milk) Double-blind

26 wk 90 mg/d 56 mg/d 0 mg/d

Lumbar spine BMC, BMD

90 mg: ↑ 2% (P < 0.05) Control: slight ↓ Women in high dose group began study with lower BMD and BMC

Reference 11 Perimenopausal Caucasian 42–62 y 69

SPI (40 g/d) +SPI –SPI Whey control Double-blind

24 wk 80 mg/d (aglycone form) 4.4 mg/d 0 mg/d

Lumbar spine BMD, BMC

% change in BMC and BMD +SPI ↓ 0.2% (P = 0.7) ↑ 0.6% (P = 0.5) –SPI ↓ 0.7% (P = 0.1) ↓ 0.6% (P = 0.3) Control ↓ 1.3% (P = 0.004) ↓ 1.7% (P = 0.004) Treatment effect of SPI+ on % change in BMD (5.6%, P = 0.023), BMC (10.1%, P = 0.0032) Continued

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TABLE 19.1 (Cont.) Reference 12 Postmenopausal Taiwanese 40–57 y 37

Isoflavone supplements No control

26 wk 150 mg/d

Calcaneous BMD

No significant change in calcaneous BMD No control group

Reference 13 Postmenopausal Caucasian(?) <65 y 46

Extracted isoflavone (red clover) supplement No control Double-blind

26 wk 28.5 mg/d 57 mg/d 85.5 mg/d

Proximal radius and ulna BMD

% change in BMD: 28.5 mg/d (NS) 57 mg/d ↑ 4.1% (P = 0.002) 85.5 mg/d ↑ 3.0% (P = 0.023)

Reference 14 Premenopausal Chinese 30–40 y 116

Usual soy food intake

164 wk (38 mo) Quartiles(mg/d): 1 = 1.4 2 = 3.75 3 = 5.89 4 = 15.16

Lumbar spine BMD

% ↓ greater (P < 0.05) in lowest (–3.5%) vs. highest (–1.1%) quartile of isoflavone intake Soy isoflavone intake (plus lean body mass, physical activity, energy-adjusted calcium intake, and follow-up time) accounted for 24% of variance in BMD

Reference 15 Premenopausal Caucasian 21–25 y 28

SPI (90 g/d) +SPI –SPI

52 wk 90 mg/d (60% aglycone form)

Lumbar spine BMC, BMD TB BMD No change in BMC or BMD at any site

Abbreviations: SPI, soy protein isolate; +SPI, isoflavone-rich soy; -SPI, isoflavone-poor soy; NS, not significant; TB, trabecular.

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ment or is an artifact of the bone-remodeling transient (18), although the longer-term study in Asian women (14) suggests true bone-sparing. A clinical trial for at least 2 and preferably 3 y is necessary to determine whether soy isoflavones will affect the remodeling balance, tipping it in favor of bone formation rather than resorption. Biochemical Markers of Bone in Response to Phytoestrogens Biochemical markers of bone serve as indices of change in bone turnover, reflecting increases or decreases in rates of resorption and formation. Several markers found either in blood or urine can be measured by enzyme-linked immunosorbent assay (ELISA), high-pressure liquid chromatography (HPLC), or radioimmunoassay (RIA or IRMA) procedures. The advantages of biochemical markers of bone are that the method is noninvasive, may predict (albeit imperfectly) the rate of bone loss in menopausal women, may predict the response to some antiresorptive therapies (19), and may be performed more frequently than bone density scans. In a research setting, measurements of bone markers are typically made at baseline and then again at one or more times during the course of the study. In a clinical setting, bone markers may be measured at baseline and then a few weeks after the initiation of treatment to determine whether a patient has experienced a therapeutic response. The primary limitation of bone markers is that circadian rhythms affect circulating concentrations and hence biologic variability is sufficiently great to necessitate large differences in the markers to detect a response to therapy (19). Additional limitations are that some markers are not sensitive or specific (i.e., bone- vs. nonbone-derived biomarkers) enough to detect small changes over time, the renal clearance capacity of the patient greatly influences values for certain blood- and urine-derived markers, sample procurement and measurement are not standardized, and the overall metabolic status of the patient at the time of sample collection was not considered. Nevertheless, increasing concentrations of both formation and resorption markers are associated with more rapid bone loss, and differences in these rates correspond to clinically significant differences in fracture risk (20). Existing data indicate that biochemical markers can aid in determining which women are at greater risk of rapid bone loss and fracture (21). The most valuable biomarkers (19) for bone formation are serum bone-specific alkaline phosphatase, osteocalcin, and the N-terminal polypeptide of procollagen I; for bone resorption, the most suitable markers are serum N-telopeptide and C-telopeptide of type I collagen and urinary pyridinoline and deoxypyridinoline collagen cross-links. The reader is referred to a review by the International Osteoporosis Foundation on the use of biochemical markers of bone turnover in osteoporosis (22). Few studies have been published examining the response of biochemical markers of bone turnover to isoflavone-rich soy protein or extracted isoflavones in humans. This first section reviews studies using soy protein as the treatment and the next section covers trials using extracted isoflavones, illustrating inconsistent results regarding the biochemical markers.

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The first group to include a measure of bone turnover as part of a study designed to examine hot flushes was that of Murkies and co-workers (23). The diet of postmenopausal women was supplemented with either wheat or soy flour (45 g/d) for 12 wk. Urinary hydroxyproline, a nonspecific marker of bone resorption, increased over time in the wheat flour (P < 0.05) but not in the soy flour group; however, the difference between groups was not significant (P = 0.47). Washburn et al. (24) also reported on alkaline phosphatase activity, a nonspecific bone formation marker, in a study designed to examine the effects of soy protein on cardiovascular disease risk factors and menopausal symptoms. In this randomized crossover (double-blind) trial, 51 subjects consumed isocaloric supplements for 6 weeks each: (i) a single dose of 20 g soy protein (34 mg isoflavones); (ii) split (two) dose of 20 g soy protein (34 mg isoflavones); or (iii) 20 g complex carbohydrate. Alkaline phosphatase (nonspecific marker of bone formation) activity decreased (P < 0.05) in women consuming either soy diet compared with the carbohydrate-supplemented group. The authors suggest that this decline may have reflected a beneficial effect of soy, whereas these results are difficult to interpret because they did not measure any bone resorption marker. Crossover designs have an inherent drawback due to the potential for carry-over or contamination in the outcomes of interest. Knight and colleagues (25) conducted a randomized (double-blind) placebo-controlled 12-wk study, designed to examine the effects of soy protein (40 g/d) on menopausal symptoms in 24 women. They reported no differences in serum alkaline phosphatase (nonspecific bone formation marker) or pyridinoline cross-links (bone resorption marker) between the isoflavone-(77.4 mg/d aglycone components) and casein-treated (control) groups. In contrast to these studies, a 24-wk trial in perimenopausal women (n = 69) described above (11) did not find a decline in bone resorption (cross-linked N-telopeptides) during the course of treatment. Repeated measures ANCOVA indicated that treatment per se had no significant effect on either cross-linked N-telopeptides (P = 0.12) or bone-specific alkaline phosphatase (P = 0.32), but both time (P < 0.005) and baseline value (P ≤ 0.0001) were significant. Yet, cohort (group of subjects that began the study at the same time) had a significant effect on N-telopeptides (P = 0.0089), but not on bone-specific alkaline phosphatase (P = 0.56), suggesting that cohort may reflect a seasonal influence on bone resorption. Wangen and co-workers (26) conducted a randomized, cross-over 3-mo SPI (63 g/d) study to examine the dose-response effect of isoflavones on bone markers in 14 premenopausal and 17 postmenopausal women. On a per kilogram body weight basis, expressed as aglycone units, the daily dose was 10 ± 1.1, 64 ± 9.2, or 128 ± 16 mg/d in the premenopausal women, and was 7 ± 1.1, 65 ± 11, or 132 ± 22 mg/d in the postmenopausal women. No effects of treatment on osteocalcin were observed in the premenopausal women, but during the early follicular phase, deoxpyridinoline cross-links increased in both isoflavone diets; during the periovulatory phase, serum insulin-like growth factor-I (IGF-I) increased in the low (64 mg/d) compared with high (128 mg/d) isoflavone diets. In postmenopausal women, the low (65 mg/d) and high (132 mg/d) isoflavone diets decreased bone-specific alkaline phosphatase. The high isoflavone

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group experienced a trend toward a decrease in osteocalcin and in IGF-I. Compared with baseline, all three diets increased bone-specific alkaline phosphatase, osteocalcin, and IGF-I, although the increases in the last-mentioned two markers were not significant at the highest isoflavone dose. The authors suggest that although isoflavones modestly affected markers of bone turnover, the changes were small and not likely clinically relevant. A recent study (27) examined the effect of consuming dietary whole-soy foods (~60 mg/d) for 12 wk on serum bone-specific alkaline phosphatase, osteocalcin, and urinary cross-linked N-telopeptides in 42 postmenopausal women. Serum bone-specific alkaline phosphatase did not change, whereas osteocalcin increased (P < 0.03) and N-telopeptides decreased (P < 0.02) from baseline to wk 12. This study suggests that soy foods may decrease bone resorption while maintaining bone formation. However, the lack of a placebo control in this study makes interpretation difficult, given the biologic variability of bone turnover markers. In contrast, Clifton-Bligh and colleagues (13), in the study described above, used isoflavones extracted from red clover and reported no differences among the doses of isoflavones and no significant changes in urinary deoxypyridinoline (bone resorption marker) from baseline to 6 mo. To determine the effects on early climacteric symptoms, Scambia and co-workers (28) randomly assigned subjects (n = 39) to a standardized soy extract (50 mg/d of isoflavones) or placebo treatment for 6 wk and then provided conjugated equine estrogen (0.625 mg/d) for 4 wk. They did not observe soy-related changes in serum osteocalcin (bone formation marker), and estrogen-related changes were not modified by soy extract. At 15 geographical sites, Upmalis and colleagues (29) used a soy isoflavone extract (50 mg/d of isoflavones) compared with placebo to determine the effects on climacteric symptoms in postmenopausal women (n = 177). They reported no treatment effect (change) in either serum osteocalcin or urinary N-telopeptides, but they found a reduction in the number and severity of hot flushes. Overall, evidence is greater for effects of soy isoflavones in maintaining bone than for preventing bone resorption, but the data are limited and somewhat contradictory or unclear. Without definitive results, conclusions about the effect of soy isoflavones on bone turnover in humans are difficult to draw. Some of this discrepancy has likely resulted from differences in study design (i.e., isoflavone dose, duration of study, specific biochemical markers used) that do not allow reasonable comparisons, and some may relate to the wide day-to-day variability of these markers, particularly in early menopausal women. Current State of Understanding Preliminary data that soy protein containing isoflavones may favorably affect bone mass must be substantiated by better-designed clinical trials. In fact, the existing controversy over whether isoflavones alone or soy protein plus the isoflavones are required to exert skeletal effects must be resolved. This issue has become even more important because of the widespread use of isoflavone-enriched supplements.

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As mentioned earlier, studies of longer duration are warranted to determine whether the purported effect is actually due to the treatment or is simply an artifact of the bone-remodeling transient (18). Although the actions of isoflavones may be distinct from those of estrogens, which have antiresorptive skeletal effects, evidence exists that isoflavones exert estrogen-like effects in human cells due to their unique structure. Depending upon the tissue and species, isoflavones may act as weak estrogen agonists or as weak estrogen antagonists; in bone tissue, they act predominantly as weak agonists of estrogens. Further studies are required to corroborate the skeletal effects of isoflavones and to determine how they spare bone tissue in the face of estrogen deficiency. Until such data are published and concurrence is reached, evidencebased medicine should not recommend isoflavones to treat or prevent osteoporosis. Although the use of soy foods as a substitute for estrogen or hormone replacement therapy cannot be recommended, health professionals should suggest that the public increase consumption of soy foods as part of a healthy diet because of the excellent nutrient profile and other health benefits of soy products. Gaps in Knowledge/Future Research Directions A major gap in our knowledge relates to the interindividual biologic variability in response to the isoflavone preparations provided in studies. Some individuals apparently are high excretors and others are low excretors of isoflavones, likely corresponding to higher and lower isoflavone bioavailability, respectively (30). Systemic bioavailability, as determined by comparing area under the curve, is greater for the β-glycosides than their corresponding aglycones (31), which may be related to gut motility and gut microflora (30). One explanation for the biologic variability is that an individual’s intestinal microflora governs the capacity to convert daidzein to equol (32). Another explanation is that dietary fat intake decreases the capacity of gut microflora to synthesize equol (33), but that interindividual variation in equol excretion may also reflect different hormonal patterns (34). Related to the issue of biologic variability is that of differences among various commercial soy isoflavone supplements (31) and the need to clearly define the isoflavone source in a given study. For example, the aglycones (genistein, daidzein, glycitein), unlike their corresponding β-glycosides (genistin, daidzin, glycitin), do not require hydrolytic cleavage before absorption and hence the former require less time (4–7 vs. 8–11 h) to attain peak plasma concentrations (31). Whether soy isoflavones from food or from supplements will exert similar effects on the skeleton is not yet known. Given the differences in pharmacokinetics and bioavailability among isoflavones, it cannot be assumed that all isoflavones behave similarly. Studies must carefully define the type, quantitative dose, and administered frequency of isoflavones provided to subjects. Another gap relates to the estrogen status of study subjects because circulating estrogens may influence the response to isoflavone treatment. Studies with postmenopausal women should include measures of circulating estrone and 17 β-estra-

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diol; those with normally cycling premenopausal women should include these measures at menses, at mid-cycle (near ovulation), and during the mid-luteal phase; those with men should also include circulating estrogens. In prospective studies, a reasonable approach would be to take circulating estrogen concentrations as well as baseline bone mass values into account when monitoring response to treatment. Because little information exists about the effects of soy isoflavones in elderly women or in men at risk for osteoporosis, long-term human studies with these groups using both soy foods and isolated isoflavones should be undertaken. Finally, the potentially negative side effects of long-term use of isolated isoflavones comprise another major gap. Although data suggest that isoflavones are the primary bone-active components of soy, studies should be conducted on both soy foods and extracted isoflavones, particularly because the public has access to isoflavone supplements. It is easier to design and carry out long-term studies using extracted isoflavones because this intervention should not perturb eating habits and body weight, thus allowing better long-term compliance and less potential confounding by extraneous factors. However, dietary intake of soy protein itself may promote health benefits unrelated to bone. Clearly, a long-term, dose-response study in humans designed to corroborate the findings reviewed herein and to examine potential mechanisms is warranted. Treatment regimens should be devised and conveyed to the public if and when researchers confirm a favorable effect of soy and/or its isoflavones on bone in mid-life women.

Summary and Conclusions The few studies that have reported a positive skeletal response in peri- and postmenopausal women to soy phytoestrogens suggest that perhaps 60–90 mg/d of isoflavones may be needed for bone protection, translating into ~2–3 servings of traditional soy foods. Further data are required to determine an optimal dose-response curve for humans. Understanding of the mechanism of isoflavones on bone cells needs further refinement, especially with regard to long-term use. Although the use of isoflavones as drugs in medical therapy is on the horizon, greater research efforts are required to establish the effectiveness and safety of isoflavones in humans. References 1. Anderson, J.J.B., and Garner, S.C. (1998) Phytoestrogens and Bone, Balliere’s Clin. Endocrinol. Metab. 12, 1–15. 2. Messina, M., Gugger, E.T., and Alekel, D.L. (2001) Soy Protein, Soybean Isoflavones, and Bone Health: A Review of the Animal and Human Data, in Handbook of Nutraceuticals and Functional Foods (Wildman, R.E.C., ed.) pp. 77–98, CRC Press LLC, Boca Raton. 3. Arjmandi, B.H. (2001) The Role of Phytoestrogens in the Prevention and Treatment of Osteoporosis in Ovarian Hormone Deficiency, J. Am. Coll. Nutr. 20 (Suppl. 5), 398S–402S, 417S–420S.

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4. Scheiber, M.D., and Rebar, R.W. (1999) Isoflavones and Postmenopausal Bone Health: A Viable Alternative to Estrogen Therapy? Menopause 6, 233–241. 5. Doren, M., and Samsioe, G. (2000) Prevention of Postmenopausal Osteoporosis with Oestrogen Replacement Therapy and Associated Compounds: Update on Clinical Trials Since 1995, Hum. Reprod. Update 6, 419–426. 6. Miodini, P., Fioravanti, J., Di Fronzo, G., and Cappelletti, V. (1999) The Two PhytoOestrogens Genistein and Quercetin Exert Different Effects on Oestrogen Receptor Function, Br. J. Cancer 80, 1150–1155. 7. Vidal, O., Kindblom, L.-G., and Ohlsson, C. (1999) Expression and Localization of Estrogen-Receptor-β in Murine and Human Bone, J. Bone Mineral Res. 14, 923–929. 8. Setchell, K.D.R. (1995) Non-Steroidal Estrogen of Dietary Origin: Possible Role in Health and Disease, Metabolism and Physiological Effects, Proc. Nutr. Soc. N.Z. 20, 1–21. 9. Dalais, F.S., Rice, G.E., Wahlqvist, M.L., Grehan, M., Murkies, A.L., Medley, G., Ayton, R., and Strauss, B.J.G. (1998) Effects of Dietary Phytoestrogens in Postmenopausal Women, Climacteric 1, 124–129. 10. Potter, S.M., Baum, J.A., Teng, H., Stillman, R.J., Shay, N.F., and Erdman, J.W., Jr. (1998) Soy Protein and Isoflavones: Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68, 1375S–1379S. 11. Alekel, D.L., St. Germain, A., Peterson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in the Lumbar Spine of Perimenopausal Women, Am. J. Clin. Nutr. 72, 844–852. 12. Hsu, C.-S., Shen, W.W., Hsueh, Y.-M., and Yeh, S.-L. (2001) Soy Isoflavone Supplementation in Postmenopausal Women. Effects on Plasma Lipids, Antioxidant Enzyme Activities, and Bone Density, J. Reprod. Med. 46, 221–226. 13. Clifton-Bligh, P.B., Baber, R.J., Fulcher, G.R., Nery, M.-L., and Moreton, T. (2001) The Effect of Isoflavones Extracted from Red Clover (Rimostil) on Lipid and Bone Metabolism, Menopause 8, 259–265. 14. Ho, S.C., Chan, S.G., Yi, Q., Wong, E., and Leung, P.C. (2001) Soy Intake and the Maintenance of Peak Bone Mass in Hong Kong Chinese Women, J. Bone Miner. Res. 16, 1363–1369. 15. Anderson, J.J.B., Chen, X.W., Boass, A., Symons, M., Kohlmeirer, M., Renner, J.B., and Garner, S.C. (2002) Soy Isoflavones: No Effects on Bone Mineral Content and Bone Mineral Density in Healthy, Menstruating Young Adult Women After One Year, J. Am. Coll. Nutr., in press. 16. Pines, A., Katchman, H., Villa, Y., Mijatovic, V., Dotan, I., Levo, Y., and Ayalon, D. (1999) The Effect of Various Hormonal Preparations and Calcium Supplementation on Bone Mass in Early Menopause. Is There a Predictive Value for the Initial Bone Density and Body Weight? J. Intern. Med. 246, 357–61. 17. Davis, J.W., Novotny, R., Wasnich, R.D., and Ross, P.D. (1999) Ethnic, Anthropometric, and Lifestyle Associations with Regional Variations in Peak Bone Mass, Calcif. Tissue Int. 65, 100–105. 18. Heaney, R.P. (1994) The Bone-Remodeling Transient: Implications for the Interpretation of Clinical Studies of Bone Mass Change, J. Bone Miner. Res. 9, 1515–1523. 19. Souberbielle, J.C., Cormier, C., and Kindermans, C. (1999) Bone Markers in Clinical Practice, Curr. Opin. Rheumatol. 11, 312–319. 20. Ross, P.D. (1999) Predicting Bone Loss and Fracture Risk with Biochemical Markers: A Review, J. Clin. Densitom. 2, 285–294.

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21. Garnero, P. (2000) Markers of Bone Turnover for the Prediction of Fracture Risk, Osteoporos. Int. 11 (Suppl. 6), S55–S65. 22. Delmas, P.D., Eastell, R., Garnero, P., Seibel, M.J., and Stepan, J., Committee of Scientific Advisors of the International Osteoporosis Foundation (2000) The Use of Biochemical Markers of Bone Turnover in Osteoporosis, Osteoporos. Int. 11(Suppl. 6), S2–S17. 23. Murkies, A.L., Lombard, C., Strauss, B.J.G., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Post-Menopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 24. Washburn, S., Burke, G., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–11. 25. Knight, D.C., Howes, J.B., Eden, J.A., and Howes, L.G. (2001) Effects on Menopausal Symptoms and Acceptability of Isoflavone-Containing Soy Powder Dietary Supplementation, Climacteric 4, 13–18. 26. Wangen, K.E., Duncan, A.M., Merz-Demlow, B.E., Xu, X., Marcus, R., Phipps, W.R., and Kurzer, M.S. (2000) Effects of Soy Isoflavones on Markers of Bone Turnover in Premenopausal and Postmenopausal Women, J. Clin. Endocrinol. Metab. 85, 3043–3048. 27. Scheiber, M.D., Liu, J.H., Subbiah, M.T.R., Rebar, R.W., and Setchell, K.D.R. (2001) Dietary Inclusion of Whole Soy Foods Results in Significant Reductions in Clinical Risk Factors for Osteoporosis and Cardiovascular Disease in Normal Postmenopausal Women, Menopause 8, 384–392. 28. Scambia, G., Mango, D., Signorile, P.G., Anselmi-Angeli, R.A., Palena, C., Gallo, D., Bombardelli, E., Morazzoni, P., Riva, A., and Mancuso, S. (2000) Clinical Effects of a Standardized Soy Extract in Postmenopausal Women: A Pilot Study, Menopause 7, 105–111. 29. Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L., and Lamia, C.A. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: A Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 30. Zhang, Y., Wang, G.-J., Song, T.T., Murphy, P.A., and Hendrich, S. (1999) Urinary Disposition of the Soybean Isoflavones Daidzein, Genistein, and Glycitein Differs Among Humans with Moderate Fecal Isoflavone Degradation Activity, J. Nutr. 129, 957–962. 31. Setchell, K.D.R., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., and Heubi, J.E. (2001) Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements, J. Nutr. 131, 1362S-1375S. 32. Lampe, J.W., Skor, H.E., Li, S., Wähälä, K., Howald, W.N., and Chen, C. (2001) Wheat Bran and Soy Protein Feeding Do Not Alter Urinary Excretion of the Isoflavan Equol in Premenopausal Women, J. Nutr. 131, 740–744. 33. Rowland, I.R., Wiseman, H., Sanders, T.A., Adlercreutz, H., and Bowey, E.A. (2000) Interindividual Variation in Metabolism of Soy Isoflavones and Lignans: Influence of Habitual Diet on Equol Production by the Gut Microflora, Nutr. Cancer 36, 27–32. 34. Duncan, A.M., Merz-Demlow, B.E., Xu, X., Phipps, W.R., and Kurzer, M.S. (2000) Premenopausal Equol Excretors Show Plasma Hormone Profiles Associated with Lowered Risk of Breast Cancer, Cancer Epidemiol. Biomark. Prev. 9, 581–586x

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Chapter 20

Skeletal Effects of Phytoestrogens: Rodent Models: Diet Bahram H. Arjmandi and Brenda J. Smith Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK

Introduction Osteoporosis is a debilitating and costly disease that afflicts millions of American women and men. Aside from drug therapies, certain lifestyle and nutritional factors may be important in maintaining or improving the skeletal health of both sexes. Among the existing and potential therapeutic options, medications that are categorically termed selective estrogen receptor modulators (SERMs) may hold promise as viable alternatives for the treatment of osteoporosis. SERM are defined as a group of compounds that behave as estrogen agonists in certain tissues, while acting as antagonists in others (1). Recent reports indicate that phytoestrogens exert their effects in a SERM-like manner (2,3). Phytoestrogens are nonsteroidal plant compounds of diverse structures found in many fruits, vegetables, and grains. Hence, food sources rich in phytoestrogens may provide postmenopausal women with yet an additional practical and safe alternative therapy. Additionally, because estrogen therapy may not be feasible in men, SERMs or SERM-like compounds that exert estrogen-like effects on bone but not on other tissues (3,4), could be of benefit to men as well. The plant food sources high in phytoestrogens/phytochemicals are numerous and include soybeans, flaxseeds, and certain other fruits and vegetables high in polyphenolic compounds. This chapter focuses on animal findings to date regarding the effects of soy protein or its isoflavones on bone. Additional statements concerning the role of other food sources rich in phytochemicals that may also positively affect bone are discussed briefly. Bone-Modulating Effects of Soy Until the early twentieth century, it was assumed that estrogens were produced exclusively by animals, however, the principle that plants can also produce estrogen-like molecules was established by 1966 (5). Now, it is recognized that certain plants and plant products contain these phytoestrogens. One group of such compounds reported to have estrogenic activity is the flavonoids (6). This group of compounds includes isoflavones, which are found in a limited number of plants

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and plant products. Soybeans, or more specifically isolated soy proteins, are considered a rich source of the isoflavones genistin and daidzin, which are converted to genistein and daidzein by the gut microflora. In recent years, there has been rising interest regarding the soy isoflavones and their influence not only on sex hormone metabolism, but also other biological activities including cholesterol-lowering properties (7,8), anticarcinogenic effects (9), and more recently, their protective role in bone health (10–13). The protective role of soy protein, its isoflavones, or their combination on bone in ovarian hormone–deficient models of osteoporosis is unclear (Table 20.1) (11–28). In these studies, the bone mineral density (BMD), biomechanical properties, the rate of bone formation and bone resorption, as assessed by biochemical markers and/or histomorphometry were unchanged, decreased, or increased. From the studies that show positive effects of soy on bone it is unclear whether the bone protective effect of soy protein is due to its amino acid composition(17), nonprotein constituents such as isoflavones (12,15), or a combination of these factors (18). It is imperative that researchers in this field be sensitive to the fact that definitive conclusions can be drawn only when there are similarities in the animal models (i.e., age, strain, and gender), experimental designs, treatment regimens, and outcome variables being used to assess the effectiveness of any intervention. Evidence for the Effectiveness of Soy Protein and Its Isoflavones on Bone in Animal Models Female Rat Models. The primary risk factor for osteoporosis in woman is ovarian hormone deficiency, which occurs at the onset of natural or surgical menopause. Several animal models, including rats, dogs, pigs, and monkeys are used to study the etiology, prophylaxis, and treatment for osteoporosis (29). However, because nonprimates do not naturally undergo menopause, the ovariectomized rat model has been used widely to mimic ovarian hormone deficiency-related bone loss (29). The ovariectomized rat model appears to be responsive to dietary treatments including phytoestrogens as well as providing practical advantages, which include a relatively short lifespan, genetic definition, and feasibility. Although the process of bone remodeling differs significantly between rats and humans, studies by Kalu et al. (30) and others (29,31) indicate that the mature ovariectomized rat is a useful model for investigating various aspects of ovarian hormone deficiency-related bone loss. Hence, the use of the ovariectomized rat model to evaluate the effectiveness of soy or its isoflavones in preventing or reversing bone loss should be considered appropriate. Among the early studies using ovariectomized Sprague-Dawley (SD) rats was a study by our laboratory indicating that soy protein had positive effects on bone. The findings of that study (11) showed that the replacement of casein by soy protein in the diet prevented bone loss due to ovariectomy with vertebral bone density similar to that of the estrogen-treated group. Nonspecific markers of bone forma-

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TABLE 20.1 Representative Animal Studies Examining the Effects of Soy Protein and Soy Isoflavones (Iso) on Bone Mineral Density (BMD) and Biomarkers of Bone Formation and Bone Resorptiona Reference

Treatment(s)

(14)

Ovx-lactating SD rats received genistein-rich Iso at three doses: 0.5, 1.6, or 5.0 mg/d 3-mo-old SD ovx rats were fed either casein- or soy protein–based diet d 3-mo-old SD ovx rats were fed either casein or soy protein with or without Iso 4-mo-old SD osteopenic ovx rats were fed either casein or soy protein with or without Iso 6-mo-old SD ovx rats received several doses of isoflavones with either soy protein or casein 12-mo-old SD ovx osteopenic rats received various doses of isoflavones after induction of fracture 2- to 3-mo-old SD glucocorticoid–induced osteopenic

(11)

(12)

(15)

22,23)

(24)

(25)

Treatment duration (d)

BMD

Bone markers

14

Genistein dose at 0.5 mg ↑ femoral ash weight

Not assessed

35

↑ Femoral and 4th lumbar bone density in rats consuming soy-based diet ↑ Femoral BMD in group consuming soy with isoflavones

No change

65

Slightly ↑ femoral but not 4th lumbar BMD

56

Isoflavones with or without soy protein did not prevent ovx-induced lose of BMD No change in BMD but BMC was increased

Soy–Iso had higher urinary excretion of hydroxyproline than all the other Ovx groups Not assessed

35

100

28

Soy protein with L-arginine prevented the glucocorticoid-

No change

None of the isoflavone doses altered the ovx-induced rise in biomarkers of bone turnover Soy protein with L-arginine reduced serum pyridinoline Cont.

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TABLE 20.1 (Continued) Reference

Treatment(s)

(25)

male rats were fed soy protein with or without L-arginine 2-mo-old SD ovx rats were injected daily with genistein at 1, 5, or 25 µg/kg body 3-mo-old SD orchidectomized rats were fed soy with or without isoflavones

(26)

(27)

Treatment duration (d)

21

65

BMD

Bone markers

induced lose of whole-body, spine, and pelvis BMC Genistein treated groups at 5 and 25 µg/g body had tibial BMD similar to sham Orchidectomy-induced bone density loss was not prevented by soy protein, irrespective of its isoflavone content BMD was not assessed

without altering serum ALP

(28)

Adult ovx cynomolgus monkeys were fed soy protein ± estrogen

210

(19)

7-mo-old Wistar ovx rats were fed casein-based diets with 4 levels of Iso: 0, 20, 40, or 80 mg per kg body 7-mo-old Wistar osteopenic ovx rats were fed casein-based diets with 4 levels of Iso: 0, 20, 40, or 80 mg per kg body 12-mo-old Wistar ovx rats were fed a casein-based diet with either genistein or daidzein at 10 µg/g body weight

91

All doses of Iso ↑ total femoral BMD

84

No change in BMD

90

Only daidzein prevented the ↓ in L2-L5 and femur BMD

(20)

(16)

Genistein at 5 µg/g body ↑ serum OC; no change in resorption markers No change

Not assessed; but histomorphometry revealed that soy protein alone increased rate of bone turnover All Iso levels maintained ovxinduced higher serum OC; Iso 40 and 80 ↓ Dpd Isoflavones dose-dependently ↓ serum OC and urinary Dpd Daidzein ↓ OC and urinary Dpd

aAbbreviations: ovx, ovariectomized; SD, Sprague-Dawley; BMC, bone mineral content; OC, osteocalcin; Dpd, deoxypyridinoline; ALP, alkaline phosphatase activity; Soy-Iso, isoflavone-deplete soy protein.

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tion, i.e., serum alkaline phosphatase (ALP) activity and bone resorption (i.e., serum tartrate-resistant acid phosphatase activity) were greater in ovariectomized rats consuming soy protein than in sham-operated control rats. Results from that study and similar studies (12,26) indicate that the beneficial effects of soy protein result from stimulation of bone formation rather than suppression of bone resorption. To determine whether soy protein itself or its isoflavones exert beneficial effects on bone, we conducted a study using 95-d-old SD rats (12). Similar to our earlier findings (11), ovariectomy resulted in greater bone turnover, as indicated by higher serum ALP activity, serum insulin-like growth factor-I, insulin-like growth factor binding protein-3 concentrations, and urinary hydroxyproline. These increases were not affected by soy with either normal or reduced isoflavone content. Histomorphometry revealed a greater bone formation rate with ovariectomy, which was not ameliorated by the soy diets. Although it appears that the bone protective effects of soy protein are related to its nonprotein constituents, isoflavones, the effects of other components such as saponins and phytic acid, which are also present in soy protein, cannot be ruled out. Omi et al. (17) reported that female osteopenic rats fed soy milk had greater BMD and mechanical bone strength than did casein-fed controls. The authors speculated that this beneficial effect might have been due to enhanced intestinal calcium absorption. Our recent findings support this notion as evidenced by the fact that the synthetic isoflavone, ipriflavone (32), and soy isoflavones (33) can enhance intestinal calcium absorption. Therefore, the positive effect of soy protein or its isoflavones may be due in part to improved calcium absorption and calcium homeostasis (22). To further investigate whether isoflavones, irrespective of soy protein, can exert a positive effect on bone, we carried out a study (34) in which two doses (125 and 250 mg isoflavones/kg diet) were added to casein-based diets of ovariectomized rats. These doses of mixed isoflavones delivered comparable amounts of genistein and diadzein, which have been reported to produce positive effects on bone (14,21,35). As expected, ovariectomy reduced the 4th lumbar BMD (P < 0.006) compared with sham animals. This ovariectomy-associated bone loss was not attenuated by either dose of isoflavones. Other indices of bone turnover were not affected by these doses of isoflavones beyond that of ovariectomy. These results indicate that isoflavones may play an important role in protecting bone only in the context of soy protein, at least in the rat model of postmenopausal osteoporosis. Recent findings of Picherit and colleagues (19) support the bone protective role of isoflavones independent of soy protein. The authors (19) reported that isoflavones dose-dependently prevented ovariectomy-induced bone loss in a rat model. However, the same group of investigators did not find similar beneficial effects of isoflavones in reversing bone loss in ovariectomized osteopenic rats (20), although isoflavones were able to reduce the rate of bone turnover. Hence, one can speculate that a longer treatment period with isoflavones would have reversed bone

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loss. Whether the magnitude of effects of isoflavones on bone in these animal studies by Picherit et al. (19,20) would have been greater had isoflavones been given in conjunction with soy protein remains to be determined. On the other hand, studies investigating the effects of individual soy isoflavones, genistin and daidzin, support the important role of these naturally occurring compounds in bone health regardless of the dietary protein source. For instance, 2 wk of genistin treatment (1.0 mg/d) in lactating ovariectomized rats was effective in maintaining trabecular bone tissue compared with ovariectomized control animals (14). Furthermore, in the same report, genistin stimulated ALP activity of an osteoblast-like cell line, suggesting a positive effect on bone formation. In another study, Fanti et al. (26) reported that genistein maintained both cortical and trabecular bones in ovariectomized rats, and the bone-sparing effect of genistein appeared to be biphasic. Although it is believed that genistin is the most potent of the soy isoflavones, a recent study by Desir et al. (16) reported that daidzin is more efficient than genistin in preventing the ovariectomy-induced increase in bone turnover and decrease in BMD. Clearly, this demonstrates that there are uncertainties concerning which isoflavones play a more important role in skeletal health. Use of a single isoflavone may not necessarily be the approach to be taken, and future studies should address whether the combination of isoflavones exerts a more pronounced effect on bone. As Messina et al. (36) indicated, using a single chemical, such as an individual isoflavone or a combination of a few, may hinder, rather than facilitate our understanding of their role in bone health. As with evaluation of many therapeutic agents, not all studies are in agreement concerning the osteoprotective effects of isoflavones. The findings of recent studies by Cai et al. (22,23) using 6-mo-old virgin SD rats, indicated that isoflavones given in conjunction with either soy protein or casein did not prevent trabecular or cortical bone loss. However, the investigators (22,23) noted that soy protein, irrespective of its isoflavone content, reduced urinary loss of calcium, reconfirming the beneficial effects of low sulfur-containing protein such as soy on calcium homeostasis. Furthermore, the findings of these investigators (22,23) may have varied from those of others (11,12) in part because of the use of older rats compared with young adults. It should be noted that these disagreements should not discourage the scientific community at large from pursuing the role of phytochemicals and functional foods in skeletal health. Whether estrogen can reduce the incidence of fracture, the main indicator of healthy bones, remains controversial after decades of research. Because the ultimate measure of success of a therapeutic or dietary intervention should be based on reduced incidence of fracture, it should be noted that ovariectomized rats can also be used as a feasible and appropriate model of fracture healing. As previously described, ovariectomized rats experience an osteopenia associated with increased rates of bone turnover, similar to the osteoporotic state of postmenopausal women (37). In spite of the higher bone formation rate, bone repair in ovariectomized rats has been found to be impaired (38–40). Strates and Nimmi (40) used segmental defects in rat fibula grafted with demineralized

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bone matrix cylinders to study bone repair in ovariectomized and sham-operated control rats. They found that the mineral contents of bone repair sites in ovariectomized rats were significantly lower than in controls. Walsh et al. (38), using a fracture model, found deficiency in mechanical properties of the healing bone in ovariectomized rats compared with sham-operated controls. These findings suggest that the hormone deficiency resulting from ovariectomy impairs the ability of bone to heal itself independently of the ovariectomy-induced disruption in bone remodeling. In fact, the actions are in opposite directions, i.e., increasing new bone formation in bone remodeling, but decreasing it in bone repair. Aside from the work of our laboratories, we are not aware of other reports in which the effects of phytochemicals have been studied in the fracture healing process. In a recent study (24), we investigated the effect of dietary vitamin E and soy isoflavones on the outcome of bone repair in ovariectomized rats. Female SD rats (12 mo old) were ovariectomized and fed standard diet for a period of 120 d to mimic ovarian hormone deficiency–associated osteoporosis. Thereafter, the bone repair model was created in both fibulae of each rat by osteotomy. Immediately after fracture induction, treatment began, with one group of ovariectomized rats receiving 525 mg vitamin E + 1000 mg isoflavones/kg diet. Rats were killed 100 d after osteotomy to evaluate the outcome of bone repair by bend testing. We found that the combination of isoflavones and vitamin E can improve the material properties of fractured bone. Although it is too early to claim that soy or its isoflavones enhance the fracture healing processes, the osteopenic rat model can be considered a valuable animal model for investigating the effects of treatments under these conditions. Male Rat Models. In the United States, men suffer from one third of all hip fractures and one half of all symptomatic vertebral fractures (41). Although the etiology of male osteoporosis shares some commonalities with female osteoporosis (42), it is not clear whether available treatment options that are directed mainly toward postmenopausal osteoporosis are also effective in preventing or treating osteoporosis in men (43). Similarly, the effects of phytochemicals on male osteoporosis may differ substantially from the effect in women. Although the pathogenesis of male osteoporosis may not be due entirely to androgen deficiency, the orchidectomized rat model has been characterized as an appropriate and practical model for studying age-related bone loss in men (44,45). Among the earliest observations indicating the beneficial effects of soy protein on bone were those made by Kalu and colleagues (46) (Fig. 20.1). These investigators reported that feeding soy protein instead of casein to old F344 male rats prevented age-associated bone loss. This positive effect of soy protein on bone was credited to its amino acid pattern. The implications of the findings of Kalu et al. (46) are at least two-fold: (i) soy can potentially prevent bone loss in men, and (ii) soy can prevent age-related bone loss. However, since that time, there have been a limited number of studies conducted in which the effects of soy or other phytochemical-rich plant compounds have been examined on male bone.

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Femur density (g/cm3)

Age (mo) Fig. 20.1. The effects of aging and dietary manipulation on femur density (g/cm3).

Each time point represents means ± SEM. The data indicate that soy protein has a positive effect on femoral bone density compared with casein. Reprinted with permission from the Endocrine Society (46).

A study by our laboratory (27) investigated the role of soy protein with or without its isoflavones on bone in male SD rats. Rats, aged 90 d, were either shamoperated or orchidectomized and fed casein- or soy-based protein diets for 65 d. Although femoral BMD was unaffected by soy diets, the yield force was significantly higher in rats that received soy treatments. The findings of this study indicated that soy protein may ameliorate male bone quality without altering BMD. In a more recent study (47), we examined the dose-dependent effects of soy isoflavones alone or in conjunction with soy protein on BMD and bone mineral content (BMC) in gonadal hormone–deficient aged male rats. For this purpose, 13mo-old male F344 rats were orchidectomized and immediately placed on treatments for 180 d. Treatments consisted of semipurified diets in which the sole protein source was either isoflavone-depleted soy protein, soy protein with normal isoflavone content, soy protein with added isoflavones, casein (control), or casein with added isoflavones. Orchidectomy significantly (P < 0.05) reduced wholebody BMD as well as BMD and BMC of the tibia and the 4th lumbar vertebra. Orchidectomy also increased (P < 0.05) urinary deoxypyridinoline (Dpd), a specific marker of bone resorption. However, the change in whole-body BMD over the course of the study was significantly improved in the group that consumed soy protein with isoflavones. Soy with isoflavones also prevented the orchidectomyinduced increase in urinary Dpd excretion. BMD of the tibia tended (P < 0.1) to be higher in orchidectomized rats fed soy compared with those fed the casein-based

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diet with the equivalent isoflavone level. Although BMD of the 4th lumbar vertebra was not affected by dietary treatment, BMC was significantly (P <0.05) higher in all orchidectomized rats fed the soy-based diets. Serum ALP activity tended to be higher in soy-fed rats compared with rats fed the casein-based diet with equivalent isoflavone levels. From the findings of this study (47), it can be concluded that soy isoflavones may be beneficial to bone in a male rat model of osteoporosis and that the combination of soy isoflavones with soy protein may offer greater benefits to bone than isoflavones alone. Soy protein or its isoflavones may also play a role in other types of male osteoporosis. For example, a study by Clementi and colleagues (25) confirms this notion. These investigators (25) reported that soy can prevent cyclosporineA–induced vertebral bone loss in male rats. The above findings suggest that soy protein or its isoflavones within the same species may behave differently depending on the strain and age of the rats, and the bone of interest. Wang et al. (48) recommended that SD rats ≥9 mo old be used as an ideal small animal model for studying age-related bone loss. These investigators have also suggested the use of 4th lumbar vertebra and femoral neck as the clinically relevant bone sites for determining the cause of bone loss and the efficacy of treatment. Other Animal Models. There are a limited number of studies that have used animal models other than rats to investigate the effects of phytochemicals on bone. These animal models offer additional alternatives depending on the forms of osteoporosis that are being studied. However, it should be emphasized that no animal models can precisely reproduce all aspects of the human conditions. Nonetheless, they are useful for studying certain aspects of osteoporosis, its etiology, and treatments. In studying bone biology, mouse models have become popular because of the relative ease in which their genome can be manipulated. However, with respect to phytochemicals, e.g., isoflavones, studies are warranted to establish the usefulness of the transgenic models. Although the bones of nonhuman primates, canines, pigs, and sheep are similar to humans in terms of the basic structural units and remodeling patterns (29), large animals may not be considered ideal models in terms of feasibility and cost. On the basis of the overall physiology, the nonhuman primate models are the closest to human. The old world monkeys, in particular, are unique among large animal models that are used for bone-related investigations. For a more detailed discussion of nonhuman primates as models for studying human osteoporosis, the readers are referred to two recent articles by Black et al. (49) and Jerome and Peterson (50). Studies in which the efficacy of soy protein, its isoflavones, or other phytochemicals have been tested using monkeys are very limited. Similar to the findings of Cai et al. in rats (22,23), soy protein isolate has not been shown to reduce the ovariectomy-induced rate of bone turnover in cynomolgus macaques (28). In fact, histomorphometry data indicated that ovariectomized monkeys fed a soy protein diet for 7 mo actually experienced an increase in bone turnover on the endocortical

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surface compared with the casein/lactalbumin-fed controls. These data raise an important issue of whether there are species differences with respect to the metabolism of soy protein and its isoflavones. For example, many feline species lack the enzyme necessary to conjugate isoflavones, resulting in infertility (51), whereas rodents are exposed to very high doses of isoflavones from the soy meal added to commercial feed, and yet these extreme levels have not resulted in noticeable breeding problems (3). Although the limited studies using monkeys as a model show no apparent positive effects of soy protein or its isoflavones on bone, the rodent model may overestimate the bone-protective effects of phytochemicals. Ultimately, long-term human clinical trials are required to determine whether isoflavones are effective in defined segments of the population, e.g., postmenopausal women and aging men and women. Evidence for Bone Protective Effects of Other Food Sources with Bioactive Components Up to this point, we have focused on the effects of soy protein or its isoflavones on bone. The remainder of this chapter will be devoted to the discussion of other food sources rich in phenolic compounds that may also effect bone metabolism. Flaxseed. Flaxseed is by far the richest source of lignans among edible plant foods that are reported to have both weak estrogenic and antiestrogenic activities (52). Lignans are structurally similar to tamoxifen, which has beneficial effects on bone (53). The polyunsaturated fatty acid content of flaxseed (54), especially α-linolenic acid (18:3n-3) may decrease the rate of bone resorption by inhibiting the biosynthesis of prostaglandins (55). Lignans present in flaxseed may also possess antioxidant properties. Oxygen-derived free radicals, which are formed by a number of phagocytes including monocytes, macrophages, and neutrophils, have been reported to increase in chronic inflammatory diseases, aging, and osteoporosis. In vivo and in vitro findings indicate that free radicals generated in the bone environment enhance osteoclast formation and bone resorption. Hence, flaxseed may reduce the rapid rate of bone loss experienced in ovarian hormone deficiency, in part, by enhancing antioxidant status. There is a paucity of research in this area using animal models. In an early evaluation of flaxseed or its lignans on mature male and female rat bone, it was neither beneficial nor harmful (56,57). However, feeding flaxseed or its lignans to growing female rats appeared to have beneficial effects on bone strength although these benefits did not persist into adulthood (57). Whether longer-term, dose-dependent studies using whole flaxseed or its components, e.g., lignans or flaxseed oil, may show a positive influence on BMD and BMC remains to be explored. Prunes. Another plant food source rich in phenolic compounds and flavonoids with potential osteoprotective properties is prunes (Prunus domestica L.). Prunes are rich in phenolic compounds such as neochlorogenic acid and chlorogenic acid (58) and are ranked as having the highest oxygen radical absorbance capacity

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(ORAC) among the commonly consumed fruits and vegetables (59). It is conceivable that phenolic compounds present in prunes may play an important role in protecting bone by scavenging free radicals and preventing oxidative damage to bone. We have observed that inclusion of prunes in the diet prevents the ovariectomy-induced BMD loss in rats (60). The histomorphometric findings also confirm the efficacy of prunes in preventing bone loss as indicated by significantly higher trabecular bone area and percentage of bone surface. We also evaluated the ability of prunes to reverse bone loss using osteopenic ovarian hormone–deficient rats. Prunes, as low as 5 g/100 g of the diet, were able to restore the femoral bone density to the level of nonosteopenic rats (61). The same dose of prunes also effectively increased 4th lumbar bone density. Whether the positive effects of prunes on bone in ovariectomized rats can also be observed in postmenopausal women should be investigated. The findings of a shortterm clinical trial (62) by our group, assessing the regular consumption of prunes on bone biomarkers, supports the beneficial effects of prunes on bone. In that study, prune supplementation for 3 mo significantly increased bone-specific ALP activity, a marker of bone formation, by nearly 6% while reducing the urinary marker of bone resorption by 11% (albeit not significantly) in postmenopausal women not receiving hormone replacement therapy. These findings indicate that prunes also have the ability to increase bone formation in postmenopausal women, thus decreasing the risk for osteoporotic fractures. Further studies are required to identify the active compounds present in prunes, the lowest effective dose, and the mechanisms of action. Grapes. Resveratrol, which is a bioflavonoid found naturally in the skins of most grape cultivars, has been reported (63) to antagonize the dioxin-induced inhibition of osteogenesis in bone-forming cultures. The influence of resveratrol on bone, an estrogen receptor (ER) positive tissue, may be due in part to its ability to bind and activate ER (64). There are a few studies that link moderate alcohol consumption to better BMD in humans (65,66). Although moderate alcohol consumption may indirectly influence bone metabolism via modulation of circulating estrogen, the findings of epidemiologic studies (65,67) suggest that the beneficial effects of alcoholic beverage may be due to wine consumption, particularly red wine. Red wine has high levels of phenolic compounds, e.g., resveratrol, that may favorably influence bone metabolism (65). Feeding resveratrol to stroke-prone spontaneously hypertensive ovariectomized rats has also been shown to prevent ovariectomyinduced decreases in femoral strength. These beneficial effects may be associated, in part, with the ability of resveratrol to enhance bone formation (68). These limited studies bring forth evidence that grapes or their phytochemicals may play an important role in protecting skeletal health. Safety Although the use of phytochemicals as alternative or complementary medicine has gained popularity, the safety issues for a number of these supplements remain unresolved. For instance, although the epidemiologic observations in Asian women

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consuming high isoflavone-containing foods indicate lower rates of breast (69) and endometrial (70) cancer, these observations are based on whole-food consumption and not soy protein or its isolated nonprotein constituents, e.g., isoflavones. With respect to soy, our animal studies (11,12) and those of other investigators indicate that, unlike estrogens, animals fed soy protein with isoflavones or individual isoflavones have no uterotrophic response. Therefore, the risks associated with estrogens should not necessarily be extended to isoflavones. From a human standpoint, although limited controlled investigations exist, the message concerning the estrogenicity of soy components is unclear. For example, a short-term dietary study of soy intake in premenopausal women with benign and malignant breast conditions was shown to stimulate breast tissue proliferation (72). In comparison, a 4-wk study by Baird et al. (73) of whole soy or texturized soy protein consumption by postmenopausal women did not induce any clear estrogenic response. Hence, the safety aspects of isolated compounds from soy require more scrutiny by welldesigned animal studies followed by long-term, controlled clinical trials. These types of studies should address questions such as whether long-term intake of these compounds from soy in women with benign, malignant, or no evidence of breast conditions has stimulatory or inhibitory effects on breast tissue proliferation.

Summary and Conclusions From the review of the literature to date, certain phytochemicals including soy isoflavones and other foods rich in phenolic compounds may have modest effects on bone. These findings are weakened by several key issues. The human data are limited by the fact that there are few long-term studies that have addressed both the bone protective efficacy and the safety issues. Animal data have shown varied results due to discrepancies in the dose of the phytochemicals, the duration of the treatment period, and the age, species, and strain of the animals being studied. Future directives on the role of phytochemicals in bone health are to address numerous questions including whether the bioactive compounds of interest, independent of their whole food, are able to prevent bone loss or restore bone mass. Another question is whether the consumption of these phytochemicals on a regular basis is necessary to observe the beneficial effects on bone. Researchers in the field are just beginning to address the health benefits and safety concerns of natural bioactive compounds. Future studies using appropriate models of osteoporosis are necessary to address the numerous questions regarding the potential skeletal benefits, the mechanisms of action, and the safety of these natural foods or their bioactive components. References 1. Cosman, F., and Lindsay, R. (1999) Selective Estrogen Receptor Modulators: Clinical Spectrum, Endocr. Rev. 20, 418–434. 2. Brezinski, A., and Debi, A. (1999) Phytoestrogens: The “Natural” Selective Estrogen Receptor Modulators, Eur. J. Obstet. Gynecol. Reprod. Biol. 85, 47–51.

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3. Setchell, K.D.R. (2001) Soy Isoflavones—Benefits and Risks from Nature’s Selective Estrogen Receptor Modulator (SERMs), J. Am. Coll. Nutr. 20, 354S–362S. 4. Jordan, V.C., Gaptsur, S., and Morrow, M. (2001) Selective Estrogen Receptor Modulation and Reduction in Risk of Breast Cancer, Osteoporosis, and Coronary Heart Disease, J. Natl. Cancer Inst. 93, 1449–1457. 5. Riddle, J.M., and Estes, J.W. (1992) Oral Contraceptives in Ancient and Medieval Times, Am. Sci. 80, 226–233. 6. Miksicek, R.J. (1993) Commonly Occurring Plant Flavonoids Have Estrogenic Activity, Mol. Pharmacol. 44, 37–43. 7. Merz-Demlov, B.E., Duncan, A.M., Wangen, K.E. Xu, X., Carr, T.P., Phipps, W.R., and Kurzer, M.S. (2000) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic, Premenopausal Women, Am. J. Clin. Nutr. 71, 1462–1469. 8. Baum, J.A., Teng, H., Erdman, J.W., Weigel, R.M., Kleinm, B.P., Persky, V.W., Freels, S., Surya, P., Bakhit, R.M., Ramos, E., Shay, N.F., and Potter, S.M. (1998) Long Term Intake of Soy Protein Improves Blood Lipid Profiles and Increases Mononuclear Cell Low Density Lipoprotein Messenger RNA in Hypercholesterolemic, Postmenopausal Women, Am. J. Clin. Nutr. 68, 545–551. 9. Messina, M.J. (1999) Legumes and Soybeans: Overview of Their Nutritional Profiles and Health Effects, Am. J. Clin. Nutr. 70, 439S–450S. 10. Delmas, P.D., Bjarnason, N.H., Mitlak, B.H., Ravoux, A.C., Shah, A.S., Huster, W.J., Draper, M., and Christiansen, C. (1997) Effects of Raloxifene on Bone Mineral Density, Serum Cholesterol Concentrations, and Uterine Endometrium in Postmenopausal Women, N. Engl. J. Med. 337, 1647–? 11. Arjmandi, B.H., Alekel, L., Hollis, B.W., Amin, D., Stacewica-Sapuntzakis, M., and Gou, P. (1996) Dietary Soy Protein Prevents Bone Loss in an Ovariectomized Rat Model of Osteoporosis, J. Nutr. 126, 161–167. 12. Arjmandi, B.H., Birnbaum, R., Goyal, N.V., Getlinger, M.J., Juma, S., Alekel, L., Hasler, C.M., Hollis, B.W., Drum, M.L., and Kukreja, S.C. (1998) Bone-Sparing Effect of Soy Protein in Ovarian Hormone Deficient Rats Is Related to Its Isoflavone Content, Am. J. Clin. Nutr. 68, 1364S–1368S. 13. Alekel, D.L., St Germain, A., Peterson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in the Lumbar Spine of Perimenopausal Women, Am. J. Clin. Nutr. 72, 679–680. 14. Anderson, J.J., Ambrose, W.W., and Garner, S.C. (1998) Biphasic Effects of Genistein on Bone Tissue in the Ovariectomized, Lactating Rat Model, Proc. Soc. Exp. Biol. Med. 217, 345–350. 15. Arjmandi, B.H., Getlinger, M.J., Goyal, N.V., Alekel, L., Hasler, C.M., Juma, S., Drum, M.L., and Kukreja, S. (1998) Role of Soy Protein with Normal or Reduced Isoflavone Content in Reversing Bone Loss Induced by Ovarian Hormone Deficiency in Rats, Am. J. Clin. Nutr. 68, 1358S–1363S. 16. Desir, C., Picherit, C., Coxam, V., Katicoulibali, S., Davicco, M., Lebecque, P., and Barlet, J. (1998) Daidzein Is More Efficient than Genistein to Prevent Osteopenia in Ovariectomized Rats, Bone 23, S573 (abstr.) 17. Omi, N., Aoi, S., Murata, K., and Ezawa, I. (1994) Evaluation of the Effect of Soybean Milk and Soybean Milk Peptide on Bone Metabolism in the Rat Model with Ovariectomized Osteoporosis, J. Nutr. Sci. Vitaminol. 40, 201–211. 18. Wangen, K.E., Duncan, A.M., Merz-Demlov, B.E., Xu, X., Marcus, R., Phipps, W.R., and Kurzer, M.S. (2000) Effects of Soy Isoflavones on Markers of Bone Turnover in

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Premenopausal and Postmenopausal Women, J. Clin. Endocrinol. Metab. 85, 3043– 3048. Picherit, C., Chanteranne, B., Bennetau-Pelissero, C., Davicco, M.J., Lebecque, P., Barlet, J.P., and Coxam, V. (2001) Dose-Dependent Bone-Sparing Effects of Dietary Isoflavones in the Ovariectomized Rat, Br. J. Nutr. 85, 307–316. Picherit, C., Bennetau-Pelissero, C., Chanteranne, B., Labecque, P., Davicco, M.J., Barlet, J.P., and Coxam, V. (2001) Soybean Isoflavones Dose-Dependently Reduce Bone Turnover but Do Not Reverse Established Osteopenia in Adult Ovariectomized Rats, J. Nutr. 131, 723–728. Picherit, C., Coxam, V., Bennetau-Pelissero, C., Kati-Coulibaly, S., Davicco, M.J., Leecque, P., and Barlet, J.P. (2000) Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats, J. Nutr. 130, 1675–1681. Cai, D.J., Glasier, J., Turner, C., and Weaver, C.M. (2001) Comparative Effects of Soy Isoflavones, Soy Protein and 17β-Estradiol on Calcium and Bone Metabolism in Adult Ovariectomized Rats—I. Analysis of Calcium Balance, Bone Densitometry and Mechanical Strength, J. Bone Miner. Res. 16, S531–?. Cai, D.J., Cullen, D.M., Peyton, A.C., and Weaver, C.M. (2001) Comparative Effects of Soy Isoflavones, Soy Protein and 17β-Estradiol on Trabecular and Cortical Bone in Adult Ovariectomized Rats—II. A Histomorphometric Analysis, J. Bone Miner. Res. 16, S536. Chakkalakal, D., Novak, J.R., Fritz, E.D., Akhter, M., Lucas, E.A., Khalil, D.A., Juma, S., El-Osta, M., Stoecker, B.J., and Arjmandi, B.H. (2001) Vitamin E Improves Bone Repair in Ovariectomized Rats in a Dose-Dependent Manner, J. Bone Miner. Res. 16, S533. Clementi, G., Fiore, C.E., Mangano, N.G., Cutuli, V.M., Pennisi, P., Caruso, A., Prato, A., Matera, M., and Amico-Roxas, M. (2001) Role of Soy Diet and L-Arginine in Cyclosporin-A-Induced Osteopenia in Rats, Pharmacol. Taxicol. 88, 16–19. Fanti, O., Faugere, Z., Schmidt, G.J., Fanti, O., Faugere, M.C., Gang, Z., Schmidt, J., Cohen, D., and Malluche, H.H. (1998) Systemic Administration of Genistein Partially Prevents Bone Loss in Ovariectomized Rats in a Non-Estrogen-Like Mechanism, Am. J. Clin. Nutr. 68, 1517S (abstr.). Juma, S., and Arjmandi, B.H. (1997) Effects of Soy Protein on Bone in Gonadal Hormone Deficiency, FASEB J. 11, A572 (abstr.). Lees, C.J., and Ginn, T.A. (1998) Soy Protein Isolate Diet Does Not Prevent Increased Cortical Bone Turnover in Ovariectomized Macaques, Calcif. Tissue Int. 62, 557–558. Rodgers, J.B., Monier-Faugere, M.C., and Malluche, H. (1993) Animal Models for the Study of Bone Loss After Cessation of Ovarian Function, Bone 14, 369–377. Kalu, D.N., Liu, C.C., Hardin, R.R., and Hollis, B.W. (1989) The Aged Rat Model of Ovarian Hormone Deficiency Bone Loss, Endocrinology 124, 7–16. Wronski, T.J., and Yen, C.-F. (1991) The Ovariectomized Rat as an Animal Model for Postmenopausal Bone Loss, Cells and Materials 1 (Suppl.), 69–74. Arjmandi, B.H., Khalil, D.A., and Hollis, B.W. (2000) Ipriflavone, a Synthetic Phytoestrogen, Enhances Intestinal Calcium Transport In Vitro, Calcif. Tiss. Int. 67, 225–229. Arjmandi, B.H., Khalil, D.A., and Hollis, B.W. (2002) Soy Protein Enhances Intestinal Calcium Transport In Vitro, Calcif. Tissue Int. (in press). Arjmandi, B.H. (2001) The Role of Phytoestrogens in the Prevention and Treatment of Osteoporosis in Ovarian Hormone Deficiency, J. Am. Coll. Nutr. 20, 398S–402S.

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35. Fanti, O., Monier-Faugere, M.C., Geng, Z., Schmidt, J., Morris, P.E., Cohen, D., and Malluche, H.H. (1998) The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats, Osteoporos. Int. 8, 274–281. 36. Messina, M., Lampe, J.W., Birt, D.F., Appel, L.J., Pivonka, E., Berry, B., and Jacobs, D.R. (2001) Reductionism and the Narrowing Nutrition Perspective: Time for Reevaluation and Emphasis on Food Synergy, J. Am. Diet. Assoc. 101, 1416–1419. 37. Kalu, D.N. (1991) The Ovariectomized Rat Model of Postmenopausal Bone Loss [Review], Bone Miner. 15, 175–192. 38. Walsh, W.R., Sherman, P., Howlett, C.R., Sonnabend, D.H., and Ehrich, M.G. (1997) Fracture Healing in a Rat Osteopenia Model, Clin. Orthop. 342, 218–227. 39. Chakkalakal, D.A., Strates, B.S., Mashoof, A.A., Garvin, K.L., Novak, J.R., Fritz, E.D., Mollner, T.J., and McGuire, M.H. (1999) Repair of Segmental Bone Defects in the Rat: An Experimental Model of Human Fracture Healing, Bone 25, 321–332. 40. Strates, B., and Nimni, M. (1994) Skeletal Repair as a Function of Aging and PostOvariectomy. Poster Presentation, American Association of Animal Science, San Francisco. 41. National Institutes of Health (1998) Osteoporosis in Men. National Institutes of Health, Osteoporosis and Related Bone Diseases, National Resource Center, Washington, DC. 42. Bilezikian, J.P., Kurland, E.S., and Rosen, C.J. (1999) in Osteoporosis in Men (Orwoll, E.S., ed.) pp. 395–41, Academic Press, New York. 43. National Osteoporosis Foundation (1999) Men and Osteoporosis. National Osteoporosis Foundation, Washington, DC. 44. Erben, R.G., Eberle, J., Stahr, K., and Goldberg, M. (2000) Androgen Deficiency Induces High Turnover Osteopenia in Aged Male Rats: A Sequential Histomorphometric Study, J. Bone Miner. Res. 15, 1085–1098. 45. Vanderschueren, D., Vandenput, L., Boonen, S., Van Herck, E., Swinnen, J.V., and Bouillon, R. (2000) An Aged Rat Model of Partial Androgen Deficiency: Prevention of Both Loss of Bone and Lean Body Mass by Low-Dose Androgen Replacement, Endocrinology 141, 1642–1647. 46. Kalu, D.N., Masoro, E.J., Yu, B.P., Hardin, R.R., and Hollis, B.W. (1988) Modulation of Age-Related Hyperparathyroidism and Senile Bone Loss in Fischer Rats by Soy Protein and Food Restriction, Endocrinology 122, 1847–1854. 47. Khalil, D.A., Juma, S., Lucas, E.A., Galloway, D.S., Hammond, L.J., Soung, D., and Arjmandi, B.H. (2002) Dose-Dependent Effects of Soy Isoflavones in Conjunction with Soy Protein or Casein as a Protein Source on Bone in an Aged Rat Model of Male Osteoporosis, to be presented at the 16th Annual Meeting of the Experimental Biology, New Orleans, LA. 48. Wang, L., Banu, J., McMahan, C.A., and Kalu, D.N. (2001) Male Rodent Model of Age-Related Bone Loss in Men, Bone 29, 141–148. 49. Black, A., Tilmont, E.M., Handy, A.M., Scott, W.W., Shapses, S.A., Ingram, D.K., Roth, G.S., and Lane, M.A. (2001) A Nonhuman Primate Model of Age-Related Bone Mass: a Longitudinal Study in Male and Premenopausal Female Rhesus Monkeys, Bone 28, 295–302. 50. Jerome, C.P., and Peterson, P.E. (2001) Nonhuman Primate Models in Skeletal Research, Bone 29, 1–6. 51. Setchell, K.D.R., Gosselin, S.J., Welsh, M.B., Johnston, J.O., Balistreri, W.F., Kramer, L.W., Dresser, B.L., and Tarr, M.J. (1987) Dietary Estrogens—A Probable Cause of Infertility in Liver Disease in Captive Cheetahs, Gastroenterology 93, 225–233.

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52. Ayers, D.C., and Loike, J.U. (1990) Lignans: Chemical, Biological, and Clinical Properties, Cambridge University Press, Cambridge. 53. Turner, R.T., Wakley, G.K., Hannon, K.S., and Bell, N.H. (1988) Tamoxifen Inhibits Osteoclast Mediated Resorption of Trabecular Bone in Ovarian Hormone-Deficient Rats, Endocrinology 122, 1146–1150. 54. Jeffrey, N.M., Sanderson, P., Sherrington, E.J., Newsholme, E.A., and Calder, P.C. (1996) The Ratio of n-6 to n-3 Polyunsaturated Fatty Acids in the Rat Diet Alters Serum Lipid Levels and Lymphocyte Functions, Lipids 31, 737–745. 55. Tashjian, A.H., Voelkel, E.F., Levine, L., and Goldhaber, P. (1972) Evidence That the Bone Resorption-Stimulating Factor Produced by Mouse Fibrosarcoma Cells Is Prostaglandin E2: A New Model for the Hypercalcemia of Cancer, J. Exp. Med. 136, 1329–1343. 56. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Flaxseed and Its Purified Lignan Reduces Bone Strength in Young but Not Older Male Rats, J. Toxicol. Environ. Health A.63, 53–65. 57. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Purified Lignan from Flaxseed (Linum usitatissimum) Alters Bone Development in Female Rats, Br. J. Nutr. 86, 499–505. 58. Donovan, J.L., Meyer, A.S., and Waterhouse, A.L. (1998) Phenolic Composition and Antioxidant Activity of Prunes and Prune Juice (Prunus domestica), J. Agric, Food Chem. 46, 1247–1252. 59. Lucas, E.A., Juma, S., Stoecker, B.J., and Arjmandi, B.H. (2000) Prune Suppresses Ovariectomy-Induced Hypercholesterolemia in Rats, J. Nutr. Biochem. 11, 255–259. 60. Arjmandi, B.H., Wang, C., Zhang, Y., Lucas, E., Soliman, A., Juma, S., and Stoecker, B.J. (1999) Prune: Its Efficacy in Prevention of Ovarian Hormone Deficiency-Induced Bone Loss, J. Bone Miner. Res. 14, SU334 (abstr.). 61. Deyhim, F., Lucas, E., Brusewitz, G., Stoecker, B.J., and Arjmandi, B.H. (1999) Prune Dose-Dependently Reverses Bone Loss in Ovarian Hormone Deficient Rats, J. Bone Miner. Res. 14, S394 (abstr.). 62. Arjmandi, B.H., Khalil, D.A., Lucas, E.A., Georgis, A., Stoecker, B.J., Hardin, C., Payton, M.E., and Wild, R.A. (2002) Dried Plums Improve Indices of Bone Formation in Postmenopausal Women, J. Women’s Health Gend. Based Med. 11, 61–68. 63. Singh, S.U.N., Casper, R.F., Fritz, P.C., Sukhu, B., Ganss, B., Girard, Jr. B., Savouret, J.F., and Tenenbaum, H.C. (2000) Inhibition of Dioxin Effects on Bone Formation In Vitro by a Newly Described Aryl Hydrocarbon Receptor Antagonist, Resveratrol, J. Endocrinol. 167, 183–195. 64. Gehm, B.D., McAndrews, J.M., Chien, P.Y., and Jameson, J.L. (1997) Resveratrol, a Poly-Phenolic Compound Found in Grapes and Wine Is an Agonist for the Estrogen Receptor, Proc. Natl. Acad. Sci. USA 94, 14138–14143. 65. De Lorimier, A.A. (2000) Alcohol, Wine, and Health, Am. J. Surg. 180, 357–361. 66. Ganry, O., Baudoin, C., and Fardellone, P. (2000) Effects of Alcohol Intake on Bone Mineral Density in Elderly Women. The EPIDOS Study, Am. J. Epidemiol. 151, 773–780. 67. Van Theil, D.H., Galvao-Teles, G., Monterio, E., Rosenblum, E., and Gavaler, J.S. (1991) The Phytoestrogens Present in De-Ethanolized Bourbon Are Biologically Active: A Preliminary Study in Postmenopausal Women, Alcohol Clin. Exp. Res. 15, 822–823.

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68. Mizutani, K., Ikeda, K., Kawai, Y., and Yamori, Y. (1998) Resveratrol Stimulates the Proliferation and Differentiation of Osteoblastic MC3T3-E1 Cells, Biochem. Biophys. Res. Commun. 253, 859–863. 69. Wynder, E.L., Fujita, Y., Harris, R.E., Hirayama, T., and Hiyama, T. (1991) Comparative Epidemiology of Cancer Between the United States and Japan: A Second Look, Cancer 67, 746–763. 70. Messina, M.J., and Loprinzi, C.L. (2001) Soy for Breast Cancer Survivors: A Critical Review of the Literature, J. Nutr. 131, 3095S–2108S. 71. Murrill, W.B., Brown, N.M., Manzolillo, P.A., Barnes, S., and Lamartiniere, C.A. (1996) Prepubertal Genistein Exposure Suppresses Mammary Cancer and Enhances Gland Differentiation in Rats, Carcinogenesis 17, 1451–1457. 72. McMichael-Phillips, D.F., Harding, C., Morton, M., Roberts, S.A., Howell, A., Potten, C.S., and Bundred, N.J. (1998) Effects of Soy Protein Supplementation on Epithelial Proliferation in the Histologically Normal Human Breast, Am. J. Clin. Nutr. 68, 1431S– 1436S. 73. Baird, D.D., Umbach, D.M., Lansdell, L., Hughes, C.L., Setchell, K.D., Weinberg, C.R., Haney, A.F., Wilcox, A.J., and Melachlan, J.A. (1995) Dietary Intervention Study to Assess Estrogenicity of Dietary Soy Among Postmenopausal Women, J. Clin. Endocrinol. Metab. 80, 1685–1690.

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Chapter 21

Phytoestrogens and Cancer: Epidemiologic Evidence Anna H. Wu University of Southern California, Keck School of Medicine, Department of Preventive Medicine, Los Angeles, CA

Overview Until the 1990s, the effects of phytoestrogens (namely, the isoflavonoids, the coumestans, and the lignan precursors) on cancers had received relatively little attention. However, there is now substantial interest in this class of compounds because of the purported anticancer, antiestrogenic, and antioxidant properties of isoflavones and lignans, the two main sources of phytoestrogens (1–5). A diet rich in isoflavones and lignans is compatible with a plant-based rich diet, typical of the traditional Asian diet. Isoflavones are consumed largely from soybeans and its products, whereas lignan precursors (secoisolariciresinol and matairesinol) are found in a large number of foods including flaxseeds, whole-grain products, fruits and berries, vegetables and select beverages. Currently, the potential beneficial or adverse effect of specific phytoestrogens in the etiology of various cancers is unclear (2,6). This chapter examines the epidemiologic evidence regarding soy (and isoflavone) and lignan intake and the risks of hormone-related and other cancers. Because information on isoflavone intake can be provided by assessment of intake of soybeans and its products, most of the studies on this topic are, in fact, based on consumption patterns of soy foods. However, because it is difficult to assess intake of lignans through food group analysis, few epidemiologic studies with such information exist. The terms isoflavones and lignans will be used when their intakes were specifically assessed. In the first section of this chapter, the general process by which soy (and isoflavone) and cancer relationships have been investigated in epidemiologic studies will be discussed. Questionnaires and biomarkers that have been used to measure intake of soy (or isoflavones) and lignans will be covered. These methodologic issues are presented to provide a general framework within which to evaluate the site-specific epidemiologic evidence. In the second section of this chapter, relevant studies on soy, lignans and risk of cancers of the breast, prostate, endometrium, thyroid, lung, colorectum, and stomach will be summarized. The overall results and the quality of the evidence will be discussed.

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Analytic Epidemiologic Studies Observational Studies Two main types of analytic epidemiologic studies have been used to investigate the relationship between soy intake and cancer in human populations. In cohort studies, intake of soy and other exposures of interest are assessed at the time of study enrollment, typically before the diagnosis of cancer. In some cohort studies, information on the various exposures of interest are also updated periodically. In casecontrol studies, soy intake is assessed after the diagnosis of cancer and the exposure histories of case patients are compared with those of control subjects. In most studies, controls are selected from the general population or are hospital/clinic patients who have conditions other than the one under investigation. An inherent limitation of the case-control study design is that information on the exposures of interest is collected after the diagnosis, and recall bias cannot be precluded (7). Selection bias is another concern, especially when hospital controls are used. Of the two types of observational analytic study designs, cohort studies are generally regarded as more reliable (8). Assessment of Intake of Soy (Isoflavones) and Lignans by Questionnaire Publications of the phytoestrogen content of various foods in Asian and non-Asian populations (9–18) and the development of appropriate food-frequency questionnaires (FFQ) have allowed more accurate estimates of intakes of soy (isoflavones) and lignans in different populations. FFQ are the most frequently used and preferred method for assessing usual diets in observational epidemiologic studies (8). Ideally, the FFQ should include all of the relevant foods so that intake of energy, isoflavones, lignans, or other nutrients of interest can be estimated accurately. A central concern is the validity of the FFQ method in assessing the aspect of diet (e.g., isoflavones or lignans) it has been designed to measure. This involves comparison with a second source that is assumed to provide an unbiased estimate. Several FFQ have been used to assess the intake of isoflavones among Asian and non-Asian populations in the United States (19–21, unpublished data) and Asia (22–25) (Table 21.1). Correlation coefficients of 0.4–0.6 were found between urinary excretion of isoflavone and FFQ assessments of isoflavone intake (21,23–25). These studies confirm that intake of isoflavones is highest in Japan and China, intermediate among Asians in Singapore and the United States, and considerably lower among non-Asians in the United States. The median intake of isoflavones in these studies was ~40 mg, 10–20 mg, and 1–2 mg/d, respectively (Table 21.1). The main sources of isoflavones were also identified in these populations. For example, tofu, miso, and natto were the main sources of isoflavones in Japan (25), whereas tofu, other processed soy products, fresh soybeans, soybean sprouts, soy milk, and dry soybeans were important contributors in China (24). For Chinese in Singapore,

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TABLE 21.1 Levels and Sources of Isoflavone Intake in Asian and Non-Asian populations, Based on Food-Frequency Questionnaires (FFQ) Median intake of genistein (GN), daidzein (DZ), and isoflavones (I) (mg/d)

Urinary excretion of isoflavones

Tofu, doughnuts, soy milk, white bread

GN: 0.78; DZ: 0.75; I: 1.65a

Not available

Modified Block FFQ

Not available

Japanese-Americans: GN: 8.8; DZ: 5.7 Caucasians GN: 0.21; DZ: 0.11

JapaneseAmericans (µmol/L) GN: 2.6 DZ: 4.9 Caucasians GN 0.25 DZ: 0.55

Modified FFQ for Multiethnic Cohort Study in Hawaii and Los Angeles (14 soyfoods)

Tofu, soymilk, miso

I: 10.6 mg/1000 Kcal

Not available

Location/ (Ref.)

n Race/Ethnicity

United States San Francisco Bay area/(19,27)

447 women (African-American, Latina, Caucasian)

Modified Block FFQ

Los Angeles area/(21)

51 JapaneseAmerican women; 18 Caucasian women

Los Angeles County/(unpublished Data)

594 women (Chinese, Japanese and Filipinos)

Diet assessment

Main sources of isoflavones

Continued

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TABLE 21.1 (Cont.) Median intake of genistein (GN), daidzein (DZ), and isoflavones (I) (mg/d)

Urinary excretion of isoflavones

Tofu, soybean cake, and yong tau foo

I: 14.1

(in nmol/mg creatinine) GN: 0.73 DZ: 1.37 I: 5.42

86-food item FFQ

Tofu, other processed soy products, fresh soybeans, soybean sprouts, soymilk, dry soybean seed

GN: 17.9 DZ: 18.0 I: 39.3a

(in nmol/mg creatinine) GN: 2.17 DZ: 4.50 I: 10.96a

FFQ for the Japan Public Health Center-based Prospective Study

Tofu, miso, natto

GN: 25.5 DZ: 15.3

(in µmol/d) GN: 9.9 DZ: 12.5

Location/ (Ref.)

n Race/Ethnicity

Asia Singapore/ (22,23)

147 Chinese

FFQ for Singapore Chinese Health Study (6 groups of soyfoods)

Shanghai, China/(24)

60 women

Japan/(25)

215 men and women

Diet assessment

Main sources of isoflavones

aTotal isoflavones included genistein, daidzein and others (e.g., biochanin A, formononetin, glycitein, equol, and O-desmethylangolensin). Intake values based on all control subjects in Reference 27.

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the main sources of isoflavones were tofu, soybean cake, and yong tau foo, a local dish that contains a mixture of soy products (22). Tofu and soy milk were important sources of isoflavones among Asians (unpublished data) and non-Asians in the United States but there were also hidden sources of isoflavones in foods such as doughnuts and white bread (19). Horn-Ross and co-workers (20) identified up to 18 other foods consumed in the West that contain low amounts of isoflavones. Although the overall contribution of hidden sources of isoflavone intake was negligible (i.e., <0.5 mg isoflavones/d) (19), this study was conducted before the 1999 Food and Drug Administration approval of a cardiovascular health claim for foods containing soy protein. If the consumption of soy-fortified foods in the United States increases substantially, this may complicate the assessment of isoflavone intake, particularly in studies conducted in non-Asian populations. In three of the studies reviewed below, intake of lignan precursors (secoisolariciresinol, matairesinol) was estimated by FFQ. In a study conducted in Texas, the median daily intake of lignans was 0.5 mg (26) compared with somewhat lower levels (0.1–0.2 mg) in studies conducted in California (27). However, levels of enterolactone (the main circulating lignan) in urine/serum were not measured in these questionnaire-based studies, making it difficult to compare these intake levels of lignans with published data. Most previous cross-sectional studies on lignans determined the correlation between intake of select lignan-rich foods and urinary or serum levels of enterolactones (28–31). Additional studies are required to determine whether FFQ assessment can provide valid and reproducible measures of lignan intake. Assessment of Intake of Soy and Lignans Using Biomarkers Interest in the health effects of isoflavones also led to studies that were designed to identify determinants of their absorption and metabolism and to characterize their exposure in the population. Individual variability exists in the metabolism of isoflavones; excretion rates vary with types and amounts of soy food consumed, other components in the diet, and other factors such as intestinal microflora (32–37). Nonetheless, agreement exists that urinary isoflavone levels are a good indicator of short-term soy food intake whether spot (22), overnight (24,38), or 24-h urine specimens (21,25) were collected. These biomarkers of isoflavone exposure represent a more direct measure of bioavailability of these compounds than dietary assessment but they primarily reflect recent consumption, i.e., over the 48-h period before specimen collection (39). Until recently, the assessment of human exposure to lignans has been based largely on urinary and serological levels of these compounds (40), reflecting the history of the discovery of these mammalian lignans (41). More recent interest in this group of compounds is demonstrated by the development of FFQ and nutrient databases on lignans. There is convincing evidence that serum levels of lignans, particularly enterolactone, can be measured reliably and that measurements of this compound are useful markers of lignan intake (42). In cross-sectional studies,

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excretion of lignans was significantly correlated with amounts of fruits and vegetables consumed (30). Similarly, in controlled experimental (31) and intervention settings (43), urinary and serum levels of enterolactones changed in a dose-dependent manner with increases in intake of lignan-rich foods. Similar to isoflavones, levels of enterolactone concentrations in serum and urine reflect only recent intake of its precursors over the several days before specimen collection and it is also influenced by intestinal microflora (40). Although there is tremendous appeal to the use of biochemical markers of isoflavone and lignan exposures in epidemiologic studies, a limitation of case/control comparisons of these biomarkers is that the biological specimens are collected after the diagnosis of cancer. The results are meaningful only if the intake of foods rich in soy and lignans is not influenced by cancer diagnosis. Cohort-nested, casecontrol studies that have collected suitable biological specimens before the diagnosis of cancer are preferred.

Specific Cancer Sites In this section, we have summarized results from cohort and case-control studies in which intake of soy (isoflavones) or lignans was assessed by questionnaires or biomarkers in urine or blood specimens. Specifically, we have considered human studies (excluding abstracts) reported in the English language in which the intake of isoflavones (genistein, daidzein, glycitein), foods rich in isoflavones (soybean or its products), lignans (enterolactone, enterodiol), or phytoestrogens was assessed for individual participants. These studies were found in the MEDLINE database or they were referenced in specific studies. We have not considered studies in which the assessment of phytoestrogens was based on intake of vegetables. We also have not attempted to review dietary studies to determine whether pinto beans or refried beans, as a marker of lignan intake, are associated with cancer risk. For each cancer site, studies are categorized by type of design (cohort vs. casecontrol study) and by geographic area (Asia vs. non-Asia). Details regarding sample sizes, sources of cases and controls (or noncases), specific soy food (or exposure variable), levels of intake, and corresponding relative risk (RR) with 95% confidence intervals (CI) (or P-values), and covariates controlled for in analyses are shown. We have paid particular attention to the assessment of soy intake and the levels of intake because these differences may explain, in part, some of the heterogeneity in study results. Breast Cancer Potential benefits and risks of soy on breast health have been reviewed in detail recently (44,58). Despite a large number of mechanistic studies on soy and breast cancer that have been conducted in the last decade, there are still relatively few epidemiologic studies on this question. During the 1990s, seven epidemiological studies on soy and breast cancer were published (Table 21.2). Three case-control

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TABLE 21.2 Summary of Epidemiologic Studies on Dietary Intake of Soy and Risk of Breast Cancer Location/ (Ref.)

n cases (ca)/ n controls (co)

Cohort studies Japan/(45)

241 deaths

Nagasaki/Hiroshima Japan/(46)

Case-control studies Asia Singapore/(47,48)

427 incident cases

109 ca/207 co (premenopausal) 91 ca/213 co (postmenopausal)

Soy intake intake

Relative risk (95% CI)

Japanese cohort of 29 health center districts

Miso Daily or more
1.00 0.85 (0.68–1.06)

The Radiation Effects Research Foundation’s Life Span Study—Cohort in Hiroshima/Nagasaki

Tofu ≤1/wk 2–4/wk 5+/wk Miso ≤1/wk 2–4/wk 5+/wk

Sources of cases/controls

Hospital-based study (2 hospitals), noncancer hospital controls

Total soy products(g/d) <20.3 20.3–54.9 55.0+

Adjusted factors Age

1.00 0.99 (0.80–1.24) 1.07 (0.78–1.47)

Age, calendar period, age at time of bombing, radiation dose, and city

1.00 1.03 (0.81–1.31) 0.87 (0.68–1.12)

Pre-

Post-

Age, age at first birth

1.0 1.0 0.6 (0.3–1.2) 0.9 (0.4–1.9) 0.4 (0.2–0.9)a 1.1 (0.5–2.3) Continued

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TABLE 21.2 (Cont.) Location/ (Ref.)

n cases (ca)/ n controls (co)

Soy intake

Relative risk (95% CI)

Japan/(49)

607 ca/ 15,084 co (premenopausal) 445 ca/6215 co (postmenopausal)

Aichi Cancer Center Hospital for cases and controls (noncancer patients)

Bean curd ≤3/mo 1–2 /wk 3+/wk Miso Occasionally or never

Pre1.0 0.9 (0.7–1.2) 0.8 (0.6–1.0) 1.0 1.2 (1.0–1.4)

Tianjin and Shanghai, China/(50)

834 ca/834 co

Population-based study

Per 18 g soy protein/da

1.0 (0.7–1.4)

Shanghai, China/ (51,52)

1459 ca/ 1556 co

Population-based study

Adult intake Soy protein (g/wk) Occasionally ≤35.0 ≤58.8 ≤91.0 >91.0 Adolescent Intake Total soy foods (g/d) <2.20 2.20–4.41 >4.41–6.61 >6.61–11.01 >11.01

Sources of cases/controls

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Adjusted factors Post1.0 0.9 (0.6–1.2) 1.0 (0.7–1.3) 1.0 1.0 (0.8–1.2)

1.00 0.76 (0.49–1.16) 0.82 (0.54–1.26) 0.88 (0.58–1.36) 0.66 (0.43–1.02)

1.00 0.75 (0.60–0.93) 0.69 (0.55–0.87) 0.69 (0.55–0.86) 0.51 (0.40–0.65)a

Age

Energy intake, relevant menstrual and reproductive factors Age, education, relevant menstrual and reproductive factors, intake of meats, fish, and total energy

Age, education, relevant menstrual and reproductive factors, intake of rice and wheat products

Case-control studies United States/Canada Asians in California and Hawaii/(53) 597 ca/966 co

Population-based study

Caucasians in California and Canada/(54)

140 ca/222 co (premenopausal)

Hospital-based study

Non-Asians in SF Bay Area/(27)

1326 ca/ 1657 co

Population-based study

Soy products (times/y) <13 13–42 43–54 55–119 120+

1.0 0.9 (0.7–1.3) 0.9 (0.6–1.2) 0.8 (0.5–1.1) 0.7 (0.4–1.0)a

Tofu <1/wk 1+/wk

1.0 0.5 (0.2–1.1)

Total isoflavones (mg/d) <1.05 1.05 to <1.65 1.65 to <2.77 2.77+

P for trend < 0.05; CI, confidence interval; Pre, premenopausal; Post, postmenopausal. aRange between the 5th and 95th percentile among controls.

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1.0 1.1 (0.87–1.4) 1.1 (0.93–1.5) 1.0 (0.79–1.3)

Age, location, Asian ethnicity, menopausal status, and migration history

Age, body mass index, alcohol, energy and various menstrual and reproductive factors Age, race/ethnicity, relevant menstrual and reproductive factors and daily energy intake

studies, conducted among chinese in Singapore (mainly premenopausal women) (47,48), Asian-Americans in California and Hawaii (53), and Caucasians in the United States and Canada (54) are supportive of a reduced risk of breast cancer in association with high soy intake. However, no significant association between soy intake and breast cancer risk was observed in case-control studies conducted in Japan (49) and China (50) and in two cohort studies conducted in Japan (45,46). In 2001, two case-control studies designed specifically to study the role of soy and breast cancer were published. Both studies were large and well-designed; they carefully assessed the intake of soy and other dietary factors, and considered both dietary and nondietary confounders in their analyses (27,51). Intake of total soy protein (51) and isoflavones (27) was also estimated (Table 21.2). Horn-Ross and coworkers (27) conducted a population-based case-control study that included 1326 women diagnosed with an incident breast cancer (35% Latina, 31% AfricanAmericans, and 34% Caucasians) and 1657 population controls (42% Latina, 28% African-Americans, and 30% Caucasians). Usual intake of specific phytoestrogenic compounds was assessed using a modified version of the Block FFQ and a nutrient database for phytoestrogens developed specifically for this population (18). Cases and controls were compared in terms of intake of total isoflavones (genistein, daidzein, biochanin A, formonetin), coumestans (coumestrol), total lignans (matairesinol, secoisolariciresinol), and total phytoestrogens (isoflavones, lignans and coumestans). There were no significant case-control differences in intake of any of the specific isoflavones, lignans, or coumestans or total lignans, total isoflavones or total phyotestrogens (27). Results were unchanged when the analyses were conducted separately for the three racial groups and for pre- and postmenopausal women. However, the intake of total isoflavones (median, 1.65 mg/d) among non-Asians was at least 15 times lower than intake in Japan (41 mg/d) (25) or Shanghai, China (33 mg/d) (51) (see below). Intake of total phytoestrogens among non-Asians in the San Francisco Bay Area was equally low (median, 2.03 mg/d) (27). Dai and co-workers (51) conducted a population-based case-control study in Shanghai, China that included 1459 incident breast cancer cases and 1556 agematched controls. Information on usual adult dietary intake was collected using a comprehensive quantitative FFQ that included some 80 foods or food groups. Usual consumption of six groups of soy foods (soy milk, tofu, dried soybeans, soy products other than tofu, fresh soybeans and soybean sprouts), covering >90% of the soy foods consumed in this study area was determined. In addition, intake of soy (i.e., tofu, soy milk and soy products other than tofu) during adolescence was assessed (52). During adult life, 96.2% of cases and 96.9% of controls were weekly consumers of soy (51). Compared with nonweekly consumers, weekly consumers showed a 22% (95% CI, 0.51–1.16) reduced risk of breast cancer. Women in the highest decile group compared with those in the lowest decile intake group displayed a 30% [adjusted odds ratio (OR) = 0.66, 95% CI, 0.46–0.95] reduced risk of breast cancer. Intake in the lowest decile group was ≤2.6 g soy protein/d (~12 mg of isoflavones) compared with at least 19.87 g soy protein (~79 mg isoflavones/d)

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in the highest decile group. However, the dose-response relationship between soy food intake and risk was not smooth (P for trend = 0.28). The inverse association with soy intake was stronger and was significant when the analysis was restricted to subjects (724 cases and 1015 controls) who reported no change in their intake of soy foods. In this population, there was a significant inverse association between soy food intake during adolescence and breast cancer risk after adjustment for various dietary and nondietary risk factors (52). The OR for the lowest to highest quintiles of total soy food intake were 1.0 (baseline), 0.75, 0.69, 0.69, and 0.51, (P for trend <0.001). The inverse association was observed for each of the soy foods examined and existed for both pre- and postmenopausal women. The association with adolescent soy food intake also remained unchanged after adjustment for usual adult soy food intake. These results (52) concur with data from animal studies (55–58), emphasizing that timing of soy exposure, particularly early life exposure, may be important in the development of breast cancer. In addition, there have been three studies with data on urinary isoflavone excretion and breast cancer risk (59–61) (Table 21.3). In two of these studies, risk in relation to urinary enterolactone (the main lignan) was also determined (59,61). In a fourth study, serum levels of enterolactone in breast cancer patients and control subjects were compared (62). Three of these were retrospective case-control studies (59,60,62) and a fourth was a nested case-control study (61). There is a suggestion of a reduction in risk of breast cancer in association with high urinary excretion of isoflavone (59–61). High excretion of equol (a metabolite of daidzein) was associated with a significantly reduced risk in one study (59), but levels of genistein were not measured and thus the assessment of isoflavones was incomplete. In two studies, high urinary (59) or serological (62) enterolactone levels were associated with a significantly lower risk of breast cancer. However, breast cancer cases actually had higher urinary enterolactone levels than controls in a nested case-control study in which urine specimens were collected before breast cancer diagnosis (61). Because the evidence for a protective effect of isoflavones and enterolactone was weakest in this nested case-control study (61), additional studies using these biomarkers are warranted. Until now, the epidemiologic studies included either self-reports of intake or biomarkers of exposure, but not both sources of information. The evidence will be strengthened if an association is demonstrated with both reported dietary intake of isoflavones and lignans in conjunction with biomarkers of exposures in the same study. Endometrial Cancer The effect of phytoestrogens on the risk of endometrial cancer has not been adequately studied. We are aware of only one case-control study that specifically investigated the effect of soy on endometrial cancer. Goodman and co-workers (63) conducted a case-control study in Hawaii to examine the role of soy, legumes, and other dietary factors in the risk of endometrial cancer. A total of 332 endome-

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TABLE 21.3 Summary of Epidemiologic Studies on Urinary or Serological Levels of Phytoestrogens (PE) and Risk of Breast Cancer

Location/ (Ref.) Australia/(59)

Shanghai, China/(60)

Finland/(62)

Netherlands/(61)

n cases (ca)/ n controls (co) Sources of subjects 144 ca/144 co Hospital-based cases, population controls

60 ca/60 co Population-based

194 ca /208 co Hospital-based cases, population controls 88 ca/ 268 co Nested case-control study; Population-based breast screening project

aP

Urinary isoflavone levels Daidzein (µmol/d) ≥ 1.3 vs. ≤ 0.60 Equol (µmol/d) ≥ 0.185 vs. ≤ 0.07 Daidzein (nmol/cr) ≥ 7.61 vs. ≤ 2.81 Genistein (nmol/cr) ≥ 4.09 vs. ≤ 0.98 Total isoflavones (nmol/cr) ≥ 18.66 vs. ≤ 5.58

RR (95% CI) for highest vs. lowest level 0.47 (0.17–1.33) 0.27 (0.10–0.69)a

Enterolactone (µmol/d) ≥ 5.25 vs. ≤ 1.45b Enterodiol (µmol/d) ≥ 0.48 vs. ≤ 0.17

RR (95% CI) for highest vs. lowest level

Adjusted factors

0.36 (0.15–0.86)

Age at menarche, alcohol, total fat intake

0.73 (0.33–1.64)

0.54 (0.22–1.32) Not available 0.70 (0.27–1.84)

Age at first pregnancy and physical activity levels

0.50 (0.19–1.31)

Not available

Genistein/creatinine (µmol/mol)c ≥ 112.3 vs. ≤ 10.2

Ligan levels in urine or serum*

0.83 (0.4–1.51)

*Enterolactone (nmol/L) ≥ 34.8 vs ≤ 6.19b

0.38 (0.18–0.77)a

Enterolactone/ creatinine (µmol/mol) ≥ 656 vs. ≤ 7.16 1.43 (0.79–2.59)

Various menstrual and reproductive factors None

for trend <0.05. RR, relative risk; CI, confidence interval. to calculations presented by Pietinen (62), the urinary enterolactone levels among controls in the study by Ingram (59) was equivalent to ~15.8 nmol/L in serum and the serum levels of enterolactone in the Australia and Finnish studies were comparable. cThe mean urinary genistein levels among control subjects were 0.81 nmol/mg creatinine, about one third the genistein levels of control subjects in the study by Zheng (60). bAccording

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trial cancer cases (25% Caucasians, 17% Hawaiians, 58% Asians) and 511 population controls (23% Caucasians, 17% Hawaiians, 60% Asians) were interviewed in person. A detailed FFQ was administered and intake of tofu, soybeans, and miso was specifically queried. There was a significant trend of decreasing risk with increasing intake of tofu. The OR for endometrial cancer associated with the three upper quartiles compared with the lowest quartile of tofu intake was 0.65, 0.70, and 0.53, respectively (P for trend = 0.04). This pattern of association was also found for tofu and other soy products, and all legumes combined (i.e., including tofu) after adjustment for total energy and nondietary risk factors. The inverse associations were observed in both Asians and non-Asians. Intake of legumes was not associated with the risk of endometrial cancer in a case-control study conducted in Shanghai, China (64). It is unclear whether soy foods were specifically included under “legumes” in this study. Thyroid Cancer The soybean and its products have been considered goitrogenic in humans and animals (65). Specific concerns have been raised because of reports of goiter and hypothyroidism in infants receiving soy-containing formula (66). However, among pre- and postmenopausal women who added soy to their diet in short-term intervention studies (67,68), there is little evidence of any significant effects of soy on thyroid function (based on serum levels of thyroid stimulating hormones, total thyronine, and triiodothyroine). The association between intake of soy foods and risk of thyroid cancers has been investigated in three epidemiologic studies, two conducted in Japan (45,69) and one in the United States (70). Risk of thyroid cancer mortality (based on 55 deaths) was reduced nonsignificantly (RR = 0.88, 95% CI, 0.55–1.42) in association with daily intake of miso soup compared with nondaily intake in a cohort analysis (45). In a hospital-based, case-control study conducted in Japan, tofu and miso intake did not differ significantly between female patients with thyroid cancer (n = 94) and female outpatients without cancer (n = 22,666). The OR was 1.4 (95% CI, 0.7–2.8) for high vs. low tofu intake and 0.9 (95% CI, 0.4–2.1) for high vs. low miso intake (70). The third study was a population-based, case-control study conducted in the San Francisco Bay Area that included 608 women (49% Caucasians, 36% Asians, 15% other) diagnosed with incident thyroid cancer and 558 control subjects (52% Caucasians, 35% Asians, 13% other) (70). The methods used to assess intake of phytoestrogens (isoflavones, coumestans, and lignans) were similar to those these investigators used in a study of breast cancer described above (27). The risk of thyroid cancer tended to decrease with increasing quintile of isoflavone intake; the age- and race-adjusted OR were 1.0, 1.2, 1.2, 0.9, 0.6 (P for trend = 0.02). These findings were weakened after further adjustment for various dietary and nondietary risk factors (adjusted OR were 1.0, 1.1, 1.3, 1.0, 0.7, P for trend = 0.13). Similarly, there was an inverse association between risk and intake of total lignans (adjusted OR by quintile levels of intake were 1.0, 1.0, 0.7, 0.8,

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0.7, P for trend = 0.07). Because intake of isoflavones contributed substantially to the total phytoestrogen index, the risk pattern associated with intake of phytoestrogens was similar to that observed for intake of isoflavones. The respective adjusted OR were 1.0, 0.9, 1.2, 0.9, 0.6 (P for trend = 0.14). Thus, there is little evidence in that study that soy intake increased the risk of thyroid cancer. However, intake of isoflavones and lignans was low in this population; the daily median intake was ~2 and 0.1 mg, respectively. Studies are warranted to confirm whether an inverse association exists between intake of phytoestrogens and thyroid cancer, especially in populations with substantially higher intake levels. Prostate Cancer Circumstantial evidence exists in support of the hypothesis that high soy intake may be associated with a lower risk of prostate cancer (71). In an international ecologic analysis, an inverse correlation between intake of soy products and prostate cancer mortality rate has been reported (72). However, in an ecologic analysis conducted in 47 prefectures in Japan, soy intake (fermented and nonfermented soy foods combined) was positively associated with prostate cancer mortality rates (73). Although compelling evidence exists that androgens play a critical role in the development of prostate cancer (74), few lifestyle determinants of androgens have been identified. The effects of soy on androgen levels have not been well studied. In a cross-sectional study of men in Japan, an inverse association between soy intake and serum levels of estradiol, estrone, and testosterone was observed (75). In contrast, in an 8-wk intervention study in men, soymilk supplementation did not significantly influence serum testosterone levels, whereas serum estrone levels were reduced (76). Eight epidemiologic studies with data on soy intake and prostate cancer risk have been published (Table 21.4). Results from studies conducted in Asia are not supportive of a protective role of soy. However, assessment of soy intake in both studies conducted in Japan was incomplete, based solely on intake of miso (45,77). In a case-control study conducted in 12 Chinese cities (78), intake of soy foods did not differ significantly between control subject (85 grams of soy foods/d) and prostate cancer patients (71 grams/d). In contrast, three cohort (79–81) and three case-control studies (26,82,83) conducted in the United States/Canada suggest that the risk of prostate cancer may be reduced in association with high intake of soy foods (mainly tofu and soymilk) (Table 21.4). Although these “positive” results are suggestive, in two studies, intake of soy was very low (<1 mg of isoflavones/d) (26) or likely to be very low (82). In two other studies, the effect of soy on prostate cancer risk, may be related in part to the influence of legume intake on risk in these populations (81, 83). In a population-based case-control study conducted by Kolonel and co-workers (83), there was a trend of decreasing risk with increasing intake of soy products (P for trend = 0.06) in all subjects and in each of the four racial/ethnic groups. However, a comparably strong and consistent pattern of reduced risk was found in association with intake of legumes, suggesting that

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TABLE 21.4 Summary of Epidemiologic Studies on Dietary Intake of Soy and Risk of Prostate Cancer Location/ (Ref.)

n cases (ca)/ n controls (co)

Cohort studies Asia Japan/(45) 183 prostate cancer deaths

Case-control studies Asia Japan/(77) Hospital-based study of 100 cases, 2 control groups (100 patients with BPH, other hospital patients) China/(78)

Hospital-based study of 133 cases, 265 neigbhorhood controls

Cohort studies United States Hawaii/(79) 174 incident prostate cancers, Japanese Americans

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Soy intake

RR (95% CI)

Miso Daily or more
1.0 1.45 (1.09–1.94)

Miso No Ordinary High

BPH controls 1.0 1.47 (0.7–3.3) 1.29 (0.6–2.9)

Soy (7 foods)

70.7 g/d for cases vs. 83.6 g/d for controls (P = 0.16)

Miso ≤1/wk 2–4/wk 5+/wk Tofu ≤1/wk 2–4/wk 5+/wk

Hospital controls 1.0 1.40 (0.6–3.2) 0.64 (0.3–1.3)

1.00 1.19 (0.80–1.76) 1.24 (0.51–3.04) 1.00 0.78 (0.53–1.14) 0.35 (0.08–1.43)

Adjusted actors

Role of legumes and other vegetables

Age

No data on legumes; no association with dark greenyellow vegetables

None

No data on legumes; low intake of β-carotene increased risk significantly by twofold

None

No case/control differences in legumes. Intake of vegetables significantly less in cases

Age

No data on legumes or total vegetables

Continued

TABLE 21.4 (Cont.) Location/ (Ref.)

n cases (ca)/ n controls (co)

Soy intake

RR (95% CI)

Adjusted actors

Role of legumes and other vegetables

Seventh-Day Follow-up between Adventist/(80) 1976 and 1982; 180 histologically confirmed prostate cancers

Vegetarian protein products ≤1/wk 1.00 1–4/wk 0.83 (0.59–1.16) 0.67 (0.40–1.12) 5+/wk

Age

Significantly reduced risk associated with high intake of beans, lentils and peas

Seventh-Day Follow-up between (81)/Adventist 1976 and 1982; a subgroup was followed 1983–1992; 225 incident prostate cancers

Soy milk Never 1/d

Age, body mass index, intake of coffee, whole fat, milk, eggs, citrus fruits, and age at first marriage

No data on legumes

1.0 0.9 (0.5–1.4) 0.7 (0.4–1.4) 0.3 (0.1–0.9)a

Case-control studies United States/Canada Texas/(26) 83 ca/ 107 co Hospital-based study, cases from MD Anderson

Estimated intake of specific

Any genistein 0.71 (0.39–1.30) Any daidzein

Age, family history of prostate cancer,

No data on legumes; no significant associations with lignans and other phytoestrogens

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Cancer Center, controls were from screening clinic

isoflavones, lignans and other phytoestrogens

Canada/(82)

1623 ca/1623 co Population-based study

Tofu or soybean None 1.0 Some 0.8 (0.6–1.1)

Age, province of residence, race, years since quitting smoking, pack-years, body mass index, family history of cancer, income, and other dietary factors

No significant association with intake of lentils/beans, and total vegetables. Risk increased with intake of cereals and fruits

California and Hawaii/ (83)

1619 ca/1618 co Population-based study

Total soy products (miso, soy beans tofu and aburage) None (g/d) 0.1–0.5 0.6–18.0 18.1–39.4 >39.4

Age, education, ethnicity, geographic area, and energy

Significantly reduced risk associated with intake of legumes without soy as well as with total legumes (i.e., including soy)

aP

0.57 (0.31–1.05)

1.00 0.75 (0.60–0.94) 0.75 (0.55–1.02) 0.85 (0.61–1.19) 0.62 (0.44–0.89) P for trend = 0.06

for trend <0.05. RR, relative risk; CI, confidence interval; BPH, benign prostatic hyperplasia.

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alcohol intake and total energy

legumes (not limited to soy products) may protect against prostate cancer. In a cohort analysis of Seventh Day Adventists, a reduced risk in association with intake of soy milk was reported (81). In a previous analysis of prostate cancer in this Adventist population, risk was reduced significantly in association with high intake of beans, lentils, and peas, whereas the reduced risk associated with high intake of vegetarian protein products was not significant (80). Thus, it is important to investigate not only the role of all relevant soy foods but also legumes on prostate cancer risk in this Adventist and other populations. Finally, reasons for the inconsistent risk patterns associated with intake of miso and tofu in a cohort study of Japanese-American men in Hawaii are not clear (79). Investigations of the separate and combined effects of fermented and nonfermented soy foods on prostate cancer risk, controlling for the potential confounding effects of legumes and other dietary factors, are warranted.

Lung Cancer Substantial evidence from observational epidemiologic studies exists showing that high intake of fruits and vegetables is associated with a reduced risk of lung cancer (84). The specific constituents in fruits and vegetables that confer protection are not known, but specific carotenoids (85) and isothiocyanates (86) are among the leading candidates. Because a plant-based diet may include intake of soy, there is growing interest to determine whether soy food intake is associated with lung cancer risk (Table 21.5). Intake of soy foods and risk of lung cancer were investigated in eight epidemiologic studies including three studies from Japan (45,92,93) and five studies among Chinese residing in China (88–90), Hong Kong (87) and Singapore (91). Four (87, 89–91) of the five studies conducted in Chinese populations showed a reduced risk of lung cancer in association with high intake of soy. Intake of all soy foods combined, and separately for tofu or soy bean paste, did not differ significantly between Northern Chinese women with lung cancer and control subjects (88). In contrast, in a smaller study of lung cancer in Northern Chinese men and women, there is some suggestion that risk was reduced in association with high intake of nonfermented soy foods, but not with fermented soy paste (90). High soy intake was significantly inversely associated with risk among male tin miners in Yunnan, China, most of whom were smokers (89). However, these findings on soy were not adjusted for vegetable intake, which had significant protective effects in this population. In Singapore, the association between soy intake and lung cancer risk differed by smoking status. Lung cancer risk of female smokers was not influenced by soy intake, but lung cancer risk among never smokers was reduced significantly in association with high soy intake (91). In Hong Kong, risk of lung cancer among female never smokers was also reduced in association with soy intake; female smokers were not included in this study (87). In Japan, lung cancer mortality was not associated with intake of miso in a cohort analysis (45). However,

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TABLE 21.5 Summary of Epidemiologic Studies on Dietary Intake of Soy and Risk of Lung Cancer

Location/ (Ref.)

n cases (ca)/ n controls (co) Gender, smoking status, cell type Soy intake

Cohort studies Asia Japan/(45) 1454 male and 463 female lung cancer deaths Case-control studies Asia Hong Kong/ Hospital-based (87) 88 ca/137 co all female nonsmokers Northern China/(88)

Yunnan, China/(89)

RR (95% CI)

Miso Daily or more
1.0 1.0 (0.97–1.15 )

Tofu/soy products Low Medium High

1.00 0.91 (0.43–1.93) 0.51 (0.21–1.24)

Population-based 965 ca/959 co, all females; 417 ca/602 co never smokers

Soybean products (times/y) <153 153–365 365–485 >485

1.00 0.7 (0.5–0.9) 0.9 (0.7–1.2) 1.0 (0.8–1.3)

Population-based 428 ca/1011 co, all males; 9 ca/72 co nonsmokers

Tofu (times/mo) Q1 (<8) Q2 Q3 Q4 (>15.9)

1.00 0.85 0.60 0.44a

Adjusted factors

Role of vegetables

Age and sex

Significantly decreased risk with intake of dark-yellow vegetables

Age, number of births and education

Intake of other vegetables associated with reduced risk

Age, education, smoking status, study area

No association with intake of vegetables or fruits

Age, respondent type, study site and education

Significantly reduced risks with high intake of vegetables

Continued Copyright 2002 by AOCS Press. All rights reserved.

TABLE 21.5 (Cont.)

Location/ (Ref.) Northern China/(90)

Japan/(92)

n cases (ca)/ n controls (co) Gender, smoking status, cell type Soy intake Hospital-based 227 ca/227 co men and women, 81 ca/115 co nonsmokers

Hospital-based 245 ca/489 co men (M) 87 ca/176 co women (W) never smokers: 10 ca/66 co (M), 49ca/145 co (W); cell type, among M: 115 SCC,106ADC; among W, 19 SCC, 58 ADC

Soybean products (g/d) Q1 (<5.5) Q2 Q3 Q4 (>21.9) Salted fermented soypaste (g/d) Q1 (<2.7) Q2 Q3 Q4 (>10.9) Miso soup
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RR (95% CI)

1.0 1.0 (0.6–1.6) 0.9 (0.5–1.7) 0.6 (0.4–1.1)

Adjusted factors

Role of vegetables

Cigarettes/day, duration of smoking, income

No association with intake of vegetables

Education, smoking and history of preious lung disease

No significant case/control differences in intake of green-yellow vegetables, in men and women

1.0 1.3 (0.7–2.3) 1.4 (0.8–2.0) 0.7 (0.4–1.2) Men 1.0 2.3 (1.0–5.2) 3.2 (1.4–7.2) 3.8 (1.7–8.5)a

Women 1.0 3.3 (0.8–3.4) 5.5 (1.4–21.7) 4.0 (1.0–15.9)a

1.0 0.7 (0.5–1.04)

1.0 1.0 (0.5–1.9)

1.0 0.7 (0.4–1.2) 0.6 (0.4–0.98)

1.0 1.2 (0.5–3.2) 1.1 (0.5–2.4)

Japan/(93)

5+/wk Miso soup

Singapore/ (91)

aP

Hospital-based study Aichi Cancer Center M: 367 ADC, 381 SCC/ small cell vs. 2964 co W: 240 ADC, 57 SCC/ small cell vs. 1189 co

Tofu <1/wk 1–2/wk 3–4/wk 5+/wk Miso soup Almost never Occasionally 1 time/d 2+ times/d

Men (ADC) 1.00 1.33 (0.95–1.87) 1.27 (0.88–1.84) 1.24 (0.83–1.85)

Men (SCC) 1.00 1.10 (0.78–1.55) 1.09 (0.76–1.57) 1.23 (0.84–1.81)

1.00 1.93 (0.90–4.14) 1.65 (0.78–3.51) 1.40 (0.63–3.11)

1.00 2.24 (0.98–5.11) 2.40 (1.07–5.38) 2.50 (1.08–5.79)

Tofu <1/wk 1–2 /wk 3–4 /wk 0.52 (0.30–0.91)a 3.00 (0.72–12.6) passive smoking, Almost never Occasionally 1 time/d 2+ times/d

Women (ADC) Women (SCC) 1.00 1.00 0.89 (0.55–1.43) 3.68 (0.99–13.6) 0.93 (0.56–1.52) 2.86 (0.73–11.2) former, current), consistent with other 1.00 1.00 0.79 (0.36–1.72) 0.51 (0.12–2.07) 0.90 (0.42–1.93) 0.45 (0.11–1.77) 0.69 (0.30–1.59) 0.51 (0.11–2.42)

All women Hospital-based study 127 ca/100 co smokers 176 ca/663 co never smokers; 126 of 176 lung cancers in never smokers were ADC

Smokers

Soy foods (servings/wk) <2.2 2.2 to <5.4 5.4+ Soy isoflavonoids (mg/d) <9.87 9.87+ to 24.5 >24.5

Never smokers

1.0 1.00 1.58 (0.81–3.09) 0.57 (0.38–0.86) 1.53 (0.76–3.11) 0.53 (0.34–0.81)a

1.00 1.00 1.55 (0.78–3.08) 0.57 (0.38–0.86) 1.30 (0.64–2.61) 0.56 (0.37–0.85)a

Age, season and year of visit, occupation, prior lung diseases, smoking (never, former, current), passive smoking, consumption of green vegetables and meat

High intake of green vegetables associated with decreased risk of ADC and SCC in men. High intake of carrots associated with decreased risk of ADC and SCC in women. Results were less consistent with other vegetables and fruits.

Age, place of birth, first degree relative with history of cancer. For smokers, adjusted for duration and intensity of smoking. For nonsmokers, adjusted for passive smoking

Among female smokers, high intake of total vegetables ↓ risk significantly. Among female never smokers, no significant effect of vegetables but high intake of fruits ↑ risk significantly.

for trend <0.01; RR, relative risk; CI, confidence interval; SCC, squamous cell carcinoma; ADC, adenocarcinoma; M, men; W, women.

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results on soy intake and lung cancer risk are conflicting in two case-control studies, conducted in Okinawa (92) and Nagoya, Japan (93) (Table 21.5). Soy intake patterns were presented in only one study (92); it is not possible to determine the extent to which intake of fermented and nonfermented soy foods differed in the two populations. The overall results appear discordant by smoking status (91), type of soy foods (90,92,93), and possibly by gender and histologic type (93). Some of the differences in results may be explained, in part, by heterogeneity in study design (populationbased vs. hospital-based) and small numbers in some of the subgroup analyses. One study included only men (89), whereas three studies were conducted in women only (87,88,91). Smokers and nonsmokers were included in most studies, and smoking habits were controlled for in the analysis. One study was restricted to nonsmokers only (87), and two studies conducted analyses stratified by smoking status (91,92). The cell type distributions (adenocarcinoma vs. nonadenocarcinoma) also differed and were considered in few studies. In addition, most of the studies did not consider other dietary factors (e.g., vegetable intake) in the analyses. Because of the importance of smoking and intake of plant foods, careful control of these factors is required to determine whether soy has any significant effect on lung cancer risk. Colorectum Cancer Epidemiologic data on the risk of colorectal cancer and polyps and soy intake are shown in Table 21.6. In studies conducted in Asia (45,94–96,98), there is little consistent evidence that intake of tofu, soybeans, or fermented soy foods had any consistent or significant effects on the risk of colon or rectal cancers. This lack of association was confirmed in a large case-control study that was conducted among Caucasians and Asians in Hawaii (100). In that study, tofu intake was not associated with risk of these tumors in men and women and in Asians and non-Asians. Any protective effect was observed only in association with high intake of legumes and soy products among women (100) (Table 21.6). Similarly, there is little evidence that risk of colorectal polyps is associated with soy intake in studies conducted in Japan (97) and California (101). Interestingly, in the ecologic study conducted by Nagata et al. (73), these investigators reported significant positive correlations between colorectal cancer mortality rates and soy product intake (in grams of soy foods and soy protein and in mg of isoflavones) in men and women in Japan after controlling for mean age, total energy, proportion of current smokers, animal fat, and alcohol intake.

Stomach Cancer There are at least 15 studies with data on fermented soy foods and risk of stomach cancer and 11 studies with data on nonfermented soy foods and risk of this tumor site. We conducted a detailed review of these studies (102) and found that 10 of the 15 studies showed that high intake of fermented soy foods was associated with an increased risk of stomach cancer. However, one study, based on stomach cancer

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TABLE 21.6 Summary of Epidemiologic Studies on Dietary Intake of Soy and Risk of Colon and Rectal Cancer/Polyps Location/ (Ref.) Cohort studies Asia Japan/(45)

n cases (ca)/ n controls (co)

Soy intake

RR (95% CI)

552 colon ca 563 rectal ca deaths

Miso Daily or more
Colon 1.00 1.13 (0.97–1.32)

Rectum 1.00 1.04 (0.89–1.21)

Age and sex

Tofu <1/wk 1–3/wk 4+/wk Miso 1+/d
Colon 1.00 1.64 1.08

Rectum 1.00 1.38 1.63

Age and sex

1.0 0.54

1.00 2.05a

Miso soup <1/d 1/d 2+/d Soybean products (excluding miso) <5/wk 5–7/wk 8+/wk

Colon 1.0 1.5 (0.6–3.6) 1.9 (0.8–4.4)

Rectum 1.0 0.7 (0.4–1.4) 0.8 (0.4–1.6)

1.0 0.6 (0.3–1.0) 0.6 (0.3–1.3)

1.0 0.8 (0.5–1.2) 0.4 (0.2–0.9)a

Case-control studies Asia Japan/(94) Hospital-based study 93 colorectal ca (42 colon, 51 rectum) 186 co

Japan/(95)

Hospital-based study 181 colorectal ca (79 colon, 102 rectum) 653 population co

Adjusted factors

Age and sex

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TABLE 21.6 (Cont.) Location/ (Ref.)

n cases (ca)/ n controls (co)

Northern China/(96)

Hospital-based 336 colorectal ca (111 colon, 225 rectum) 336 controls

Japan/(98)

Japan/(97)

Soy intake

Bean products(tofu, dried bean curd, bean sprouts) <5.5 g/d >5.5 6 g/d

Hospital-based 94 proximal colon co 137 distal colon co 201 rectum ca 31,782 outpatient co

Soybean paste soup Daily vs. less (men) (women) Tofu Daily vs. less (men) (women)

Hospital-based 187 adenoma ca 1557 co with normal colonoscopy

Miso soup <1/d 1/d 2+/d

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RR (95% CI)

Adjusted factors

Colon (no association in men and women). Rectum (no association in women).

None

Rectum (male) 1.0 0.57 Proximal 1.2 0.8

Distal 0.7 0.8

Rectum 0.8 1.1

0.9 1.3

1.7 0.6

1.2 0.9

1.0 0.99 (0.68–1.42) 0.87 (0.55–1.37)

Age

Smoking, alcohol use, rank and body mass index

Case-control studies United States Hawaii/(99) Hospital-based, Japanese 179 colorectum ca 357 co Hawaii/(100)

California/(101)

aP

Population-based 687 male case/co pairs; 494 female case/co pairs

Hospital-based 488 colorectal polyps and 488 co

Fermented soybeans: above vs. below average intake

1.6 for colorectum combined

Tofu (g/d) Q1 (none) Q2 Q3 Q4 (≥24.7) Legumes and soy products (g/d) Q1 (≤10.6)b Q2 Q3 Q4 (46.44)

Men 1.0 1.1 (0.7–1.6) 1.1 (0.7–1.7) 1.0 (0.6–1.6)

Women 1.0 1.1 (0.7–1.8) 0.9 (0.5–1.6) 0.9 (0.5–1.5)

1.0 0.8 (0.5–1.2) 0.8 (0.5–1.1) 0.8 (0.5–1.2)

1.0 0.6 (0.4–1.0) 0.7 (0.4–1.1) 0.5 (0.3–0.9)a

Tofu of soybeans (serving/wk) None <0.5 1+

for trend <0.05. RR, relative risk; CI, confidence interval. females, Quartile 1 was ≤9.3 and Quartile 4 was ≥43.6 g/d.

bIn

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1.0 0.89 (0.49–1.45) 0.55 (0.27–1.11)

Age, family history of colorectal cancer, alcoholic drinks/wk, pack-years of cigarette smoking, lifetime recreational activity, body mass index 5 y earlier, total energy, eggs and calcium

Race, body mass index, physical activity, smoking, energy, saturated fat, dietary fiber, folate, β-carotene, and vitamin C

mortality in Japan, found a significantly reduced risk in association with high miso intake (45). Results from this single study were clearly heterogeneous. Exclusion of this single study from the pooled analysis yielded a combined risk estimate of 1.26 (95% CI, 1.11–1.43) for stomach cancer in association with high intake of fermented soy products. In contrast, in 9 of 11 studies, the risk of stomach cancer was reduced in association with high intake of nonfermented soy foods; the pooled risk estimate was 0.72 (95% CI, 0.63–0.82). Similar results were found in analyses conducted separately in case-control and cohort studies (102). Our further analyses suggest that fermented and nonfermented soy foods may be associated with salt and fruit/vegetable intake, respectively. Both high intake of salt and low intake of plant-based foods are directly associated with stomach cancer risk (103). Although a few of the studies (104–106) on soy and stomach cancer have considered the possible confounding role of salt and fruit/vegetable intake, these potential confounders were not considered in most of the studies (102). Two studies from Japan offered additional insights into the association between soy intake and stomach cancer mortality/prognosis. Huang and colleagues (107) examined the influence of various dietary factors, smoking, and alcohol use on prognosis of gastric cancer in Japan. After controlling for age, gender, histological grade, and stage of disease, hazard ratios were significantly decreased in association with frequent intake (>3 times per week) of three foods, i.e., raw vegetables, tofu, and chicken. In contrast, there was no association between risk of dying from stomach cancer and intake of miso soup. These results are intriguing and would be strengthened if the analyses on tofu and vegetables mutually controlled for each other and factors (e.g., smoking and alcohol use) that were associated with increased risks of mortality. In an ecologic analysis, Nagata et al. (73) reported that stomach cancer mortality rate among Japanese men was significantly inversely correlated with intake of total intake of soy protein but that the association was much weaker in women. Soy intake in this analysis included miso, tofu, fried tofu, soybeans, soy milk, and yuba. It would be informative to reanalyze these data separately for fermented and nonfermented soy foods.

Summary and Conclusions There have been ~50 epidemiologic studies reporting results on intake of soy or isoflavones or blood or urine levels of isoflavones and risk of a cancer site. No definitive conclusion can be reached for any of the cancer sites at this time. For breast and prostate cancers, studies conducted in Asia (particularly those published in the 1990s) tended to be less supportive of a “protective” role of soy than studies conducted in Western countries in which intake of soy is substantially lower. Several possible explanations may help to clarify these discrepant results. Misclassification bias due to incomplete assessment of soy intake may be a more serious problem for studies conducted in Asia than those conducted in the West. Because there are few soy foods available in the West until recently, intake of tofu (although incomplete) is a good sur-

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rogate for total soy food intake. However, in Asia, the sources of soy intake are more diverse and intake is substantially higher. Thus, incomplete assessment of soy intake will result in more serious misclassification bias. With the publication of more accurate estimates of isoflavone intakes and sources of soy in various populations (19–25), it is now possible to qualitatively rate published studies in terms of the completeness of soy assessment. For example, for studies conducted in Japan, soy intake based only on tofu or miso intake alone is likely to result in considerable misclassification and the direction of such bias is unclear. Little is known about the correlation between intake of fermented and nonfermented soy foods but their effect on risk may differ for some sites (e.g., prostate, lung, and stomach). Fermented and nonfermented soy foods may be markers of different dietary factors that influence risk of a cancer site. In our review of studies on stomach cancer and soy food intake, intake of fermented soy foods was positively associated with intake of foods that are high in salt, whereas intake of nonfermented soy foods was positively associated with intake of fruits/vegetables. Correlates of soy food (fermented and nonfermented sources) intake may differ in different populations. As shown in the tables, known or suspected risk factors were not controlled in many of the studies. Careful consideration of potential dietary and nondietary confounders is clearly important. Although it is important to be complete in the assessment of soy intake, investigation of risk patterns by intake of the main groups of soy foods (i.e., fermented vs. nonfermented) and by total isoflavones or soy protein may help clarify whether soy intake is important in itself or whether eating habits (or lifestyle habits) associated with eating certain types of soy foods is important. Understanding dose-response relationships between intake of soy and cancer risk continues to be a challenge in epidemiologic studies. Because soy intake is uniformly high in Asia, it may be particularly difficult to identify a truly unexposed group, and thus the effect of soy may be diluted. This is demonstrated in a large breast cancer case-control study from Shanghai, China (51). Less than 4% of subjects in this population consumed soy less than weekly so that although a reduced risk was observed with soy intake, a smooth trend of decreasing breast cancer risk with increasing amounts of soy protein consumed was not observed. In contrast, in studies conducted in the West, the generally low soy intake precluded meaningful investigations of dose-response relationships. In addition, if the greatest difference in risk is between consumers and nonconsumers of soy, does this imply that any amount of intake is protective or is soy a marker of other lifestyle habits? This is particularly important because the intake levels of soy differ considerably in studies conducted in Asia vs. those conducted in the West. Issues related to the timing of exposure to soy have been largely ignored in most epidemiologic studies because questions are typically focused on usual adult intake patterns. As suggested by the results of Shu and co-workers (52), soy intake during adolescence appears to have profound and lasting protective effects on breast cancer risk. Confirmation of these results for breast cancer is warranted. Investigations of timing of soy exposure in other cancer sites should provide additional insights regarding the potential health effects of this group of foods.

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Acknowledgments Dr. Anna H. Wu is supported, in part, by the California Breast Cancer Research Program (3PB-0102, 5PB-0018) and the Susan G. Komen Breast Cancer Foundation (BASIC9900328).

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63. Goodman, M.T., Wilkens, L.R., Hankin, J.R., Lyu, L.C., Wu, A.H., and Kolonel, L.N. (1997) Association of Soy and Fiber Consumption with the Risk of Endometrial Cancer, Am. J. Epidemiol. 146, 294–306. 64. Shu, X.O., Zheng, W., Potischman, N., Brinton, L.A., Hatch, M.C., Gao, Y.T., and Fraumeni, J.F., Jr. (1993) A Population-Based Case-Control Study of Dietary Factors and Endometrial Cancer in Shanghai, People’s Republic of China, Am. J. Epidemiol. 137, 155–165. 65. Divi, R.L., Chang, H.C., and Doerge, D.R. (1997) Anti-Thyroid Isoflavones from Soybean: Isolation, Characterization, and Mechanisms of Action, Biochem. Pharmacol. 54, 1087–1096. 66. Van Wyk, J.J., Arnoid, M.B., Wynn, J., and Pepper, F. (1959) The Effects of a Soybean Product on Thyroid Function in Human, Pediatrics 24, 752–760. 67. Duncan, A.M., Merz, B.E., Xu, X., Nagel, T.C., Phipps, W.R., and Kurzer, M.S. (1999) Soy Isoflavones Exert Modest Hormonal Effects in Premenopausal Women, J. Clin. Endocrinol. Metab. 84, 192–197. 68. Duncan, A.M., Underhill, K.E.W., Xu, X., Lavlleur, J., Phipps, W.R., and Kurzer, M.S. (1999) Modest Hormonal Effects of Soy Isoflavones in Postmenopausal Women, J. Clin. Endocrinol. Metab. 84, 3479–3483. 69. Takezaki, T., Hirose, K., Inoue, M., Hamajima, N., Kuroishi, T., Nakamura, S., Koshikawa, T., Matsuura, H., and Tajima, K. (1996) Risk Factors of Thyroid Cancer Among Women in Tokai, Japan, J. Epidemiol. 6, 140–147. 70. Horn-Ross, P.L., Hoggatt, K.J., and Lee, M.M. (2002) Phytoestrogens and Thyroid Cancerc Risk: The San Francisco Bay Area Thyroid Cancer Study, Cancer Epidemiol, Biomark. Prev. 11, 43–49. 71. Adlercreutz, H., Markkanen, H., and Watanabe, S. (1993) Plasma Concentrations of Phyto-Estrogen in Japanese Men, Lancet 342, 1209–1210. 72. Herbert, J.R., Hurley, T.G., Olendzki, B.C., Teas, J., Ma, Y., and Hampl, J.S. (1998) Nutritional and Socioeconomic Factors in Relation to Prostate Cancer Mortality: A Cross-National Study, J. Natl. Cancer Inst. 90, 1637–47. 73. Nagata, C. (2000) Ecological Study of the Association Between Soy Product Intake and Mortality from Cancer and Heart Disease in Japan, Int. J. Epidemiol. 29, 832–836. 74. Ross, R.K., and Schottenfeld, D. (1996) Prostate Cancer, in Cancer Epidemiology and Prevention, 2nd edn., Schottenfeld, D., and Fraumeni, J.F., Jr., eds.) pp. 1180–1206, Oxford University Press, New York. 75. Nagata, C., Takatsuka, N., Shimizu, H., Hayashi,H., Akamatsu, T., and Murase, K. (2001) Effect of Soymilk Consumption on Serum Estrogen and Androgen Concentrations in Japanese Men, Cancer Epidemiol. Biomark. Prev. 10, 179–184. 76. Nagata, C., Inaba, S., Kawakami, N., Kakizoe, T., and Shimizu, H. (2000) Inverse Association of Soy Product Intake with Serum Androgen and Estrogen Concentrations in Japanese Men, Nutr. Cancer 36, 14–18. 77. Oishi, K., Okada, K., Yoshida, O., Yamaba, H., Ohno, K., Hayes, R.B., and Schroeder, F. H. (1988) A Case-Control Study of Prostatic Cancer with Reference to Dietary Habits, Prostate 12, 179–190. 78. Lee, M.M., Wang, R.T., Hsing, A.W., Gu, F.L., Wang, T., and Spitz, M. (1998) CaseControl Study of Diet and Prostate Cancer in China, Cancer Causes Control 9, 545–552.

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79. Severson, R.K., Nomura, A.M.Y., Grove, J.S., and Stemmermann, G.N. (1989) A Prospective Study of Demographics, Diet, and Prostate Cancer Among Men of Japanese Ancestry in Hawaii, Cancer Res. 49, 1857–1860. 80. Mills, P.K., Beeson, L., Phillips, R.L., and Fraser, G.E. (1989) Cohort Study of Diet, Lifestyle, and Prostate Cancer in Adventist Men, Cancer 64, 598–604. 81. Jacobsen, B.K., Knutsen, S.F., and Fraser, G.E. (1998) Does High Soy Milk Intake Reduce Prostate Cancer Incidence? The Adventist Health Study (United States), Cancer Causes Control 9, 553–557. 82. Villeneuve, P.J., Johnson, K.C., Kreiger, N., Mao, Y., & The Canadian Cancer Registries Epidemiology Research Group (1999) Risk Factors for Prostate Cancer: Results from the Canadian National Enhanced Cancer Surveillance System, Cancer Causes Control 10, 355–367. 83. Kolonel, L.N., Hankin, J.H., Whittemore, A.S., Wu, A.H., Gallagher, R.P., Wilkens, L.R., John, E.R., Howe, G.R., Dreon, D.M., West, D.W., and Paffenbarger, R.S. Jr., (2000) Vegetables, Fruits, Legumes and Prostate Cancer: A Multiethnic Case-Control Study, Cancer Epidemiol. Biomark. Prev. 9, 795–804. 84. Ziegler, R.G., Mayne, S.T., and Swanson, C.A. (1996) Nutrition and Lung Cancer, Cancer Causes Control 7, 157–177. 85. Michaud, D.S., Feskanich, D., Rimm, E.B., Colditz, G.A., Speizer, F.E., Willett, W.C., and Giovannucci, E. (2000) Intake of Specific Carotenoids and Risk of Lung Cancer in 2 Prospective US Cohorts, Am. J. Clin. Nutr. 72, 900–907. 86. Verhoeven, D.T.H., Goldbohm, R.A., van Poppel, G., Verhagen, H., and van den Brandt, P.A. (1996) Epidemiological Studies on Brassica Vegetables and Cancer Risk, Cancer Epidemiol. Biomark. Prev. 5, 733–748. 87. Koo, L.C. (1988) Dietary Habits and Lung Cancer Risk Among Chinese Females in Hong Kong Who Never Smoked, Nutr Cancer 11, 155–172. 88. Wu-Williams, A.H, Dai, X.D., Blot, W., Xu, Z.Y., Sun, X.W., Xiao, H.P., Stone, B.J., Yu, S.F., Feng, Y.P., Ershow, A.G., Sun, J., Fraumeni, J.R., Jr., and Henderson, B.E. (1990) Lung Cancer Among Women in North-East China, Br. J. Cancer 62, 982–987. 89. Swanson, C.A., Mao, B.L., Li, J.Y., Lubin, J.H., Yao, S.X., Wang, J.Z., Cai, S.K., Hou, Y., Luo, Q.S., and Blot, W.J. (1992) Dietary Determinants of Lung Cancer Risk: Results From A Case-Control Study in Yunnan Province, China, Int. J. Cancer 50, 876–880. 90. Hu, J., Johnson, K.C., Mao, Y., Xu, T., Lin, Q., Wang, C., Zhao, F., Wang, G., Chen, Y., and Yang Y. (1997) A Case-Control Study of Diet and Lung Cancer in Northeast China, Int. J. Cancer 71, 924–931. 91. Seow, A., Poh, W.T., Teh, M., Eng, P., Wang, Y.T., Tan, W.C., Chia, K.S., Yu, M.C., and Lee, H.P. (2002) Diet, Reproductive Factors and Lung Cancer Risk Among Chinese Women in Singapore: Evidence for a Protective Effect of Soy in NonSmokers, Int. J. Cancer 97, 365–371. 92. Wakai, K., Ohno, Y., Genka, Y., Ohmine, K., Kawamura, T., Tamakoshi, A., Lin, Y., Nakayama, T., Aoki, K., and Fukuma, S. (1999) Risk Modification in Lung Cancer By A Dietary Intake of Preserved Foods and Soyfoods: Findings from A Case-Control Study in Okinawa, Japan, Lung Cancer 25, 147–159. 93. Takezaki, T., Hirose, K., Inoue, M., Hamajima, N., Yatabe, Y., Mitsudomi, T., Sugiura, T., Kuroishi, T., and Tajima, K. (2001) Dietary Factors and Lung Cancer

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Risk in Japanese: With Special Reference to Fish Consumption and Adenocarcinomas, Br. J. Cancer 84, 1199–1206. Tajima, K., and Tominaga, S. (1985) Dietary Habits and Gastro-Intestinal Cancers: A Comparative Case-Control Study of Stomach and Large Intestinal Cancers in Nagoya, Japan, Jpn. J. Cancer Res. (Gann) 76, 705–716. Hoshiyama, Y., Sekine, T., and Sasaba, T. (1993) A Case-Control Study of Colorectal Cancer and Its Relation To Diet, Cigarettes, and Alcohol Consumption in Saitama Prefecture, Japan, Tohoku J. Exp. Med. 171, 153–165. Hu, J., Liu Y., Yu, Y., Zhao, T., Liu, S., and Wang, Q. (1991) Diet and Cancer of the Colon and Rectum: A Case-Control Study in China, Int. J. Epidemiol. 20, 362–367. Kono, S., Imanishi, K., Shinchi, K., and Yanai, F. (1993) Relationship of Diet To Small and Large Adenomas of the Sigmoid Colon, Jpn. J. Cancer Res. 84, 13–19. Inoue, M., Tajima, K., Hirose, K., Hamajima, H., Takezaki, T., Hirai, T., Kata, T., and Ohno, Y. (1995) Subsite-Specific Risk Factors for Colorectal Cancer: A HospitalBased Case-Control Study in Japan, Cancer Causes Control 6, 14–22. Haenszel, W., Berg, J.W., Segi, M., Kurihara, M., and Locke, F.B. (1980) LargeBowel Cancer in Hawaiian Japanese, J. Natl. Cancer Inst. 51, 1765–1779. Le Marchand, L., Hankin, J.H., Wilkens, L.R., Kolonel, L.N., Englyst, H.N., and Lyu, L.C. (1997) Dietary Fiber and Colorectal Cancer Risk, Epidemiology 8, 658–665. Witte, J.S., Longnecker M.P., Bird, C.L., Lee, E.R., Frankl, H.D., and Haile, R.W. (1996) Relation of Vegetable, Fruit, and Grain Consumption to Colorectal Adenomatous Polyps, Am. J. Epidemiol. 144, 1015–1025. Wu, A.H., Yang, D., and Pike, M.C. (2000) A Meta-Analysis of Soyfoods and Risk of Stomach Cancer: The Problem of Potential Confounders, Cancer Epidemiol. Biomark. Prev. 9, 1051–1058. Nomura, A. (1996) Stomach Cancer, 2nd edn., pp. 707–724, Oxford University Press, New York. Hoshiyama, Y., and Sasaba, T (1992) A Case-Control Study of Stomach Cancer and Its Relation to Diet, Cigarettes, and Alcohol Consumption in Saitama Prefecture, Japan. Cancer Causes Control 3, 441–448. Lee, J.K., Park, B.J., Yoo, K.Y., and Ahn, Y.O. (1995) Dietary Factors and Stomach Cancer: A Case-Control Study in Korea, Int. J. Epidemiol. 24, 33–41. Ji, B.T., Chow, W.H.,. Yang, G., Jin, F., Gao, Y.T., and Fraumeni, J.F., Jr. (2001) Correspondence re: A.H. Wu, et al., A Meta-Analysis of Soyfoods and Risk of Stomach Cancer: The Problem of Potential Confounders, Cancer Epidemiol. Biomarker. Prev. 10, 570–571. Huang, X.-E., Tajima, K., Hamajima, N., Kodera, Y., Yamamura, Y., Xiang, J., Tominaga, S., and Tokudome, S. (2000) Effects of Dietary, Drinking, and Smoking Habits on the Prognosis of Gastric Cancer, Nutr. Cancer 38, 30–36.

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Chapter 22

Flaxseed Lignans: Health Benefits, Bioavailability, and Safety Lilian U. Thompson and Wendy E. Ward Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada

Introduction The three major types of phytoestrogens are the isoflavones, lignans, and coumestans. This chapter will focus on the lignans and their health benefits, including their ability to reduce the risk of cancer, cardiovascular disease, diabetes, kidney disease, osteoporosis, menopausal symptoms, as well as their bioavailability and safety. Lignans are ubiquitous components of plants with levels depending on the species and tissue type, including seeds, fruits, roots, leaves, flowers, and stems. They are dimeric compounds formed by the coupling of two monomeric C6C3 moieties derived from the phenylpropanoid pathway (1). Some plant lignans are metabolized by the bacterial flora in the colon of humans and animals to the mammalian lignans enterolactone (EL) and enterodiol (ED). The plant lignan precursors of ED and EL were earlier thought to be only secoisolariciresinol diglucoside (SDG) and matairesinol (2; Fig. 22.1). More recently, other precursors of mammalian lignans have been identified, including 7-hydroxymatairesinol, lariciresinol, isolariciresinol, arctigenin, trachelogenin, syringaresinol, and pinoresinol (3; Fig. 22.1). After their discovery in the early 1980s, the mammalian lignans were hypothesized to be responsible in part for the beneficial effect of plant foods on health (2), but only in recent years have studies supporting this hypothesis been conducted. Using an in vitro fermentation system with human fecal inoculum to simulate human colonic fermentation, Thompson et al. (4) found that mammalian lignan precursors are present in 66 different plant foods in the vegetarian diet. However, flaxseed, also called linseed, is the richest source of mammalian lignan precursors, with values 75–800 times higher than other plant foods including other oilseeds, cereal grains, legumes, fruits, and vegetables. This was confirmed in studies in which the plant lignans were analyzed directly (5). In the absence of commercially available mammalian lignan precursors, therefore, flaxseed has been used as a model system with which to determine the potential health benefits of lignans. Nevertheless, because flaxseed is also rich in dietary fiber and α-linolenic acid (ALA, an n-3 fatty acid), which also have health benefits, the effect of flaxseed

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Fig. 22.1. Chemical structure of mammalian lignans and their precursors.

cannot be attributed solely to the lignans that it contains, unless the results are also compared with that of its purified major lignan precursor (SDG).

Cancer Breast Cancer Epidemiology. Adlercreutz and co-workers (6,7) first observed that the urinary excretion of mammalian lignans was lower in breast cancer patients than in healthy postmenopausal omnivorous and vegetarian women, suggesting that lignans may have a protective effect against breast cancer. Ingram et al. (8) supported this result when the median EL concentration (1.97 mol/24 h) in urine samples of 144 women

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with newly diagnosed breast cancer was found to be lower than that of 144 controls (3.10 µmol/24 h). After adjustment for confounding variables, a threefold reduction in breast cancer risk was observed for women with the highest vs. the lowest quartile of EL excretion (P < 0.013 for trend). More recently, Pietienen et al. (9) measured serum EL from 194 cases and 208 controls and observed higher (P < 0.033) levels of EL among controls (26 nmol/L) vs. cases (20 nmol/L). A significant reduction in breast cancer risk from the highest vs. lowest quartile of EL was also observed. Because in retrospective studies the urinary lignans may be influenced by metabolic consequences of the disease rather than its possible cause, Tonkellar et al. (10) measured lignans in urinary specimens taken 1–9 y before breast cancer was diagnosed. However, no association between EL excretion and breast cancer risk was elucidated. The mean levels of EL measured in two urine samples taken on different days were 576 and 566 µmol/mol creatinine in the cases and controls, respectively. Hence, epidemiologic studies are not unanimous in their support of the role of lignans in reducing breast cancer risk. Animal Studies: Initiation, Promotion, Progression, and Metastasis Stages of Carcinogenesis. To determine the role of lignans in reducing breast cancer risk, our laboratory has been testing the effect of lignans on the development of breast cancer when provided at the various stages of carcinogenesis. Our early feeding studies used flaxseed as the lignan source because the mammalian lignans ED and EL as well as their major precursors, SDG and matairesinol, are not available commercially (11–13). Feeding 5 or 10% flaxseed or defatted flaxseed for 4 wk followed by injection with the mammary carcinogen dimethylbenzanthracene (DMBA) resulted in significant reductions in nuclear aberration and cell proliferation (11). A negative correlation was observed between urinary lignan excretion and the number of nuclear aberrations in the mammary epithelial cells, indicating that the lignans may protect against the initiation of cancer. Indeed, when rats were fed a 5% flaxseed diet during the preinitiation stage (4 wk before DMBA injection) and the high-fat (20% corn oil) basal diet at the promotion stage (20 wk after DMBA injection), a lower tumor incidence and multiplicity were observed compared with the control (12). Similar results were observed when 5% flaxseed was fed at both the preinitiation and promotion stages of carcinogenesis. However, when the treatment was reversed, i.e., rats were fed the basal high-fat diet at the preinitiation stage and the 5% flaxseed at the promotion stage, the flaxseed group had a tumor incidence and multiplicity that did not differ significantly from the control, but the tumor volumes were significantly lower (12). In agreement, feeding rats 2.5 or 5% flaxseed when the primary tumors were already established, i.e., at 13 wk post-DMBA treatment, resulted in large significant reductions (~75%) in the primary tumor volumes (13). These results suggest that the effect of flaxseed is dependent on the stage of carcinogenesis. Because the effect of flaxseed may not all be due to its lignans, SDG was isolated from flaxseed (14) and tested for its independent effect on carcinogenesis.

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Rats fed SDG at 1.5 mg/d, equivalent to the amount in a 5% flaxseed diet, starting 1 wk after DMBA treatment (promotion stage), had significantly lower tumor multiplicity and number of tumors per group compared with control (15). The tumor volume and incidence were also reduced although not significantly. When fed for 7 wk starting at a time when the tumors were already established, i.e., 13 wk postDMBA, SDG reduced the established tumor volume by 50%, and also reduced the total number and size of new tumors that appeared (13). The total volume, i.e., new plus established tumor volume, in the SDG-treated rats was significantly lower than in the control group. The results with SDG feeding did not differ significantly from that with feeding 2.5 or 5% flaxseed, indicating that the effect of flaxseed is due in part to the lignans that it contains. In a follow-up study, rats were fed for 22 wk the control basal diet, 2.5 or 5.0% flaxseed, or equivalent amounts of SDG, i.e.. low-dose SDG (LSDG) at 0.7 mg/d or high-dose SDG (HSDG) at 1.4 mg/d, starting 1 wk after injection with the carcinogen methyl nitrosourea (MNU) (16). The 5% flaxseed group produced the lowest tumor size. The LSDG group had the highest tumor multiplicity and the HSDG group, the lowest. However, all of the treatment groups demonstrated significantly decreased tumor invasiveness and grade, indicating that both the flaxseed and the SDG diets delayed the progression of MNU-induced mammary tumorigenesis. Because it is unclear whether the growth inhibitory effects of lignans can be demonstrated in human tumors and also because the DMBA- or MNU-induced mammary tumors do not metastasize, we conducted a study to determine the effect of flaxseed lignans on the growth and metastasis of human tumors cells in athymic mice (unpublished data). Athymic mice were injected with the human cancer cells MDA-MB-435, which are known to metastasize; 8 wk later when the tumors were already established, the mice were divided into two groups and fed either the 10% flaxseed or the control diet for 7 wk. The flaxseed diet significantly reduced the tumor growth rate and the metastasis incidence, particularly in the lymph nodes and lungs. Because MDA-MB-435 cells are estrogen receptor negative, the results indicate that the effect was not hormone related. Although it remains to be determined whether the effect is due to the lignans or other components of flaxseed, in vitro studies have shown that ED and EL at physiologic levels (1–5 µmol/L) can reduce the invasion, adhesion, and migration of human tumor cells, steps involved in the metastasis process (unpublished data). Animal Studies: Effect of Early Exposure to Lignans. It has been suggested that the risk of mammary cancer depends on the state of cellular differentiation in the mammary gland structure at the time of carcinogen exposure (17–19). The less differentiated, highly proliferating terminal end bud (TEB) structure is more susceptible to carcinogens than the more differentiated alveolar buds and lobules. Hence, the lower the number of TEB at the time of carcinogen exposure [postnatal day (PND) 50 in rats], the lower the risk of mammary cancer. When a 5 or 10%

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flaxseed diet was fed to rat dams during pregnancy and lactation, a significant reduction in the number of TEB was observed in the mammary gland of their offspring compared with those fed the control diet (20). No significant difference was observed between groups when the flaxseed was fed to the offspring after weaning only. When a 10% flaxseed diet or equivalent amount of SDG was fed to the mothers only during lactation, a reduction in TEB was observed among offspring (21) similar to that observed with flaxseed or SDG feeding during pregnancy and lactation (20). The effect of SDG did not differ from that of the 10% flaxseed, indicating that the effect of flaxseed on the mammary gland structures is primarily due to its lignans. The results also suggest that the reduction in TEB in the mammary gland of the offspring occurred primarily during suckling via the transfer of lignans through mother’s milk. Studies with radiolabeled SDG (3H-SDG) have confirmed the transfer of lignans from the mother to the offspring (22). Studies are now in progress to determine whether the offspring exposed to flaxseed or purified lignans through the mother’s milk during lactation will have a lower tumor incidence when they are injected with carcinogens at PND 50. If the results are positive, early exposure to flaxseed or its purified lignans during suckling may be one strategy for reducing breast cancer risk. Clinical Study. The purified lignans have not been tested for their effects on breast cancer in women. However, feeding 25 g flaxseed in a muffin formulation to patients with newly diagnosed breast cancer from the time of diagnosis to the time of surgery resulted in significantly lower tumor cell proliferation, measured as Ki67 and CerB labeling indices in their tumor biopsy tissues, compared with those fed the placebo muffin without flaxseed (23). This result agrees with the observations in rats, which showed tumor regression or reduced tumor growth when flaxseed or SDG was fed after tumors were already established (13). Although feeding SDG in human studies remains to be done, on the basis of the results of the above-mentioned animal studies, the effect of flaxseed among breast cancer patients may be due in part to its lignans. Colon Cancer Limited work has been done on the role of flaxseed lignans on colon cancer. Serraino and Thompson (24) fed azoxymethane (AOM)-treated rats 5 or 10% flaxseed or defatted flaxseed for 28 d and observed a 50% reduction in the number of aberrant crypts and aberrant crypt foci compared with the controls fed the basal diet. Aberrant crypts have been suggested to be early biomarkers of colon cancer risk (25). The fact that defatted flaxseed produced an effect that did not differ significantly from the full-fat flaxseed suggests that the effect is not due to the oil content of flaxseed. The results also suggest that a high level of intake may not be necessary because the same effect was observed at the 5 and 10% levels. Hence, in another study, rats were fed, for 100 d, the basal diet without or with supplementa-

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tion with 2.5 or 5% flaxseed or defatted flaxseed or SDG (1.5 mg/d) equivalent to the amount consumed in the 5% flaxseed diet (26). All flaxseed and SDG diets resulted in significant reductions in aberrant crypt multiplicity. Because the 5% flaxseed diet produced results that did not differ significantly from the SDG-supplemented diet and the urinary lignan excretion also had a significant negative relationship with aberrant crypt multiplicity, the results indicate a role of lignans from flaxseed in colon carcinogenesis. This was supported by an in vitro study that showed significant reductions in cell proliferation of four human colon tumor cell lines (27). The reduction in cell proliferation does not appear to be estrogen related because the growth of these tumor cell lines is not estrogen dependent. Interestingly, flaxseed and SDG increased the cecal β-glucuronidase activity in rats (28) in direct relationship to the intake level and to urinary lignan excretion (26,28). Other studies have indicated that increased β-glucuronidase activity is a marker of increased colon cancer risk because this enzyme increases the enterohepatic circulation of carcinogens, mutagens, and estrogens (29). However, it does not seem to be the case in the studies discussed above. β-Glucuronidase activity is likely a marker of bacterial activity and, depending on what components this enzyme helps circulate, its increased activity could be protective or nonprotective. Because lignans are protective, the increased β-glucuronidase activity relates more to a reduction in aberrant crypt multiplicity, a biomarker of colon cancer risk. Prostate Cancer The incidence of prostate cancer is lower in Hong Kong and Portugal than in the United Kingdom (30). It is of interest that the prostatic fluid levels of phytoestrogens including the lignans EL and ED and the isoflavones daidzein and equol in 17–22 men tested in these countries were higher in Hong Kong and Portugal than in the United Kingdom (31). These data provide evidence of a potential role of lignans in the prevention or treatment of prostate cancer. In vitro studies have shown that ED and EL can inhibit the growth of the prostate cancer cell lines PC-3, DU145, and LNCaP (32). EL was more effective than ED [50% inhibitory concentration (IC50) = 57 and 100 µmol/L, respectively], but both are less potent than genistein (IC50 = 25 µmol/L). Using the transgenic adenocarcinoma mouse prostate (TRAMP) model, the effect of a 5% flaxseed diet in the development of prostatic neoplasia and metastasis mice was determined (33). The tumor malignancy score, tumor weight, incidence of lung and lymph node metastasis, and cell proliferation were higher and the apoptotic index was lower in the control compared with the flaxseed group, indicating that 5% flaxseed suppressed the growth and development of prostate cancer in the TRAMP mouse. In a recent study, patients with newly diagnosed prostate cancer who supplemented their low-fat diets (≤ 20% of energy) with 30 g flaxseed from the time of diagnosis to the time of surgery (average 34 d) experienced significant reductions in tumor proliferative index, free androgen index, serum total testosterone, and

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total cholesterol, and an increase in the tumor apoptosis index (34), indicating better patient prognosis. Matched historic cases consuming a high-fat diet, without flaxseed supplement, did not show similar changes. However, because two variables were introduced into the diet, i.e., low fat and flaxseed, it is not clear whether the beneficial effect was due to the lowering of the dietary fat or the lignans and other components in flaxseed. Moreover, because this study was not a randomized, placebo-controlled trial, further study is warranted to confirm the role of lignans on prostate cancer in humans. Skin Cancer Mice were fed 2.5, 5, or 10% flaxseed 2 wk before and 2 wk after injection with the melanoma cell line B16BL6 directly into the blood stream to determine its effect on lung metastasis (35). A dose-dependent reduction in lung tumor number and area was observed. A repeat of this experiment using SDG at levels equivalent to the amount in the 2.5, 5, or 10% flaxseed diet (i.e. 73, 147, and 293 µmol/kg diet) showed similar results (36). Again, this finding indicates that the effect of flaxseed is due in part to its SDG.

Cardiovascular Disease Epidemiology. In a prospective, nested, case-control study of middle-aged men from Eastern Finland, the relationship between serum EL concentration and the risk of acute coronary events was determined (37). Cases (167 men with average 7.7 y of follow-up to acute coronary event) and matched controls (167 men) were from a cohort of 2005 men with no clinical coronary heart disease at baseline. Cases were found to have lower baseline serum EL (18.2 nmol/L) compared with controls (23.5 nmol/L). After adjustment for the most strongly predictive risk factors, a 65.3% lower risk was observed in men with the highest quartile of serum EL than men in the lowest quartile. Although a significant reduction in heart disease was indicated in this study, it should be noted that the difference in serum EL concentration (5.3 nmol/L) between the cases and control was very small. Considering the large variability in EL production commonly observed among subjects (38,39), it is not known whether the levels equivalent to the difference in serum level observed in this study would be effective in reducing cardiovascular risk factors in an intervention study. Animal Studies. Prasad (40) fed laboratory diet pellets with or without flaxseed (7.5 g/kg body weight), 1% cholesterol, or flaxseed plus 1% cholesterol to rabbits. After 8 wk of consumption, the high-cholesterol diet increased serum total cholesterol and the oxygen free radical–producing activity of polymorphonuclear leukocytes (PMNL-CL) without changing serum triglyceride levels. These were reflected in the marked development of aortic atherosclerosis. However, flaxseed reduced

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the aortic arteriosclerosis by 46% as well as the PMNL-CL without reducing serum cholesterol. This effect of flaxseed was thought to be due to either its lignan or ALA content because lignans have antioxidant (41–43) and antiplatelet activating factor (PAF) activities, whereas ALA can suppress the production of interleukin-1, tumor necrosis factor, leukotriene B4, and oxygen free radicals by PMNL and monocytes (40). However, a repeat of this experiment using flaxseed with very low ALA content (2–3%) instead of the high ALA (>50%) still showed a reduction in aortic arteriosclerosis by 69% without equivalent changes in serum lipids (44). Hence ALA was not responsible for the antiatherogenic effect of flaxseed. On the other hand, when SDG [15 mg/(kg body.d)] was fed using the same experimental design (45), a decrease in serum cholesterol (33%), low density lipoprotein (LDL) cholesterol (35%), aortic arteriosclerosis (73%), and lipid peroxidation products, and an increase in high density lipoprotein (HDL) cholesterol and antioxidant reserve were observed, indicating that SDG and its metabolites play a role in the reduction of cardiovascular disease risk. Clinical Studies. Cunnane et al. (46) fed nine healthy women 50 g flaxseed (raw or incorporated into bread) for 4 wk and observed a significant reduction in LDL (9%) and total cholesterol (18%). A follow-up study in 5 healthy men and 5 healthy women fed 50 g flaxseed in muffin formulation vs. placebo muffin showed significant reductions in total cholesterol (6%) and LDL cholesterol (9%) (47). In 22 men and 7 postmenopausal hyperlipidemic women fed the National Cholesterol Education Program (NCEP) Step II diet supplemented with 50 g partially defatted flaxseed to provide 20 g fiber/d or 20 g fiber from wheat bran, significant reductions in total cholesterol (4.6%), LDL cholesterol (7.6%), apo-B lipoprotein (5.4%) and apo-A lipoprotein (5.8%) were observed (48). Evidently, flaxseed can influence lipid metabolism in humans. However, these clinical studies did not test the direct effect of lignans nor did they differentiate the effect of lignans from that of the lipid or fiber components. Hence, it remains to be seen whether the effect of lignans observed in the rabbit model can be reproduced in humans.

Diabetes Because SDG and its ED and EL metabolites have been shown to have antioxidant activities (41–43) and because reactive oxygen species are believed to be involved in the development of diabetes (49), SDG was tested for its effect on streptozotocin (STZ)-induced diabetes in rats (50). At 22 mg/kg body, SDG reduced the development of diabetes by 75%, and this effect appears to be related to its antioxidant activity as indicated by an accompanying reduction in serum and pancreatic malonaldehydes and oxygen free radical–producing activity of the white blood cells, and an increase in pancreatic antioxidant reserve. When fed to diabetes prone rats (BioBreeding rats), a model of human type 1 diabetes (insulin-dependent diabetes mellitus), SDG at 22 mg/kg body reduced the development of diabetes by 71%

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(51). The development of diabetes was also reduced by 80% when SDG (40 mg/kg body) was fed to Zucker diabetic fatty (ZDF)/Gmi-fa/fa female rats, a model of human type 2 diabetes (52). Similar to the effect seen in STZ-treated rats, the effect of SDG was related to its ability to reduce oxidative stress (50,51). No clinical studies have yet been conducted to determine the effect of SDG on diabetes.

Kidney Disease In the MRL/lpr mice model of lupus nephritis, a 15% flaxseed diet, compared with the laboratory diet, has been shown to reduce mortality and proteinuria, to preserve renal function (glomerular filtration rates), and inhibit lymphadenophathy, splenic T-cell proliferation, and PAF-induced platelet aggregation (53,54). Although both flaxseed oil and flaxseed are able to retard progression in renal injury, flaxseed was more effective than flaxseed oil, indicating that the effect of flaxseed was due to the additive or synergistic effect between the flaxseed oil and other components of flaxseed such as the lignans (55). When SDG was fed at 600 or 1200 µg/d, a doserelated delay in the onset of proteinuria with preservation of glomerular filtration rate and renal size was observed (56). These findings indicate that SDG has a therapeutic effect in lupus nephritis. Nine lupus nephritis patients were fed 15, 30, or 45 g flaxseed/d sequentially for 4 wk with a 5-wk washout period between each intervention (57). The interventions resulted in dose-related improvements in renal function as indicated by reduced PAF-induced platelet aggregation, serum creatinine, proteinuria, and increased creatinine clearance. These results led to a 2-y nonplacebo-controlled crossover study in which 23 lupus patients were randomized to eat 30 g ground flaxseed daily for 1 y followed by the reverse treatment after a 12-wk washout period (58). The results, however, are not conclusive because only 15 patients completed the study and, of these, only 9 were compliant. Although a decreasing trend for urine creatinine and microalbumin was observed during the flaxseed period, it was not significant. Clinical studies using the SDG still must be conducted to confirm the renoprotective effect observed using the lupus mouse model. Flaxseed has also been tested for its effect in polycystic kidney disease using the Han: SPRD-cy rat model (59,60). The 10% flaxseed diet fed to the rats for 8 wk from weaning ameliorated the interstitial nephritis in this disease compared with the effect of pair-fed control chow diet. However, it is not clear whether this effect is due to the lignans present in the flaxseed.

Menopausal Symptoms Although hormone replacement therapy (HRT) is proven to provide relief from hot flushes (61), there is no definitive conclusion regarding whether consuming a diet rich in lignans and/or flaxseed prevents or reduces the severity of menopausal symptoms (62,63). Most studies investigating the ability of phytoestrogens to pre-

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vent or reduce the severity of menopausal symptoms have studied the effect of feeding soy protein (containing isoflavones), purified isoflavones, or red clover extract and have reported mixed findings (64–70). With respect to flaxseed interventions, outcome measures include the frequency of hot flushes, vaginal cell maturation, vaginal dryness, and/or measurement of specific serum sex hormones (62,63,71). A randomized controlled trial in women (n = 145) who either consumed a phytoestrogen-rich diet or a diet devoid of phytoestrogens for 12 wk, reported no difference in menopausal scores (62). The phytoestrogen-rich diet consisted of a variety of foods abundant in lignans and isoflavones such as flaxseed, tofu, and miso that substituted for ~25% of a subject’s total energy intake. Although the menopausal symptom scoring system that was used included information pertaining to a variety of menopausal symptoms, further analysis revealed that if specific symptoms such as frequency of hot flushes and vaginal dryness were assessed individually, women consuming diets rich in phytoestrogens had a significantly lower number of hot flushes as well as less vaginal dryness (62). Supplementation with flaxseed (45 g), soy (45 g of soy grit enriched bread), or wheat (45 g) for 4 wk each resulted in no significant difference in the incidence of hot flushes between flaxseed and soy groups (71). Interestingly, hot flushes were lowest when women were consuming the wheat (71). Another study supplemented women (n = 25) with three different interventions for 2 wk each, in succession, for a total supplementation period of 6 wk (25 g flaxseed, 45 g soy flour, and 10 g dried red clover seeds/d). There was a significant reduction in follicle stimulating hormone over the 6-wk intervention (63). Furthermore, vaginal cell maturation was increased after a 2-wk supplementation with either flaxseed or soy flour but not with red clover. However, the differences in the levels of follicle stimulating hormone and vaginal cell maturation were small and it is questionable whether these differences are clinically significant. Interpretation of these findings is complicated by the fact that there was no true control group nor was there a washout period separating the three different interventions (flaxseed, soy flour, or dried red clover seeds). It is important to recognize that studies that assess menopausal symptoms are particularly challenging to conduct because there is a “placebo” effect that is speculated to account for up to a 40% reduction in the number and severity of hot flushes. Also, it is not feasible or realistic to conduct a crossover trial because menopausal symptoms decline over time, suggesting that parallel studies are most appropriate (64). There are no reported studies that have compared the effectiveness of feeding flaxseed and/or purified lignan with the gold standard treatment, HRT, in reducing the number and severity of hot flushes or any other menopausal symptoms.

Bone Metabolism There is evidence that feeding isoflavones to ovariectomized rodents attenuates the reduction in bone mass that accompanies the withdrawal of estrogen (72–76).

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Because lignans share a similar chemical structure with isoflavones and endogenous estrogen, and may thereby exert an estrogenic effect, there is a biological basis on which to hypothesize that lignans have hormonal effects on bone tissue. Animal Studies: Bone Development. On the basis of the fact that maximizing the attainment of peak bone mass may protect against bone loss and ultimately, the risk of fragility fracture during aging, we conducted studies to determine whether exposure to flaxseed or purified lignan during early, hormone-sensitive stages of development altered bone mass and biomechanical bone strength among male and female rats. Female offspring were exposed to the equivalent quantity of SDG, in a 5 or 10% flaxseed diet from the start of suckling through PND 21 or continuously from birth through PND 50 or 132 (77). Femur bone mineral content (BMC), bone mineral density (BMD), and biomechanical bone strength were not different at PND 50 among female offspring exposed to lignans only during suckling. However, rats that were continuously exposed to lignans at either the 5 or 10% level from the start of suckling through PND 50 had a greater femur biomechanical strength compared with the other groups. Because this effect on femur strength did not persist into adulthood (PND 132), it is hypothesized that the elevation in endogenous estrogen after PND 50 and through to adulthood diluted the positive effect of early exposure to lignans on femur strength. Male offspring that were exposed to a 10% flaxseed diet or the equivalent quantity of lignan precursor, SDG, from the start of suckling through PND 21 (end of suckling) had a similar femur BMC and BMD and biomechanical strength at young adulthood (PND 50) or adulthood (PND 132) compared with rats exposed to a diet devoid of lignans during suckling (78). In contrast, male offspring that received lignans, in the form of flaxseed, through mother’s milk during suckling and then through their diet until PND 50 had weaker femurs than the other rats, but BMC and BMD were not different. Because this was not a lignan-mediated effect, fatty acids in flaxseed may have influenced specific regulators of bone metabolism (79,80). The reduced femur strength was not observed at adulthood. Flaxseed and lignan appears to be safe with respect to bone development in male rats as assessed by bone mass and biomechanical strength. Young female rats (3–4 wk old) exposed to a 5 or 10% flaxseed diet or a 6.2% flaxmeal diet had lower bone alkaline phosphatase activity after 56 d of feeding (81). The authors commented that the lower bone alkaline phosphatase activity, used as a measure of zinc status, is in agreement with an earlier report that reduced femur zinc is observed among rats fed flaxseed (82). It is hypothesized that the effect on bone alkaline phosphatase activity and femur zinc content is due to the phytate or fiber content of flaxseed or flaxmeal in developing rats. Other measures such as bone mass and bone strength were not measured. Whether diets containing lignan provide protection against bone loss and fracture during aging, particularly when production of endogenous estrogen by the ovaries has stopped, remains to be determined.

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Clinical Study: Postmenopausal Women. One study has reported the effects of flaxseed supplementation on biochemical markers of bone metabolism among postmenopausal women (83). Feeding 38 grams of flaxseed, in breads or muffins for 6 wk, significantly reduced serum tartrate-resistant acid phosphatase, a marker of osteoclastic activity, without altering serum levels of bone-specific alkaline phosphatase activity, a marker of osteoblastic activity (83). It is hypothesized that this effect is due to the lignan component in flaxseed but confirmation of this effect is an area for future investigation. Ultimately, larger feeding trials of longer duration are required to assess changes in BMC, BMD and most importantly, fracture risk.

Lignan Metabolism and Bioavailability Lignan Metabolites. It is established that plant lignans such as SDG and matairesinol are converted to the mammalian lignans ED and EL by the bacterial flora in the colon (2; Fig. 22.1). Hence, production of mammalian lignans from precursors has been estimated using in vitro fermentation of the food or substrates with human fecal inoculum (3,4). The mammalian lignans undergo enterohepatic circulation with a portion reaching the kidney and excreted in the urine (2). Thus, urinary excretion of ED and EL has been used as an indicator of production and availability of mammalian lignans in animals and humans (2–16,20–24,26,37–39). Although ED and EL are the main mammalian lignans analyzed in urine, other metabolites have recently been identified (84). ED has been shown to be metabolized in vitro by rat liver microsomes to three aromatic and four aliphatic monohydroxylated compounds and the EL to six aromatic and six aliphatic monohydroxylated compounds (84). Many of these metabolites have also been detected in the bile of bile duct–catheterized rats administered ED or EL (10 mg/kg body) intraduodenally, and in the urine of rats gavaged with ED or EL or fed a diet containing 5% flaxseed (85). In four human subjects fed flaxseed (16 g) for 5 d, however, only the aromatic monohydroxylated metabolites of ED and EL were detected (86). These metabolites were estimated to represent <5% of the total urinary lignans in the human subjects (86) or <3% of the parent lignans ED or EL fed to rats (85). In contrast, when we fed radiolabeled SDG (3H-SDG) to rats (87), only the ED, EL, secoisolariciresinol (SECO; aglycone of SDG), and four other unidentified metabolites, none of which had mass spectra that matched those observed by Metzler and colleagues (84–86), were found in the urine. Two of the unknown metabolites were observed in the urine of rats fed nonradioactive flaxseed or SDG (87). The other metabolites from SDG, ED or EL, although formed, may have been present at too low a level to be detectable. When a large volume of urine was analyzed, one of the monohydroxylated ED metabolites reported by Jacobs et al. (84–86) was detected in the urine of the flaxseed- but not the SDG-fed rats (87). However, the use of large urine volumes for analysis to detect the minor lignan metabolites decreases the accuracy and sensitivity of detection for the major metabolites (i.e., ED, EL, and SECO).

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Dose-Response. When rats were fed diets containing 2.5, 5, or 10% flaxseed, the urinary excretion of total lignans (ED + EL + SECO) was linear up to 5% and then started to level off, suggesting a threshold response (14). A similar pattern of results was observed when SDG was gavaged at levels (1.2, 2.2, and 4.4 mol SDG/d) equivalent to the amounts consumed in the 2.5, 5, and 10% flaxseed diet (14). The urinary lignans leveled off after an intake of 2.2 mol/d. In a lupus nephritis mouse model gavaged with various levels of SDG, however, no leveling off of urinary lignan levels at high SDG intake was observed (56). Furthermore, most of the urinary lignan was SECO, whereas in many studies, ED and EL are the major metabolites and SECO, if detected, is very low in concentration. It is not clear whether the result in this mouse model is unique or whether it is a consequence of the lupus disease. Nonetheless, unlike in other studies, it suggests that the effect of SDG in the lupus mouse model (53–56) may be due more to SECO than to ED or EL. In agreement with the rat study, a dose-related linear increase in urinary lignan (ED + EL + SECO) excretion was observed in premenopausal women fed 0, 5, 15, and 25 g flaxseed (39) and in postmenopausal women fed 0, 5, and 10 g flaxseed (88). The intake of 5% flaxseed diet by rats is equivalent to an intake of ~25 g flaxseed by humans, thus no leveling off of urinary lignan excretion was observed. Body Distribution of Metabolites after Acute vs. Chronic Intake. The body distribution and excretion of the SDG metabolites was determined at various times for up to 48 h after rats were gavaged with 3H-SDG (89). After 48 h, most of the recovered radioactive dose was excreted i.e., >60% of recovered dose in the feces and 28–32% of recovered dose (at least 70% of the absorbed dose) in the urine. The total recovery did not vary with acute (single treatment) vs. chronic (after treatment with 1.5 mg SDG/d for 10 d) intake, but there was a delay in the fecal excretion with chronic intake. The fecal excretion was almost complete after 12 h in the acute group, whereas the fecal radioactivity in the chronic group was negligible after 12 h and approached the level excreted in the acute group only after 24 h. This was attributed to increased enterohepatic circulation of the lignans after chronic treatment, which in turn was related to increased β-glucuronidase activity due to the lignans (26,28). The urine radioactivity excreted after 24 h (12%) was in agreement with a previous report of 11.4% recovery of ED and EL in rats fed 1.5 mg SDG/d for 2 wk (14), suggesting that the recovered activity is primarily metabolites of SDG. Later analysis of the urine confirmed that the majority of the radioactivity in the samples were from ED, EL, and SECO with minor amounts from four unidentified lignan metabolites (88). Radioactivity was detected in all analyzed tissues (heart, liver, kidney, spleen, lung, adipose, mammary gland, ovaries, uterus, skin, muscle, brain, stomach, small intestine, cecum, and colon), but the highest levels were found in those involved in lignan metabolism, i.e., gastrointestinal tissues (3–7% of recovered dose), the liver, and kidney (levels 5 times higher than other nongastrointestinal tissues) (89).

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Although appreciable amounts were also detected in estrogen-sensitive tissues such as the uterus and ovary, low levels were found in the mammary gland, indicating that the effect of lignans is not only through their direct binding to this tissue. Chronic intakes increased the radioactivity level in the liver and adipose tissue but not in the other tissues. The radioactivity level in the blood was <1% of recovered dose, most of which was present in the plasma (89). Plasma lignan concentration was estimated to be ~1 µmol/L in rats fed 1.5 mg/d SDG, a value ~3000 times higher than peak estrogen levels in rats. The level peaked 9 h after 3H-SDG intake and remained steady up to 24 h in the chronic group but had dropped, although still not lower than that at the 12-h time point, in the acute group (87). Similar results were obtained in premenopausal women fed 25 g raw flaxseed once (acutely) or daily for 8 d (chronic) in which plasma levels peaked at 9 h postconsumption (39). The plasma lignan level in the women was higher on d 8 than on d 1 of intake. The plasma concentration stabilized by the eighth day of intake suggesting that consumption of flaxseed or other lignan sources, once a day, may be sufficient to maintain plasma lignan concentrations. Effect of Processing. When flaxseed was incorporated into pancake, bread, muffin, and pizza dough, the mammalian lignan production from these products after in vitro fermentation was directly related to the level of flaxseed (6.2–13.2%) used in the formulation despite differences in the cooking times (10–40 min) and temperature (190–205°C) of processing (90). Furthermore, the urinary lignan excretion was the same in premenopausal women fed 25 g flaxseed either as raw seed or in a muffin or bread formulation (39). These data indicate that flaxseed lignans are stable during the processing of the above products (90). In contrast, only a 73–75% recovery of the SDG was observed in bread loaves baked with 0, 4, 8, or 12% added flaxseed meal (91), whereas almost complete recovery was observed when 82% pure SDG was incorporated into bread.

Safety Reproductive Indices. The safety of exposure to flaxseed during early life is of concern because lignans can be transferred to nursing offspring via mother’s milk (22) and have the potential to act as hormonal disruptors, affecting reproductive indices. Hence, we studied in rats the effect of maternal feeding of a 10% flaxseed diet during pregnancy and lactation and observed estrogenic effects on reproductive indices among female and male offspring (22,92). Reproductive indices measured included anogenital distance, age and weight at puberty onset, length of estrous cycle, and sex organ weights. Female offspring exposed to the 10% flaxseed diet had a decreased anogenital distance and an earlier onset of puberty. In addition, lengthened estrus cycles were observed at PND 50 and 132. Among males, exposure to the 10% flaxseed diet in utero and during suckling resulted in estrogenic effects including a

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lower birth weight and reduced weight gain. Also, sex organs were heavier than controls at adulthood (PND 132). Similar effects were observed among female and male offspring that were exposed to the 10% flaxseed diet in utero and during suckling and subsequently fed the same diet that their mothers received through to adulthood (PND 132). These findings suggest that early exposure, during the in utero and suckling periods, altered reproductive indices. In contrast, there were no differences in reproductive indices at PND 50 or 132 among female or male offspring that were fed flaxseed diets postweaning, thus providing evidence that pregnancy and/or lactation were the hormone-sensitive period in which flaxseed and its lignans exerted their effect. A subsequent study showed that exposure of offspring to a 5 or 10% flaxseed diet or the equivalent quantity of SDG in these flaxseed diets only during suckling did not alter any reproductive indices in male and female rats (93). Together, these studies support the conclusion that in utero and not suckling is the critical developmental period during which exposure to purified lignan or lignan in flaxseed can alter reproductive indices. Other studies have tested the effect of exposure to higher levels of flaxseed (20 or 40%) and also flaxmeal (13 or 26% defatted flaxseed) among male offspring during suckling through adulthood (PND 91) on reproductive indices including spermatogenesis and the histology of sex organs (94,95). At these levels of flaxseed or flaxmeal, a significant increase in serum luteinizing hormone and increases in cauda epididymal weight and cauda epidymal sperm numbers expressed per gram of epididymus were observed (95) but not in histological changes in spermatogenesis (94). The fact that similar effects were observed with the flaxseed and flaxmeal suggests that the lignan component mediated these changes (95). Future investigation is required to determine whether the changes observed are manifested throughout aging (i.e., increased risk of developing specific diseases). In addition, breeding studies are required to ultimately determine whether fertility is affected. Genotoxicity The lignans were tested for genotoxicity because of their chemical structural similarity to diethylstilbestrol, a known carcinogen (96). EL, ED, matairesinol, and SECO were tested at 200 µmol/L on cell-free microtubule assembly and at 100 µmol/L in cultured male Chinese hamster V79 cells at five different genetic end points, i.e. disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the hypoxanthine phosphoribosyltransferase (HPRT) locus (97). Results showed no aneuploidogenic and clastogenic potential at the levels of lignans used. In contrast, using the same methods, the isoflavones genistein and coumestrol have been shown to be genotoxic (98).

Summary and Conclusions Epidemiologic, animal, clinical, and in vitro studies provide evidence that lignans may protect against the development of several diseases, or attenuate the disease

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process. However, further studies are still required before a definitive conclusion regarding the health benefits of lignans can be made. Because there is a paucity of clinical intervention trials and the fact that many diseases (e.g., cancer, cardiovascular disease, or osteoporosis) have long latency periods, clinical feeding trials of longer duration (i.e., several years) are required to identify whether changes in specific biomarkers of diseases are manifested as a reduction in disease development. In addition, for certain diseases, it is imperative that investigators feed lignans as isolated compounds and compare these effects to those observed with feeding lignan-rich foods such as flaxseed. Such studies will elucidate the lignan-specific mechanisms of action, without confusion regarding whether observed effects are due to other biologically active components such as ALA in flaxseed. Moreover, as more lignans are being isolated and identified, feeding trials involving these lignans may reveal further health benefits and mechanisms of action. The metabolism and availability of lignans in a 2.5–10% flaxseed diet or of equivalent levels of purified lignans (i.e., SDG), as seen in animal and human studies, are sufficiently high to produce the health benefits. Exposure of offspring to lignans at the above levels during lactation or postweaning produced no adverse effects on reproductive indices. ED and EL have no genotoxic effects in in vitro studies although the genotoxicity of the other lignan metabolites has yet to be tested. These data indicate that moderate intake of the major lignans is safe. However, only long-term multigenerational studies in animals and then in humans will be able confirm this. Acknowledgments The cited works of the authors were funded by the Natural Sciences and Engineering Research Council, Health Canada, Flax Council, Saskatchewan Flax Development Commission, American Institute for Cancer Research, Cancer Research Society, Canadian Breast Cancer Research Foundation, National Cancer Institute, and National Institute of Nutrition (postdoctoral fellowship to W.W.).

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55. Ingram, A.J., Parbtani, A., Clark, W.F., Spanner, E., Huff, M.W., Philbrick, D.J., and Holub, B.J. (1995) Effects of Flaxseed and Flax Oil Diets in a Rat-5/6 Renal Ablation Model, Am. J. Kidney Dis. 25, 320–329. 56. Clark, W.F. (2000) A Novel Treatment for Lupus Nephritis: Lignan Precursor Derived from Flax, Lupus 9, 429–436. 57. Clark, W.F., Parbtani, A., Huff, M.W., Spanner, E., de Salis, E., Chin-Yee, I., Philbrick, D., and Holub, B. (1995) Flaxseed: A Potential Treatment for Lupus Nephritis, Kidney Int. 48, 475–480. 58. Clark, W.F., Kortas, C., Heidenheim, A.P., Garland, J., Spanner, E., and Parbtani, A. (2001) Flaxseed in Lupus Nephritis: A Two-Year Nonplacebo-Controlled Crossover Study, J. Am. Coll. Nutr. 20, 143–148. 59. Ogborn, M.R., Nitschman, E., Bankovic-Calic, N., Buist, R., and Peeling, J. (1998) The Effect of Dietary Flaxseed Supplementation on Organic Anion and Osmolytic Content and Excretion in Rat Polycystic Kidney Disease, Biochem. Cell Biol. 76, 553–559. 60. Ogborn, M.R., Nitschmann, E., Weiler, H., Leswick, D., and Bankovic-Calic, N. (1999) Flaxseed Ameliorates Interstitial Nephritis in Rat Polycystic Kidney Disease, Kidney Int. 55, 417–423. 61. MacLennan, A., Lester, S., and Moore, V. (2001) Oral Oestrogen Replacement Therapy Versus Placebo for Hot Flushes [Cochrane Review]. Cochrane Database Syst. Ref. 1, CD002978. 62. Brzezinski, A., Adlercreutz, H., Shaoul, R., Rosler, A., Shmueli, A., Tanos, V., and Schenker, J.G. (1997) Short-Term Effects of Phytoestrogen-Rich Diet on Postmenopausal Women, Menopause 4, 89–94. 63. Wilcox, G., Wahlqvist, M.L., Burger, H.G., and Medley, G. (1990) Oestrogenic Effects of Plant Foods in Postmenopausal Women, Br. Med. J. 301, 905–906. 64. St Germain, A., Peterson, C.T., Robinson, J.G., and Alekel, D.L. (2001) IsoflavoneRich or Isoflavone-Poor Soy Protein Does Not Reduce Menopausal Symptoms During 24 Weeks of Treatment, Menopause 8, 17–26. 65. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 66. Washburn, S., Burke, G.L., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13. 67. Baber, R., Templemen, C., and Moreton, T. (1997) A Study of Clover Extract as a Treatment of Menopausal Symptoms, Proc. Aust. Menopause Soc. Congr., p. 73. 68. Fugh-Berman, A., and Kronenberg, F. (2001) Red clover (Trifolium pratense) for Menopausal Women: Current State of Knowledge, Menopause 8, 333–337. 69. Baber, R., Templeman, C., Morton, T., Kelly, G.E., and West, L. (1999) Randomized Placebo-Controlled Trial of an Isoflavone Supplement and Menopausal Symptoms in Women, Climacteric 2, 85–92. 70. Knight, D., Howes, J.B., and Eden, J.A. (1999) The Effect of Promensil, an Isoflavone Extract on Menopausal Symptoms, Climacteric 2, 79–84. 71. Dalais, F.S., Rice, G.E., Wahlqvist, M.L., Grehan, M., Murkies, A.L., and Medley, G., (1998) Effects of Dietary Phytoestrogens in Postmenopausal Women, Climacteric 1, 124–129. 72. Arjmandi, B.H., Alekel, L., Hollis, B.W., Amin, D., Stacewicz-Sapuntzakis, M., Guo, P., and Kukreja, S. C. (1996) Dietary Soybean Protein Prevents Bone Loss in an Ovariectomized Rat Model of Osteoporosis, J. Nutr. 126, 161–167.

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73. Arjmandi, B.H., Birnbaum, R., Goyal, N.V., Getlinger, M.J., Juma, S., Alekel, L., Hasler, C.M., Drum, M.L., Hollis, B.W., and Kukreja, S.C. (1998) Bone-Sparing Effect of Soy Protein in Ovarian Hormone-Deficient Rats Is Related to Its Isoflavone Content, Am. J. Clin. Nutr. 68 (Suppl. 6), 1364S–1368S. 74. Fanti, P., Monier-Faugere, M. C., Geng, Z., Schmidt, J., Morris, P. E., Cohen, D., and Malluche, H.H. (1998) The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats, Osteoporos. Int. 8, 274–281. 75. Ishimi, Y., Arai, N., Wang, X., Wu, J., Umegaki, K., Miyaura, C., Takeda, A., and Ikegami, S. (2000) Difference in Effective Dosage of Genistein on Bone and Uterus in Ovariectomized Mice, Biochem. Biophys. Res. Commun. 274, 697–701. 76. Picherit, C., Coxam, V., Bennetau-Pelissero, C., Kati-Coulibaly, S., Davicco, M. J., Lebecque, P., and Barlet, J.P. (2000), Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats, J. Nutr. 130, 1675–1681. 77. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Purified Lignan from Flaxseed Alters Bone Development in Female Rats, Br. J. Nutr. 86, 499–505 78. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Flaxseed and Its Purified Lignan Reduces Bone Strength in Young but Not Older Male Rats, J. Toxicol. Environ. Health 63, 53–65. 79. Watkins, B.A., Shen, C.L., Allen, K.G., and Seifert, M.F. (1996) Dietary (n-3) and (n-6) Polyunsaturates and Acetylsalicylic Acid Alter Ex Vivo PGE2 Biosynthesis, Tissue IGF-I Levels, and Bone Morphometry in Chicks, J. Bone Miner. Res. 11, 1321–1332. 80. Watkins, B.A. (1998) Regulatory Effects of Polyunsaturates on Bone Modeling and Cartilage Function, World Rev. Nutr. Diet. 83, 38–51. 81. Babu, U.S., Mitchell, G.V., Wiesenfeld, P., Jenkins, M.Y., and Gowda, H. (2000) Nutritional and Hematological Impact of Dietary Flaxseed and Defatted Flaxseed Meal in Rats, Int. J. Food Sci. Nutr. 51, 109–117. 82. Kaup, S.M., Hight, S.C., Ahn, S.M., and Rader, J.I. (1994) Flaxseed and Mineral Metabolism in Rats, in Proc. 55th U.S. Flax Inst., pp. 29–38, U.S. Flax Institute, Fargo, ND. 83. Arjmandi, B.H., Juma, S., Lucas, E.A., Wei, L.L., Venkatesh, S., and Khan, D.A. (1998) Effects of Flaxseed Supplementation on Bone Metabolism in Postmenopausal Women, in Proc. 57th U.S. Flax Inst., pp. 65–74, U.S. Flax Institute, Fargo, ND. 84. Jacobs, E., and Metzler, M. (1999) Oxidative Metabolism of the Mammalian Lignans Enterolactone and Enterodiol by Rat, Pig, and Human Liver Microsomes, J. Agric. Food Chem. 47, 1071–1077. 85. Niemeyer, H.B., Honig, D., Lange-Bohmern, A., Jacobs, E., Kulling, S.E., and Metzler, M. (2000) Oxidative Metabolites of the Mammalian Lignans Enterodiol and Enterolactone in Rat Bile and Urine, J. Agric. Food Chem. 48, 2910–2919. 86. Jacobs, E., Kulling, S.E. and Metzler, M. (1999) Novel Metabolites of the Mammalian Lignans Enterolactone and Enterodiol in Human Urine, J. Steroid Biochem. Mol. Biol. 68, 211–218. 87. Rickard, S.E., and Thompson, L.U. (2000) Urinary Composition and Postprandial Blood Changes in 3H-Secoisolariciresinol Diglucoside (SDG) Metabolites in Rats Do Not Differ Between Acute and Chronic SDG Treatments, J. Nutr. 130, 2299–2305. 88. Hutchins A.M., Martini, M.C., Olson, B.A., Thomas, W., and Slavin, J.,L. (2000) Flaxseed Influences Urinary Lignan Excretion in a Dose-Dependent Manner in Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 9, 1113–1118.

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89. Rickard, S.E., and Thompson, L.U. (1998) Chronic Exposure to Secoisolariciresinol Alters Lignan Disposition in Rats, J. Nutr. 128, 615–623. 90. Nesbitt P.D., and Thompson, L.U. (1997) Lignans in Homemade and Commercial Products Containing Flaxseed, Nutr. Cancer 29, 222–227. 91. Muir, A.D., and Westcott, N.D. (2000) Quantitation of the Lignan Secoisolariciresinol Diglucoside in Baked Good Containing Flaxseed or Flax Meal, J. Agric. Food Chem. 48, 4048–4052. 92. Tou, J.C., Chen, J., and Thompson, L.U. (1999) Dose, Timing, and Duration of Flaxseed Exposure Affect Reproductive Indices and Sex Hormone Levels in Rats, J. Toxicol. Environ. Health 56, 555–570. 93. Ward, W.E., Chen, J., and Thompson, L.U. (2001) Exposure to Flaxseed or Its Purified Lignan During Suckling Only or Continuously Does Not Alter Reproductive Indices in Male and Female Offspring, J. Toxicol. Environ. Health 64, 567–577. 94. Sprando, R.L., Collins, T.F., Wiesenfeld, P., Babu, U.S., Rees, C., Black, T., Olejnik, N., and Rorie, J. (2000) Testing the Potential of Flaxseed to Affect Spermatogenesis: Morphometry, Food Chem. Toxicol. 38, 887–892. 95. Sprando, R.L., Collins, T.F., Black, T.N., Olejnik, N., Rorie, J.I., Scott, M., Wiesenfeld, P., Babu, U.S., and O’Donnell, M. (2000) The Effect of Maternal Exposure to Flaxseed on Spermatogenesis in F(1) Generation Rats, Food Chem. Toxicol. 38, 325–334. 96. Metzler M., Kulling, S.E., Pfeiffer, E., and Jacobs, E. (1998) Genotoxicity of Estrogens, Z. Lebensm.-Unters.-Forsch A 206, 367–373. 97. Kulling, S.E., Jacobs, E., Pfeiffer, E., and Metzler, M. (1998) Studies on the Genotoxicity of the Mammalian Lignans Enterolactone and Enterodiol and Their Metabolic Precursors at Various Endpoints In Vitro, Mutat. Res. 416, 115–124. 98. Kulling, S.E., Rosenber, B., Jacobs, E., and Metzler, M. (1999) The Phytoestrogens Coumestrol and Genistein Induce Structural Chromosomal Aberrations in Cultured Human Peripheral Blood Lymphocytes, Arch. Toxicol. 73, 50–54.

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Chapter 23

Phytoestrogens, Estrogens and Risk of Colon Cancer Maurice R. Bennink and Elizabeth A. Rondini Food Science and Human Nutrition, Michigan State University, East Lansing, MI

Introduction The intestine has not been considered a target tissue for estrogens because estrogen receptors (ER) were not initially found in the cytoplasm (1) or on plasma membranes (2) of intestinal epithelium. Even so, in 1946, Bullough (3) reported that mitosis in mouse colon varied during the estrous cycle and that estrogen stimulated mitosis in the colon mucosa. Thirty-three years later, Hoff and Chang (4) verified that estrogen influences colon epithelial cell kinetics in the descending mouse colon. Another indication that sex hormones may influence colon mucosa and thus colon cancer was the observation that male rats have a higher incidence of chemically induced colon cancer than females (5). The long-standing observation that the age-adjusted colon cancer incidence rates are higher in men than in women in the United States (6–8) also suggests that sex hormones influence colon cancer. In addition, men with the inherited condition, hereditary nonpolyposis colorectal cancer (HNPCC), have been found to have a higher penetrance of colon cancer than women (9–10). This led to the suggestion that some of the sex-related differences in the incidence of colon cancer, and in particular certain phenotypes, may be the result of prolonged estrogen exposure (11–13). Lastly, with the availability of more advanced techniques, it has now been demonstrated that ER are present in the colon mucosa. Thus, it seems reasonable that estrogen or estrogen-like substances may influence colon cancer. Epidemiology Studies: Female Reproductive Factors and Hormone Replacement Therapy McMichael and Potter (11) initially proposed a protective effect of either high fertility or exogenous hormonal exposure in women against the development of colon cancer. To date, most epidemiologic data support the hypothesis that hormone replacement therapy (HRT) protects against colon cancer (12,14–23). However, the data are less supportive for a role of other reproductive factors, i.e., age at menarche, age at menopause, or parity, in modulating colon cancer susceptibility (20,21,24–34). Using meta-analysis, Grodstein et al. (14) analyzed 18 epidemiology studies (10 prospective and 8 case-control) specifically for postmenopausal hormone use and risk of colon and rectal cancer in women. Compared with never-users, 13 of 18

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studies reported a decreased risk for ever-users of HRT with a summary relative risk (RR) of 0.80 (95% confidence interval [CI], 0.74–0.86). When the studies that included premenopausal women in the reference group as never-users were excluded, the RR was 0.76 (95% CI, 0.7–0.82). The protective effect of postmenopausal estrogen use is weak for duration of use (15–17,19,21,35) but the association appears to be stronger in current rather than ever-users (14,15,17,23,35). Also, users of HRT had less rectal cancer (RR 0.81, 95% CI, 0.72–0.92) compared with never-users (14). There is evidence to suggest that certain tumor genotypes may be particularly estrogen responsive. Breivik et al. (13) examined genetic alterations at three different loci in colon tumors in relation to age, gender, and location of the tumor (proximal colon vs. distal colon). They found that microsatellite instability (MIN), a mutator phenotype associated with some forms of hereditary colon cancer, was significantly more proximally located, and present with a much lower frequency among young women compared with men in the same age group and with older women. Slattery et al. (12) examined the influence of gender and estrogen-related factors on MIN status. They found that women were more likely to have MIN+ tumors at an older age and this association was confined to the proximal colon. Women with a history of pregnancy, oral contraceptive use, or HRT use were at a reduced risk of having MIN+ tumors. In addition, women with a higher body mass index and consequent increase in estrogen are at lower risk for developing MIN+ tumors. The authors proposed that the decrease in circulating estrogens that occurs during menopause may lead to a decrease in estrogen receptor expression and subsequently an increase in the incidence of MIN+ tumors (12). The epidemiologic data strongly support the hypothesis that estrogen inhibits one or more steps in the carcinogenic process and is therefore associated with a decrease in cancer risk, in part due to a decrease in MIN+ tumors. MIN+ tumors occur at a much higher frequency in the proximal colon than in other regions. However, there has been a substantial decline in the incidence of distal colon cancer, particularly among Caucasian women (7) since 1985. Some of this decline has been attributed to an increase in HRT, which suggests that HRT may potentially influence both MIN+ and MIN tumors. The anti-colon cancer mechanism of estrogen is not known, but it may be related to a decrease in cell proliferation in the colon mucosa (see next section). Estrogenic Actions on Colon Mucosa: Animal Models Weyant et al. (36) examined the effect of ovariectomy on intestinal carcinogenesis in MinAPC mice, a genetic model of inherited colon cancer. They found a significant increase (P = 0.0004) in intestinal tumor development in untreated, ovariectomized mice compared with sham-operated controls. Treatment of ovariectomized mice with estrogen pellets was sufficient to suppress tumorigenesis to control levels in the small intestine (P = 0.85). Ovariectomy was associated with a decrease in

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the amount of estrogen receptor α and an increase in estrogen receptor β protein (see next section) in the intestinal tissue. Estrogen has also been shown to modulate epithelial cell kinetics in the colon of mice. Hoff and Chang (4) examined the effect of ovariectomy and estrogen treatment on cell kinetics in the distal colon of female mice. They found that 3 wk after bilateral ovariectomy, there was a significant reduction in crypt size compared with intact mice (27.4 vs. 35.1 cells, P < 0.001). This was due primarily to a reduction in the differentiated cell compartment. After a single or multiple doses of estrogen treatment, the number of proliferating (3H-thymidine labeled) cells decreased compared with the untreated ovariectomized mice. Estrogen administration, however, was not sufficient to restore crypt size or the size of the differentiated cell compartment back to levels comparable to those of intact mice. More studies are required to clarify the role of estrogen in cell cycle control in the colon mucosa and to determine whether the action of estrogen is mediated through ER and, therefore estrogen-responsive genes. Estrogen Receptor and in the Colon and in Colon Cancer Cell Lines Normal human colon mucosa contains both the ERα and the ERβ subforms, with ERβ as the predominant form of estrogen receptor in the colon (37–40). ERα protein is present at low levels in normal and malignant tissue, with no variation related to gender (39,41). Thus, Foley et al. (39) suggested that the ERα does not play a major role in colon carcinogenesis. They found that ERβ protein is down-regulated in colon tumors compared with normal adjacent mucosa for both men and women (39). In addition, they reported that there were no differences in mRNA between normal and malignant tissue and there were no differences due to gender (39). They further suggested that the reduction of ERβ protein was due to post-transcriptional alterations. On the other hand, Campbell-Thompson et al. (40) reported that the mRNA for ERβ in malignant tissue was significantly decreased compared with normal adjacent tissue for women, but not for men. It should be noted that for men, the tumor tissue had less mRNA for ERβ than normal mucosa, but the differences were not large enough for the small sample size to achieve statistical significance. Also, ERβ mRNA in normal colon tissue was not altered by the age of the women. This is a very new area of research. These discrepancies will most likely be resolved in the future as more studies are conducted and as the mRNA specificity and methodologies continue to improve. Colon cancer cell lines, like normal colon mucosa, express predominantly the ERβ subform of the ER (40,42,43). The colon cancer cell lines HT-29, Caco-2, T84, DLD-1, Colo320, SW480, and LoVo all express ERβ (40,42,43), but two groups reported that cell growth is not influenced by estradiol at physiologic concentrations of 1–10 nmol/L (43,44). In contrast, Fiorelli et al. (42) reported that estradiol at 1 and 100 pmol/L stimulated growth of HCT8 cells, whereas 10–1000 nmol/L E2 inhibited growth of HCT 116 and DLD-1. Furthermore, from 1 pmol/L to 1000 nmol/L of estradiol inhibited growth of LoVo cells.

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The normal role for ERβ in colon mucosa is not known. Moreover, there are three isoforms of the ERβ in normal colon mucosa, i.e., ERβ1, ERβ2, and ERβ5 (38), which adds to the complexity for determining ER-mediated functions. Most colon cancer cell lines have depressed levels of ERβ compared with nontransformed cells. It is not clear at this time whether there is a selective loss of one isoform or if more than one isoform is depressed in transformed cells. Nevertheless, interpretation of results regarding estrogen and phytoestrogen action on these cell lines is tenuous due to our incomplete understanding of the role of the ERβ and the various isoforms. Ideally, normal, nontransformed colonocytes with normal levels of ERβ isoforms would be used for such studies. However, it is not currently possible to grow normal colonocytes in culture for more than a few replications before terminal differentiation occurs. Estrogen Receptor β Affinity for Phytoestrogens Plants produce compounds that bind to both ERα and ERβ and are therefore considered plant estrogens or “phytoestrogens.” These phytoestrogens are chemically divided into three main classes: flavonoids, coumestans, and lignans (45). All plant-produced phytoestrogens function as agonists to the ERα and ERβ receptors when assayed by using estrogen-responsive reporter gene constructs (45). The binding affinity of some phytoestrogens is higher for ERβ; however, there are differences in the degree of activation with genistein, for example, leading to only a partial agonist activity on ERβ (45,46). It should be mentioned that in these systems, the effect of ligand on estrogen response elements would be through homodimerization of the estrogen receptor. Agonist/antagonist-induced responses may depend on the relative proportion of ERα:ERβ in tissues (47) as well as ligandindependent and estrogen response element (ERE)-independent pathways (48–49). The concentration necessary for genistein and coumestrol to cause 50% activation of ERβ is ~6 nmol/L (45). Individuals who consume soy products frequently have plasma levels of genistein in the 10–1000 nmol/L range (50). Moreover, the colon mucosa is exposed to high concentrations of phytoestrogens in the luminal contents. Both dietary phytoestrogens and phytoestrogens from bile pass to the colon, are decongugated by the microflora, and are then available for absorption into the mucosal cells. Sung et al. (51) estimated that the colon mucosa may be exposed to as much as 100 µmol/L lignans. Similar arguments could be made for the soy isoflavones genistein and daidzein. Therefore, both genistein and coumesterol would be expected to exert strong activation of ERβ in colon mucosa if the phytoestrogen is consumed. People consuming soy products or isoflavone supplements would clearly have sufficient genistein in the colon and blood to elicit an estrogenic response via the ERβ. Much less is known about customary intakes of coumesterol, but the intake is most likely considerably less than that of genistein. Daidzein, biochanin A, apigenin, kaempferol, and naringenin are moderately strong ERβ agonists (45). Considering the intake of these phytoestrogens when

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legumes, fruits and vegetables are consumed, they could easily activate the ERβ in colonocytes. Quercetin and formononetin are relatively weak ERβ agonists (45), but quercetin could be physiologically important to the colon given the amounts of quercetin consumed. It is not known whether the plant and/or mammalian lignans are ERβ agonists. Phytoestrogens and Colon Cancer: In Vitro Studies The effect of phytoestrogens on the growth of colon cancer cells expressing ERβ has been studied. Genistein at 10 µmol/L caused a slight decrease in the growth of Colo 205 (52), Colo 320, HT 29, and LoVo (43) cells. Wang et al. (52) reported a slight inhibition in growth of Colo205 by 1 and 10 µmol/L biochanin A and a 8 and 20% reduction by 1 and 10 µmol/L of enterolactone, respectively. They did not find any growth inhibition by formononetin, daidzein, or coumesterol. Sung et al. (51) reported that enterodiol and enterolactone inhibited growth of LS 174T, HCT-15, T84, and Caco 2 cells by 20–85% at 100 µmol/L. It should be noted that if the concentrations of phytoestrogens have to be >1 µmol/L to be effective, the physiologic/biochemical effect is most likely not due to activation of the ERβ, but due to other actions such as inhibition of tyrosine kinase activity. The studies using colon cancer cell lines demonstrate that many phytoestrogens can be bound by the ERβ and cause transactivation. But there is little evidence to suggest that binding of phytoestrogens to the ERβ has any effect on cell growth. As discussed above, the true potential for phytoestrogen action on the colon epithelium may not be realized if colon cancer cell lines are the model because of down-regulation of one or more of the ERβ isoforms that occurs in transformed cells. Nakayama (44) found that the 50% inhibitory concentration (IC50) for tamoxifen by itself was ~15 mmol/L for the DLD-1, DLd-1/5FU, or DLD-/FdUrd cell lines. Quercitin by itself did not inhibit growth of these cells. However, the IC50 was 2–4 nmol/L for tamoxifen when these cell types were cultured in the presence of 10 mol/L quercitin. Moreover, the cell lines DLD-1/5FU and DLD-1/FdUrd are normally insensitive to 5FU, but culturing these cell in 1–10 nmol/L concentrations of tamoxifen plus 10 mol/L quercitin significantly increased the sensitivity to 5FU. This observation has great implications for adjuvant treatment for individuals with 5FU-resistant colon cancer Phytoestrogens and Colon Cancer Risk: Clinical Study One clinical intervention study was conducted to determine whether eating isolated soy protein containing phytoestrogens would alter biomarkers of colon cancer risk (53). Subjects at moderate risk of developing colon cancer (i.e., the subjects had colon polyps or cancer surgically removed from the colon before the study) were fed powdered supplements. The supplements had either 38 g of soy protein containing 70 mg of total isoflavones or 38 g of casein protein with no isoflavones. Biopsies of the colon mucosa were taken before the study started and after subjects

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had consumed the protein supplements for 1 y. There was a downward shift in the proliferation zone in the mucosa crypts and an increase in cell differentiation in subjects that consumed the supplement containing soy protein with isoflavones (Table 23.1). This indicates that they were at a significantly lower risk of developing colon cancer. For the subjects consuming the control supplement with casein for 1 y, there was no change in the proliferation zone or in cell differentiation. This indicates that the colon cancer risk was unchanged for the control subjects. Phytoestrogens and Colon Cancer Risk: Animal Studies Foods rich in isoflavones and lignans have been fed to laboratory animals to determine whether the foods containing phytoestrogens would inhibit chemically induced colon cancer. Feeding rats soy protein that contained isoflavones, i.e., isolated soy protein (54), full-fat soy flour (55), or defatted soy flour (56–57), significantly reduced azoxymethane (AOM)-induced colon cancer. The studies with fullfat or defatted soy flour have been repeated at least twice, included a large number of animals, and produced a consistent inhibition of colon tumorigenesis compared with either casein or soy concentrate with few isoflavones (Table 23.2). However, two studies did not find a reduction in chemically induced colon cancer when soy with isoflavones were fed. Davies et al. (58) fed isolated soy protein with isoflavones and found no difference in tumorigenesis compared with isolated soy protein without isoflavones. McIntosh et al. (59) reported that feeding soybean oil meal, which contains isoflavones, caused a nonsignificant (P = 0.12) increase in 1,2-dimethylhydrazine (DMH)-induced tumor incidence. Flaxseed and rye bran contain significant amounts of lignans. Feeding rye bran was found to reduce AOM-induced tumor incidence in rats and tumor number in MinAPC mice. Davies et al. (58) reported that feeding a 30% rye bran diet to F344 male rats resulted in a significant reduction in the incidence of AOM-induced tumors (16.7%) compared with rats fed soy protein with low isoflavone content (72.7%) or high isoflavone content (75%). Feeding rye bran (10%) to MinAPC mice significantly reduced polyp number throughout the small intestine and in particuTABLE 23.1 Cell Proliferation in Colon Mucosa Biopsies Before and After Consuming Casein or Soy Supplements for One Yeara Casein (n = 13)

Labeling index Proliferation zone aSource:

Soy protein isolate (n = 29)

Before

After

Before

After

0.285 0.486

0.282 0.473

0.308 0.528

0.257b 0.452b

Reference 53. cell nuclear antigen labeling index and proliferation zone decreased for the group consuming the soy supplement (P < 0.05), but not for the group consuming casein.

bProliferating

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TABLE 23.2 Colon Tumor Incidence, Number and Weight in Rats Fed Casein, Soy Concentrate, Defatted Soy Flour, or Full-Fat Soy Floura

aSource:

Diet

n

Tumor incidence (%)

Casein Soy concentrate Defatted soy flour Full-fat soy flour

80 80 98 73

68b 68b 43c 44c

Number of tumors/ rat/

Tumor weight (mg/rat)

1.43b 1.11b 0.67c 0.78c

68c 131b 36d 71c

Compiled from References 55–57. within a column with different superscripts are different (P < 0.05).

b–dMeans

lar, the distal small intestine (60). In two studies, flaxseed was fed sufficiently long to allow tumors to develop; the other studies utilizing flaxseed measured aberrant crypts as predictors of colon tumorigenesis. Gilbert (61) reported that feeding flax did not inhibit intestinal tumorigenesis (experimental details were not provided). Similarly, Bennink et al. (55) fed a diet containing 16.8% full-fat flaxseed and did not find a reduction in any aspect of tumorigenesis (tumor incidence, multiplicity, or burden) compared with controls (Table 3). Feeding 5 or 10% full-fat flaxseed or defatted flaxseed for 4 wk reduced AOM-induced aberrant crypt foci (ACF) by 48–57% (62). However, in a follow-up study, feeding 2.5 or 5% full-fat or defatted flax seed for 100 d did not produce a significant decrease in ACF (63). However, the average number of aberrant crypts per focus was significantly less for rats fed the flaxseed. Although soy, flax, and rye are rich sources of phytoestrogens, these foods also contain many other bioactive microconstituents that have been postulated to inhibit cancer. Thus, it cannot be concluded from the studies cited above that phyTABLE 23.3 Colon Tumor Incidence, Number and Weight in Rats Fed Full-Fat Flaxseed, Soy Concentrate, Defatted Soy Flour, Genistin, or a Mixture of Isoflavonesa

Diet Full-fat flaxseed Soy concentrate Defatted soy flour Soy concentrate plus genistin Soy concentrate plus isoflavone mixture aSource:

n

Tumor incidence (%)

Number of tumors/ rat/

Tumor weight (mg/rat)

29 30 29 26 24

62c 73c 48d 89b 63c

1.00b,c 1.20b 0.83c 1.54b 1.29b

124c 209b 105c 103c 81c

Modified from Reference 53; flaxseed data have been added. within a column with different superscripts differ (P < 0.05); the tumor incidence for the soy concentrate plus genistin group tended to differ at P = 0.06. b–dMeans

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toestrogens modulate colon tumorigenesis. Pure isoflavones or lignans were fed to determine whether phytoestrogens in soy, flax, or rye alter colon cancer in vivo. Genistein, genistin (the glycoside form of genistein), a mixture of isoflavones, and secoisolariciresinol diglycoside were fed to rats to determine whether the phytoestrogens would alter various aspects of chemically induced colon carcinogenesis. Dietary genistein at concentrations of 75 and 150 µg/g inhibited AOM-induced ACF in male rats by 29 and 36% (P < 0.05), respectively (64). Helms and Gallaher (65) found that feeding genistein (372 µg/g) reduced DMH-induced ACF by 35%. Thiagarajan et al. (66) fed 150 µg/g genistein and found a 43% reduction in the number of ACF. In contrast to the above, Gee et al. (67) fed genistein (250 µg/g) for 1 wk before injection of DMH and found a threefold increase in ACF. If the genistein was fed after DMH administration, there was no difference in ACF compared with control rats. Jenab and Thompson (63) reported that administering 1.5 mg secoisolariciresinol diglycoside/d by gavage for 100 d inhibited ACF formation in the distal colon. Collectively, these studies suggested that feeding purified phytoestrogens inhibits the very early stages of colon cancer promotion. Because ACF are very early lesions in the multistage process of colon cancer, and because most studies found a reduction in ACF when genistein was fed, it was hypothesized that dietary genistein would reduce AOM-induced colon tumorigenesis. However, Rao et al. (68) found that feeding genistein (250 µg/g diet) increased rather than decreased AOM-induced colon carcinogenesis in male F344 rats. Tumor incidence was similar in the control and genistein-fed rats, but the number of tumors per rat (1.35 vs. 2.03; P < 0.027) and the number of tumors/tumor-bearing rat (1.75 vs. 2.63; P < 0.004) increased. Bennink et al. (57) fed genistin (equivalent to 500 µg/g genistein, the amount contained in the defatted soy flour diet that inhibited colon tumorigenesis [55,56]) and found an increase in tumor incidence (P = 0.06, Table 23.3), which confirmed the report by Rao et al. (68) that dietary genistein increases colon carcinogenesis. Feeding a mixture of soy isoflavones (genistin, daidzin, and glycitin, in amounts comparable to what is found in defatted soy flour) did not inhibit or increase colon tumorigenesis (57). Whether secoisolariciresinol diglycoside would increase colon tumorigenesis similarly to genistein/genistin remains to be determined.

Summary and Conclusions The substantial difference in colorectal cancer incidence between females and males strongly suggest that sex hormones are important determinants of colon cancer risk. Epidemiologic studies also suggest that estrogens are protective against development of colon cancer. Although the colon is not typically considered an estrogen-responsive tissue, estrogen has been shown to exert physiologic effects on epithelial indices in the colon. Estrogen receptor β has now been detected in the colon, raising the possibility that some of the effects of estrogen may be mediated through it. Plants contain a variety of estrogens (phytoestrogens) that have been shown in in vitro systems to bind ERβ with a high affinity and to increase tran-

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scription of reporter genes when fused to estrogen response elements. There are at least three different ERβ isoforms found in colonocytes, but little is known about the degree of activation of the various isoforms when phytoestrogens are bound. Moreover, homodimers or heterodimers of the ER can form, ligand-independent pathways can be activated, and/or estrogen response element–independent pathways can be activated. Thus, an extremely complex control system exists, of which we know very little. Colon cancer cell lines have been cultured in the presence of phytoestrogens with little apparent consequence except at exceptionally high concentrations (10–100 µmol/L). Lack of information about the amounts of the various isoforms present in the cell lines and the other limitations addressed above constrain the usefulness of these data. Foods that contain high amounts of phytoestrogens including soy, flaxseed, and rye have been shown to inhibit tumorigenesis in rodents. Purified genistein and secoisolariciresinol diglycoside reduced very early precancerous lesions in the colon, but are not sufficient by themselves to decrease tumorigenesis. Although epidemiologic data suggest a role for estrogens in colon cancer inhibition, the experimental evidence to date does not indicate that phytoestrogens also inhibit tumorigenesis. Whether the appropriate study has not been conducted or whether phytoestrogens are simply unable to inhibit colon cancer is not known. It should be noted that almost all of the animal experiments evaluating phytoestrogen efficacy for cancer inhibition have been done with male rodents, thereby excluding the possibility of detecting gender-related differences in response to phytoestrogens. Lastly, the observation that genistein increased chemically induced colon cancer alerts us to the possibility that phytoestrogens consumed as pure compounds at levels found in food matrices may have adverse consequences. References 1. Jensen, E.V., and Desombre, E.R. (1973) Estrogen-Receptor Interaction, Science 182, 126–134. 2. Pietras, R.J., and Szego, C.M. (1977) Specific Binding-Sites for Estrogen at Outer Surfaces of Isolated Endometrial Cells, Nature 265, 69–72. 3. Bullough, W.S. (1946) Mitotic Activity in The Adult Female Mouse, Mus musculus L. A Study of Its Relation to the Oestrus Cycle in Normal and Abnormal Conditions, Phil. Trans. R. Soc. London Series B. Biol. Sci. 231, 453–516. 4. Hoff, M.B., and Chang, W.W.L. (1979) Effect of Estrogen on Epithelial-Cell Proliferation and Differentiation in the Crypts of the Descending Colon of the Mouse— Autoradiographic Study, Am. J. Anat. 155, 507–516. 5. Odagiri, E., Jibiki, K., Kato, Y., Nakamura, S., Oda, S.I., Demura, R., and Demura, H. (1985) Steroid-Receptors in Dimethylhydrazine-Induced Colon Carcinogenesis, Cancer. 56, 2627–2634. 6. Chu, K.C., Tarone, R.E., Chow, W-H., Hankey, B.F., and Ries, L.A.G. (1994) Temporal Patterns of Colorectal Cancer Incidence, Survival, and Mortality from 1950 Through 1990, J. Natl. Cancer Inst. 86, 997–1006.

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22. Jacobs, E.J., White, E., and Weiss, N.S. (1994) Exogenous Hormones, Reproductive History, and Colon-Cancer (Seattle, Washington, USA), Cancer Causes Control 5, 359–366. 23. Newcomb, P.A., and Storer, B.E. (1995) Postmenopausal Hormone Use and Risk of Large-Bowel Cancer, J. Natl. Cancer Inst. 87, 1067–1071. 24. Broeders, M.J.M., Lambe, M., Baron, J.A., and Leon, D.A. (1996) History of Childbearing and Colorectal Cancer Risk in Women Aged Less Than 60: An Analysis of Swedish Routine Registry Data 1960–1984, Int. J. Cancer 66, 170–175. 25. Fernandez, E., La Vecchia, C., Franceschi, S., Braga, C., Talamini, R., Negri, E., and Parazzini, F. (1998) Oral Contraceptive Use and Risk Of Colorectal Cancer, Epidemiology 9, 295–300. 26. Platz, E.A., Martinez, M.E., Grodstein, F., Fuchs, C.S., Colditz, G.A., Stampfer, M.J., and Giovannucci, E. (1997) Parity and Other Reproductive Factors and Risk of Adenomatous Polyps of the Distal Colorectum (United States), Cancer Causes Control 8, 894–903. 27. Peipins, L.A., Newman, B., and Sandler, R.S. (1997) Reproductive History, Use Of Exogenous Hormones, and Risk of Colorectal Adenomas, Cancer Epidemiol. Biomark. Prev. 6, 671–675. 28. Troisi, R., Schairer, C., Chow, W.H., Schatzkin, A., Brinton, L.A., and Fraumeni, J.F. (1997) Reproductive Factors, Oral Contraceptive Use, and Risk of Colorectal Cancer, Epidemiology 8, 75–79. 29. Martinez, M.E., Grodstein, F., Giovannucci, E., Colditz, G.A., Speizer, F.E., Hennekens, C., Rosner, B., Willett, W.C., and Stampfer, M.J. (1997) A Prospective Study of Reproductive Factors, Oral Contraceptive Use, and Risk of Colorectal Cancer, Cancer Epidemiol. Biomark. Prev. 6, 1–5. 30. Slattery, M.L., Mineau, G.P., and Kerber, R.A. (1995) Reproductive Factors and ColonCancer—The Influences of Age, Tumor Site, and Family History on Risk (Utah, United-States), Cancer Causes Control 6, 332–338. 31. Kvale, G., and Heuch, I. (1991) Is the Incidence of Colorectal-Cancer Related to Reproduction—A Prospective-Study of 63,000 Women, Int. J. Cancer 47, 390–395. 32. Newcomb, P.A., Taylor, J.O., and Trentham-Dietz, A. (1999) Interactions of Familial and Hormonal Risk Factors for Large Bowel Cancer in Women, Int. J. Epidemiol. 28, 603–608. 33. Davis, F.G., Furner, S.E., Persky, V., and Koch, M. (1989) The Influence of Parity and Exogenous Female Hormones on the Risk of Colorectal-Cancer, Int. J. Cancer 43, 587–590. 34. Talamini, R., Franceschi, S., Dal Maso, L., Negri, E., Conti, E., Filiberti, R., Montella, M., Nanni, O., and La Vecchia, C. (1998) The Influence of Reproductive and Hormonal Factors on the Risk of Colon and Rectal Cancer in Women, Eur. J. Cancer 34, 1070–1076. 35. Troisi, R., Schairer, C., Chow, W.H., Schatzkin, A., Brinton, L.A., and Fraumeni, J.F. (1997) A Prospective Study of Menopausal Hormones and Risk of Colorectal Cancer (United States), Cancer Causes Control 8, 130–138. 36. Weyant, M.J., Carothers, A.M., Mahmoud, N.N., Bradlow, H.L., Remotti, H., Bilinski, R.T., and Bertagnolli, M.M. (2001) Reciprocal Expression of ER Alpha and ER Beta Is Associated with Estrogen-Mediated Modulation of Intestinal Tumorigenesis, Cancer Res. 61, 2547–2551.

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37. Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz, S., Lagercrantz, J., Fried, G., Nordenskjold, M., and Gustafsson, J.A. (1997) Human Estrogen Receptor Beta-Gene Structure, Chromosomal Localization, and Expression Pattern, J. Clin. Endocrinol. Metab. 82, 4258–4265. 38. Moore, J.T, McKee, D.D., Slentz-Kesler, K., Moore, L.B., Jones, S.A., Horner, E.L., Su, LJ., Kliewer, S.A., Lehmann, J.M., and Wilson, T.M. (1998) Cloning and Characterization of Human Estrogen Receptor β Isoforms, Biochem. Biophys. Res. Commun. 247, 75–78. 39. Foley, E.F., Jazaeri, A.A., Shupnik, M.A., Jazaeri, O., and Rice, L.W. (2000) Selective Loss of Estrogen Receptor Beta in Malignant Human Colon, Cancer Res. 60, 245–248. 40. Campbell-Thompson, M., Lynch, I.J., and Bhardwaj, B. (2001) Expression of Estrogen Receptor (ER) Subtypes and ER Beta Isoforms in Colon Cancer, Cancer Res. 61, 632–640. 41. Singh, S., Sheppard, M.C., and Langman, M.J.S. (1993) Sex-Differences in the Incidence of Colorectal-Cancer—An Exploration of Estrogen and Progesterone Receptors, Gut 34, 611–615. 42. Fiorelli, G., Picariello, L., Martineti, V., Tonelli, F., and Brandi, M.L. (1999) Functional Estrogen Receptor Beta in Colon Cancer Cells, Biochem. Biophys. Res. Commun. 261, 521–527. 43. Arai, N., Strom, A., Rafter, J.J., and Gustafsson, J.A. (2000) Estrogen Receptor Beta mRNA in Colon Cancer Cells: Growth Effects of Estrogen and Genistein, Biochem. Biophys. Res. Commun. 270, 425–431. 44. Nakayama, Y., Sakamoto, H., Satoh, K., and Yamamoto, T. (2000) Tamoxifen and Gonadal Steroids Inhibit Colon Cancer Growth in Association with Inhibition of Thymidylate Synthase, Survivin and Telomerase Expression Through Estrogen Receptor Beta Mediated System, Cancer Lett. 161, 63–71. 45. Kuiper, G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van Der Saag, P.T., Van Der Burg, P., and Gustafsson, J.A. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor Beta, Endocrinology 139, 4252–4263. 46. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J-A., and Nilsson, S. (1998) Differential Response of Estrogen Receptor α and Estrogen Receptor β to Partial Estrogen Agonists/Antagonists, Mol. Pharmacol. 54, 105–112. 47. Hall, J.M., and McDonnell, D.P. (1999) The Estrogen Receptor Beta-Isoform (ER Beta) of the Human Estrogen Receptor Modulates ER Alpha Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens, Endocrinology 140, 5566–5578. 48. Hall, J.M., Couse, J.F., and Korach, K.S. (2001) The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling, J. Biol. Chem. 276, 36869–36872. 49. Paech, K., Webb, P., Kuiper, G., Nilsson, S., Gustafsson, J.A., Kushner, P.J., and Scanlan, T.S. (1997) Differential Ligand Activation of Estrogen Receptors ER Alpha and ER Beta at AP1 Sites, Science 277, 1508–1510. 50. Adlercreutz, H., Markkanen, H., and Watanabe, S. (1993) Plasma-Concentrations of Phyto-Estrogens in Japanese Men, Lancet 342, 1209–1210. 51. Sung, M.K., Lautens, M., and Thompson, L.U. (1998) Mammalian Lignans Inhibit the Growth of Estrogen-Independent Human Colon Tumor Cells, Anticancer Res. 18, 1405– 1408. 52. Wang, W.Q., Liu, L.Q., Higuchi, C.M., and Chen, H.W. (1998) Induction of NADPH: Quinone Reductase by Dietary Phytoestrogens in Colonic Colo205 Cells, Biochem. Pharmacol. 56, 189–195.

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53. Bennink, M.R. (2001) Dietary Soy Reduces Colon Carcinogenesis in Human and Rats. Nutrition and Cancer Prevention: New Insights into the Role of Phytochemicals, Adv. Exp. Med. Biol. 492, 11–17. 54. Hakkak, R., Korourian, S., Ronis, M.J.J., Johnston, J.M., and Badger, T.M. (2001) Soy Protein Isolate Consumption Protects Against Azoxymethane-Induced Colon Tumors in Male Rats, Cancer Letters. 166, 27–32. 55. Bennink, M.R., Om, A.S., and Miyagi, Y. (2000) Inhibition of Colon Cancer (CC) by Wheat Bran, Soy Flour, and Flax, FASEB J. 14, A217 (abstr.). 56. Bennink, M.R., and Om, A.S. (1998) Inhibition of Colon Cancer (CC) by Soy Phytochemicals but Not by Soy Protein, FASEB J. 12, A3808 (abstr.). 57. Bennink, M.R., Om, A.S., and Miyagi, Y. (1999) Inhibition of Colon Cancer (CC) By Soy Flour but Not by Genistin or a Mixture of Isoflavones, FASEB J. 13, A50 (abstr.). 58. Davies, M.J., Bowey, E.A., Adlercreutz, H., Rowland, I.R., and Rumsby, P.C. (1999) Effects of Soy or Rye Supplementation of High-Fat Diets on Colon Tumour Development in Azoxymethane-Treated Rats, Carcinogenesis 20, 927–931. 59. McIntosh, G.H., Regester, G.O., Leleu, R.K., Royle, P.J., and Smithers, G.W. (1995) Dairy Proteins Protect Against Dimethylhydrazine-Induced Intestinal Cancers in Rats, J. Nutr. 125, 809–816. 60. Mutanen, M., Pajari, A.M., and Oikarinen, S.I. (2000) Beef Induces and Rye Bran Prevents the Formation of Intestinal Polyps in Apc(Min) Mice: Relation to BetaCatenin and PKC Isozymes, Carcinogenesis 21, 1167–1173. 61. Gilbert, J.M. (1987) Experimental Colorectal-Cancer as a Model of Human-Disease, Ann. R. Coll. Surg. Engl. 69, 48–53. 62. Serraino, M., and Thompson, L.U. (1992) Flaxseed Supplementation and Early Markers of Colon Carcinogenesis, Cancer Lett. 63, 159–165. 63. Jenab, M., and Thompson, L.U. (1996) The Influence of Flaxseed and Lignans on Colon Carcinogenesis and Beta-Glucuronidase Activity, Carcinogenesis 17, 1343–1348. 64. Pereira, M.A., Barnes, L.H., Rassman, V.L., Kelloff, G.V., and Steele, V.E. (1994) Use of Azoxymethane-Induced Foci of Aberrant Crypts in Rat Colon to Identify Potential Cancer Chemopreventive Agents, Carcinogenesis 15, 1049–1054. 65. Helms, J.R., and Gallaher, D.D. (1995) The Effect of Dietary Soy Protein Isolate and Genistein on the Development of Preneoplastic Lesions (Aberrant Crypts) in Rats, J. Nutr. 125, 802S. 66. Thiagarajan, D.G., Bennink, M.R., Bourquin, L.D., and Kavas, F.A. (1998) Prevention of Precancerous Colonic Lesions in Rats by Soy Flakes, Soy Flour, Genistein, and Calcium, Am. J. Clin. Nutr. 68, 1394S–1399S. 67. Gee, J.M., Noteborn, H., Polley, A.C.J., and Johnson, I.T. (2000) Increased Induction of Aberrant Crypt Foci by 1,2-Dimethylhydrazine in Rats Fed Diets Containing Purified Genistein or Genistein-Rich Soya Protein, Carcinogenesis 21, 2255–2259. 68. Rao, C.V., Wang, C.X., Simi, B., Lubet, R., Kelloff, G., Steele, V., and Reddy, B.S. (1997) Enhancement of Experimental Colon Cancer by Genistein, Cancer Res. 57, 3717–3722.

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Chapter 24

Phytoestrogen Actions in the Breast and Uterus Charles E. Wooda, Stephen Barnesb, and J. Mark Clinea aDepartment

of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Comparative Medicine Clinical Research Center, Winston-Salem, NC bDepartment

of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL

Introduction Phytoestrogens are a diverse group of naturally occurring compounds present in a wide variety of grains, legumes, fruits, and vegetables (1). The isoflavones, lignans, and coumestans are the predominant classes of phytoestrogens in the human diet, and soy is the major source of phytoestrogen supplementation in the United States. Soy-based foods, rich in the isoflavones genistein and daidzein, are important components of the Asian diet but are relatively scarce in the typical Western diet; however, consumption of soy foods by health conscious individuals in the United States is rapidly increasing (2). Interest in phytoestrogens as cancer-preventive agents has grown recently alongside the emerging awareness that diet is important in the etiology of many chronic diseases. The focus on phytoestrogens is based in part upon a number of epidemiologic studies suggesting important health benefits from a soy-based diet. Notable examples include migrant studies showing that women of Japanese origin have higher breast cancer rates when they migrate to the United States and adopt Western diets and lifestyles, compared with the rates in Japan [see, e.g. (3,4)]. Several case-control studies of soy intake and breast cancer risk also point to a protective effect of soy, particularly in premenopausal women (5). In addition to these observational findings, growing evidence from a number of in vitro and animal studies has indicated that phytoestrogens, and soy isoflavones in particular, may have consequential effects, both beneficial and adverse, relevant to our understanding of diet and cancer (6). A particularly challenging problem in the field of women’s health is the need for a safe form of estrogen replacement for women at high risk for breast cancer. Soy phytoestrogens (SPEs), as compounds with demonstrated estrogen agonist and antagonist, hormone-modulating, and antiproliferative actions, are being actively explored as natural alternatives to traditional estrogen replacement therapy in postmenopausal women (7). This interest is supported by the recent consensus statement on estrogen deficiency in women surviving breast cancer, which recommended phytoestrogens as potentially important new agents for long-term treatment of breast cancer sur-

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vivors (8). The following review examines the various cancer-related actions and effects of phytoestrogens, with emphasis on soy isoflavones, and explores their role in the prevention and management of breast and uterine cancer. Phytoestrogens: Mechanisms of Action in the Breast and Uterus Phytoestrogens have a wide range of biochemical and biological actions, many of which relate to their potential role in cancer. Selected cancer-related effects of SPE are listed in Table 24.1. Phytoestrogen activities are divided into three major categories, which reflect their roles as hormonal, nonhormonal, and hormone-modulating agents. Estrogen-Like Actions. Phytoestrogens are nonsteroidal compounds that have structural and functional similarities to endogenous estrogens. In vitro, phytoestrogens have been demonstrated to bind estrogen receptors (ER) competitively, induce expression of estrogen markers, stimulate proliferation of estrogen-sensitive tumor cells, and exhibit inhibition by antiestrogens such as tamoxifen (9–13). Collectively, phytoestrogens display a wide range of estrogenic potencies, although they are generally considered to be weaker than mammalian estrogens. The soy isoflavone genistein, for example, has ~10 times less affinity for ER-α relative to 17β-estradiol (E2) in cell-free competitive binding assays (11) and estimated estrogenic activities 100–1000 times lower than E2, based on stimulation of alkaline phosphatase activity in endometrial (Ishikawa) (14) and MCF-7 cells (15). TABLE 24.1 Phytoestrogen Activities Relevant to Cancer Preventiona Estrogen-like actions Selective ER-β agonism Estrogen antagonism MCF-7 cell proliferation Inhibition by antiestrogens Modulation of sex steroid metabolism Decreased serum estradiol, 16-hydroxyestrogens Inhibition of enzymes of estradiol biosynthesis Sulfatase/sulfotransferase inhibition Enhanced SHBG synthesis Antiproliferative/Apoptotic actions Inhibition of protein tyrosine kinase Antioxidant activity Inhibition of DNA topoisomerase Induction of tumor cell differentiation, G2/M arrest Induction of TGF-β1, p21WAF1/CIP1, wt p53 Suppression of COX, c-fos, heat shock proteins Inhibition of angiogenesis aER,

estrogen receptor; SHBG, steroid hormone binding globulin; TGF, transforming growth factor; COX, cyclooxygenase.

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The recent discovery of a second estrogen subtype, called ER-β (16), has opened up an exciting new area of research on the actions and regulation of estrogen in the body. Phytoestrogens typically act as agonists for both ER subtypes, ER-α and ER-β (11,13). However, certain compounds, including genistein, have binding affinities for ER-β that are 20–30 times greater than those for ER-α (11). ER-α and ER-β are expressed differently throughout the body. Although ER-α mRNA is more abundant in the uterus, ER-β protein expression has recently been shown to be significantly increased in human breast cancers (17). These differences suggest a potential mechanism for tissue-specific phytoestrogen effects on cancer risk. Genistein exhibits biphasic effects in vitro that are variably mediated by ER [see, e.g. (10,12,13,15)]. For example, at concentrations <5 µmol/L, genistein stimulates proliferation of estrogen-dependent MCF-7 breast cancer cells (10,13) but not hormone-insensitive MDA-MB-27 cells (12); however, at concentrations >5–10 µmol/L, genistein inhibits growth and initiates cell death of both estrogen-sensitive (13) and estrogen-insensitive breast cancer cells (18). Antiestrogens such as hydroxytamoxifen have also been shown to inhibit the proliferative effects of genistein (9,13). This evidence suggests that the low-dose stimulatory effects of genistein are an ER-mediated process, whereas the higher-dose inhibitory actions are independent of ER. Antiproliferation and Proapoptotic Effects. SPEs have potent antiproliferative and proapoptotic effects on various cancer cell lines. One of the earliest reported cellular actions of genistein was inhibition of protein tyrosine kinase (PTK) (19). PTK is important for many cell growth–signaling pathways, and its inhibition may regulate cancer cell proliferation in several ways. For example, genistein has been shown to significantly counteract growth stimulation of MCF-7 cells by estradiol, transforming growth factor (TGF)-α, insulin-like growth factor (IGF)-I, and IGFII, all of which promote breast cancer cell growth through PTK activation (12). In another example, genistein was shown to bind epidermal growth factor (EGF) in MDA-MB-231 and BT-20 breast cancer cells and subsequently inhibit EGF receptor–associated tyrosine kinase and multiple protooncogene PTKs, thus inducing apoptosis (20). Other antiproliferative and/or proapoptotic actions of genistein include the following: irreversible interaction with DNA topoisomerases during replication, leading to DNA strand breaks and apoptosis (21–25); induction of cancer cell differentiation (23,26–28); increased arrest of breast cancer cells in the G2-M stage of the cell cycle (29–32); induction of the inhibitory cell cycle regulator p21WAF1/CIP1 (18); reduced expression of heat shock proteins, which would otherwise protect cancer cells from undergoing apoptosis (33); induction of the wild-type tumor suppressor p53 (32); and suppression of induced transcriptional activity of cyclooxygenase (34). Additionally, genistein is reported to increase expression of TGF-β1 in normal human mammary epithelial cells at concentrations <5 µmol/L (35,36). TGF-β1 is an inhibitory growth factor that regulates a variety of pathways in cellular proliferation and differentiation [see, e.g. (37)].

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High doses of dietary isoflavones have also been shown to significantly reduce pulmonary metastatic tumors in mice injected with B16BL6 melanoma cells (38), supporting previous studies reporting an antimetastatic effect of soy (39,40). Possible mechanisms for this effect include inhibition of endothelial cell proliferation and angiogenesis (31,41) and PTK [see, e.g. (19)], which may interrupt integrin-mediated cell adhesion (42). Shao et al. (31) also found decreased expression of matrix metalloproteinase 9 and increased tissue inhibitor of metalloproteinases (TIMP) with genistein treatment of breast cancer cells; these effects would tend to impede tumor cell invasion and metastasis. SPEs may also inhibit tumor initiation through various antioxidative effects. In vitro studies have shown genistein at concentrations of 15–30 µmol/L to be a potent inhibitor of phorbol ester-induced hydrogen peroxide production in normal human leukocytes and HL-60 leukemia cells (43). Similarly, genistein, daidzein, and several flavones have shown significant inhibition of superoxide anion production by phorbol acetate-activated HL-60 cells (44). These cells produce the proinflammatory oxidants, peroxynitrite and hypochlorous acid, as part of their respiratory burst. Isoflavones rapidly react with these oxidants to produce 3′-nitroisoflavones and several isomers of chloroisoflavones, respectively (45,46). Activated neutrophils completely convert genistein (10 µmol/L) to metabolic products in 30 min, suggesting that at the level of target cells in inflammatory tissue, the local metabolites may exert a very different effect compared with that observed in cell culture (46). The antioxidant effects of SPEs have been demonstrated in both in vitro and in vivo studies. Using Cu2+-induced oxidation of LDL, Hodgson et al. (47) showed that although daidzein and genistein had antioxidant effects in the low µmol/L range, for the daidzein metabolites equol and O-desmethylangolensin, the antioxidant effect occurred at an order of magnitude lower (100 nmol/L). This may be an underestimate of antioxidant effects because it was shown recently that genistein acts synergistically with vitamin C (48,49). Genistein is also reported to induce expression of the antioxidant protein metallothionein in human colon cancer cells at 100 µmol/L (50), and a recent clinical study revealed that administration of soy isoflavones in men and women leads to a 60% reduction in serum markers of DNA oxidation (51). It is unclear whether these higher-dose antiproliferative, proapoptotic, and antioxidative mechanisms are responsible for the epidemiologic link between soy intake and decreased risk of hormone-dependent cancers. A typical vegetarian or high-soy diet providing a daily isoflavone content of 20–75 mg results in average total plasma isoflavone concentrations of ~0.5 µmol/L in Japanese men (52) and women (53), although peak serum isoflavone concentrations from a high-soy meal may transiently rise above 3 µmol/L (54). Interestingly, a recent study by Urban et al. (55) revealed total mean fasting isoflavone concentrations of ~1.5 µmol/L in American men consuming a daily soy beverage with 69 mg of isoflavones, suggesting a difference between Japanese and American soy consumers. These concentrations given to cells in vitro would not have profound anticancer effects.

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However, because of the large differences in gene expression profiles (56), cell phenotype, endocrinology, and paracrine milieu between cultured cells in vitro and normal or neoplastic cells in vivo, the predictive value of dose effects in cell culture experiments is limited. For example, large differences have been found between measured amounts of isoflavone intake and excretion, reflecting extensive metabolic conversion in animals and humans; certain isoflavone metabolites, many of which have not even been identified, may have important biological effects, and important metabolite differences may exist between in vitro and in vivo cell populations (46) (as well as between species and even individuals). Concentration of isoflavones within specific tissues may also occur, as demonstrated by a recent tissue distribution study using [14C] genistein in rats (57), but local tissue isoflavone concentrations are not well explored. Clearly, experimental work in humans or animal subjects is required to better determine the clinical relevance of in vitro studies. Sex Steroid Hormone Modulation. 17β-Estradiol (E2) is an important hormone in breast and uterine carcinogenesis. Increased lifetime estrogen exposure is associated with elevated risk for breast cancer (58), and exogenous estrogen increases endometrial cancer risk (59). Phytoestrogens, by affecting sex steroid production and/or interconversion, may exert significant effects on both circulating and local tissue concentrations of estradiol. Data from several human studies suggest that dietary intake of soy isoflavones may diminish circulating ovarian hormones in premenopausal women. For example, daily consumption of ~3 mg/kg body of soy milk isoflavones for 1 mo by healthy premenopausal women significantly decreased serum estradiol and luteal phase progesterone without affecting gonadotropin levels (60,61). However, this may be more of a soy effect rather than an isoflavone effect because a recent study by the same authors revealed similar hormonal decreases using an isoflavone-free soy diet (62). Other studies in premenopausal women using daily isoflavone doses of 1 mg/kg have found either an increase in serum estradiol (63,64) or no change (65–67). Recently, two studies reported that dietary soy isoflavones may modulate serum ratios of different estrogen metabolites, reducing the production of mutagenic forms such as 16-hydroxyestrogen that can damage DNA and induce tumors (68,69). SPEs have also been reported to elevate serum steroid hormone binding globulin (SHBG) levels, potentially reducing the bioavailable estradiol in circulation (70). Increased SHBG has been observed in studies of premenopausal women consuming soy milk and purified soy isoflavones (72); however, several studies examining intake of whole-soy products or other phytoestrogen-containing foods found no effects on SHBG levels in either premenopausal (63,64,66,73) or postmenopausal (64,74) women, possibly reflecting competing effects between isoflavones and other compounds in soy. Highly variable or conflicting outcomes in such studies may also reflect individual differences in human responses to phytoestrogens. For example, a recent study by Duncan et al. (75) found significantly lower plasma steroid hormone concentrations in premenopausal women excreting

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equol, a potent soy isoflavone that is assimilated and absorbed only by certain individuals. This finding was present independent of soy isoflavone consumption, emphasizing the importance of constitutive individual differences in phytoestrogen metabolism in addition to intake. In postmenopausal women, virtually all estradiol is synthesized peripherally from adrenal precursors via pathways depicted in Figure 24.1. Breast tumor cells typically have increased levels of estradiol, which is generally attributed to in situ production [see, e.g. (76)]. Multiple biochemical and in vitro studies have shown that a variety of phytoestrogens may modulate key enzymes involved in peripheral (extraovarian) biosynthesis of estradiol. Table 24.2 shows the 50% inhibitory concentration (IC50) values for selected phytoestrogens against aromatase, the enzyme responsible for converting testosterone to estradiol and androstenedione to estrone. Although certain lignan and flavonoid compounds demonstrate potent antiaromatase action (77–83), soy isoflavones (genistein, daidzein, and equol) appear to have little or no inhibition against aromatase (77,84–86). This in vitro evidence does not preclude indirect mechanisms of aromatase inhibition by soy, for example, via induction of TGF-β1. In contrast, isoflavones show strong inhibition of certain enzymes in the hydroxysteroid dehydrogenase (HSD) family, as seen in

Fig. 24.1. Pathways of estradiol biosynthesis. The 17β-HSD family has recently been shown to contain multiple isozymes, which catalyze either reductive (e.g., E1 to E2) or oxidative (e.g., E2 to E1) reactions. Abbreviations: DHEA, dehydroepiandrosterone; HSD, hydroxysteroid dehydrogenase.

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TABLE 24.2 IC50 Values of Selected Phytoestrogens Against Aromatasea Enzyme

Study/ Ref.

Selected phytoestrogens

IC50 (mol/L)

Aromatase

(84)

Biochanin A Genistein

94.5 nid

(82)

Apigenin Chrysin Hesperetin 7-Hydroxyflavone Apigenin Coumestrol Genistein Daidzein Flavone Equol

0.9 1.1 1 0.2 2.9 25 nid nid 375 850

Apigenin Equol Daidzein Genistein Coumestrol Enterolactone Isoflavone (O-DMA) Enterolactone Equol Daidzein Chrysin Daidzein Genistein Equol 7-Hydroxyflavone Chrysin Isoflavone 7,8-Benzoflavone Chrysin

84 793 >>1000 3500 17 74 nid 14 150 >100 4.6 nid nid nid 0.5 100 >200 0.07 0.5

(77)

(85)

(79)

(80)

(86)

(78)

(81)

Tissue and substrate Human placental microsomes Androstenedione AG = 6.5 Human placental microsomes Androstenedione AG = 0.4 Human placental microsomes Androstenedione AG = 1

Human placental microsomes Androstenedione AG = 130 Rainbow trout ovarian aromatase Androstenedione AG = 39 Human preadipocytes Androstenedione AG = 5 Human placental microsomes Androstenedione Human preadipocytes Androstenedione

Human placental microsomes Androstenedione AG = 2.2 Human placental microsomes Androstenedione

aIC

50 is the concentration at which 50% inhibition of the compound occurs; nid, no inhibition detected; AG, IC50 value for aminoglutethimide (AG), a pharmaceutical aromatase inhibitor used as a positive control in several studies; O-DMA, O-desmethylangolensin.

Table 24.3. Genistein, for example, significantly inhibits reductive 17β-HSD activity (converting estrone to estradiol) at concentrations ≤1 µmol/L (77,87,88) and 3βHSD at concentrations ≤10 µmol/L (83,89). Structural comparisons suggest that this inhibition is associated with the presence of a phenolic B ring in the 3-position of the pyran ring, also present in daidzein, coumestrol, and biochanin A (83). It should be noted that 11 different types of 17β-HSD have now been characterized

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TABLE 24.3 IC50 Values of Selected Phytoestrogens Against Enzymes Modulating Estradiol Biosynthesisa Enzyme

Study/ Ref.

Selected phytoestrogens

IC50 (mol/L)

17β-HSD

(77)

Coumestrol

0.2

(88)

Apigenin 7-Hydroxyflavone Genistein Daidzein Genistein

0.3 0.9 1 10 0.06

(87)

Biochanin A Daidzein 7-Hydroxyflavone Genistein (type 1)

0.06 0.07 >10 ~1.0

(92)

Coumestrol Genistein (type 5)

~1.0 >20

Daidzein

>20

(83)

Genistein

2.9

(89)

Daidzein Biochanin A Coumestrol Biochanin A

10 10 >50 7.5

Genistein Daidzein

10 11

Daidzein-sulfate Genistein Daidzein Daidzein-4′-O-sulfate Daidzein-7,4′-O-sulfate Daidzein

8.6 >25 >50 6 1.5 nid

(100)

3′,4′-OH-Flavone 3′,4′,7-OH-Isoflavone Quercetin Genistein Daidzein Quercetin

0.02 0.08 0.1 0.3 0.4 0.1

(EST)

Estradiol

3β-HSD

Sterol sulfatase

(95)

(89)

Sulfotransferases

(95) (SULT1A1)

aIC 50

Tissue and substrate Human placental microsomes Androstenedione

P. testosteronii purified β-HSD Testosterone

Wild-type T-47D breast cancer cells Estrone Purified recombinant 17β-HSD type 5 Androstenedione Human placental microsomes DHEA

Bovine adrenal microsomes DHEA Human platelets Estrone sulfate Hamster liver microsomes DHEA(-S) Human platelets Estradiol

Human mammary epithelial cells

is the concentration at which 50% inhibition of the compound occurs; nid, no inhibition detected; HSD, hydroxysteroid dehydrogenase; GSF, genital skin fibroblasts; DHEA(-S), dehydroepiandrosterone (-sulfate); SULT1A1, phenol sulfotransferase; EST, estrogen sulfotransferase.

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(90). Of those with well-defined functions, types 1, 3, 5, and 7 are considered to catalyze the conversion of inactive 17-keto-steroids into their active 17β-hydroxyforms (namely, testosterone and estradiol), whereas types 2 and 4 catalyze the reverse reactions (91). It seems likely that significant differences in inhibition could exist between specific 17β-HSD isotypes, as indicated by studies using types 1 and 5 (77,92). However, no published studies have specifically compared phytoestrogen effects on multiple 17β-HSD types, and it is currently unclear how isoenzyme interactions in vivo may affect estradiol biosynthesis. Modulation of the sulfation and sulfohydrolysis pathways represents another potentially important mechanism of phytoestrogen action. Sulfohydrolysis of estrone sulfate is reported to be a major source of estrogen in breast tumors (93,94). Although phytoestrogens have not demonstrated strong inhibitory effects in vitro on estrone sulfatase activity (95,96), their in vivo effects may be more potent. Significant amounts of dietary isoflavones such as daidzein circulate in sulfated forms (97,98), and these sulfoconjugates may act as competitive inhibitors for estrone sulfatase (99). An intriguing recent finding indicates potent inhibition of estrogen sulfotransferase (EST) (100) and phenol sulfotransferase (SULT1A1) (95) by various flavones and isoflavones. Sulfotransferases are widely involved in the metabolism, elimination, and bioactivation of numerous compounds, including endogenous hormones and xenobiotics [see, e.g. (101)]. EST is the only known sulfotransferase expressed in normal human breast epithelial cells, whereas SULT1A1 is reportedly the major enzyme responsible for estrone sulfation in human breast carcinoma cells (102,103). Phytoestrogen inhibition of these enzymes, therefore, could theoretically increase local tissue estradiol concentrations and increase the risk of breast cancer. However, sulfotransferases have a wide range of other potentially carcinogenic actions, including activation of heterocyclic amine procarcinogens (104); when activated, these compounds may bind genomic DNA and potentiate breast cancer (105). On the basis of their dietary abundance, it seems improbable that many of the individual phytoestrogens listed in Tables 24.2 and 24.3 would reach serum concentrations high enough to exert a significant physiologic effect. Nevertheless, these data suggest an additive mechanism by which phytoestrogens, when consumed as part of a diet rich in legumes and vegetables, may affect hormone-dependent cancers. Additionally, given the wide variation in experimental values and the complexity of possible interactions, these data also highlight the need for in vivo studies on phytoestrogen modulation of steroidogenic enzymes. Effects of Phytoestrogens in the Breast The effect of soy on the breast is controversial. Demographic and epidemiologic observations indicate a weak protective effect of soy consumption on breast cancer risk, particularly for premenopausal women (5), and SPEs prevent the formation of breast tumors in rats (106). However, studies of breast cancer cells in vitro and in mice (107), and short-term evaluation of surrogate risk markers in the breasts of women (108,109) suggest the opposite, i.e., that breast cells and breast cancer cells

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are stimulated by soy isoflavones. These contrasting findings have yet to be reconciled. Clinical studies are underway to address such questions as secondary prevention effects in patients with prior breast cancer and interactions of SPEs with tamoxifen. However, interpretation of human trials is often limited by highly individualized responses to phytoestrogens based on differences in age of exposure, hormonal status, gastrointestinal metabolism, dose and duration of treatment, and interactions with other dietary components. Untangling these interactions is an important challenge for future research on phytoestrogens and breast cancer. Regarding dietary variation, it is also important to consider other nonphytoestrogen compounds in soy with potential chemoprotective properties. These compounds, including saponins (110), protease inhibitors (111), phytic acid, and phytosterols (112), may make important independent and/or synergistic contributions to the anticancer effects of soy. As an example, genistein induces proliferation of estrogen-sensitive breast cancer cells in ovariectomized nude mice (107) but strongly inhibits chemically induced mammary tumor formation when added with soy protein (113). As purified soy phytoestrogen additives and supplements increase in popularity, more specific clinical guidelines are clearly warranted to address such issues. Cell-Culture Studies. Recent in vitro and in vivo studies suggest that the growth of normal and neoplastic breast epithelium is promoted by phytoestrogens (107, 114,115). As described previously, the widely used MCF-7 breast cancer cell line responds with proliferation to doses of genistein in the low micromolar range (<5–10 µmol/L) and growth inhibition at higher doses (>10 µmol/L). The ER dependence of this proliferative effect was demonstrated by the experiments of Wang et al. (10). Subsequently, genistein was found to inhibit growth of ER-negative MDA-MB-468 cells with an IC50 of 8 µmol/L (116). The adverse proliferation-inducing effect on ER-positive breast cancer cells is seen in both in vitro and in vivo studies (107) and may indicate risk of tumor promotion in women with inapparent or recurrent ER-positive breast cancer. However, it is important to note that MCF-7 cells are cancerous, transformed cells; therefore, animal inoculation studies with these cells address tumor growth promotion but not the initiating step in carcinogenesis. Also, by using athymic mice, the inflammatory response to what should be “foreign” cells is suppressed, thereby eliminating local metabolism of genistein. Further, there are important differences between MCF-7 cells and normal breast epithelial cells. For example, MCF-7 cells secrete less of the inhibitory growth factor TGF-β1 than noncancerous epithelial cells and have increased growth with exposure to low doses of genistein; conversely, genistein induces a dose-dependent increase in TGF-β1 in normal human mammary epithelial cells and inhibits growth in these cells (35). The basis for certain differences between normal and neoplastic cells may lie in the paracrine regulation of the breast. For example, in normal breast epithelial cells, sex steroid receptor expression and proliferation rarely occur in the same cells, implying paracrine signaling or temporal separation; in contrast, in breast tumors, the receptors and proliferation occur

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together (117). Other possible mechanisms for context-specific effects of genistein may depend on cellular expression of coactivators and corepressors. Transient transfection studies in HeLa cervical cancer cells have shown that genistein binds with steroid receptor coactivator protein-1 and possibly cAMP response element binding protein coactivator, and these associations may augment ER-α–mediated estrogen-like responses to genistein (118). Genistein may also have differentiationinducing effects on breast cancer cells. Constantinou et al. (119) demonstrated that lipid and casein production was induced by genistein in MCF-7 and MDA-MB468 breast cancer cells, indicating differentiation to a secretory phenotype. Animal Studies. A large number of rodent carcinogenesis studies have shown a protective effect of soy or SPEs against chemically induced mammary neoplasms. These studies are reviewed elsewhere by several authors (6,120,121). Of considerable recent interest is the observation that soy supplementation further increased the antitumor effect of tamoxifen (122), thus confirming the original observation by Gotoh et al. (123). Neonatal exposure of rats to genistein produces a protective effect against mammary cancer later in life (124); a similar finding was made for genistein exposure at puberty, and this effect has been correlated with a proportional decrease in carcinogen-sensitive undifferentiated terminal end buds in the mammary gland (125). This type of differentiating effect may mimic the wellknown protective effect of early pregnancy and may also be induced by estrogens such as diethylstilbestrol (DES) (126). Genistein concentrates in the rat mammary gland, reaching concentrations approximately twice those in serum (127); therefore, effects on the breast may exceed those anticipated on the basis of serum concentrations. This may be a beneficial phenomenon because the antiproliferative effects of soy occur at relatively high concentrations, which may be difficult to attain in vivo (107). However, it is worthwhile to note that the growth of cancerous cells injected into nude mice has been promoted by administration of dietary genistein (107) and that this is a dosedependent effect in the range of 15–300 µg/g of the diet (~2.5–50 mg/kg body) (128). In the widely used dimethylbenzanthracene (DMBA) rat model, dietary soy supplementation has been shown to inhibit tumorigenesis but induce a slight yet significant increase in cellular proliferation once tumors are present (129). This result is in contrast to the work of Hawrylewicz et al. (120), who found diminished growth of recurrent mammary tumors in N-methylnitrosourea (NMU)-treated rats fed soy protein isolate (SPI) after removal of the primary tumor; histopathology of secondary tumors in soy-fed rats revealed a significant shift to more benign tumor types compared with casein-fed rats. Other studies of normal breast tissue in rats have indicated that the effects of dietary estrogens and estrogen replacement therapy are interactive and dose dependent, such that SPEs may not alter breast proliferation at low doses of estrogens but decrease breast proliferation in combination with higher estrogen doses (130,131). This evidence is supported by studies using the cynomolgus macaque model showing that dietary SPEs can antagonize the pro-

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liferation-inducing effect of estradiol on the mammary gland and uterus of monkeys (132). Recent studies using the DMBA model have shown that this estrogen-antagonist effect on the mammary gland also results in antagonism of tumorigenesis (129). Studies of Women. Observational, case-control studies of women suggest a breast cancer-protective effect of SPEs. These studies were reviewed recently (133), and the latest epidemiologic findings are found elsewhere in this book. In general, the protective effect of soy phytoestrogen intake is stronger in premenopausal women (134), and associations are strengthened in those studies for which interindividual variation in phytoestrogen intake and metabolism were controlled by measuring urinary isoflavone concentrations (135,136). Of recent interest is a study by Shu et al. (137) which found decreased risk of pre- and postmenopausal breast cancer in women who consumed tofu as teenagers. This finding emphasizes the importance of early exposure effects in support of Lamartiniere et al. (106,124), who showed enhanced mammary gland differentiation in rats with prepubertal genistein exposure. Few studies have addressed the effect of phytoestrogens on biomarkers of breast cancer risk in nonneoplastic human breast tissue. A recent cross-sectional study of mammographic density and soy intake indicates that a greater percentage of the breast occupied by radiographically dense tissue is associated with higher soy intake in women of Caucasian and Hawaiian ethnicity but not in women of Chinese or Japanese descent (138); it is currently unclear how this finding relates to breast cancer risk. Some concern was raised by the finding of Petrakis et al. (108), namely, a small increase in the amount of nipple aspirate fluid (NAF) obtainable from premenopausal women consuming 38 g of soy protein isolate (SPI) per day, containing 38 mg of genistein. However, NAF volume continued to increase after discontinuation of soy consumption, suggesting the effect may have been due to the repeated aspirations rather than the soy supplementation, and the magnitude of the increase in NAF was small (~20 µL, or less than half a drop of fluid). Of greater concern is the presence of hyperplastic epithelial cells in the NAF; however, this effect also continued beyond cessation of soy supplementation, and validation of this cytologic end point is lacking. Further interesting data regarding potential effects in women lies in the work of Hargreaves et al. (109), who examined the effect of short-term (2-wk) treatment with a SPI containing 45 mg/d of isoflavones, given as a dietary supplement before excision of a breast lesion. In this study, soy supplementation increased expression of progesterone receptor and lowered apolipoprotein D levels, both of which are estrogenic changes, but did not increase cellular proliferation or alter expression of ER-α or the antiapoptotic marker bcl-2. Interpretation of this study is difficult because nine different types of benign and malignant breast tissues, ranging from normal breast to invasive carcinoma, were included in the study, and the distribution of these diagnoses within the study population was not described. There are also well-documented paracrine effects of neoplasms within the breast that could have confound-

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ed the results reported. Perhaps the most interesting effect found in this study was the concentration of isoflavones in breast fluid; mean serum concentrations of isoflavones in soy-treated women were ~250 ng/mL (1 µmol/L), whereas concentrations in breast fluid were ~800 ng/mL (3 µmol/L). Furthermore, some untreated women had low serum concentrations but high breast concentrations, possibly reflecting accumulation of isoflavones in the breast over time (109). The difficulties encountered in the conduct and interpretation of these studies in women point to the need for further preclinical evaluation of soy phytoestrogen effects and validation of breast cancer risk biomarkers. Effects of Phytoestrogens in the Uterus Available epidemiologic evidence suggests that consumption of soy-based products may decrease endometrial cancer risk in women (139), although the role of diet in endometrial cancer is not well explored. Animal studies have shown that SPEs such as genistein and daidzein may antagonize certain estrogen-induced uterine changes. On the other hand, various phytoestrogens have also been shown to induce a wide range of potentially adverse effects in the uterus, depending on species, age, dose, and conditions of exposure. Of particular clinical concern is whether phytoestrogens may induce endometrial hyperplasia and/or neoplasia in women, in a manner similar to unopposed estrogens. Although unconfirmed by human and monkey data, rodent studies indicate that SPEs stimulate estrogen-sensitive genes and increase uterine weight at pharmacologic doses [see, e.g. (140)]. However, unlike estradiol, these compounds do not appear to significantly stimulate endometrial proliferation or induce growth of transplanted endometrial tumor cells in ovariectomized animals (141). These findings suggest that SPEs do not function simply as weak physiologic estrogens in the uterus but have unique effects. Cell Culture Studies. In one of the few published uterine cell culture studies, human endometrial adenocarcinoma cells (Ishikawa cells) treated with genistein, equol, and daidzein exhibited half-maximal (EC50) estrogen-specific alkaline phosphatase activities at concentrations of ~0.01, 0.1, and 0.5 µmol/L, respectively (14), suggesting that estrogenic stimulation by isoflavones may occur at the low concentrations attained in tissues of people consuming a high-soy diet. In another experiment using RUCA-1 rat endometrial adenocarcinoma cells, genistein and daidzein induced a significant increase in expression of the estrogen-sensitive marker complement C3 at 1 µmol/L but also induced expression of fibronectin, an antiestrogen marker (142). It should be noted that proliferation markers were not assayed in these experiments. In a cell culture study using cervical cancer cell lines HeLa and ME-180, genistein inhibited cell growth at IC50 values of 35 and 60 µmol/L, respectively, and suppressed invasion of HeLa cells into a surrogate membrane in a dose-dependent manner (143). Animal Studies. The estrogenic effects of phytoestrogens on the reproductive tract have been recognized for over half a century, arising from observations of fer-

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tility loss in sheep foraging on isoflavonoid-rich clover (144,145). Since that time, numerous rodent studies have reported adverse reproductive effects of neonatal exposure to the more potent phytoestrogens, including persistent estrus (146), premature anovulatory syndrome (147), delayed vaginal opening (148), cervicovaginal adenosis, uterine squamous metaplasia (149), and increased rates of uterine adenocarcinomas in mice (150). However, these effects occurred with the use of pharmacologic doses of the more potent phytoestrogens (most of which were administered subcutaneously, bypassing the liver); despite widespread use of soybased infant formulae, there is little evidence to date that phytoestrogens are responsible for any human developmental or reproductive problems (151). Rodent studies have shown a dose-dependent uterotropic effect of various phytoestrogens. As shown in Figure 24.2, genistein administered subcutaneously to immature mice may induce increases in uterine wet weight similar to those seen with DES and estradiol, but at concentrations 1000 times greater than estradiol and 50,000 times greater than DES (152). Similarly, evaluation of available rat and mouse studies assessing uterine responses to genistein revealed a dose-dependent uterotropic effect; relevant data are presented in Figures 24.3 and 24.4. Significant increases in uterine weight are reported in each of seven studies in which genistein [≥50 mg/(kg body⋅d)] was administered by gavage or injection (141,153–158). In contrast, only two of five studies using dietary genistein >50 mg/(kg body ⋅ d) found a significant increase in uterine weight (157,159–162), suggesting that dietary isoflavone exposures may produce different effects than pharmacologic administration of purified compounds. Of the 10 studies using maximum isoflavone doses <50 mg/(kg body⋅d) (163–172), only two (171,172) found significant increases in uterine weight. When evaluating phytoestrogen effects in these rodent studies, several considerations are worth mention. The first of these involves dose. Although genistein clearly increases uterine weight in rodents, the exposures at which these effects were observed are rarely attained in human diets. A daily genistein dose of 50 mg/kg is ~25 times greater than the estimated exposure from a traditional Asian diet (63) and >2500 times greater than the exposure from a Western diet (173). Infants receiving strictly soybased formulas could potentially reach this level of effective exposure (141,154), although precocious puberty is not an outcome in soy-fed infants. A second consideration involves differences in isoflavone metabolism between humans and rodents. In rats, the predominant circulating phytoestrogen after consumption of soy-containing laboratory diet is the isoflavan equol, a metabolite of daidzein. Equol is present at concentrations (2–4 µmol/L) that are 6–7 times greater than those of genistein (400 nmol/L) and daidzein (300 nmol/L), a result of lower renal clearance (174). In contrast, only one third of humans have blood equol concentrations >10 nmol/L. This may make assessments of soy effects in humans based upon rodent models problematic. For example, the lower than expected daidzein and genistein blood concentrations for a given dose (in mg/kg body) in rodents could lead to an underestimation of the estrogenicity of these compounds. On the other

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Uterine wt./body wt. ratio × 100

Treatment (µg/kg)

Fig. 24.2. Immature uterotropic bioassay of several phytoestrogens. Immature mice were injected subcutaneously with each compound for three consecutive days and killed on d 4. Uterine wet weights and body weights were determined and the ratio was plotted. ■, estradiol; ●, diethylstilbestrol (DES); ▲, genistein; ◆, coumestrol; ■, naringenin; O, taxifolin. (Reprinted from Ref. 152 with permission from Elsevier Science.)

hand, the presence of equol, itself estrogenic, may stimulate a different spectrum of gene transcription responses. It is not known whether the phytoestrogen-induced increases in uterine weight in rodents correspond to increased cancer risk. Uterine weight is widely used in animal studies to assess estrogenic responses, but it is a nonspecific indicator of endometrial changes, and without histologic or immunohistochemical evaluation, it is difficult to assess whether the observed “uterotropic” effects of high-isoflavone doses in rodents consist of fluid accumulation or actual hyperplasia. Soy isoflavones have not induced endometrial hyperplasia in studies of ovariectomized rats, monkeys, and menopausal women (175), but concern still exists regarding other estrogenlike mechanisms of uterine carcinogenesis. Prenatal and neonatal exposure to synthetic estrogens such as DES has been associated with several reproductive abnormalities, including uterine carcinoma (150). Recently, neonatal exposure to genistein at 50 mg/(kg body⋅d) was associated with a 35% incidence of uterine adenocarcinomas in CD-1 mice at 18 mo, comparable to that produced by an equipotent dose of DES (154). However, in a separate study, genistein and daidzein displayed significant inhibitory effects on the development of estrogen-related endometrial adenocarcinoma and atypical endometrial hyperplasia when given monthly with estradiol for 30 wk to mice at ~33 mg/kg body; additionally, genistein and daidzein were shown to significantly inhibit estrogen-induced expression of c-fos and c-jun, genes related to cellular proliferation and differentiation (176). These studies highlight the importance of timing of exposure in determining uterine responses and also the need for endometrial assessment in phytoestrogen studies.

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Percent change in uterine weight

Isoflavone dose (mg/kg) Fig. 24.3. Dose-dependent uterotropic effects of soy isoflavones in rats. Data com-

Percent change in uterine weight

piled from 20 studies evaluating soy isoflavone effects on uterine weights. Solid symbols indicate significant differences in uterine weight vs. controls. Asterisks denote in utero exposure. Plus signs denote prepubertal exposure.

Isoflavone dose (mg/kg) Fig. 24.4. Dose-dependent uterotropic effects of soy isoflavones in mice. Data com-

piled from four studies evaluating soy isoflavone effects on uterine weights. Solid symbols indicate significance difference in uterine weight vs. controls. Asterisks denote in utero exposure. Plus signs denote prepubertal exposure.

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Genistein and daidzein have been shown to modulate expression of several estrogen-sensitive markers in the normal rat uterus, including complement C3, clusterin, androgen receptor, ER (140,141), and insulin-like growth factor I (IGF-I) (177). However, it remains to be determined whether genistein and other phytoestrogens stimulate all the same estrogen-responsive genes regulated by a physiologic estrogen such as 17β-estradiol (E2). The idea that endometrial effects are differentially modulated through (phyto)estrogen-specific patterns of gene expression was suggested by a recent study by Diel et al. (140). Gene expression profiles for multiple estrogen-sensitive genes were obtained in rat uteri after exposure to ethinyl estradiol, several xenoestrogens, and one phytoestrogen (daidzein); interestingly, the compounds all induced increases in uterine weight but exhibited strikingly different gene expression fingerprints. In a subsequent study in rats using a malignant uterine allograft model, subcutaneous injection of genistein at 50 mg/(kg body ⋅ d) for 28 d significantly up-regulated certain estrogen-responsive genes but produced no significant agonistic activity (in contrast to estradiol) in ectopic syngeneic estrogen-sensitive RUCA-1 endometrial adenocarcinoma cells (141). The availability of genome-wide DNA microarray chips will help greatly in answering this question of genomic-based variation in phytoestrogen effects, although the true answer really lies at the protein level. Uterotropic effects of soy isoflavones have not been observed in nonhuman primate studies. Ovariectomized cynomolgus macaques fed SPI for 6 mo at the equivalent of 148 mg/d in women displayed no significant increase in uterine weight or endometrial proliferation, as measured by Ki67 expression; additionally, in monkeys treated with both SPI and estradiol, the SPEs antagonized estradiolinduced proliferation (132). This antiproliferative effect of soy was not accompanied by down-regulation of progesterone receptor expression, suggesting an ERindependent mechanism (J.M. Cline, unpublished data). Anthony et al. (178) also found no significant changes in uterine weights of rhesus macaques fed a soybased diet with ~10 mg isoflavones/(kg body⋅d) for 6 mo. Studies of Women. Endometrial cancer risk is markedly lower in Asian women relative to women in North America, similar to breast cancer [see, e.g. (179)]. The one known case-control study of dietary soy intake and endometrial cancer risk showed an ~50% reduction in endometrial cancer risk between the lowest and highest quartiles of soy intake (139). As with nonhuman primates, SPEs in women have not been associated with overt uterotropic effects. Two studies using histologic evaluations of endometrial biopsies found no significant changes in women consuming soy isoflavone diets (65,72). However, these studies were limited by small sample size, short duration, relatively low doses of isoflavones (1–2 mg/(kg body⋅d), and a lack of quantitative endometrial assessment. In a more recent study, ingestion of 50 mg/d of purified red clover isoflavones by 30 perimenopausal women for 3 mo did not induce changes in endometrial proliferation, as determined by immunohistochemical staining of the Ki67 proliferation marker on monthly biopsies (180).

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Vaginal Changes. Relatively few studies have examined the effects of soy isoflavones on the vaginal epithelium, but available rodent studies suggest that the vagina, like the uterus, may exhibit estrogenic responses at higher isoflavone doses. Ovariectomized rats given soy protein doses of 1.6 and 16.5 mg/(kg body⋅d) for 2 mo did not have increased vaginal epithelial maturation (170); however, in rats given oral genistein doses of 50 and 100 mg/(kg body⋅d), significant increases in vaginal epithelial height were observed (141). Ovariectomized cynomolgus macaques fed ~10 mg/(kg body ⋅ d) of isoflavones for 6 mo did not have significantly increased vaginal cornification (181), and several randomized trials in women consuming soy supplements or soy flour have revealed no significant maturative effects of soy isoflavones on the vaginal epithelium (74,182).

Summary and Conclusions Phytoestrogens are naturally occurring compounds widely present in the human diet that may exert a diverse range of effects relevant to the prevention of breast and uterine cancer. Over the past decade, much emphasis has been placed on the actions of phytoestrogens at the ER. This remains an active field of study with current projects investigating such topics as phytoestrogen interactions with ER-β, receptor coactivator effects, and differential expression of estrogen-responsive genes. Phytoestrogens, and soy isoflavones in particular, also have critical mechanisms of action occurring independently of the ER, including antioxidation, effects on growth factor-induced signal transduction, and modulation of estradiol metabolism. Moving forward, these nonhormonal actions, and the complex determinants of these actions in vivo, represent important lines of inquiry. Despite progress in our mechanistic understanding of phytoestrogens, consensus regarding their application to cancer prevention is lacking, due in part to concern over potential estrogen-like actions in the breast and uterus. Endocrine-modulating, antiproliferative, and cell-cycle effects of phytoestrogens are largely dose and context dependent for the breast and uterus, and general statements regarding aggregate benefit or risk of phytoestrogen consumption cannot yet be made. Further research is required in the following areas: to define clinical conditions and doses for cancer-preventive and harmful effects; to elucidate tissue-specific paracrine and receptor-mediated effects; to examine interindividual differences in phytoestrogen metabolism and receptor status; and to evaluate phytoestrogen interactions with estradiol, enzymes of estradiol biosynthesis, antiestrogens, and other dietary components. It is important that these questions be addressed in the process of making dietary recommendations on human phytoestrogen consumption. Acknowledgments The authors would like to thank Dr. Thomas Clarkson and Dr. Mary Anthony for their valuable editorial assistance. We also gratefully acknowledge Robin Barnhardt and Michelle Browne for their help in compiling information for Figures 24.3 and 24.4.

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Chapter 25

Induction of Apoptosis by Genistein: Potential Applications in Cancer Prevention and Treatment Andreas I. Constantinou Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, IL, and Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus

Introduction Chemically, genistein is an isoflavone (4′,5,7-trihydroxyisoflavone) found in considerable quantities in subterranean clover (Trifolium subterraneum L. var. Dwalganup) and soybeans (Glycine max). Genistein was identified as a phytoestrogen after the observation that ewes in Western Australia grazing on pastures dominated by subterranean clover experienced infertility and other reproductive disorders. Synthetic genistein, given at doses up to 15 mg/d, affected the fertility of both male and female mice, confirming that this was the causal agent responsible for the reproductive disorders associated with soy consumption (1). Genistein was subsequently shown to compete with estradiol in MCF-7 cells for the estrogen receptor (ER) and to elicit other typical estrogen-induced responses such as altering the expression of estrogen and progesterone receptors. Genistein’s binding affinity for the ER is 200 times lower than that of estradiol, whereas daidzein’s binding affinity to ER is ~1000 times lower than that of estradiol (2). At these low concentrations (10–200 nmol/L), genistein stimulates expression of the estrogen-responsive pS2 gene, whereas prolonged exposure or a higher genistein dose reduces ER mRNA levels (5). The plasma concentration of genistein varies widely among the human populations that regularly consume soy products as part of their diet. With the exception, perhaps, of infants consuming soy-based formulas (6), the plasma concentrations of genistein in adults is generally <0.2 µmol/L (1 µmol/L = 1 × 10–6 mol/L) (7–9). This review will focus on biological responses of mainly cultured tumor cells, at genistein concentrations that clearly exceed those that can be reached in human tissues. Therefore, this paper specifically evaluates the molecular pathways that lead to apoptosis in cultured tumor cells that generally require pharmacologic concentrations of genistein ranging from 25 to 500 µmol/L (see Table 25.1). The biological effects of genistein depend on its concentration but also on whether the target cells express the estrogen receptors. In vitro, genistein competes with estradiol for receptor binding (3); it binds to the ERα and ERβ receptors with Ki of 2.6 and 0.3 nmol/L, respectively (10-12) and inhibits estrogen-stimulated cell

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TABLE 25.1 Effects of Genistein on the Molecular Pathways Leading to Cell Cycle Arrest and/or Apoptosisa Modulation of molecular target

Downstream effector and cellular response

Inhibition of PTK activity in cell-free systems Inhibition of PTK activity in intact cells Inhibition of the Src-family PTK activity

Not applicable Reduced tyrosine phosphorylation of phosphoproteins Inhibition of Lyn and Lck kinase activity associated with decreased tyrosine phosphorylation leading to apoptosis Inhibition of translation of proteins required for proliferation; antimitogenic Activation of intrinsic apoptotic pathway, or activation of stress pathway Prevents release of NFB and translocation to the nucleus; apoptosis Up-regulation of P21WAF1 and G2/M block ‘’Cleavable complex” formation and/or abnormal chromosomal segregation; DNA breakage; apoptosis

Inhibition of in situ phosphorylation of S6K by EGFR Bax/Bcl-2: increased, or unchanged Indirect inhibition of IκB phosphorylation Inhibition of Cdc2 and Cdk2 kinase activities Inhibition of Topo II activity aAbbreviations:

Concentration µmol/L

Reference

25–30 120 185–220 0.35b 20–25

21 34 36 43,44 23,40,42

25–150

29,49–52,65

25–50

60

25–50 100–300

32,68,69 77,78,79,81,83,90

PTK, protein tyrosine kinase; EGFR, epidermal growth factor receptor; Topo, topoisomerase was introduced as a conjugate with B-43 overexpressing Lyn or Lck kinases. In contrast, micromolar concentrations of unconjugated genistein did not affect the phosphorylation status of tyrosine-phosphorylated proteins or the enzymatic activity of Lyn kinase.

bGenistein

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growth in vitro (2). Genistein induced both DNA synthesis and cellular proliferation at concentrations ranging from 0.1–10 µmol/L in estrogen-dependent tumor cells (MCF-7), but not in estrogen-independent (MDA-MB-231) tumor cells (13). On the other hand, genistein may provide antiproliferative (and chemopreventive effects) in ERβ-expressing normal cells. Populations consuming predominantly plant-based diets tend to have lower incidence and mortality rates of several cancers than do populations consuming mainly animal-based diets. This difference is more striking when Asian populations deriving a large percentage of their daily energy intake from soybeans are compared with people from Western Europe and the United States who consume little or no soy products (14,15). Genistein and daidzein (4′,7-dihydroxyisoflavone) are the most abundant isoflavones in soy, found in concentrations of 1–3 mg/g dry weight; >95% of the isoflavone content of soy is contributed by genistein and daidzein (16). The average nonvegetarian American consumes 1–5 mg of isoflavones daily (~50% is genistein), compared with ~29 mg for residents of Japan (17). Bioanalytical and animal studies provide additional evidence for the involvement of soy isoflavones in protecting against hormonally regulated cancers. Men with a low risk of prostate cancer have a higher urinary excretion of soy isoflavones than men with a high risk of prostate cancer (7). Urinary phytoestrogen levels are also decreased in women with breast cancer (18). Soybean diets and soy isoflavones have also been shown to offer protection against mammary tumor development in animal models of cancer (18). Although several components of soy, including the protease inhibitors, saponins, and phytic acid, have demonstrated cancer chemopreventive effects, genistein is the most investigated component of soy, not only for its potential to be effective in cancer chemoprevention but also for its potential as a chemotherapeutic drug and genotoxic agent. In addition to the estrogen receptors, genistein appears to act on several targets within the cell, including inhibition of protein tyrosine kinase (PTK), topoisomerase II (topo II), ribosomal S6 kinase (RS6K), epidermal growth factor receptor (EGFR), and Src kinases (21–23). Normal and tumor cells respond to these inhibitory effects of genistein by altering their program of cell differentiation and their rates of proliferation, cycling, and apoptosis (24–26). The main objective of this article is to evaluate the effects of genistein on the pathways that lead to programmed cell death (apoptosis) or survival. The effects of genistein on the key regulators of cellular cycling are also discussed because an overlap exists between the pathways that lead to apoptosis and those that lead to cell cycle arrest. Of particular interest is the effect of genistein as an inducer of topo II-mediated DNA breakage or a modulator of tyrosine phosphorylation, because these can, respectively, trigger or control the pathways that lead to apoptosis. This article is not intended to provide a comprehensive review of the literature, but merely to highlight research data that may have applications in the prevention or treatment of cancer.

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Apoptotic Pathways Apoptosis is induced in response to a wide variety of cellular stimuli. At least two separate pathways are involved, i.e., the intrinsic, also called the mitochondriadependent pathway, and the extrinsic, also known as the death receptor pathway. These two pathways of apoptosis, which are redundant and interdependent, have been described in detail (27,28). Both general and brief descriptions of the two pathways are given below. The intrinsic pathway is controlled by the Bcl-2 family of proteins. The >15 members of this family of proteins are divided into those that promote survival (i.e., Bcl-2, Bcl-Xl) and those that promote death (i.e., Bax, Bak). Because these proteins form heterodimers with each other, their relative abundance controls the release of cytochrome c into the cytoplasm. Cytochrome c released from the mitochondrion binds to Apaf-1, which is oligomerized, and recruits and activates caspase-9, which in turn activates the effector caspase-3. The extrinsic pathway involves external signaling via membrane receptors, such as Fas and tumor necrosis factor (TNF). Upon binding of death ligands (i.e., FasL and TNF) to the extracellular cysteine-rich ligand-binding domain of their corresponding death receptors, they activate a series of intracellular signaling events that begin the apoptotic cascade. In the case of Fas, upon binding of the ligands, the intracellular portion of three death receptors, known as death domains, become associated with each other. This clustering allows Fas-associated death domain (FADD), which acts as an adapter protein, to bind. The death effector domain (DED), found within FADD, recruits procaspase 8, which becomes activated through self-cleavage and activates the effector caspase that commits the cell to apoptosis by breaking down the cellular structure. Genistein can trigger both the intrinsic and extrinsic pathways of apoptosis. With some exceptions, the literature supports the theory that PTK inhibition is the key mediator of this effect. A large number of tumor cell lines have been reported to undergo apoptosis, including breast (29), lung (30), prostate (25), leukemia (26), colon (31), bladder (32), and head and neck (33). The molecular targets of genistein, their downstream effects, and cellular responses are summarized in Table 25.1. The effects of genistein on PTK must be examined carefully because these control the signaling pathways that lead to cell cycle delay and are also crucial in the pathways that lead to apoptosis. Effect of Genistein on Protein Tyrosine Kinases Genistein was identified as a PTK inhibitor by Akiyama et al. (21,34), who demonstrated that the soy isoflavone inhibits, in cell-free systems, the tyrosine kinase activities of epidermal growth factor receptor (EGFR), v-Src, v-Abl, and Gag-fes with 50% inhibitory (IC50) doses ranging from 2.5–30 µmol/L. The inhibition is competitive with respect to ATP and noncompetitive with phosphate receptor. Subsequent studies determined that members of the Src family of kinases, Lck,

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Lyn, Fyn, and Blk, are effectively inhibited by genistein (35). However, not all PTK activities are inhibited by genistein; for example, P94 PTK was not inhibited by genistein, suggesting specificity among PTK (28). The popularity of genistein is partially due to its high specificity for PTK. Genistein discriminates between serine/threonine and tyrosine kinases, in contrast to other flavonoids such as quercetin that do not. Genistein was found to lack inhibitory activity against cAMP-dependent kinase, protein kinase C, and phosphorylase kinase (21). Because of its specificity for PTK, genistein is used as a research tool for exploring the role of tyrosine phosphorylation in a variety of cellular processes. Although genistein is effective in inhibiting PTK at concentrations <30 µmol/L in cell-free systems, it is emphasized that genistein is less effective in cultured cells. The concentration of genistein that is necessary to inhibit the growth of cell lines by 50% (IC50) generally ranges from 5 to 50 µmol/L in most tumor cell lines (pharmacological doses). The inhibition of PTK in cultured cells, generally requires supra-pharmacological doses ranging from 50–500 µmol/L. For example, genistein doses of 111 µmol/L were necessary to prevent the autophosphorylation of EGFR in A431 cells by 50% (21,34), and doses of 185–222 µmol/L were required to inhibit EGFR tyrosine phosphorylation in MDA-MB-468 breast cancer cells (36,37). Genistein concentrations of up to 74 µmol/L failed to inhibit the tyrosine phosphorylation of Raf and phospholipase Cγ, whereas the tyrosine phosphorylation induced by phosphoinoside 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) was resistant to up to 135 µmol/L genistein (38). The p70 ribosomal S6 kinase (S6K) is a mitogen-activated Ser/Thr kinase that phosphorylates the S6 protein of the 40S ribosome. Treatment of fibroblast cells with genistein (and herbimycin A) completely blocks integrin-mediated activation of S6K, indicating a requirement for tyrosine kinase activity (39). However, under these conditions, the tyrosine phosphorylation of focal adhesion kinase (FAK) and c-Src were largely unaffected. The data suggest that a tyrosine-sensitive tyrosine kinase may be required for integrin-dependent activation of S6K. In an earlier study in NIH-3T3 cells, the 40S ribosomal protein was shown to undergo phosphorylation by the EGFR within minutes of stimulation by EGF (23). This phosphorylation that increases S6K activity was demonstrated to be inhibited by 15 µmol/L of genistein in the same cells. The in situ S6 phosphorylation was also prevented by similar genistein concentrations (12–19 µmol/L), which inhibited the mitogenic effect mediated by EGF or by insulin. These results suggest that, although high genistein concentrations (111 µmol/L) are necessary to inhibit the tyrosine kinase activity of EGFR in cultured cells, the in situ phosphorylation of specific substrates such as S6K may be inhibited at much lower concentrations. The effects of genistein in inhibiting the phosphorylation of S6K at the moderate concentration of 25 µmol/L were also reproduced in monkey kidney epithelial CV-1 cells (40). These effects of genistein on S6K activity may account for its antiproliferative effects, i.e., S6K regulates the translation of mRNA containing 5-poly-pyrimidine tracts such as those encoding for elongation factor 1 or insulin-like growth factor II.

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Through its effects on S6K, genistein may effectively inhibit the translation of proteins that are required for DNA synthesis or mitosis and progression through the G1 phase. On the basis of this scheme, genistein is expected to cause cell cycle delay in the G1 phase of the cell cycle. A genistein-induced block in the transition from G1 to the S phase was observed in mouse B16-F1 melanoma cells and BALB/c 3T3 fibroblast cells and prostate cancer LNCaP cells (41,42). However, in most cell types, the cycle is delayed in the G2/M phase. Generally, higher concentrations of genistein were associated with G2/M block rather than G1 block. The effects of genistein on cell cycle checkpoint may possibly be cell type specific and dose dependent, and different molecular targets may come into play at the higher concentrations. Substantially stronger evidence supports the theory that the inhibition of PTK (by genistein) can account mechanistically for the induction of apoptosis. The genistein doses that induce apoptosis are within the range of the doses that inhibit tyrosine phosphorylation in cultured tumor cells (50–500 µmol/L). These concentrations cannot be reached in the serum of animals or humans by consuming soy or by administering genistein. The use of immunoconjugates, however, allows the delivery of therapeutic doses of genistein to the targeted tumor cells, where it induces apoptosis by interacting directly with the PTK domains of the receptor tyrosine kinases. By immunoconjugating genistein to a monoclonal antibody, the isoflavone can be delivered directly to the cancer cells expressing PTK-associated–specific antigen and may induce apoptosis. Genistein conjugated with B43, a monoclonal antibody targeting the B-cell specific receptor CD19, provided an effective treatment of BCP leukemia in mice. The mode of action involved inhibition of the CD19-associated Src-family of kinases and induction of apoptosis (43). Cancer cells express several members of the Src PTK family that share most of their substrates. Inhibitors that are specific for a single member of the Src family of kinases are less effective because Src PTK may compliment each other. Genistein as a general inhibitor of the Src-family of kinases was found to be promising in the treatment of the most common form of childhood cancer. The B43-genistein immunoconjugate also showed promise in a clinical study of seven children and eight adults with B-lineage acute lymphocytic leukemia and chronic lymphocytic leukemia that failed previous chemotherapy regimens (44). Administration of B43-genistein for up to 10 d showed an acceptable toxicity profile and the ability to elicit objective responses (44). Based on the same principle, recombinant EGF was conjugated with genistein. The conjugate was capable of binding to EGFR, entering the cells (MDA-MB-231 expressing EGFR), and inhibiting the PTK activity of the EGFR (36). How does inhibition of PTK lead to apoptosis of cancer cells? The human EGFR consists of an extracellular domain, a single transmembrane domain, and a cytoplasmic domain with PTK activity. Upon binding of EGF to the EGFR receptor, dimerization with itself or other members of the ErbB family of transmembrane PTK takes place, resulting in the phosphorylation and activation of the PTK domain (45).

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EGFR is expressed at high levels in many types of cancer cells, including breast, and this is associated with excessive proliferation, metastasis, and poor relapse-free survival (46). The EGF-genistein conjugate allows delivery of more genistein molecules to the cancer cells (expressing the EGFR), thereby effectively increasing the intracellular concentration to suprapharmacologic levels. Upon binding of the genistein-EGF conjugate to the EGFR, the isoflavone comes in direct contact with EGFR tyrosine kinase. One possibility is that the localization of the genistein molecule in close proximity to the ATP-binding domain of the EGFR PTK may increase the effective binding constant. Another possibility is that genistein inactivates the PTK activity of Src kinase, which is physically and functionally associated with EGFR (45,47). Similar activities may occur with the B43-genistein immunoconjugate because CD19 is also physically associated with the Src family of PTK (48). This approach allows selective delivery of genistein to its target enzymes, where it reaches concentrations sufficient to block membrane-associated PTK activity. The advantage of the approach is the elimination of nonspecific toxic effects in normal tissues that do not express the targeted receptor kinase. The Effect of Genistein on the Ratio of Bcl-2/Bax Family of Proteins Bcl-2 is a protooncogene that promotes neoplastic expansion and provides selective survival advantage by preventing cell death rather than promoting DNA replication. It is a 26-kDa intracellular integral membrane protein found primarily in the nuclear envelope, endoplasmic reticulum, and mitochondrial membrane. Bax promotes apoptosis in two ways: (i) by binding to Bcl-2 and inhibiting its antiapoptotic activity, and (ii) by directly targeting mitochondria and inducing release of caspase-activating proteins (49). Thus, Bax protein is an apoptosis promoter, whereas Bcl-2 is an apoptosis inhibitor. Up-regulation of Bcl-2 and down-regulation of Bax are associated with cancer progression (30). An imbalance in these proteins may modulate downstream proteins involved in apoptosis. Antiapoptotic proteins that act like Bcl-2 include BclXl, Bcl-W, Mcl-1, and Bfl-1. Proapoptotic, Bax-like proteins include Bak, Bad, Bid, and Bik. In the following paragraphs, we will examine the question “Is the Bax/Bxl-2 ratio, or the ratio of proapoptotic/antiapoptotic proteins altered in response to treatment of cancer cells with genistein concentrations that trigger apoptosis?” Our laboratory addressed the question four years ago in MCF-7 cells that were treated with a high dose (150 µmol/L) of genistein (29). In that study, we did not evaluate the Bax levels, but a decrease was found in the Bcl-2 protein levels, which apparently increased the Bax/Bcl-2 ratio. The decrease in Bcl-2 protein level was delayed (48 h post-treatment) and modest (46% of the control levels). Several other studies in a variety of tumor cell types reported increases in the Bax/Bcl-2 ratio in response to genistein, which is consistent with the appearance of apoptosis. Nakagawa et al. (50) reported that genistein-induced apoptosis in DD-762, Sm-MT,

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MCF-7, and MDA-MB-231 breast cancer cells was accompanied by up-regulation of Bax protein, down-regulation of Bcl-Xl protein, and activation of caspase-3. Up-regulation of Bax and p21WAF1 expression and down-regulation of Bcl-2 and c-erbB-2 were evident in MDA-MB-435 and 435.eB; this effect was induced by genistein and caused the cells to undergo apoptosis (51). Because 435.eB cells were established by transfecting c-erbB-2 cDNA into MDA-MB-435 cells, Li et al. (51) concluded that genistein may inhibit invasion and metastasis of breast cancer cells. In HN4 squamous cell carcinoma cells, Alhasan et al. (33) reported up-regulation of Bax, with modest down-regulation of Bcl-2 protein expression, in response to genistein treatment. Up-regulation of Bax and down-regulation of Bcl2 were also reported in MDA-MB-231 cells that were treated with genistein doses that induced apoptosis (52). However, these same cells were found to be resistant to genistein-induced apoptosis in another study . Although the above studies support genistein-induced changes in the Bax/Bcl-2 ratio that are consistent with proapoptotic effects, the changes are generally delayed and nondramatic. Three other studies found either no changes or changes that were not consistent with proapoptotic effects. Thus, Bax became elevated in MCF-7 cells in response to treatment with 25 µmol/L genistein (a concentration that caused apoptosis) but it was accompanied by increased levels of Bcl-2; it was concluded that the elevated Bcl-2 protein might neutralize the proapoptotic effect of Bax (54). The mechanism of genistein-induced apoptosis was thought to rely largely on the stress pathway rather than the pathway mediated by the Bcl-2 family of proteins. In MCF-7 cells, an initial decrease of Bcl-2 was evident in response to 50 µmol/L genistein, followed by an increase; no significant changes in Bax protein were evident (53). Similar results were reported in LNCaP cells in which 20 µmol/L genistein were ineffective in changing the Bcl-2 or Bax levels, although there was an increase in p21WAF1 levels that was associated with an antiproliferative response (42). Apparently, conflicting data exist concerning the effect of genistein on the levels of proapoptotic and antiapoptotic proteins and their mRNA. Obviously, not all cell types that undergo apoptosis in response to genistein are expected to respond by increasing their Bax/Bcl-2 ratio because there are multiple pathways that lead to apoptosis. However, conflicting results have been reported even in the same cell line (i.e., MCF-7). These conflicting results may be due to the genistein doses being used because the studies that used >50 µmol/L of genistein were more likely to report changes in the levels of Bcl-2 and Bax than those that used ≤50 µmol/L (see Table 25.1). Another reason for the conflicting reports may be due to the fact that simple expression of Bcl-2 may not be enough to functionally protect cells from apoptosis. The genistein-induced changes in the protein levels of Bcl-2 may be very modest and other (possibly stronger) changes that may be taking place at another level of Bcl-2 regulation have not been examined. Phosphorylation is shown to modulate the function of Bcl-2 and its ability to form heterodimers with Bax (55). Hyperphosphorylation on multiple sites, such as that induced by taxol, has been shown to

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inhibit the antiapoptotic potential of Bcl-2 and to be associated with death (56). We reported previously that genistein increased Bcl-2 phosphorylation in MCF-7 cells within 30 min of treatment (24). The possibility that genistein, as a protein tyrosine kinase inhibitor, modulates Bcl-2 phosphorylation indirectly, e.g., by inactivating a Bcl-2 phosphatase, merits further investigation. The Effect of Genistein on NF-κB Nuclear factor-κB (NF-κB) is a transcription factor that protects cells from apoptosis. NF-κB is known to translocate to the nucleus and activate a broad spectrum of genes, including growth factors, cell adhesion molecules, chemokines, and interferons (57). NF-κB is believed to prevent apoptosis by blocking activation of caspase-8, which activates caspase-3, thus intercepting the death receptor (extrinsic) pathway of apoptosis (58). After stimulation with an inducer such as TNF-α, IκB undergoes phosphorylation by activated IκB kinase (IKK), ubiquitinated, and then degraded by the 26S proteosome. The free NF-κB complex is then translocated into the nucleus, binds to a DNA consensus sequence, and transactivates genes (59). Thus, the activation of NF-κB is primarily a post-translational event involving the release of NF-κB complex from the IκB-inhibitory protein. Davis et al. demonstrated that administration of 50 µmol/L genistein to LNCaP cells (androgen-sensitive) and PC3 cells (androgen-insensitive) reduced NF-κB DNA binding in prostate cancer cells. In the same study, genistein also blocked reactive oxygen species such as H2O2 or DNA-damaging agent, i.e., TNF-α-mediated NF-B induction. The results of this study suggested that prostate tumor cells exposed to genistein had decreased NF-κB activity, which favors the induction of apoptosis. Apparently, genistein blocks the phosphorylation of the inhibitory protein IκBα. The dephosphorylated IκBα is no longer released from the complex, preventing NF-κB translocation into the nucleus and gene transactivation (60). Because IκBα is phosphorylated on serine residues, this observation suggests that genistein mediates this effect indirectly through an unidentified tyrosine kinase upstream of IKK. The Effect of Genistein on Cell Cycle Progression Genistein at moderate doses (5–50 µmol/L) was demonstrated to inhibit cell growth and delay or block the progression of the cell cycle of breast cancer cells in the G2/M phase. Estrogen receptor (ER)+ (MCF-7, T47-D, ZR75.1) and ER– (MDA-MB-468, MDA-MB 231, BT20) breast cancer cell lines (24,46,58,59–64, were reported to be equally delayed in the G2/M phase in response to genistein treatment. Apparently, the ER is not involved in genistein-induced cell cycle delay and growth inhibition, although it seems to be involved in growth stimulation by low (0.1–1 µmol/L) genistein concentrations (13). Accumulation in the G2/M phase of the cell cycle was also reported in HL-60 promyelocytic leukemia and MOLT-4 lymphocytic leukemia cells (24); HN4 squamous cell carcinoma (65); H460 non-small cell lung cancer cells (33); PC-3M prostate metastatic cancer cells

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(66); and TSGH 8301, TL4, and J82 high-grade human bladder cancer cells (32). Genistein can induce cell cycle arrest and growth inhibition in tumor cells generally at lower doses than those required to induce apoptosis (Table 25.1). Understanding the mechanism by which genistein induces G2/M cell cycle arrest may provide better opportunities for therapeutic interventions. Up-regulation of p21WAF1 after genistein treatment was evident in neoplastic PC-3-M, HN4, MDA-MB-468, MCF-7, H460 (67–69), and nonneoplastic MCF-10F cells (71). p21WAF1 was initially identified as a p53-inducible gene that mediates cell cycle arrest at the G1 phase (71) after DNA damage. In addition to being induced by p53, p21WAF1 is also induced via a p53-independent mechanism in various cell lines stimulated for growth arrest and differentiation (72). Also, p21WAF1 can participate in arresting cells in the G2/M phase of the cell cycle (67,73-75). In TL4 and J82 bladder cancer cell lines, Su et al. (32) determined that genistein caused a dose-dependent induction of G2/M cell cycle arrest and inhibition of Cdc2 kinase activity. Up-regulation of p21WAF1 was evident in both wild-type p53-expressing H460 and mutant p53-expressing H322 lung cancer cell lines, suggesting that genistein mediates its antiproliferative effects through p53-independent pathways (68). Interestingly, in the above study, lung cancer cells treated with genistein showed increased expression of endogenous wild-type p53, whereas the level of the mutant p53 protein remained unchanged. In p53-null human prostate cancer cells, genistein treatment at 100 µmol/L resulted in arrest at the G2/M phase of the cell cycle, which was related to upregulation of p21WAF1 and down-regulation of cyclin B1 (69). Furthermore, it was determined through the use of deletion mutants that the response to genistein could be localized to the 300 base pairs proximal to the transcription start site, a region that seems to be controlled by the Sp1 transcription factor. Down-regulation of cyclin B1 expression and reduced activities (which were not associated with reduced protein levels) of Cdk2 and Cdc2 kinase were part of the cascade of molecular events that led to G2/M arrest in PC-3-M cells (69). Collectively, these reports suggest that genistein may exert its effect on cell cycle progression via two pathways: (i) by inducing a significant decrease in G2/M cyclin B1, and (ii) by inducing an increase in p21WAF1 expression that leads to increased binding with Cdc2 and Cdk2, resulting in a marked decrease of their kinase activities. With only a few exceptions, general agreement exists in the literature regarding the cascade of molecular events that lead to cell cycle arrest in response to treatment with moderate doses of genistein. Through a p53-independent transcriptional regulation of p21WAF1, genistein may cause G2/M arrest, leading to inhibition of tumor cell proliferation. No evidence exists to suggest that ERα is involved, nor do any reports indicate involvement of ERβ. The Effect of Genistein on Topoisomerase II-Mediated DNA Cleavage Topoisomerases constitute a family of conserved essential enzymes that resolve topological problems during DNA replication transcription and recombination. The

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mammalian type-I enzyme (or topo I) is an ATP-independent DNA single-strand endonuclease and ligase that functions mainly during transcription. The mammalian type II enzyme (or topo II) is represented by two isoforms (α and β) that are ATP-dependent DNA double-stranded endonucleases and ligases. Topo II α is a major component of the chromosomal matrix that decatenates double-stranded DNA during replication. The expression of topo IIα is cell cycle regulated and proliferation dependent, whereas the expression of topo I and topo IIβ are constant throughout the cell cycle and independent of proliferation. Genistein was initially mistakenly reported to inhibit the catalytic activities of both topo I and topo II, apparently due to the use of a nonspecific assay and impure enzyme preparations (76). Markovitz et al. (77) observed that 9-hydroxyellipticine–resistant Chinese hamster lung cells (DC-3F/9-OH-E) were markedly more resistant to genistein than the parental cell line DC-3F. Because the DC-3F/9-OHE cells have been shown to have an altered DNA topoisomerase II, this observation suggested that genistein may inhibit topo II. When we used purified topo II, in a highly specific assay (the topo II unknotting assay), we found that genistein inhibited the topo II catalytic activity with an IC50 of ~110 µmol/L (22). Furthermore, genistein failed to inhibit topo I, demonstrating its specificity toward topo II. Inhibition of topo II generally takes place by either stabilization of a transient reaction intermediate between the topo II and DNA (called the cleavable complex) or the hindering of its formation by interfering with the religation step; on the basis of their mode of action, topo II inhibitors are distinguished into catalytic inhibitors or poisons, respectively (78). A variety of cytotoxic antitumor drugs such as 4′-(9acridinylamino)methanesulfon-m-anisidide (m-AMSA) and demethylepipodophyllotoxin ethylidene-β-D-glucoside (VP-16 or etoposide) exert their effects by stabilizing the cleavable complex. The ability of genistein to stabilize the cleavable complex and produce topo IIdependent DNA cleavage in vitro was found to be comparable to the above antitumor agents; consequently, genistein can be classified as a topo II poison (79). Topo II poisons produce characteristic patterns of plasmid DNA fragmentation depending on whether they intercalate to DNA. When the plasmid cleavage patterns of genistein (nonintercalator) were compared with those of m-AMSA (intercalator) and VP-16 (nonintercalator), it was confirmed that all three target topo II (80). However, mechanistic differences were evident on the DNA cleavage/religation, DNA strand passage, or ATP hydrolysis components of the topoisomerization reaction. Stabilization of topo II at the cleavable complex stage with topo II poisons (e.g., VP-16) initiates double strand breaks, G2/M block, caspase 3 activation, and endonucleolytic DNA cleavage characteristic of apoptosis. Most of the evidence presently available suggests that apoptosis, in response to cleavable complex formation, is induced via p53-independent mechanisms. Conflicting reports have appeared concerning whether the cleavable complex is required for the induction of apoptosis by topo II inhibitors. A pathway proposed by Boland et al. (81) that

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involves deregulation of NFκB suggests that the cleavable complex is required for apoptosis. Specifically, it was determined that the topo II poison, mitoxandrone, activated NFκB and stimulated IκB degradation in HL-60 cells, which led to caspase 3 activation. This IκB depletion was absent in HL60/MX2 cells that express a truncated form of topo IIα and lack topo IIβ. Topo II catalytic inhibitors that do not stabilize the cleavable complex, such as ICRF 187 and merbarone, did not deplete IκB, but rather protected against mitoxandrone-induced depletion. Genistein-induced DNA breakage and apoptosis in HT-29, SW-1116, and SW-620 colon cancer cells was found to be associated with cell cycle arrest in G2/M (82). Aclarubicin, a topo II catalytic inhibitor, reduced genistein-induced DNA breaks but did not prevent apoptosis. These data suggested that, in colon cancer cells, topo II serves as the enzymatic target of genistein, and topo II-mediated DNA cleavage is not required for the induction of apoptosis. Indeed, topo II catalytic inhibitors that do not stabilize the cleavable complex, such as merbarone, ICRF-187, and fostriecin, have been reported to cause G2/M block, caspase-3-like protease activation, and apoptosis (83,84). Because normal topo II activity is essential for chromosomal segregation, the catalytic inhibitors of topo II are expected to cause abnormalities in sister chromatid segregation during the mitotic phase of the cell cycle. These abnormalities can result in chromosomal breakage and apoptosis (85). On the basis of this scenario, the formation of the cleavable complex by topo II poisons (including genistein) can be dissociated from the downstream signals that lead to catastrophic DNA damage and apoptosis. Another possibility that must be considered in evaluating the interactions of genistein with topo II is that sublethal doses of genistein may cause permanent genetic alterations. If these alterations involve key regulatory genes such as transcription factors, oncogenes, or tumor suppressor genes, carcinogenesis may be initiated. The carcinogenic effects of etoposide chemotherapy in the case of acute myeloid leukemia (AML) is an example of this delayed adverse effect of topo II poison–induced genetic toxicity. The determination that certain tissues, including the mammary gland, accumulate higher levels of genistein than those found in serum (86), supports the possibility that genistein may cause delayed adverse effects by producing low levels of DNA damage either by stabilizing the interactions between DNA and topo II or by promoting abnormal chromosomal segregation.

Conclusions Numerous studies have reported that genistein can induce apoptosis in breast cancer cell lines, and a few studies have demonstrated that normal cells resist genistein-induced apoptosis and growth arrest (65,87). Furthermore, proliferating cells may be more susceptible to the antiproliferative or apoptotic effects of genistein (88). The general conclusion is that genistein may find applications both as a cancer therapeutic drug and a cancer chemopreventive agent. Although these effects of

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genistein can be of value in cancer treatment, it should be stressed that the apoptotic effects of genistein are generally evident at concentrations >50 µmol/L. These concentrations are generally below the physiologic serum levels that had been reported in human subjects consuming soy-rich diets (0.2–5 µmol/L). Cell cycle delay/growth arrest and differentiation that can be obtained at moderate concentrations (5–50 µmol/L) may find applications in cancer chemoprevention. Only the estrogenic/antiestrogenic effects of genistein are evident at physiologically relevant concentrations. Unfortunately, no evidence exists to suggest that the estrogen receptor (at least ERα) is involved in the apoptotic pathway. The manuscripts reviewed here rather suggest that, mechanistically, apoptosis could be the result of up-regulation of death signals (i.e., Bax, p53) or inhibition of proliferation signals (i.e., Src family of PTKs, NFκB, Cyclin B1). A third possible mechanism is through inhibition of topo II, which stabilizes the topo II-DNA cleavable complex, although the cleavable complex may not be required for the induction of apoptosis. In summary, to use genistein-induced apoptosis as a therapeutic approach, we must find ways to deliver the isoflavone to its molecular targets at effective concentrations. One confirmed method of achieving this is by conjugating the isoflavone to antibodies or ligands, that target tumor cells (44,89). Acknowledgments We are thankful to Kevin Grandfield for editing the manuscript and LynAriane Morgan Lucas for her assistance. Expenses associated with the writing of this article were provided by National Institutes of Health grant CA96517.

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Chapter 26

Phytoestrogens: Diabetic Nephropathy Tammy J. Stephenson and James W. Anderson Department of Nutrition & Food Science, Metabolic Research Group, Department of Internal Medicine, Graduate Center for Nutritional Sciences, University of Kentucky and VA Medical Center, Lexington, KY

Introduction Dietary intake plays a key role in the prevention and treatment of both early and end-stage renal disease (ESRD). Specifically, protein-restricted diets are often prescribed to patients with diabetes, who are at risk for developing renal disease. Compliance with such a diet is generally poor; thus, if the diet is not followed carefully, it is an ineffective treatment against kidney damage. Interestingly, research over the past century has found that a diet rich in soy foods compared with a diet dense in animal foods may control blood sugars, manage high blood pressure, prevent and treat diabetic nephropathy, and improve nutritional status in ESRD patients. At this time, it appears that it is a combination of both the soy protein and isoflavones that mediates changes in renal function. However, it is only recently that attention has been focused on the potential role of phytoestrogens, specifically the isoflavones genistein and daidzein, in renal disease. This chapter will review the limited research that examines the role that phytoestrogens play in preventing and treating diabetic nephropathy.

Soy Food Consumption and Diabetic Nephropathy Diabetes is the leading cause of ESRD in the United States. Today >40 % of new ESRD cases can be attributed to diabetes and this number continues to rise. The clinical course of diabetic nephropathy is distinguished by a series of stages, or renal changes, leading up to ESRD (1,2). Early incipient diabetic nephropathy is characterized by microalbuminuria and glomerular hyperfiltration and is a reversible stage in the progression to ESRD (3,4). Histologically, there is a thickening of the glomerular basement membrane and mesangeal matrix expansion. Unfortunately, nearly 80% of individuals with Type 1 diabetes and untreated incipient nephropathy develop overt nephropathy, a nonreversible condition. Macroalbuminuria, histological evidence of diffuse or nodular glomerulosclerosis, tubulointerstitial fibrosis, and glomerular hypofiltration are distinctive of overt nephropathy (5). Overt nephropathy progresses to renal failure or ESRD in the majority of diabetic individuals afflicted with this condition.

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Poor glycemic control, high diastolic blood pressure, the presence of microalbuminuria or proteinuria, more severe retinopathy, and history of impaired tactile sensation or temperature insensitivity are associated with renal damage (3). Normalization of blood glucose, hypertensive treatment, and restriction of dietary protein are the primary interventions for those at risk for diabetic nephropathy. Since first suggested by Dr. Richard Bright over 150 years ago, significant research has supported the recommendation to restrict dietary protein to effectively slow the progression of nephropathy (6). Consumption of 0.6–1.0 g/(kg body ⋅ d) of protein appears to improve markers of renal damage and slow progression toward organ failure in diabetic individuals. Decreased albumin excretion rates, normalization of renal hyperfiltration, and decreased rate of decline in glomerular filtration rate (GFR) have all been reported with this moderate protein restriction (7). However, compliance with such a diet has habitually been very poor and therefore is of limited benefit. In reality, most Americans consume nearly two times their recommended dietary allowances for protein every day and cutting back on protein is habitually difficult to do (8). For this reason, recent research has begun to evaluate the potential renal affects of substituting soy protein-rich foods for animal proteinrich foods to prevent and treat diabetic nephropathy. Preliminary findings suggest that a plant-based, soy food-dense, diet may be superior to a traditional animal food-based diet in the prevention and treatment of diabetic renal disease. The soy protein hypothesis states that substitution of soy protein for animal protein in persons with diabetic nephropathy results in less hyperfiltration and glomerular hypertension with resultant protection from diabetic nephropathy (9). Indeed, this hypothesis has been supported with clinical trials that have shown that animal protein intake significantly increases renal blood flow and glomerular filtration rate, two contributors to diabetic nephropathy, compared with soy protein intake (10,11). Eight intervention trials in humans have specifically evaluated the effects of a plant-based diet on renal function. Three of these studies evaluated renal function in individuals with diabetes; however, they included only small numbers of subjects and therefore interpretation from these trials was limited (9,12,13). We recently completed a 5-mo soy foods intervention trial in individuals with Type 1 diabetes (n = 14) and clinical evidence of early incipient nephropathy (Table 26.1). After a 4-wk run-in period to assess baseline characteristics (Baseline) subjects were trained to follow a soy food-based diet for 8 wk (Soy Diet). Soy foods were provided to participants in the form of soy patties, soy milk, soy pasta, soy chocolate drinks, soy snack bars, sweet green soybeans, and roasted soy nuts. After the 8 wk of soy intervention, subjects then resumed their habitual, animal protein-rich diet for 8 wk (Control Diet). Dietary counseling was provided throughout the course of the trial, and 3-d food records were collected from all participants biweekly. GFR was improved in 11 of the 12 hyperfiltering subjects during the Soy Diet intervention period. On average, GFR was reduced by 10% after 8 wk of consuming a soy protein-based diet (Table 26.2). Similarly, total and low density lipopro-

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TABLE 26.1 Baseline Characteristics of Fourteen Type 1 Diabetes Subjectsa Variable Gender Race Age (y) Duration of diabetes (y) Diabetes therapy Body weight (kg) Body mass index (kg/m2) GFR [mL/(min ⋅ 1.73m2)] Dietary protein intake (g/kg body) aValues

7 M, 7 W 12 Caucasians, 1 Jamaican, 1 African-American 29.9 ± 2.4 (18–49) 15.1 ± 2.3 (2–30) 4 insulin pumps, 10 multiple daily injections 79.2 ± 5.0 (52–120) 26.6 ± 1.5 (21–43) 151.9 ± 8.2 (120–206) 1.32 ± 0.12 (0.4–2.2)

are means ± SEM (range); GFR, glomerular filtration rate.

tein (LDL)-cholesterol were lowered by 7 and 9%, respectively, during the Soy Diet. Serum triglycerides were numerically lower after 8 wk of soy consumption compared with Baseline levels. Microalbuminuria was detected in only 3 of the 14 study participants. Two of the three subjects experienced a drop in microalbuminuria when following the Soy Diet. Urine sodium, urine urea nitrogen, and urine creatinine excretion were assessed from overnight and 24-h specimen collections. No significant differences in excretion rates were found between treatment periods. Interestingly, total dietary intake was significantly less during the Control Diet compared with either the Baseline or the Soy Diet. Total energy, dietary fat, dietary cholesterol, and dietary sodium were all significantly lower during the Control Diet. These preliminary findings suggest that consumption of a soy food-rich diet for 8 wk reverses hyperfiltration and improves dyslipidemia in diabetic individuals. In addition, a soy diet may potentially reduce microalbuminuria, but too few subjects were available for careful assessment of this parameter. The mechanisms by which a soy diet beneficially affects renal health in diabetic nephropathy require further study. From the preliminary evidence, it appears that it is both the protein and isoflavone components of soy foods that contribute to the renal protective TABLE 26.2 Laboratory Parameters at Baseline, After 8 wk of Soy Food Consumption (Soy Diet), and After 8 wk of Control Dieta Baseline GFR [mL/(min ⋅ 1.73 Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) Conjugated dienes (µmol/L) m2)]

aValues bP

159 ± 7.7 181 ± 10.1 115 ± 9.3 45.1 ± 3.0

Soy diet 7.4b

143 ± 168 ± 8.0b 105 ± 7.7b 41.0 ± 4.7

are means ± SEM; GFR, glomerular filtration rate; LDL, low density lipoprotein. < 0.05 by ANOVA.

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Control diet 161 ± 10.0 187 ± 9.4 117 ± 8.6 43.7 ± 3.9

affects in diabetic nephropathy. Differences in amino acid composition between animal and soy protein may contribute to varying vasodilatory effects of the afferent arterioles of the glomeruli, but further research is required to define these differences (12,14). While phytoestrogen-specific effects on renal function require further in-depth study; we will review selected aspects of this current research.

Phytoestrogen Effects on Diabetic Nephropathy From preliminary evidence, we hypothesize that phytoestrogens act in at least three specific ways: (i) they mediate renal afferent and efferent arteriole reactivity; (ii) they modulate nitric oxide activity; and (iii) they have important antioxidant activities. Cutting-edge research points to the exciting potential effects of isoflavone metabolites on vascular biology (15). Phytoestrogens Mediate Renal Arteriole Reactivity Phytoestrogens are believed to play a role in mediating vascular reactivity; thus, they may affect afferent and efferent renal arteriole properties. Research has found that isoflavones, such as genistein and daidzein, induce endothelium-independent relaxation of coronary arteries through a mechanism involving calcium antagonism (16). Indeed, in ways very similar to estrogen, genistein has vascular activity and can accentuate acetylcholine-induced vasodilation (17). Arteries from female monkeys fed a high-isoflavone soy-based diet for 6 mo were found to dilate in response to acetylcholine, whereas arteries from monkeys fed a low-isoflavone soy-based diet actually constricted. Hence, the phytoestrogens were enhancing the dilator response to acetylcholine of atherosclerosic arteries in female monkeys (18). In addition to the acetylcholine-mediated effects of isoflavones, genistein may mediate vascular reactivity through several other pathways. For example, genistein has been shown in vitro to be a tyrosine kinase inhibitor (19–21). The concentrations of genistein (2.6–26 µmol/L) that induce vascular smooth muscle relaxation are within the inhibitor concentration values for genistein against tyrosine protein kinases. We hypothesize that through one, or a combination of these mechanisms, renal delete arterioles are relaxed. It is this relaxation of efferent arterioles that then induces a reduction in glomerular pressure and GFR, thus protecting the kidneys of individuals with diabetes. Phytoestrogens Mediate Nitric Oxide Activity Like acetylcholine, nitric oxide may also be mediating vascular changes that have been described in diabetes individuals and early nephropathy (22,23). Indeed, nitric oxide with activity has been implicated in the pathogenesis of glomerular hyperfiltration (24–26) and in enhanced permeability to macromolecules that leads to microalbuminuria (27,28). Nitric oxide appears to preferentially induce afferent arteriolar dilation and glomerular enlargement (29). Interestingly, the isoflavone

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genistein appears to modulate the enzymatic control of nitric oxide in vitro (30). It appears that nitric oxide production may be suppressed after an isoflavone-rich soy protein meal and therefore may reduce renal afferent arteriole dilation. This vascular change may then reduce renal blood flow, glomerular pressure, and GFR. Phytoestrogens Act as Antioxidants Several animal models of renal disease have confirmed the protective effects of low-fat diets and lipid-lowering agents on the extent of glomerular damage and proteinuria (31–34). Dyslipidemia plays a central role in the rate of decline of diabetic nephropathy (35). Interestingly, Dubois et al. (36) showed a close temporal relationship between hypertriglyceridemia and nephropathy in men with noninsulin-dependent diabetes mellitus. Experimental data indicate that dyslipidemia favors the development of focal glomerulosclerosis (37) and foam cells in the glomeruli resembling those of atherosclerosis (38). In addition, LDL cholesterol has been shown to increase adherence of monocytes to endothelial cells; these monocytes might then play a role in the initiation of vascular injury in the glomeruli (39). Dyslipidemia may also promote platelet aggregation and stimulate the release of platelet-derived growth factor, which may induce proliferation of mesangial and smooth muscle cells (40). A diet rich in soy foods has been shown to improve hyperlipidemia in both healthy and diabetic individuals. D’Amico and Gentile (41) found that soy consumption improved dyslipidemia in patients with diabetic nephropathy. We found that total and LDL-cholesterol were significantly reduced in our Type 1 diabetic subjects after 8 wk of isoflavone-rich soy food intervention. These findings are consistent with those of Type 2 proteinuric diabetic subjects who experienced a 9% reduction in total and LDL-cholesterol and a 23% improvement in triglycerides while consuming a soy protein-based diet (9). Oxidation of mesangial cells may also promote glomerular injury (42). Antioxidant therapy may therefore prevent this injury. Soy isoflavones have been shown to inhibit the oxidation of LDL and may thus prevent this oxidative-induced glomerular injury (43–47). Tikkanen and collaborators (48) evaluated the effects of soybean phytoestrogen intake on LDL oxidation in a study of six healthy volunteers. Subjects consumed two soy bars daily for 2 wk; each bar contained 12 mg genistein, 7 mg daidzein, and 7.1 g soy protein. Compared with the control diet, the soy diet led to a significant lag phase in the LDL oxidation curve. Similarly, lipid peroxidation levels were reduced by 17% in nonhuman primates fed an isoflavone-rich soy protein isolate compared with monkeys fed a comparable casein and lactoalbumin protein diet (49). To further evaluate the antioxidant potential of phytoestrogens in our Type 1 diabetic subjects, LDL-oxidation was measured in seven of our participants. In these seven subjects, conjugated dienes, a marker of LDL-oxidation, were numerically, but not significantly lower during the Soy Diet, suggesting a potential antioxidant effect. LDL-oxidation will be measured in additional subjects so that we can better ascertain the potential antioxidant potential of soy isoflavones in diabetic nephropathy.

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Summary and Conclusions Consumption of a soy food-rich diet yields many benefits in the prevention and treatment of diabetic nephropathy. Preliminary evidence suggests that substituting soy foods for animal foods reverses hyperfiltration and dyslipidemia in diabetic individuals with incipient nephropathy. It appears to be the marriage of the soy protein and isoflavones that mediates many of the renal protective effects of a soy-rich diet. At this time, we hypothesize that the phytoestrogen-specific roles include modulation of vascular health through acetylcholine or nitric oxide mechanisms and antioxidant activity. Indeed, further research is required to assess the role of phytoestrogens more fully in both diabetic nephropathy and other forms of renal disease. References 1. Mogensen, C.E. (1997) How to Protect the Kidney in Diabetic Patients with Special Reference to IDDM, Diabetes 46, S104–S111. 2. Mogensen, C.E., Christiansen, C.K., and Vittinghus, E. (1983) The Stages in Diabetic Renal Disease with Emphasis on the Stage of Incipient Nephropathy, Diabetes 32, 64–78. 3. American Diabetes Association (2000) Diabetic Nephropathy, Diabetes Care 23, S69–S72. 4. Chiarelli, F., Verrotti, A., Mohn, A., and Morgese, G. (1997) The Importance of Microalbuminuria as an Indicator of Incipient Diabetic Nephropathy: Therapeutic Implications, Ann. Med. 29, 439–445. 5. Osterby, R. (1992) Glomerular Structural Changes in Type 1 (Insulin-Dependent) Diabetes Mellitus: Causes, Consequences, and Prevention, Diabetologia 35, 803–812. 6. Pedrini, M.T., Levey, A.S., Lau, J., Chalmers, T.C., and Wang, P.H. (1996) The Effect of Dietary Protein Restriction on the Progression of Diabetic and Nondiabetic Renal Diseases: A Meta-Analysis, Ann. Intern. Med. 124, 627–632. 7. Hansen, H.P., Christensen, P.K., Tauber-Lassen, E., Klausen, A., Jensen, B.R., and Parving, H.H. (1999) Low-Protein Diet and Kidney Function in Insulin-Dependent Diabetic Patients with Diabetic Nephropathy, Kidney Int. 55, 621–628. 8. Anderson, J.W. (1993) Why Do Diabetic Individuals Eat So Much Protein and Fat? Med. Exerc. Nutr. Health 2, 65–68. 9. Anderson, J.W., Blake, J.E., Turner, J., and Smith, B.M. (1998) Effects of Soy Protein on Renal Function and Proteinuria in Patients with Type 2 Diabetes, Am. J. Clin. Nutr. 68, 1347S–1353S. 10. Nakamura, H., Yamazaki, M., Chiba, Y., Tani, N., Momotsu, T., Kamoi, K., Ito, S., Yamaji, T., and Shibata, A. (1990) Acute Loading with Proteins from Different Sources in Healthy Volunteers and Diabetic Patients, J. Diabet. Complicat. 38, 136–140. 11. Kontessis, P., Jones, S., Dodds, R., Trevisan, R., Nosadini, R., Fioretto, P., Borsato, M., Sacerdoti, D., and Viberti, G.C. (1990) Renal, Metabolic and Hormonal Responses to Ingestion of Animal and Vegetable Proteins, Kidney Int. 79, 173–180. 12. Kontessis, P., Bossinakou, I., Sarika, L., Iliopoulou, E., Papantoniou, A., Trevisan, R., Roussi, D., Stipsanelli, K., Grigorakis, S., and Souvatzoglou, A. (1995) Renal, Metabolic, and Hormonal Responses to Proteins of Different Origin in Normotensive, Nonproteinuric Type 1 Diabetic Patients, Diabetes Care 18, 1233–1240.

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13. Jibani, M.M., Bloodworth, L.L., Foden, E., Griffiths, K.D., and Galpin, O.P. (1991) Predominately Vegetarian Diet in Patients with Incipient and Early Clinical Nephropathy. Effects on Albumin Excretion Rate and Nutritional Status, Diabet. Med. 8, 949–953. 14. Nakamura, H., Takasawa, M., Kasahara, S., Tsuda, A., Momotsu, T., Ito, S., and Shibata, A. (1989) Effects of Acute Protein Loads of Different Sources on Renal Function in Patients with Diabetic Nephropathy, Tohoku J. Exp. Med. 159, 153–162. 15. Chin-Dusting, J.P.F., Fisher, L.J, Lewis,T.V., Piekarska, A., Nestel, P.J., and Husband, A. (2001) The Vascular Activity of Some Isoflavone Metabolites: Implications for a Cardioprotective Effect, Br. J. Pharmacol. 133, 595–605. 16. Figtree, G.A., Griffiths, H., Lu, Y.Q., Webb, C.M., MacLeod, K., and Collins, P. (2000) Plant-Derived Estrogens Relax Coronary Arteries In Vitro by a Calcium Antagonistic Mechanism, J. Am. Coll. Cardiol. 35, 1977–1985. 17. Mendelsohn, M.E., and Karas, R.H. (1999) The Protective Effects of Estrogen on the Cardiovascular System, N. Engl. J. Med. 340, 1801–1811. 18. Honore, E.K., Williams, J.K., Anthony, M.S., and Clarkson, T.B. (1997) Soy Isoflavones Enhance Coronary Vascular Reactivity in Atherosclerotic Female Macaques, Fertil. Steril. 67, 148–154. 19. Yang, S.G., Saifeddine, M., and Hollenberg, M.D. (1992) Tyrosine Kinase Inhibitors and the Contractive Action of Epidermal Growth Factor-Urogastrone and Other Antagonists in Gastric Smooth Muscle, Can. J. Physiol. Pharmacol. 70, 85–93. 20. Jin, N., Siddiqui, R.A., English, D., and Rhoades, R.A. (1996) Communication Between Tyrosine Kinase Pathway and Myosin Light Chain Kinase Pathway in Smooth Muscle, Am. J. Physiol. 271, H1348–H1355. 21. Steusloff, A., Paul, E., Semenchuk, L.A., Di-Salvo, J., and Pfitzer, G. (1995) Modulation of Ca2+ Sensitivity in Smooth Muscle by Genistein and Protein Tyrosine Phosphorylation, Arch. Biochem. Biophys. 320, 236–242. 22. Chiarelli, F., Cipollone, F., Romano, F., Tumini, S., Costantini, F., di Ricco, L., Pomilio, M., Pierdomenico, S.D., Marini, M., Cuccurullo, F., and Mezzetti, A. (2000) Increased Circulating Nitric Oxide in Young Patients with Type 1 Diabetes and Persistent Microalbuminuri: Relation to Glomerular Hyperfiltration, Diabetes 49, 1258–1263. 23. Pugliese, G., Tilton, R.G., and Williamson, J.R. (1991) Glucose-Induced Metabolic Imbalances in the Pathogenesis of Diabetic Vascular Disease, Diabetes Metab. Rev. 7, 35–39. 24. Corbett, J.A., Tilton, R.G., Chang, K., Hassan, K.S., Ido, Y., Wang, J.L., Sweetland, M.A., Lancaster, J.R., Williamson, J.R., and McDaniel, M.L. (1992) Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction, Diabetes 41, 552–6. 25. Graier, W.F., Wascher, T.C., Lackner, L., Toplak, H., Krejs, G.J., and Kukovetz, W.R. (1993) Exposure to Elevated D-Glucose Concentrations Modulates Vascular Endothelial Cell Vasodilatory Response, Diabetes 42, 1497–1505. 26. Bank, N., and Aynedjian, H.S. (1993) Role of EDRF (Nitric Oxide) in Diabetic Renal Hyperfiltration, Kidney Int. 43, 1306–1312. 27. Tolins, P., Shultz, P.J. Raij, L., Brown, D.M., and Mauer, S.M. (1993) Abnormal Renal Hemodynamic Response to Reduced Renal Perfusion in Diabetic Rats: Role of Nitric Oxide, Am. J. Physiol. 265, F886–F895. 28. Reyes, A.A., Karl, I.E., Kissane, J., and Klahr, S. (1993) L-Arginine Administration Prevents Glomerular Hyperfiltration and Decreases Proteinuria in Diabetic Rats, J. Am. Soc. Nephrol. 4, 1039–1045.

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29. Sugimoto, H., Shikata, K., Matsuda, M., Hayashi, Y., Hiragushi, K., Wada, J., and Makino, H. (1998) Increased Expression of Endothelial Cell Nitric Oxide Synthase (ECNOS) in Afferent and Glomerular Endothelial Cells Is Involved in Glomerular Hyperfiltration of Diabetic Nephropathy, Diabetologia 41, 1426–1434. 30. Dong, Z., Qi, X., Xie, K., and Fidler, I.J. (1993) Protein Tyrosine Kinase Inhibitors Decrease Induction of Nitric Oxide Synthase Activity in LipopolysaccharideResponsive and Lipopolysaccharide-Nonresponsive Murine Macrophages, J. Immunol. 151, 17–24. 31. Keane, W.F., Mulkahy, W.S., Kasiske, B.L., Kim, Y., and O’Donnell, P. (1991) Hyperlipidemia and Progressive Renal Disease, Kidney Int. 39, 41–48. 32. Schreiner, G.F. (1991) Dietary Treatment of Immunologically Mediated Renal Disease, Kidney Int. 39, 49S–56S. 33. Barcelli, U.O. (1991) Effect of Dietary Prostaglandin Precursors on the Progression of Renal Disease in Animals, Kidney Int. 39, 57–64. 34. Fujisawa, K., Yagasaki, K., and Funabiki, R. (1995) Reduction of Hyperlipidemia and Proteinuria Without Growth Retardation in Nephritic Rats by a Methionine-Supplemented, Low-Soy Diet, Am. J. Clin. Nutr. 61, 603–606. 35. Mulec, H., Johnson, S., and Bjorck, S. (1990) Relationship Between Serum Cholesterol and Diabetic Nephropathy, Lancet 335, 1537–1538. 36. Dubois, D., Chanson, P., and Timsit, J. (1994) Remission of Proteinuria Following Correction of Hyperlipidemia in NIDDM Patients with Nondiabetic Glomerulopathy, Diabetes Care 17, 906–908. 37. Peric-Golia, L., and Peric-Golia, M. (1983) Aoratic and Renal Lesions in Hypercholesterolemic Adult Male Virgin Sprague-Dawley Rats, Atherosclerosis 46, 57–65. 38. Wellman, K., and Volk, B.W. (1970) Renal Changes in Experimental Hypercholesterolemia in Normal and Subdiabetic Rats, Lab. Investig. 22, 144–155. 39. Alderson, L.M., Endemann, G., Lindseay, S., Pronczuk, A., Hoover, R.L., and Hayes, K.C. (1986) LDL Enhances Monocyte Adhesion to Endothelial Cells In Vitro, Am. J. Pathol. 123, 334–342. 40. Thomas, W.A., and Kim, D.N. (1983) Atherosclerosis as a Hyperplastic and/or Neoplastic Process, Lab. Investig. 48, 245–255. 41. D’Amico, G., and Gentile, M.G. (1993) Influence of Diet on Lipid Abnormalities in Human Renal Disease, Am. J. Kidney Dis. 22, 151–157. 42. Wheeler, D.C., and Chana, R.S. (1994) Oxidation of Low-Density Lipoprotein by Mesangial Cells May Promote Glomerular Injury, Kidney Int. 45, 1628–1636. 43. Jenkins, D.J.A., Kendall, C.W.C., and Garsetti, M. (2000) Effect of Soy Protein Foods on Low-Density Lipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity— A Controlled Crossover Trial, Metabolism 49, 537–543. 44. Anderson, J.W., Diwadkar, V.A., and Bridges, S.R. (1998) Selective Effects of Different Antioxidants on Lipoproteins from Rats, Proc. Soc. Exp. Biol. Med. 218, 376–381. 45. Hodgson, J.W.M., Croft, K.D., and Puddey, I.B. (1996) Soybean Isoflavonoids and Their Metabolic Products Inhibit In Vitro Lipoprotein Oxidation in Serum, J. Nutr. Biochem. 7, 664–669. 46. Madani, S., Prost, J., and Belleville, J. (2000) Dietary Protein Level and Origin (Casein and Highly Purified Soybean Protein) Affect Hepatic Storage, Plasma Lipid Transport, and Antioxidative Defense Status in the Rat, Nutrition 16, 368–375.

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47. Meng, Q.H., Lewis, P., Wähälä, K., Adlercreutz, H., and Tikkanen, M.J. (1999) Incorporation of Esterfied Soybean Isoflavones with Antioxidant Activity into Low Density Lipoprotein, Biochim. Biophys. Acta 1438, 369–376. 48. Tikkanen, M.J., Wähälä, K., Ojala, S., Vihma, V., and Adlercreutz, H. (1998) Effect of Soybean Phytoestrogen Intake on Low Density Lipoprotein Oxidation Resistance, Proc. Natl. Acad. Sci. USA 95, 3106–3110. 49. Wagner, J.D., Cefalu, W.T., Anthony, M.S., Litwak, K.N., Zhang, L., and Clarkson, T.B. (1997) Dietary Soy Protein and Estrogen Replacement Therapy Improve Cardiovascular Risk Factors and Decrease Aortic Cholesteryl Ester Content in Ovariectomized Cynomolgus Monkeys, Metabolism 46, 698–705.

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Chapter 27

Hormonal Effects of Phytoestrogens in Premenopausal Women Alison M. Duncana, William R. Phippsb, and Mindy S. Kurzerc aDepartment

of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada bDepartment

of Obstetrics and Gynecology, University of Rochester, Rochester, NY

cDepartment

of Food Science and Nutrition, University of Minnesota, St. Paul, MN

Introduction Phytoestrogens are naturally occurring plant chemicals that consist of four classes, including isoflavones, lignans, coumestans, and resorcyclic acid lactones. The majority of research regarding the hormonal effects of phytoestrogens in premenopausal women has focused on isoflavones, and to a lesser extent, lignans. The major dietary sources of isoflavones are soybean products (1), whereas lignans are more ubiquitous in the plant kingdom, with exceptionally high concentrations in flaxseed (2). Both isoflavones and lignans undergo metabolism by intestinal microflora before absorption. The two major isoflavones in soy, the isoflavone conjugates genistin and daidzin, are metabolized in the gut to their aglycones, genistein and daidzein (3,4). Genistein may then be further metabolized to p-ethylphenol, whereas daidzein may be further metabolized to O-desmethylangolensin (ODMA) and/or equol (3,4). Both the isoflavones and their metabolites are then absorbed to varying degrees (3,4). The two major plant lignans, secoisolariciresinol and matairesinol, are metabolized to the mammalian lignans, enterodiol and enterolactone, which are then absorbed (2). Both isoflavones and lignans are excreted in the urine (5) and also undergo enterohepatic circulation via the bile (3). Since the majority of the human studies involve soy, this review will focus on soy isoflavones, while mentioning the lignans where relevant. Isoflavones are structurally similar to selective estrogen receptor modulators, and as such, can bind to the estrogen receptor, showing a greater affinity to estrogen receptor β (6). As weak estrogens, isoflavones have been shown to act as estrogen agonists or antagonists, depending on the levels of endogenous estrogen present (7). A common hypothesis in premenopausal women is that the weakly estrogenic isoflavones will compete with the more potent endogenous estrogens, thereby exerting a net antiestrogenic effect. The relevance of this theory is the potential for soy isoflavone consumption to prevent estrogen-dependent diseases such as breast and endometrial cancer.

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When the worldwide incidence of breast cancer is considered, the rates are lowest in Asian women and highest in Caucasian women (8). Interestingly, the incidence of breast cancer in Asian migrants and their offspring approach Western rates, suggesting that environmental influences are more important than genetic factors (9). A growing body of evidence suggests that soy foods, which are consumed in high amounts by Asian women, may play a role in the worldwide variation in breast cancer incidence (4,7,10). Epidemiologic studies in premenopausal women have reported a protective effect of soy consumption against cancer of the endometrium (11) and breast (12–17), although others have been unable to detect relationships between breast cancer and either soy (18–20) or lignan (20) consumption. To avoid limitations due to a lack of focus on soy or isoflavone consumption, as well as the general limitations of food intake questionnaires (7), other epidemiologic studies have measured phytoestrogen concentrations in biological fluids as an alternative. These studies have detected inverse relationships between breast cancer risk in premenopausal women and serum concentrations of enterolactone (21), as well as urinary concentrations of total isoflavones (22), glycitein (22), and enterolactone (23). Results for urinary equol have been inconsistent, with one study observing a significant negative association between breast cancer risk and urinary equol (23) and another study observing no significant relationship (22). Studies have been unable to detect significant associations between breast cancer risk and urinary concentrations of genistein (23), daidzein (22,23), or enterodiol (23) in premenopausal women. Since the etiology of breast cancer is related to reproductive hormones and their metabolism (24,25), investigators have studied the hormonal effects of phytoestrogens in premenopausal women as a possible mechanism by which they may lower breast cancer risk. The purpose of this review is to summarize the studies performed in the last decade that have provided evidence that phytoestrogens, particularly soy isoflavones, exert small hormonal effects in premenopausal women. Study Design Issues Before reviewing these studies, it is pertinent to consider the various study design factors that complicate their direct comparison. Studies have varied on numerous factors including sample size, ethnicity of subjects, randomization, phytoestrogen source, phytoestrogen dose, length of intervention, control used, and end points evaluated (Table 27.1). Effects on Menstrual Cycle Parameters Menstrual cycle parameters include the lengths of the total cycle as well as the follicular and luteal phases. To monitor lengths of menstrual cycle phases, investigators must include a method for determining day of ovulation in their study design. Recently, an epidemiologic study reported a significant positive association between soy consumption and menstrual cycle length in premenopausal women

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TABLE 27.1 Premenopausal Phytoestrogen Intervention Studiesa Ref.

n

Soy food studies 27,28 6

Design

Treatment

Length

Iso dose (mg/d)

TVP

1 MC

23, 45

Iso-free soy, miso Soymilk

1 MC

0, 25

1 mo

SPI

Control

End points evaluated

200

Soy-free diet (1 MC) Soy-free diet (1 MC) Baseline sample

6 mo

~70

Usual diet (3 mo) Usual diet (n = 29, 3 MC) SPI (10 mg Iso, 3 MC) Usual diet (n = 23, 2 wk) Usual diet (2 MC) SPI (10 mg Iso, 3 MC)

MC and phase lengths, plasma hormones MC and phase lengths, plasma hormones MC length, plasma hormones Plasma hormones, nipple aspirate fluid MC length, plasma hormones Urinary estrogens

28

3, 5

29

6

51

14

NR crossover Metabolic ward NR crossover metabolic ward NR Metabolic ward NR crossover

30

31

R two-arm

Soymilk

3 MC

109

68

12

R crossover

SPI

3 MC

65, 129

71

28

R two-arm

TVP

2 wk

45

32

36

R crossover

SPI

2 MC

38

31

14

R crossover

SPI

3 MC

64, 128

Nipple aspirate fluid, breast histology MC and phase lengths, plasma and urinary hormones MC and phase lengths, plasma hormones, endometrial histology Continued

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TABLE 27.1 (Cont.) Ref.

n

Design

Treatment

Length

Iso dose (mg/d)

Control

End points evaluated

35

20

NR crossover

3 MC

32

Usual diet (2 MC)

69

8

1 MC

158

10

Soymilk

1 MC

154

Soymilk (<4.5 mg Iso, 1 MC) Baseline sample

MC and phase lengths, plasma hormones Urinary estrogens

33 34

9

NR crossover Metabolic ward NR Metabolic ward NR

Soymilk, tofu, soybean peas Soymilk

Soymilk

1 MC

<5

Baseline sample

Flax studies: 37 18

R crossover

Flax

3 MC

—

Usual diet (3 MC)

70

R crossover

Flax

2 MC

—

Usual diet (2 MC)

Isoflavone extract studies: 36 19 NR crossover

Soy extract

1 mo

40

36

Soy extract

1 mo

50

20 mg Iso (n = 20, 1 mo) Baseline data

16

3

aAbbreviations:

NR

MC and phase lengths, plasma hormones MC and phase lengths, plasma hormones

MC and phase lengths, plasma hormones Urinary estrogens

MC length, plasma hormones MC length, plasma hormones

Iso, isoflavone; NR, nonrandomized; TVP, texturized vegetable protein; MC, menstrual cycle; SPI, soy protein isolate; R, randomized.

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(26). This is consistent with an intervention study by Cassidy and colleagues (27), who observed an increase of 1.5 d in total length of the cycle, largely due to a significant increase of 2.5 d in follicular phase length in women consuming texturized vegetable protein (TVP). The same investigators observed no significant effects of an isoflavone-free product on cycle or phase lengths, suggesting that the isoflavones in the TVP were responsible for the prolonged cycle and follicular phase lengths (28). Although other soy food or soy protein studies have not reported significant changes in total or phase lengths (28–35), it is noteworthy that none observed a decrease and many observed increases that approached significance. For example, nonsignificant increases of 5.4 and 1.9–3.5 d were observed in women consuming miso (28) and soy milk (29,30), respectively. It is possible that studies unable to demonstrate a significant increase were insufficiently powered to overcome the interindividual variability in menstrual cycle lengths and/or were not long enough to demonstrate an effect. Consistent with this are observations by Watanabe et al. (36), who reported a 3-d increase in cycle length in 60% of subjects consuming an isoflavone extract, and Phipps et al. (37), who found a significant increase of 1.2 d in luteal phase length in women consuming lignan-rich flaxseed. Although small, it is possible that such an increase in menstrual cycle length over many years could reduce risk of breast cancer. Supportive of this is a case-control study that reported significantly shorter menstrual cycle lengths (average of 2.2 d) in breast cancer patients compared with controls (38). An increased menstrual cycle length would reduce the cumulative number of menstrual cycles over a lifetime, thereby reducing breast cancer risk through a reduced overall exposure to estrogen. Since cycle length variation is most often due to variation in follicular phase length (39), the proportionally greater time spent in the follicular phase, when levels of estrogen and progesterone as well as breast tissue mitotic activity are lowest (40,41), is thought to be protective against breast cancer. In addition, regular ovulatory cycles increase breast cancer risk (42) and anovulatory cycles, in some cases, are thought to protect against breast cancer (43), with both factors related to exposure to luteal phase hormones. These observations suggest that even in the absence of significant increases, the small increase in menstrual cycle length observed by consumption of isoflavone-rich soy and lignan-rich flaxseed may protect against breast cancer risk over long periods of time. Effects on Plasma Hormones Estrogens. The potential mechanisms by which phytoestrogens alter plasma estrogens could include an effect on pituitary gonadotropins, which in turn can influence estrogen concentrations, direct effects on the ovary, and/or effects on enzymes involved in estrogen synthesis and metabolism. In vitro, isoflavones (daidzein, equol, ODMA) and lignans (enterolactone, enterodiol) inhibit aromatase, the rate-limiting enzyme in estrogen biosynthesis (44–46), and genistein

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inhibits 17β-hydroxysteroid oxidoreductase, the enzyme that converts estrone to the more potent estradiol (47). Epidemiologic studies report negative correlations between soy consumption and plasma estradiol in premenopausal Japanese women (48), as well as between urinary isoflavones and plasma free estradiol in premenopausal Finnish women (49,50), although intervention studies have been unable to consistently confirm these observations. Five studies, ranging in isoflavone intake from 5 to 200 mg/d, observed a significant decrease in plasma estradiol during the follicular phase (33,34), mid-cycle (d 12–14) (29), and luteal phase (29,33–35) after consumption of various soy products. In contrast, two studies (45 and 70 mg isoflavones/d) observed a significant increase in estradiol after soy consumption (27,51), one study observed a positive correlation between urinary daidzein and luteal phase estradiol after consumption of isoflavone extract (40 mg isoflavones/d) (36), and six studies observed no significant effect of soy (5–128 mg isoflavones/d) (28,30–32) or flax (37) consumption on plasma estradiol. Plasma estrone was positively associated with soy consumption during the luteal phase, but not the follicular phase, in a recent epidemiologic study in premenopausal women (26). Intervention studies have found no significant effects of soy (31,32) or flax (37) consumption on plasma estrone during the luteal phase. In contrast, plasma estrone during the follicular phase was significantly decreased in women consuming soy milk containing 109 mg isoflavones (30) and soy protein containing 128 mg isoflavones (31), but was unchanged in women consuming soy protein containing 38 mg isoflavones (32) or flax (37). Finally, plasma estrone-sulfate during the follicular and luteal phases was not significantly affected by soy protein containing either 64 or 128 mg isoflavones in premenopausal women (31). Given the inconsistent effects of phytoestrogen consumption on plasma estrogens, the relevance to breast cancer is unclear. Lower plasma estrogens are thought to be protective due to the role of estrogens in the etiology of breast cancer (24,25) and Asian women, who consume relatively large amounts of soy, have lower plasma estrogens compared with Western women (52–54). Apart from study design differences, it is noteworthy that three studies that focused on Asian populations (30,35,48) consistently observed a decrease in plasma estrogens with soy consumption. In particular, the small decrease in plasma estradiol observed by Wu et al. (35), was observed only in their subset of Asian subjects and not in the Caucasian subjects. It is possible that an effect on plasma estrogens requires a long-term consumption of soy and is therefore more easily detected in women who have been consuming soy their whole lives. Gonadotropins. The activity of the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), is most relevant midcycle when they surge before ovulation in response to positive feedback by estrogen (40). Since high concentrations of estrogen, particularly estradiol, are responsible for the normal midcycle gonadotropin surge, its suppression by soy isoflavones may reflect an antie-

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strogenic effect on the hypothalamic-pituitary-ovarian axis. Two intervention studies in premenopausal women did observe a significant suppression of midcycle plasma LH and FSH after consumption of soy providing 45 and 64 mg isoflavones/d (27,31). On the other hand, 0, 5, 23, 25, and 154 mg/d did not significantly alter plasma LH or FSH levels (28,33,34). Finally, peak levels of LH and FSH were decreased in three women who consumed an isoflavone extract (36). Attenuated midcycle gonadotropin surges could result in a weakened luteal phase, as reflected by delayed peak progesterone (27,28), decreased plasma progesterone (29,33,34), and decreased luteal phase estradiol, (29,33–35). Progesterone. Although decreased progesterone could result from suppressed gonadotropins, it could also result from a direct inhibition of synthesis, as indicated by in vitro data reporting that a high concentration of genistein inhibits progesterone synthesis in bovine granulosa cells (55). Despite this, the majority of studies have not detected a significant change in luteal phase progesterone after consumption of soy foods (27,28,31,32,35,51), flax (37), or isoflavone extract (36). A significant decrease in progesterone was observed in three studies by the same investigators (29,33,34), although one study did not standardize the blood sampling to ovulation, raising the possibility that the effect was due to a delayed progesterone peak secondary to an altered cycle length (29). The other two studies differed in isoflavone dose (154 vs. <5 mg/d), suggesting that either the effect was not due to the isoflavones in the soy milk or 5 mg of isoflavones is sufficient to exert an effect on plasma progesterone (33,34). A delay in number of days to the midluteal progesterone peak was observed in women consuming TVP (27) and miso (28). Although there does not appear to be a consistent significant effect of phytoestrogen consumption on plasma progesterone, it is relevant that of the eight studies unable to detect a significant effect on plasma progesterone (27,28,31,32,35–37, 51), six observed a nonsignificant decrease ranging in magnitude from 6.8 to 16.8% (27,31,32,35,36,51). Sex Hormone Binding Globulin. Increased concentrations of plasma sex hormone binding globulin (SHBG) would reflect an estrogenic effect because estrogen is known to stimulate SHBG synthesis (56), although the result would be decreased concentrations of free estrogens (56). Epidemiologic studies have correlated plasma SHBG positively with urinary isoflavones and negatively with plasma free estradiol in premenopausal Finnish women (49,50). In addition, in vitro studies report that isoflavones (57,58) and enterolactone (59) stimulate SHBG synthesis by HepG2 cells. On the other hand, another epidemiologic study found no significant association between soy consumption and plasma SHBG (48). Finally, human intervention studies provide no support because those that measured plasma SHBG found no significant effects of either soy (27,28,30–32,35,51) or flax (37) consumption. The exception is a significant increase in plasma SHBG observed in three women who consumed an isoflavone extract containing 50 mg isoflavones/d for 1 mo (36).

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Androgens. Androgens may affect breast cancer risk directly by stimulating growth of breast cancer cells (60) or indirectly through aromatization to estrogens (61). Although a negative correlation was observed between plasma free testosterone and urinary enterolactone in premenopausal Finnish women (49,50), evidence of effects of phytoestrogen consumption on plasma androgens is weak and inconclusive (27,31,32). On the other hand, plasma testosterone was significantly increased by consumption of flax (37) and by isoflavone extract (statistical significance not reported) (36). Plasma dehydroepiandrosterone (DHEA)-sulfate was significantly decreased in two soy intervention studies (29,31); however, effects were modest and other studies found no significant effect of soy (32) or flax (37) consumption. Moreover, there was no significant effect of soy consumption on plasma DHEA (31). Plasma androstenedione was decreased in women consuming an isoflavone extract (statistical significance not reported) (36), yet a soy intervention study was unable to detect a significant effect (31). The few human studies that have examined the effect of phytoestrogen consumption on plasma androgens have produced variable results and preclude any sound conclusions. Other Plasma Hormones. Other plasma hormones evaluated in phytoestrogen intervention studies include prolactin and thyroid hormones. Although decreased prolactin may be considered protective for breast cancer (62), no significant effects of soy (31,32) or flax consumption (37) have been observed. There is increasing concern regarding the potential for soy to interfere with thyroid gland function. Animal studies suggest that soy is goitrogenic (63) and increases plasma T4 (64), and in vitro studies show that isoflavones may inhibit thyroid peroxidase (65); however, human data are limited. Plasma free T3 was significantly decreased in women consuming soy protein, yet there were no significant effects on any other thyroid hormones including total T3, free or total T4, and thyroid stimulating hormone (31). Plasma T4 increased in the follicular phase and decreased in the luteal phase in three women consuming an isoflavone extract and there were no apparent changes in plasma T3 (36). Effects on Urinary Hormones Effects of phytoestrogen consumption on urinary hormones in premenopausal women have focused on estrogens and their metabolites. Estrogen metabolism has been hypothesized to influence breast cancer risk through the formation of urinary metabolites suggested to be genotoxic, such as the 16- and 4-hydroxylated estrogens (66,67). Evidence of an effect of phytoestrogen consumption on urinary estrogens in premenopausal women is limited but convincing. Xu et al. (68) reported that moderate and high isoflavone (65 or 129 mg) soy protein significantly decreased urinary estradiol, estrone, estriol, total estrogens, as well as the genotoxic metabolites, 16α-hydroxyestrone, 4-hydroxyestradiol, 4-hydroxyestrone, and significantly increased the 2/16α-hydroxyestrone ratio, compared with very low

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isoflavone (10 mg) soy protein. Other studies have focused only on the 2/16αhydroxyestrone ratio, also observing an increase after both soy (69) and flax (70) consumption, although one soy intervention study was unable to detect a significant effect (32). Although there are only four reports of the effects of phytoestrogen consumption on urinary estrogens, the data are convincing and clearer than the plasma data. It is possible that one way in which phytoestrogens protect against breast cancer risk is by altering estrogen metabolism away from genotoxic metabolites toward inactive metabolites. Effects on Functional Indicators of Estrogen Action In an attempt to evaluate the hormonal effects of soy isoflavones on reproductive tissues, researchers have sampled breast and endometrial tissue in studies of premenopausal women. Although there was no significant effect of soy consumption on the endometrium (31), studies evaluating functional breast end points have suggested a small estrogenic response (51,71). Petrakis et al. (51) hypothesized that 6 mo of soy consumption by 14 healthy premenopausal women would decrease breast nipple aspirate fluid (NAF) volume, but instead they observed a significant increase as well as the appearance of breast cell hyperplasia in 30% of the subjects, both indicative of an estrogenic response. Conversely, the same study reported a significant decrease in breast secretion of GCDFP-15, a glycoprotein associated with breast cancer (51). In another study focusing on the premenopausal breast, 2 wk of soy supplementation did not significantly affect breast cell proliferation, progesterone receptor expression, estrogen receptor status, apoptosis, mitosis, or the expression of Bcl-2, but there was a significant increase in the estrogen-dependent protein pS2 (71). Although the data are not consistent enough for definitive conclusions, the small estrogenic effects of soy supplementation observed on the premenopausal breast warrants caution and justifies longer-term studies on the effects of soy on breast physiology. Urinary Isoflavone Metabolism and Plasma Hormones Concentrations of urinary isoflavones are consistently increased in premenopausal women in a dose-dependent manner after consumption of isoflavone-rich soy (27,33,35,36,51,68). As expected, there is a certain degree of interindividual variation in urinary excretion of isoflavones and their metabolites. Of particular interest is the up to 1500-fold variation found in urinary excretion of a specific isoflavone metabolite, equol (68,72–76). Equol, produced by the gut microflora from daidzein, is unique among the isoflavone metabolites because it is produced by only 30–40% of the population (72–74,76,77). Equol is also one of the most potent isoflavones, with a higher binding for the estrogen receptor than its precursor daidzein (78), and the ability to exert a more estrogenic response than daidzein in

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estrogen-dependent breast cancer cells (79) and an endometrial tumor line (80). Furthermore, equol has a longer half-life than either genistein or daidzein (73), suggesting that the ability to produce equol may maximize exposure to the soy isoflavones. The physiologic relevance of the ability to produce and excrete equol has not been determined, although urinary equol excretion has been shown to be inversely correlated with risk of breast cancer (23). A recent study reported that premenopausal equol excretors have plasma hormone profiles associated with a lowered risk of breast cancer, specifically lower estrogens, androgens and higher SHBG and progesterone (81). In addition, the increase in follicular phase length after TVP consumption observed by Cassidy et al. (27) was greatest in subjects who had the highest urinary equol concentrations. Given the uniqueness of equol, there is growing interest in understanding why the variation occurs and whether it can be manipulated. Setchell et al. (82) first proposed that the variation could be due to factors that may be influenced by diet, such as composition of the intestinal microflora, intestinal transit time, or the redox level in the large intestine. Studies comparing habitual dietary intakes of equol excretors and nonexcretors are limited and have produced inconsistent results. Some studies have shown higher consumption of carbohydrate and dietary fiber and lower consumption of fat in equol excretors (74,76), whereas others have shown no significant differences (77,81,83). A recent intervention study in premenopausal women reported that neither wheat bran nor long-term soy protein feeding was able to alter urinary excretion of equol (77). Further research is required to gain a better understanding of equol and its effects on reproductive hormones in premenopausal women.

Summary and Conclusions The hormonal effects of phytoestrogens observed in cell culture and animal studies (10) have not been as clearly elucidated in humans. Over the past decade, there have been numerous intervention studies evaluating the effects of soy, flax, or isoflavone extract consumption on various hormonal end points including menstrual cycle parameters, plasma and urinary hormones, and functional indicators of estrogen action in endometrial and breast tissue (see Table 27.1). Unfortunately, results have been inconsistent, making it challenging to draw definitive conclusions. Only four studies have focused specifically on isoflavones, by evaluating effects of an isoflavone-free soy product (28,34), using an isoflavone doseresponse study design (31), or using an isoflavone extract (36). When the various hormonal end points are considered, there appears to be some evidence for a significant lengthening of the menstrual cycle by phytoestrogen consumption (26,27,37), providing possible protection against breast cancer over a lifetime. Effects on plasma hormones are quite variable, although two studies observed a consistent decrease in midcycle gonadotropins (27,31), again providing evidence for protection against breast cancer. Arguably most convincing are the data report-

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ing that phytoestrogen consumption can alter estrogen metabolism in a direction beneficial for breast cancer risk (68–70). In contrast, there have been preliminary data showing an estrogenic effect of soy supplementation on the breast (51,71), raising the need for further investigation. Overall, these data provide support for hormonal effects of phytoestrogens in premenopausal women, both estrogenic and antiestrogenic. Future research should focus on specific soy components, variability in phytoestrogen metabolism, and effects of phytoestrogens on specific target tissues. References 1. Reinli, K., and Block, G. (1996) Phytoestrogen Content of Foods A Compendium of Literature Values, Nutr. Cancer 26, 123–148. 2. Axelson, M., Sjövall, J., Gustafsson, B.E., and Setchell, K.D.R. (1982) Origin of Lignans in Mammals and Identification of a Precursor from Plants, Nature 298, 659–660. 3. Setchell, K.D.R., and Adlercreutz, H. (1988) Mammalian Lignans and PhytoOestrogens. Recent Studies on Their Formation, Metabolism and Biological Role in Health and Disease, in Role of the Gut Flora in Toxicity and Cancer, pp. 315–345, Academic Press, London. 4. Setchell, K.D.R., and Cassidy, A. (1999) Dietary Isoflavones: Biological Effects and Relevance to Human Health, J. Nutr. 129, 758S–767S. 5. Adlercreutz, H., van der Wildt, J., Kinzel, J., Attalla, H., Wähälä, K., Mäkelä,T., Hase, T., and Fotsis, T. (1995) Lignan and Isoflavonoid Conjugates in Human Urine, J. Steroid Biochem. 52, 97–103. 6. Kuiper, G.G.J.M., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., and Gustafsson, J.-Å. (1997) Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors α and β, Endocrinology 138, 863–870. 7. Messina, M.J., Persky, V., Setchell, K.D.R., and Barnes, S. (1994) Soy Intake and Cancer Risk: A Review of the In Vitro and In Vivo Data, Nutr. Cancer 21, 113–131. 8. Parkin, D.M. (1989) Cancers of the Breast, Endometrium and Ovary: Geographic Correlations, Eur. J. Cancer Clin. Oncol. 25, 1917–1925. 9. Ziegler, R.G., Hoover, R.N., Pike, M.C., Hildesheim, A., Nomura, A.M.Y., West, D.W., Wu-Williams, A.H., Kolonel, L.N., Horn-Ross, P.L., Rosenthal, J.F., and Hyer, M.B. (1993) Migration Patterns and Breast Cancer Risk in Asian-American Women, J. Natl. Cancer Inst. 85, 1819–1827. 10. Fournier, D.B., Erdman, J.W., and Gordon, G.B. (1998) Soy, Its Components, and Cancer Prevention: A Review of the In Vitro, Animal, and Human Data, Cancer Epidemiol. Biomark. Prev. 7, 1055–1065. 11. Goodman, M.T., Wilkens, L.R., Hankin, J.H., Lyu, L.-C., Wu, A.H., and Kolonel, L.N. (1997) Association of Soy and Fiber Consumption with the Risk of Endometrial Cancer, Am. J. Epidemiol. 146, 294–306. 12. Nomura, A., Henderson, B.E., and Lee, J. (1978) Breast Cancer and Diet Among the Japanese in Hawaii, Am. J. Clin. Nutr. 31, 2020–2025. 13. Hirayama, T. (1986) A Large Scale Cohort Study on Cancer Risks by Diet with Special Reference to the Risk Reducing Effects of Green-Yellow Vegetable Consumption, in Diet, Nutrition and Cancer, pp. 41–53, Japanese Scientific Society Press, Tokyo.

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31. Duncan, A.M., Merz-Demlow, B.E., Nagel, T.C., Xu, X., Phipps, W.R., and Kurzer, M.S. (1999) Soy Isoflavones Exert Modest Hormonal Effects in Premenopausal Women, J. Clin. Endocrinol. Metab. 84, 192–197. 32. Martini, M.C., Dancisak, B.B., Haggans, C.J., Thomas, W., and Slavin, J.L. (1999) Effects of Soy Intake on Sex Hormone Metabolism in Premenopausal Women, Nutr. Cancer 34, 133–139. 33. Lu, L.-J.W., Anderson, K.E., Grady, J.J., Kohen, F., and Nagamani, M. (2000) Decreased Ovarian Hormones During a Soya Diet: Implications for Breast Cancer Prevention, Cancer Res. 60, 4112–4121. 34. Lu, L.-J.W., Anderson, K.E., Grady, J.J., and Nagamani, M. (2001) Effects of an Isoflavone-Free Soy Diet on Ovarian Hormones in Premenopausal Women, J. Clin. Endocrinol. Metab. 86, 3045–3052. 35. Wu, A.H., Stanczyk, F.Z., Hendrich, S., Murphy, P.A., Zhang, C., Wan, P., and Pike, M.C. (2000) Effects of Soy Foods on Ovarian Function in Premenopausal Women, Br. J. Cancer 82, 1879–1886. 36. Watanabe, S., Terashima, K., Sato, Y., Arai, S., and Eboshida, A. (2000) Effects of Isoflavone Supplement on Healthy Women, Biofactors 12, 233–241. 37. Phipps, W.R., Martini, M.C., Lampe, J.W., Slavin, J.L., and Kurzer, M.S. (1993) Effect of Flax Seed Ingestion on the Menstrual Cycle, J. Clin. Endocrinol. Metab. 77, 1215–1219. 38. Olsson, M., Landin-Olsson, M., and Gullberg, B. (1983) Retrospective Assessment of Menstrual Cycle Length in Patients with Breast Cancer, in Patients with Benign Breast Disease, and in Women Without Breast Disease, J. Natl. Cancer Inst. 70, 17–20. 39. Aksel, S. (1981) Hormonal Characteristics of Long Cycles in Fertile Women, Fertil. Steril. 36, 521–523. 40. Hadley, M.E. (1996) Hormones and Female Reproductive Physiology, in Endocrinology, 4th edition, pp. 412–437, Prentice Hall, New Jersey. 41. Ferguson, D.J.P., and Anderson, T.J. (1981) Morphological Evaluation of Cell Turnover in Relation to the Menstrual Cycle in the ‘Resting’ Human Breast, Br. J. Cancer 44, 177–181. 42. Henderson, B.E., Ross, R.K., Judd, H.L., Krailo, M.D., and Pike, M.C. (1985) Do Regular Ovulatory Cycles Increase Breast Cancer Risk? Cancer 56, 1206–1208. 43. Pike, M.C., and Spicer, D.V. (2000) Hormonal Contraception and Chemoprevention of Female Cancers, Endocr. Relat. Cancer 7, 73–83. 44. Adlercreutz, H., Bannwart, C., Wähälä, K., Mäkelä, T., Brunow, G., Hase, T., Arosemena, P.J., Kellis, J.T., Jr., and Vickery, L.E. (1993) Inhibition of Human Aromatase by Mammalian Lignans and Isoflavonoid Phytoestrogens, J. Steroid Biochem. Mol. Biol. 44, 147–153. 45. Campbell, D.R., and Kurzer, M.S. (1993) Flavonoid Inhibition of Aromatase Enzyme Activity in Human Preadipocytes, J. Steroid Biochem. Mol. Biol. 46, 381–388. 46. Wang, C., Mäkelä, T., Hase, T., Adlercreutz, H., and Kurzer, M.S. (1994) Lignans and Flavonoids Inhibit Aromatase Enzyme in Human Preadipocytes, J. Steroid Biochem. Mol. Biol. 50, 205–212. 47. Mäkelä, S., Poutanen, M., Lehtimäki, J., Kostian, M.-L., Santti, R., and Vihko, R. (1995) Estrogen-Specific 17β-Hydroxysteroid Oxidoreductase Type 1 (E.C. 1.1.1.62) as a Possible Target for the Action of Phytoestrogens, Proc. Soc. Exp. Biol. Med. 208, 51–59. 48. Nagata, C., Kabuto, M., Kurisu, Y., and Shimizu, H. (1997) Decreased Serum Estradiol Concentration Associated with High Dietary Intake of Soy Products in Premenopausal Japanese Women, Nutr. Cancer 29, 228–233.

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49. Adlercreutz, H., Höckerstedt, K., Bannwart, C., Bloigu, S., Hämäläinen, E., Fotsis, T., and Ollus, A. (1987) Effect of Dietary Components, Including Lignans and Phytoestrogens, on Enterohepatic Circulation and Liver Metabolism of Estrogens and on Sex Hormone Binding Globulin (SHBG), J. Steroid Biochem. 27, 1135–1144. 50. Adlercreutz, H., Höckerstedt, K., Bannwart, C., Hämäläinen, E., Fotsis, T., and Bloigu, S. (1988) Association Between Dietary Fiber, Urinary Excretion of Lignans and Isoflavonic Phytoestrogens, and Plasma Non-Protein Bound Sex Hormones in Relation to Breast Cancer, in Progress in Cancer Research and Therapy. Hormones and Cancer 3, vol. 35, pp. 409–412, Raven Press, New York. 51. Petrakis, N.L., Barnes, S., King, E.B., Lowenstein, J., Wiencke, J., Lee, M.M., Miike, R., Kirk, M., and Coward, L. (1996) Stimulatory Influence of Soy Protein Isolate on Breast Secretion in Pre- and Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 5, 785–794. 52. Bernstein, L., Yuan, J.M., Ross, R.K., Pike, M.C., Hanisch, R., Lobo, R., Stanczyk, Y.T., and Henderson, B.E. (1990) Serum Hormone Levels in Pre-Menopausal Chinese Women in Shanghai and White Women in Los Angeles: Results from Two Breast Cancer Case-Control Studies, Cancer Causes Control 1, 51–58. 53. Key, T.J.A., Chen, J., Wang, D.Y., Pike, M.C., and Boreham, J. (1990) Sex Hormones in Women in Rural China and in Britain, Br. J. Cancer 62, 631–636. 54. Goldin, B.R., Adlercreutz, H., Gorbach, S.L., Woods, M.N., Dwyer, J.T., Conlon, T., Bohn, E., and Gershoff, S.N. (1986) The Relationship Between Estrogen Levels and Diets of Caucasian American and Oriental Immigrant Women, Am. J. Clin. Nutr. 44, 945– 953. 55. Kaplanski, O., Shemesh, M., and Berman, A. (1981) Effects of Phyto-Estrogens on Progesterone Synthesis by Isolated Bovine Granulosa Cells, J. Endocrinol. 89, 343–348. 56. Anderson, D.C. (1974) Sex-Hormone Binding Globulin, Clin. Endocrinol. 3, 69–96. 57. Loukovaara, M., Carson, M., Palotie, A., and Adlercreutz, H. (1995) Regulation of Sex Hormone-Binding Globulin Production by Isoflavonoids and Patterns of Isoflavonoid Conjugation in HepG2 Cell Cultures, Steroids 60, 656–661. 58. Mousavi, Y., and Adlercreutz, H. (1993) Genistein Is an Effective Stimulator of Sex Hormone-Binding Globulin Production in Hepatocarcinoma Human Liver Cancer Cells and Suppresses Proliferation of These Cells in Culture, Steroids 58, 301–304. 59. Adlercreutz, H., Mousavi, Y., Clark, J., Höckerstedt, K., Hämäläinen, E., Wähälä, K., Mäkelä, T., and Hase, T. (1992) Dietary Phytoestrogens and Cancer: In Vitro and In Vivo Studies, J. Steroid Biochem. Mol. Biol. 41, 331–337. 60. Lippman, M., Bolan, G., and Huff, K. (1976) The Effects of Androgens and Antiandrogens on Hormone-Responsive Human Breast Cancer in Long-Term Tissue Culture, Cancer Res. 36, 4610–4618. 61. Secreto, G., Toniolo, P., Pisani, P., Recchione, C., Cavalleri, A., Fariselli, G., Totis, A., Di Pietro, S., and Berrino, F. (1989) Androgens and Breast Cancer in Premenopausal Women, Cancer Res. 49, 471–476. 62. Ingram, D.M., Nottage, E.M., and Roberts, A.N. (1990) Prolactin and Breast Cancer Risk, Med. J. Aust. 153, 469–473. 63. Wilgus, H.S., Jr., Gassner, F.X., Patton, A.R., and Gustavson, R.G. (1941) The Goitrogenicity of Soybeans, J. Nutr. 22, 43–52. 64. Forsythe, W.A., III (1995) Soy Protein, Thyroid Regulation and Cholesterol Metabolism, J. Nutr. 125, 619S–623S.

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65. Divi, R.L., Chang, H.C., and Doerge, D.R. (1997) Anti-Thyroid Isoflavones from Soybean, Biochem. Pharmacol. 54, 1087–1096. 66. Fishman, J., Osborne, M.P., and Telang, N.T. (1995) The Role of Estrogen in Mammary Carcinogenesis, Ann. N.Y. Acad. Sci. 768, 91–100. 67. Hayes, C.L., Spink, D.C., Spink, B.C., Cao, J.Q., Walker, N.J., and Sutter, T.R. (1996) 17β-Estradiol Hydroxylation Catalyzed by Human Cytochrome P450 1B1, Proc. Natl. Acad. Sci. USA 93, 9776–9781. 68. Xu, X., Duncan, A.M., Merz, B.E., and Kurzer, M.S. (1998) Effects of Soy Isoflavones on Estrogen and Phytoestrogen Metabolism in Premenopausal Women, Cancer Epidemiol. Biomark. Prev. 7, 1101–1108. 69. Lu, L.-J.W., Cree, M., Josyula, S., Nagamani, M., Grady, J.J., and Anderson, K.E. (2000) Increased Urinary Excretion of 2-Hydroxyestrone but Not 16α-Hydroxyestrone in Premenopausal Women During a Soya Diet Containing Isoflavones, Cancer Res. 60, 1299–1305. 70. Haggans, C.J., Travelli, E.J., Thomas, W., Martini, M.C., and Slavin, J.L. (2000) The Effect of Flaxseed and Wheat Bran Consumption on Urinary Estrogen Metabolites in Premenopausal Women, Cancer Epidemiol. Biomark. Prev. 9, 719–725. 71. Hargreaves, D.F., Potten, C.S., Harding, C., Shaw, L.E., Morton, M.S., Roberts, S.A., Howell, A., and Bundred, N.J. (1999) Two-Week Dietary Soy Supplementation Has an Estrogenic Effect on Normal Premenopausal Breast, J. Clin. Endocrinol. Metab. 84, 4017– 4024. 72. Kelly, G.E., Nelson, C., Waring, M.A., Joannou, G.E., and Reeder, A.Y. (1993) Metabolism of Dietary (Soya) Isoflavones in Human Urine, Clin. Chim. Acta 223, 9–22. 73. Kelly, G.E., Joannou, G.E., Reeder, A.Y., Nelson, C., and Waring, M.A. (1995) The Variable Metabolic Response to Dietary Isoflavones in Humans, Proc. Soc. Exp. Biol. Med. 208, 40–43. 74. Lampe, J.W., Karr, S.C., Hutchins, A.M., and Slavin, J.L. (1998) Urinary Equol Excretion with a Soy Challenge: Influence of Habitual Diet, Proc. Soc. Exp. Biol. Med. 217, 335– 339. 75. Lampe, J.W., Gustafson, D.R., Hutchins, A.M., Martini, M.C., Li, S., Wähälä, K., Grandits, G.A., Potter, J.D., and Slavin, J.L. (1999) Urinary Isoflavonoid and Lignan Excretion on a Western Diet: Relation to Soy, Vegetable and Fruit Intake, Cancer Epidemiol. Biomark. Prev. 8, 699–707. 76. Rowland, I.R., Wiseman, H., Sanders, T.A.B., Adlercreutz, H., and Bowey, E.A. (2000) Interindividual Variation in Metabolism of Soy Isoflavones and Lignans: Influence of Habitual Diet on Equol Production by the Gut Microflora, Nutr. Cancer 36, 27–32. 77. Lampe, J.W., Skor, H.E., Li, S., Wähälä, K., Howald, W.N., and Chen, C. (2001) Wheat Bran and Soy Protein Feeding Do Not Alter Urinary Excretion of the Isoflavan Equol in Premenopausal Women, J. Nutr. 131, 740–744. 78. Shutt, D.A., and Cox, R.I. (1972) Steroid and Phytoestrogen Binding to Sheep Uterine Receptors In Vitro, J. Endocrinol. 52, 299–310. 79. Brienholt, V., and Larsen, J.C. (1998) Detection of Weak Estrogenic Flavonoids Using a Recombinant Yeast Strain and a Modified MCF7 Cell Proliferation Assay, Chem. Res. Toxicol. 11, 622–629. 80. Markiewicz, L., Garey, J., Adlercreutz, H., and Gurpide, E. (1993) In Vitro Bioassays of Non-Steroidal Phytoestrogens, J. Steroid Biochem. Mol. Biol. 45, 399–405. 81. Duncan, A.M., Merz-Demlow, B.E., Xu, X., Phipps, W.R., and Kurzer, M.S. (2000) Premenopausal Equol Excretors Show Plasma Hormone Profiles Associated with Lowered Risk of Breast Cancer, Cancer Epidemiol. Biomark. Prev. 9, 581–586.

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82. Setchell, K.D.R., Borriello, S.P., Hulme, P., Kirk, D.N., and Axelson, M. (1984) Nonsteroidal Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Disease, Am. J. Clin. Nutr. 40, 569–578. 83. Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T., Hämäläinen, E., Hasegawa, T., and Okada, H. (1991) Urinary Excretion of Lignans and Isoflavonoid Phytoestrogens in Japanese Men and Women Consuming a Traditional Japanese Diet, Am. J. Clin. Nutr. 54, 1093–1100.

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Chapter 28

Phytoestrogens: Effects on Menopausal Symptoms Fabien S. Dalais Department of Epidemiology and Preventive Medicine, Monash University, The Alfred Hospital, Prahran, VIC 3181 Australia

Introduction The notion that estrogens from plants may have effects similar to those of traditional hormone replacement therapy (HRT) in alleviating the symptoms of menopause is interesting. Over the past 15 years, there have been a number of clinical trials examining the effects of these phytoestrogens on menopausal symptoms and, to date, there are no firm conclusions. This chapter will present the latest scientific evidence and illustrate some of the limitations experienced to date. Hormone Replacement Therapy and Menopause It is generally accepted that HRT in its various forms is efficient in reducing the symptoms of menopause among other benefits (1). Common symptoms of menopause in the Western world include hot flashes, night sweats, mood swings, depression, anxiety, and vaginal dryness. However, HRT does have adverse effects, such as an increased risk of breast cancer for long-term users, the possibility of deep vein thromboses, and an increased risk of endometrial cancer with estrogen alone HRT (1). Additionally, compliance is poor, ranging from 10 to 50% (2). This lack of compliance is often due to the above adverse effects; in addition, however, many women seek a more natural alternative to HRT. Cross-Cultural Experiences of the Menopause Menopausal symptoms are experienced worldwide, but the prevalence of these symptoms varies widely among different communities. This was highlighted in a cross-cultural study examining the prevalence of menopausal symptoms in seven South-East Asian countries (3). The study surveyed the menopause experience in Hong Kong, Indonesia, Korea, Malaysia, the Philippines, Singapore, and Taiwan. It showed that the incidence of one of the most common menopausal symptoms in Western countries, hot flashes, varied markedly, with Singapore the lowest at 19.6% and the Philippines the highest at 60%. On average, between 80 and 85% of Western women experience hot flashes (4). Additionally, the incidence of hot flashes is equally low in other South-East Asian countries (5). The reasons for these marked differences could arise from cultural factors, but more importantly they could also be due to dietary differences. Many of these cul-

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tures consume foods high in phytoestrogens, notably soy-based foods, which are high in isoflavones (a class of phytoestrogens) (6,7). This was highlighted by Adlercreutz et al. (8), who showed that the urinary excretion of isoflavones was 10-100 times higher in Japanese women than in their American and Finnish counterparts. This led him to hypothesize that this high intake of estrogen-like compounds may act similarly to HRT and be responsible for the low incidence of menopausal symptoms such as hot flashes in Asian communities (8). With this hypothesis in mind, what follows is a review of the clinical trials examining the effects of phytoestrogens on menopausal symptoms. These trials have focused on the role of soy and its constituent isoflavones in hot flashes and vaginal dryness (also including changes in vaginal cytology) as primary symptoms of menopause. Phytoestrogens and Menopausal Symptoms Wilcox et al. (9) were the first to demonstrate a physiologic effect of phytoestrogens in menopausal women. This trial came at a time when the hypothesis had not yet been developed; it examined the role of soy as flour, flaxseed (linseed) as seeds, and red clover sprouts on vaginal maturation index as a measure of estrogenicity because the superficial cells in the vagina are highly responsive to estrogen. Postmenopausal women (n = 25) were randomized in a Latin-square design to receive one of three high-phytoestrogen diets. After 6 wk of dietary supplementation, there was a significant increase in vaginal cell maturation as well as a significant decrease in follicle stimulating hormone (FSH). In 1995, Murkies et al. (10) randomized 58 postmenopausal women to a diet supplemented with soy flour as the active group or one supplemented with wheat flour as the placebo group for a 12-wk period. At the end of the trial, there was a significant decrease of 40% in hot flash rate in the soy group and a significant decrease of 25% in the placebo group. Baird et al. (11) examined the effects of a soy-supplemented diet compared with a normal low-phytoestrogen diet on vaginal cytology, luteinizing hormone (LH), FSH, sex hormone binding globulin (SHBG), and estradiol in 97 postmenopausal women for 4 wk. There were no significant changes in any of the parameters measured, possibly due to the short duration of the trial. Brzezinski et al. (12) conducted a 12-wk trial of a phytoestrogen-rich compared with a phytoestrogen-poor diet in 165 postmenopausal women. The phytoestrogenrich diet consisted of soy drinks, tofu, miso, and flaxseed. There was a significant increase in SHBG, and a significant decrease in hot flash score and vaginal dryness score in the phytoestrogen-rich group. Although there was a significant decrease in hot flash score in the phytoestrogen-poor diet, it was not as significant as that for the phytoestrogen-rich diet. No other significant changes were demonstrated. In a randomized, double-blind, placebo-controlled, crossover trial of 52 postmenopausal women, Dalais et al. (13) investigated the effects of a high-soy, highflaxseed compared with a high-wheat placebo diet. The high consumption of the

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above ingredients was achieved by specially designed breads containing high levels of soy, flaxseed, and wheat. After two dietary interventions of 12 wk, there were significant decreases in hot flash rate in the wheat (51%) and flaxseed (41%) groups, but not in the soy group. However there was a significant improvement in vaginal cytology of 103% in the soy group. In a 12-wk trial of 104 postmenopausal women, Albertazzi et al. (14) showed a 45% reduction in hot flash rate in women consuming 60 g of isolated soy protein (76 mg of isoflavones) compared with a 30% decrease in the placebo group. Unlike the previous studies, this study demonstrated a significant difference between the groups, and not only within the group. To date there have been two published studies using an isoflavone extract from red clover. In the first study, postmenopausal women were randomized to placebo, 40 mg or 160 mg of isoflavones for 12 wk (15). Decreases in hot flash rate to the order of 46, 44, and 26% were observed in the placebo, 40 and 160 mg groups, respectively. There was a correlation between the urinary excretion of isoflavones (particularly daidzein) and the flash response. The second study was carried out in 51 postmenopausal women (16). The results were similar to the first study with a negative correlation between increasing level of urinary daidzein and hot flash rate, but there were no significant differences in menopausal symptoms in either group after 12 wk (crossover) of intervention. In a randomized, double-blind, placebo-controlled, crossover study of 51 perimenopausal women, Washburn et al. (17) used three different dietary supplements to elucidate whether there was a difference between a high-phytoestrogen diet consumed once daily or twice daily compared with a control supplement. The study participants were randomized to either 20 g of soy protein (34 mg of isoflavones) in a single dose, 20 g of soy protein split into two doses, or a control supplement. They were randomized to one of the three diets for 6 wk, and subsequently to the remaining two diets. Significant improvements in the severity of menopausal symptoms were observed in the twice daily group compared with the placebo group. In a 12-wk, double-blind, randomized study, Scambia et al. (18) evaluated the activity of a soy extract containing 50 mg of isoflavones (for the first 6 wk), followed by soy extract in combination with conjugated equine estrogens (Premarin) (for the following 4 wk), compared with placebo. At wk 6, there was a significant decrease in the mean number and the severity of hot flashes compared with baseline in the soy group, but no significant changes were detected in the placebo group. Kotsopoulos et al. (19) examined the effects of 40 g of isolated soy protein (containing 118 mg of isoflavones) in 94 postmenopausal women for 12 wk. This double-blind, placebo-controlled trial demonstrated significant improvements in libido, facial hair, and dry skin in both soy and placebo groups, but there was a significant improvement in vaginal dryness in the soy group only. In the largest study published to date, 177 postmenopausal women were randomized to receive either a soy isoflavone extract containing 50 mg of isoflavones or a placebo extract for 12 wk (20). Within 2 wk of randomization, decreases in

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incidence and severity of hot flashes could be observed in the soy group. At 6 wk duration, there was a significant difference in hot flash rate between the soy and placebo group (P = 0.03), but that difference did not persist to 12 wk (P = 0.08). Although most studies to date have concentrated on healthy peri- or postmenopausal women, Quella et al. (21) reported no effect of 150 mg/d of isoflavones on hot flashes in breast cancer survivors in a 4-wk crossover study. It is worth mentioning that 68% of study participants were taking tamoxifen, thus possibly affecting study results. In a recent study, 69 perimenopausal women were recruited to determine the effects of isolated soy protein with isoflavones (80.4 mg/d), isolated soy protein with low levels of isoflavones (4.4 mg/d) and whey protein as the control group on menopausal symptoms (22). Women were randomized to one of the three groups for 24 wk. Although there were significant decreases in hot flashes and night sweats in all groups, there were no treatment effects on the change in hot flashes and night sweat frequency and severity. With these studies in mind (Table 28.1), there are a number of limitations that prevent us making any firm conclusions. It is well known that hot flashes are an extremely variable and subjective end point, thus making the assessment of the effects of phytoestrogens difficult. The more significant effects observed are seen in the studies with the greater number of study participants (12,14). The hypothesis on phytoestrogens and menopausal symptoms is based on the fact that levels of estrogen are lower in postmenopausal women, therefore the phytoestrogens would have a better effect. Although the majority of studies on phyto-estrogens and menopause have been carried out in postmenopausal women, it could be that perimenopausal women would respond better due to their higher frequency of symptoms. It is also possible that women, whether peri- or postmenopausal, would respond more to phytoestrogens if they had more symptoms to start with. The majority of studies to date have used soy flour, soy foods, soy protein, or isoflavone tablets. It would appear that the isoflavones in their soy matrix, whether as soy food or soy protein have more of an effect than in isolation. Additionally, many of the traditional soy foods and other specially manufactured high-soy foods for trials, are often low in fat and contain a number of other essential nutrients as well as phytonutrients, thus offering additional nutritional benefits to menopausal women.

Summary and Conclusions As time progresses and menopausal women become more aware of the potential benefits of phytoestrogens, it becomes harder to keep subjects and researchers blinded. With the current literature at hand, we may be able to say that phytoestrogens have a mild effect on menopausal symptoms, but until we address some of the limitations listed above and have larger trials as well as determine a potential mode of action and a specific dosage, we will not be able to say definitively that phyto-

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TABLE 28.1 Summary of Clinical Trials Examining the Effects of Phytoestrogens on Menopausal Symptoms, Based on the Source of Phytoestrogen Used Number of study Duration Isoflavone Ref. participants (wk) dose

Effect Hot flashes

Soy foods (including soy flour and soy bread) (9) 25 6 NAa (10)

58

12

NA

(11) (12)

97 165

4 12

NA NA

(13)

52

12 × 2

52 mg

No significant change in soy group

12

76 mg

Soy protein (14) 104

40% decrease in flash rate in soy group, compared with 25% decrease in control group More significant decrease in hot flash score in soy group

Vaginal dryness Significant increase in vaginal cell maturation No change

No change More significant decrease in vaginal score in soy group Significant increase in vaginal cell maturation in soy group

(17)

51

6×3

34 mg

(19)

94

12

118 mg

A significant 45% decrease in hot flash rate in soy group compared with 30% in control group Significant improvement in the severity of menopausal symptoms in a soy group compared with placebo No significant differences

(22)

69

24

4.4 or 80 mg

Significant decreases in all groups but no treatment effect

Isoflavone extract from soy (18) 39 6

50 mg

(20)

177

12

50 mg

No change

(21)

177

4×2

150 mg

Significant decrease in number and severity of hot flashes in soy group Significant difference between the groups detected at 6 wk, but not at study completion No significant differences

No significant differences

No change

Isoflavone extract from red clover (15) 37 12 40 or 160 mg (16) 51 12 x 2 40 mg aNA,

not available.

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No significant differences

Significant improvement in vaginal dryness in soy group

No change

estrogens have benefits similar to those of traditional HRT in alleviating menopausal symptoms without the side effects of HRT. References 1. Barrett-Connor, E., and Grady, D. (1998) Hormone Replacement Therapy, Heart Disease and Other Considerations, Annu. Rev. Public Health 19, 55–72. 2. Hahn, R.G. (1989) Compliance Considerations with Estrogen Replacement: Withdrawal Bleeding and Other Factors, Am. J. Obstet. Gynecol. 161, 1854–1858. 3. Boulet, M.J., Oddens, B.J., Lehert, P., Vemer, H.M., and Visser, A. (1994) Climacteric and Menopause in Seven South-East Asian Countries, Maturitas 19, 157–176. 4. Sturdee, D.W. (1997) Clinical Symptoms of Estrogen Deficiency, Curr. Obstet. Gynaecol. 7, 190–196. 5. Lock, M. (1991) Contested Meanings of the Menopause, Lancet. 337, 1270–1272. 6. Astuti, M., Meliala, A., Dalais, F.S., and Wahlqvist, M.L. (2000) Tempe, a Nutritious and Healthy Food from Indonesia, Asia Pac. J. Clin. Nutr. 9, 322–325. 7. Nagata, C., Takatsuka, N., Kurisu, Y., and Shimizu, H. (1998) Decreased Serum Total Cholesterol Concentration Is Associated with High Intake of Soy Products in Japanese Men and Women, J. Nutr. 128, 209–213. 8. Adlercreutz, H., Hamalainen, E., Gorbach, S.L., and Goldin, B.R. (1992) Dietary Phyto-Oestrogens and the Menopause in Japan, Lancet 339, 1233. 9. Wilcox, G., Wahlqvist, M.L., Burger, H.G., and Medley, G. (1990) Oestrogenic Effects of Plant Foods in Postmenopausal Women, Br. Med. J. 301, 905–906. 10. Murkies, A.L., Lombard, C., Strauss, B.J., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Post-Menopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 11. Baird, D.D., Umbach, D.M., Lansdell, L., Hughes, C.L., Setchell, K.D.R., Weinberg, C.R., Haney, A.F., Wilcox, A.J., and Mc Lachlan, J.A. (1995) Dietary Intervention Study to Assess Estrogenicity of Dietary Soy Among Postmenopausal Women, J. Clin. Endocrinol. Metab. 80, 1685–1690. 12. Brzezinski, A., Adlercreutz, H., Shaoul, R., Rosler, A., Tanos, V., Schenker, J.G. (1997) Short-Term Effects of Phytoestrogen-Rich Diet on Postmenopausal Women, Menopause 4, 89–94. 13. Dalais, F.S., Rice, G.E., Wahlqvist, M.L., Grehan, M., Murkies, A.L., Medley, G., Ayton, R., and Strauss, B.J.G. (1998) Effects of Dietary Phytoestrogens in Postmenopausal Women, Climacteric. 1, 124–129. 14. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–11. 15. Knight, D.C., Howes, J.B., and Eden, J.A. (1999) The Effect of Promensil, an Isoflavone Extract, on Menopausal Symptoms, Climacteric 2, 79–84. 16. Baber, R.J., Templeman, C., Morton, T., Kelly, G.E., and West L. (1999) Randomized Placebo-Controlled Trial of an Isoflavone Supplement and Menopausal Symptoms in Women, Climacteric 2, 85–92. 17. Washburn, S., Burke, G.L., Morgan ,T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13.

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18. Scambia, G., Mango, D., Signorile, P.G., Anselmi Angeli, R.A., Palena, C., Gallo, D., Bombardelli, E., Morazzoni, P., Riva, A., and Mancuso, S. (2000) Clinical Effects of a Standardized Soy Extract in Postmenopausal Women: a Pilot Study, Menopause 7, 105–111. 19. Kotsopoulos, D., Dalais, F.S., Liang, Y.L., McGrath, B.P., and Teede, H.J. (2000) The Effects of Soy Protein Containing Phytoestrogens on Menopausal Symptoms in Post Menopausal Women, Climacteric 3, 161–167. 20. Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L., and Lamia, C.A. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: a Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 21. Quella, S.K., Loprinzi, C.L., Barton, D.L., Knost, J.A., Sloan, J.A., LaVasseur, B.I., Swan, D., Krupp, K.R., Miller, K.D., and Novotny, P.J. (2000) Evaluation of Soy Phytoestrogens for the Treatment of Hot Flashes in Breast Cancer Survivors: A North Central Cancer Treatment Group Trial, J. Clin. Oncol. 18, 1068–1074. 22. St Germain, A., Peterson, C.T., Robinson, J.G., and Alekel, D.L. (2001) IsoflavoneRich or Isoflavone-Poor Soy Protein Does Not Reduce Menopausal Symptoms During 24 Weeks of Treatment, Menopause 8, 17–26.

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Chapter 29

Use of Soy Isoflavones as an Alternative to Traditional Hormone Replacement Therapy Mara Z. Vitolinsa, Mary S. Anthonya,b, and Gregory L. Burkea aDepartments of Public Health Sciences and bPathology, Wake Forest University School of Medicine, Winston-Salem, NC

Introduction This chapter will seek to provide an overview of the potential for soy isoflavones to serve as a viable alternative to traditional hormone replacement therapy (HRT). It is important to state that the expectations of an “optimal hormone replacement therapy” are extremely high. It is anticipated that an intervention would be either neutral or reduce the incidence of reproductive cancer (i.e., breast, endometrial), reduce ischemic heart disease incidence, reduce osteoporosis/fracture incidence, reduce the prevalence of vasomotor symptoms, enhance quality of life, and have no untoward side effects. It should be noted that drug development for any other new agent does not have such challenging expectations in scope or scale of outcomes. Thus, any evaluation of the appropriateness of a hormone replacement agent must consider the risk/benefit balance. In this chapter, we will briefly summarize the current literature pertaining to advantages and disadvantages of traditional HRT followed by a summary of the current literature on soy and isoflavones effects on chronic disease risk using data from cross-cultural studies, observational studies, and clinical trials. Benefits from Traditional Hormone Replacement Therapy A number of studies have found beneficial effects of estrogen therapy on lipid and lipoproteins levels, osteoporosis, and vasomotor symptom relief in peri- and postmenopausal women. Conjugated equine estrogens, the most commonly used form of ERT in the United States, lower low density lipoprotein cholesterol (LDL-C) concentrations while increasing high density lipoprotein cholesterol (HDL-C)(1). It should be noted that the addition of even a low dose of medroxyprogesterone acetate (to reduce endometrial proliferation and risk of uterine cancer) offsets the HDLC–raising effect of unopposed conjugated equine estrogen, but does not substantially weaken its LDL-C lowering effect (2,3). It is important to note that precise estimates of the effects of HRT on coronary heart disease (CHD) morbidity and mortality risk have been, and remain the subject of several clinical trials [e.g., the Hormone Replacement Study (HERS), the Estrogen Replacement Angiographic (ERA) trial, and the Women’s Health Initiative (WHI)].

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A clear beneficial effect of traditional HRT for the treatment and prevention of osteoporosis has been observed in both observational studies and clinical trials (4). Numerous observational studies and trials have observed a stabilization of bone mass in women using HRT. Similarly, a strong and immediate reduction in both the number and severity of vasomotor symptoms has been observed in women using HRT (5). There are biological mechanisms through which estrogen could potentially lead to improvements in cognition and reduction of risk for dementia. Studies conducted to date have been fraught with methodologic problems and have therefore produced mixed results (6). Although definitive trials are underway (e.g., the ongoing Women’s Health Initiative Memory Study) and should help clarify what, if any, benefits HRT may have on memory and cognition in postmenopausal women, traditional HRT shows substantial promise for beneficial effects on cognitive function. Risks from Traditional Hormone Replacement Therapy Use As with any drug, women must weigh the beneficial effects of HRT against potential negative effects. All forms of animal estrogens have potent estrogen agonist effects on the endometrium requiring the co-administration of a progestin to protect women with uteri from endometrial cancer. However, withdrawal bleeding often accompanies progestin replacement. Progestins can also cause weight gain, breast tenderness, bloating, and depressive mood swings (7,8). These estrogens are also agonists for breast tissue and appear to increase occurrence of breast cancer, especially when used for longer periods of time (9). The lack of enthusiasm for HRT is due to both the symptomatic side effects of the medication (e.g., continued menstrual bleeding, breast tenderness) and a concern for increased risk of uterine and breast cancer. There is some concern that adding a progestin to ERT may not be equivalent to estrogen alone in favorably affecting CHD rates; however, this remains to be shown in studies with women (10). Schairer et al. (11) reported that estrogen + progestin replacement therapy (PERT) increased the risk of developing breast cancer more than estrogen alone. For secondary prevention of cardiovascular disease (CVD), analysis of the HERS Study data revealed no overall effect after 4 y of treatment with conjugated estrogen plus medroxyprogesterone acetate (MPA) on the risk of nonfatal myocardial infarction and death from CHD among women diagnosed with coronary atherosclerosis (12). The ERA study group reported similar findings. Neither estrogen alone nor estrogen plus MPA affected the progression of coronary atherosclerosis in the postmenopausal women who participated in the trial (13). This suggests that HRT should not be prescribed to women with known CVD as a therapy for secondary prevention. Data on the long-term use of HRT for women without CVD is forthcoming from the Women’s Health Initiative Trial. Traditional HRT in the form of conjugated equine estrogens has been used in an effort to reduce vasomotor symptoms, morbidity and mortality in peri- and postmenopausal women. Data suggesting an increase in breast cancer risk with long-

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term HRT use and an increase in thromboembolic events early in the course of HRT treatment have likely contributed to the relatively low prevalence of longterm use of traditional HRT in post-menopausal women; only ~20% of U.S. women continue HRT for >5 y (14). Some of the uncertainties concerning the risk/benefit ratio of traditional HRT have encouraged investigators to seek other options for reduction of the chronic disease burden in postmenopausal women. Soy isoflavones represent one example of compounds that have been investigated as an alternative to traditional HRT. Cross-Cultural Studies Evaluating Potential Effects of Soy Consumption

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Interest in dietary soy consumption and isoflavones was originally motivated by the known differences in chronic disease risk observed in Asian compared with Western countries. Given the fact that dietary intake of soy is much higher in Asia than in Western countries, it is possible that many of the differences in chronic disease outcomes could be attributable, at least in part, to the differences in dietary intake. Asian women have been observed to have a markedly lower incidence of CHD, osteoporosis-related fractures, and reproductive cancer (breast and endometrial) compared with Western women. In fact, the cumulative mortality rate for CHD in native Japanese women between the ages of 40 and 69 y was more than eightfold lower than the cumulative mortality rate of Caucasian women in the United States as shown in Figure 29.1 (15). This is much greater than the expected protection obtained by using conventional HRT. Migrant studies of Japanese can provide information about the effect of environmental factors (such as diet) on chronic disease risk. Japanese women who migrated to Hawaii and those born in the United States (i.e., consuming

Fig. 29.1. Age-adjusted

coronary heart disease mortality rates in U.S. Caucasian women and Japanese women, 40–69 y old. Data adapted from Beaglehole (15).

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more “Westernized” diets) lose some of their protection from chronic diseases compared with women living in Japan (16,17). Although there are many potential reasons for these differences, it has been hypothesized that one potential explanation for the differences in chronic disease risk is related to the different sources of protein consumed in these countries, soy proteins with intact isoflavones (Japan) vs. animal protein (United States). Certainly, other dietary intake differences, including the fact that the traditional Japanese diet is lower in fat content than the typical “Western” diet, may explain a significant portion of the reduction in cardiovascular and breast disease between the two cultures. However, the greater inclusion of vegetable protein, principally derived from soybeans, in the traditional Japanese diet may have important implications in the reduction of risk of chronic diseases. The “estrogenic” component of soybeans (genistein and daidzein and a by-product of their digestion, equol) may play an important role in the protection against breast and endometrial cancer and CVD (18–20). Soy and Menopausal Symptoms Peri- and postmenopausal women often experience disruptive vasomotor symptoms (hot flashes and night sweats) as a result of the drop in estrogen secretion that accompanies menopause. These vasomotor symptoms can result in dramatic changes in a woman’s quality of life and is one of the main reasons why women initiate HRT therapy. Soy isoflavones have been shown to affect the hypothalamic-pituitary axis of both women and animals. However, it does not reduce plasma levels of luteinizing hormone (LH) or follicle-stimulating hormone (FSH) in rodents or women, but does blunt LH and FSH response to gonadotropin-releasing hormone in both ovariectomized rats and women (21–24). Sex hormone-binding globulin (SHBG) concentrations have not been affected consistently by diets high in soy (25). In some studies, soy decreased plasma concentrations of gonadal steroids, likely by increasing excretion via the gut and away from the kidney (26). Additionally, soy may also decrease both ovarian and adrenocortical secretion of gonadal steroids and their precursors. The result of lower plasma concentrations of gonadal steroids may be an increase in menopausal symptoms; however, direct effects of the isoflavone component of soy appears to be either neutral or may have a modest effect on relief of vasomotor symptoms (27–29). Isoflavones have been found to bind to estrogen receptors (ER). The identification of estrogen receptor β (ERβ) has shed more light on the estrogenic activities of genistein and daidzein. Kuiper et al. (30) found that the binding affinity of genistein to ERβ was ~20 times greater than to ERα. Kuiper et al. (31) also compared isoflavone ER binding ability with that of estradiol and found the binding affinity of genistein to ERα at 4% and to ERβ at 87% compared with 17β-estradiol at 100%. The isoflavone daidzein binds to ERα at 0.1%, and to ERβ at 0.5%. Previous research using animal models suggested that isoflavones might have the ability to act as estrogen agonists

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when estrogen concentrations are low and act as an antagonist when estrogen concentrations are high. This fascinating evidence on the estrogenic activity of soy isoflavones has stimulated research in a multitude of areas. Table 29.1 summarizes the results of several studies published on the effects of soy and soy isoflavones on vasomotor symptoms (hot flashes). Six of the eleven studies utilized soy foods or soy protein isolate (SPI) (27–29,32–34). Murkies et al. (27) was one of the first to report the potential of soy foods to reduce vasomotor symptoms of menopause. Murkies and colleagues noted reductions of hot flashes by 40% in participants consuming 45 g of soy flour for 12 wk compared with the control group who experienced a 25% reduction while consuming wheat flour. Washburn et al. (28) reported a study of postmenopausal women (n = 43) who participated in a crossover trial in which the treatments consisted of 20 g SPI containing 34 mg isoflavones as one serving, 20 g of SPI in a split serving (half in the morning, half in the afternoon), or a carbohydrate-based control (placebo). The authors found significant reductions in the severity of symptoms in the split-serving ISP group. Albertazzi et al. (29) conducted a study in Italy using 60 g of soy protein containing 76 mg isoflavones and reported that this treatment significantly reduced hot flashes by 45% compared with the control group (casein). St. Germain et al. (32) reported that after 24 wk of high isoflavone (80.4 mg/d), low isoflavone (4.4 mg/d) soy protein treatment or whey protein control (n = 69), no differences were observed among the groups in the relief of vasomotor or other menopausal symptoms.

TABLE 29.1 Soy/Isoflavone Effects on Hot Flushes: Summary of Published Studiesa

Reference (27) (29) (33) (28) (34) (32) (38) (39) (37) (36) (35)

Isoflavones (mg)

Source

Tx length (wk)

Change from baseline (%)

~70 76 ~53 34 118 80 40 40 160 150 50 50

Soy flour SPI Soy grits SPI SPI SPI Pills Pills Pills Pillsc Pills Pills

12 12 12 6 12 24 12 12 12 12 6 12

↓40 ↓45 ↓22 — ↓6b ↓49 ↓21 ↓29 ↓34 ↓26 ↓45 ↓28

aAbbreviations:

Tx, treatment; SPI, soy protein isolate. hot flush severity as determined by self-reported score from mild (1) to severe (3). cGiven in split dose. bIndicates

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Difference from placebo (%) ↓15 ↓15 ↑29 ↓22b ↓4b ↓2 ↓4 ↑6 ↑1 ↑8 ↓19 ↓8

The other studies listed in Table 29.1 utilized isoflavone pills (35–39). The data from these studies are mixed. Upmalis et al. (35) investigated the effect of isolated soy isoflavones in a pill on hot flashes and endometrial tissue. Postmenopausal women (n = 177) received either 50 mg isoflavones or a placebo for 12 wk. The isoflavone groups experienced a significant reduction in average hot flash severity, and hot flash frequency was also significantly reduced compared with the control. They also reported that endometrial thickness did not change. Scambia and colleagues (36) conducted a pilot study to evaluate the effectiveness of a standardized soy extract and placebo given alone or in combination with conjugated equine estrogens (CEE). After 6 wk of soy extract or placebo treatment, both groups had significant reductions in hot flushes and the soy extract group had a greater decrease in the number of hot flushes compared with the placebo group (P < 0.01). When CEE was added to the soy extract or placebo treatment, there were even greater reductions in mean number of hot flushes, and no significant difference between the groups. No effect was noted on vaginal cytology or endometrial thickness. Quella et al. (37) reported that the use of soy pills in a double-blind clinical crossover study did not alleviate the hot flashes experienced by the participants who were breast cancer survivors. In summary, randomized clinical trials have shown mixed results on the effect of soy isoflavones on menopausal symptoms. Although soy may reduce vasomotor symptoms of menopause, it is clear that it is not as powerful as traditional HRT for symptom relief. Soy and Cardiovascular Disease Replacing animal protein with soy protein in the diet reduces plasma lipid and lipoprotein concentrations in hypercholesterolemic individuals (40–42) and reduces arterial plaque accumulation in various animal models (43–45). Soy has been shown to have beneficial effects on CVD risk factors. A meta-analysis conducted by Anderson et al. (46) of 38 clinical trials found that consumption of soy protein reduced LDL-C and plasma triglycerides by 13 and 10%, respectively, and increased HDL-C by 2%. Similar improvements in plasma lipids and lipoprotein levels have been observed in trials conducted in postmenopausal women. Washburn et al. (28) studied perimenopausal women 45–55 y old in a crossover study (n = 43) to investigate the effect of a supplement containing 20 g of SPI with 34 mg of isoflavones on CVD risk factors. Significant declines in total cholesterol (6%) and LDL-C (7%) were noted for soy supplementation compared with the placebo treatment. No significant changes were noted for HDL-C and triglycerides. The group also reported a significant decline in diastolic blood pressure, of ~5 mm Hg, for the split-serving soy treatment compared with placebo. Wangen et al. (47) evaluated postmenopausal women (n = 18) for three 93-d treatment periods in a crossover study, while they were consuming SPI containing 7 mg isoflavone/d (control), 65 mg isoflavone/d (low dose) or 132 mg isoflavone/d (high dose).

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Compared with placebo, women receiving the high-isoflavone treatment experienced an LDL-C reduction of 6.5%; the ratio of LDL-C:HDL-C was 8.5 and 7.7% lower after the low- and high-isoflavone treatment, respectively. Baum et al. (48) studied moderately hypercholesterolemic (total cholesterol concentrations 240–300 mg/dL at baseline) postmenopausal women (n = 81) who were randomized to receive either 56 or 90 mg of phytoestrogens (in 40 g soy protein) or casein/nonfat dry milk (40 g protein/placebo). After following the National Cholesterol Education Program Step 1 diet for 2 wk and throughout the 6-mo treatment period, both soy groups were found to have significantly lower LDL and VLDL cholesterol and higher HDL compared with placebo; no dose response was noted. Teede et al. (49) recently evaluated the cardiovascular effects of soy isoflavones by measuring blood pressure, lipids, vascular function, and endothelial function of 213 participants (105 postmenopausal women, 108 men). The 3-mo treatment included consumption of either 40 g soy protein with 118 mg isoflavones or casein protein (40 g). The group reported a significant increase in urinary isoflavones and a significant reduction in both systolic and diastolic blood pressure levels and blood lipids. In the women, no differences were found in vascular function between treatment groups. The authors did note two potential adverse effects, however, namely, an increase in lipoprotein(a) concentrations and a decline in endothelial function in the male participants. Isoflavone pills do not appear to have the same benefits on plasma lipid concentrations as soy protein with its naturally occurring isoflavones. Several studies have been conducted using isoflavone pills with postmenopausal women; however, most have reported no significant effects on total cholesterol, lipoprotein, or triglyceride levels (50–52). One study recently published by Clifton-Bligh et al. (53) reported favorable effects on apolipoprotein B (apo B) and HDL-C. Postmenopausal women in this trial (n = 46) were randomized to receive 28, 57, or 85 mg of isoflavones on a daily basis for 6 mo. They reported that compared with baseline, HDL-C increased significantly in all groups and apo B decreased significantly; however, these lipid changes were independent of the dose of isoflavones given. The authors themselves suggested a cautious interpretation of their results because they did not include a control group (i.e., no isoflavones). The beneficial improvements that soy protein exerts on plasma lipoprotein concentrations has resulted in the FDA approval of a health claim for 25 g/d of soy protein as part of a diet low in saturated fat and cholesterol for reducing heart disease risk (54). Soy with isoflavones can improve endothelial-mediated vascular reactivity in postmenopausal women (55). Three recent studies have evaluated the effects of isoflavones without soy protein on vascular function (51,52,56); two were conducted in postmenopausal women (51,52). The first was a crossover study of menopausal women (n = 17) who consumed 80 mg isoflavones (isolated from red clover). Nestel et al. (51) reported improved systemic arterial compliance, a measure of arterial elasticity, by 23% in the isoflavone compared with the placebo period. The second study, by Simons et al. (52) randomized postmenopausal women (n = 20) with evidence of endothelial dysfunction and observed no improvement in

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flow-mediated endothelium-dependent dilation with pills containing 80 mg isoflavone. Thus, isoflavones appear to affect vascular function, but the effects may be more consistent when isoflavones are given with soy protein. Soy Effects on Endometrial and Breast Tissue Epidemiologic and laboratory-based data support a benefit of soy isoflavones for reproductive cancer prevention. Cross-cultural comparisons of cancer incidence provide evidence for this relationship (Fig. 29.2) (57,58). The rates of endometrial and breast cancer are lower in Japan, where soy is a dietary staple, compared with rates in the United States, where very little soy is consumed. Data from migrant studies also support environmental factors, including dietary intake, as important in the pathogenesis of breast cancer because the rate of breast cancer increases among Japanese who immigrated to the United States (59,60). It is important to note that although these data do not directly implicate soy or its isoflavones, they provide a rationale for further exploration of this issue in experimental studies. Other observational studies in the literature also support a soy/cancer association. Several studies found markedly reduced risk of breast cancer with increasing soy consumption (60–63). The mechanism of action of soy’s antineoplastic effects is not entirely understood, but may reside in the effects of the soybean isoflavones on tyrosine kinase, antioxidant effects, or in lowering plasma and urinary concentrations of estradiol and its metabolites by increasing gut excretion (20,64). Other mechanisms by which the isoflavones may affect cancer initiation and progression include estrogen agonist/antagonism, antioxidant effects, topoiso(B) Endometrical cancer incidence

Rate/100,000

Rate/100,000

(A) Breast cancer incidence

Age (y) Fig. 29.2. (A) Age-adjusted breast cancer incidence rates in women 35–74 y old in the

United States and Japan. Adapted from Ursin et al. (55). (B) Age-specific endometrial cancer rates in U.S. Caucasian and Japanese women. Adapted from Parkin et al. (56).

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merase inhibition, aromatase inhibition, angiogenesis inhibition, and apoptosis promotion (65). Animal studies present compelling evidence for an isoflavone and cancer association. Barnes et al. (66) reported a reduction in carcinogen-induced mammary number and size among rats treated with SPI with the isoflavones intact compared with those fed the isolate with the isoflavones removed. Lamartiniere et al. (67) treated rats neonatally with genistein and found that they were protected against carcinogen-induced mammary tumors later in life. Foth and Cline (68) fed soy protein with isoflavones or a casein-based diet to nonhuman primates and reported no significant increases in cellular proliferation in the mammary gland and endometrium. Conversely, studies of MCF-7 breast cancer cells and one human study suggest that soy isoflavones may contribute to breast cell proliferation and potentially increase breast cancer risk (69). For endometrial tissue, Scambia et al. (36) and Upmalis et al. (35) both reported that no effects were seen on the endometrial tissue of soy isoflavone extract–treated participants. The research regarding soy and cancer prevention in humans is ongoing. On balance, the current literature suggests that there may be a beneficial effect of soy intake on cancer prevention. However, until more is known, it seems prudent for postmenopausal women to incorporate soy foods into their diets not as a cancer chemoprevention strategy per se but as a healthy dietary source of protein. Soy and Osteoporosis Osteoporotic hip fractures occur less frequently in Japanese than in Americans (70). The protection from hip fractures could be explained by lower fall rates, more favorable bone geometry at the hip, and perhaps soy protein consumption (71). The evidence regarding the efficacy of soy protein and isoflavones on preservation of bone density is mixed. Three human studies that evaluated the effect of soy protein on bone mineral density are reported here. All three evaluated total body and lumbar spine density and were conducted with postmenopausal women of varying ages. Potter et al. (72) randomized postmenopausal women (n = 66) to 40 g of soy protein diets containing 2 levels of isoflavones for 6 mo and reported a 2.5% increase (P < 0.05) in lumbar spine BMD in the group ingesting 90 mg total isoflavones each day, but no effect on femoral neck BMD, and no effect of a 56 mg isoflavone dose. A trial conducted by Alekel et al. (73) reported a smaller decrease in spine BMD (–0.23 %) in perimenopausal women consuming 80 mg of isoflavones per day compared with the reduction observed in the whey control group (–1.28%). In a study presented only in abstract form, early postmenopausal women (n = 65) were randomized to SPI containing one of three levels of isoflavones (~4, 52, or 96 mg/d isoflavones). No significant difference was seen for either the spine or femoral neck BMD at 9 mo in the group consuming 96 mg isoflavones/d compared with the control group (–1.7 vs. –0.83%) (74). The effect of soy protein on biomarkers of bone metabolism has also been studied and the results have been mixed. One study showed that 60 mg isoflavones

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reduced urinary excretion of cross-linked deoxypyridinolines (69 vs. 53 nmol/mmol creatinine, P < 0.01) (75). Another study in which postmenopausal women (n = 42) consumed soy milk (60 mg of isoflavones) for 12 wk showed a significant reduction in urinary N-terminal cross-linked peptide and an increase in serum osteocalcin, suggesting reduced bone turnover (76). Two other studies did not find any effects on bone turnover markers (73,74) The short length of soy intervention, the age of the participants, and their menopausal status can explain some of the inconsistency of the findings of these studies. Additionally the selection of the control group “treatment” (placebo) may have played a role in these results. Potter et al. (72) and Alekel et al. (73) used a milk protein control, whereas Gallagher et al. (74) used isoflavone-devoid (alcohol-washed) soy protein. If some of the effects of soy on bone are related to a protein effect rather than an isoflavone effect, then one might not expect to find differences between groups fed isoflavone-devoid soy vs. isoflavone-intact SPI. This hypothesis is consistent with the results of Alekel’s study, which reported a difference between whey and isoflavone-intact soy, with the isoflavone-devoid soy group intermediate. On the basis of these data, it appears that there is either no effect or only a mildly beneficial effect from soy and isoflavone consumption on menopausal bone maintenance and health. Soy and Cognitive Function As is the case for traditional HRT, the presence of documented soy effects on the brain remains uncertain. One study regarding effects of soy on cognition in postmenopausal women was conducted in King County, WA (77). It was a cross-sectional study of Japanese-American women ≥65 y old that found that current users of HRT who ate tofu three or more times per week did not have significantly better cognitive function than the no-HRT, low-tofu consumption group. In a longitudinal study of Asian men, White et al. (78) found that those who consumed larger amounts of tofu earlier in life were subsequently noted to have lower scores on cognitive tests and were more likely to be diagnosed with Alzheimer’s disease later in life compared with men who ate relatively small amounts of soy. On the other hand, two recently reported clinical trials showed beneficial effects of soy protein and isoflavones on cognitive function and memory (79,80). Given the current data, continued investigations regarding the efficacy and safety of soy isoflavones on the brain are warranted.

Conclusion In an attempt to compare the effects of soy isoflavones and traditional HRT, the current scientific data are summarized in Table 29.2. Beneficial effects on CVD risk factors are seen with both soy isoflavones and traditional HRT. Certainly menopausal symptom relief and positive effects on osteoporotic burden are significantly better with traditional HRT than with soy. Conversely, there appear to be no adverse effects of soy on breast cancer, nor do there appear to be adverse effects on

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TABLE 29.2 Comparison of Soy/Isoflavones and Hormone Replacement Therapy (HRT)

Outcome Cardiovascular disease Menopausal symptoms Osteoporosis Breast cancer Endometrial cancer Cognition Miscellaneous adverse symptoms

Soy/Isoflavone + + or none + or none + or none None +/0/– None

Traditional HRT + or none +++ +++ Negative Negative ++ or none Numerous negative

endometrial cancer; unlike ERT/HRT. Although the data evaluating effects on cognition are mixed for both soy and ERT/HRT, there appear to be more studies suggesting a beneficial effect for traditional HRT, possibly because of a longer period of study. Finally, there are more adverse symptoms observed with traditional HRT compared with soy isoflavones (e.g., bleeding, breast tenderness, headaches). Although some areas of research are promising, in aggregate, soy isoflavones do not meet the criteria of the optimal HRT. Research shows there are relatively few known side effects and many potential benefits to increasing soy consumption. The “typical” U.S. diet previously contained very little, if any, soy. However, current marketplace changes in soy food availability have greatly increased the amount of soy consumed in the U.S. Food manufacturers have recognized that soy has the advantage of being an inexpensive, abundant, well-tolerated food source, and is being sought by consumers. On the basis of the current evidence, we suggest that a healthy diet should contain one serving of soy per day. This recommendation is currently in agreement with the existing recommendations of the American Heart Association, the National Cancer Institute, and the American Dietetic Association. We believe that the use of isoflavone supplements is premature due to a paucity of data on both the efficacy and safety of these agents and the greater potential for intake of unsafe megadoses. We agree with an evidence-based consensus opinion published by the North American Menopause Society, which concluded that health care providers can recommend consumption of whole foods with isoflavones to menopausal women, keeping in mind, however, that the health effects in humans at this time cannot be attributed solely to the isoflavones (81). There are many unanswered questions pertaining to the use of soy isoflavones as an alternative to traditional HRT. Future studies could seek to evaluate the effects of the different components in soy at differing dosages (e.g., equol, genistein) on menopausal outcomes. Other studies could focus on side-by-side comparisons of isoflavone pills with soy protein containing isoflavones for CVD risk factors, osteoporosis, and cognitive function. Studies evaluating the risk-benefit ratio from a combination of soy and HRT could ascertain whether the combination has a more favorable effect on reproductive cancer risk and other chronic disease out-

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comes. Additionally, studies of greater duration that can evaluate the longer-term effects on disease outcomes should be considered. References 1. Luciano, A.A., Turksoy, R.N., Carleo, J., and Hendrix, J.W. (1988) Clinical and Metabolic Responses of Menopausal Women to Sequential Versus Continuous Estrogen and Progestin Replacement Therapy, Obstet. Gynecol. 71, 39–43. 2. Walsh, B.W., Schiff, I., Rosner, B., Greenberg, L., Ravnikar, V., and Sacks, R.M. (1991) Effects of Postmenopausal Estrogen Replacement on the Concentrations and Metabolism of Plasma Lipoproteins, N. Engl. J. Med. 325, 1196–1204. 3. The Writing Group for the PEPI Trial (1995) Effects of Estrogen/Progestin Regimens on Heart Disease Risk Factors in Postmenopausal Women: The Postmenopausal Estrogen/ Progestin Interventions (PEPI) Trial, J. Am. Med. Assoc. 273, 199–208. 4. Ettinger B. (1988) Prevention of Osteoporosis: Treatment of Estradiol Deficiency, Obstet. Gynecol. 72, 12S–17S. 5. Brenner PF. (1988) The menopausal syndrome, Obstet. Gynecol. 72, 6S–11S. 6. Yaffe, K., Sawaya, G., Lieberburg, I., and Grady, D. (1998) Estrogen Therapy in Postmenopausal Women: Effects on Cognitive Function and Dementia, J. Am. Med. Assoc. 279, 688–695. 7. Ravnikar, V.A. (1987) Compliance with Hormone Therapy, Am. J. Obstet. Gynecol. 156, 1332. 8. Ravnikar, V.A. (1992) Compliance with Hormone Therapy: Are Women Receiving the Full Impact of Hormone Replacement Therapy’s Preventive Health Benefits? Women Health Issues 2, 75–82. 9. Collaborative Group on Hormonal Factors in Breast Cancer (1997) Breast Cancer and Hormone Replacement Therapy: Collaborative Reanalysis of Data from 51 Epidemiological Studies of 52,705 Women with Breast Cancer and 108,411 Women Without Breast Cancer, Lancet 350, 1047–1059. 10. Woodruff, J.D., and Pickar, J.H. for the Menopause Study Group. (1994) Incidence of Endometrial Hyperplasia in Postmenopausal Women Taking Conjugated Estrogens (Premarin) with Medroxyprogesterone Acetate or Conjugated Estrogens Alone, J. Obstet. Gynecol. 170, 1213–1223. 11. Schairer, C., Lubin, J., Troisi, R., Sturgeon, S., Brinton, L. and Hoover, R. (2000) Menopausal Estrogen and Estrogen-Progestin Replacement Therapy and Breast Cancer Risk, J. Am. Med. Assoc. 283, 485–491. 12. Hulley, S., Grady, D., and Bush, T. (1998) Randomized Trial of Estrogen Plus Progestin for Secondary Prevention of Coronary Heart Disease in Postmenopausal Women, J. Am. Med. Assoc. 280, 605–613. 13. Herrington, D.M., Reboussin, D.M., Brosnihan, K.B., Sharp, P.C., Shumaker, S.A., Snyder, T.E., Furberg, C.D., Kowalchuk, G.J., Stuckey, T.D., Rogers, W.J., Givens, D.H., and Waters, D. (2000) Effects of Estrogen Replacement on the Progression on Coronary-Artery Atherosclerosis, J. Am. Med. Assoc. 343, 522–529. 14. Brett, K.M., and Madans, J.H. (1997) Use of Postmenopausal Hormone Replacement Therapy: Estimates from a Nationally Representative Cohort Study, Am. J. Epidemio1. 145, 536–545.

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15. Beaglehole, R. (1990) International Trends in Coronary Heart Disease Mortality, Morbidity, and Risk Factors, Epidemiol. Rev. 12, 1–15. 16. Gordon, T. (1967) Further Mortality Experience Among Japanese Americans, Pub. Health. Rep. 82, 973–984. 17. Dunn, J.E., Jr. (1975) Cancer Epidemiology in Populations of the United States with Emphasis on Hawaii and California and Japan, Cancer Res. 35, 3240–3245. 18. Adlercreutz, H. (1990) Western Diet and Western Diseases: Some Hormonal and Biochemical Mechanisms and Associations, Scand. J. Clin. Lab. Investig. 50 (Suppl.), 3–23 19. Messina, M., and Barnes, S. (1991) The Role of Soy Products in Reducing Risk of Cancer, J. Natl. Cancer Inst. 83, 541–546. 20. Messina, M.J., Persky, V., Setchell, D.R., and Barnes, S. (1994) Soy Intake and Cancer Risk: A Review of the In Vitro and In Vivo Data, Nutr. Cancer 21, 113–131. 21. Hughes, C.L. (1988) Effects of Phytoestrogens on GnRH-Inducted Luteinizing Hormone Secretion in Ovariectomized Rats, Reprod. Toxicol. 1, 179–181. 22. Hughes, C.L., Kaldas, R.S., Wisinger, A.S., McCants, C.E., and Basham, K.B. (1991) Acute and Subacute Effects of Naturally Occurring Estrogens on Luteinizing Hormone Secretion in the Ovariectomized Rat: Part 1, Reprod. Toxicol. 5, 127–132. 23. Hughes, C.L., Chakinala, M.M., Reece, S.G., Miller, R.N., Schomberg, D.W., Jr., and Basham, K.B. (1991) Acute and Sub Acute Effects of Naturally Occurring Estrogens on Luteinizing Hormone Secretion in the Ovariectomized Rat: Part 2, Reprod. Toxicol. 5, 133–137. 24. Nicholls, J., Lasley, B.L., Gold, E.B., Nakajima, S.T., and Schneeman, B.O. (1994) Phytoestrogens in Soy and Change in Pituitary Response to GnRH Challenge Test in Women, J. Nutr. 125 (Suppl.), 803. 25. Clarkson, T.B., Anthony, M.S., and Hughes, C.L., Jr. (1995) Estrogenic Soybean Isoflavones and Chronic Disease: Risks and Benefits, Trends Endocrinol. Metab. 6, 11–16. 26. Goldin, B., Adlercreutz, H., Gorbach, S.L., Woods, M.N., Dwyuer, J.T., Conlon, T., Buhn, E., and Gershoff, S.N. (1986) The Relationship Between Estrogen Levels and Diets of Caucasian American and Oriental Immigrant Women, Am. J. Clin. Nutr. 44, 945–953. 27. Murkies, A.L., Lombard, C., Strauss, B.J.G., Wilcox, G., Burger, H.G., and Morton, M.S. (1995) Dietary Flour Supplementation Decreases Postmenopausal Hot Flushes: Effect of Soy and Wheat, Maturitas 21, 189–195. 28. Washburn, S., Burke, G.L., Morgan, T., and Anthony, M. (1999) Effect of Soy Protein Supplementation on Serum Lipoproteins, Blood Pressure, and Menopausal Symptoms in Perimenopausal Women, Menopause 6, 7–13. 29. Albertazzi, P., Pansini, F., Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998) The Effect of Dietary Soy Supplementation on Hot Flushes, Obstet. Gynecol. 91, 6–10. 30. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A.(1996) Cloning of a Novel Receptor Expressed in Rat Prostate and Ovary, Proc. Natl. Acad. Sci. USA 93, 5925–5930. 31. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.-A.(1998) Interaction of Estrogenic Chemicals and Phyotestrogens with Estrogen Receptor β, Endocrinology 139, 4252–4263.

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32. St. Germain, A., Peterson, C.T., Robinson, J.G.B., and Alekel, D.L. (2001) IsoflavoneRich or Isoflavone-Poor Soy Protein Does Not Reduce Menopausal Symptoms During 24 Weeks of Treatment, Menopause 8, 17–26. 33. Dalais, F.S., Rice, G.E., Wahlquist M.L., Grehan, M., Murkies, A.L., Medley, G., Ayton, R., and Strauss, B.J.G. (1998) Effects of Dietary Phytoestogens in Postmenopausal Women, Climacteric 1, 124–129. 34. Kotsopoulos, D., Dalais, F.S., Liang, Y-L., McGrath, B.P., and Teede, H.J. (2000) The Effects of Soy Protein Containing Phytoestrogens on Menopausal Symptoms in Postmenopausal Women, Climacteric 3, 161–167. 35. Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L., and Lamia, C.A. (2000) Vasomotor Symptom Relief by Soy Isoflavone Extract Tablets in Postmenopausal Women: a Multicenter, Double-Blind, Randomized, Placebo-Controlled Study, Menopause 7, 236–242. 376 Scambia, G., Mango, D., Signorile, P.G., Anselmi Angeli, R.A., Palena, C., Gallo, D., Bombardelli, E., Morazzoni, P., Riva, A., and Mancuso, S. (2000) Clinical Effects of a Standardized Soy Extract in Postmenopausal Women: A Pilot Study, Menopause 7, 71–75. 37. Quella, S.K., Loprinzi, C.L., Barton, D.L., Knost, J.A., Sloan, J.A., LaVasseur, B.I., Swan, D., Krupp, K.R., Miller, K.D., and Novotny, P.J. (2000) Evaluation of Soy Phytoestrogens for the Treatment of Hot Flashes in Breast Cancer Survivors: A North Central Cancer Treatment Group Trial, J. Clin. Oncol. 18, 2792–2793. 38. Baber, R.J., Templeman, C., Morton, T., Kelly, G.E., and West, L. (1999) Randomized Placebo-Controlled Trial of an Isoflavone Supplement and Menopausal Symptoms in Women, Climacteric 2, 85–92. 39. Knight, D.C., Howes, J.B., and Eden, J.A. (1999) The Effect of Promensil, an Isoflavone Extract, on Menopausal Symptoms, Climacteric 2, 79–84 40. Sirtori, C.R., Agradi, E., Conti, F., Mantero, O., and Gatti, E. (1977) Soybean-Protein Diet in the Treatment of Type II Hyperlipoproteinaemia, Lancet 1, 275–277. 41. Sirtori, C.R., Gatti, E., Mantero, O., Conti, F., Agradi, E., Tremoli, E., Sirtori, M., Fraterrigo, L., Tavazzi, L., and Kritchevsky, D. (1979) Clinical Experience with the Soybean Protein Diet in the Treatment of Hypercholesterolemia, Am. J. Clin. Nutr. 32, 1645–1658. 42. Potter, S.M. (1998) Soy Protein and Cardiovascular Disease: The Impact of Bioactive Components in Soy, Nutr. Rev. 56, 231–235. 43. Adams, M.R., Golden, D.L., Anthony, M.S., Register, T.C., and Williams, J.K. (2002) The Inhibitory Effect of Soy Protein Isolate on Atherosclerosis in Mice Does Not Require the Presence of LDL Receptors or Alteration of Plasma Lipoproteins, J. Nutr. 132, 43–49. 44. Anthony, M.S., Clarkson, T.B., Bullock, B.C., and Wagner, J.D. (1997) Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet-Induced Coronary Artery Atherosclerosis of Male Cynomolgus Monkeys, Arterioscler. Thromb. Vasc. Biol. 17, 2524–2531. 45. Ni, W., Tsuda, Y., Sakono, M., and Imaizumi, K. (1998) Dietary Soy Protein Isolate, Compared with Casein, Reduces Atherosclerotic Lesion Area in Apolipoprotein EDeficient Mice, J. Nutr. 128, 1884–1889. 46. Anderson, J.W., Johnstone, M.B., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 47. Wangen, K.E., Duncan, A.M., Xu, X., and Kurzer, M.S. (2001) Soy Isoflavones Improve Plasma Lipids in Normocholesterolemic and Mildly Hypercholesterolemic Postmenopausal Women, Am. J. Clin. Nutr. 73, 225–31.

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48. Baum, J.A., Teng, H., Erdman, J.W., Weigel, R.M., Klein, B.P., Persky, V.W., Freels, S., Surya, P., Bakhit, R.M., Ramos, E., Shay, N.F., and Potter, S.M. (1998) Long-Term Intake of Soy Protein Improves Blood Lipid Profiles and Increases Mononuclear Cell Low-Density-Lipoprotein Receptor Messenger RNA in Hypercholesterolemic, Postmenopausal Women, Am. J. Clin. Nutr. 68, 545–551. 49. Teede, H.J., Dalais, F.S., Kotsopoulos, D., Liang, Y.L., Davis, S., and McGrath, B.P. (2001) Dietary Soy Has Both Beneficial and Potentially Adverse Cardiovascular Effects: A Placebo-Controlled Study in Men and Postmenopausal Women, J. Clin. Endocrinol. Metab. 86, 3053–3060. 50. Howes, J.B., Sullivan, D., Lai, N., Nestel, P., Pomeroy, S.,West, L., Eden, J.A., and Howes, L.G. (2000) The Effects of Dietary Supplementation with Isoflavones from Red Clover on the Lipoprotein Profiles of Post Menopausal Women with Mild to Moderate Hypercholesterolaemia, Atherosclerosis 152, 143–147. 51. Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff P., Behrsing J., Cameron J.D., and West L. (1999) Isoflavones from Red Clover Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal Women, J. Clin. Endocrinol. Metab. 84, 895–898. 52. Simons, L.A., von Konigsmark, M., Simons, J., and Celermajer, D.S. (2000) Phytoestrogens Do Not Influence Lipoprotein Levels or Endothelial Function in Healthy, Postmenopausal Women, Am. J. Cardiol. 85, 1297–1301. 53. Clifton-Bligh, P.B., Baber, R.J., Fulcher, G.R., Nery, M.L., and Moreton, T. (2001) The Effect of Isoflavones Extracted from Red Clover (Rimostil) on Lipid and Bone Metabolism, Menopause 8, 259–265. 54. Food and Drug Administration (1999) Food Labeling, Health Claims, Soy Protein, and Coronary Heart Disease, Fed. Reg. 57, 699–733. 55. DuBroff, R. and Decker, P. (1999) Soy Phytoestrogens Improve Endothelial Dysfunction in Postmenopausal Women, North American Menopause Society Meeting Abstracts 99, 085, 53. 56. Walker, H.A., Dean, T.S., Sanders, T.A.B., Jackson G., Ritter J.M., and Chowienczyk, P.J.(2001) The Phytoestrogen Genistein Produces Acute Nitric Oxide-Dependent Dilation of Human Forearm Vasculature with Similar Potency to 17β-Estradiol, Circulation 103, 258–262. 57. Ursin, G., Bernstein, L., and Pike, M.C. (1994) Breast Cancer, Cancer Surv. 19–20, 241–264. 58. Parkin, D.M., Muir, C.S., Whelan, S.L., Gao, Y.T., Ferlay, J., and Powell, J. (1992) Cancer Incidence in Five Continents, Vol VI. IARC Scientific Publications, No. 120. International Agency for Research on Cancer. World Health Organization, Geneva. 59. Shimizu, H., Ross, R.K., Berstein, L., Yatani, R., Henderson, B.E., and Mack, T.M. (1991) Cancers of the Prostate and Breast Among Japanese and White Immigrants in Los Angeles County, Br. J. Cancer 63, 963–966. 60. Wu, A.H., Ziegler, R.G., Horn-Ross, P.L., Nomura, A.M., West, D.W., Kolonel, L.N., Rosenthal, J.F., Hoover, R.N., and Pike, M.C. (1996) Tofu and Risk of Breast Cancer in Asian-Americans, Cancer Epidemiol. Biomark. Prev. 5, 901–906. 61. Hirose, K., Tajima, K., Hamajima, N., Inoue, M., Takezaki, T., Kuroisha, T., Yoshida, M., and Tokudome, S. (1995) A Large-Scale, Hospital-Based Case-Control Study of Risk Factors of Breast Cancers According to Menopausal Status, Jpn. J. Cancer Res. 86, 146–154. 62. Yuan, J-M., Wang, Q-S., Ross, R.K., Henderson, B.E., and Yu, M.C. (1995) Diet and Breast Cancer in Shanghi and Tianjin, China, Br. J. Cancer 71, 1353–1358.

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63. Ingram, D., Sanders, K., Kolybaba, M., and Lopez, D. (1997) Case-Control Study of Phyto-Oestrogens on Breast Cancer, Lancet 350, 990–994. 64. Xu, X., Duncan, A.M., Wangen, K.E., and Kurzer, M.S. (2000) Soy Consumption Alters Endogenous Estrogen Metabolism in Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 9, 781–786. 65. Barnes, S., Peterson, T.G., and Coward, L. (1995) Rationale for the Use of GenisteinContaining Soy Matrices in Chemoprevention Trials for Breast and Prostate Cancer, J. Cell. Biochem. 22, 181–187. 66. Barnes, S., Peterson, G., Grubbs, C., and Setchell, K. (1994) Potential Role of Dietary Isoflavones in the Prevention of Cancer, in Diet and Cancer: Markers, Prevention, and Treatment, (Jacobs, M.M., ed.) pp. 135–147, Plenum Press, New York. 67. Lamartiniere, C.A., Moore, J., Holland, M., and Barnes, S. (1995) Neonatal Genistein Chemoprevents Mammary Cancer, Proc. Soc. Exp. Biol. Med. 208, 120–123. 68. Foth, D., and Cline, J.M. (1998) Effects of Mammalian and Plant Estogens on Mammary Glands and Uteri of Macaques, Am. J. Clin. Nutr. 68 (Suppl.), 1413S–1417S. 69. Petrakis, N.L., Barnes, L., King, E.B., Lowenstein, J., Wiencke, J., Lee, M.M., Miike, R., Kirk, M., and Coward, L. (1996) Stimulatory Influence of Soy Protein Isolate on Breast Secretion in Pre- and Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 5, 785–794. 70. Ross, P.D., Norimatsu, H., and Davis, J.W. (1991) a Comparison of Hip Fracture Incidence Among Native Japanese, Japanese Americans, and American Caucasians, Am. J. Epidemiol. 133, 801–809. 71. Nakamura, T., Turner, C.H., and Yoshikawa, T. (1994) Do Variations in Hip Geometry Explain Differences in Hip Fracture Risk Between Japanese and White Americans? J. Bone Miner. Res. 9, 1071–1076. 72. Potter, S.M., Baum, J.A., Teng, H.Y., Stillman, R.J., Shay, N.F., and Erdman, J.W. (1998) Soy Protein and Isoflavones; Their Effects on Blood Lipids and Bone Density in Postmenopausal Women, Am. J. Clin. Nutr. 68, 61–65. 73. Alekel, D.L., St. Germain, A., Pererson, C.T., Hanson, K.B., Stewart, J.W., and Toda, T. (2000) Isoflavone-Rich Soy Protein Isolate Attenuates Bone Loss in the Lumbar Spine of Perimenopausal Women, Am. J. Clin. Nutr. 72, 844–852. 74. Gallagher, J.C., Rafferty, K., Haynatzka, V., and Wilson, M. (1998) The Effect of Soy Protein on Bone Metabolism, Am. J. Clin. Nutr. 68 (Suppl.), 1518S. 75. Pansini, F., Bonaccorsi, G., and Albertazzi, P. (1997) Soy Phytoestrogens and Bone. (abstract #97) The North American Menopause Society, 8th Annual Meeting, Boston, MA. 76. Scheiber, M.D., Liu, J.H., Subbiah, M.T., and Rebar, R.W., and Setchell, K.D. (2001) Dietary Inclusion of Whole Soy Foods Results in Significant Reductions in Clinical Risk Factors for Osteoporosis and Cardiovascular Disease in Normal Postmenopausal Women, Menopause 8, 384–392. 77. Rice, M.M., LaCroix, A.Z., Lampe, J.W., van Belle, G., Kestin, M., Drinkwater, B.L., Graves, A.B., and Larson, E.B. (2000) Soy Consumption and Bone Mineral Density in Older Japanese American Women in King County, Washington, J. Nutr. 130, 686S. 78. White, L.R., Petrovitch, H., Ross, G.W., Masaki, K., Hardman, J., Nelson, J., Davis, D., and Markesbery, W. (2000) Brain Aging and Midlife Tofu Consumption, J. Am. Coll. Nutr. 19, 242–255. 79. Duffy, R.,Wiseman, H., and File, S. (2002) Dietary Soya Improves Memory in Humans, Am. J. Clin. Nutr. 132, 587S.

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80. Kritz-Silverstein, D. Von Muhlen, D., and Barrett-Conner, E. (2002) The Soy and Postmenopausal Health in Aging (SOPHIA) Study: Overview and Baseline Cognitive Function, Am. J. Clin. Nutr. 132, 586S. 81. Anonymous (2000) The Role of Isoflavones in Menopausal Health: Consensus Opinion of the North American Menopause Society, Menopause 7, 215–229.

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Chapter 30

Deleterious Effects of Genistein Follow Exposure During Critical Stages of Development Retha R. Newbolda, Wendy Jeffersona, Elizabeth Padilla-Banksa, and Bill Bullockb aDevelopmental

Endocrinology Section, Laboratory of Molecular Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC bDepartment

of Pathology, Wake Forest University, School of Medicine, Wake Forest University, Winston-Salem, NC

Introduction Genistein, the principal isoflavone in soy, interacts with estrogen receptors and multiple molecular targets. This phytoestrogen is found in high levels in soy products including soy-based infant formulas and dietary soy supplements. In adults, soy consumption has been reported to decrease the incidences of breast and prostate cancers, protect against osteoporosis and cardiovascular disease, and alleviate some unwanted symptoms of menopause (1–10). However, the actual role of genistein in these potential beneficial effects is unclear because diets high in soy contain multiple agents that may contribute to these findings; further, high-soy diets are also associated with low energy and fat intake, both of which are welldocumented cancer protective factors. Thus, the possibility of soy isoflavones including genistein in reducing chronic disease risks seems to be inconclusive, with the most promising benefits occurring in the prevention of cardiovascular disease (11). Despite all of the hypothesized beneficial effects of soy genistein, the potential for deleterious effects has not received equal attention. It is well known that the developing fetus and neonate are uniquely sensitive to perturbation with estrogenic chemicals; the toxic effect of prenatal exposure to diethylstilbestrol (DES) is a classic example. Therefore, it is not surprising that there are increasing concerns about the adverse consequences of this phytoestrogen if exposure occurs in infants and young children (12–16). This early exposure is of particular importance because 25% of infants in the United States are fed soy-based formulas. Studies have shown that infants fed soymilk formulas have plasma isoflavone levels that are orders of magnitude higher than those of infants fed human or cow’s milk, but the possible long-term effects of these relatively high levels of phytoestrogens during infancy remain unknown. A recently published epidemiologic study by Strom et al. (20) found differences between young adults who were fed soy formula as infants vs. those fed cow’s milk; problems reported in the soy formula group

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included increased incidence of multiple births, stillborn deliveries, preterm deliveries, and greater discomfort with menstruation. Because this cohort was composed of young people in their 20s, these reported differences warrant longer follow-up to determine both whether the severity increases with age or other problems develop. Unfortunately, no other long-term epidemiologic studies exist that have a similar sizeable cohort for study. However, a recent study by North et al. (21) reports that male infants, born to vegetarian mothers consuming high levels of phytoestrogens, have an increased incidence of hypospadias, suggesting that phytoestrogens do indeed play a role in the deleterious effects on the developing reproductive system. Additional evidence for adverse effects of genistein can be seen with experimental studies that show that exposure of pregnant dams to genistein enhanced 7, 12-dimethyl benz(a)anthracene (DMBA)-induced mammary tumors in their female offspring (22). Furthermore, adverse effects of soy-containing foods and soy components on reproductive processes of experimental animals and humans have been reported (16,23–29). Using a well-established experimental animal model of developmental exposure, the effects of genistein on representative estrogen target tissues of outbred CD-1 mice, derived from the breeding colony at The National Institute of Environmental Health Sciences (NIEHS), were evaluated by treating neonates on d 1–5 of life with genistein dissolved in corn oil as previously described for other estrogenic substances (30). The doses were administered as subcutaneous injections [0.5, 5.0, and 50 mg/(kg ⋅ d)] and compared with DES at 0.001 mg/(kg ⋅ d), which is equal in estrogenicity to the highest dose of genistein [50 mg/(kg ⋅ d)]. Although the subcutaneous route of administration in our experimental animal model differs from the usual dietary exposure in humans, we showed that the maximal circulating serum levels of genistein (conjugated + aglycone) in the high dose group were comparable to those reported from dietary exposures in rats and in human infants consuming soy-based formulas (31). Alterations observed in the ovary, reproductive tract (oviduct, uterus, cervix, and vagina), and mammary gland after development exposure to relevant levels of genistein were discussed. Comparison of the Estrogenicity of Genistein and DES in Neonates To compare genistein to DES, a well-studied environmental estrogen, doses were chosen on the basis of data obtained from an immature uterotrophic bioassay that directly compared both compounds over a wide dose range (32). Genistein at a dose of 50 mg/kg was estrogenic in the uterotropic bioassay, demonstrating a doubling of uterine wet weight. For a direct comparison of the effects of DES to genistein, a dose of 0.001 mg/kg of DES was chosen because it was similar in estrogenicity to that of 50 mg/kg of genistein in immature mice. To verify that these doses were similar in estrogenicity in the neonatal mouse model, mice were subcutaneously injected on neonatal d 1–5 with DES or genistein in corn oil at a dose of 0.001 or 50 mg/kg [0.002 or 100 µg/(pup ⋅ d)], respectively. A

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minimum of eight female pups per dose times two compounds was used. On the afternoon of d 5 after the last injection, mice were killed, body weights and uterine weights recorded, and uterine wet weight to body weight ratios calculated; the results of this assay are shown in Figure 31.1. Both compounds at the doses tested caused a significant increase in uterine wet weight compared with controls, as determined by Fisher’s exact test. In fact, they both gave a similar magnitude of response and were therefore chosen as appropriate comparative doses for study. Because soy-based infant formula contains varying amounts of genistein (18,19), lower doses of 0.5 and 5 mg/kg were included in the study to ensure that a wide range of relevant exposure levels of genistein were covered. Ovary

Uterine wet wt/body wt ratio × 100

Genistein, long recognized for its estrogenic activity, has been shown to exhibit a 20-fold higher relative binding affinity for estrogen receptor (ER)β than ERα (33). A similar binding preference for ERβ has been reported for other phytoestrogens, including coumestrol and naringenin (34). However, the role of ERα and ERβ, and the extent to which each contributes to the actions and possible toxicity of genistein exposure has only recently come under investigation. An additional confounding factor unique to the study of genistein is its well-described tyrosine kinase inhibitory properties. In fact, genistein is currently marketed as a laboratory reagent to effectively inhibit tyrosine specific protein kinase activity (35), which is independent of its genomic interaction with the ER (36,37). Genistein has also been reported to have genotoxic activity (38). Given the binding preference of genistein for ERβ, which is expressed more abundantly in the ovary relative to other tissues (39,40), the actions of genistein

Treatment [mg/(kg ⋅ d)]

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Fig. 31.1. Comparison of estrogenicity of genistein and diethylstilbestrol (DES) in neonates. Outbred CD-1 female mice were treated by subcutaneous injection on d 1–5 with DES [0.001 mg/(kg ⋅ d)] or genistein [50 mg/(kg ⋅ d)]. Body weights and uterine weights were determined on d 5 after the last injection. Data are plotted as a ratio of uterine wet weight to body weight. Data demonstrate a similar magnitude of response.

exposure on the ovary warranted further investigation. In the experimental mouse model, an array of effects of neonatal genistein exposure on the maturing ovary was seen that may be categorized as follows: (i) biochemical effects such as the induction of ectopic expression of ERα in granulosa cells; (ii) morphological effects such as induction of multioocyte follicles (MOF) in the ovary; and (iii) functional effects such as altered ovarian response to superovulation treatment. Detailed information on these ovarian alterations has been described (41). Furthermore, using the gene-targeted ERα-null (αERKO) and ERβ-null (βERKO) mice (42) as well as the nonestrogenic tyrosine-kinase inhibitor (lavendustin A), both the estrogenic and tyrosine-kinase inhibitory properties of genistein and the contribution of each pathway to the ultimate effects in the ovary were evaluated. In the initial study, CD-1 mice were treated on neonatal d 1–5 with genistein at doses of 0.5, 5, or 50 mg/(kg ⋅ d). Ovaries were collected on d 5, 12, and 19 and were either fixed in cold 10% formalin for ERα and ERβ immunohistochemistry (IHC) or frozen for ribonuclease protection assays (RPA) analysis. An additional group of CD1 mice was treated with lavendustin A, a tyrosine kinase inhibitor, at doses of 0.5 or 5 mg/(kg ⋅ d) on d 1–5. The ovaries from these mice were also collected for ERα and ERβ IHC and processed using techniques previously described (40). Total RNA was isolated from pooled ovaries on d 5, 12, and 19 and assayed for differences in ERα or ERβ mRNA expression by RPA. These data are summarized in Table 31.1. ERα mRNA was dramatically increased on d 5 in the genistein TABLE 31.1 Ribonuclease Protection Assay (RPA) Analysis of Estrogen Receptor (ER)α and ERβ mRNA in the Developing Ovary After Neonatal Genistein (Gen) Exposure Fold induction over control

Treatmenta (mg/kg)

ERα (% cyc)b

ERβ (% cyc)b

ERα

ERβ

d5

Control Gen 0.5 Gen 5 Gen 50

2.50 7.26 4.12 1.10

5.27 8.04 6.71 2.81

1.0 2.9c 1.6 0.4

1.0 1.5 1.3 0.5

d 12

Control Gen 0.5 Gen 5 Gen 50

2.32 1.05 5.16 2.11

10.62 6.64 10.96 6.91

1.0 0.5 2.2c 0.9

1.0 0.6 1.0 0.7

d 19

Control Gen 0.5 Gen 5 Gen 50

0.49 0.55 0.77 0.43

10.90 11.42 15.22 9.71

1.0 1.1 1.6 0.9

1.0 1.0 1.4 0.9

Age

aOutbred CD-1 mice were treated on d 1-5 with varying doses of genistein. Ovaries were collected, frozen, and RNA isolated. ERα and ERβ mRNA transcripts were detected by RPA as described in text. bCyclophilin was assayed for normalization of each sample and the percentage of ERα and ERβ was calculated. cSignificant increases over control.

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0.5 mg/(kg ⋅ d) group and slightly increased in the 5 mg/(kg ⋅ d) group. At 12 d, ERα mRNA was increased in the genistein 5 mg/(kg ⋅ d) group. By 19 d, little ERα expression in the ovary could be detected (40). A slight increase in ERβ mRNA was found at 5 d of age in the lower doses of genistein, although the difference became less apparent with age because ERβ was abundantly expressed in all treatment groups by 19 d of age. To localize the ER subgroups in specific ovarian cellular compartments, tissue sections from control and genistein-treated mice were immunostained for ERα and ERβ protein. At 19 d, ERβ was strongly expressed in the granulosa cells of both control and genistein-treated mice (40); there was a slight increase in immunostaining at low doses of genistein compared with controls. Thus, ERβ by RPA and IHC appeared to not be altered by genistein treatment. In addition, mice treated with the tyrosine kinase inhibitor, lavendustin, also showed the same pattern of ERβ expression as control and genistein-treated mice. ERα was seen predominately in interstitial and thecal cells of control mice with little or no detectable immunostain in granulosa cells (Fig. 31.2A). However, ERα was localized differently after neonatal genistein treatment. These mice showed induction of ERα expression in granulosa cells, especially at the lower doses of exposure (Fig. 31.2B). Induction of ERα protein in the granulosa cells

Fig. 31.2. Estrogen receptor (ER)α immunohistochemistry of ovaries from 19-d-old mice

after neonatal exposure to genistein. CD-1 mice were treated on d 1–5 with corn oil as control or genistein 0.5 mg/(kg ⋅ d). Ovaries were collected, fixed in cold formalin, embedded in paraffin, and cut at 4 µm. Ovary sections were immunostained for ERα as described in the text. (A) Control ovary shows ERα predominantly in the interstitial and thecal cells. (B) Genistein ovary shows an induction of ERα in the granulosa cells.

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was also seen in lavendustin-treated mice suggesting that the tyrosine kinase inhibition activity of genistein was involved in this effect (41). As a functional analysis of the ovary, the ovulatory capacity of mice treated neonatally with genistein was determined using standard methods (43). At 21 d of age, mice were weaned; on the next day (d 22), all mice received a single subcutaneous injection of 2.2 IU pregnant mares’ serum gonadotropin (PMSG) (Sigma) followed 48–52 h later with 3.2 IU human chorionic gonadotropin (hCG) (Sigma). Mice were killed 16–20 h after the hCG injection, oviducts removed, and the oocyte/cumulus mass extracted from the oviducts; oocytes were counted after enzymatic disassociation from the surrounding cumulus. The data in Table 31.2 represent the sum of two experiments, each of which included 7–8 mice/treatment group. Mice treated with the lowest dose of genistein as neonates exhibited a slightly increased ovulatory response compared with untreated controls (33.9 ± 3.3 vs. 23.2 ± 2.8), whereas mice treated with higher doses of genistein yielded a reduced number of ovulated oocytes (16.5 ± 1.8). The underlying mechanisms for this low dose effect of genistein, however, are unclear. Because mice exposed to the lowest dose of genistein are apparently ovulating more oocytes, they may possibly exhibit premature depletion of oocytes, resulting in early reproductive senescence. In fact, our laboratory has previously shown a similar phenomenon in mice exposed prenatally to low doses of DES; these mice had an increased number of corpora lutea at 2 mo of age but a marked decrease by 6 mo of age relative to agematched controls, thus demonstrating early reproductive senescence (44). It is interesting to note that the dose of genistein that induced the highest level of ERα expression in the granulosa cells is the same dose that caused the highest number of oocytes following superovulation. Because estrogen action is known to be an antiatretic factor in the ovary (45) and clearly facilitates ovulation as demonstrated by ERKO mice (42,46), the elevated expression of ERα within the granulosa cells may allow a greater number of follicles to be available for ovulation durTABLE 31.2 Number of Oocytes Ovulated After Superovulation in Mice Treated Neonatally with Genistein (Gen) Treatment [mg/(kg ⋅ d)]a Control Gen 0.5 Gen 5 Gen 50 aMice

Mice (n)

Oocytes (n)

15 16 14 16

23.2 ± 2.8 33.9 ± 3.3b 17.9 ± 1.4 16.5 ± 1.8

were treated neonatally on d 1–5 with genistein at varying doses. At 22 d of age, mice were treated with pregnant mares’ serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as described in the text. Mice were killed 16–20 h after the last injection; ovaries/oviducts were collected and incubated with hyaluronidase; oviducts were then ruptured and oocytes counted. Data are expressed as the mean ± SEM of the number of oocytes per mouse. bP < 0.05 compared with controls using Dunnett’s test.

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ing gonadotropin treatment. Although the increased number of ovulated oocytes at low doses is very interesting, of equal importance is the decrease in oocytes at the higher doses especially because these doses are in the range of typical human infant exposure levels. Clearly, neonatal genistein exposure is adversely affecting ovarian function at all doses studied in the experimental animal model. Another interesting finding was the appearance of multioocyte follicles (MOF) after neonatal exposure to genistein (40,41,47). An example of MOF can be seen in Figure 31.3. The ovaries from 8 mice/treatment group were evaluated for the presence of MOF, a rare finding in outbred CD-1 mice. A summary of the data is given in Table 31.3. A dose-related increase in the number of mice that exhibited this phenotype was found with increasing doses of genistein. In addition, the multiplicity (greatest number of MOF observed per tissue section) of these follicles was increased; the greatest number of MOF in a single section was 8 in the genistein 50 mg/(kg ⋅ d) group. The oocytes in the MOF generally appeared to be similar in size and usually two per follicle, but three or four oocytes in a single follicle were occasionally observed. Because lavendustin-treated mice did not have MOF (Table 31.3), this genistein-induced phenotype is probably not due to its tyrosine kinase inhibitor activity but rather to its estrogenic activity. Further support for the role of its estrogenic activity in the formation of MOF can be seen from studies of pre- and neonatal exposure to DES, which resulted in the appearance of MOF in the mouse ovary (48–50). The neonatal ovary was shown to be uniquely susceptible to the effect of DES compared with adult tissue, which did not develop MOF when treated with DES; the MOF effect was specific

Fig. 31.3. Morphology of multioocyte follicles after neonatal genistein treatment: example. This is an ovary from a mouse treated with genistein [50 mg/(kg ⋅ d)]; it was collected on d 19, fixed with 10% cold formalin, embedded in paraffin, and cut at 6 µm. Ovarian tissue sections were stained with hematoxylin and eosin for evaluation. A double oocyte follicle as well as a triple oocyte follicle can be seen (arrows).

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TABLE 31.3 The Incidence of Multioocyte Follicles in Immature Mice After Neonatal Exposure to Genistein (Gen) or Lavendustin (Lav) Treatment [mg/(kg ⋅ d)]a

Incidence of multioocyte folliclesb (%)

Multiplicityc

0/8 (0) 1/8 (12.5) 2/8 (25) 6/8 (75) 0/8 (0) 0/8 (0)

0 2 4 8 0 0

Control Gen 0.5 Gen 5 Gen 50 Lav 0.5 Lav 5 aMice

were treated subcutaneously on neonatal d 1–5 of life. Ovaries were collected on d 19 and evaluated by light microscopy to determine the presence of multioocyte follicles. Three sections at three different levels per mouse were screened. bThe numerator is the number of mice that showed the presence of at least one multioocyte follicle and the denominator is the total number of mice screened per treatment. cThe highest number of multioocyte follicles observed in a single section from that treatment group.

to estrogen action and not induced by similar treatments with progesterone or testosterone (51,52). Furthermore, the induction of MOF by DES appeared to result from a direct action on the ovary because neonatal ovaries exposed to DES in culture and then transplanted to adult control hosts also exhibited MOF (52). Because other estrogens like DES induced the MOF phenotype, the estrogenic role of genistein’s activity via the estrogen receptor was investigated using ER knockout (ERKO) mice. Both αERKO and βERKO mice on a C57/Bl6 background (42) were treated neonatally with genistein as described for CD-1 mice. Ovaries were collected on d 19 and examined for the presence of MOF. αERKO mice showed an incidence of MOF similar to that of CD-1 mice as well as wildtype mice of the same strain (C57/BL6) treated with genistein (Table 31.4). However, βERKO mice do not show MOF after neonatal genistein treatment, suggesting that the formation of MOF is mediated through ERβ- but not ERα-associated pathways (41). The occurrence of MOF in the mouse ovary may not be unique to estrogen exposure but may result from other ER-associated pathways. Recent descriptions of mice lacking growth differentiated factor-9 (GDF-9) or bone morphogenetic protein 15 (BMP-15) (53), both oocyte-secreted growth factors, or mice lacking insulin-like growth factor receptor 2 (IRS-2), a member of the IGF signaling pathway (54), exhibited an increased incidence of MOF. Also, mice overexpressing the inhibin gene (55) had an increased incidence of MOF. Therefore, the mechanisms underlying the development of MOF are obviously complex and probably involve multiple pathways. Although the origin of MOF has not been determined, they likely result from a failure of primary follicular cells to differentiate and surround primordial oocytes during early stages of follicle organization. Genistein acting via ERβ may alter granulosa cell response to the oocyte-secreted growth factors just

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TABLE 31.4 The Incidence of Multioocyte Follicles in Estrogen Receptor α Knockout (αERKO) and βERKO Mice After Neonatal Exposure to Genistein (Gen) Treatmenta [mg/(kg ⋅ d)] Control Gen 0.5 Gen 5 Gen 50

αERKO

Wild-type

βERKO

Incidenceb

Multiplicityc

Incidence

Multiplicity

Incidence

Multiplicity

1/11 1/11 9/11 11/11

1 1 3 10

1/3 2/4 4/6 —

1 1 4 —

1/2 0/4 0/5 1/3

1 0 0 2

aMice were treated subcutaneously on neonatal d 1–5 of life. Ovaries were collected on d 19 and evaluated by light microscopy to determine the presence of multioocyte follicles. Three sections at three different levels per mouse were screened. bThe numerator is the number of mice that showed the presence of at least one multioocyte follicle and the denominator is the total number of mice screened per treatment. cThe greatest numbers of multioocyte follicles observed in a single section from that treatment group.

mentioned or it may alter the expression of proteases or adhesive proteins involved in oocyte-granulosa cell interactions. More study is required to examine the mechanisms by which genistein causes MOF and to determine whether MOF occur in human females who were fed soy-based formula as infants. The Strom epidemiology study (20), which reported an increased incidence in twinning, suggests that indeed MOF may be a feature in humans after developmental exposure to genistein. Together, the data demonstrate that neonatal genistein exposure produces multiple effects on the morphology and function of the mouse ovary. The mechanisms by which genistein causes such effects is just beginning to be elucidated. The induction of ERα expression in the granulosa cells of the ovary appears to be associated with the tyrosine kinase inhibition properties of genistein rather than its estrogen actions, although indirect effects secondary to estrogenization of the hypothalamic-pituitary axis cannot be ruled out. In contrast, the induction of MOF in the ovary, which appears to be a direct ovarian effect and unrelated to the changes in ERα expression, is dependent on the presence of functional ERβ within the ovary. In addition to these ovarian effects, other alterations were also observed after neonatal exposure to genistein. Comparison of ovarian abnormalities observed in mice treated neonatally with genistein [50 mg/(kg ⋅ d)] or DES [0.001 mg/(kg ⋅ d)] is shown in Table 31.5. The number of mice without corpora lutea was higher in genistein and DES-treated groups at 6–8 wk and 4–6 mo of age. This suggests altered estrous cycles and subfertility. At 18 mo of age, cystic ovaries were common in all treatment groups (46% in controls, 41% in the genistein group, and 58% in the DES group) and did not appear to be related to treatment. However, corpora lutea were absent in 100% (17/17) of the genistein-treated mice, whereas only 33% (4/12) of the DES-treated mice lacked corpora lutea; all 13 of the control mice had corpora lutea. In the DES-treated mice, 17% (2/12) had ovarian stromal tumors;

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TABLE 31.5 Incidence of Selected Abnormalities in Mice Treated Neonatally with Diethylstilbestrol (DES) or Genistein (Gen) Treatment a

Ovary

Reproductive tract

Control DES 0.001 mg/(kg ⋅ d)

6–8 wk old 0/17 No CL (0)b 6/11 No CL (55)

Genistein 50 mg/(kg ⋅ d)

18/19 No CL (95)

Control

4–6 mo old 0/12 No CL (0)

DES 0.001 mg/(kg ⋅ d)

3/16 No CL (19)

6/16 Uterine hyperplasia (38) 6/16 Uterine squamous metaplasia (38) 15/16 Excess vaginal keratinization (94)

Genistein 50 mg/(kg ⋅ d)

5/9 No CL (56)

11/19 Uterine hyperplasia (58) 4/19 Uterine squamous metaplasia (21) 9/19 Excess vaginal keratinization (47)

Control

18 mo old 0/13 No CL (0)b 6/13 Cysts (46)

0/17 Uterine hyperplasia (0) 3/14 Uterine hyperplasia (21) 6/14 Uterine squamous metaplasia (43) 13/14 Excess vaginal keratinization (93) 10/20 Uterine hyperplasia (50) 4/20 Uterine squamous metaplasia (20) 10/20 Excess vaginal keratinization (50) 0/12 Uterine hyperplasia (0) 0/12 Uterine squamous metaplasia (0) 0/12 Excess vaginal keratinization (0)

0/13 PPL (0)c 3/16 CEH (19)d 0/16 Uterine squamous metaplasia (0) 0/16 Uterine adenocarcinoma (0) 0/16 Excess vaginal keratinization (0)

DES 0.001 mg/(kg ⋅ d)

4/12 No CL (33) 7/12 Cysts (58) 2/12 Ovarian stromal tumors (17)

5/10 PPL (50) 7/13 CEH (54) 5/13 Uterine squamous metaplasia (3 4/13 Uterine adenocarcinoma (31) 5/13 Excess vaginal keratinization (38)

Genistein 50 mg/(kg ⋅ d)

17/17 No CL (100) 7/17 Cysts (41)

14/14 PPL (100) 8/17 CEH (47) 11/17 Uterine squamous metaplasia (64) 1/17 Uterine atypical hyperplasia (5) 6/17 Uterine adenocarcinoma (35) 8/17 Excess vaginal keratinization (47)

CD-1 mice were treated by subcutaneous injections on d 1–5 of neonatal life with 0.001 mg/(kg ⋅ d) [0.002 µg/(pup ⋅ d)] of DES or 50 mg/(kg ⋅ d) [100 µg/(pup ⋅ d)] of genistein; these are approximately equal estrogenic doses. Mice were allowed to age and were killed at 6 wk, 4 mo, and 18 mo. bCL, corpora lutea. cPPL, progressive proliferative lesion of the oviduct. dCEH, cystic endometrial hyperplasia. eThe 18-mo-old data are summarized from Reference 16. aOutbred

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ovarian tumors were not seen in the control or genistein treatment groups. These data suggest that some of the effects of genistein are similar to those of DES, but others such as lack of corpora lutea, may be even more severe with genistein than equal estrogenic doses of DES. Reproductive Tract Oviduct. Abnormalities in the oviduct were seen in both the DES and genistein treatment groups at 18 mo of age but not at earlier ages (Table 31.5). As described previously with DES (56,57), progressive proliferative lesion (PPL) of the oviduct was seen in 100% of the aged genistein-treated mice. In PPL, the pattern of tubal plications of oviductal mucosa was distorted and characterized by lack of plications or irregularities in the size and shape of the mucosal folds relative to the control mice. The mucosal folds had an adenomatous (gland-like) appearance but maintained connection with the oviductal lumen. This abnormality in proliferation was termed “PPL” because it did not spread along the serosal surface or metastasize. Although sections were not always available through the entire length of the oviduct, at 18 mo of age, PPL was observed in ~50% (5/10) of DES-treated mice, whereas all (14/14) of the genistein-treated mice had this abnormality. These data suggest that genistein can have estrogenic effects similar to those of DES in the oviduct and further, that these effects can appear more severe at equal estrogenic doses. The enhanced effects of genistein on the oviduct may be due to its multiple pathways of activity. Although the role of PPL in subsequent disease processes and impaired fertility is uncertain, this abnormality was diagnosed in women who were exposed prenatally to DES (58). Because experimental animal data suggest a high incidence in PPL in genistein-treated groups, this abnormality should be followed closely in young women who were fed soy formula as infants; PPL may be a common finding of genistein exposure across species and may progress with age. Uterus/Cervix/Vagina. The range of abnormalities in the reproductive tract of mice aged 6–8 wk, 4–6 mo and 18 mo exposed neonatally to genistein or DES is shown in Table 31.5. At 6–8 wk and 4–6 mo of age, uterine hyperplasia and squamous cell metaplasia were typical findings in DES- and genistein-treated mice but not controls; uterine hyperplasia was higher in genistein-treated mice compared with DES at both ages, but squamous cell metaplasia was higher in DES-treated mice compared with genistein. Excessive vaginal keratinization was also higher in the DES-treated mice compared with genistein treatment; this vaginal phenotype, associated with excessive estrogen exposure, increased in severity with age, as did uterine squamous metaplasia. Data from genistein- and DES-treated mice at 18 mo of age have been reported (16). At this age, the incidence of cystic endometrial hyperplasia (CEH) of the uterus was similar in both DES and genistein treatment groups [54% (7/13) and 47% (8/17), respectively]. Although a low incidence of CEH occurred in aged con-

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trol mice [19% (3/16)], genistein caused a significantly increased incidence of this lesion, suggesting that CEH was related to estrogen treatment. Interestingly, unlike the younger ages, the incidence of uterine squamous cell metaplasia was higher in the genistein-treated mice than in the DES-treated mice; squamous cell metaplasia of the uterus occurred in 64% (11/17) of the genistein group and in 38% (5/13) of the DES group but not in controls, again suggesting that this lesion was definitely treatment related. Furthermore, other severe pathologies occurred after neonatal exposure to both DES and genistein. Atypical hyperplasia of the uterus occurred in 5% (1/17) of the genistein-treated mice at 18 mo of age. Most importantly, 35% (6/17) of the genistein-treated mice had cellular alterations that progressed to uterine adenocarcinoma (Fig. 31.4A and B). These tumors were usually well differentiated and characterized by irregularly shaped glands with little intervening stroma. Some tumors extended through the myometrium to the serosal surface of the uterus. Mitotic figures and nuclear pleomorphism were frequently observed. In

Fig. 31.4. Uterine adeno-

0.5 mm

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carcinoma in a mouse treated neonatally with genistein. (A) Well-differentiated invasive uterine adenocarcinoma in an 18-mo-old mouse treated neonatally with genistein [50 mg/(kg ⋅ d)] on d 1–5 of life. Neoplastic endometrial tissue is present between strands of smooth muscle. Note the irregularly shaped glands with little intervening stroma and eosinophilic material in the lumen. (B) Higher magnification of uterine adenocarcinoma. There is much nuclear pleomorphism, and mitotic figures are frequent. The irregularly shaped endometrial glands are packed together between bundles of smooth muscle.

comparison, at an equal estrogenic dose of DES [0.001 mg/(kg ⋅ d)], 31% (4/13) had uterine adenocarcinomas. Similar malignant lesions were not observed in control mice of this strain. These data show the induction of benign and malignant reproductive tract lesions, including uterine adenocarcinoma in mice treated neonatally with genistein. The dose of genistein was within the range to which humans may be exposed in soy infant formulas based on levels reported by Setchell (18). Most importantly, mice exposed to an equal estrogenic dose of DES had a similar incidence of uterine adenocarcinoma. The association of estrogenicity and carcinogenicity in the neonate is supported by results of other studies comparing metabolites of estradiol (59); on the basis of the d 5 uterotropic bioassay, compounds with the highest estrogenic potency in the neonatal mouse uterus showed the highest incidence of uterine adenocarcinoma after neonatal exposure. The comparative tumor induction ability of genistein and DES provides further support for an association between the estrogenic activity of the compound in the neonate and the incidence of uterine adenocarcinoma after neonatal exposure. Because genistein is readily available to many infants during the first few years of life as a component of soy-based formulas, and the amounts used in the experimental mouse model are similar to the level of human exposure (19,31), it is essential to determine whether genistein causes similar adverse reproductive effects in humans as described in the mouse model. However, some of these effects, e.g., increased incidence of neoplasia, may not be apparent until much later in life. Mammary Gland Although soy products have been hypothesized to play a role in reducing the risk of breast cancer, the effects of genistein on the development of the mouse mammary gland were examined at 5 and 6 wk of age after neonatal exposure, considering the adverse effects observed in reproductive tract tissue and the ovary after a similar exposure period. Mammary glands (number 4) were removed, fixed in Carnoy’s fixative, and stained with carmine for whole-mount analysis. Following a procedure described by Ball (60), the number of terminal end buds (TEB) in the peripheral leading edge of the differentiating ductal mass was counted (3 glands per treatment group per age) (Fig. 31.5A). TEB are responsive to local and systemic hormones that drive ductal morphogenesis (60). The cap cells of the terminal end buds (TEB) are normally present during neonatal and prepubertal-pubertal growth periods of mice, but they are thought to play a role in mammary gland carcinogenesis. Control mice exhibited an increase in TEB from 5 to 6 wk of age, indicating active ductal morphogenesis. Mice treated with the lowest dose of genistein [0.5 mg/(kg ⋅ d)] showed a similar increase in the number of TEB from 5 to 6 wk but the numbers were higher overall than those of controls at both ages. Strikingly, the genistein 5 mg/(kg ⋅ d) dose group showed a different pattern of

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5 wk 6 wk

Treatment [mg/(kg ⋅ d)] 5 wk 6 wk

Treatment [mg/(kg ⋅ d)] Fig. 31.5. Mammary gland differentiation in mice treated neonatally with genistein. Mice were treated on neonatal d 1–5 with varying doses of genistein. Mammary glands were collected at 5 and 6 wk of age and stained with carmine as described in the text. (A) Terminal end buds (TEB) >0.03 mm were counted for a minimum of 3 mice per treatment group. Numbers represent mean ± SEM. Mice in the genistein 0.5 mg/(kg ⋅ d) group showed a slight increase in the number of TEB at both ages. A dramatic increase in TEB was apparent in the 5 mg/(kg ⋅ d) genistein group at 5 wk of age with a subsequent decrease at 6 wk of age. Mice in the 50 mg/(kg ⋅ d) genistein group showed abnormal morphology compared with the other groups, but the number of TEB was similar to controls. (B) Measurement of the length on the furthest elongated duct from the lymph node was determined for a minimum of 3 mice per treatment group at 5 and 6 wk of age. The expanding mammary ductal mass increased after exposure to genistein at the 5 mg/(kg ⋅ d) dose at 5 wk of age and the 0.5 mg/(kg ⋅ d) dose at 6 wk of age. The high dose of genistein showed less expansion at both ages.

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mammary gland development, with more TEB present at 5 wk of age and less at 6 wk of age, suggesting an advance in differentiation at this dose. The highest dose of genistein [50 mg/(kg ⋅ d)] showed a number of TEB similar to that of controls, but mammary gland morphology was abnormal with smaller ducts and less ductal branching. As another indication of mammary development and differentiation, the length of the furthest elongated duct from the lymph node was measured (3 glands per treatment group per age) (Fig. 31.5B). These measurements showed that the expanding mammary ductal mass increased after exposure to genistein at the 5 mg/kg dose at 5 wk, and at the 0.5 and 5 mg/(kg ⋅ d) doses at 6 wk of age (Fig. 31.6). The 50 mg/(kg ⋅ d) dose at both time points showed a shorter expansion into the fat pad than controls, and the amount of mammary gland in the fat pad was less than all of the other treatment groups. This stunted development was evident throughout the life of the mice, remaining apparent even at 18 mo of age (Fig. 31.7). The lactational function of this hypoplastic mammary gland was not tested due to poor fertility experienced by this dosed group. Rats treated neonatally with genistein have been reported to have low progesterone levels (61). Because progesterone is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty (62), the abnormal pathology of the highest dose of genistein may be due to low levels of progesterone in these mice. Further indication of progesterone’s role in this abnormal morphology is demonstrated in progesterone receptor knockout mice (PRKO), which have severely limited mammary gland development similar to our high-dose genistein-treated mice (63). Although little is known about the effects of genistein on the immature mouse mammary gland, exposure just before puberty to high doses of genistein [~25–1000 mg/(kg ⋅ d)] was reported to result in a reduction of carcinogen (DMBA)-induced mammary cancer (61,64). These protective effects of genistein were attributed to advanced mammary gland differentiation and fewer TEB, thus making the tissue less susceptible to carcinogens. However, understandably, premature development and differentiation of the mammary gland is not recommended as a prevention for breast cancer. Another report from the same laboratory described a similar reduction in DMBA-induced mammary cancer after neonatal treatment with high doses of genistein (65). Again, although this study suggested protection against mammary cancer, other effects on the reproductive system such as alterations in the estrous cycle and a significant reduction in the number of corpora lutea, were not favorable. Low numbers of corpora lutea suggest that these mice exhibit some level of subfertility. Indeed, high doses such as those reported (65) do contribute to persistent vaginal cornification, lack of cyclicity, and infertility. In addition, a recent report of Hilakivi-Clark et al. (22) showed that lower doses [~0.067–1 mg/(kg ⋅ d)] given prenatally resulted in high incidences of DMBA-induced mammary cancer. Therefore, the effects of genistein on the mammary gland are complicated because of dose dependency and timing of exposure. Certainly any potential benefits of

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6 wk

Genistein 50

Genistein 5

Genistein 0.5

Control

5 wk

Fig. 31.6. Mammary gland growth and differentiation. Mammary glands were collected at

5 and 6 wk of age and stained with carmine as described in the text. The lymph node in each picture is used for orientation. Growth in the fat pad is in the direction of the arrows. Panels A, C, E, and G are from 5-wk-old mice. Panels B, D, F, and H are from 6-wk-old mice. The treatments are indicated on the figure. Terminal end buds can be seen in all treatment groups. The expanding mammary ductal mass increased after exposure to genistein at the 5 mg/(kg ⋅ d) dose at 5 wk of age and the 0.5 mg/(kg ⋅ d) dose at 6 wk of age. Mice in the 50 mg/(kg ⋅ d) genistein group showed abnormal morphology with less branching and less expansion into the fat pad compared with control mice at the same age.

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A. Control

B. Genistein

Fig. 31.7. Mammary gland morphology in aged mice after neonatal treatment with

genistein. Mice were treated on neonatal d 1–5 with varying doses of genistein. Mammary glands were collected at 18 mo of age, fixed and stained with carmine as described in the text. (A) Control; (B) genistein 50 mg/(kg ⋅ d). Note the lack of differentiation and in particular, the lack of branching in the genistein-treated mouse.

genistein on the mammary gland, if shown to be helpful in breast cancer prevention, have to be considered in combination with its adverse effects on reproductive tract tissues and the ovary before determining an appropriate risk/benefit analysis for genistein treatment. Russo and Russo established the rodent mammary gland as a useful tool for testing carcinogenic compounds due to the susceptibility of mammary tissue to hormonedependent neoplasia (66). Because rodent lesions are similar to those found in humans (66), additional experimental studies with genistein are warranted to try to clarify conflicting rodent data, which report both beneficial and adverse effects on the mammary gland. Any possible beneficial effects of genistein regarding breast cancer protection must be weighed against possible adverse effects especially because existing hormoneresponsive cancers may be exacerbated by the estrogenic activity of genistein. Furthermore, prenatal and early neonatal treatment of human subjects with genistein should be approached with utmost caution because of the toxic and carcinogenic effects of genistein on differentiating reproductive tract tissues.

Conclusions The findings of the present study raise concerns over consumption of phytoestrogens in soy-based formulas and other soy-based products that are fed to infants and young children. Certainly, additional studies are required to determine potential adverse effects in humans exposed to high quantities of phytoestrogens during critical stages of neonatal or early development, but precautionary measures are warranted before continuing to expose a susceptible population to the toxicity of estrogenic substances especially in the absence of medical necessity. Unfortunately, lessons learned from prenatal exposure to DES, a previously prescribed estrogen, serve

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as a poignant reminder of the drastic effects of xenoestrogen exposure during critical stages of differentiation. Ongoing National Toxicology Program (NTP) studies designed to address multigenerational effects of genistein (67), will help to determine the risks of developmental exposure to genistein and other endocrine-disrupting chemicals. The potential risks and benefits of substances like genistein must be thoroughly assessed during development so that any permanent adverse effects can be identified. The short- and long-term effects of developmental exposure should be included in the evaluation of potential adverse or beneficial effects of substances such as genistein. References 1. Adlercreutz, H., and Mazur, W. (1997) Phyto-Oestrogens and Western Diseases, Ann. Med. 29, 95–120. 2. Barnes, S. (1998) Evolution of the Health Benefits of Soy Isoflavones, Proc. Soc. Exp. Biol. Med. 217, 386–392. 3. Clarkson, T.B., Anthony, M.S., Williams, J.K., Honore, E.K., and Cline, J.M. (1998) The Potential of Soybean Phytoestrogens for Postmenopausal Hormone Replacement Therapy, Proc. Soc. Exp. Biol. Med. 217, 365–368. 4. Cassidy, A. (1996) Physiological Effects of Phyto-Oestrogens in Relation to Cancer and Other Human Health Risks, Proc. Nutr. Soc. 55, 399–417. 5. Liu, J., Burdette, J.E., Xu, H., Gu, C., van Breemen, R.B., Bhat, K.P., Booth, N., Constantinou, A.I., Pezzuto, J.M., Fong, H.H., Farnsworth, N.R., and Bolton, J.L. (2001) Evaluation of Estrogenic Activity of Plant Extracts for the Potential Treatment of Menopausal Symptoms, J. Agric. Food Chem. 49, 2472–2479. 6. Kurzer, M.S., and Xu, X. (1997) Dietary Phytoestrogens, Annu Rev. Nutr. 17, 353–381. 7. Messina, M. (1994) To Recommend or Not to Recommend Soy Foods, J. Am. Diet. Assoc. 94, 1253–1254. 8. Messina, M., Barnes, S., and Setchell, K.D. (1997) Phyto-Oestrogens and Breast Cancer, Lancet 350, 971–972. 9. Mäkelä, S., Poutanen, M., Lehtimaki, J., Kostian, M.L., Santti, R., and Vihko, R. (1995) Estrogen-Specific 17β-Hydroxysteroid Oxidoreductase Type 1 (E.C. 1.1.1.62) as a Possible Target for the Action of Phytoestrogens, Proc. Soc. Exp. Biol. Med. 208, 51–59. 10. Setchell, K.D., and Cassidy, A. (1999) Dietary Isoflavones: Biological Effects and Relevance to Human Health, J. Nutr. 129, 758S–767S. 11. Anderson, J.J., Anthony, M.S., Cline, J.M., Washburn, S.A., and Garner, S.C. (1999) Health Potential of Soy Isoflavones for Menopausal Women, Public Health Nutr. 2, 489–504. 12. Goldman, L.R., Newbold, R., and Swan, S.H. (2001) Exposure to Soy-Based Formula in Infancy, J. Am. Med. Assoc. 286, 2402–2403. 13. Bingham, S.A., Atkinson, C., Liggins, J., Bluck, L., and Coward, A. (1998) PhytoOestrogens: Where Are We Now? Br. J. Nutr. 79, 393–406. 14. Fort, P., Moses, N., Fasano, M., Goldberg, T., and Lifshitz, F. (1990) Breast and SoyFormula Feedings in Early Infancy and the Prevalence of Autoimmune Thyroid Disease in Children, J. Am. Coll. Nutr. 9, 164–167. 15. Sheehan, D.M. (1998) Herbal Medicines, Phytoestrogens and Toxicity: Risk/Benefit Considerations, Proc. Soc. Exp. Biol. Med. 217, 379–385.

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16. Newbold, R.R., Banks, E.P., Bullock, B., and Jefferson, W.N. (2001) Uterine Adenocarcinoma in Mice Treated Neonatally with Genistein, Cancer Res. 61, 4325–4328. 17. American Academy of Pediatrics, Committee on Nutrition (1998) Soy Protein-Based Formulas: Recommendations for Use in Infant Feeding, Pediatrics 101, 148–153. 18. Setchell, K.D., Zimmer-Nechemias, L., Cai, J., and Heubi, J.E. (1997) Exposure of Infants to Phyto-Oestrogens from Soy-Based Infant Formula, Lancet 350, 23–27. 19. Setchell, K.D., Zimmer-Nechemias, L., Cai, J., and Heubi, J.E. (1998) Isoflavone Content of Infant Formulas and the Metabolic Fate of These Phytoestrogens in Early Life, Am. J. Clin. Nutr. 68, 1453S–1461S. 20. Strom, B.L., Schinnar, R., Ziegler, E.E., Barnhart, K.T., Sammel, M.D., Macones, G.A., Stallings, V.A., Drulis, J.M., Nelson, S.E., and Hanson, S.A. (2001) Exposure to SoyBased Formula in Infancy and Endocrinological and Reproductive Outcomes in Young Adulthood, J. Am. Med. Assoc. 286, 807–814. 21. North, K., and Golding, J. (2000) A Maternal Vegetarian Diet in Pregnancy Is Associated with Hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood, Br. J. Urol. 85, 107–113. 22. Hilakivi-Clarke, L., Cho, E., Onojafe, I., Raygada, M., and Clarke, R. (1999) Maternal Exposure to Genistein During Pregnancy Increases Carcinogen-Induced Mammary Tumorigenesis in Female Rat Offspring, Oncol. Rep. 6, 1089–1095. 23. Cassidy, A., Bingham, S., and Setchell, K.D. (1994) Biological Effects of a Diet of Soy Protein Rich in Isoflavones on the Menstrual Cycle of Premenopausal Women, Am. J. Clin. Nutr. 60, 333–340. 24. Cassidy, A., Bingham, S., and Setchell, K. (1995) Biological Effects of Isoflavones in Young Women: Importance of the Chemical Composition of Soyabean Products, Br. J. Nutr. 74, 587–601. 25. Hughes, C.L., Jr., Kaldas, R.S., Weisinger, A.S., McCants, C.E., and Basham, K.B. (1991) Acute and Subacute Effects of Naturally Occurring Estrogens on Luteinizing Hormone Secretion in the Ovariectomized Rat: Part 1, Reprod. Toxicol. 5, 127–132. 26. Nagata, C., Kabuto, M., Kurisu, Y., and Shimizu, H. (1997) Decreased Serum Estradiol Concentration Associated with High Dietary Intake of Soy Products in Premenopausal Japanese Women, Nutr. Cancer 29, 228–233. 27. Xu, X., Duncan, A.M., Merz, B.E., and Kurzer, M.S. (1998) Effects of Soy Isoflavones on Estrogen and Phytoestrogen Metabolism in Premenopausal Women, Cancer Epidemiol. Biomark. Prev. 7, 1101–1108. 28. Xu, X., Duncan, A.M., Wangen, K.E., and Kurzer, M.S. (2000) Soy Consumption Alters Endogenous Estrogen Metabolism in Postmenopausal Women, Cancer Epidemiol. Biomark. Prev. 9, 781–786. 29. Whitten, P.L., Lewis, C., and Naftolin, F. (1993) A Phytoestrogen Diet Induces the Premature Anovulatory Syndrome in Lactationally Exposed Female Rats, Biol. Reprod. 49, 1117–1121. 30. Newbold, R.R., Bullock, B.C., and McLachlan, J.A. (1990) Uterine Adenocarcinoma in Mice Following Developmental Treatment with Estrogens: A Model for Hormonal Carcinogenesis, Cancer Res. 50, 7677-7681. 31. Doerge, D.R., Twaddle, N., Padilla-Banks, E., Jefferson, W.J., and Newbold, R. (2002) Pharmacokinetic Analysis in Serum of Genistein Administered Subcutaneously to Neonatal Mice, Cancer Lett., in press.

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32. Jefferson, W.N., and Newbold, R.R. (2000) Potential Endocrine-Modulating Effects of Various Phytoestrogens in the Diet, Nutrition 16, 658–662. 33. Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J.A. (1997) Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors Alpha and Beta, Endocrinology 138, 863–870. 34. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., and Gustafsson, J.A. (1998) Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor Beta, Endocrinology 139, 4252–4263. 35. Saunders, P.T., Fisher, J.S., Sharpe, R.M., and Millar, M.R. (1998) Expression of Oestrogen Receptor Beta (ER Beta) Occurs in Multiple Cell Types, Including Some Germ Cells, in the Rat Testis, J. Endocrinol. 156, R13–R17. 36. Makarevich, A., Sirotkin, A., Taradajnik, T., and Chrenek, P. (1997) Effects of Genistein and Lavendustin on Reproductive Processes in Domestic Animals In Vitro, J. Steroid Biochem. Mol. Biol. 63, 329–337. 37. Sar, M., and Welsch, F. (1999) Differential Expression of Estrogen Receptor-Beta and Estrogen Receptor-Alpha in the Rat Ovary, Endocrinology 140, 963–971. 38. Kulling, S.E., Rosenberg, B., Jacobs, E., and Metzler, M. (1999) The Phytoestrogens Coumoestrol and Genistein Induce Structural Chromosomal Aberrations in Cultured Human Peripheral Blood Lymphocytes, Arch. Toxicol. 73, 50–54. 39. Couse, J.F., Lindzey, J., Grandien, K., Gustafsson, J.A., and Korach, K.S. (1997) Tissue Distribution and Quantitative Analysis of Estrogen Receptor-α (ERα) and Estrogen Receptor-β (ERβ) Messenger Ribonucleic Acid in the Wild-Type and ERα-Knockout Mouse, Endocrinology 138, 4613–4621. 40. Jefferson, W.N., Couse, J.F., Banks, E.P., Korach, K.S., and Newbold, R.R. (2000) Expression of Estrogen Receptor Beta Is Developmentally Regulated in Reproductive Tissues of Male and Female Mice, Biol. Reprod. 62, 310–317. 41. Jefferson, W.J., Couse, J.F., Padilla-Banks, E., Korach, K.S., and Newbold, R. (2002) Neonatal Exposure to Genistein Induces Estrogen Receptor-a Expression and MultiOocyte Follicles in the Maturing Mouse Ovary: Evidence for ER β-Mediated and NonEstrogenic Actions, Biol. Reprod., in press. 42. Couse, J.F., and Korach, K.S. (1999) Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocrine Rev. 20, 358–417. 43. Rafferty, K.A. (1970) Methods in Experimental Embryology of the Mouse, Johns Hopkins Press, Baltimore. 44. Newbold, R.R., Bullock, B.C., and McLachlan,J.A. (1983) Exposure to Diethylstilbestrol During Pregnancy Permanently Alters the Ovary and Oviduct, Biol. Reprod. 28, 735–744. 45. Kaipia, A., and Hsueh, A.J. (1997) Regulation of Ovarian Follicle Atresia, Annu. Rev. Physiol. 59, 349-363. 46. Schomberg, D.W., Couse, J.F., Mukherjee, A., Lubahn, D.B., Sar ,M., Mayo, K.E., and Korach, K.S. (1999) Targeted Disruption of the Estrogen Receptor-Alpha Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult, Endocrinology 140, 2733–2744. 47. Jefferson, W.N., Padilla-Burgos, E., Miller, C., and Newbold, R.R. (1998) Endocrine Modulating Effects of Various Phytoestrogens, in Human Diet and Endocrine

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63. Lydon, J.P., DeMayo, F.J., Funk, C.R., Mani, S.K., Hughes, A.R., Montgomery, C.A., Jr., Shyamala, G., Conneely, O.M., and O'Malley, B.W. (1995) Mice Lacking Progesterone Receptor Exhibit Pleiotropic Reproductive Abnormalities, Genes Dev. 9, 2266–2278. 64. Hilakivi-Clarke, L., Onojafe, I., Raygada, M., Cho, E., Skaar, T., Russo, I., and Clarke, R. (1999) Prepubertal Exposure to Zearalenone or Genistein Reduces Mammary Tumorigenesis, Br. J. Cancer 80, 1682–1688. 65. Lamartiniere, C.A., Moore, J., Holland, M., and Barnes, S. (1995) Neonatal Genistein Chemoprevents Mammary Cancer, Proc. Soc. Exp. Biol. Med. 208, 120–123. 66. Russo, J., and Russo, I.H. (2000) Atlas and Histologic Classification of Tumors of the Rat Mammary Gland, J. Mammary Gland Biol. Neoplasia 5, 187–200. 67. Delclos, K.B., Bucci, T.J., Lomax, L.G., Latendresse, J.R., Warbritton, A., Weis, C.C., and Newbold, R.R. (2001) Effects of Dietary Genistein Exposure During Development on Male and Female CD (Sprague-Dawley) Rats, Reprod. Toxicol. 15, 647–663.

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Chapter 31

Evaluation of Phytoestrogen Safety and Toxicity in Rodent Models That Include Developmental Exposure K. Barry Delclos Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR

Introduction The potential effect of exposure to endocrine active agents in the environment has been a topic of much scientific and regulatory concern in recent years. The consequences of diethylstilbestrol (DES) treatment of pregnant women stand as the clearest example of the potential adverse effects of developmental estrogen exposure and support the relevance of animal data to the human experience (1,2). With the recent interest in the potential consequences of developmental exposure to synthetic environmental estrogens, more attention has been focused on evaluating whether and under what circumstances adverse effects from exposure to ubiquitous phytoestrogens might occur. It can be argued that there is much evidence that the consumption of vegetables, fruits, and grains containing phytoestrogens is beneficial to health and that exposure to phytoestrogens has occurred throughout human history without major toxic consequences. However, in recent years, products containing phytoestrogens derived from flaxseed, red clover, and soy have begun to be increasingly marketed and consumed for potential health benefits by many segments of the population, including women of childbearing age. In addition, over the past several decades, a substantial number of infants have consumed soy infant formula as their major source of nutrition. Because many manifestations of toxicity resulting from developmental exposure may not be evident until later in life, the safety assessment of these agents after exposure during developmental stages is an issue of considerable importance. Swan (3) has pointed out the difficulty in detecting rare or subtle clinical adverse events in epidemiologic studies, even with potent agents such as DES given at pharmacologic doses. With the exception of the induction of goiter in iodine-deficient infants receiving soy formula [see references in (4,5)], exposure to dietary phytoestrogens during development has not been clearly linked to any human health problem. Several groups of investigators have suggested possible relationships of developmental phytoestrogen exposure to adverse effects in humans including hypospadias (6), autoimmune disease (7), and infantile leukemia (8), although these links remain speculative. The use of the term “phytoestrogen” might be taken to imply that these compounds exert their biological effects via

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estrogen receptors, but the possible role of other molecular targets must be considered. The DNA damage reported by Strick et al. (8), for example, results from the inhibition of topoisomerase II by bioflavonoids, and soy-induced goiter likely results from inhibition of thyroid peroxidase by soy components, including isoflavones (4,5,9). In the case of the isoflavone genistein, interaction with estrogen receptors does occur at lower concentrations than does interaction with many other molecular targets (10), although inhibition of thyroid peroxidase by genistein has been clearly demonstrated in rats at low dietary dose levels (5). Studies conducted in developing rodents have indicated potential beneficial and adverse effects that can be used to guide human studies to establish clearly the safety of dietary phytoestrogens. Although the term “developmental” toxicity has sometimes been confined to toxicities resulting from exposures occurring during embryonic development (11), the term here will be applied as used by Selevan et al. (12), that is, “…the occurrence of adverse effects on the developing organism that may result from exposure prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation.” A variety of exposure regimens have been used to evaluate the safety and toxicity of phytoestrogens in developing rodents. These include exposures during pregnancy, the neonatal period, the prepubertal period, or a time period that spans part or all of these developmental stages. In some cases, experimental designs that include developmental exposures to phytoestrogens continue treatment into adulthood, so that it is not possible to determine to what extent developmental exposure contributed to the observed effects or whether there was a critical window of exposure. The majority of studies involving developmental exposures to phytoestrogens have focused on effects on tissues of the reproductive tract, but the broader effects of estrogens on the development of multiple organ systems have been appreciated for some time, and research on phytoestrogen effects in developing organisms has begun to include other systems, such as the immune and nervous systems and bone. Differences in the test animals used (species, strain, and source), dosing regimens, basal diets, and statistical power are among the experimental variables that complicate comparisons across studies, although definite patterns of potentially affected end points have emerged from the rodent studies. Only relatively recently has information on blood and tissue levels of phytoestrogens, particularly the soy isoflavones, in treated experimental animals become available to aid in the comparison of animal studies and in the extrapolation of results to humans. Importantly, these studies have shown that substantial transfer of isoflavones occurs to the fetus and nursing pup from treated dams (13–15). With regard to developmental exposures, several studies have shown that various phytoestrogens bind weakly to α-fetoprotein (16–20), a protein that serves to protect developing rodents from high endogenous estrogen levels. This interaction has been shown to modify the effects of certain phytoestrogens in vitro (18), but whether protein binding affects the activity of phytoestrogens in vivo, particularly at higher exposure levels, has not been established.

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The occurrence, chemistry, and metabolism of the major phytoestrogens have been reviewed extensively in this volume and elsewhere. In this chapter, the focus will be on the phytoestrogens for which a significant body of literature in rodent models has accumulated. Specifically, data on the following compounds will be considered: soy and its component or derivative phytoestrogens, genistein, daidzein, and equol; flaxseed and the component mammalian lignan precursor secoisolariciresinol diglucoside (SDG); the coumestan, coumestrol; and the resorcylic acid lactone fungal metabolite, zearalanone, and its metabolite α-zearalanol (or zeranol), which is also used as a veterinary drug in beef cattle in the United States. Although zearalenone is often not considered along with the phytoestrogens but rather separately as a mycoestrogen, it is included here because it is consumed as a contaminant of plant material. Effects of Developmental Phytoestrogen Exposure on Female Reproductive Tract and Mammary Gland Field reports on reproductive problems in domestic and captive animals served to focus attention on potential safety issues associated with phytoestrogens by indicating the disruption of reproductive processes in females. Much of the research on safety and toxicity of phytoestrogens in rodent models of development has also focused on the female reproductive tract and mammary gland. Effects of developmental exposures to phytoestrogens or phytoestrogen-containing foods observed in female rodents, some of which can be considered adverse and others neutral or beneficial, include birth weight and litter size decreases, acceleration or delay of puberty as measured by age or body weight at vaginal opening, altered length of estrous cycles, changes in serum sex hormones, altered ovarian, uterine, and vaginal histology, altered mammary gland development, alterations of sexually dimorphic structures in the brain, altered pituitary responsiveness to gonadotropin-releasing hormone (GnRH), altered fertility, and cancer. The occurrence, magnitude, and direction of these effects have been shown to depend on the timing and magnitude of the dose administered, as will be discussed below. Early Sexual Development and Puberty in Females. Although the sex of rodents and other mammals is genetically determined, the early hormonal environment can affect the development of the reproductive organs and other sexually distinct structures to result in animals that show variation in the degree of masculine and feminine structural and functional features. The demonstration that the sexual characteristics and behavior of rodent pups are influenced by the sex of neighboring fetuses during gestation indicates that very slight differences in the fetal hormone environment can influence sexual development (21–23). Depending on the exposure and evaluation regimen used, sex ratios of litters can be altered by selective toxicity to parental sperm, selective embryotoxicity, or selective maternal infanticide. In the case of rodent studies of phytoestrogens/mycoestrogens, alter-

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ation of sex ratios in litters from exposed parents have been reported only in the case of pregnant NMRI mice treated with subcutaneous (sc) injections of 150 mg/kg zeranol on gestation day (GD) 9 and 10 in which the percentage of males was significantly lower in treated litters (24). More subtle effects on the sexual features of pups born to phytoestrogen-exposed parents have been reported. One marker of masculinization/feminization widely used in reproductive toxicity studies is anogenital distance. In males, androgens, and in particular dihydrotestosterone, promote the growth of the perineal region so that at a given age, whether near-term fetus, newborn, adolescent, or adult, the distance is greater in males than in females. Variations in anogenital distance within a sex have been correlated with behavioral differences in both mice and rats (22,25). Exogenous estrogens, as well as antiandrogens, have been reported to affect anogenital distance, and although there are various mechanisms whereby estrogens might exert this effect such as alterations in testosterone or androgen receptor levels, the mechanisms behind estrogen effects on AGD have not been established. Because this measurement is sensitive to body size, an adjustment is generally made for body weight at the time of measurement, often by using an AGD:body weight ratio (anogenital distance index, AGDI) or other adjustments (26). Several developmental studies with phytoestrogens have reported effects in both directions on anogenital distance in females (Table 32.1), although this end point is not consistently altered in all studies in which it has been included. Both 5% flaxseed or an amount of SDG (the precursor of enterolactone and enterodiol) equivalent to that found in the 5% flaxseed diet increased the AGDI in female pups (27), as did the administration of dietary genistein (1000 µg/g) from GD 1 to postnatal day (PND) 56 (28) or a soycontaining diet from GD 0 to GD 20 (29). In the latter case, the effect of the diet on AGDI was seen in preterm pups, but not in PND 3 pups, although the AGD itself was increased in the neonates. Interestingly, a 10% flaxseed diet decreased both AGD and AGDI in newborn female rat pups, an effect opposite to that seen with the 5% flaxseed diet (27). Levy et al. (30) also reported a decreased AGD in female pups born to dams that had been treated with 5 mg genistein by sc injection on GD 16–20. No information on hormone or hormone receptor levels was available for those studies, and although the mechanisms behind these observations and their significance are unclear, the results suggest that phytoestrogens are able to masculinize or hyperfeminize female pups depending on the dosing conditions. Similar conclusions can be drawn from reports of the effects of developmental exposure to phytoestrogens on the achievement of puberty in female rodents. In rats, the timing of vaginal opening, which is measured by the age or the body weight at which vaginal patency is achieved and occurs near the time of the first ovulation, is the most readily measured marker of the attainment of puberty in females. The timing of the onset of puberty is complex and can be altered by the levels or timing and pattern of release of controlling hormones from the hypothalamus and pituitary as well as direct effects on the ovaries (31). In most cases, developmental exposure to phytoestrogens has been reported to accelerate the time of

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TABLE 32.1 Effects of Developmental Phytoestrogen Treatments on Anogenital Distance (AGD) and Vaginal Opening in Female Rodents: Examples of Differences Resulting from Dose and Timing of Dosea Compound

Species/ Strain

Dose (route)

Timing of dose

AGD

AGD/Body weight

Vaginal opening

Reference

Flaxseed Flaxseed SDG Genistein Genistein Genistein Zearalanone Zearalanone Zearalanone

Rat/SD Rat/SD Rat/SD Rat/SD Rat/SD Rat/SD Mouse/ICR Mouse/ICR Mouse/ICR

5% (diet) 10% (diet) 1.5 mg/d(gavage)b 1000 µg/g (diet) 5 mg (sc) 25 mg (sc) 30 µg (ip) 20 µg, 30 µg (ip) 30 µg (ip)

GD 0–PND 21 GD 0–PND 21 GD 0–PND 21 GD 0–PND 56 GD 16–GD 20 GD16–GD 20 PND 10 PND 1–3 PND 1–5

No change Decreased No change NR Decreased No change NR NR NR

Increased Decreased Increased Increased NR NR NR NR NR

Delayed Accelerated Delayed Accelerated Delayed No change Accelerated Delayed Delayed

(27,32,33) (27,32,33) (27,33) (28) (30) (30) (34) (34) (34)

aAbbreviations: bDose

SD, Sprague-Dawley; GD, gestational day; PND postnatal day; SDG, secoisolariciresinol diglucoside; NR, not reported; sc, subcutaneous; ip, intraperitoneal. equivalent to the amount of SDG consumed per day in the 5% flaxseed diet.

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vaginal opening, although delays have also been observed (Table 32.1). Differences in the direction of this effect might be explained by differences in dose and/or timing of exposure. In the case of flaxseed, gestational and lactational exposure of Sprague-Dawley rats to a diet containing 5% flaxseed or a level of SDG equivalent to that present in 5% flaxseed resulted in a delayed vaginal opening, whereas 10% flaxseeed resulted in accelerated vaginal opening (27,32,33). Flaxseed contains potentially active ingredients other than lignans, but the fact that SDG was active, whereas flaxseed oil, which does not contain lignans, was not active argues that the lignans were responsible for the effects of flaxseed on the timing of puberty. Ito et al. (34) reported that zearalenone administered sc to ICR mice in single or multiple doses between birth and PND 10 resulted in an acceleration or delay in vaginal opening depending on the timing of exposure (Table 32.1). Levy et al. (30) administered genistein by sc injection to pregnant dams on GD 16–20 at 2 dose levels [5 and 25 mg, or ~12 and 60 mg/(kg⋅d)] and found a delay in vaginal opening only at the lower dose. Other studies in mice or rats with injected, gavage, or dietary genistein or with dietary SPI have generally reported an acceleration of vaginal opening or no effect (28,35–37). Similarly, coumestrol treatments either via the diet or sc injections to neonates or through gestation and lactation result in an acceleration of puberty (38–41). Thus, early exposure of female rodents to phytoestrogens can alter the timing of puberty. Puberty has been reported to be occurring earlier in girls in the United States, and although the reasons for and potential psychological effect of early puberty have not been definitively established (42,43), there could clearly be long-term health concerns given the association of lifetime estrogen exposure and breast cancer (44). Although this is an important end point to be considered in any human study of the health effects of phytoestrogens, no data exist at present indicating that phytoestrogens alter the timing of puberty in humans, and, in fact, existing data suggest that this end point is not altered in girls given soy formula (45). Just as in humans, many other factors in addition to exposure to endogenous estrogens can alter the timing of puberty in rodents, and factors in the diet other than phytoestrogens can exert effects on sexual development mediated through the central nervous system (46,47). Vaginal Cytology, Estrous Cycles, and Ovarian and Uterine Histology. High doses of estrogens or aromatizable androgens administered to female rodents in the perinatal period have long been known to induce infertility and persistent vaginal cornification in animals well before the time when nontreated animals would enter reproductive senescence (48,49). Several phytoestrogens have been shown to mimic more potent estrogens in inducing the anovulatory syndrome in rodents when administered via the diet or by injection (38–41,50–53). In the case of coumestrol and zearalanone, it has been shown that the persistent vaginal cornification produced by lower doses is dependent on the presence of ovaries (41,54), whereas higher doses were able to induce persistent cornification in ovariectomized animals (34,40,52), implying a direct effect of the compound on the vagina.

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Prolonged cycles and increased time in estrus have also been observed in animals that are still cycling (27,32,33,55,56). Low and high flaxseed diets were reported to have differing effects on the cycles of animals treated with 5 or 10% flaxseed during gestation and lactation, with acyclic animals in the 5% group showing persistent diestrus and acyclic animals in the 10% group showing persistent estrus (27). As with the effects of these diets on vaginal opening and AGD discussed above and on the mammary gland, discussed below, these cycle effects are consistent with an antiestrogenic effect of the low-dose diet and an estrogenic effect of the high-dose diet. Although the precise mechanisms involved in the antiestrogenic and estrogenic actions of the differing doses of flaxseed are not established, the 10% dietary dose increased circulating estrogen levels, whereas the 5% dose did not (32,33) As expected from the effects on the estrous cycle and vaginal cytology, altered ovarian histopathology has been observed in older animals after developmental treatment with phytoestrogens. Polyovular, hemorrhagic and atretic follicles, and decreased corpora lutea are among the effects observed in the ovaries after phytoestrogen treatment (40,41,52,55–57). Two recent reports on the ovarian toxicity of orally administered genistein are of particular interest because they suggest significant adverse effects on the ovary at relatively low doses, although this has not been a consistent finding in studies that have included an examination of the ovaries in genistein-treated animals (Table 32.2). Awoniyi et al. (57) treated pregnant Sprague-Dawley rats with dietary genistein at 5 µg/g feed (reported ingested dose of 50 µg/d) from GD 17 through weaning at PND 21 or through killing at PND 70. They observed atretic follicles, secondary interstitial glands, and cystic rete ovarii in genistein-exposed rats regardless of whether the exposure was terminated at PND 21 or PND 70 and suggested that this could indicate compromised fertility. Nagao et al. (58) gave genistein at 12.5–100 mg/(kg⋅d) by gavage and necropsied the rats at 21 d and 18 wk of age. In the older rats, all dose groups showed an increase of abnormal cycles and a decreased fertility index (number of rats pregnant/number of rats copulated). Some rats in the 50 or 100 mg/(kg⋅d) dose groups showed atrophic ovaries with atretic follicles and no corpora lutea, but lesions were not observed in the lower-dose groups. As noted by other authors (10,15,59,60), phytoestrogen (specifically, isoflavones) serum levels in rodents have been reported to be within ranges reported in humans despite ingestion of doses substantially higher than those reported for humans. For example, data from recent studies conducted at the National Center for Toxicological Research (35) indicated that dietary dose levels of 100–250 µg genistein/g feed result in ingested doses, depending on the stage of life, that are close to the 12.5 mg/kg body dose used by Nagao et al. (58), yet serum levels of genistein for the 100 µg/g feed dose level are in the range reported for humans consuming a soy diet with intakes closer to 1–2 mg/(kg⋅d) (59). The dietary dose of 5 µg/g feed was selected by Awoniyi et al. (57) to give an ingested level similar, on a mg/(kg body⋅d) basis, to that expected for humans consuming a normal soy-containing diet. However, this level of

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TABLE 32.2 Reported Reproductive Tract Effects of Genistein in Female Sprague-Dawley Rats: Examples of Variability in Responses Under Different Developmental Exposure Conditionsa Dose (route)

Timing of dose

Lowest effective dose

Observations

Reference

12.5, 25, 50, 100 mg/(kg⋅d) (gavage)

PND 1–5

12.5 mg/(kg⋅d)

Increase in abnormal cycles, decreased fertility index

(58)

12.5, 25, 50, 100 mg/(kg⋅d) (gavage)

PND 1–5

50 mg/(kg⋅d)

Atrophic ovaries, atretic follicles, absence of corpora lutea

(58)

5 µg/g (diet)

GD 17–PND 21

~50 µg/d

Atretic follicles, cystic rete ovary, or PND 70 increased secondary ovarian interstitial glands

(57)

500 mg/(kg⋅d) (sc)

PND 2, 4, 6

500 mg/(kg⋅d)

Atretic follicles, reduced corpora lutea

(55)

500 mg/(kg⋅d) (sc)

PND 16, 18, 20

500 mg/(kg⋅d)

No adverse effects in ovary, prolonged time in estrus phase of cycle

(36,56)

25 and 250 µg/g (diet)

GD 0–PND 21

—

No adverse effects

(13)

5, 25, 100, 250, 625, 1250 µg/g (diet)

GD 7–PND 50

Ovarian degeneration (cycle asynchrony observed at 625 µg/g feed)

(35)

aAbbreviations: bIntake

1250 µg/g feedb

PND postnatal day; GD, gestational day; sc, subcutaneous. varied from ~70 to 200 mg/(kg⋅d), depending on the life stage.

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dietary genistein would be expected to give extremely low serum and tissue levels of genistein in line with those found in humans consuming Western diets, which lack significant amounts of soy, and far lower than animals consuming standard laboratory diets (5,15). In addition, Thigpen et al. (61) and Brown and Setchell (15) pointed out the high levels of isoflavones present in soy-containing diets, and the latter authors reported high serum levels in newborns and adult animals consuming these diets. Brown and Setchell (15) found conjugates of equol, a metabolite of daidzein, to be the major isoflavone in serum of animals consuming soy meal, whereas levels of genistein and daidzein were considerably lower. Doerge et al. (5,62,63) showed that levels of genistein reached in the serum of animals consuming a soy-containing diet are identical to those fed similar levels of the aglycone genistein in the diet. Brown and Setchell (15) calculated that a lactating rat dam ingesting Purina 5001 chow is consuming 90–140 mg isoflavones/(kg body⋅ d). The Purina 5001 diet contains a particularly high isoflavone level (~600 µg/g feed) relative to most other commonly used rodent diets, but this example illustrates the fact that rats and mice consuming standard laboratory chows are consuming high levels of isoflavones, well within the range expected to produce biological effects. One important factor in comparing these exposures may be that the soycontaining diets contain soy meal, a product quite different from the more highly refined soy protein isolate (SPI) or soy extracts consumed in many human products or the pure aglycones used in many animal studies. In this regard, Pollard and colleagues (64,65) showed recently in adult Lobund-Wistar rats, a strain susceptible to spontaneous and chemically induced tumors of the prostate-seminal vesicle complex, that SPI protects against the development of tumors, whereas a diet containing soy meal with a slightly higher content of isoflavones did not provide protection. The authors suggest that the protective effect of the SPI is due to the estrogenic activity of the isoflavones and that soy meal contains an inhibitory activity not present in the isolate. Similar conclusions were reached by Hilakivi-Clarke et al. (66) in a mammary tumor model in rats. Helferich and colleagues (67–69) recently reported comparable stimulation of the growth of estrogen-responsive MCF-7 cells in vivo in athymic mice by genistein, or similar doses of isoflavone administered as genistin or SPI, but not soy flour. Taken together, these data suggest that serum levels of isoflavones, although providing an excellent measure of internal dose, may not predict response in all cases and that a greater understanding of the potential modulating effects of other components of the matrix in which isoflavones are administered is required. The conclusion that reproductive effects of isoflavones observed in test animals may be of little concern given the fact that these effects are not seen in animals consuming soy meal–containing diets may not be warranted for certain end points. In any case, the results of Awoniyi et al. (57) and Nagao et al. (58) concerning effects of genistein on female fertility at low doses after developmental exposure require further evaluation and replication. In recent short-term dose range finding studies of genistein administered in a soy-free diet to Sprague-Dawley rats from GD 7 to

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PND 50 at levels ranging from 5 to 1250 µg/g feed, ovarian toxicity similar to that seen in the above studies was seen only at the high dose level (35). Similarly, Fritz et al. (13) detected ovarian toxicity only at high doses of genistein and Kang et al. (70) reported no ovarian lesions with maternal exposures of 0.4 and 4 mg/(kg body ⋅d). Multigenerational and chronic toxicity studies being conducted under the auspices of the National Toxicology Program at the National Center for Toxicological Research are using dietary genistein at 5 µg/g feed, the dose used by Awoniyi et al. (57), as the low dose, and a wide range of end points, including ovarian histopathology, are currently being evaluated. Early and late effects on the uterus of mice and rats after treatments on PND 1–5, 1–10, or 10–14 (the time of uterine gland genesis in rats) with phytoestrogens have been reported. Both coumestrol and equol (100 µg/injection) increased uterine weight soon after treatment and induced premature development of uterine glands and a transient increase in lumenal epithelial cell height (71,72). Later depression of uterine weight was observed along with decreased estrogen receptor levels in coumestrol- but not equol-treated rats. When C57BL mice dosed with coumestrol on PND 1–5 were evaluated at 13 mo of age, doses ≥50 µg induced squamous metaplasia, but no uterine adenocarcinomas were seen (52). Newbold et al. (73) recently reported that genistein administered by sc injection on PND 1–5 at 50 mg/kg produced uterine adenocarcinomas in mice with an incidence similar to that of an equally estrogenic dose of DES. This dose resulted in low µmol/L serum levels of genistein (Doerge et al., unpublished data) and clearly demonstrates an adverse estrogenic effect of genistein after developmental exposure. Female Mammary Glands. One of the major forces driving phytoestrogen research is the potential modulating effects of phytoestrogens on estrogen-related cancers, particularly breast cancer. Although cancers, including breast cancer, are generally diseases of aging, there is compelling evidence suggesting that early and total lifetime hormone exposures are related to breast cancer risks. A series of studies from the laboratory of Lamartiniere (13,36,55,56) showed that neonatal or prepubertal exposure to genistein, either from high doses (500 mg/kg body) administered by sc injection or via the diet at 25 or 250 µg/g, can reduce the incidence of mammary cancer induced by subsequent treatment with DMBA. Reproductive toxicity was induced by the high-dose injection treatment (55), but this was dependent on the timing of the dose [neonatal treatment induced toxicity, prepubertal treatment did not (36)], and protection from mammary tumorigenesis was seen in the absence of toxicity. Hilakivi-Clarke et al. (74) also showed protection from DMBAinduced mammary cancer by prepubertal treatment with genistein or zearalenone (~1 mg/kg for both compounds). At this lower dose level, tumor multiplicity, rather than incidence, was depressed, and the tumors in the phytoestrogen-treated animals were not malignant, as they were in controls. This well-known and widely used animal model has provided many insights into the induction and modulation of mammary carcinogenesis. In this model, the timing of carcinogen exposure

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(~PND 50) is critical, presumably because proliferative activity in the gland increases the sensitivity to the carcinogen due to fixation of DNA damage. It has been determined that prepubertal exposure of the glands to genistein or other estrogen agonists accelerates the differentiation of the mammary glands, so that at the time of carcinogen treatment, the number of proliferating terminal end buds is decreased and the number of more differentiated lobules is increased. Lamartiniere’s group has demonstrated, using the high-dose prepubertal exposure regimen, that early stimulation (PND 21) of proliferation may be mediated, at least in part, by up-regulation of EGF receptors and of transforming growth factor-α (TGF-α) (75). This system is subsequently down-regulated in the mammary gland at the time of carcinogen exposure (75). Hilakivi-Clarke et al. (66) also indicated that selective changes in the levels of estrogen receptors α and β in the mammary gland, with estrogen receptor-α decreased and estrogen receptor-β increased, may be involved in the protective effects of phytoestrogens. The results achieved in this model appear to be highly dependent on the timing of exposure to both the carcinogen and the phytoestrogen, however. Hilakivi-Clarke et al. (76) found that in utero exposure to genistein increased tumor incidence when 20, 100, or 300 µg of genistein was administered by sc injection to dams on GD 15–20, although 20 µg of the more potent estrogen zearalenone was not effective. In mice, in utero exposure to genistein (sc injection, 20 µg on GD 15–20) increased the density of terminal end buds at PND 35 and 46, whereas similar treatment with 2 µg of zearalenone showed this effect at PND 35, but not at PND 46, when the gland showed an increase in differentiated structures (terminal ducts and alveolar buds) (53). The authors proposed that the difference between genistein and zearalenone may result from the fact that zearalanone produced a significant increase in persistent estrus, consistent with its higher estrogenic potency than genistein, and with the fact that it does not preferentially bind to estrogen receptor β as does genistein. Yang et al. (77) also found that in utero exposure to genistein combined with exposure to carcinogen at an earlier time when an increase in differentiation was not evident in genistein-treated rats, did not inhibit and in fact slightly enhanced tumor multiplicity. Thus, in rodent mammary cancer models, the timing of estrogen exposure during development can have a profound effect on the response to carcinogens. How well these developmental rodent models translate to the human situation, in which the nature and timing of carcinogen exposure is not clear, remains to be determined. In addition to the timing of the dose, the dose level can also alter the effect of phytoestrogens on the mammary gland. Tou et al. (33) demonstrated that prepubertal exposure to both 5 and 10% flaxseed decreased the number of terminal end buds in the mammary gland; however, the higher dose, but not the lower one, increased the number of alveolar buds (that is, increased differentiation). As with the observations on the effects of these flaxseed diets on AGD, vaginal opening, and estrous cycles discussed above, the authors suggested that the lower dose was acting through an antiestrogenic mechanism, whereas the higher dose acted as an estrogenic stimulus.

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Effects of Developmental Exposure to Phytoestrogens on Males Early Sexual Development and Puberty in Males. Although the study of the effects of phytoestrogens on females continues to be an important research concern, there has been an increasing appreciation of the importance of estrogens in male reproductive physiology; this has been reflected in an increased interest in the possible effects of phytoestrogen exposure on end points in males. Although several studies have looked at anogenital distance as an early marker of feminization or masculinization and/or at the attainment of puberty, as assessed by the time or body weight of balanopreputial separation (78) or by the age at testicular descent, there have been few reports of significant effects on these end points. In fact, there appear to be no published reports in which phytoestrogens have altered the time of male puberty in rodent models. Flaxseed at 10% in the diet was reported to decrease (i.e., feminize) AGD in newborn males, but body weight was also lower and there was no significant effect on the AGD adjusted for body weight (27). Levy et al. (30) reported that 5 mg (but not 25 mg) of genistein administered to dams from GD 16–20 by sc injection decreased the AGD in newborn males. On the other hand, it was reported that male pups of dams receiving a diet containing ~600 µg soy phytoestrogens/g feed throughout gestation increased the ratio of anogenital distance to body weight in male pups just before birth at GD 20.5, although no significant difference was observed on PND 3 (29). A diet containing 200 µg soy phytoestrogens/g feed had no effect. The long term biological meaning of these small and sometimes transient effects on anogenital distance is not clear. Prostate. The prostate is of considerable interest because it is a major site of morbidity and mortality in men and because of the association of soy consumption with lower risk of development of advanced prostate cancer in epidemiologic studies. Several studies involving developmental exposure to phytoestrogens have shown effects on the prostate, although clear-cut conclusions on the effects of developmental exposures to phytoestrogens on the prostate are not yet possible. In the case of flaxseed, Tou et al. (32) reported that exposure of Sprague-Dawley rats through gestation and lactation reduced or elevated prostate weight and stimulated or inhibited prostate growth depending on dose, with 10% stimulatory and 5% inhibitory. These effects were dependent on developmental exposure because exposures limited to the postweaning period had no effect. On the other hand, Sprando et al. (79) treated rats continuously through gestation and lactation and after weaning until PND 70 with 20 or 40% flaxseed or 13 or 26% flaxmeal (contains lignans but has most lipids, including α-linolenic acid, removed) and found decreased prostate weights. Awoniyi et al. (80) found that daily sc administration of 100 µg coumestrol on PND 1–5, a treatment that had substantial effects on female pups, had no effect on prostate or spermatogenesis in male rats. Males exposed from GD 7 through PND 50 to dietary genistein ranging from 5 to 1250 µg/g feed showed prostate effects only at the highest dose; at that dose, ventral

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prostate weight and secretory material were reduced and the dorsolateral prostate showed increased inflammation (35). Lund et al. (81) reported that rats exposed to a soy diet containing 600 µg/g total phytoestrogens through gestation and throughout life had decreased prostate weight relative to a phytoestrogen-free diet. Several studies involving true “development only” exposures to phytoestrogens have reported prostate data. Nagao et al. (58) did not observe changes in the rat adult ventral prostate weight, the only prostate lobe examined, at doses between 12.5 and 100 mg/(kg⋅d) on PND 1–5. Strauss et al. (82) found that although ventral prostate weight was reduced in NMRI mice injected with 50 or 500 mg/(kg⋅d) on PND 1–3, hyperplasia and abnormal prostate histology, similar to that produced by neonatal treatment with potent estrogens, was observed only in the high-dose mice as adults. On the basis of parallel studies with adult male mice, the authors of the last-mentioned study concluded that neonates were much less sensitive than adults to the estrogen-like actions of genistein and that adverse effects on the prostate in neonates did not occur at doses likely to be encountered through the diet. Kang et al. (70) dosed dams by gavage with 0.4 or 4 mg/(kg body ⋅ d) from GD 6 through PND 20 and examined the F1 offspring at several ages. The prostate weight was significantly increased in the 4 mg/(kg⋅d) dose group at PND 70, but not on PND 100, indicating a transient effect. These investigators chose these dose levels to approximate human intake levels. The 4 mg/(kg⋅d) dose approximates the doses consumed by pregnant and lactating dams receiving 25–100 µg/g genistein in the diet (35); under conditions of continuous dietary exposure, the serum levels of genistein in animals consuming 100 µg/g genistein approximated those reported in humans consuming an Asian soy-containing diet (59). Taken together with several additional studies (28,37,47,70) that used soy meal, SPI, or genistein as the test agents during development and reported no adverse effects on prostate weight and/or histology, it can be concluded at the present time that phytoestrogens can affect the prostate under certain developmental treatment conditions, particularly at the higher end of the tested dose range. Long-term consequences or benefits have yet to be demonstrated. Testes and Spermatogenesis. Another focus of attention, related to the increasing interest in the role of estrogens in spermatogenesis and the hypothesis that environmental estrogen exposure may lead to decreased sperm counts, has been the process of spermatogenesis. Awoniyi et al. (80) reported no effects on testes weight or sperm counts after administration of 100 µg coumestrol by sc injection on PND 1–5. Roberts et al. (83) found no effect of 5 µg/g dietary genistein administered from GD 17 through PND 21, PND 70, or PND 130, with all animals necropsied at PND 70 or 130, and Nagao et al. (58) found no effect on sperm counts or serum testosterone after neonatal gavage administration of genistein at doses ranging from 12.5 to 100 mg/(kg ⋅ d). Sprando et al. (79,84) found minor effects on serum luteinizing hormone (LH), cauda epididymal weight, and cauda sperm per gram tissue in rats fed flaxseed diets (20 and 40%) during pregnancy

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until necropsy at PND 70 and minor structural effects on the testes that were not considered to be adverse. On the other hand, Fisher et al. (85) reported increased testes weight at PND 75 in rats given sc injections of 4 mg genistein/kg body between PND 2 and 12. A transient reduction in efferent duct epithelial cell height was observed in early life and was abolished by PND 25. We also reported histological evidence for delayed spermatogenesis in animals exposed to dietary genistein at 1250 µg/g from GD 7 through PND 50, although testicular spermitid head counts did not differ from controls (35). Additionally, Atanassova et al. (86) found that both a soy-containing diet and genistein administered to neonates by sc injection at 4 mg/(kg⋅d) retarded spermatogenesis. On the other hand, Kang et al. (70) found no effects on sperm number or the distribution of cell types or their numbers in adult rats whose mothers were exposed to 0.4 or 4 mg/(kg body⋅ d) by gavage from GD 6 to parturition and then from PND 2 to PND 20. A preliminary evaluation of data from a multigenerational study involving dietary administration of genistein at doses of 5, 100, and 500 µg/g feed from conception to PND 140 or PND 21 suggests no significant effects on sperm motility, morphology, or counts in animals necropsied at PND 140 (Delclos et al., unpublished data). Thus, the data available to this point show equivocal evidence for the effect of developmental exposure to soy or flaxseed phytoestrogens on spermatogenesis, and adverse reproductive outcomes or other long-term testicular toxicity have not been demonstrated. Male Mammary Glands. Although there have been reports that male mammary glands may be sensitive indicators of exposure to estrogenic agents and agents that alter dopamine release (87), little attention has been paid to the effects of phytoestrogens on the development of the male mammary gland. This can be attributed largely to the fact that much of the interest in phytoestrogens revolves around the modulation of mammary carcinogenesis, which is a relatively rare disease in human males. Feeding of genistein to Sprague-Dawley rats from GD 7 through PND 50 at doses of 250–1250 µg/g resulted in hyperplasia and hypertrophy of ductal and alveolar mammary glands in male pups evaluated at PND 50 (35). The pubertal age of the animals at killing and the exposure regimen leave open the question of the long-term nature and consequences of this effect and whether continuous exposure is required to elicit this response, but on-going studies that include chronic assessment of animals dosed continuously and through weaning cover this dose range and will address these questions. Effects of Developmental Exposures to Phytoestrogens Outside the Reproductive Tract and Mammary Glands in Both Sexes Estrogens are known to affect multiple organs outside of the reproductive tract and mammary glands, some of which, such as the central nervous system and pituitary, have direct effects on reproductive behavior and function; others, such as bone, do not. These end points are increasingly being included in evaluations of develop-

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mental exposures to phytoestrogens, although relatively few data that pertain to the body of information on reproductive organ effects are available. Recent data from a series of short-term studies conducted with genistein suggest that some of the nonreproductive end points may be more sensitive (i.e., show effects at lower doses) than the reproductive end points (Table 32.3). Neuroendocrine, Neuroanatomical, and Behavioral Effects. Neuroendocrine effects of genistein, zearalenone, coumestrol, and flaxseed have been reported. Dietary flaxseed at 20 and 40% and 26% flaxmeal were reported to increase serum LH in males, but the associated reproductive effects were considered to be negligible TABLE 32.3 Summary of Effects of Dietary Genistein on Sprague-Dawley Rats in a Series of Short-Term Studiesa,b Lowest dose tested Sex

End point/effect

M/F M/F M M/F M M M/F M/F F F M F F

Increased anti-CD3–mediated splenocyte proliferation Altered spleen T- and B-cell populations Decreased bone marrow subpopulations Decreased thyroid peroxidase activityc Mammary gland hypertrophy and hyperplasia Decreased volume of SDN-POA Mineralization of renal tubules Increased hepatic 5α-reductase Mammary gland hyperplasia Vaginal dyssynchrony Epididymis, hypospermia Ovarian degeneration Increased spleen IgM-forming cell response to T-cell–dependent antigen Decreased body weight Increased consumption of salt-flavored solution Inflammation, dorsolateral prostate Decreased ventral prostate weight and secretory material Depletion and retention of elongated spermatids Decreased hepatic cytochrome P450 isozymes

M/F M/F M M M M

Lowest effective dose

(µg/g feed)

Reference

25 25 25 25 5 5 5 25 5 5 5 5 25

25 25 25 25 25 25 250 250 625 625 625 1250 1250

(114) (114) (114) (114) (35) (92) (35) (111) (35) (35) (35) (35) (114)

5 25 5 5 5 25

1250 1250 1250 1250 1250 1250

(35,95) (95) (35) (35) (35) (111)

aThe studies summarized in this table utilized three separate sets of Sprague-Dawley rats from the NCTR breeding colony. Pregnant rats were started on dose in all cases at gestational day (GD) 7. Dosing was continued throughout pregnancy and lactation, and the pups were continued on dosed feed until killing. Studies reported in References 35, 92 and 110 utilized doses of 5, 25, 100, 250, 625, and 1250 µg/g and were terminated on postnatal day (PND) 50. Studies reported in References 94 and 113 utilized doses of 25, 250, and 1250 µg/g feed and were terminated at PND 63 and 77, respectively. bAbbreviations: SDB-POA, sexually dimorphic nucleus of the medial preoptic area; Ig, immunoglobulin. cLater studies conducted on rats exposed to dietary genistein from conception to PND 140 found that thyroid peroxidase activity was significantly lower than control levels in females at the 5 µg/g dose level (5).

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(79,84). Faber and Hughes (88) administered sc injections of genistein to SpragueDawley rats from PND 1 to 10 and later examined the response (LH release) of rats of both sexes gonadectomized at PND 21 and challenged with GnRH on PND 42. Both sexes showed an increased GnRH-stimulated LH release at the low dose tested (100 µg, or ~10–20 mg/kg body) and a decreased response at the high dose (1000 µg). Zeralenone decreased the response at both the high and low doses, which were the same as the doses of genistein. The high dose of genistein also masculinized (increased) the volume of the sexually dimorphic nucleus (SDN) of the hypothalamus in females, but had no effect on that of males. This nucleus is one of several that have been identified to show differences in males and females and whose development can be altered by perinatal treatment with estrogens or aromatizable androgens. Alterations in the size of this nucleus have been associated with changes in sexual behavior in rodents (89). In a subsequent study in which only females were examined over a wider dose range, an increased response to GnRH was found at a dose of 10 µg and a decreased response was found at the high dose of 1000 µg (90). The sexually dimorphic nucleus of the medial preoptic area (SDN-POA) was increased at both 500 and 1000 µg. When pregnant dams were administered doses of 5 or 25 mg of genistein on GD 16–20, no effect on the GnRH responsiveness or the SDN-POA of the rats was noted. It is not clear whether this was due to the time of administration or the dose of genistein that reached the pups in utero. This same group also conducted similar experiments with coumestrol, giving sc injections of 0.1–10 µg on PND 1–10 to Sprague-Dawley pups (91). Basal LH was elevated at all dose levels in females and at the 1 and 10 µg levels in males, whereas the high dose reduced the response to GnRH. No effect on the SDN-POA relative to controls was noted, although the high dose gave a significantly increased volume relative to the two lower doses. It should be noted that DES at a dose that had been shown to increase the volume of the SDN-POA in females in a previous study failed to alter the volume in this study. Meredith et al. (92,93) reported that intermediate doses of dietary genistein (25, 100, and 250 µg/g feed) administered from GD 7 through PND 50 reduced (i.e., feminized) the volume of the SDN-POA in males, whereas higher doses (625 and 1250 µg/g feed) had no effect. This suggested the possibility of differing mechanisms at low and high doses, perhaps with effects on testosterone levels or aromatase at intermediate doses and direct estrogenic effects at the higher doses. Although these results are potentially interesting, it should be noted that measurements of this end point have been variable both between and within research groups (94; Scallet, A.C., personal communication). Therefore, in the absence of corresponding effects on reproductive parameters, the effects on SDNPOA volume should be interpreted with caution. Whitten et al. (38) found a blunted LH response to progesterone challenge in female rats whose mothers were exposed to 100 µg/g coumestrol from PND 1 to 10. Males did not show hormonal effects but did show behavioral effects with increased latencies to mount and ejaculate and a decreased mount and ejaculation rate (39). These studies indicate that phytoestrogens can affect the perinatal

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organization of the brain and reproductive system and produce lasting effects on reproductive behavior. In addition to effects on reproductive behavior, Flynn et al. (95) examined the effects of dietary genistein on other sexually dimorphic behaviors (open field activity, play behavior, running wheel activity, and consumption of saccharin- and sodium chloride–flavored solutions) in Sprague-Dawley rats. All of these behaviors have been shown to be sexually dimorphic and can be altered by interfering with sex hormones during the time of central nervous system development. At the highest dietary dose tested, 1250 µg/g feed, rats of both sexes consumed more salt solution than controls, an effect that was not due to an overall increase in fluid consumption because intake of plain water was not altered. This same effect was seen with three other estrogenic agents tested, and suggests that genistein was able to feminize this behavior in males and hyperfeminize the behavior in females, although the extent to which this effect was induced during the organization of the central nervous system could not be determined from the study design used. Bone. Most studies involving estrogens, including phytoestrogens, and bone end points have focused on changes that occur in bone after the attainment of peak bone mass, using the ovariectomized rat as a model of menopause. However, the peak bone mass achieved in younger animals is a major determinant of risk for later osteoporosis. Migliaccio et al. (96,97) determined that perinatal treatment of mice with DES induces changes in bone development with effects that last into adulthood, including a sustained increase in mineral apposition rate and decrease in osteoclast activity. As adults, the DES-treated mice had shorter bones with a higher bone mineral density. Fukazawa et al. (98) also reported that neonatal exposure to DES resulted in shorter bones, but reported decreased osteoblast numbers, implying decreased bone formation. On the basis of the confirmed developmental effects of DES, it is possible that environmental estrogens may affect bone development, although few studies have addressed this to this point. In a two-generation study of zearalenone, which included exposure through gestation and lactation, Becci et al. (99) noted increased trabeculation of the femur at the highest dose of 10 mg/(kg⋅d) as the only observed histological effect. Transient effects of flaxseed diets on the development of male and female rodents have been reported, with increased strength, as measured by resistance to bending, in female rats and decreased strength in male rats examined at 50 d of age, but no differences noted between treated and control animals in adulthood (100,101). Furthermore, these differences did not appear to be due to the estrogenic lignan component of flaxseed because treatment with equivalent doses of SDG did not show the differences observed with flaxseed. Given the high exposure of some infants to soy phytoestrogens, additional targeted studies on potential beneficial and adverse long-term effects of developmental phytoestrogen exposures on bone seem warranted. Kidney. Phytoestrogens have been reported to have beneficial effects on kidney function (102). In our feeding studies involving exposure of rats from GD 7 through PND 50, dietary genistein at ≥250 µg/g feed induced a significant increase

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in the incidence and/or severity of nephrocalcinosis, or mineralization of the renal tubules, in both sexes (35). This is a sex-related lesion common in untreated female rats and influenced by diet composition (103). Nephrocalcinosis is rare in untreated male rats and has been reported to be induced by estrogen treatment in males (103). We observed a treatment-related increase in nephrocalcinosis in males with ethinyl estradiol (Delclos et al., unpublished) and the weakly estrogenic nonylphenol (104), suggesting that this may be an effect related to estrogenic activity. This end point has not been evaluated with other phytoestrogens, and the extent to which developmental exposure is important to the observed effect and the long-term consequences, if any, of this mild mineralization, are under investigation. Hepatic Enzymes and Estrogen Receptors. In rats, certain cytochromes P450 are expressed in a sex-specific fashion, which results in a sexually dimorphic pattern of steroid metabolism (105,106). Steroid 5α-reductase, the enzyme responsible for converting testosterone to dihydrotestosterone, is also expressed at a higher level in female liver than in male liver (107,108). The expression of these sex-specific enzymes is developmentally regulated, and neonatal imprinting appears to be due to aromatization of testosterone to estradiol in the brain and subsequent effects on the growth hormone secretion pattern (109). Similarly, estrogens can alter estrogen receptor expression in the liver through the hypothalamic-pituitary axis via growth hormone (110). Laurenzana et al. (111) demonstrated effects of dietary genistein at 250–1250 µg/g feed from GD 7 through PND 50 on hepatic testosterone hydroxylase activities, 5α-reductase, and estrogen receptors, although the alterations could not be attributed to the estrogenic activity of genistein and did not appear to alter circulating testosterone levels. Ronis et al. (112) examined the effects of a soy diet containing ~400 µg/g isoflavones from conception to adulthood on the expression, activity, and inducibility of members of the cytochrome P450 1A family, enzymes important in carcinogen metabolism. Although no effects on basal expression or activity of the enzymes were observed, the inducibility by 3-methylcholanthrene or isosafrole was lower in the soy-fed rats than in those fed casein or whey protein. These studies did not examine the effect of developmental exposure alone on hepatic enzyme and estrogen receptor expression, but they suggest that the possibility of imprinted effects on the liver, which could alter later responses to endogenous or exogenous agents, should be considered in future studies. Immune System. Estrogens are known to influence the immune system, and developmental exposure to potent estrogens such as DES has been associated with adverse effects on the immune system in rodents and humans (2,113). Few data exist on the effects of developmental exposures to phytoestrogens on later immune responses. A battery of assays to assess the immune response in Sprague-Dawley rats administered genistein at doses of 25, 250, and 1250 µg/g feed in the diet from GD 7 through PND 77 was conducted (114). In addition to the pups, the dams were also treated for 65 d and assayed. The pups of both sexes, but not the dams

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treated only as adults, showed enhanced T-cell activity in all dose groups as measured by anti-CD3 (a T-cell receptor) antibody-mediated spleen cell proliferation. The biological consequences of this apparent developmental effect of enhanced cell-mediated immunity are not clear; they could be beneficial or harmful (by enhancing autoimmune responses) depending on the host environment. Changes in B- and T-cell populations and myelotoxicity were also seen in the pups, but not the dams, and these effects varied according to dose and sex (summarized in Table 32.3).

Summary and Conclusions Although there are some inconsistencies in the available data, rodent studies indicate that developmental exposure to phytoestrogens or phytoestrogen-containing foods can alter multiple end points in both sexes in multiple organ systems ranging from the reproductive system, for which considerable data have been generated, to the immune system, which is only beginning to be examined. The magnitude and direction of the responses are dependent on the compound, the dose, and the timing of the dose. Other experimental factors, such as the test animal, route of administration, and base diet may also affect the outcome of studies and complicate the comparison of studies across laboratories. Many, but not all of the observed effects are consistent with the conclusion that the interaction of the compounds with estrogen receptors is an important component of the mechanism of action. Although in some cases the result of phytoestrogen exposure is clearly beneficial or adverse, in other cases, the consequences are not clear and require long-term study for further definition of the dose response, critical windows of exposure, and determination of the transient or permanent nature of the effect. Even more problematic is the extrapolation of the observed effects to humans. It is well known that timing of exposure during the developmental process is critical, and that these critical exposure periods differ for rodents and humans. For developing humans, exposure to phytoestrogens can occur in utero, a time period that is well modeled in rodents. However, the time of highest exposure to phytoestrogens is the postnatal period, a time in which development of multiple systems continues, but one that is not well modeled in rodents. The phytoestrogens are potentially important weapons in the battle against a range of human diseases, but it is important to determine in targeted and carefully controlled human studies whether potentially adverse effects in developmental and adult exposure rodent studies are in fact cause for concern. Acknowledgments The work from the series of studies summarized in Table 32.3, which is also discussed throughout the text, was supported by Interagency Agreement 224-93-0001 between the National Institute for Environmental Health Sciences and the United States Food and Drug Administration and NTP Contract ES 55094 with Virginia Commonwealth University (immunotoxicology work described in Ref. 114). The opinions expressed in the manuscript reflect those of the author and do not necessarily reflect the policies of the U.S. FDA.

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66. Hilakivi-Clarke, L., Cho, E., deAssis, S., Olivo, S., Ealley, E., Bouker, K.B., Welch, J.N., Khan, G., Clarke, R., and Cabanes, A. (2001) Maternal and Prepubertal Diet, Mammary Development and Breast Cancer Risk, J. Nutr. 131, 154S–157S. 67. Allred, C.D., Allred, K.F., Ju, Y.H., Virant, S.M., and Helferich, W.G. (2001) Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of EstrogenDependent (MCF-7) Tumors in a Dose-Dependent Manner, Cancer Res. 61, 5045–5050. 68. Allred, C.D., Ju, Y.H., Allred, K.F., Chang, J., and Helferich, W.G. (2001) Dietary Genistin Stimulates Growth of Estrogen-Dependent Breast Cancer Tumors Similar to That Observed with Genistein, Carcinogenesis 22, 1667–1673. 69. Hsieh, C.Y., Santell, R.C., Haslam, S.Z., and Helferich, W.G. (1998) Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-Positive Human Breast Cancer (MCF-7) Cells In Vitro and In Vivo, Cancer Res. 58, 3833–3838. 70. Kang, K.S., Che, J.H., and Lee, Y.S. (2002) Lack of Adverse Effects in the F1 Offspring Maternally Exposed to Genistein at Human Intake Dose Level, Food Chem. Toxicol. 40, 43–51. 71. Medlock, K.L., Branham, W.S., and Sheehan, D.M. (1995) Effects of Coumestrol and Equol on the Developing Reproductive Tract of the Rat, Exp. Biol. Med. 208, 67–71. 72. Medlock, K.L., Branham, W.S., and Sheehan, D.M. (1995) The Effects of Phytoestrogens on Neonatal Rat Uterine Growth and Development, Exp. Biol. Med. 208, 307–313. 73. Newbold, R.R., Banks, E.P., Bullock, B., and Jefferson, W.N. (2001) Uterine Adenocarcinoma in Mice Treated Neonatally with Genistein, Cancer Res. 61, 4325–4328. 74. Hilakivi-Clarke, L., Onojafe, I., Raygada, M., Cho, E., Skaar, T., Russo, I., and Clarke, R. (1999) Prepubertal Exposure to Zearalenone or Genistein Reduces Mammary Tumorigenesis, Br. J. Cancer 80, 1682–1688. 75. Brown, N.M., Wang, J., Cotroneo, M.S., Zhao, Y.X., and Lamartiniere, C.A. (1998) Prepubertal Genistein Treatment Modulates TGF-, EGF and EGF-Receptor mRNAs and Proteins in the Rat Mammary Gland, Mol. Cell. Endocrinol. 144, 149–165. 76. Hilakivi-Clarke, L., Cho, E., Onojafe, I., Raygada, M., and Clarke, R. (1999) Maternal Exposure to Genistein During Pregnancy Increases Carcinogen-Induced Mammary Tumorigenesis in Female Rat Offspring, Oncol. Rep. 6, 1089–1095. 77. Yang, J., Nakagawa, H., Tsuta, K., and Tsubura, A. (2000) Influence of Perinatal Genistein Exposure on the Development of MNU-Induced Mammary Carcinoma in Female Sprague-Dawley Rats, Cancer Lett. 149, 171–179. 78. Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. (1977) Preputial Separation as an External Sign of Pubertal Development in the Male Rat, Biol. Reprod. 17, 298–303. 79. Sprando, R.L., Collins, T.F., Black, T.N., Olejnik, N., Rorie, J.I., Scott, M., Wiesenfeld, P., Babu, U.S., and O’Donnell, M. (2000) The Effect of Maternal Exposure to Flaxseed on Spermatogenesis in F(1) Generation Rats, Food Chem. Toxicol. 38, 325–334. 80. Awoniyi, C.A., Roberts, D., Chandrashekar, V., Veeramachaneni, D.N., Hurst, B.S., Tucker, K.E., and Schlaff, W.D. (1997) Neonatal Exposure to Coumestrol, a Phytoestrogen, Does Not Alter Spermatogenic Potential in Rats, Endocrine 7, 337–341. 81. Lund, T.D., Rhees, R.W., Setchell, K.D.R., and E.D., L. (2001) Altered Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) Volume in Adult Long-Evans Rats by Dietary Soy Phytoestrogens, Brain Res. 914, 92–99.

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82. Strauss, L., Makela, S., Joshi, S., Huhtaniemi, I., and Santti, R. (1998) Genistein Exerts Estrogen-Like Effects in Male Mouse Reproductive Tract, Mol. Cell. Endocrinol. 144, 83–93. 83. Roberts, D., Veeramachaneni, D.N., Schlaff, W.D., and Awoniyi, C.A. (2000) Effects of Chronic Dietary Exposure to Genistein, a Phytoestrogen, During Various Stages of Development on Reproductive Hormones and Spermatogenesis in Rats, Endocrine 13, 281–286. 84. Sprando, R.L., Collins, T.F., Wiesenfeld, P., Babu, U.S., Rees, C., Black, T., Olejnik, N., and Rorie, J. (2000) Testing the Potential of Flaxseed to Affect Spermatogenesis: Morphometry, Food Chem. Toxicol. 38, 887–892. 85. Fisher, J.S., Turner, K.J., Brown, D., and Sharpe, R.M. (1999) Effect of Neonatal Exposure to Estrogenic Compounds on Development of the Excurrent Ducts of the Rat Testis Through Puberty to Adulthood, Environ. Health Perspect. 107, 397–405. 86. Atanassova, N., McKinnell, C., Turner, K.J., Walker, M., Fisher, J.S., Morley, M., Millar, M.R., Groome, N.P., and Sharpe, R.M. (2000) Comparative Effects of Neonatal Exposure of Male Rats to Potent and Weak (Environmental) Estrogens on Spermatogenesis at Puberty and the Relationship to Adult Testis Size and Fertility: Evidence for Stimulatory Effects of Low Estrogen Levels, Endocrinology 141, 3898–3907. 87. Cardy, R.H. (1991) Sexual Dimorphism of the Normal Rat Mammary Gland, Vet. Pathol. 28, 139–145. 88. Faber, K.A., and Hughes, C.L. (1991) The Effect of Neonatal Exposure to Diethylstilbestrol, Genistein, and Zearalenone on Pituitary Responsiveness and Sexually Dimorphic Nucleus Volume in the Castrated Adult Rat, Biol. Reprod. 45, 649–653. 89. Rhees, R.W., Al-Saleh, H.N., Kinghorn, E.W., Fleming, D.E., and Lephart, E.D. (1999) Relationship Between Sexual Behavior and Sexually Dimorphic Structures in the Anterior Hypothalamus in Control and Prenatally Stressed Male Rats, Brain Res. Bull. 50, 193–199. 90. Faber, K.A., and Hughes, C.L. (1993) Dose-Response Characteristics of Neonatal Exposure to Genistein on Pituitary Responsiveness to Gonadotropin Releasing Hormone and Volume of the Sexually Dimorphic Nucleus of the Preoptic Area (SDNPOA) In Postpubertal Castrated Female Rats, Reprod. Toxicol. 7, 35–39. 91. Register, B., Bethel, M.A., Thompson, N., Walmer, D., Blohm, P., Ayyash, L., and Hughes, C. (1995) The Effect of Neonatal Exposure to Diethylstilbestrol, Coumestrol, and Beta-Sitosterol on Pituitary Responsiveness and Sexually Dimorphic Nucleus Volume in the Castrated Adult Rat, Exp. Biol. Med. 208, 72–77. 92. Meredith, J.M., Bennett, C., Delclos, K., Weis, C., Newbold, R., and Scallet, A.C. (2000) Ethinyl Estradiol and Genistein, but Not Vinclozolin, Decrease the Volume of the SDN-POA in Male Rats, Soc. Neurosci. Abstr. 26, 1369 (abstr.). 93. Ferguson, S.A., Scallet, A.C., Flynn, K.M., Meredith, J.M., and Schwetz, B.A. (2000) Developmental Neurotoxicity of Endocrine Disrupters: Focus on Estrogens, Neurotoxicology 21, 947–956. 94. Meredith, J.M., Bennett, C., and Scallet, A.C. (2001) A Practical Three-Dimensional Reconstruction Method to Measure the Volume of the Sexually-Dimorphic Central Nucleus of the Medial Preoptic Area (MPOC) of the Rat Hypothalamus, J. Neurosci. Methods 104, 113–121. 95. Flynn, K.M., Ferguson, S.A., Delclos, K.B., and Newbold, R.R. (2000) Effects of Genistein Exposure on Sexually Dimorphic Behaviors in Rats, Toxicol. Sci. 55, 311–319.

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96. Migliaccio, S., Newbold, R.R., Bullock, B.C., McLachlan, J.A., and Korach, K.S. (1992) Developmental Exposure to Estrogens Induces Persistent Changes in Skeletal Tissue, Endocrinology 130, 1756–1758. 97. Migliaccio, S., Newbold, R.R., Teti, A., Jefferson, W.J., Toverud, S.U., Taranta, A., Bullock, B.C., Suggs, C.A., Spera, G., and Korach, K.S. (2000) Transient Estrogen Exposure of Female Mice During Early Development Permanently Affects Osteoclastogenesis in Adulthood, Bone 27, 47–52. 98. Fukazawa, Y., Nobata, S., Katoh, M., Tanaka, M., Kobayashi, S., Ohta, Y., Hayashi, Y., and Iguchi, T. (1996) Effect of Neonatal Exposure to Diethylstilbestrol and Tamoxifen on Pelvis and Femur in Male Mice, Anat. Rec. 244, 416–422. 99. Becci, P.J., Johnson, W.D., Hess, F.G., Gallo, M.A., Parent, R.A., and Taylor, J.M. (1982) Combined Two-Generation Reproduction-Teratogenesis Study of Zearalenone in the Rat, J. Appl. Toxicol. 2, 201–206. 100. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Flaxseed and Its Purified Lignan Reduces Bone Strength in Young but Not Older Male Rats, J. Toxicol. Environ. Health. Part A 63, 53–65. 101. Ward, W.E., Yuan, Y.V., Cheung, A.M., and Thompson, L.U. (2001) Exposure to Purified Lignan from Flaxseed (Linum usitatissimum) Alters Bone Development in Female Rats, Br. J. Nutr. 86, 499–505. 102. Velasquez, M.T., and Bhathena, S.J. (2001) Dietary Phytoestrogens: A Possible Role in Renal Disease Protection, Am. J. Kidney Dis. 37, 1056–1068. 103. Ritskes-Hoitinga, J., and Beynen, A.C. (1992) Nephrocalcinosis in the Rat: A Literature Review, Prog. Food Nutr. Sci. 16, 85–124. 104. Latendresse, J.R., Newbold, R.R., Weis, C.C., and Delclos, K.B. (2001) Polycystic Kidney Disease Induced in F(1) Sprague-Dawley Rats Fed para-Nonylphenol in a Soy-Free, Casein-Containing Diet, Toxicol. Sci. 62, 140–147. 105. Kamataki, T., Maeda, K., Yamazoe, Y., Nagai, T., and Kato, R. (1983) Sex Difference of Cytochrome P-450 in the Rat: Purification, Characterization, and Quantitation of Constitutive Forms of Cytochrome P-450 from Liver Microsomes of Male and Female Rats, Arch. Biochem. Biophys. 225, 758–770. 106. Waxman, D.J., Ko, A., and Walsh, C. (1983) Regioselectivity and Stereoselectivity of Androgen Hydroxylations Catalyzed by Cytochrome P-450 Isozymes Purified from Phenobarbital-Induced Rat Liver, J. Biol. Chem. 258, 11937–11947. 107. Pak, R.C., Tsim, K.W., and Cheng, C.H. (1985) The Role of Neonatal and Pubertal Gonadal Hormones in Regulating the Sex Dependence of the Hepatic Microsomal Testosterone 5-Reductase Activity in the Rat, J. Endocrinol. 106, 71–79. 108. Dannan, G.A., Guengerich, F.P., and Waxman, D.J. (1986) Hormonal Regulation of Rat Liver Microsomal Enzymes. Role of Gonadal Steroids in Programming, Maintenance, and Suppression of Delta 4-Steroid 5 Alpha-Reductase, Flavin-Containing Monooxygenase, and Sex-Specific Cytochromes P-450, J. Biol. Chem. 261, 10728– 10735. 109. Lund, J., Zaphiropoulos, P.G., Mode, A., Warner, M., and Gustafsson, J.A. (1991) Hormonal Regulation of Cytochrome P-450 Gene Expression, Adv. Pharmacol. 22, 325–354. 110. Sahlin, L. (1995) Dexamethasone Attenuates the Estradiol-Induced Increase of IGF-I mRNA in the Rat Uterus, J. Steroid Biochem. Mol. Biol. 55, 9–15. 111. Laurenzana, E.M., Weis, C., Bryant, C., Newbold, R.R., and Delclos, K. (2002) Effect of Dietary Administration of Genistein, Nonylphenol or Ethinyl Estradiol on Hepatic Testosterone Metabolism, Cytochrome P-450 Enzymes, and Estrogen Receptor Alpha Expression, Food Chem. Toxicol. 40, 117–127.

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112. Ronis, M.J., Rowlands, J.C., Hakkak, R., and Badger, T.M. (2001) Inducibility of Hepatic CYP1A Enzymes by 3-Methylcholanthrene and Isosafrole Differs in Male Rats Fed Diets Containing Casein, Soy Protein Isolate or Whey from Conception to Adulthood, J. Nutr. 131, 1180–1188. 113. Holladay, S.D., and Smialowicz, R.J. (2000) Development of the Murine and Human Immune System: Differential Effects of Immunotoxicants Depend on Time of Exposure, Environ. Health Perspect. 108, 463–473. 114. National Toxicology Program (1998) Final Report, Protocol E2122.14: Immunotoxicity of Genistein in Male and Female Sprague-Dawley Rats, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

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Chapter 32

The Health Consequences of Soy Infant Formula, Soy Protein Isolate, and Isoflavones Thomas M. Badger, Martin J.J. Ronis, Reza Hakkak, and Sohelia Korourian Arkansas Children’s Nutrition Center, Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR

Introduction The American Academy of Pediatrics (AAP) recommends breast-feeding over formula feeding (1). For infants who cannot tolerate breast milk or infants of mothers who are not able or willing to breast-feed, milk-based formula is recommended over soy-based formula, even though there have been no demonstrated advantages of cow’s milk–based formula. The AAP specifically states: “In term infants whose nutritional needs are not being met from maternal breast milk or cow’s milk-based formulas, isolated soy protein–based formulas are safe and effective alternatives to provide appropriate nutrition for normal growth and development.” The preference toward cow’s milk formula over soy formula appears to be based on the lack of confirmed advantages of soy-based formula, coupled with its much shorter safety history, rather than any deficiency. This chapter will discuss what is known about the safety issues of infant formula and the health effects of soy foods. Soy Formulas In the 1920s, infant formulas began to gain in popularity, and the percentage of mothers who breast-fed declined over the next few decades. The United States Federal Drug Administration (FDA) has established safety, quality, and nutrient requirement standards for infant formulas, and requires labeling with nutritional information. Thus, all commercial infant formulas are highly regulated in the United States. Although feeding soy foods to infants has a long history in Asia, it was not until 1909 that a preliminary report on the use of soy formula was published in the U.S. (2). In 1929, Hill and Stuart (3) were the first to report the use of soy formula for infants with allergies to cow’s milk protein. Since those early reports, the quality and acceptability of soy formula have improved tremendously. There are several reasons pediatricians or parents select soy formula for their infants, including the following: (i) milk protein allergies; (ii) postdiarrhea lactose intolerance; (iii) galactosemia; (iv) primary lactase deficiency; and (v) maintenance of vegetarian lifestyle. Furthermore, the popular press has been an influential source of informa-

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tion on the potential benefits of dietary factors (such as those found in soy) in preventing chronic diseases, leading more Americans to use soy formulas. The number of U.S. infants who consume soy formula each year has been estimated from marketing data at 25% of the nearly 4 million newborns in the United States (1). Hospital discharge records suggest a similar figure of 20–25%. For example, hospital discharge records obtained from the Arkansas Department of Health and Human Services indicated that during the years 1994 through 1998, an average of 21% of all infants born in Arkansas hospitals were fed soy formula, and this increased to 25% by 6 mo. The time at which infants are first fed soy formula and the duration of soy feeding vary. It is not uncommon for infants to be breastfed before being switched to soy formula and some infants are switched from milkbased diets to soy formula and vice versa, for a variety of reasons. Breast milk has been used as a “target infant food” for formulation of infant formula, and where practical, the composition of formula mimics breast milk. There are, of course, compounds in human breast milk that are absent in formulas. Some of these may have health benefits, but they are not included in infant formulas, primarily because of cost. Because cow’s milk does not have the same composition as human milk, there are significant differences between milk-based formula and human milk. This is especially true of milk proteins, for which the ratio of casein to whey proteins (including the globulin proteins) differs substantially (4). Similarly, the composition of soy formula is not the same as human milk, nor is it the same as cow’s milk formulas. However, all “over-the-counter” formulas sold in the United States meet the FDA nutrient requirements. Soy Isoflavones Commercially available soy formula marketed in the United States is made with soy protein isolate (SPI). SPI has several phytochemicals associated with it, including isoflavones. The soy phytochemicals found in soy formula are not found in high concentrations in human or cow’s milk (5). The soy isoflavones are in the form of glucosides in the soybean and in the isolated soy protein used in infant formula; however, they are found as aglycones, glucuronides, and sulfates in body fluids. Breast Milk. The composition of breast milk can be influenced by the diet of the lactating mother, and consuming soy foods during lactation can result in isoflavones in breast milk. But, data from Setchell et al. (5,6) and Franke and Custer (7) suggest that breast milk of women who do not consume appreciable amounts of soy foods (i.e., most American women) is nearly devoid of isoflavones, and even women who consume soy foods have very low concentrations of isoflavones (~45 µg/L) in their breast milk (7). This means that breast-fed infants are not exposed to appreciable levels of soy isoflavones during nursing, even in women who are high soy consumers. Although very little research has been pub-

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lished on the bioavailability of soy isoflavones in human breast milk, there is some speculation that it may be greater than that of soy formula (7). Still, the absolute soy isoflavone levels reaching infants through breast-feeding are thought to be a minor source of infant exposure. On the other hand, soy infant formulas have ~32–47 mg/L of total isoflavones, and infants consume between 6 and 9 mg/(kg ⋅ d) total isoflavones, resulting in plasma total isoflavone concentrations ranging from 0.6 to 7 µg/mL (5). Plasma. Isoflavones bind to estrogen receptors and function as either estrogen agonists, antagonists, or selective estrogen receptor modulators, depending upon the tissue, the cell type, isoflavone concentration, and other conditions such as hormonal status and age (8–12). Although soy isoflavones are considered much weaker than the primary ovarian estrogen, 17-β-estradiol (E2), circulating isoflavone concentrations can become much greater than those of E2 (Tables 33.1 and 33.2). Typically, plasma E2 concentration are greater in women than men, and range from 8–80 pg/mL in the follicular phase of the menstrual cycle to 100–600 pg/mL at midcycle. Plasma E2 concentrations increase in maternal and cord blood to 0.8–40 ng/mL and are relatively high in newborns (100–200 pg/mL). Plasma E2 concentrations decrease in neonates to <10 pg/mL and remain low until puberty. Thus, the typical peak circulating levels of endogenous estrogens in infants and children are below ~10 pg/mL or 0.04 nmol/L, whereas the typical peak E2 concentration at midcycle for women would be 0.4–2.4 nmol/L. Setchell (5) reported the concentrations of circulating total isoflavones in infants fed soy formula to be 0.55 to 1.8 µg/mL (~2–7 µmol/L) range. These high blood isoflavone concentrations (and presumably tissue concentrations) are of interest because humans do not normally have high circulating concentrations of gonadal steroids until after puberty. This raises the question of potential short- and long-term health effects of these phytoestrogens during infant development. Although circulating endogenous estrogens are low until puberty, phytoestrogens can attain very high concentrations early in human and animal development. For example, the total maternal plasma isoflavonoid concentrations of Japanese TABLE 33.1 Estimated Plasma Estradiol Concentrations as Influenced by Hormonal Status Hormonal status Menstrual cycle Follicular phase Midcycle Luteal Menopause Gestational

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Plasma estradiol (nmol/L) 0.3 2.4 0.7 0.07 150..

TABLE 33.2 Effects of Age and Hormonal Status on the Levels of Serum Isoflavones Serum isoflavone (nmol/L) Age and/or hormonal stage Pregnancy Midcycle Fetusa Infants Breast-fed Milk formula Soy formula

Asian

American

800 800 800

<25 <25 <25

50 <25 NAb

<25 <25 7000

aCord bNot

blood and amniotic fluid. applicable to the majority of Asians.

women who consumed their normal diets ranged from 19–744 nmol/L and the mean concentrations of cord blood and amniotic fluid at birth were 299 nmol/L and 223 nmol/L (Table 33.2), respectively (13). Thus, isoflavonoid phytoestrogens are transferred from the maternal to the fetal compartment in substantially high concentrations. For comparison, cord blood contains ~150 pmol/L and 800 nmol/L of E2 and isoflavones, respectively (5,13). It should also be noted that the women in the Aldercreutz study (13) did not eat during their labor and the blood sampling was taken at the hospital during delivery. This means the isoflavone concentrations reported in that study are likely to be substantially lower than the maximal concentrations attained throughout the day. Therefore, accounting for the timing of sample collection in relation to meals and the differences in methodology between laboratories, the isoflavone concentrations of infants fed soy formula (5) and the fetuses of Asian women (13) could be within the same general concentration range (i.e., low µmol/L range). Isoflavone Potency. Relative to E2, isoflavones such as genistein have been reported to be less potent. However, potency has been studied mainly in in vitro systems or in laboratory animals. The true potency in target cells of human infants is very difficult to ascertain, and has never been reported. The estimates of potency differences between E2 and genistein in in vitro systems range from 3 to 400,000 times less for genistein (9,10,14,15), but a potency factor of 1:1000 is often used to compare E2 to genistein. Even if one were to consider isoflavones as being 1000 times less potent than E2, infants fed soy formula have formidable circulating concentrations of these phytoestrogens. For example, as mentioned above, plasma E2 concentrations are in the range of 0.7–2.4 nmol/L at midcycle of women, compared with total plasma isoflavone concentrations of 2–7 µmol/L in infants fed soy formula, a factor of approximately 1:5000. Thus, given the uncertainty about the potency of isoflavones in infants, the high plasma isoflavone con-

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centrations in infants raises the possibility that these isoflavones could exert estrogenic effects. A scientific debate has developed and centers upon the idea that isoflavones in soy formula may have adverse effects (16–20). Although the newborn has somewhat high plasma E2 concentrations, these decrease shortly after birth and children are not exposed to high concentrations of endogenous estrogens until puberty; even then, the concentrations are in the pmol/L range for girls and less for boys. Infant Exposure to Isoflavones. Relevant to this question is infant exposure to soy isoflavones in different countries throughout the world. Although Asian populations have consumed soy foods for centuries without major adverse health effects, the exposure to soy phytochemicals throughout the life cycle differs substantially between Asia and the United States. The most obvious difference is the extremely low soy food consumption in the United States compared with Asia. But, perhaps an even greater distinction is that the infant fed soy formula is the single exception to the low soy food intake in the U.S. and these infants are exposed to significant levels of soy only between birth and weaning. This is because U.S. women consume very low levels of soy and thus their infants are not exposed to the influence of substantial levels of soy components in utero. Furthermore, because soy is not a main component of the American diet, children fed soy formula are not likely to consume appreciable levels of soy during the remainder of their life. Compare this with most Asians who consume high levels of soy throughout their entire lives. Even before the typical weaning to solid foods, Asian infants are often fed soy foods, such as fermented soy paste. However, the major direct exposure of Asian children to soy starts at weaning. The typical Asian mother breastfeeds her infant or uses milk-based formula, because soy infant formulas are not fed with as high a frequency as in the United States. As mentioned above, the other important lifecycle exposure period for soy (or soy components) is in utero, because there is maternal to fetal transport of soy components across the placenta. Debate on the Safety of Soy Infant Formula Central to the debate on the safety of soy formulas are reports of adverse health effects of estrogens and estrogenic compounds, and the relevancy of those effects to the possible health consequences of soy formula. The major worry revolves around possible adverse effects of phytoestrogen isoflavones on endocrine sensitive systems of reproduction. Perhaps the most well-known complication of isoflavones is clover disease, the infertility first reported in 1946 in Austrialian sheep that grazed on clover with a high isoflavone content (21). Other, more subtle reproductive disorders related to phytoestrogen consumption in sheep have been recently reviewed (22). Clover disease has been the single most cited “adverse health effect” by opponents of soy formula for infants and soy food consumption by women. However, the clover disease situation is not applicable to infants con-

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suming soy formula. The maturity of the ovine reproductive tract, the sheep digestive system, the type of isoflavone in clover, and the effective isoflavone doses achieved in the sheep that developed clover disease are so different from the infant fed soy formula that very little parallel can be drawn between clover disease and any potential adverse effects from consuming soy infant formula. These sheep were reproductively mature and seasonal breeders, two extremely important factors that do not exist in human infants. Furthermore, the concentrations of equol, a potent isoflavone, were approximately six orders of magnitude greater in sheep than could be achieved in infants. Equol is formed by gastrointestinal bacteria that are not present in infants at sufficient levels to produce detectable circulating concentrations (5), whereas the sheep, because it is a ruminant animal, has sufficient bacteria to produce high concentrations of equol. Some investigators have mistakenly compared the abnormalities in estrogen target tissues caused by the potent synthetic estrogen diethylstilbestrol (DES) to potential effects that occur with consumption of dietary phytoestrogens in soy infant formula (19,20). However, there are absolutely no data to suggest that consumption of soy foods has any connection to birth defects or reproductive disorders later in life in humans. In fact, quite the contrary; Asians have consumed high levels of soy during pregnancy for centuries with no reported or suspected birth defects associated with soy. Furthermore, DES is often studied in neonatal rodents. Investigators conducting such studies suggest that the observed adverse effects of DES on the reproductive tract of these rodents may model the effects in infants fed soy formula. This is highly unlikely for several reasons, including potency and mechanistic differences between isoflavones and DES, and development differences between humans and rodents. For example, the neonatal rodent and postnatal human are not at equivalent morphological stages of development (23) and the neonatal rodent does not model the infant human (18). Most investigators agree that the developmental stages of the late gestational human fetus and early neonatal rodent are more equivalent than neonatal infants and neonatal rodents. The reported isoflavonoid exposure concentrations of Japanese fetuses (based on cord blood and amniotic fluid concentrations) were as high as 0.8 µmol/L (13) and, as discussed earlier in this chapter, it is likely that peak plasma isoflavone concentrations were actually closer to the 2–7 µmol/L reported for infants fed soy formula (5). To our knowledge, there have been no reports of soy food–associated adverse health effects for Japanese newborns, suggesting that perinatal exposure to the high isoflavone concentrations achieved in utero or by soy infant formula is not likely to result in adverse health effects, especially not the type of abnormalities ascribed to DES. Furthermore, DES is a powerful steroid with actions much different from those of the less potent isoflavones that circulate in infants. Thus, the validity of treating rodents with high levels of powerful drugs in the neonatal stage during critical periods of imprinting and relating that to the health effects of human infants who are fed soy formula is questionable at best. In the United States, foods come under a different regulatory process than drugs. Similarly, food supplements and other products marketed under the classifi-

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cation of herbals do not currently require the rigorous proof of efficacy, safety, and content before marketing that the FDA requires of new drugs. On the other hand, infant formulas are regulated by the FDA and monitored by advocate groups, such as the American Academy of Pediatrics, for effects on growth, development, safety, and general health. Thus, infant formulas are monitored by both government agencies and the advocacy groups most attuned to child safety, development, and health. It may be instructive to examine the evidence for health effects of soy throughout the world. Consumption of soy foods has been a practice for centuries in Asia. Similarly, millions of American infants have been fed soy formula over the past three decades with an adverse-health-effects rate no greater than that of children who were breast-fed or fed milk-based formula. There are so many factors that go into determining the health status of a population that is difficult to assign the contribution of any one environmental component to health status, unless it is some adverse effect such as a nutrient deficiency or toxicity. There are many examples of such adverse effects, including the following: (i) iodine deficiency and thyroid diseases; (ii) iron deficiency and anemia; (iii) folate deficiency and neurotube defects; (iv) lead exposure and brain disorders; or (v) alcohol exposure and fetal alcohol syndrome. So, what are the data on the safety of soy foods in general and on soy formula in particular? There are no human data to support toxicity of soy foods as marketed in the United States, especially as relates to reproductive competency. In countries in which soy has been consumed at the greatest daily intake for centuries, the population has increased at rates often exceeding the national ability to support usual population needs, such as food supplies or health care. Women who consume soy foods that produce the high circulating concentrations of isoflavones are capable of conceiving, taking the pregnancy to term, delivering normal infants, normally lactating, and otherwise caring for their infants. Because these women consumed soy foods before pregnancy, and continued eating soy during pregnancy and during lactation, soy food consumption during pregnancy and lactation does not appear to have adverse effects on reproduction or early human development. Thus, there is no reported epidemiologic evidence to suggest that soy foods have adverse affects at these important critical times during which other hormones and drugs, such as DES or alcohol, were reported to have damaging developmental and health effects. Furthermore, multiple generations of people have consumed soy foods without long-term adverse effects of early exposure to soy when these are consumed in the context of a normal diet. When dealing with health effects of specific foods or dietary components, it is essential to consider the entire lifestyle of the population under study. The Asian lifestyle differs significantly from that of the United States. The diet of Asians who have not become Westernized contains much less red meat, less fat, more rice, more fish, and more soy. In addition, Asians exercise more and consume less total energy, especially fat calories. Under these conditions, Asian populations have not

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developed reproductive disorders that have been reported to decrease population dynamics. In fact, overpopulation rather than infertility is more the issue in countries that consume large amounts of soy. Furthermore, Asians have lower risks of heart disease, certain cancers, and bone diseases than Americans. Thus, overall, if other conditions such as sufficient food supplies, better immunization, and better access to modern health care were combined with the exercise and dietary components of the Asian lifestyle, which includes high soy intake, the risk of diseases would be predicted to be even lower. Soy foods and Purified Soy Isoflavones In relation to the data supporting the health effects of soy formula, there are two specific areas of the soy industry to consider, i.e., processed soy foods such as tofu and soy formula, and specific components of the soybean, such as isoflavones. Purified soy isoflavones are a relatively new product in the American market place. They are part of the phytochemical by-products of soy protein processing. Genistein is probably best known; it is often marketed alone, but daidzein and mixtures of soy isoflavones and/or other phytochemicals extracted from soy proteins are marketed also. Although many claims have been made, the health effects of purified isoflavones or the mixtures of soy phytochemicals have not been well characterized. Similarly, the bioavailability and safety of soy isoflavones in humans under controlled conditions have not been determined. Thus, there is no evidence that they adversely or beneficially affect the crucial events necessary for development of the female reproductive system in preparation for conception, implantation, maintenance of pregnancy, delivery of a child, or lactation. There are also no human data on the possible effects of purified soy isoflavones on cancer initiation or promotion in either women or men. It is important to reiterate that soy infant formula is made from soy protein isolate (SPI), and is balanced with the proper amino acids, vitamins, and minerals to account for bioavailablity and nutrient requirements of the infant. Currently, there are no approved infant formulas made with any protein to which high levels of the purified isoflavones are added, nor are there ever likely to be any. There are >100 animal studies on the effects of injected or orally gavaged purified isoflavones or diets made with purified soy isoflavones. These types of studies are important because the commercial availability of isoflavones is increasing throughout the world. The health consequences of these products are not known and thus additional studies are required. However, it is a mistake to assume that the effects of purified isoflavones are necessarily the same as those of soy foods or SPI. How does consumption of diets high in soy foods by adults relate to infants consuming soy formula? There are many obvious differences between infants and adults that may be important in determining the health consequences of diet. For example, adults consume a more varied diet than infants, and this is true in all countries. Infants, regardless of whether breast-fed or formula-fed, consume essentially the same diet

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continuously for an extremely long period (months). Furthermore, the neonatal period is one of the most important in human development. Virtually all organs are still developing, and each physiologic system is at a different stage of development and function. In addition, the bioavailability of dietary factors may be quite different in infants than adults. For example, it is well known that the bacterial flora of infants differs substantially from that of adults. This is important for soy foods, because the bacteria type and titer play a significant role in metabolism and absorption of soy phytochemicals, such as the isoflavones. Isoflavone glucosides such as genistin and daidzin are not absorbed intact; they require deconjugation by gut bacteria before absorption as the aglycones, genistein and daidzein, respectively. Another good example is the production of the potent isoflavone equol from daidzein, which is accomplished by gut bacteria as mentioned earlier in relation to clover disease in sheep. The type and titer of infant gut bacteria may have implications for metabolism of other isoflavones as well. In addition, the integrity of the infant intestine differs from that of adults, and the implications of gut development, either good or bad, with respect to soy infant formula have not been fully explored. Because soy isoflavone aglycones are reconjugated to form glucuronides and sulfates by first-pass metabolism in the intestine mucosa, the intestine is a key organ in infants fed soy formula. Much emphasis has been placed on the isoflavone effects of soy foods. But when this has been examined carefully, the isoflavone contribution to some beneficial health effects has actually been shown to be minor. For example, the FDA has permitted the use of a health claim for potential soy food–induced reduction of cardiovascular disease through lowering serum cholesterol. This claim is based upon research using SPI, the same protein source used in infant formula. Studies in both animals and humans have demonstrated that SPI intake reduces serum cholesterol (24). However, the most recent data demonstrate that although the maximal cholesterol-lowering effects are produced with the intact SPI, near maximal effects (<70%) are attained with SPI processed to contain extremely low levels of isoflavones (25). A similar story is emerging in mammary cancer. It is well established that Asians have 5–8 times less mammary cancer than Americans (26). We have studied dimethylbenz(a)anthracene (DMBA)-induced mammary cancers in female rats and azoxymethane (AOM)-induced colon cancers in male rats, and found that SPI reduces the incidence of both cancers (27,28). Interestingly, Constantinou et al. (29) fed SPI that had been extracted to remove the isoflavones and found that reduced mammary cancer occurred in the absence of high levels of isoflavones. This is analogous to the cholesterol-lowering effects of SPI (25). We conducted similar studies and also found that rats fed AIN-93G diets made with SPI that was processed to remove isoflavones (SPI–) had reductions of DMBA-induced incidence of mammary gland adenocarcinomas (Fig. 33.1) at the same levels as previously reported for SPI that contained isoflavones (27). Although isoflavone levels vary somewhat among SPI lots, a typical isoflavone content profile per kg diet in our SPI diets would be 488, 344, and 83 mg for genistein, daidzein and glycitein, respectively, and for the SPI– diet 8, 4, and 2 mg, respectively.

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Fig. 33.1. The incidence of dimethylbenz(a)anthracene (DMBA)-induced mammary

tumors in female rats fed AIN-93G diets made with either casein (casein) or soy protein isolate (SPI–). SPI– is soy protein isolate processed to contain very low levels of phytochemicals; the diet made with this protein contained 8 and 4 mg/g genistein and daidzein, respectively. The data are the means SEM (n > 38/group).

Thus, there are two important messages in these latter studies. First, contrary to finding adverse effects of SPI, many studies point to potential health benefits; one of these findings, the cardiovascular benefits, has resulted in a FDA-approved health claim provision. Second, the effects of SPI appear to not only be positive, but the contribution of the protein appears to have more importance than previously thought. The protein itself is an important factor, and thus studies conducted with the purified isoflavones in the absence of the soy protein have little relevance to the effects of soy formula. There is a long history of the safety and health benefits of soy foods in Asia. This history involves foods made with soybeans, not purified isoflavone aglycones. There is virtually no human history published on the health effects of people consuming high levels of purified soy isoflavones. Although it is important to study the effects of purified isoflavones in adults, especially given the appearance of purified genistein and mixed soy isoflavones now marketed across the United States, these results have little bearing on the health effects of soy infant formula. There is no evidence that the effects of purified isoflavones are the same as those of either SPI or soy infant formula. In fact, results from several studies have shown that SPI effects differ substantially from isoflavone effects.

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In this regard, there are two types of studies that require comment. One involves studies commissioned by the National Toxicology Program (NTP) to evaluate the blood and tissue concentration of genistein aglycone and its metabolites. In those studies, rats were fed diets containing different levels of purified genistein in the absence of soy protein or were given genistein by oral gavage. Genistein was present in the fetal brains as aglycones and present in the serum mainly as glucuronides (30–32). Although these studies may be important to determine the possible effects of consuming high levels of pure genistein, they likely have little relevance to infants fed soy formula. For example, infants are fed a balanced formula that contain isoflavones in a different molecular form and in a protein matrix quite different from those of the NTP studies using purified genistein with no soy protein. In addition, the serum and tissue genistein metabolites of the rats fed diets made with purified genistein differ substantially from urinary, plasma, and tissue profiles of rats or adult humans fed the SPI used in infant formula (33–35). This suggests that the bioavailability, pharmacokinetics, and metabolism of purified aglycone isoflavones consumed as part of a diet devoid of soy protein may be quite different from the metabolism of the more complex isoflavones associated with soy protein. Furthermore, these results, when combined with the data demonstrating that the cholesterol-lowering effects of SPI and the reduction in chemically induced cancers by SPI (25,29, Fig. 33.1) indicate that there are substantial differences in the biological effects of purified isoflavones and foods containing SPI. These data further suggest that studies of high levels of purified isoflavones have little bearing on the health effects of soy formula. The second type of study often quoted as demonstrating that soy has adverse effects are those using immunocompromised and gonadectomized rodents that have been implanted with estrogen receptor (ER) positive human breast cancer lines (36–38). This research has very nicely demonstrated that ER tumor cells placed in animals without ovaries will grow in response to feeding either genistein or soy protein. These effects were predictable because in the absence of endogenous ovarian factors (such as estrogens, progestins, inhibins, and other important modulating factors) and any immune system, the soy isoflavones or their metabolites act as estrogens and promote tumor growth, exactly as demonstrated in cell cultures in vitro. In fact, these animals essentially serve as “in vivo incubators.” This does not happen, however, in other experimental paradigms that more closely mimic the human situation in which the ovary is still present, whether menopause has occurred or not, and where there is a functional immune system. For example, Hawrylewicz et al. (39) reported that SPI inhibited promotion of mammary cancers in rats having intact ovarian and immune systems. There have been no studies that have demonstrated an increased risk of breast cancer in postmenopausal women who consume soy. More importantly, although the studies in ovariectomized rodents raise the possibility of adverse effects of soy intake in adult postmenopausal women (who may also be immunocompromised), they do not apply to soy infant formula.

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Growth and Development of Children Fed Soy Formula By far, the most convincing data about the safety and efficacy of soy infant formula comes from the health, growth, and development of the millions of infants fed these formulas over the >25 years of their existence. Although soy formulas have existed since the 1960s, the current formulations have been fed to infants for shorter periods. Because infants are growing rapidly, even small deficiencies in nutrient levels and balance will have adverse effects on growth rates and serum chemistries. There have been several studies demonstrating that soy formula supports normal growth and development in term infants (40–44). When growth was studied over the first year of life, it was found that body weight gains and body length of infants were virtually the same, whether they were fed soy formula, milk-based formula, or breast-fed (45). Long-Term Effects. Recently, Strom et al. (46) reported on 811 young adults who were fed either milk-based formula or soy formula as infants. In that study, men and women between the ages of 20 and 34 y were studied to determine the longterm health consequences of early soy intake. There were a few significant differences between the milk formula- and soy formula-fed groups, but no differences were found in growth, development, puberty, reproductive function, pregnancy outcomes, or a host of other parameters. In fact, this and all the other studies cited above indicate that soy formula is not only safe, but is also very effective in supporting optimal growth and development of term infants. However, it should be noted that this study has three major deficiencies. First, the number of subjects was low and this reduces the chances of detecting biologically important differences in many outcomes between groups. This subject number does not allow one to determine whether the incidence of pregnancy outcomes, such as the following: higher preterm deliveries (12.7% in soy vs. 8.1% in milk), higher stillborn deliveries (3.8% in soy vs. 0% in milk), or higher multiple birth (5.4% in soy vs. 3.6% in milk) are biologically meaningful. Second, the subjects were too young to determine their risk of developing chronic diseases that occur mainly later in life. Third, the population selected for study was limited to mainly white, well-educated Midwestern Americans and may not be applicable to a wider population. Nonetheless, these data add to the already large database suggesting that soy formula is safe and effective in promoting normal growth and development of term infants. Multigenerational Animal Studies. We also studied the effects of feeding the same SPI used in infant formulas to several generations of rats with the idea of establishing a situation similar to that among Asians who have high levels of soy intake throughout their lives. One issue related to soy infant formula is the longterm health consequences of early consumption of these formulas. We fed AIN93G diets (made with SPI+) throughout their life and found that male and female rats have the same breeding efficiency as rats fed commercial diets or AIN-93G

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diets made with casein (47). The numbers of offspring, gender ratios, birth weights, birth lengths, health, and general appearance of soy-fed rats were the same as those of casein-fed rats. The only major effect was that vaginal opening occurred 1 d earlier in soy-fed rats (Fig. 33.2). Because an earlier pubertal age has not been reported in children of Asian countries in which soy intake is high, the practical consequence of this finding is unclear. Other indices of estrogenicity in SPI-fed rats, such as weights of secondary sex organs (e.g., uterus, prostate, seminal vesicles) were found to be normal (47). Similarly, serum concentrations of the endogenous gonadal steroids estrone and estradiol (Fig. 33.3) and the mammary gland morphology, development, differentiation, or estrogen receptors (48,49) did not differ consistently between SPI-fed and casein-fed rats.

Conclusions Asians have been consuming high levels of soy foods for centuries, and these foods have been eaten throughout the life cycle. Because Asian women continue to eat soy foods during pregnancy, their fetuses are exposed to whatever components of the soybean can be transported across the placenta, and those infants who breast-feed are exposed only to those soy components that cross into the breast

Fig. 33.2. The effects of diet on puberty of female rats. Rats were fed AIN-93G diets made with either soy protein isolate (Soy) or Casein (Casein) for two generations as previously described (47). The data are the percentage of rats (n = 40/group) having vaginal opening. The day vaginal opening occurs is the primary measure of puberty in rats.

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Estradiol (pg/mL)

Estrone (pg/mL)

Fig. 33.3. The effects of soy protein isolate (SPI) on serum estrone and estradiol concentrations (n = 10/group). Rats were fed diets containing either SPI+ or casein from weaning until age 50 d.

milk. Upon weaning, Asian children consume increasing amounts of soy foods and continue to do so throughout their lives. Asians are more likely to breast-feed than formula-feed their infants, and milk-based formula far outsells soy formula. Thus, newborn infants are the only segment of the Asian population least likely to consume high levels of soy foods. In the United States, on the other hand, the segment of the population that consumes the most soy are the millions of infants fed formula made with SPI. Before birth and after weaning, the vast majority of Americans are not exposed to appreciable levels of soy foods, other than foods that have small amounts of processed soy components. The plasma of soy-fed infants contains high concentrations of isoflavones (and probably other soy phytochemicals). On the basis of in vivo and in vitro data, these isoflavones are clearly within the concentration range to activate both estrogen receptors (ERα and ERβ). Thus, soy isoflavones could act as estrogen agonists, antagonists, or selective estrogen receptor modulators, depending upon the conditions. However, multigenerational animal studies and studies in infants who consumed SPI suggest that SPI is safe throughout the life cycle, and especially during early development. Furthermore, SPI diets support normal growth and development in human infants, and recent data suggest that there are no long-term adverse effects of early exposure to soy formula. The major cautionary note concerning health effects of soy formula is that there is only one study on the long-term health effects and there are no data beyond early adulthood. Thus, it is not known whether any adverse effects will develop with aging. Results from the vast majority of both animal and human research indicate positive

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health benefits from soy foods (50). The potential health benefits include the following: (i) improved cardiovascular health; (ii) reduced risk of certain cancers; and (iii) improved bone health. Further research is required to confirm the results of the few studies that have been conducted and new studies are indicated to investigate the more subtle potential effects that could occur during development or that could surface later in life because previous studies were not designed to address those areas. Acknowledgments This work was funded by the Arkansas Children’s Nutrition Center, an ARS program of the USDA. We would like to thank Protein Technologies International for supplying the SPI used in our studies. We would also like to acknowledge the work of Dr. Craig Rowlands on mammary gland development and the excellent technical assistance of Terry Fletcher, Matt Ferguson, Kim Hale, and Jamie Badeaux.

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13. Adlercreutz, H., Yamad, T., Wähälä, K., and Watanabe, S. (1999) Maternal and Neonatal Phytoestrogens in Japanese Women During Birth, Am. J. Obstet. Gynecol. 180, 737–743. 14. Farmakalidis, E. and Murphy, P.A. (1985) Isolation of 6′′-O-Acetylgenistein from Toasted Defatted Soyflakes, J. Agri. Food. Chem. 33, 385–389. 15. Setchell, K.D.R., and Aldercreutz, H. (1988) Mammalian Ligands and Phytochemicals: Recent Studies on Their Formation, Metabolism and Biological Role in Health and Disease, in The Role of Gut Microflora in Toxicity and Cancer, (Rowland, I.A., ed.) pp. 315–345, Academic Press, New York. 16. Irvine, C., Fitzpatrick, M., Robertson, I., and Woodhams, D. (1995) The Potential Adverse Effects of Soybean Phytoestrogens in Infant Feeding, N.Z. Med. J. 108, 208–209. 17. Robertson, I.G.C. (1995) Phytoestrogens: Toxicity And Regulatory Recommendations, Proc. Nutr. Soc. N.Z. 20, 35–42. 18. Sheehan, D.M. (1997) Isoflavone Content of Breast Milk and Soy Formula: Benefits and Risks, Clin. Chem. 43, 850–852. 19. Newbold, R. (1995) Cellular and Molecular Effects of Development Exposure to Diethylstilbesterol: Implications for Other Environmental Estrogens, Environ. Health Perspect. 103, 83–87. 20. Newbold, R. (2001) Effects of Developmental Exposure to Genistein, a Soy Phytoestrogen, in an Experimental Animal Model, J. Nutr. 132, 576S. 21. Bennetts, H.W. Underwood, E.J., and Shier, F.L. (1946) A Specific Breeding Problem of Sheep on Subterranean Clover Pastures in Western Australia, Aust Vet. J. 22, 2–12. 22. Adams, N.R. (1990) Permanent Infertility in Ewes Exposed to Plant Oestrogens, Aust. Vet. J. 67, 197–201. 23. Ojeda, S.R., Andrews, W.W. Advis, J., and Smith-White, S. (1980) Recent Advances in the Endocrinology of Puberty, Endocr. Rev. 1, 228–257. 24. Vitolins, M.Z., Anthony, M.B., and Burke, G.L. (2001) Soy Protein Isoflavones, Lipids and Arterial Disease, Curr. Opin. Lipidol. 12, 433–437. 25. Anthony, M.S., Blair, R.M., and Clarkson, T.B.(2002) Neither Isoflavones Nor the Alcohol-Extracted Fraction Added to Alcohol-Washed Soy Protein Isolate Restores the Lipoprotein Effects of Soy Protein Isolate, J. Nutr. 132, 581S. 26. Pisani, P., Parkin, D.M., and Ferlay, J. (1993) Estimates of the Worldwide Mortality of Eighteen Major Cancers in 1985: Implications for Prevention and Projections of Future Burden, Int. J. Cancer 55, 891–903. 27. Hakkak, R., Korourian, S., Shelnutt, S.R., Lensing, S., Ronis, M.J.J., and Badger, T.M. (2000) Diets Containing Whey Proteins or Soy Protein Isolate Protect Against 7,12Dimethylbenz(a)anthracene-Induced Mammary Tumors in Female Rats, Cancer Epidemiol. Biomark. Prev. 9, 113–117. 28. Hakkak, R., Korourian, S., Ronis, M.J.J., Johnson, J., and Badger, T.M. (2001) Soy Protein Isolate Consumption Protects Against Azoxymethane-Induced Colon Tumors in Male Rats, Cancer Lett. 166, 27–32. 29. Constantinou, A.I., Rossi, H., Nho, C.-W., Jeffereys, E.H., Xu, X., van Breemen, R.B., and Pezzuto, J.M. (2000) Soy Protein Isolate Depleted of Isoflavones Prevents DMBAInduced Mammary Tumors in Female Rats, Proc. Am. Assoc. Cancer Res. 14, 310. 30. Doerge, D.R., Churchwell, M.I., Chang, H.C., Newbold, R.R., and Delclos, K.B. (2001) Placental Transfer of the Soy Isoflavone Genistein Following Dietary and Gavage Administration to Sprague Dawley Rats, Reprod. Toxicol. 15, 22–29.

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31. Chang, H.C., Churchwell, M.I., Delclos, K.B., Newbold, R.R., and Doerge, D.R. (2000) Mass Spectrometric Determination of Genistein Tissue Distribution in Diet-Exposed Sprague Dawley Rats, J. Nutr. 130, 1963–1970. 32. Holder, C.L., Churchwell, M.I., and Doerge, D.R. (1999) Quantitation of Soy Isoflavones, Genestein and Daidzein, and Conjugates in Rat Blood Using LC/ES-MS, J. Agric. Food Chem. 47, 3764–3770. 33. Cimino, C., Shelnutt, S.R., Ronis, M.J.J., and Badger, T.M. (1999) An LC-MS Method to Determine Concentrations of Isoflavones and Their Sulfate and Glucuronide Conjugates in Urine, Clin. Chim. Acta 287, 69–82. 34. Shelnutt, S.R., Cimino, C., Wiggins, P.A., and Badger, T.M. (2000) Urinary Pharmacokinetics of the Glucuronide and Sulfate Conjugates of Genistein and Daidzein, Cancer Epidemiol. Biomark. Prev. 9, 413–419. 35. Shelnutt, S.R., Cimino, C., Wiggins, P.A., Ronis, M.J.J., and Badger, T.M. (2002) Pharmacokinetics of the Glucuronide and Sulfate Conjugates of Genistein and Daidzein Following a Soy Meal in Men and Women, Am. J. Clin. Nutr., in press.( 36. Hsieh, C.Y., Santell, R.C., Haslam, S.Z., and Helferich, W.G. (1998) Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-Positive Human Breast Cancer (MCF-7) Cells In Vitro and In Vivo, Cancer Res. 58, 3833–3838. 37. Santell, R.C., Kieu, N., and Helferich, W.G. (2000) Genistein Inhibits Growth of Estrogen-Independent Human Breast Cancer Cells in Culture but Not in Athymic Mice, J. Nutr. 130, 1665–1669. 38. Allred, C.D., Allred, K.F., Young, H.J., Suzanne, M.V., and Helferich, W.G. (2001) Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of EstrogenDependent (MCF-7) Tumors in a Dose-Dependent Manner, Cancer Res. 61, 5045–5050. 39. Hawrylewicz, E.J., Zapata, J.J., and Blair W.H. (1995) Soy and Experimental Cancer: Animal Studies, J. Nutr. 125, 698S–708S. 40. Fomon, S.J. (1993) Nutrition of Normal Infants, (Craven, L., ed.) Mosby, St. Louis. 41. Businco, I., Bruno, G., and Giampieto, P.G. (1992) Allergenicity and Nutritional Adequacy of Soy Protein Formulas, J. Pediatr. 121, 821–828. 42. Churella, H.R., Borschel, M.W., Thomas, M.R., Breem, M., and Jacobs, J. (1994) Growth and Protein Status of Term Infants Fed Soy Protein Formulas Differing in Protein Content, J. Am. College Nutr. 13, 262–267. 43. Kohler, L., Meeuwisse, G., and Mortensson, W. (1984) Food Intake and Growth of Infants Between Six and Twenty-Six Weeks of Age on Breast Milk, Cow’s Milk Formula, or Soy Formula, Acta Paediatr. Scand. 73, 40–48. 44. Graham, G.G., Placko, R.P., and Morals, E. (1970) Dietary Protein Quality in Infants and Children. VI. Isolated Soy Protein Milk, Am. J. Dis. Child. 120, 419–423. 45. Lasekan, J.B., Ostrom, K.M., Jacobs, J.R., Blatter, M.M., Ndife, L.I., Gooch, W.M., and Cho, S. (1999) Growth of Newborn, Term Infants Fed Soy Formulas for 1 Year, Clin. Pediatr. 38, 563–571. 46. Strom, B.L., Schinnar, R., Ziegler, E.E., Barnhart, K., Sammel, M., Macones, G., Stallings, V., Hanson, S.A., and Nelson, S.E. (2001) Follow-Up Study of a Cohort Fed Soy Based Formula During Infancy, J. Am. Med. Assoc. 286, 807–814. 47. Badger, T.M., Ronis, M.J.J., and Hakkak, R. (2001) Developmental Effects and Health Aspects of Soy Protein Isolate, Casein, and Whey in Male and Female Rats, Int. J. Toxicol. 20, 165–174.

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48. Rowlands, J.C., Brewer, T.L., Hakkak, R., and Badger, T.M. (2000) Effects of SoyProtein Isolate or Whey Protein on Rat Mammary Epithelium, FASEB J. 14, A240 (Abstr.). 49. Rowlands, J.C., Hakkak, R., Till, R., and Badger, T.M. (2001) Increased Expression of Progesterone Receptor in the Mammary Terminal End Buds in Rats Fed Soy Protein Isolate or Whey Protein Hydrolysate, FASEB J. 15, A280 (Abstr.). 50. Friedman, M., and Brandon, D.L. (2001) Nutritional and Health Benefits of Soy Proteins, J. Agri. Food Chem. 49, 1069–1086.

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Chapter 33

Public Health Implications of Dietary Phytoestrogens Joel Rotsteina, and G. Sarwar Gilanib Toxicological Evaluation Section aChemical Health Hazard Assessment Division, Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Canada bNutrition

Research Division, Bureau of Nutritional Sciences, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Canada

Introduction Phytoestrogens are compounds found in plants and are structurally similar to estrogen. This class of chemicals includes isoflavones, coumestans, and lignans. Phytoestrogens can be found in a large variety of foods, including grains, cereals, nuts, vegetables, and fruits. They are most abundant in soybeans, flaxseed, and several types of berries. Although excellent work has been conducted on coumestans and lignans (1–3), this chapter will discuss isoflavones exclusively. In the human diet, isoflavones are found in significant amounts only in soy-based foods. In Asian populations, for whom soy-based foods are a substantial part of the diet, consumption of isoflavones has been estimated to be in the range of 20–50 mg/person/d. In contrast, soy-based foods are not a significant part of Western diets, and the consumption of isoflavones by Western peoples is estimated to be considerably less than 1 mg/person/d. This difference in exposure may be significant because the consumption of soy-based foods has been associated with lower incidences of prostate and breast cancer, cardiovascular disease, and menopausal symptoms in Asian populations relative to Western peoples (2). These associations have been the basis for a relatively extensive database on isoflavones derived from epidemiologic, clinical, and experimental studies. Isoflavones include the chemicals genistein, daidzein, and glycitein. Genistein and daidzein are the predominant isoflavones and together make up greater than 90% of the total isoflavones in soy-based foods. Isoflavones are unusual among natural substances in that their potential to cause physiologic effects has been examined in a variety of subpopulations, including the fetus, the infant, men and women. This chapter will highlight the literature that has examined these subpopulations, with details on their exposure to isoflavones and descriptions of the potential health hazards and health benefits of isoflavone consumption.

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Phytoestrogens and the Fetus Exposure Fetal exposure to isoflavones is a consequence of the dietary habits of the mother. This was investigated in a study (4) in which the concentrations of isoflavonoids, i.e., genistein and daidzein plus their respective metabolites, O-desmethylangolensin and equol, were determined in maternal plasma, cord plasma, and amniotic fluid at the time of delivery. The seven healthy pregnant women in the study had been consuming a traditional Japanese diet. This diet contained 20–50 mg of isoflavones/d (0.3–0.8 mg/kg bw/d) (5), which is substantially more than the less than 1 mg present in daily Western diets (<0.08 mg/kg bw/d) (6). The results showed that isoflavonoids pass from the maternal circulation into the amniotic fluid through the placenta. At delivery, the fetus is exposed to isoflavonoids at concentrations similar to those in the mother’s plasma (range of total isoflavonoids in amniotic fluid: 56–799 vs. 19–744 nmol/L in mother’s plasma; Table 34.1). During pregnancy, however, fetal exposure to isoflavonoids may be much higher than these levels. Given that isoflavones have a circulating half-life of <8 h and that most mothers did not eat for at least 2 h before delivery, the fetus is probably exposed to even greater concentrations of isoflavones than those observed here. The study also showed that the mother and fetus conjugate isoflavones in similar proportions, such that >90% of the isoflavonoids are glucuronidated and <5% of them are the free form. The authors suggested that, if these compounds are like estrogen, only free and not conjugated isoflavones would be able to cross the placental barrier. If this is so, then the fetus must be able to conjugate free isoflavones. Animal data are consistent with these findings. In rats, oral administration of the aglycone form of genistein results in the aglycone form of genistein crossing the placenta and the conjugated form circulating in the fetal blood and tissues (7,8). Health Effects For many generations, Asian populations have consumed diets that are rich in soy foods and therefore rich in isoflavones. Given the long history of safe use, it is unlikely that women in this population are placing their fetuses at risk by consuming soy products. There is no evidence in humans of an adverse effect to the fetus when the mother consumes significant amounts of isoflavones in food, whether in an Asian or Western diet. However, one epidemiologic study (9) involving the assessment of nearly 8000 boys, found an association between a mother’s vegetarian diet and the development of hypospadia (a male developmental anomaly, in which the urethra opens on the underside of the penis). The authors conclude that vegetarian mothers have greater exposure to isoflavones than omnivore mothers, and that these compounds may explain the developmental defect. Yet the proportion of boys with hypospadia whose mothers drank soy milk or ate soy foods regu-

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TABLE 34.1 Summary of Fetus and Infant Exposure Studies

Subjects

Treatment length (d)

Subjects n

Fetus

270–154

7

Infant

112–154

Infant

56–154

Test material

Dose of isoflavonesa (mg/subject)

(mg/kg bw/d)

Traditional Japanese diet

~20

ND

7

Soy-based infant formula

28–47

4.5–8.0

7

Cow’s milk– based formula

ND

ND

7

Breast milkb

ND

ND

4

Soy-based infant formula

43–48

6–10

aDoses

Effects Fetal exposure to total isoflavonoids: 56–799 nmol/L (similar to maternal exposure). Fetal conjugation results in >90% of isoflavones in glucuronidated form (similar to maternal pattern). Plasma concentration of isoflavones: 684 ng/mL (genistein, ~2530 nmol/L) and 295 ng/mL (daidzein, ~1160 nmol/L). Plasma concentration of isoflavones: 3.2 ng/mL (genistein, ~11.6 nmol/L) and 2.1 ng/mL (daidzein, ~8.1 nmol/L). Plasma concentration of isoflavones: 2.8 ng/mL (genistein, ~10.2 nmol/L) and 1.4 ng/mL (daidzein, ~5.86 nmol/L). Plasma concentration of isoflavones: 1010–3440 nmol/L (genistein plus daidzein). No free isoflavones were detected; all isoflavones were in the glucuronidated form.

Reference 4

20

23

described as mg/kg bw/d were generally estimated on the basis of a body weight of 60 kg. isoflavone concentration in breast milk was determined to be 5–15 ng/mL (21). This results in the consumption rate of 0.005–0.01 mg of isoflavones/(infant/d). ND, not determined. bThe

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larly was not statistically different from the proportion of boys with hypospadia whose mothers did not consume these foods. The lack of an association between hypospadia and soy brings the authors’ conclusion into question because soy is the most significant source of isoflavones in the diet. Evidence for developmental toxicity is stronger in animal studies, which have shown that isoflavones can cause changes in estrogen-sensitive tissues. For example, subcutaneous injections of genistein to pregnant rats at doses as low as 0.1 mg/kg bw/d during d 15 through 20 of gestation showed that the pups were more susceptible to the development of mammary gland cancer when later exposed to chemical carcinogens (10,11). Dietary exposure of dams to genistein at doses as low as 11 mg/kg bw/d, from gestation d 7 through weaning, with pups continuing the same diet as the dams until postnatal d 50 (termination), resulted in microscopic changes in the mammary gland tissue and reproductive tissues of pups. In the mammary gland, ductal/alveolar hyperplasia and hypertrophy were observed in the female progeny. Additional histologic changes in female progeny at doses of 70 mg/kg bw/d and higher included abnormal cellular maturation in the vagina and abnormal antral follicles in the ovaries. In male progeny, delayed spermatogenesis was seen when dams had received doses ≥70 mg/kg bw/d. Both sexes showed developmental delays, such as late eye opening and ear unfolding, at ≥70 mg/kg bw/d (12). Similarly designed studies, in which the dam and pups had been administered genistein in the diet, examined developmental end points in greater detail. Generally, the findings showed that genistein in the dose range of 60–100 mg/kg bw/d can cause slight developmental delays or accelerations, minor weight changes to reproductive organs, and minor alterations to some sex-related behaviors (13–15). In addition, studies have shown that genistein in the range of 10–40 mg/kg bw/d has no significant effect on these developmental parameters (13,14,16,17). This range of intake is greater than ten times the amount consumed by an adult human intake from a traditional Asian diet (1 mg/kg bw/d). In contrast to the many studies with genistein, relatively few studies have been conducted with daidzein alone. In a recent study (18), rat dams were fed daidzein up to 66 mg/kg bw/d for 2 wk before mating, throughout pregnancy, lactation, and weaning; then the pups continued to consume the parental diet until postnatal d 50. No significant adverse effects were observed in the daidzein-fed pups with respect to survival or growth when compared with the control pups. Histologic examination of the reproductive tracts of daidzein-fed female pups at postnatal d 50 revealed no anomalies in the vagina, uterus, ovaries, or mammary gland. These findings with daidzein contrast with the effects of genistein at similar doses, examined in other studies (12). These results suggest that equal doses of different isoflavones do not have equal biological effects. In an interesting abstract (19), an experiment in rats in which soy milk was consumed daily over five generations of rats was described. The study compared rats fed a control diet, or a diet containing cow’s milk, soy milk or a cow-soy milk

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mix. Rats fed soy milk consumed 24 mg isoflavones/d, equivalent to ~80 mg/kg bw/d in adult rats. There were no effects with soy milk treatment in treated rats with respect to reproductive outcomes and histology of the reproductive tracts when compared with control rats. This finding suggests that over multiple generations of exposure to isoflavones in the diet, there is no cumulative toxicity at a dose that causes no adverse effect over a single generation.

Phytoestrogens and the Infant Exposure Infants who are fed soy-based infant formula are the subpopulation most heavily exposed to isoflavones from the diet. Setchell (20) examined the isoflavone composition of five different brands of commercially available soy-based infant formulas. The findings showed that the soy-based formulas contained concentrations of total isoflavones (genistein plus daidzein) between 32 and 47 µg/mL. This is much greater than the 5–15 ng/mL present in breast milk from mothers consuming a Western diet or the 50–150 ng/mL of women who have consumed soy products during lactation (21). On the basis of this concentration range in the soy formula, it was calculated that an average 4-mo-old infant fed a soy-based formula consumed between 4.5 and 8.0 mg/kg bw of total isoflavones daily. Setchell (20) compared the plasma concentrations of isoflavones in 4-mo-old infants fed soy-based formula, cow’s milk formula, or human breast milk (n = 7/group). It was determined that the 4-mo-old infants consuming soy-based formula had mean plasma concentrations of genistein and daidzein of 684 ± 443 and 295 ± 60 ng/mL, respectively. These values were significantly greater than the concentrations of genistein and daidzein found in the plasma of infants fed cow’s milk formula (3.2 ± 0.7 and 2.1 ± 0.3 ng/mL, respectively) or human breast milk (2.8 ± 0.7 and 1.4 ± 0.1 ng/mL, respectively) (Table 34.1). Further, equol, a metabolite of daidzein that is more estrogenic than daidzein itself, was present in the plasma of some infants in the concentration range of 3–5 ng/mL. Equol was observed in this concentration range regardless of which formula the infant was consuming. This may reflect the immaturity of the infant gut flora; gut bacteria are thought to be critical for the metabolism of daidzein to equol (22). Subsequent work (23) determined that >90% of the isoflavones are in the glucuronidated form in the plasma of infants consuming soy-based formula. An in vitro study has shown that the glucuronidated forms of genistein and daidzein are ~50 times less able to bind to the estrogen receptor than their respective free forms and are therefore less likely to be as estrogenic as the free form (24). Health Effects General Health. The paradox of high isoflavone exposure during infancy was expressed by Setchell (20) who stated “that phyto-oestrogens circulate in soy for-

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mula–fed infants at concentrations that are 13,000–22,000 times higher than plasma oestradiol concentrations, which ranges from 40 to 80 pg/mL in the newborn. Even allowing for their weak oestrogenic activity, dietary isoflavones must have some biological activity in the infant.” The article also points out that the plasma concentrations of isoflavones in infants fed these formulas is an order of magnitude greater than in adults who consume a traditional Japanese diet. Although tens of millions of infants have consumed soy-based formula over the last 50 years, no adverse biological effect has been demonstrated in spite of the high concentration of isoflavones in these infants. Several studies show that babies who consume methionine-supplemented soybased formula grow normally during infancy (25–29). In a retrospective study (30), 248 subjects who had been fed soy-based formula were compared with 563 subjects fed cow’s milk formula, 20–34 y after infancy. The individuals were assessed with regard to pubertal maturation, menstrual and reproductive history, height, weight, and general health. No significant differences were seen between groups of men or women when more than 30 outcomes were compared. It was observed that women who had been fed soy-based formula had a small increase but statistically significant, in the length of the menstrual bleeding period (0.37 d) compared with the cow’s milk formula group. This group also reported greater discomfort during menstruation. No other differences were shown to be significant. Overall, the clinical events observed were considered to be relatively minor. Critics of the study point out that some of the outcomes were too subjective, in that they depended on the subject’s memory of biological events. For example, as an indication of pubertal maturation, males were asked at what age their voice changed. It was also thought that the study size was too small to be definitive. For example, the incidence of cervical cancer was higher in the women fed soy-based formula as infants (4 of 128 women; 3.1%) compared with women fed cow’s milk formula (3 of 268 women; 1.1%). The difference was not significant but might have become so with a greater number of subjects. Goldman (31) suggested that the study would have been improved if breast-fed infants had been included, so that the natural background could be used as a comparison. In spite of these limitations, the study is an important contribution to the weight of evidence supporting the safety of soy-based infant formula. Thyroid. Another area of concern has been the association of soy-based formula with thyroid hypertrophy, or goiter. Several decades ago, about a dozen cases of thyroid hypertrophy were reported in infants fed soy-based formula (32–37). These thyroid growths regressed once the soy-based formulas were no longer fed to the infant. Most of these cases were associated with the formula of one manufacturer. During the 1960s, manufacturing practices improved, and the change most relevant to controlling thyroid hypertrophy was the addition of adequate amounts of iodine to the formulas. Since then, only one case report has appeared in the literature (38). The case was noteworthy because the infant was born with an abnormal thyroid,

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and the soy-based formula exacerbated the infant’s condition. The case served as a reminder to physicians to avoid feeding soy-based formula under such medical conditions (39). Interestingly, a series of animal and in vitro studies (40,41) have elegantly shown that genistein and daidzein irreversibly inhibit thyroid peroxidase. This enzyme is required to catalyze the tyrosine iodination necessary to synthesize thyroid hormone (TH). It was thought that this mechanism of inhibition would result in isoflavones decreasing thyroid function by preventing the production of adequate amounts of hormone and that this would subsequently lead to thyroid hypertrophy. However, further studies showed that dietary doses of isoflavones sufficient to inhibit thyroid peroxidase in rats failed to alter levels of the thyroid hormones T3 and T4 or thyroid-stimulating hormone (TSH), and did not lead to a hypothyroid effect (42). Premature Breast Development. Many years ago, a series of reports observed an increased incidence of premature thelarche associated with the ingestion of soybased infant formula (43–46). Hypothetically, the early breast development observed in the girls less than 8 y old was due to the estrogenic effect of the isoflavones present in soy-based infant formula. The event was unusual in that it was observed only in Puerto Rico and not reported elsewhere. Puerto Ricans living in Philadelphia were not affected, and therefore it was not considered a problem of genetic propensity. In addition, other ethnic groups living in Puerto Rico were also affected by the condition (45). An alternative explanation for the phenomenon, which took into account local environmental factors, was recently published (47). These authors found significant concentrations of phthalate esters in the serum of 41 Puerto Rican girls with premature thelarche, whereas none were found in 35 control girls. This new finding is consistent with the estrogenic activity of phthalate esters (48) and with the possibility that excessive amounts of these substances may have contaminated the infant formula (49). No other human studies have suggested an association between soy-based formula consumption and anomalous breast development. Cancer Development. One human study (30) examined the incidence of cancers among adults who consumed soy-based formula as infants. The incidence of cancers in this group was not significantly different from that of adults who had been fed milk formula. No other study has examined this issue. Nevertheless, after more than 50 y of consumption, no association has been observed between soy-based formula consumption and cancer development. This lack of association is supported by observations made in Korea. Soy-based infant formula manufactured and consumed in Korea contains five times the amount of isoflavones of North American varieties (50). Yet after 30 y of soy-based infant formula consumption, Koreans do not have an increased incidence of cancers that have been associated with the consumption of these foods.

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The animal data, however, clearly show the carcinogenic potential of genistein. A recent study demonstrated that one daily subcutaneous injection of genistein for 5 d at a dose of 50 mg/kg bw/d to neonatal mice generated uterine adenocarcinoma 18 mo postadministration (51). The study demonstrates the potential effects of this isoflavone, but the relevance to humans is not clear. The route of administration makes a significant difference to the metabolism of the test material; consequently, it is likely to affect the biological activity of the substance. When injected, genistein enters the blood as the relatively biologically active aglycone or free form (52). Ingested genistein is absorbed from the gastrointestinal tract and enters the circulation in a glucuronidated form (53), which has been shown in an in vitro system to be 50 times less able to bind to the estrogen receptor than the aglycone form (24). In contrast to the above findings, genistein administered to lactating rats through the diet at doses of ~13 mg/kg bw/d or given to neonatal rats as repeated subcutaneous injections at doses ranging from 1 to 500 mg/kg w, reduced the number of tumors per mammary gland when the mature rats were challenged with a chemical carcinogen (54–56). Dietary administration of daidzein at doses of ~13 mg/kg bw/d to lactating female rats, however, had no anticarcinogenic effect on newborn females in the same model for chemically induced mammary gland carcinogenesis (18).

Phytoestrogens and the Adult Exposure Adults are exposed to isoflavones mainly through their diets (1,6,57), although dietary supplements provide an additional route of exposure. Individuals consuming a Western diet ingest less than 0.08 mg/kg bw/d, whereas persons eating an Asian diet could eat 1 mg/kg bw/d. In contrast, supplement capsules have been reported to contain isoflavones in amounts ranging from 2.8 to 58.0 mg/capsule (58). Therefore, one high-potency capsule could contain the daily intake of isoflavones from a traditional Asian diet. Health Effects Infertility. A major concern arising from the ingestion of isoflavones is the possibility of infertility. The earliest interest in isoflavones was the discovery that the infertility in a flock of sheep was the result of these animals eating clover containing high amounts of isoflavones (59). Since that time, phytoestrogens have been observed to cause infertility in wildlife and laboratory animals (60–63). However, there are no reported incidents of infertility in humans associated with phytoestrogen consumption. People consuming an Asian diet, which contains high amounts of isoflavones, do not have overt infertility problems. A partial explanation for the absence of infertility in humans may be due to the fact that isoflavone consumption in animals is much greater on a body weight basis than in adult humans.

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Infertility in Men. In a recent study (64), some reproductive parameters were assessed in men (n = 15) who took a supplement containing 40 mg of isoflavones daily for 2 mo (0.67 mg/kg bw/d). Blood and semen samples were obtained twice during the 2-mo test period, as well as 2 mo before and 4 mo after supplementation. Concentrations of sex hormones (estradiol and testosterone), gonadotropins [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] and isoflavones (genistein and daidzein) were analyzed in the blood samples. Semen samples were analyzed for ejaculate volume, sperm concentration, total sperm count, motility, and morphology. The isoflavone concentrations of genistein and daidzein in the plasma reached ~1 and 0.5 µmol/L, respectively. No significant change was observed in any of the parameters measured. Thus, in the short term, phytoestrogen consumption at a daily dose of 0.67 mg/kg body does not seem to significantly affect these parameters of male reproductive health. Consistent with the above work were the findings of a controlled study (65) in which healthy men (n = 12) consumed 60 g of soy with or without isoflavones every day for 1 mo (equal to 45 mg isoflavones/d; 0.75 mg/kg bw/d). No significant effect was observed in the serum concentrations of androgen, LF, or FSH. In another study (66), healthy men (n = 6) consumed soy milk with every meal for 1 mo (equivalent to 3.33 mg isoflavones/kg bw/d). Neither serum testosterone nor estradiol levels were affected compared with baseline values. In a randomized controlled study (67), healthy Japanese men (n = 35) consumed 400 mL of soymilk each day for 2 mo (equivalent to 90 mg isoflavones; 1.5 mg/kg bw/d) or maintained their regular diet with no soy products. There were no significant differences between the groups with respect to serum concentrations of estradiol, testosterone, or sex hormone-binding globulin (SHBG). There was a slight decrease in the serum concentration of estrone in the soy milk group compared with its own baseline. Overall, there is no evidence that dietary amounts of isoflavones decrease male fertility or significantly alter major sex hormones in men (Table 34.2). Infertility in Women. There is epidemiologic evidence that Japanese women, who consume relatively large amounts of soy, have a lengthened menstrual cycle compared with women consuming Western diets (32 vs. 28 d) (68). These women also have plasma estrogen concentrations that are 20–30% less than their Western counterparts (69). Yet, there is no indication that Japanese women are less fertile than their Western counterparts. Consistent with the above observation is a study (70) in which the effects of soy consumption on menstrual cycle parameters were reported (Table 34.2). After a 4 mo baseline period, six healthy women stayed in a metabolic suite for at least 1 mo and consumed a controlled diet. Starting on d 1 of their menstrual cycles, the women ate 60 g of soy protein (45 mg isoflavones; 0.75mg/kg bw/d) daily for the entire month. The women were monitored for at least another 2 mo after the diet intervention. Compared with their preintervention cycle durations, the women were observed to have an increase of 2.5 d in the length of follicular phase of their

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TABLE 34.2 Summary of Infertility Studies in Men and Womena Subjects n

Men

60

15

Isoflavone supplement

40

0.67

Men

30

12

45

0.75

Men

30

6

100

3.33

Men

60

25

90

1.5

Women Pre-M

30

6

Soy protein (60 g) Soy milk (36 oz) Soy milk (400 mL) Soy protein (60 g)

45

0.75

Subjects

Test material

Dose of isoflavonesa

Treatment length (d)

(mg/subject) (mg/kg bw/d)

Effects No effect on blood concentrations on estradiol, testosterone, FSH, LH. No effect on ejaculate volume, sperm concentration total sperm count motility or morphology. No effect on blood concentration of androgen, LH or FSH. No effect on blood concentration of testosterone or estradiol. No effect on blood concentration of estradiol, testosterone or SHBG. Follicular phase of menstrual cycle increased (2.5 d) in length. Decreased blood concentration (>50%) of LH and FSH during peak times. Estradiol concentration increased (>40%) during follicular phase.

Reference 65

65 66 67 70,77

Continued Copyright 2002 by AOCS Press. All rights reserved.

TABLE 34.2 (Cont.)

Subjects

Treatment length (d)

Subjects n

Women Pre-M

30

6

Women Pre-M

90

14

Women Pre-M

30

9

Test material

Soy milk (36 oz)

Soy protein powder (53 g protein)

Soy milk (36 oz)

aAbbreviations:FSH,

Dose of isoflavonesa (mg/subject) (mg/kg bw/d)

~200 (~100 daidzein plus ~100 genistein) 10, 64, 128

3.3

5

<0.1

0.15, 1, 2

Effects No effect on blood concentration of SHBG, testosterone, or progesterone. Blood concentration of estradiol decreased (~50%) during different phases. Progesterone concentration decreased (35%) during luteal phase. No effect on menstrual cycle length. No effect on plasma concentration of LH, FSH, or estradiol. No effect on plasma concentration of testosterone, cortisol, TH, TSH, insulin, or SHBG. No effect on endometrial histology. Decreased (20%) concentration of estradiol and decreased (33%) Progesterone during entire menstrual cycle. No effect on LH, FSH or SHBG.

Reference

71

72

73

follicle-stimulating hormone; LH, luteinizing hormone; pre-M, premenopausal; SHBG, sex hormone–binding globulin; TH, thyroid hormone; TSH, thyroid-stimulating hormone. bDoses described as mg/(kg bw/d) were generally estimated on the basis of a body weight of 60 kg.

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menstrual cycle when consuming soy (15 ± 0.9 vs. 17.5 ± 2.3 d) and a delay in menstruation of 1.5 d (27 ± 2.4 vs. 29 ± 2.0 d; not significant). Five of the six women had an increase in their menstrual cycle length, with the increases ranging from 1 to 5 d. The remaining subject had a decrease in cycle length of 2 d. After the soy treatment, the mean menstrual cycle length returned to the baseline value. In addition, while consuming the soy diet, the peak plasma concentrations of LH and FSH decreased 60 and 50%, respectively, compared with baseline values. No effects were observed on the plasma concentrations of SHBG, testosterone, or progesterone. Estradiol concentrations were increased greater than 40% during the follicular phase of the cycle during the soy diet. In another study (71), six healthy women consumed doses of 3.3 mg isoflavones/ kg bw/d over the course of a single menstrual cycle (Table 34.2). It was shown that the entire menstrual cycle length increased during isoflavone consumption compared with baseline lengths (28.3 vs. 31.8 d). Plasma estradiol concentrations were decreased 31–81% during the different phases of the cycle. Plasma progesterone concentration was decreased 35% during the luteal phase. Two to three cycles after ingesting soymilk, the mean cycle length returned to the baseline value. Both studies showed large individual variation in the parameters examined. For example, in the second study (71), one subject had a 12-d increase in her menstrual cycle length during the isoflavone consumption period. Such an extreme change biased the results of the study. In a further investigation (72), healthy women (n = 14) were enrolled in a randomized, crossover design feeding intervention study in which subjects consumed diets containing isoflavones at doses 0.15, 1 or 2 mg/kg bw/d (Table 34.2). Diets were consumed for three menstrual cycles plus 9 d, starting at d 2 of menses. Each diet was followed by a 3-wk washout period before a new diet was initiated. The results showed no significant effects on the length of the total menstrual cycle or any of the phases. There was a slight decrease in the plasma concentrations of LH and FSH during the preovulation phase. No dose-dependent changes were observed in the plasma concentrations of LH, FSH, or estradiol. No effects were observed on plasma concentrations of testosterone, cortisol, TH, TSH, insulin, or SHBG. The investigators also examined endometrial biopsies, which were sampled during the luteal phase of the third cycle of each of the diets. No effect was observed on endometrial histology. All of these findings must be reconsidered in light of a recent paper (73) in which the effect of an isoflavone-free soy diet was assessed on ovarian hormones in premenopausal women (Table 34.2). Nine women consumed a diet containing soy but with only a total of less than 5 mg of isoflavones (<0.1 mg/kg bw/d). The diets were consumed for a single menstrual cycle. The consequence of the soy diet was a 20% reduction in circulating level of estradiol throughout the entire menstrual cycle, and a decrease of 33% in progesterone levels, compared with the subjects consuming the Western diets at home. No effects were observed on LH, FSH, or SHBG. Although preliminary, this study suggests the possibility that dietary levels

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of isoflavones may not be responsible for the biological activities observed with soy consumption. Cancer. In the development of breast and prostate cancer, sex hormones are thought to play a significant role (74,75). However, diet is believed to affect the production, metabolism, and mechanism of action of hormones in these disease processes (76). Low incidences of these cancers in populations that consume relatively large amounts of isoflavones suggest the possibility that this component of food may modulate the development of both of these hormone-sensitive cancers (2,77). Prostate Cancer. The epidemiologic evidence of an association between increased isoflavone consumption and decreased incidence of prostate cancer is relatively consistent. However, at least one prospective study involving more than 122,000 Japanese men showed a decreased incidence of prostate cancer with an increased consumption of green and yellow vegetables but no association with soy consumption (78). On the other hand, a prospective study examining nearly 8000 Japanese men over a 20-y period, showed a dose-response trend for tofu consumption and a decreased incidence of prostate cancer (79). Another prospective study examined the relationship between soy milk consumption and prostate cancer incidence (80). In that study, the incidence of prostate cancer was assessed in more than 12,000 Seventh-Day Adventist men during a 5-y follow-up period. The result showed that there was a 70% reduction in the relative risk of prostate cancer for those men who consumed soy milk more than once a day (3 of 223 men) compared with those men who never drank soy milk (190 of 10,875 men). The authors of the paper estimated that the men who consumed soy milk ingested ~10 mg of genistein and 7 mg of daidzein daily (equivalent to 0.28 mg of isoflavones/kg bw/d). In a case-control study, phytoestrogen intake from several different foods was assessed with respect to prostate cancer risk (81). In that study, 83 cases and 107 controls were assessed for phytoestrogen intake. The analysis showed an inverse relationship between prostate cancer development and the consumption of coumesterol and daidzein. The inverse relationship was statistically weaker with the ingestion of genistein and prostate cancer development. The daily total isoflavone intake was estimated to be 1.2 mg/person (0.02 mg/kg bw/d). Compared with an Asian diet (1.0 mg/kg bw/d), the daily amount of isoflavones consumed in that study was relatively small, suggesting that the observed effect may be due to other factors. There are few clinical studies that have examined the role of isoflavones in the development or progression of prostate cancer. In one study, the concentrations of lignans and isoflavones were determined in plasma and prostatic fluid from normal, healthy Portuguese, Chinese, and British men consuming their traditional diets (82). With 20 men/group, the results showed that the prostatic fluid of men from Hong Kong in which the incidence of prostate cancer is lower than in the

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other two countries, contained a relatively high mean concentration of daidzein (70 ng/mL) and equol (172 ng/mL) compared with samples from Portuguese (4.6 and 1.72 ng/mL, respectively) and British men (11.3 and 0.5 ng/mL, respectively). In a second study (83), a randomized, double-blind, crossover study was performed in elderly men (n = 34), who had elevated serum concentrations of prostate specific antigen (PSA) and the soluble component of the protooncogene p105erbB2 (p105) (Table 34.3). The men consumed one of two soy drinks daily for a period of 6 wk, and then drank the second beverage each day for an additional 6 wk. Both beverages contained 20 g of isolated soy protein. However one drink contained 42 mg of genistein and 27 mg of daidzein (equal to 1.2 mg of isoflavones/kg bw/d) and the second beverage provided 2.1 and 1.3 mg of the isoflavones, (equal to 0.06 mg of isoflavones/kg bw/d) respectively. The results showed no decrease in the concentrations of PSA or p105 with either treatment. These data suggest that at the modest doses tested and for the short duration of this test, these isoflavones had no effect on these biomarkers of prostate cancer development (Table 34.3). In rodent models for prostate cancer, when soy protein isolates containing isoflavones or genistein alone are added to the diet, they have been shown to reduce the incidence of chemically induced and naturally occurring prostate cancers (84–87). Breast Cancer. Epidemiologic evidence shows that in places in which soy consumption is high, breast cancer incidence is low (88). However, when the epidemiology of soy consumption is examined more closely, the association is weak or absent (89). Some cohort studies show a reduction of breast cancer cases with soy consumption (90), and others have shown no association with soy consumption (91,92). Similarly, some case-control studies have given results that show a decreased incidence of breast cancer with soy consumption (93–96), and other studies do not show a decreased incidence (97,98). There are many clinical studies that have assessed the effect of soy consumption on breast cancer development. The studies that have been performed have been of short duration and have examined variables that are associated with breast cancer development, but not actual cancer formation. Most of them examined hormonal changes as a result of diet supplementation with soy. These results have been mixed, in that some studies show that soy consumption decreases serum estradiol concentrations (71,99); in other studies, no effect was observed (100, 101). In one study (102), it was observed that individuals who can metabolize daidzein to equol had a hormone pattern that was associated with lower breast cancer risk (e.g., lower concentrations of estrone, estrone-sulfate, or testosterone) compared with the pattern of individuals who did not excrete equol. Other clinical studies have examined the effect of soy protein consumption on the breast tissue itself. In one study (101), 24 healthy women; 14 premenopausal and 10 postmenopausal) consumed a soy protein isolate daily for 6 mo (80 mg isoflavones/person/d; 1.3 mg/kg bw/d) (Table 34.3). Nipple aspirate fluid (NAF) volume, fluid color, NAF cytology, and gross cystic disease fluid protein (GCDFP-

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TABLE 34.3 Summary of Clinical Studies on Prostate and Breast Cancera Treatment length (d)

Subjects n

Men

42

34

Women

180

14 pre-M 10 post-M

Women

14

~30 pre-M

Subjects

Test material Isolated soy protein (20 g) Isolated soy protein (38 g) Soy supplement (60 g)

aAbbreviations: bDoses

Dose of isoflavonesb (mg/subject)

(mg/kg bw/d)

3.4 or 69

0.06 or 1.2

80

1.3

45

0.75

PSA, prostate specific antigen; pre-M, premenopausal; post-M, postmenopausal. described as mg/kg bw/d were generally estimated on the basis of a body weight of 60 kg.

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Effects

Reference

No effect on concentrations of PSA or p105 with either treatment.

83

Increased (2–6×) volumes of nipple aspirate fluid volume. Altered histology of nipple aspirate fluid cells. Nipple aspirate fluid contained less apolipoprotein D and increased pS2 concentrations. No effect on breast epithelial cell proliferation, estrogen or progesterone receptor status.

101

103

15) concentration were used as indications of estrogenic activity in the breast. NAF samples were taken once each month, beginning 2 mo before the start of the ingestion of soy and continuing for 3 mo after the end of the soy trial period. The entire study was conducted over 12 mo. The results showed that NAF volumes increased two- to sixfold during the time that soy was consumed compared with the pretreatment period, which suggested increased estrogenic activity. However, this finding was observed only in premenopausal woman and not in the postmenopausal women. The NAF cytology of 4 of 14 premenopausal and 3 of 10 postmenopausal women showed epithelial hyperplasia on 1–5 occasions during the time these subjects were ingesting soy protein or afterward. Only one premenopausal women had this finding before the soy protein treatment. The GCDFP-15 concentrations showed a moderate decrease in the premenopausal women when they were consuming soy but no significant change was observed in the postmenopausal women. There was no significant change in the color of NAF fluid during the study. The authors concluded that the consumption of soy protein was an estrogenic stimulus to breast tissue on the basis of the findings of increased NAF volume and the presence of hyperplasia of epithelial cells in NAF. Overall, the biological significance of the findings was not clear because the NAF volumes ranged widely among individuals, and one or two women with high values could have greatly influenced the results. In another clinical study (103), randomized groups of premenopausal women (n = ~30) consumed a daily dietary soy supplement containing 45 mg isoflavones (equal to 0.75mg/kg bw/d) or a control diet for 14 d (Table 34.3). NAF was collected before and after soy treatment. Apolipoprotein D (also known as GCDFP-24) and pS2 are two proteins whose expression is under estradiol control. Their concentrations were measured in NAF as an indication of estrogenicity. The results showed that after soy supplementation, NAF contained significantly lower concentrations of GCDFP-24 and increased pS2 levels compared with pretreatment levels. These findings suggest that soy consumption generated an estrogenic effect. However, soy supplementation did not affect breast epithelial cell proliferation, estrogen, or progesterone receptor status, apoptosis, mitosis, or Bcl-2 expression. These authors concluded that dietary soy produces a very mild estrogenic effect. The actual health consequences of this effect are not clear. Animal studies exploiting models of chemically induced mammary gland carcinogenesis, have shown protective effects of soy consumption in rodents (104). However, studies with genistein show that these effects vary with the timing and route of exposure to the isoflavone (11,54,55,105,106). For example, in utero exposure to genistein administered subcutaneously to dams increases the susceptibility of the pups to develop mammary cancer when subsequently treated with a chemical carcinogen; however, feeding soy containing the same amount of genistein to the dams has no effect on tumor susceptibility (107). Neonatal and prepubescent exposures to genistein or soy appear to be the most protective to rodents when exposed to a chemical carcinogen. This result is interesting considering a recent epidemiologic finding in which increased consumption of soy during ado-

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lescence was associated with a decreased incidence of breast cancer in pre- and postmenopausal women (108). Menopausal Symptoms. Epidemiologic evidence suggests that some symptoms of menopause are less common in countries in which the population consumes large amounts of isoflavones compared with people who have low dietary intakes (109). It is suggested that during menopause, isoflavones may act to replace the estrogens that the body no longer produces in sufficient quantity. About twelve clinical studies have been conducted, and they have examined the effect of isoflavones on vaginal cytology and the incidence of symptoms such as hot flushes, night sweats, mood swings, lost libido, and urinary frequency. The most common symptom measured is the hot flush. These are discrete events and can be monitored by the subjects themselves. Two studies have been conducted using supplements of isoflavone isolated from red clover (Table 34.4). In a blind, randomized, controlled study (110), thirtysix postmenopausal women consumed tablets of the placebo, 40, or 160 mg of isoflavones every day for 12 wk. Each supplement tablet contained a total of 40 mg of total isoflavones (the exact amount of each isoflavone was not provided in the paper). In the groups that consumed the placebo, 40, or 160 mg of isoflavones, the rate of hot flushes decreased 46, 44 and 26%, respectively. Only the highest dose group showed a significant decrease in their rate of hot flushes compared with the control group. However, other symptoms of menopause were not significantly different for the women consuming the supplements. In a randomized, blind, placebo-controlled crossover study (111), fifty-one postmenopausal women consumed a 40-mg isoflavone tablet derived from red clover supplement or a placebo, daily for 12 wk. After a 1-mo “washout period,” the subjects previously consuming supplements began to ingest the placebo and vice versa for an additional 12 wk. There were no significant differences in menopausal symptoms between the groups. Other clinical studies (112–114) have used soy flour or isolated soy protein as the test material. Generally, these studies involved less than 100 subjects who consumed 0.5–2.5 mg isoflavones/kg bw/d for periods of 4–12 wk. At the higher dosages, these amounts are similar to the intakes of individuals consuming a traditional Asian diet. Generally the studies showed a modest improvement in one or two of the parameters examined. One of the larger studies (115), was a double-blind, randomized, placebo-controlled trial involving 104 postmenopausal women (Table 34.4). Fifty-one subjects consumed 60 g of isolated soy protein (76 mg isoflavones; 1.3 mg/kg bw/d) daily and 53 women ingested a placebo of casein. Both groups were treated for 12 wk. The women ingesting soy experienced a 45% reduction in the mean number of hot flushes/24 h compared with a 30% reduction in the placebo group. Although statistically significant, the actual decrease in hot flushes was a reduction from an initial 11 hot flushes over 24 h, down to 6 in the soy group compared with 8 in the placebo group after 12 wk.

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TABLE 34.4 Summary of Clinical Studies on Menopausal Symptom and Isoflavone Exposure

Subjects

Treatment length (d)

Subjects n

Women

84

12

Women

84

51

Women

84

~52

Women

84

~88

Women

168

~24

aDoses

Test material Isoflavone supplement Isoflavone supplement Isolated soy protein (60 g) Soy isoflavone extract Whey, soy (depleted of isoflavones) or soy protein (40 g)

Dose of isoflavonesa (mg/subject)

(mg/kg bw/d)

0, 40, 160

0, 0.67, 2.7

0 or 40

0 or 0.67

0 or 76

0 or 1.3

0 or 50

0 or 0.8

0, 4.4, 80.4

0, 0.07, 1.34

described as mg/kg bw/d were generally estimated on the basis of a body weight of 60 kg.

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Effects All groups showed a decrease in the rate of hot flashes (46, 44, and 26%, respectively). No difference between the two groups with respect to the rate of hot flashes. Women consuming isoflavones had a greater decrease in the rate of hot flashes than women ingesting placebo (30 vs. 45%). No difference between the two groups with respect to the rate of hot flashes. No difference among any of the groups with respect to the rate of hot flashes.

Reference 110 111 115

116

117

Another large, double-blind, randomized, placebo-controlled trial (116), involved a group of 177 postmenopausal women, who consumed a soy isoflavone extract containing 50 mg of isoflavones (0.8 mg/kg bw/d) or a placebo, daily for 12 wk (Table 34.4). The investigators assessed the daily incidences of hot flushes and their intensity. In addition, endometrial thickness, vaginal histology and plasma concentrations of SHBG and FSH were evaluated. Within 2 wk of treatment, decreases in the incidence and severity of hot flushes were observed in the soy group but not in the control group. However, with time, both groups showed decreases in the mean number of hot flushes. The decrease was greater in the soy group than in the control group after 6 wk, but the difference was not significant at 12 wk. The incidence of hot flushes decreased from ~8.6 to 6.5/d. There were no other differences observed between the two groups during the study. In an interesting smaller study (117) investigators examined the change in menopausal symptoms in groups of 24 postmenopausal women after consumption of soy protein containing isoflavones (80.4 mg of isoflavones/d; 1.34 mg/kg bw/d), soy protein depleted of isoflavones (4.4 mg of isoflavones/d; 0.07 mg/kg bw/d), or whey protein for 24 wk (Table 34.4). At the end of the study, all treatment groups showed significant reductions with respect to frequency, duration, or severity of hot flushes or night sweats compared with baseline values. The lack of difference between the groups treated with soy containing high or low amounts of isoflavones suggests that if soy has an effect on menopausal symptoms, the isoflavones may not be the active component. Osteoporosis. Estrogen deficiency increases bone loss and the likelihood of fractures. In menopausal women, estrogen therapy decreases bone loss and reduces the incidence of fractures (118). Hypothetically, due to their estrogenic properties, phytoestrogens may act similarly to estrogen. Support for this hypothesis is provided from the results of two prospective studies (119,120) that showed that the intake for 6 mo of either soy or soy protein isolates containing ~80–90 mg/d of isoflavones (1.3–1.5 mg/kg bw/d) by postmenopausal women was associated with a significant (P < 0.05) increase in bone mineral content and bone density, compared with their respective control groups. In a recent clinical study (121) 46 postmenopausal women consumed a supplement derived from red clover (containing formononetin, genistein, biochanin, and daidzein) in daily doses of 28.5, 57 or 85.5 mg/d (0.48, 0.95 and 1.43 mg isoflavones/kg bw/d) for 6 mo. The results showed an increase in bone mineral density at the mid and high doses. Although encouraging, these studies are preliminary. Bone remodeling takes between 30 and 80 wk to occur (122), and these studies were comparatively short term. Therefore, the actual benefits to bone health are inconclusive. The studies are consistent in that they have shown a modest benefit to bone health (a reduction in the rate of bone mineral loss). This benefit may lead to decreased osteoporosis and a decrease in the incidence of fractures, but to demonstrate this possibility further, studies of longer duration and with greater numbers of subjects are required.

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Cardiovascular Disease. On the basis of the observation that men and postmenopausal women are at the greatest risk for cardiovascular disease (CVD), estrogen is thought to play a mitigating role in this condition (118). Estrogens have been shown to improve the risk factors associated with lipoprotein profile and vascular function. Similarly, phytoestrogens have been tested with respect to several of these risk factors. Although early studies were promising, it seems that the cholesterol-lowering effect of soy isoflavones may be due almost entirely to the soy protein (123). With regard to vascular function, randomized, double-blind, placebo-controlled clinical trials have found no effect on blood pressure (124) or endothelial function (125) from the consumption of isoflavone supplements. In two small clinical studies (126,127), it was demonstrated that isoflavone supplements can improve systemic arterial compliance, and thus reduce the risk of CVD. Cognitive Function. In clinical studies, hormone replacement therapy has been shown to slow the decline of cognitive function in postmenopausal women (128). Therefore, it was assumed that phytoestrogens might function similarly because of their estrogenic activity. Some work has been consistent with this assumption. In a randomized controlled study (129) involving a total of 27 men and premenopausal women, the daily consumption of soy containing 100 mg of isoflavones (1.7 mg/kg bw/d) for 10 wk improved memory compared with diets containing 0.5 mg of isoflavones (<0.01 mg/kg bw/d). In contrast, a retrospective study (130,131) involving nearly 4000 JapaneseAmerican men found an association between high tofu consumption during midlife and decreased cognitive function in later life. High tofu consumption was defined as eating tofu ≥2 times/wk. A commentary on the study suggested that the men who were high tofu consumers also spent childhood in greater poverty than low tofu consumers. In their opinion, the nutritionally deficient diet of their childhood was likely more responsible for the decline in cognitive function than the consumption of tofu. They also point out that age, strokes, and other factors were much stronger predictors of decline than tofu intake.

Discussion The health risk assessment of isoflavones is complicated because of many factors. For example, most studies were performed using soy-based foods as a source of isoflavones and not pure chemicals. Soy-based foods contain a large number of biologically active substances in addition to isoflavones. These substances include soy protein, phytic acid, saponins, protease inhibitors, phytosterols, and vitamin E (132). Their presence makes the interpretation of the studies difficult because the phenomena observed may be due to the activity of these other substances and not isoflavones. Alternatively, the other substances may increase or decrease isoflavone activity (133,134). For example, the presence of certain carbohydrates in soy-based foods can promote the growth of gut bacteria and lead to enhanced daidzein metab-

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olism with greater equol production. The consequence of increased amounts of equol could be greater estrogenic activity (133). Interpretation of these studies is further complicated by the fact that soy-based foods contain several types of isoflavones. Genistein, daidzein, and glycitein and all their precursors, metabolites, and conjugated forms are not equal in terms of their estrogenic properties (3). Therefore, comparing the effects of total isoflavones used in different studies is not always appropriate because the putative estrogenicity of the soy food can vary depending on the relative levels and the forms of the isoflavone compounds present. Further, at least one isoflavone has demonstrated biological properties in addition to estrogenicity. Genistein has been shown to act as a tyrosine kinase inhibitor (135), an inhibitor of topoisomerase activity (136), and an inhibitor of angiogenesis (137). Some or all of these activities might affect the observations made in individuals consuming soy food. Consequently, the amount of genistein present in a food might be the single most significant factor in producing the effects seen in an experiment using a mix of isoflavones. Other complications in evaluating studies with soy-based foods or pure substances relate to individual metabolic variation. For example, daidzein can be metabolized to equol, a more estrogenic substance, but this occurs in only about one third of the human population (22,138,139). Therefore, findings may be biased if these individuals are not considered separately. Generally, this issue is not addressed in clinical studies.

Conclusions In spite of the limitations of the studies, some assessment of the role of isoflavones in public health can be made. As stated in the Introduction, these natural substances are unusual because their effect on health has been examined in several subpopulations. Each of these groups possesses a distinct biology; as a result, isoflavones may interact with each subpopulation to generate unique biological outcomes. Generally, the fetus is considered most sensitive subpopulation to toxic substances. The data summarized here suggest that in Asia, fetuses are exposed to amounts of isoflavones similar to those in their pregnant mothers. No apparent toxicities have been associated with these diets, which have been consumed over many generations. Therefore, the consumption of dietary isoflavones by the mother seems to have no adverse effect on the health of the fetus. However, the use of isoflavone supplements by pregnant women is another issue. The effect of supradietary amounts on the health of the fetus is not known. Even at dietary levels, supplement use may pose a problem. Specifically, the ingestion of supplements results in a bolus dose of the entire day’s intake. This form of consumption may result in peaks of elevated concentrations of isoflavones in the blood above those created by consuming food containing isoflavones. The effect of these elevated concentra-

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tions of isoflavones on pregnant women or infants is not known. Therefore, the use of isoflavone supplements even at dietary amounts cannot be considered safe without further investigation. Infants constitute another subpopulation very sensitive to toxins. As detailed in previous sections, breast-fed infants and those receiving milk-based formula are exposed to only minute quantities of isoflavones. Even mothers who eat a substantial amount of isoflavones pass only very small amounts through their breast milk to their infants, and these are in the less biologically active glucuronidated forms (140). In contrast, infants fed soy-based formula consume larger amounts of isoflavones per kilogram of body weight than any other subpopulation (4.5–8 mg/kg bw/d). Several studies have shown that these infants grow normally throughout infancy. A recent retrospective study examining adults who consumed soy-based formula as infants revealed no significant differences in their health compared with those adults who consumed cow’s milk formula as infants. Other follow-up studies are being conducted and should give further insight into the long-term effects of soy-based infant formula feeding. However, after more than 40 y of consumption by millions of infants, there is no evidence of overt toxicity associated with soy-based infant formula, which lends strong support to their safe use. Government health agencies, pediatric societies, and dietetic associations strongly recommend breast-feeding infants for optimal nutrition (141). However, they consider soy-based formula appropriate for infant use for specific medical or personal reasons. At present, the presence of isoflavones in soy-based infant formula is not a safety concern. In adult men and women, there is no evidence that dietary isoflavones adversely affect fertility. As mentioned in previous sections, reproductive toxicities including infertility have been observed in wildlife, farm, and laboratory animals. These animals consumed large amounts of phytoestrogens for prolonged periods of time before these toxicities were observed. Further, the metabolism of isoflavones by animals can be different from that of humans. For example, daidzein can be metabolized to the more estrogenic compound equol in some animals. When rats consume a standard laboratory diet, they might ingest daidzein at doses between 1.5 and 5.0 mg/kg bw/d (142), and this can generate plasma concentrations of equol of between 2000 and 4000 nmol/L (143). In contrast, when daidzein is consumed in either a Western (<0.1 mg/kg bw/d) or Asian (1.0 mg/kg bw/d) diet, only about one third of humans develop plasma concentrations of equol >10 nmol/L. This difference between the two species in their ability to produce estrogenic equol may lead to different biological outcomes, despite similar isoflavone exposure. In men, the association between isoflavone consumption and a decreased risk of prostate cancer development is weak. Overall, the epidemiologic data suggest a decreased incidence of prostate cancer with increased isoflavone consumption, but the dosages appear to be too low to be of significant protective value (0.02–0.28 mg/kg bw/d). Completed clinical studies are too limited to contribute to the assess-

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ment of a protective effect. There is therefore insufficient scientific evidence to conclude that isoflavones provide a benefit in the prevention of prostate cancer. Similarly, in women, the association between increased isoflavone consumption and a decreased incidence of breast cancer is weak. The epidemiologic data are inconsistent, with some studies finding no effect and other studies suggesting a decreased incidence of breast cancer. No study has shown an increase of breast cancer incidence with increased isoflavone consumption. The results of clinical studies are also equivocal, with the more recent studies showing only minimal hormonal changes with soy consumption. At present, there is insufficient scientific evidence to conclude that isoflavones provide protection from breast cancer development. The hypothesis that isoflavones may relieve menopausal symptoms is based largely on epidemiologic data. About a dozen small-size clinical studies have been performed; overall, the effects have been very modestly positive. However, these are difficult studies to interpret because symptoms such as hot flushes improve with time. Large-size clinical trials are required to demonstrate clear benefits before recommendations can be made. Because estrogen replacement therapy reduces bone loss in postmenopausal women, it has been hypothesized that phytoestrogens may do the same. Although preliminary data are encouraging, the clinical studies performed to date are too short in duration to determine whether isoflavones can reverse bone loss and, more importantly, reduce the incidence of bone fractures. Longer-term studies are ongoing and their results should help determine the usefulness of these substances in the treatment of osteoporosis. With regard to cardiovascular disease, the most recent data suggest that the component of soy that improves the outcome of this disease is largely the soy protein and not the isoflavones. The use of isoflavones may provide some benefit, but the data are insufficient to recommend the use of these substances to treat or prevent the condition. Although a large epidemiologic study found an association between the consumption of soy in middle age and an increased rate of dementia in old age, the study did not account for all confounders, such as the overall nutrition of the subjects during childhood. The results of the study contradict the findings of a smaller intervention study. Further, on a population basis, the incidence of dementia in Japan is not greater than that in the United States (144). This study is not strong enough to recommend against soy consumption with regard to cognitive function. Overall, there is insufficient evidence to recommend that the public increase its intake of isoflavones to treat or prevent disease. Further study is required to substantiate the beneficial effects of isoflavones on disease processes. This additional evidence must include clinical trials with larger group sizes, longer durations, and better controlled conditions. These studies are necessary to establish the safety of isoflavones and to validate their effectiveness in improving disease conditions.

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In humans, the suggestion of adverse effects of dietary isoflavones has been based largely on observations from animal studies. The concerns are countered by a long history of safe use in all subpopulations. This conclusion may not necessarily apply to the use of isoflavone supplements. As argued earlier, the use of a supplement is equal to a bolus dose, which could lead to levels of isoflavones in the blood that exceed a safe concentration. The putative dangers are greater when it is considered how easily a supradietary dose can be achieved. This pharmacologic aspect of supplements is a concern and requires further investigation to establish safe levels of consumption. References 1. Mazur, W. (1998) Phytoestrogen Content in Foods, Baillieres Clin. Endocrin. Metabol. 12, 729–742. 2. Adlercreutz, H. (1998) Human Health Phytoestrogens, in Reproductive and Developmental Toxicology, (Korach, K.S., ed.) pp. 299–371, Marcel Dekker, New York. 3. Whitten, P.L., and Patisaul, H.B. (2001) Cross-Species and Interassay Comparisons of Phytoestrogen Action, Environ. Health Perspect. 109, 5–20. 4. Adlercreutz, H., Yamada, T., Wähälä, K., and Watanabe, S. (1999) Maternal and Neonatal Phytoestrogens in Japanese Women During Birth, Am. J. Obstet. Gynecol. 180, 737–743. 5. Nagata, C., Kabuto, M., Kurisu, Y., and Shimizu, H. (1997) Decreased Serum Estradiol Concentration Associated with High Dietary Intake of Soy Products in Premenopausal Japanese Women, Nutr. Cancer 29, 228–233. 6. Coward, L., Barnes, N.C., Setcehll, K.D.R., and Barnes. S. (1993) Genistein and Daidzein, and Their β-Glycoside Conjugates: Anti-Tumor Isoflavones in Soybean Foods from American and Asian Diets, J. Agric. Food Chem. 41, 1961–1967. 7. Chang, H.C., Churchwell, M.I., Delclos, K.B., Newbold, R.R., and Doerge, D.R. (2000) Mass Spectrometric Determination of Genistein Tissue Distribution in DietExposed Sprague-Dawley Rats, J. Nutr. 130, 1963–1970. 8. Doerge, D.R., Churchwell, M.I., Chang, H.C., Newbold, R.R., and Delclos, K.B. (2001) Placental Transfer of the Soy Isoflavone Genistein Following Dietary and Gavage Administration to Sprague-Dawley Rats, Reprod. Technol. 15, 105–110. 9. North, K., and Golding, J. (2000) A Maternal Vegetarian Diet in Pregnancy Is Associated with Hypospadias, Br. J. Urol. Int. 85, 107–113. 10. Hilakivi-Clarke, L., Cho, E., Onojafe, I., Raygada, M., and Clarke, R. (1999) Maternal Exposure to Genistein During Pregnancy Increases Carcinogen-Induced Tumorigenesis in Female Rat Offspring, Oncol. Rep. 6, 1089–1095. 11. Yang, J., Nakagawa, H., Tsuta, K., and Tsubura, A. (2000) Influence of Perinatal Genistein Exposure on the Development of MNU-Induced Mammary Carcinoma in Female Sprague-Dawley Rats, Cancer Lett. 149, 171–179. 12. Delclos, K.B., Bucci, T.J., Lomax, L.G., Latendresse, J.R., Warbritton, A., Weis, C.C., and Newbold, R.R. (2001) Effects of Dietary Genistein Exposure During Development on Male and Female CD (Sprague-Dawley) Rats, Reprod. Technol. 15, 647–663. 13. Casanova, M., You, L. Gaido, K.W. Archibeque-Engle, S., Janszen, D.B., and Heck, H. (1999) Developmental Effects of Dietary Phytoestrogens in Sprague-Dawley Rats

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