Oral Presentation Abstracts Soy Isoflavones and Cognition: A Review of the Clinical Data. Bioavailability of the Cancer Preventive Soy Peptide

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1 2005 Abstracts 1 Oral Presentation Abstracts Please note: The following abstracts have not been edited for content. They appear as submitted by the authors. Sunday, October 30, 2005 Soy Isoflavones and Cognition: A Review of the Clinical Data. L. Dye and C. Hill, Human Appetite Research Unit, Institute of Psychological Sciences, University of Leeds, Leeds, LS2 9JT, UK. The aim of this presentation is to provide a detailed examination of the human experimental studies of the effects of soy derived isoflavones (soy IF) on cognitive function in females. In contrast to the vast number of studies of health effects of soy IF, there have been only a small number of intervention studies of soy IF with cognitive endpoints. There have been three intervention studies in young adults. One study (File et al., 2001) examined young men and women and found that a high soy diet (100mg/d) for 10 weeks improved verbal and non-verbal episodic memory and mental flexibility. Gender specific effects were noted for verbal fluency and planning ability, with improved performance in females on the high soy diet, but not males. Another found effects on spatial performance after consumption of 900g of soybeans within 1 week, although this study suffers from the lack of a control group (Celec et al., 2005). In the only double-blind randomised placebo-controlled trial in young adults, Hill et al. (2005) administered 68mg/d soy IF (aglycone weight, 116mg/d conjugated weight) in a soy food matrix across two consecutive menstrual cycles. An improvement in long-term verbal memory in the first and last week of the intervention was observed. There have been 4 published RCTs of soy IF in postmenopausal women varying in duration from 3-12months with a variety of intake forms and doses (ranging from 60 to 110mg/d). Three further unpublished data sets will be reported upon. Generally soy IF appear to exert stronger cognitive effects in postmenopausal women compared to premenopausal women and effects seem to be mainly confined to improvements in memory and certain aspects of frontal lobe function. Variables such as age, time of exposure, dose and administration of isoflavones may be important mediating factors in the effects observed. Whilst these studies have provided some evidence of significant effects on cognition which are more pronounced in postmenopausal than younger women, it is not possible to know whether the effects reported in these relatively short term interventions will be sustainable in the long-term. The intriguing preliminary data warrant the funding of long-term trials Absorption of Soy Bioactives Long-Term Dietary Habits Affect Soy Isoflavone Metabolism and Accumulation in Prostatic Fluid in Caucasian Men. T. Hedlund 1, P. Maroni 1, P. Ferucci 1,R. Dayton 1, S. Barnes 2, K. Jones 2, R. Moore 2, L. Ogden 1,K. Wahala 3, H. Sackett 1, and K. Gray 1, 1 University of Colorado Health Sciences Center, Aurora, CO, USA, 2 University of Alabama at Birmingham, Birmingham, AL, USA, 3 Univ. of Helsinki, Helsinki, Finland. The soy isoflavones daidzein and genistein are believed to reduce prostate cancer risk in soy consumers. However, daidzein can be metabolized by the intestinal flora to form a variety of compounds with different bioactivities. In the current study, we investigated the influence of long-term dietary habits on daidzein metabolism in healthy Caucasian men (19-65 y old). A secondary goal was to compare plasma and prostatic fluid concentrations of 5 isoflavonoids: genistein, daidzein, equol, dihydrodaidzein, and O-desmethylangolensin. Baseline plasma levels of isoflavonoids were quantitated in 45 men by HPLC/electrospray ionization/ mass spectrometry. Participants then consumed a soy beverage daily for 1 wk, and post-soy isoflavonoid levels were quantitated in plasma and prostatic fluid. Equol was the only metabolite that appeared to be influenced by routine dietary habits. Stratified analyses revealed that men who had consumed 30 mg/d soy isoflavones for at least 2 y had 5.3-times the probability of producing equol than men who had consumed 5 mg/d (P=0.014). Additionally, those men who consumed animal meat regularly had 4.7-times the probability of producing equol than men who did not consume meat (P=0.023). Equol production was not linked to age, BMI, or the consumption of yogurt, dairy, fruit or American style fast food. Daidzein and its metabolites (but not genistein) were typically present at higher levels in prostate fluid than plasma (median = 4-13 times that in plasma). In conclusion, our data suggest that the ability of Caucasian men to produce equol is favorably influenced by the long-term consumption of high amounts of soy, and the consumption of meat. Lastly, the high concentrations of isoflavonoids in prostatic fluid increases the potential for these compounds to have direct effects in the prostate. Bioavailability of the Cancer Preventive Soy Peptide Lunasin in Animals. B.O. de Lumen 1,C.Lim 1, I. Reyes 1,P. Vichayavilas 1, H. Chu 1, J. Lee 1, R. Hurwitz 1, Y. Fang 1 M. Fitch 1 and H. Jeong 2, 1 Division of Nutritional Sciences & Toxicology, University of California, Berkeley, CA, USA, 2 Andong University, Andong, Korea. Oral bioavailability of the cancer preventive soy peptide lunasin was determined in mice and rats. 3 H-lunasin was administered by gavage to male and female CD-1 mice with lunasin-enriched soy (LES) and organs were collected at 3,6,9,12, and 24 hrs post-administration. 3 H- lunasin is absorbed and distributed in the various tissues collected including those that are targets for the most common cancers such as lungs, mammary gland, prostate and colon. At 3 hrs, approximately 30% of the total dose is accounted in all the tissues collected, extrapolation to 1 hr gives a value of approximately 38% absorption. Sprague-Dawley rats were fed LES for 4 weeks. Lunasin extracted from the blood and liver of these rats are intact and bioactive. Inflammatory Diseases Dietary Soy Protein During Pregnancy and Lactation Reduces Renal Inflammation and Disease Progression in Young Adult Rat Offspring with Genetically Determined Kidney Disease. H. Aukema 1,2, L. Cahill 1,C.Peng 1, D. Sankaran 1, N. Bankovic-Calic 1, and M. Ogborn 1,2, 1 University of Manitoba, Winnipeg, Manitoba, Canada, 2 Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada. Dietary soy protein ameliorates disease progression in several ani- Oral Presentation Abstracts

2 Oral Presentation Abstracts 2 mal models of kidney disease when soy exposure is initiated at weaning or later in life. We previously have demonstrated the beneficial effects of dietary soy protein exposure initiated at weaning in the Han:SPRD-cy rat, a genetic model of renal disease characterized by renal cyst growth and the common renal pathologies of inflammation and fibrosis. Therefore, the objective of the current study was to determine whether exposure to soy protein in the diet earlier in life would have further benefits. Han:SPRD-cy rats were given either soy protein or casein based diets from 2 wk prior to conception up to weaning and/or in the post-weaning period in a 2x2 design. During young adulthood (at 70 d of age), the progression of renal disease was examined by histology. As previously shown, soy protein compared to casein in the postweaning diet reduced disease progression, as evidenced by less inflammatory cell infiltration, renal cyst growth and epithelial cell proliferation. With respect to maternal effects, renal inflammatory cell infiltration was reduced by dietary soy protein exposure in the pregnancy/lactation period alone, and was even lower when maternal exposure was combined with soy protein feeding in the post-weaning period. For cell proliferation, maternal exposure to soy protein was just as effective as post-weaning exposure in lowering renal cell hyperproliferation in diseased kidneys. Maternal soy protein compared to casein also resulted in less proteinuria, indicating better renal function. In conclusion, these studies demonstrate that maternal soy protein exposure has long-term beneficial effects on renal inflammation and disease progression in young adult offspring in this model of genetically determined renal disease. Supported by the Manitoba Instute of Child Health Isoflavonoid-Free Soy Shows Anti-Inflammatory Activity in an Experimental Model for Chronic Nonbacterial Prostatitis. M. Kauppila 1, J. Bernoulli 1,E. Yatkin* 1, N. Saarinen 2, and R. Santti 1, 1 Dept. of Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland, 2 Functional Foods Forum, University of Turku, Turku, Finland. There is emerging evidence to support a role for non-bacterial chronic inflammation in prostate carcinogenesis. Because of the fragmentary and conflicting information available about anti-inflammatory properties of soy in prostate, we studied the effects of soy in an animal model reproducing the clinical picture of men with chronic non-bacterial prostatitis. Prostatic inflammation was induced in Noble rats with combined treatment with testosterone and estradiol known to induce prostate cancer. When kept on soy-free diet, rats developed gradually perivascular, stromal, and glandular inflammation in dorsolateral prostate. The inflammatory cells consisted mainly of T-lymphocytes (CD3+ cells). At six weeks, the inflammation proceeded to glandular form and subset of T-lymphocytes, cytotoxic (CD8+) cells were observed intraepithelially. Testosterone concentration and prostate size remained within the normal range while estrogen and prolactin concentrations were elevated. At 9 weeks, a gradual decline was measured in serum testosterone concentration and prostate weight. Dietary soy reduced inflammatory changes in each category (perivascular, stromal and glandular) while body weight gain, prostate size and hormone concentrations in serum remained unaltered. When rats were kept on isoflavonoidfree soy, the content of isoflavones in urine markedly decreased, yet an equivalent decrease in the number of inflammatory foci was observed at 3 and 9 weeks. Neither soy nor isoflavonoid-free soy showed any signs of anti-estrogenic potency. If inflammatory etiology is found important in prostate carcinogenesis, anti-inflammatory therapy based on regular consumption of soy or isoflavonoid-free soy could be enhanced. Supported by Raisio Group Foundation, Raisio, Finland. Monday, October 31, 2005 Equol 6th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease A Review of the Equol Hypothesis. M. Kurzer, University of Minnesota, Saint Paul, Minnesota, USA. It has been proposed that the isoflavone metabolite equol may be largely responsible for the biological effects of soy consumption. This is of particular interest, given that only 20-40% of the population produces this metabolite after soy consumption. Studies have shown that equol producers have hormone profiles consistent with lowered risk of breast cancer, and that in some cases equol producers may respond better than non-producers when it comes to soy benefits on indicators of cardiovascular disease, bone health, cancer risk, and menopausal symptoms. At the same time, other studies have shown no differences in effects of soy consumption between subjects who produce equol and those who do not. Thus, the relevance of equol production remains controversial, although the wide variability in production is not. Since equol is produced via bacterial metabolism, it has generally been assumed that this wide variation in equol production is due to differences in colonic microflora. If equol is critically important, the large variability in equol production could explain some of the inconsistencies in results among studies that have evaluated the biological effects of soy consumption. In addition, if it is true that equol production improves response to soy, it becomes even more important to understand the factors that influence isoflavone metabolism, and to determine whether it is possible to turn an equol non-producer into a producer. This talk will briefly summarize the factors influencing equol production, its mechanisms of action, and data related to the equol hypothesis. Equol: A Comparison of the Effects of the Racemic Compound with that of the Purified S-Enantiomer on the Growth, Invasion and DNA Integrity of Breast and Prostate Cells in vitro. P. Magee 1, M. Raschke 2,C. Steiner 2, J. Duffin 1, B. Pool-Zobel 2, T. Jokela 3, K. Wahala 3, and I. Rowland* 2, 1 University of Ulster, Coleraine, UK, 2 Friedrich Schiller University, Jena, Germany, 3 University of Helsinki, Helsinki, Finland. The ability of soy isoflavones to protect against hormone-dependent cancers, such as those of the breast and prostate has been intensively studied to date. Equol, an isoflavan, is produced by approximately 30-40% of the population via the bacterial conversion of the soy isoflavone daidzein in the gut. Due to the presence of a chiral centre, equol exists as two distinct optically active isomers, R- and S-equol, though the enantiomer produced by metabolic reduction from isoflavones is known to be S-(-)equol. It has been postulated that the R- and S- enantiomers have different biological properties, given their different binding affinities for the estrogen receptor. In this study we have compared the biological effects of the purified S-equol enantiomer to that of racemic (R and S) equol on breast and prostate cancer cells in vitro. Effects on cell proliferation, invasion through matrigel and DNA integrity using the Comet assay were studied. Both racemic and S-equol inhibited the growth of the breast cancer cell-line MDA-MB-231 (>10mM) and the prostate cancer cell-lines LNCaP (>5mM) and LAPC-4 (>2.5mM) with both compounds exerting similar effects as assessed by DAPI staining. The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30mM) was, however, unable to prevent DNA damage in MCF-7 or MCF-10A breast cells follow-

3 2005 Abstracts 3 ing exposure to 2-hydroxy-4-nonenal, menadione or benzo(a)pyrene-7,8- dihydrodiol-9,10-epoxide. In contrast, DNA damage in MCF-10A cells was significantly reduced following exposure to 2-hydroxy-4-nonenal or menadione when cells were pretreated with racemic equol (10, 30mM). These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol. This study was carried out with financial support from the Commission of the European Communities specific RTD programme Quality of Life and Management of Living Resources (project no QLK ). It does not necessarily reflect its views and in no way anticipates the Commission s future policy in this area. Probiotic and Prebiotic Effects on Soy Isoflavone Metabolism, Equol, and Lipids. T. Larkin 1,3, L. Astheimer 1,3, and W. Price 3, 1 Department of Biomedical Science, University of Wollongong, Wollongong, NSW, Australia, 2 Department of Chemistry, University of Wollongong, Wollongong, NSW, Australia, 3 Smart Foods Key Centre, Wollongong, NSW, Australia. Due to the essential role of gut microflora in isoflavone metabolism, particularly that to equol, it was hypothesized that concurrent intake of soy with either a probiotic (yoghurt) or prebiotic (resistant starch) may affect isoflavone metabolism and lipids. Thirty-one hyperlipidemic men and postmenopausal women completed this randomised, crossover study of two 5-week dietary periods separated by a 4-week washout. Intake of soy alone was compared with either probiotic (soy + probiotic) or prebiotic (soy + prebiotic) treatments for effects on plasma and urinary isoflavones and plasma lipids following a test soy meal. Five weeks of soy intake significantly increased circulating plasma daidzein and genistein levels, but did not affect plasma or urinary isoflavones after the test soy meal. There was a trend for increased plasma daidzein and genistein levels with both probiotic and prebiotic treatment, but no overall significant effects. Neither treatment induced or increased equol production, though there was a trend with probiotic treatment for increased plasma equol in equol-positive subjects (n = 12). Despite a lack of significant effects on isoflavone bioavailability, total cholesterol was significantly decreased with both probiotic and prebiotic treatment (-4.7 ± 2.0%, p = and -5.5 ± 1.6%, p = 0.003, respectively) and LDL cholesterol was most significantly decreased with prebiotic treatment (-7.3 ± 2.2%, p = 0.005). Independent of the potential for probiotic or prebiotic treatment to affect isoflavone bioavailability, the synergistic action between soy and probiotic or prebiotic intake for lipid-lowering effects suggests these combinations may be useful in lipid management. Specialty Cereals Pty Ltd., Paul s Dairy, So Natural Foods, Penford Australia, Dr. Barbara Meyer, Maria de los Angeles. Urinary Excretion of Equol and the Risk of Breast Cancer in Japanese Women. C. Nagata 1, T. Ueno 2, S. Uchiyama 2, K. Urata 2, Y. Nagao 3, C. Shibuya 3, Y. Kashiki 3, and H. Shimizu 1, 1 Gifu University Graduate School of Medicine, Gifu, Japan, 2 Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical Co., Ltd., Saga, Japan, 3 Gihoku General Hospital, Gifu, Japan. The association between urinary excretion of equol and the risk of breast cancer was examined in a case-control study among Japanese women. The case subjects were 160 women newly diagnosed and histologically confirmed with breast cancer between 2000 and 2002 at a general hospital in Japan. A total of 456 controls individually matched to cases on age, menopausal status, and the date of sample collection were selected from those who attended a breast cancer mass screening at this hospital. Diet before the date of diagnosis for the cases and the date of the screening visit for the controls was assessed by a validated semiquantitative food frequency questionnaire. Spot urine samples from each subject were analyzed for urinary excretion of isoflavonoids. Urine samples from breast cancer cases were collected before cancer therapy. Dietary intake of soy products or soy isofolavones did not differ significantly between cases and controls. The percentage of women with detectable levels of equol (>1.13nmol/ml) in urine was significantly greater in controls than that in cases (19.6 vs 8.2%). Urinary levels of daidzein and genistein were significantly higher in cases than in controls. The status of excretion of equol (+ or -) was moderately positively associated with urinary levels of daidzein and genistein in controls. Urinary equol or the ability to produce equol may be associated with the risk of breast cancer. Treatment of Postmenopausal Monkeys with Equol Did Not Improve the Plasma Lipid Profile. S. Appt, T. Clarkson*, and H. Chen, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA. Background: Monkeys fed soy protein, compared to casein, have markedly improved plasma lipid profiles (increased HDLC and decreased LDL+VLDLC). Soy consumption results in high plasma equol concentrations, which might account for the large lipid benefits in monkeys compared to much smaller benefits observed in women in (only about a third of which produce equol). Materials and Methods: Fifteen postmenopausal monkeys, all fed a casein-lactalbumin based diet containing some added cholesterol (0.20 mg/cal), were randomized into a control group (n=7) or an equol group (n=8). Racemic equol was added to the diet of the equol group in an amount in which the s-anisomer would be equivalent to the amount of daidzein in our previous soy protein studies (52.4 mg/1,800 Cals). Plasma HDLC, LDL+VLDLC and triglycerides (TG) were measured at baseline, 3, 6 and 8 months. Repeated measures ANCOVA, adjusting for baseline, was used to analyze the data. Results: Postprandial total plasma equol concentrations were about 470 nmol/l, comparable to monkeys fed soy protein containing daidzein equivalent to the equol s-anisomer fed in this study. After 8 months of treatment, there were no significant effects of equol on HDLC, LDL+VLDLC or TG. Discussion: We found no evidence that equol per se is the mediator of the plasma lipid benefits of soy protein fed to monkeys. There are limitations to the study: synthetic equol may be handled differently by the gut, may act differently administered with soy protein or the effects may be due to dehydrodaidzein or tetrahydrodaidzein. Cancer Developing a Soy Food Based Intervention Among Healthy Men. G. Maskarinec 1, S. Hebshi 1, Y. Morimoto 1,S. Sharma 1, A.A. Franke 1, and F.Z. Stanczyk 2, 1 Cancer Research Center of Hawaii, Honolulu, HI, USA, 2 University of Southern California, Los Angeles, CA, USA. Increasing prostate cancer risk among men who migrated from Asia to the United States led to the hypothesis that soy foods may be protective through an effect on circulating sex steroids. This pilot study Oral Presentation Abstracts

4 4 6th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease Oral Presentation Abstracts examined the feasibility of recruiting healthy men for a soy food based intervention, developed a dietary protocol that could achieve compliance, obtained preliminary hormonal data, and assessed emotional status during the intervention. Invitations were sent to 1000 Kaiser Permanente Hawaii members, ages years, who had a normal PSA test. We received replies from 90 (9%) interested men and identified 25 eligible subjects. After a run-in period, 24 men (92% of eligible) were randomized and 23 subjects completed the study. In a cross-over design, half of the subjects completed either a high soy diet or maintained their usual diet. In the high soy diet, dairy, meat, and snacks were replaced with approximately 75mg of isoflavones as 2 servings of soy milk, tofu, or soy nuts. After 3 months and a 1-month washout period, participants crossed over to the other diet. Compliance was assessed by 24-hour recalls, soy logs, and urinary isoflavone excretion measured by high pressure liquid chromatography. Each subject donated 3 blood samples and 5 overnight urine samples and completed the Profile Of Mood States questionnaire. Testosterone and PSA were quantified in serum by highly specific immunoassays. The intervention and all laboratory analyses were completed recently. The statistical analysis will apply t-tests and mixed models to examine differences by diet while incorporating the repeated measures. This pilot stuy was supported by a developmental grant from the Cancer Research Center of Hawaii. Soy Intake, Use of Menopausal Hormones, Body Size, and Breast Cancer Risk in Asian-American Women. A. Wu 1, M. Yu 2, C. Tseng 1, and M. Pike 1, 1 University of Southern California, USA, 2 University of Minnesota, USA. We conducted a large population-based case-control study of breast cancer among Asian American women in Los Angeles County which included 1386 women with breast cancer (494 Chinese, 384 Japanese, 508 Filipino) and 1226 control women without breast cancer (515 Chinese, 331 Japanese, 380 Filipino). Participants provided detailed information on menstrual and reproductive factors, use of exogenous hormones, family history, body size, dietary history, and other lifestyle factors in a structured in-person interview. Established risk factors such as nulliparity, family history of breast cancer, and use of menopausal hormones are significant risk factors in Asian-American women as in other western populations. Risk of breast cancer is significantly inversely associated with soy intake during adolescence and adult life; the significant risk reductions were found in both premenopausal and postmenopausal women after adjustment for relevant dietary and non-dietary variables. However, further analyses in postmenopausal women showed that the benefits of soy intake are found only in certain subgroups; namely women with low body size and non-users of menopausal hormones. These results and our interpretation of the findings will be discussed. This work was supported by grants (1RB-0287, 3PB-0102, 5PB- 0018) from the California Breast Cancer Research Program. Incident breast cancer cases for this study were collected by the USC Cancer Surveillance Program (CSP), which is supported under subcontract by the California Department of Health. The CSP is also part of the National Cancer Institute s Division of Cancer Prevention and Control Surveillance, Epidemiology, and End Results Program, under contract no. N01CN25403 Antiestrogen Effects of Soybean Glyceollins in Postmenopausal Monkeys. C. Wood 1, S. Appt 1, T. Clarkson 1, A. Franke 2, S. Boue 3, M. Burow 4, and J.M. Cline* 1, 1 Wake Forest University School of Medicine, Winston-Salem, NC, USA, 2 Cancer Research Center of Hawaii, Honolulu, HI, USA, 3 Southern Regional Research Center, United States Dept. of Agriculture, New Orleans, LA, USA, 4 Tulane University School of Medicine, New Orleans, LA, USA. Estrogens have an important role in the development of breast and uterine cancer. Glyceollins are isoflavonoid pterocarpans, present in stressed soybeans, which have potential estrogen-antagonist effects. In this short-term study we used a primate model to evaluate the antiestrogen effects of glyceollin-enriched soy protein (GLY) on biomarkers for cancer risk. Thirty ovariectomized adult female cynomolgus macaques were randomized to one of three dietary treatments (expressed as equivalent doses for women): (1) estradiol (E2, 1 mg/day) + casein lactalbumin (control); (2) E2 + soy protein isolate (SPI) containing 180 mg/day isoflavones; and (3) E2 + GLY containing 189 mg isoflavones and 134 mg glyceollins (I-III). Experimental diets were given for 3 weeks. Breast and uterine measures were taken before and after each treatment. Serum glyceollin concentrations were nm (40.0% of daidzein+genistein) 4 hours after feeding and <1 nm 24 hours after feeding. Compared to baseline, E2 treatment induced significantly greater breast proliferation in the control group (p=0.02 in lobules; p=0.002 in ducts) and SPI group (p=0.04 in lobules; p=0.02 in ducts) but not in the GLY group (p=0.09 in lobules; p=0.93 in ducts). E2 significantly increased uterine area in all groups (p<0.01 for all); however, the change in uterine area was significantly lower in the GLY group versus the control (p=0.01) and SPI (p=0.049) groups. These findings demonstrate that soybean glyceollins are absorbed and rapidly cleared following dietary intake. Preliminary data suggest that glyceollins may enhance the antiestrogenic properties of standard soy protein isolate in the breast and uterus. This work was supported by the NIH/NCCAM R01-AT00639 (JMC). Phytoprevent: A European Project on the Prevention of Breast and Prostate Cancer by Phytoestrogens. I. Rowland, University of Ulster, Coleraine, UK. Phytoprevent is a 3 year multi-disciplinary project funded by the European Union with the aim of investigating the role of foods rich in phytoestrogens (isoflavones and lignans) on reduction of risk of breast and prostate cancer. In analytical studies, HPLC CoulArray methods have been validated for the analysis of food, plasma, urine and faecal and an ID-GC-MS-SIM method has been established for the measurement of plasma estrogen/phytoestrogen levels. In vitro methods were used to study effects of isoflavones on various stages of cancer. Preincubation of breast and prostate cell lines with low concentrations of isoflavones decreased hydrogen peroxide-induced DNA damage. Isoflavones were found to significantly enhance tight junction integrity in the breast cancer cell-lines MCF-7 and MDA-MB-231 suggesting a beneficial effect on tumor promotion. Isoflavones inhibited invasion of MDA-MB-231 breast cancer cells through Matrigel at concentrations as low as 2.5uM. The anti-angiogenic activity of 16 synthetic phytoestrogen metabolites has been determined. Two synthetic phytoestrogens were shown to possess anti-angiogenic activity using proliferation and differentiation assays. Studies in the Tg.NK transgenic mouse mammary cancer model showed that perinatal exposure to isoflavones had no effect on tumor development, while post weaning and life long exposure to isoflavones delayed tumor development and reduced tumor incidence. In a human intervention study (22 days) with foods containing soy, changes in urinary estrogen profile were seen in the soy consumers that were consistent with lower breast cancer risk. This study was carried out with financial support from the Commission of European Communities specific RTD programme

5 2005 Abstracts 5 Quality of Life and Management of Human Resources (Project No QLK It does not necessarily reflect its views and in no way anticipates the Commission s future policy in this area The Combination of Soy and Flaxseed or Their Phytoestrogens Can Better Reduce the Growth of Breast Tumors than Soy or Genistein Alone While Causing Little Effects on Bone Health in Ovariectomized Nude Mice. L. Thompson, K. Power, N. Saarinen, J. Chen, and W. Ward, Department of Nutritional Sciences, University of Toronto, Toronto, ON, Canada. Phytoestrogen (PE)-rich diets have been associated with a lower risk of breast cancer (BC) and osteoporosis. In previous studies in ovariectomized (OVX) mice, flaxseed (FS), rich in lignan PE, reduced the growth of human MCF-7 estrogen receptor positive (ER+) BC, while the soy isoflavone PE, genistein (GEN), stimulated tumor growth. In this study, we determined whether the combination of soy and FS or their respective PE, isoflavone genistein (GEN) or the lignans enterolactone (EL) or enterodiol (ED), can more effectively reduce the growth of ER+ BC in OVX mice than soy or GEN alone while maintaining protective effects on bone. OVX mice with established MCF-7 tumors were fed the basal diet, 20% soy protein isolate (SPI), 10% FS, or 20% SPI + 10% FS for 25 weeks. All mice exhibited tumor regression but SPI induced late stage tumor growth, which was negated by combining it with FS. SPI maintained femur bone mineral density (BMD) and strength but FS slightly attenuated this effect when combined with SPI. Using the same model, mice with MCF-7 tumors were given daily injections of GEN, EL, ED, EL+ED+GEN, or vehicle control for 22 weeks. All mice showed tumor regression but GEN caused the least effect. The effect of combined PE on bone did not differ from the control and GEN. Tumor cell proliferation and apoptosis data agreed with the observed changes in tumor size. In conclusion, combining PE or PE-rich foods resulted in a better control of tumor growth while causing little effects on bone health. Funded by NSERC Tuesday, November 1, 2005 Cardiovascular Disease Hypocholesterolemic Effect of Soy Proteins Only Due to Protein and Only in Hypercholesterolemics. Cesare R. Sirtori, Dept. of Pharmacological Sciences and University Center for Dyslipidemias, Niguarda Hospital, Milano, Italy. The concept that animal proteins may be atherogenic versus the case of vegetable proteins dates back to Ignatowsky (1908) and had a formal proof in 1977 in a model study. This was: a) carried out in hypercholesterolemic individuals (lowest starting cholesterolemia 270 mg/dl); b) on in-patients, followed in metabolic ward conditions; c) based on a direct comparison according to a cross-over protocol of an exclusively soy protein and an animal protein based diet (Sirtori et al, Lancet 1977; i: 275). In a second part of the study the addition of cholesterol was tested on similar in-patients on the soy diet, proving no change in the remarkable cholesterol lowering activity of the experimental regimen (ie around 23% LDL-C reduction). No other study ever fulfilled all these qualifications. In a larger outpatient study in Italy/Switzerland the same soy product (later found to be phytoestrogen-free) was given to 130 patients all with cholesterolemia >300 mg/dl (Descovich et al 1980; ii: 709); both similar reductions of total and LDL-C levels as well as a clear correlation between these reductions and baseline cholesterolemia were reported. A large number of studies have been, unfortunately, carried out in normolipidemic individuals with, as expectable, negative findings; in addition, essentially all published studies have been on dietary supplements. The cholesterol lowering mechanism of soy protein is by way of stimulation of LDL-receptor activity, as shown in animal (Lovati et al, J Nutr 2000; 130:2543) and in clinical studies (Lovati et al, J Clin Invest 1987; 80: 1498). LDL-receptor activity is normal in normolipidemic individuals, unlikely to respond to this treatment. The mechanism thus differs from that of statins, working on cholesterol biosynthesis and active also at low levels of cholesterolemia, and is somewhat similar to that of berberine, also an LDL-receptor upregulator (Kong et al, Nature Med 2004 ; 10: 1344). A matter of confusion in the field was the belief that phytoestrogens might be in some way responsible for cholesterol lowering. This belief was based on primate studies and also on human findings using ethanol extracted soy products. This drastic chemical treatment most likely destroys active protein components, since a more appropriate column extraction procedure maintains the hypocholesterolemic activity in the absence of phytoestrogens (Fukui et al, J Agric Food Chem 2002; 50: 5717). Due to this ill fated belief, US distributed soy proteins show grossly altered structure, as assessed from a proteomic evaluation (Gianazza et al, J Nutr 2003; 133: 9); surprisingly they still cause some reduction of cholesterolemia. A reputable meta-analysis (Anderson et al, N Engl J Med 1995; 333: 276) clearly showed that soy proteins (mainly supplements) reduce cholesterolemia to a minimal extent in normolipidemic individuals (mean reduction -5.2 mg/dl; - 3.3% ) versus a high effect in patients with marked cholesterol elevations (up to mg/dl; %). This type of findings allowed the preparation of a predictive nomogram for assessing the individual benefit of treatment: the cholesterol reductions reported in recent American studies (Lichtenstein et al, ATVB. 2002; 22: 1852; Jenkins et al, JAMA 2003; 290: 502) fit well with the nomogram. It is thus unfortunate that a recent US Report (Evidence Report Technology Assessment, No.126, Effects of Soy on Health Outcomes), pooling together all sorts of studies and eliminating many (including our early ones) came to negative conclusions. Indeed the majority of the studies were in normolipidemics, frequently treated with chemically unsuitable proteins. Superficial comments such as Evidence suggests that initial estimates on the potency of soy protein were too high have no basis: there was never an estimate, since it was clear from the onset that the cholesterol lowering activity is based first on the patient characteristics, second on the soy product and third, most likely, on the daily amount. Soy proteins are active only in hyperlipidemics; this may reduce their appeal, ie considering the present indication of statins in normolipidemic coronary patients. It leaves, however, ample space for clinical use (children, renal patients, etc.). In addition, similar to the case of milk proteins providing ACE-inhibitory peptides for hypertension (FitzGerald et al, J Nutr 2004; 134: 980S), it should not be long before soy protein varieties from adequate cultivars or fractions thereof may turn out to provide effective pill like cholesterol reducers. 7S Soy Globulin Peptide and Cholesterol Homeostasis: in vitro and in vivo Data. M. Lovati, C. Manzoni, S. Castiglioni, and E. Disconzi, Dept. of Pharmacological Sciences, University of Milano, Milano, Italy. 7S soy globulin and its alfa subunit have been shown to positively modulate LDL receptor activity and to decrease triglyceride synthesis Oral Presentation Abstracts

6 6 6th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease Oral Presentation Abstracts both in vitro (HepG2 cells) and in vivo (rats fed cholesterol-rich diet). The aim of the present investigation was to evaluate the effect of a synthetic peptide, corresponding to a sequence which differs between the alfa and alfa subunits from 7S soy globulin, on cell cholesterol homeostasis both in HepG2 cells and in rats fed casein-cholesterol diet. HepG2 cells were incubated for 24 hr in minimum essential medium (MEM) + 5% LPDS in the presence/absence of whole 7S globulin (0.5 mg/ml) or its alfa subunit (0.125 mg/ml) or the synthetic peptide (10-5 M). At the end of incubation, cell membranes and nuclei were separated by validated procedures. Aliquots of membrane and nuclear fractions were run on % SDS-PAGE gels, transferred onto nitrocellulose, and overlaid with a polyclonal antibody against SREBP-1. Cell preincubation with whole 7S soy globulin caused an increase in the mature forms of SREBP-1, both in membranes (+ 90%) and in the nuclei ( + 130%), vs that found in the control HepG2 cells (MEM + 5% LPDS). Moreover, a lower level of mature nuclear SREBP-1 was detected in HepG2 cells exposed to both the alfa subunit and the synthetic peptide, compared to that found after exposure to whole 7S globulin. The oral administration (14 days) of synthetic peptide (5 mg/kg b.w.) to caseincholesterol fed rats resulted in lower plasma lipid levels (cholesterol, - 25%; triglycerides, -28%) vs values recorded in rats fed casein-cholesterol diet alone. These results represent the first in vivo evidence of the plasma lipid-lowering properties induced by the synthetic peptide; moreover, these data suggest that alfa subunit from 7S soy globulin, and its synthetic peptide, are responsible for the direct up-regulation of LDL receptors. The mechanism of this effect is likely to be through the SREBP-2 transcriptional factor, the activity of which we are currently investigating. Supported in part by a grant from MIUR of Italy. Effect of Two Types of Soy Milk and Dairy Milk on Plasma Lipids in Hypercholesterolemic Adults: A Randomized Trial. C.D. Gardner 1, J. Morris 1, M. Messina 2, A. Kiazand 1, A. Varady 1, and A. Franke 3, 1 Stanford University, Stanford, California, USA, 2 Loma Linda University, Loma Linda, California, USA, 3 Cancer Research Center of Hawai I, 1236 Lauhala St., Honolulu, HI 96813, USA. Background The literature on the hypocholesterolemic effect of soy proteins is inconsistent. The effect of soy proteins may differ by dose and by the level of hypercholesterolemia. Objective To contrast plasma lipid responses between two commercially available soy milks, one made using whole soy beans and the other using soy protein isolate, and low-fat dairy milk. Design Randomized clinical trial, cross-over design. Patients Adults (n=28), years of age, with pre-study LDLcholesterol concentrations of mg/dl, not on lipid lowering medications and with overall Framingham risk score of< 10%. Intervention Participants were required to consume sufficient milk to provide 25 g protein/day from each milk source, preferably distributed over three times/day. The protocol included 3 four-week milk consumption phases, each separated from the next by wash-out periods of at least four weeks. Results The average LDL-C concentration at the end of each phase (mean +/- SD) was 161 +/- 20, 161 +/- 26 and 170 +/- 24 mg/dl for the whole soy bean milk, the soy protein isolate milk, and the dairy milk, respectively (p=0.9 between soy milks, p=0.02 for each soy milk vs. cow s milk,). No significant differences by type of milk were observed for HDL-cholesterol or triacylglycerols. The only pair-wise difference in total-c/hdl-c ratio was a significantly lower ratio for the soy protein isolate milk relative to the dairy milk (p=0.02). Conclusion The results of this study support a modest, 5% LDLcholesterol lowering effect of either type of soy milk, relative to dairy milk, when provided in amounts yielding 25 g protein/day to adults with elevated LDL-cholesterol concentrations for four weeks. Soy Food Effects on Serum Lipoproteins in Humans: Updated Meta-Analysis. J.W. Anderson, VA Medical Center and University of Kentucky, Lexington, KY, USA. Soy foods significantly affect serum lipoproteins by decreasing LDL-cholesterol (LDL-c) and triglycerides (TG) while increasing HDL-cholesterol (HDL-c). Since our 1995 meta-analysis, most studies have reported less impressive alterations in serum lipoproteins than the 12.9% decrease in LDL-c then reported for soy foods. Since 1995 at least 55 randomized controlled trials with soy foods have reported serum lipoprotein changes. To examine possible causes of differences in lipoprotein responses, we have analyzed these studies with a focus on LDL-c. Weighted changes for different soy foods are presented as changes from baseline (to estimate maximum effects) and net changes (adjusted for control diet changes). In 55 trials, soy protein consumption averaged 44 g/d fed for an average of 29 days. Serum LDL-c decreased 8.5% with net change of 4.5% (95% CI, %). In 16 studies soy was baked or cooked with LDL-c changes of -0.9% and net changes of -1.4% (not significant). Using soy protein once daily and <25 g/d was less effective than use of > 25 g/d and > 2x/d. In 24 studies with g/d soy protein given > 2x/d, LDL-c reduction was 9.3% with a net reduction of 5.4% (95% CI, %). These studies reported significant changes in HDL-c (+3.7%, 95% CI, %) and TG (-8.0%, 95% CI, -2.8 to -13.1%). Further subgroup analyses will be presented. Recent studies indicate that soy peptides have potent hypocholesterolemic effects and that soy proteins with more intact peptides have significantly greater hypocholesterolemia than more fragmented preparations. Soy protein extraction or baking may fragment the most active hypocholesterolemic peptides. Effect of Soy Isoflavone Protein and Soy Lecithin on Endothelial Function in Healthy Postmenopausal Women. Z. Faridi, M. Evans, V. Njike, M. Hoxley, and D. Katz, Yale Griffin Prevention Research Center, Derby, Connecticut, USA. Objective: To assess the effects of soy isoflavone protein concentrate and soy lecithin on endothelial function measured as flow mediated dilation (FMD) of the brachial artery in healthy postmenopausal women. Design: Randomized, double-blind, placebo controlled crossover trial. Subjects/Setting: 25 subjects (mean age 61 years; BMI kg/m2) were recruited from the general population of Southwestern Connecticut. Intervention: Subjects underwent endothelial function testing at baseline and following 4 weeks of randomly assigned treatments with intervening 4-week washout periods. Treatment assignments included: Soy isoflavone protein (25gm/day) and soy lecithin (20gm/day); soy isoflavone protein (25gm/day) and placebo lecithin; placebo protein and soy lecithin (20gm/day); and double placebo. Outcome Measures: FMD of the brachial artery, and serum lipid levels Results: 22 women completed the trial; pre-treatment FMD was 8.60 ± No statistically significant difference was seen in FMD between treatment assignments. However, FMD was highest after treatment with soy protein plus lecithin (7.50 ± 9.85), followed by soy protein plus placebo lecithin (5.51 ± 10.11), placebo protein plus soy lecithin (5.35 ± 6.13), and lowest after double placebo (4.53 ± 7.84).

7 2005 Abstracts 7 Soy isoflavone protein and soy lecithin significantly increased HDL/LDL relative to baseline value (soy isoflavone protein and soy lecithin, 0.64 ± 0.19; soy isoflavone protein and placebo lecithin, 0.58 ± 0.17; placebo protein and soy lecithin, 0.65 ± 0.18; baseline, 0.49 ± 0.15). Conclusion: In this sample of healthy postmenopausal women, soy isoflavone protein and soy lecithin significantly improved the lipid profile. Improvement in endothelial function could not be confirmed. The study was funded by a grant from Health & Nutrition Group of Eridania Béghin-Say America, Inc. Workshop Presentation Role of Soy Foods in the Management of Obesity and Related Chronic Diseases: Summary of a Symposium at the University of Illinois. John W. Erdman Jr., Keith Cadwallader and Barbara P. Klein, Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL, USA. Obesity among American adults has doubled over the past two decades and has nearly tripled for children and adolescents. The consequences of obesity include increased risk of a host of chronic diseases including diabetes, cardiovascular disease, cancer and bone and joint degeneration. Enhanced obesity rates are not limited to westernized countries, as most societies world-wide are now reporting higher proportions of severely overweight citizens. Soy foods have the potential for assisting in obesity management and reduction of subsequent chronic disease development. To address these issues, a two-day forum was held on May 25 and 26, 2005 at the University of Illinois featuring leading researchers from universities and the food industry. They reviewed the current state of knowledge regarding the management of obesity and related diseases and the roles of soy and its components. Presentations focused upon the state of knowledge of obesity incidence, effectiveness of weight loss programs, and impacts of obesity on cardiovascular disease, diabetes and other co-morbidities. It is well accepted that consumption of soy can improve serum lipid profiles. There is emerging evidence suggesting that soy-containing isoflavones may also affect adipogenesis and may be beneficial for diabetics. An industry panel discussed their experiences in utilization of soy foods for weight loss. This presentation will review the scientific outcomes from the symposium. In addition, gaps in current knowledge for the roles of soy in the management of obesity and related diseases will be discussed. Immune Function Soy Isoflavones Modulate Immune Function in Healthy Postmenopausal Women. T. Ryan-Borchers, J. Park, B. Chew, M. McGuire, L. Fournier, and K. Beerman, Washington State University, Pullman, WA, USA. Background: After menopause, the immune system may be compromised due to effects of aging and diminishing concentrations of estrogen, an immune-modulating hormone. Isoflavones, plant-derived compounds that possess antioxidant, anti-estrogenic and pro-estrogenic actions, may offer immunological benefits to women in this stage of life. Objective: The objective of this study was to evaluate the effects of soy isoflavones, both soymilk and supplement form, on markers of immunity and oxidative stress in postmenopausal women. Design: Postmenopausal women aged y (n = 52) enrolled in this 16 wk double-blind, placebo-controlled trial were randomized to one of three experimental groups: 1) Control ml/d cow s milk plus a placebo supplement; 2) Soymilk mg isoflavones derived from 706 ml/d soymilk plus a placebo supplement; and 3) Supplement - 70 mg isoflavones in a supplement plus 706 ml/d cow s milk. Plasma and 24-h urine samples were obtained at baseline and at 16 wk. Immune variables included lymphocyte subsets, cytokine production, as well as markers of inflammation and oxidative damage. Results: Baseline immune variables did not differ among groups. Both isoflavone interventions increased (p < 0.05) β cell populations and concentrations of plasma interferon-gamma (IFN-γ) and decreased (p < 0.05) concentrations of 8-hydroxy-2- deoxy-guanosine (8-OHdg), an oxidative marker of DNA damage. Isoflavone treatments did not influence concentrations of urinary 8-isoprostane (8-iso), plasma interleukin-2 (IL- 2), tumor necrosis factor-alpha (TNF-α) or C-reactive protein (CRP). Conclusions: Soymilk and supplemental isoflavones modulate β cell populations and IFN-γ concentrations, and appear protective against DNA damage in postmenopausal women. Supported by a grant from the Washington State Attorney General s office; soymilk provided by White Wave, Inc. and isoflavone (Novasoy ) and placebo supplements provided by the Archer Daniels Midland Co.We thank Bridgett Mathison for lab assistance; Heather Gardner, Livia Wagner and April Broderick for delivery of study treatments and data entry; and all of the women who graciously participated in this study. Effects of ImmuSoy as a Food Supplement for Altering Peanut Allergic Reactions. T. Zhang 1,W.Pan 2, M. Takebe 3, H. Sampson 1, and X. Li* 1, 1 Pediatrics, Allergy & Immunology, Mount Sinai School of Medicine, New York, NY, USA, 2 Surgery, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA, 3 Nichimo Co., Ltd., Tokyo, Japan. RATIONALE: Probiotic foods were only recently clinical trials conducted to evaluate probiotics in the treatment of atopic dermatitis and food allergy, Lactobacillus rhamnosus GG (LGG) had some effect on the prevention of early atopic disease in high-risk infants. Recent studies shown that soy as a kind of allergen-specific immunotherapy can be used to desensitize/downregulate peanut-specific response in peanutallergic mice. ImmuSoy is a koji fungus (Aspergillus oryzae) and lactobacteria soybean fermentation product, based on traditional Japanese fermentation technology. It is believed that this unique food supplement would be beneficial for the immune system when food allergy happened. We hypothesized that ImmuSoy may be effective for treating peanut allergy. METHODS: This study was undertaken in mice to investigate whether specific doses of ImmuSoy would has better effect than LGG for desensitization peanut allergic mice. Peanut allergic mice (C3H/HeJ, n=8-10/group) were fed ImmuSoy containing chow (0.5 % and 1 %), LGG 0.5% containing chow or regular chow (control) for 4 weeks beginning at week 10 following peanut sensitization, and then were challenged with peanut. Anaphylactic scores, plasma histamine, serum peanut specific-ige levels and splenocyte cytokine production to peanut stimulation were determined. RESULTS: All control mice developed anaphylaxis (median score 3.3) following peanut challenge, in contrast, 50% and 25% of low and high doses of ImmuSoy treated-mice developed anaphylaxis (median scores 1.0 and 0.33 respectively) while LGG treated mice have 80% and mean score around 2. The low and high dose ImmuSoy treated-mice showed 50.4 % and 80.7% reduction respectively in plasma histamine(lgg 20%), and 47.1% and 73.9% reduction respectively in serum IgE levels as compared to untreated mice (p<0.05)(lgg 20% P>0.05). Oral Presentation Abstracts

8 8 6th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease Oral Presentation Abstracts Furthermore, IL-4, IL-5 and IL-13 production by splenocytes of high dose ImmuSoy-treated mice were reduced by 74.1% and 77.0% (LGG 75%) whereas IFN-γ production was increased by 34.8%(LGG increased 10%) compared to control splenocytes. CONCLUSIONS: LGG of 0.5% containing chow shown some protective effects on peanut allergic mice, but when we increase the dose of LGG even to 5 times higher than ImmuSoy 1%, no dose dependent or no better effects shown (data not shown here). Compared with LGG, ImmuSoy used as a food supplement has dose dependent protective effects against peanut-induced anaphylaxis in mouse peanut allergic model, which is associated with down downregulation of Th2 responses. This approach might be a potential novel therapy for peanut allergy. Diabetes Habitual Soyfood Consumption Improves Glycemic Control Among Postmenopausal Chinese Women: A One-Year Follow-up Study. S. Ho and Y. Chen, The Chinese University of Hong Kong, Hong Kong SAR. Objectives: Previous studies show soy supplementation improves glycemic control in animals and in diabetic subjects. Little data are available on their effects among healthy adults. We examined the effect of habitual soyfood intake on changes in serum fasting glucose over a one-year followup period. Methods: This study included 173 community-based postmenopausal Chinese women aged years who had been recruited into a one-year randomized controlled trial of three doses of isolated soy isoflavones (0, 40 and 80 mg/d) on bone changes. Habitual soyfood intake was assessed using a validated food frequency questionnaire. Fasting serum glucose (FG) was measured at baseline and at the end of one-year. Results: The mean intake of habitual soy protein among the study subjects was 5.3g/d (sd = 4.46). Soy protein intake, changes in body mass index, and baseline glucose were significantly correlated with changes of FG over one year. Stepwise regression analysis showed that habitual soy protein was significantly and inversely associated with the absolute and percent changes in FG in women with initial glucose over the median (92.1mg/dl) rather than in those below the median after adjusting for supplemental isoflavones and other potential confounders (including age, years since menopause, baseline FG, changes in body mass index, dietary intakes of total fat, fruits, vegetables, and physical activities). Soy protein accounted for 14.1% (p=0.002) and 12.8% (p=0.003) of the variations of absolute and percent changes of FG, respectively; and it was estimated that a daily increase of ten-gram soy protein was associated with 13.5 mg/dl (or 9.3%) decreases in fasting glucose (p<0.001) in women with higher initial FG. Conclusion: Habitual soyfood intake improves glycemic control in women with higher baseline FG. Beneficial Metabolic Effect of Soy-Rich Diets in Young Firefighter Trainees. Y. Yamori 1,2, M. Shibata 1,M. Mori 3, N. Ishiwata 4, M. Tokoro 2, and Y. Yamamoto 1, 1 Hyogo Prefecture Health Promotion Association, Kobe, Japan, 2 Mukogawa Women s University, Nisinomiya, Japan, 3 Research Institute for Production Development, Kyoto, Japan, 4 Atomi Junior College, Tokyo, Japan. Purpose: To investigate the effect on the indices related to lifestylerelated disease risks, of continuous daily soy-rich diet intake in healthy young Japanese men. Method: A randomized, cross-over controlled dietary intervention study on metabolic effects of soy-rich diets was carried out in cooperation with 100 healthy firefighter trainees aged After informed consent was obtained, they were randomized into soy rich (S) and ordinary control (C) diet groups during the first and second intervention periods (45 and 44 days, respectively) with an interval of the washout period of 25 days. S diets consisting of common Japanese soy products, such as tofu, soymilk, etc. contained more soy protein (29.4g in average) a day than regular Japanese C diets (soy protein 8.6g), and both diets were nearly the same in daily total energy ( Kcal) and total protein ( g) intakes. Questionnaires about dietary custom, body mass index (BMI), body fat (F), blood pressure (BP), and blood and 24-hour urine were examined 4 times before and at the end of both intervention periods. Results: From among 95 male participants, 43 completed all tests including 4 times of 24-hour urine collection. S group (18) showed significant increases in urinary isoflavones (ISO: µmol) after the intervention and significant reduction in systolic and diastolic BP, fasting glucose (G), BMI and F. In contrast, C group (25), whose urinary ISO excretions were not changed, showed no significant changes in these parameters. Significant inter-group differences were noted in changes from the baseline levels of G and HOMA-IR (homeostasis model assessment insulin resistance index: G immuno reactive insulin). Conclusion: Daily intake of soy rich diets containing about 30g of soy protein decreased G and improved HOMA-IR significantly in comparison with regular diets, suggestive of the beneficial effect of soy-rich diets on insulin resistance even in young healthy men. (This study was supported by Hyogo Prefecture Government) Menopausal Symptoms The Effects of 100mg Soy Isoflavone Supplements on Menopausal Symptoms and Quality of Life: A Double- Blind, Randomised, Placebo-Controlled, Crossover Trial in British Postmenopausal Women. C. Hill 1,A. Cassidy 2, J. Powell 3, D. Talbot 3, and L. Dye 1, 1 Institute of Psychological Sciences, University of Leeds, Leeds, West Yorkshire, UK, 2 School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, East Anglia, UK, 3 Unilever R&D, Sharnbrook, Bedfordshire, UK. Research into the efficacy of soy isoflavones (soy IF) to relieve menopausal symptoms has yet to produce a unanimous conclusion. Attention has been largely focused on hot flushes, with other psychological, vaso-somatic and general somatic symptoms typically assessed before and after an intervention. The conflicting findings on these endpoints could be attributed to the methodological issues surrounding retrospective reporting and the use of psychometrically weak scales. Therefore this study adopted a prospective assessment of the severity and frequency of menopausal symptoms. Retrospective measurements of quality of life were also taken, which tapped into a non-symptomatic consideration of well being. This study employed a double-blind, randomised, placebo-controlled crossover trial of 100mg soy IF supplement over 8 weeks in 21 British postmenopausal women. No significant difference was noted between soy IF and placebo for total symptom score or for the psychological, vaso-somatic or general somatic subscales. However, specific reductions were observed in the frequency of feeling easily stirred up and the severity of early morning awakenings and sleeplessness. Daily counts and severity measures of hot flushes were also significantly reduced across all weeks of soy IF intervention, with greater efficacy in women with > 5 hot flushes/day. There were no interactions with time on treatment, with a generally lower level of these

9 2005 Abstracts 9 symptoms across all weeks of soy IF. Health, sexual, emotional and occupational quality of life was not significantly improved with soy IF, and there was no difference between treatments on the Cognitive Failures Questionnaire. However, frequency of sleep problems was significantly reduced after 4 weeks of soy IF, with no further improvements at week 8. Increases in circulating levels of oestrogen were significantly correlated with the reduction of hot flushes, with no clear relationship with other symptoms. Therefore, whilst this study was able to detect some effects on specific symptoms, generally this more methodologically rigorous approach validated findings from earlier methodologically weaker studies. Isoflavone Supplements Predominantly Containing Genistin/Genistein Reduce Hot Flushes: A Critical Analysis of Published Studies. P. Williamson-Hughes 1,B. Flickinger 1, M. Messina 2, and M. Empie 1, 1 Archer Daniels Midland Company, Decatur, IL, USA, 2 Nutrition Matters, Inc., Townsend, WA, USA. Several reviews and meta-analyses have evaluated the clinical evidence relating isoflavone treatment to menopausal hot flush symptom relief. The majority of these reviews and meta-analyses have included a range of isoflavone sources; often without differentiating the chemical identity of individual isoflavone molecules contained in the study product or the compositional ratio of isoflavones. We conducted a re-evaluation of the published literature specific to studies utilizing well characterized isoflavone-containing supplements to determine if observed effects, or lack thereof, were attributable to specific isoflavones. Eleven studies were stratified in relation to their specific isoflavone composition. Six studies described isoflavone products which provided greater than 10 milligram genistein per treatment (calculated as aglycone equivalents), administered to a total of 188 subjects. These six studies consistently reported a statistically significant effect in reducing hot flushes in post-menopausal women. In contrast, the remaining five studies (total of 167 subjects) used supplements containing high daidzein or formononetin and low genistin/genistein content; only one of these (30 subjects) reported an effective reduction in hot flush. Hence, reports finding that the relationship between isoflavone consumption and hot flush reduction inconclusive suffer from over generalization by including any product with the term isoflavones. Lack of discrimination between individual molecular entities contained in heterogeneous isoflavone sources can be misleading. In light of our observations, we conclude that higher genistein ratio extract products with a minimum genistein dose level are effective in reducing hot flushes and that evaluation of isoflavone effects should be related to content of individual isoflavones within supplements. Comparison of Isoflavones and Tibolone Regarding Vaginal Estrogenicity in Peri and Postmenopausal Women. U.D. Rohr 1 and A. Jungbauer 2, 1 AHS, 1090 Vienna, Austria, 2 Department of Biotechnology, BOKU, 1090 Vienna, Austria. Isoflavones are promising agents in chemoprevention. Clinical effects in peri- and postmenopausal women of isoflavones and tibolone in postmenopausal women are compared to binding affinities to a yeast estrogen receptor model. Tibolone a compound used in postmenopausal hormone therapy, is metabolized into 3 further active compounds. 80mg isoflavone per day were applied in 11 perimenopausal women and 36 postmenopausal women for 3 month. Competitive radio-ligand binding assays of different dilutions of the Isoflavone preparations resulted in a binding to estrogen receptor (ER)-alpha and beta in a dose-dependent manner. 2.5 mg Tibolone was applied daily in 24 post-menopausal women for 6 months. Tibolone and its delta-isomer did predominantly bind and transactivate progesterone receptor and androgen receptor, whereas its 3alpha-hydroxy- and 3beta-hydroxy-tibolone predominantly bind and transactivate ER-alpha. Isoflavones did show a preference for binding to the ER-beta. The PAP-smear test, to control for inflammation and cancer risk of the vagina and portio of the uterus, was conducted after 3 month application of either tibolone or isoflavones. Isoflavone reduced vaginal PAP-test (superfiscial cells) for perimenopausal women from to counts and for postmenopausal women from to counts. Tibolone increased cell counts in postmenopausal women in the PAP-test. Hypertrophic effects in the vagina as well as endometrium are believed to be facilitated by the ER-alpha and suppressed by ER-beta: Our in vivo results correlate with our in vitro results: ER-beta affinity of isoflavones could explain the reduction of cells in the PAP smear test and ER-alpha affinity of tibolone may be correlated to the increase of cells in the PAP smear test by tibolone. Wednesday, November 2, 2005 Can Soy Isoflavones Cause Endometrial Cancer? J.M. Cline 1, C. Wood 1, A. Franke 2, D. Bland 1,D.Foth 3 ; 1 Wake Forest University School of Medicine, USA, 2 University of Hawaii, USA, 3 University of Cologne, Germany. Estrogens markedly increase the risk of endometrial cancer in women, and uterotrophic effects of estrogens are well-documented in animal models including nonhuman primates. As many investigators have shown, high-dose soy-derived isoflavonoids have estrogen-agonist activity on the uterus in rodent models, including the induction of estrogen-dependent hyperplastic and neoplastic changes. A recent 5-year trial of isolated soy isoflavones given in pill form to postmenopausal women found a slight but significant increase in the incidence of endometrial hyperplasia. This observation and its interpretation should be carefully considered. Human observational studies show no evidence of estrogen agonist effects of soy food consumption, and several short-term trials of soy isoflavonoids in women have not shown any proliferation-inducing effect on the uterus. Similarly, our nonhuman primate studies spanning up to 3 years (~10 years of equivalent human lifespan) have shown no evidence of isoflavonoid-induced endometrial hyperplasia at dietary doses of soy isoflavonoids. We have shown a lack of uterotrophic effect in short-term nonhuman primate studies using up to 10 times the dietary exposure of soy isoflavonoids or purified equol. We have also demonstrated estrogen-antagonist effects of soy isoflavone supplementation on estrogen-induced increases in uterine size, a finding which parallels the pattern of endometrial cancer risk in the Shanghai breast cancer study. Finally, in a 2-year trial of women given soy protein isolate supplementation at 58 mg/day, we found no association between serum isoflavonoid concentrations and endometrial proliferation. These findings give us some confidence that there is not a significant uterotrophic effect of dietary soy isoflavonoids in the human or nonhuman primate endometrium, and in fact that there may be a protective effect in the presence of higher endogenous or exogenous estrogens. However, the potential remains for differences in risk:benefit ratio based on isoflavone formulation or individual characteristics. ACKNOWLEDGEMENTS The Solae Company; NIH Grants NIH HL45666; NIH AT00639 Oral Presentation Abstracts

10 Oral Presentation Abstracts 10 Osteoporosis Effects of the Phytoestrogen Genistein on Bone Loss, Cardiovascular Risk Prevention and Climacteric Symptoms: A Two Year Double Blind Placebo Controlled Study. Interim Evaluation at One Year. F. Squadrito, R. D Anna, F. Corrado, A. Gaudio, M. Atteritano, A. Bitto, D. Altavilla, and N. Frisina, University of Messina, Messina, Sicily, Italy. To confirm our previous findings on the phytoestrogen genistein (1,2) 389 postmenopausal women with a BMD at the femoral neck of <0.795 g/cm2 were randomly assigned to receive either genistein (54 mg/day) (n=198; age = 55±4; BMI= 24±3 ) or placebo (n. 191; age =54±3; BMI = 25±4) in a two years double blind study. Bone loss: Genistein reduced at 12 months the urinary excretion of deoxypiridinoline (-35±13 %; p< 0.01) and increased the serum levels of the bone-specific ALP (18±6%; p<0.01) and bone G1a protein (16±5 %). Genistein increased BMD in the femur and lumbar spine. Placebo treatment did not affect any of those parameters. Cardiovascular risk factors: By comparison with placebo, genistein treatment at 12 months decreased fasting glucose (-14%; p<0.01), fasting insulin (-51%; p<0.01) and HOMA-IR (-43%; p< 0.01) Genistein administration reduced fibrinogen (-18%; p< 0.05), urinary F(2) isoprostanes (- 24%; p<0.05) and plasma soluble ICAM (-16%; p<0.01). Climacteric symptoms: By comparison with placebo, genistein caused a reduction from baseline in both the mean number (-34%; p<0.01 vs placebo) and the mean severity score (-40%; p<0.05 vs placebo) of hot flushes. Safety assessment: After a 12-months genistein treatment no significant difference in endometrial thickness was observed and vaginal cytology (using the karyopycnotic index or the maturation value score) showed no change. Our 1-year data confirm that genistein is an effective and safe alternative in postmenopausal women. 1) Morabito N. et al. J Bone Miner Res. 2002;17(10): ) Squadrito F. et al. Am J Med. 2003;114(6): This work was supported by grant from MIUR (Ministero dell Istruzione dell Università e della Ricerca). Effects of Long Term Soy Dietary Supplementation on Bone Mineral Density. F. Lovrien 1, J. Williams 2, B. Meyer 1, and D. Erger 1, 1 Sioux Valley Hospital, Sioux Falls, South Dakota, USA, 2 University of South Dakota, Vermillion, South Dakota, USA. A few studies in postmenopausal (PMP) women suggest that a diet that includes soy may reduce bone loss. Findings in some studies may be confounded by short study periods. We examined whether a diet with soy protein for 2 y reduces losses in bone mineral density (BMD) in PMP women. Subjects 1-5 years PMP were randomly assigned: soy/hormone replacement therapy (HRT; n=34), casein/hrt (n=33), soy/no-hrt (n=28), casein/no-hrt (n=20). For 2 y, subjects ate a normal diet supplemented with 40 g soy (90 mg aglycone isoflavone) or casein daily. Isoflavones (mg/g soy): genistein, 2.1; daidzein, 1.1; glycitein, 0.2. Both supplements provided 1400 mg calcium. DEXA scans of hip and spine were taken before and after 1 and 2 years. Comparisons (% change) were made in BMD between soy/hrt and casein/hrt and between soy/no-hrt and casein/no-hrt groups with an unpaired t-test. In the spine, after 1 year BMD decreased 1.26±0.72% (mean±se) and 2.21±0.56% in soy/no-hrt subjects and casein/no-hrt subjects (NS) and 0.02±1.00% and 2.73±0.71% after 2 years (P<0.05). Changes in BMD were similar between soy/hrt and casein/hrt after 1 and 2 years. In the hip, no differences occurred after 1 or 2 y when comparisons were made between soy/hrt and casein/hrt and between soy/no-hrt and casein/no-hrt groups. Our findings indicate that long-term dietary supplementation with soy can reduce bone loss in PMP women, but this protection may be selective for certain bones. Supported by the South Dakota Health Research Foundation and the South Dakota Soybean Research and Promotion Council Effect of Soy Protein With or Without Isoflavones on Bone Mineral Density in Postmenopausal Women. J.C Gallagher, Prema Rapuri, Sue Longsdon, Jeff Detter; Creighton University Medical School, Omaha NE 68131, USA. Soy protein or its isoflavones have been suggested to have a positive effect on bone that may help to prevent the development of osteoporosis. The objective of the present study was to determine the effect of soy protein with or without isoflavones on bone mineral density (BMD) in postmenopausal women aged years over 24 months. Two hundred and thirty women were enrolled into the study. They were randomly assigned to one of the three supplement groups, 25 g soy protein without isoflavones, 25 g soy protein containing 90 mg of isoflavones (genistein and daidzein) or 25 g casein. Twenty four women did not receive any supplemental protein and served as non protein control. Spine, femoral neck, trochanter, total femur and total body BMD measurements were performed by dual energy absorptiometry at baseline and at the end (24 months) of the study.the percent change in BMD over baseline was calculated at the end of the treatment period. Data was analyzed by ANOVA and Bonferroni correction was used for multiple comparisons. There were no significant differences between the groups with respect to their age and body mass index. The baseline BMD was not significantly different between the four groups. Bone loss occurred in al three groups; our preliminary analyses indicate that the unadjusted percent change in BMD was not significantly different between the treatment groups and none of the protein treatment groups prevented bone loss. ACKNOWLEDGEMENTS This work was supported by funding from Solae Company. Cognitive Function 6th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease Cognitive Effects of Soy Isoflavones in Older Adults: Influence of Gender and ApoE Genotype. C. Gleason 1,2, S. Meade 1,2, N. Lane 1,2, T. Ohrt 1,2, and S. Asthana 1,2, 1 University of Wisconsin, Dept. of Medicine, Sect. of Geriatrics, Madison, WI, USA, 2 Madison VA GRECC, Madison, WI, USA. Evidence from basic science suggests that estrogen has neuroprotective and neuromodulatory effects; however, findings from the Women s Health Initiative Memory Study (WHIMS) have raised concerns over the feasibility of traditional hormone therapies because of the increased risk of serious adverse effects, including cognitive declines. Soy isoflavones may serve as a critically needed alternative therapy to traditional hormone therapy. The few human research projects that have examined the cognitive effects of soy isoflavones offer intriguing, but as yet preliminary support for isoflavones beneficial actions. Careful and systematic study is needed to ascertain the merit of this potential therapeutic. This randomized, placebo-controlled, double-blind design clinical pilot study evaluated the effects of soy isoflavones on cognition in older men and postmenopausal women. It was hypothesized that

11 2005 Abstracts isoflavone supplements would ameliorate age-associated cognitive declines for healthy older adults (i.e., decrease rate of decline, stabilize or enhance cognitive abilities) as compared with subjects on placebo. Data were obtained from 30 cognitively healthy older adults (50% women). Subjects received either 100 mg/day of soy isoflavones or a placebo for 6 months. Cognitive evaluations were conducted at baseline and at 1, 3, and 6 months after initiation of study medications. Cognitive data, collected while on treatment with either isoflavones or placebo were used to calculate change from baseline scores. Women on isoflavones outperformed women on placebo on a test of category fluency (χ 2 = 5.4, p=0.02). Men on treatment did not differ significantly from those on placebo in category fluency performance. The accompanying figure depicts these data. In addition to sex-differences, a genetic risk factor for Alzheimer s disease, the ApoE gene was also found to influence response to isoflavone treatment. Presence of one or two copies of the ApoE 4 allele increases a person s risk for Alzheimer s disease by three- and nine-fold, respectively. Only men without this genetic risk factor appeared to benefit from isoflavone treatment on a measure of executive function, the Trail Making Test (χ 2 = 3.94, p =0.05). Although preliminary, these data suggest that isoflavone treatment is associated with improvements in language and executive function when compared with placebo. However, these beneficial effects are moderated by subject characteristics, such as sex and ApoE genotype. NIA grant K23 AG24302 Research staff and study volunteers from the Wisconsin Comprehensive Memory Program The General Clinical Research Center: grant M01 RR03186 from the National Center for Research Resources, National Institutes of Health; Madison GRECC of the Department of Veterans Affairs; Archer Daniels Midland provided Novasoy brand isoflavones tablets and matching placebo. Prediction of Direct Anti-Oxidant Activity for Soy Isoflavones in Mammalian Brain. S. Eliuk, J. Deshane, L. Wilson, M. Kirk, S. Barnes, and H. Kim*, University of Alabama at Birmingham, Birmingham, Alabama, USA. Soy isoflavones protected against ovariectomy-induced cognitive dysfunction in rats, however the molecular basis for this is unknown. Proteomics of brains from animals given grape seed extract (GSE) or soy protein isoflavones (SPI) indicated that there were common proteins among those affected in the brains of animals that received either polyphenol mixture. Since polyphenols have been shown to have antioxidant activity, we hypothesized that dietary GSE or SPI would attenuate brain protein oxidations. We studied a transgenic mouse model of dementia, the Tg2576 mouse, which overexpresses amyloid precursor protein in the brain, and the animals become cognitively impaired over time, concomitant with the deposition of amyloid plaques in their brains. Protein carbonyls in brain homogenates were derivatized with dinitrophenylhydrazine (DNPH), then analyzed by 2D gel proteomics and Western blots with anti-dnp antibody. While the overall patterns of gel spots were similar, the 2D Western blots of brain proteins from the GSE-fed animals were reduced (20-40% of controls). Gel spots reactive with the anti-dnp antibody were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry. In view of the common proteins affected by GSE and SPI, we predict that proteins similar to those protected by GSE from oxidation will be protected by SPI and studies are ongoing to examine these effects. The DNP-reactive proteins reported here are the first protein oxidations identified, using proteomic technologies, to be affected by dietary polyphenols. GSE, SPI, and related polyphenols may have pleiotropic actions, including on gene expression, protein modifications, and anti-oxidant activity. These studies were supported in part by a subcontract of grant P50-AT00477 from Purdue University (Connie Weaver, PI), by a grant from the United Soybean Board, and by grant DAMD from the Dept of Defense Medical Research and Materiel Command Breast Cancer Research Program. The grape seed extract preparation was generously provided by Kikkoman Corporation (Chiba, Japan), and the soy protein preparations provided by Protein Technologies, Inc. (now Solae, Ltd.). The operation of the UAB Comprehensive Cancer Center Proteomics/Mass Spectrometry Shared Facility was supported in part by a grant (P30 CA13148) from the National Cancer Institute to the UAB Comprehensive Cancer Center (A. LoBuglio, PI). Purchase of proteomic and mass spectrometry instrumentation in the Shared Facility was enabled by Shared Instrumentation Grants to HK and SB from the National Center for Research Resources. Weight Control 16-Week Randomized, Controlled Trial of Soy vs. Casein Meal Replacements for Weight Management of Obese Women. J.W. Anderson 1, J. Fuller 1,E.Konz 1, and A. Tabor 2, 1 University of Kentucky, Lexington, KY, USA, 2 Revival Soy, Kernersville, NC, USA. Meal replacements (MR) are very effective adjuncts to lifestyle changes for weight loss and weight maintenance. Soy protein has specific effects on fat, glucose and lipid metabolism and promotes weight loss in animals. This randomized, controlled trial compared effects of soy meal replacements vs. casein meal replacements on body weight for obese women participating in an intensive lifestyle intervention. Volunteers consumed three MR shakes, either soy or casein, one MR entrée, and five fruits or vegetables daily. Soy protein consumption was 62 g/d with 147 mg/d of isoflavones (aglycones); casein intake was ~68 g/d. 43 subjects enrolled and 35 (17 soy, 18 casein) completed the 16- week study. Soy and casein shakes were well tolerated with good adherence. With soy, subjects lost more visceral adipose tissue and had greater reductions in LDL-cholesterol than with casein but differences were not statistically significant. Outcome changes at 16 weeks for soy and casein, respectively, with mean (SE) and significance from baseline* were: weight, -12.8% (1.4)*,-14.0% (1.2)*; DEXA total fat, -21.8% (2.4)*, -23.7% (2.0)*; visceral fat by mid-abdominal computerized tomography, -30.3% (4.2)*, -27.2% (3.7)*; LDL-cholesterol, -12.5% (4.2)*, -7.7% (4.4)*; and serum triglycerides, -12.5% (8.1), -6.7% (7.7). This intensive intervention was associated with significant weight loss averaging 17 pounds at 8 weeks and 28 pounds at 16 weeks with total fat losses averaging 23% and visceral fat losses averaging 29%. Including 3 MR shakes, either soy or casein, with one portion-control entrée and 5 fruits or vegetables is a very effective weight loss intervention. Supported in part by Revival Soy. 11 Oral Presentation Abstracts