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1999 Soy Symposium Abstracts

Dietary Soy Supplement and Menopausal Hormones and Hot Flashes.

Margo Woods, Donna Speigelman, Ellen Hertzmark, Ann LaBrode, and Christopher Longcope; Tufts University, Boston, MA, USA.

A 7-mo, double-blind, crossover study was designed to determine whether a soy supplement containing 45 mg/d of phytoestrogen would decrease the number and intensity of hot flashes and night sweats in menopausal women (ages 45–58 y) reporting more than five hot flashes daily.

An alternative to hormone replacement therapy (HRT) for the alleviation of hot flashes would be welcomed by the medical community and by many patients who do not choose to use HRT.

Eighty-five women completed the protocol that started with 2 wk of baseline data, recording menopausal symptoms, and collecting blood for the determination of baseline hormones and gonadotrophins. Women were randomly assigned to receive a soy or placebo supplement for 12 wk. A daily record of symptoms was kept and a blood sample was obtained at the end of the12 wk. One month of washout was followed by the alternative treatment for a second 12 wk and repeated data collection. Serum levels of phytoestrogens were determined in a subsample of the women (n = 7) to determine the serum levels achieved on the soy supplement to compare with levels reported in Asian women.

A control group of women (n = 45) was recruited that reported fewer than one hot flash daily for a baseline period of 2 wk to obtain data on hot flashes, hormone levels, and gonadotrophins in women in the same age category and stage of menopause who were experiencing a low level of hot-flash symptoms.

A 22% and 26% reduction in the frequency of hot flashes was reported during both the soy- and placebo-supplemented phases of the study, respectively, compared with baseline, but no difference was observed in the reported number or intensity of the hot flashes when the soy and placebo phases were compared. Endogenous hormones, however, were altered with a significant decrease in serum estradiol in the soy phase compared with baseline (P = 0.003) and compared with the placebo (P = 0.03). Decreases in sex hormone-binding globulin (P = 0.0001) and increases in follicle-stimulating hormone and luteinizing hormone (P = 0.03 for both) compared with baseline were also seen.

Levels of serum phytoestrogen achieved in the women while on the soy supplement were comparable and probably higher than levels seen in the Asian population. A significant inverse association was observed between levels of estrone sulfate and the number of hot flashes reported (P = 0.02).

Association of High Midlife Tofu Consumption with Accelerated Brain Aging.

Lon White; Pacific Health Research Institute, Honolulu, HI, USA.

This investigation used the resources of the Honolulu Heart Program, a longitudinal study of Japanese-American men established in 1965 for research on heart disease and stroke. Questions regarding frequency of consumption of tofu and 26 other foods were asked at interviews in 1965–1967 and again in 1971–1974.

Cognitive testing was done (n = 3734) and cases of dementia identified (n = 225) at the 1991–1993 examination, when participants were aged 71–93 y. Atrophy was assessed by neuroimaging (n = 574) or autopsy (n = 290). Cognitive test data were also analyzed for wives of a sample of study participants (n = 502) who had been living with the participants when their dietary interviews were done.

Poor cognitive test performance in late life was associated with higher midlife tofu consumption. An independent association of similar size and direction was apparent in wives of cohort members, with the husband's answers used as proxy for the wife's consumption.

Midlife tofu consumption was independently associated with low brain weight and with ventricular enlargement. Independent associations of more frequent midlife tofu consumption with clinically diagnosed Alzheimer's disease and with poor cognitive functioning among nondemented subjects were demonstrated. Associations generally followed a dose-response pattern, were statistically significant after all relevant and potentially confounding factors were controlled for, and remained apparent after stratifying by age or obesity.

These data suggest that regular consumption of tofu over many years in middle life may have an adverse influence on brain aging manifest as accelerated atrophy, cognitive decline, and a lowering of the threshold for the clinical manifestations of Alzheimer's disease. We speculate that these may reflect chronic suboptimal neuronal plasticity caused by isoflavone inhibition of tyrosine kinase activity, interference with estrogen-related mechanisms, or both.

Tofu Consumption and Cognition in Older Japanese American Men and Women.

M. M. Rice1,2, A. B. Graves6, S. M. McCurry3, L. Gibbons4, J. Bowen5, W. McCormick2, and E. B. Larson2; Departments of 1Epidemiology, 2Medicine, 3Psychosocial and Community Health, 4Environmental Health, and 5Neurology, University of Washington, Seattle, WA, USA; 6Department of Epidemiology and Biostatistics, University of South Florida, Tampa, FL, USA.

Several epidemiologic and basic neurobiological studies suggest that estrogen may have a beneficial influence on brain function.

We previously found a modest beneficial association between current unopposed estrogen use (ERT) and cognitive change in female participants of the Kame Project. The purpose of the present study was to determine whether the consumption of tofu, an isoflavone-rich food, influenced cognition in men and women. A secondary aim was to determine whether tofu consumption modified the association between ERT and cognition in women.

Subjects were 767 women and 634 men participating in the Kame Project. The Kame Project is a longitudinal cohort study of Japanese Americans aged 65+ y living in King County, WA. The 100-point Cognitive Abilities Screening Instrument (CASI) was measured at the baseline and 2-y follow-up examinations. Tofu consumption was categorized as low (< 1/wk), moderate (1–2/wk), and high (3+/week). All analyses were adjusted for age, education, and language spoken at the interview. In addition, baseline CASI score was adjusted for when the 2-y change in CASI score was examined and surgical menopause was adjusted for when the tofu- ERT interaction was examined.

Cross-sectional results: High tofu consumers had significantly lower CASI scores than did low and moderate consumers in the cohort as a whole (P for trend = 0.03). This trend was not significant in men or in women who had never used ERT. Among women who were current ERT users, a significant negative association was observed between tofu consumption and CASI score (P for trend = 0.04). CASI scores were higher in current ERT users than women who had never used ERT for the low and moderate tofu consumers but not the high tofu consumers.

Longitudinal results: Overall, most men and women showed slight improvements in their CASI score over the 2-y period. No associations were observed between tofu consumption and 2- y change in CASI score, although men and women who were modest tofu consumers showed the greatest improvements in their CASI scores. An important limitation to this study was that total isoflavone exposure was not measured in this cohort. Data from a sample of female Kame participants suggested that tofu accounted for only about half of the soy-derived isoflavones consumed by this population.

The cross-sectional data suggested that high tofu consumption was associated with lower cognitive scores and opposed the beneficial association between ERT and cognitive scores in women. On the other hand, the longitudinal data suggested that tofu consumption was not associated with the rate of cognitive change in older Japanese American men and women and that tofu consumption did not appear to oppose the beneficial association between ERT and cognitive change in women.

Genistein Toxicity from Dietary Exposure from Early Pregnancy Through Puberty

D. M. Sheehan1, K. B. Delclos1, D. R. Doerge1, W. S. Branham1, and R. R. Newbold2; 1 National Center for Toxicological Research, Jefferson, AR, USA; 2National Institute for Environmental Health Sciences, Research Triangle Park, NC, USA.

A dose range finding study was conducted by feeding genistein to Sprague-Dawley rats at 0, 5, 25, 100, 250, 625, and 1250 ppm in a soy-free diet, from gestation day 7 to postnatal day 50. Plasma levels ranged from about 10 nmol/L to 10 µmol/L. Adverse effects included hypertrophy and hyperplasia of the mammary ducts and acini in males at 25 ppm and in females at 625 ppm. Hypospermia at the head of the epididymis, inflammation of the dorsal prostate, and asynchronous cycles of the uterus and vagina were observed at 625 ppm. Degeneration of the ovaries and seminiferous tubules was seen at 1250 ppm. There was a dose-dependent decrease in thyroid peroxidase activity at 25–1250 ppm. Thyroid peroxidase activity is known to be inhibited in vitro by genistein, and the prevalence of autoimmune thyroiditis was reported to be increased in children fed soy infant formula.

In a separate study, genistein (1–100 µg) was injected into neonatal mice daily on postnatal days 1–5 and the mice were killed at 18 mo. Polyovular (multioocyte) follicles, uterine epithelial hyperplasia, hypoplastic uteri, and uterine adenocarcinoma were observed. These are similar to the lesions reported earlier to be induced by diethylstilbestrol in mice by using the same treatment protocol. In addition to genistein, 45 other phyto- or mycoestrogens were assayed in a uterine estrogen receptor competitive binding assay. Twenty-nine of these competed and had a wide range of relative binding affinities. The number of naturally occurring chemicals that can bind to the estrogen receptor suggests that more intense or additional effects may be expected from exposures to multiple estrogenic chemicals from plants.

These findings show a significant number of adverse effects from genistein in estrogen target organs, including malignancies. Some of these effects occur at low doses, consistent with findings from other estrogens. Additionally, genistein inhibition of thyroid peroxidase suggests the possibility that this mechanism may be responsible for goiter and autoimmune thyroiditis. The results of these studies have been used to design an ongoing multigeneration study with genistein that will examine, among other endpoints, reproductive fitness.

Long-Term Adverse Effects After Developmental Exposure to Genistein.

Retha R. Newbold, Wendy N. Jefferson, Elizabeth Padilla Banks, and Bill C. Bullock; Developmental Endocrinology Section, Laboratory of Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

Exposure to synthetic and naturally occurring estrogenic compounds results in permanent alterations in the developing organism if exposure occurs during the critical stages of differentiation. Reports from our laboratory showed that perinatal exposure of mice to diethylstilbestrol (DES) results in impaired fertility, structural malformations, and lesions of the reproductive tract. In fact, long-term consequences of exposure to DES at 2 µg/(pup·d) on days 1–5 include a high incidence (95%) of uterine carcinoma in animals older than18 mo. Doses less than 2 µg/(pup·d) also result in long-term adverse effects. Thus, the developing reproductive tract appears to be extremely sensitive to perturbation by compounds with estrogenic activity.

Because the nutritional and pharmaceutical use of phytoestrogens increased over the past few years mainly because of its reported beneficial effects, we investigated the potential risks posed by genistein if exposure occurs early in development.

We tested the possibility that developmental exposure to this compound would influence morphological, functional, and biochemical markers known to be estrogen sensitive. Uterine epithelial cell proliferation and induction of uterine lactoferrin and complement C3 were increased in response to genistein.

Although, lactoferrin was previously reported to be constitutively produced in the uteri of neonatally DES-treated mice as early as 2 mon prior to the development of uterine carcinoma, the role of this protein in the induction of neoplasia, either as a marker or contributing factor, remains to be determined. However, lactoferrin and other marker proteins are induced by developmental exposure to genistein.

The interaction of genistein with the estrogen receptors a and ß was investigated. Further, mice were followed to evaluate a potential increased risk for histological abnormalities, including uterine tumors later in life. Many of the long-term effects observed after DES treatment, including uterine adenocarcinoma, were observed after developmental exposure to genistein. Similarities between the effects of DES and genistein point to the need for further mechanistic studies in phytoestrogens and the role in long-term effects that follow developmental exposure.

Genistin, the Glycoside Form of Genistein, Stimulates Growth of Estrogen-Dependent Human Breast Cancer Cells In Vivo.

Clinton D. Allred, Kimberly F. Allred, and William G. Helferich; Division of Nutritional Science and Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL, USA.

The phytoestrogen genistein stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo.

Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to genistein in the gut. However, it is unclear whether the rate of this metabolism is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth.

We hypothesized that dietary genistin stimulates tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted MCF-7 tumors. Mice were fed either genistein at 750 ppm or genistin at 1200 ppm, which provides equimolar concentrations of genistein in both diets. Tumor size was measured weekly.

At completion of the study, half the animals were killed and tumors were collected for cell proliferation analysis. Incorporation of BrdU into cellular DNA was used as an indicator of cell proliferation.

Dietary genistin resulted in increased tumor growth rate and cell proliferation similar to that observed with genistein.

The remaining mice were switched to diets free of genistin and genistein. Removal of the isoflavones from the diet resulted in tumor regression.

In summary, genistin, like genistein, can act as an estrogen agonist to increase proliferation of estrogen-dependent breast cancer cells, and on its removal tumors regress.