Soy Studies

This paper compiles animal and human data on the biologic effects and exposure levels of phytoestrogens in order to identify areas of research in which direct species comparisons can be made. In vitro and in vivo assays of phytoestrogen action and potency are reviewed and compared to actions, dose-response relationships, and estimates of exposure in human subjects. Binding studies show that the isoflavonoid phytoestrogens are high-affinity ligands for estrogen receptors (ERs), especially ER beta, but have lower potency in whole-cell assays, perhaps because of interactions with binding proteins. Many other enzymatic actions require concentrations higher than those normally seen in plasma. In vivo data show that phytoestrogens have a wide range of biologic effects at doses and plasma concentrations seen with normal human diets. Significant in vivoresponses have been observed in animal and human tests for bone, breast, ovary, pituitary, vasculature, prostate, and serum lipids. The doses reported to be biologically active in humans (0.4--10 mg/kg body weight/day) are lower than the doses generally reported to be active in rodents (10--100 mg/kg body weight/day), although some studies have reported rodent responses at lower doses. However, available estimates of bioavailability and peak plasma levels in rodents and humans are more similar. Steroidogenesis and the hypothalamic-pituitary-gonadal axis appear to be important loci of phytoestrogen actions, but these inferences must be tentative because good dose-response data are not available for many end points. The similarity of reported proliferative and antiproliferative doses illustrates the need for fuller examination of dose-response relationships and multiple end points in assessing phytoestrogen actions.

The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.

Objectives: Women exposed prenatally to diethylstibestrol (DES) have an excess risk of clear-cell adenocarcinoma of the vagina and cervix, but the effect on the incidence of squamous neoplasia is uncertain. The purpose of the current study was to evaluate the long-term risk of developing high-grade squamous neoplasia of the genital tract among women exposed prenatally to DES.

Methods: A cohort comprising 3899 DES-exposed and 1374 unexposed daughters was followed for 13 years (1982-1995) for pathology-confirmed diagnoses of high-grade squamous intraepithelial neoplasia (HSIL) of the genital tract. Poisson regression analysis was used to compute relative risks (RR) and 95% confidence intervals (95% CI), adjusting for age, calendar year, and other covariates.

Results: The RR (95% CI) among DES-exposed versus unexposed, based on 111 cases of high-grade disease, was 2.1 (1.2-3.8). Adjustment for screening history estimated by the number of years since the last Pap smear had little effect. Risk estimates were higher with earlier intrauterine exposure; the RR (95% CI) for exposure within 7 weeks of the last menstrual period was 2.8 (1.4-5.5). Only two cases of invasive squamous cervical cancer occurred in total, precluding separate analysis

Conclusions: The findings support an association between in-utero DES exposure and high-grade squamous neoplasia, although a role for more intensive screening among DES-exposed women in the production of this excess could not be completely ruled out.

Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4+CD8 and double-positive CD4+CD8+ thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4+CD8 helper T cell lineage. Decreases in the relative percentages of CD4+CD8 thymocytes were accompanied by decreases in relative percentages of splenic CD4+CD8 cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.

Quotes

The world population explosion has pointed out the need for new and effective contraceptive agents...

...it can be seen that 565 species of plants are known to have a folkloric reputation for use as abortifacients, ecbolics, or emmenagogues. Of this 565 species, 225 have been shown to elicit a stimulant response when tested againsts uterine muscle either in vitro or in vivo.

Quotes

A number of estrogenic sterols have been reported as being derived from higher plants

If one inspects the sturctures of the estrogenic sterols, isoflavones, and coumestans, one can see a striking similarity of the skeletal structures of these compounds with the structure of the synthetic estrogen diethylstilbestrol.

The order or degree of the biological activity of each of the three main groups of natural estrogens has been investigated. The sterol estrogens were found to be of the highest order of activity followed by the coumestrols and then the isoflavones.

Abstract

We have evaluated the hypothesis of a protective effect of human milk on the development of insulin dependent diabetes mellitus (IDDM). We studied the feeding histories of 95 diabetic children and compared them with controls consisting of their non-diabetic siblings and a pair matched group of non-diabetic peers of the same age, sex, geographical location and social background. The incidence of breast feeding in diabetic children was 18%. This was similar to the control group. The duration of breast feedings was also similar among all three groups. There was no difference in the age of introduction of solid food between diabetic and non-diabetic children. Twice as many diabetic children, however, received soy containing formula in infancy as compared to control children. The mean age of onset of IDDM was not related to the type of feeding during infancy. The incidence of positive thyroid antibodies was two and one half times higher in formula-fed diabetic children that in breast-fed ones. In our studies we were unable to document any relationship between the history of breast feeding and subsequent development of IDDM in children.

Recommendations

• 1. Breast-feeding is strongly endorsed as the primary source of nutrition during the first year of life for all infants.
• 2. In families with a strong history of IDDM, particularly if a sibling has diabetes, breast-feeding and avoidance of commercially available cow’s milk and products containing intact cow’s milk protein during the first year of life are strongly encouraged.
• 3. Since the antigenicity of infant formulas and cow’s milk may be different and there is no evidence against the use of formula for infants whose mothers do not breast-feed, commercial infant formulas utilising cow’s milk protein remain the approved alternate.
• 4. The substitution of soy-based formulas for milk-based formulas is not advised for either general or high-risk infant feeding practices because of animal studies linking the ingestion of soy protein intake to the development of diabetes.

Several plant estrogens, especially coumestrol and genistein, were found to reduce the conversion of [3H]estrone to [3H] 17 beta-estradiol catalyzed by estrogen-specific 17 beta-hydroxysteroid oxidoreductase Type 1 (E.C. 1.1.1.62) in vitro. Coumestrol, the most potent inhibitor in our experiments, is the best inhibitor of the enzyme known to date. All compounds with inhibitory effects were also estrogenic. However, structural demands for 17 beta-HSOR Type 1 inhibition and estrogenicity of tested compounds in breast cancer cells (judged by increased cell proliferation) were not identical. Zearalenone and diethylstilbestrol, both potent estrogens, did not inhibit 17 beta-HSOR Type 1. Thus, changes in the estrogen molecule may discriminate between active sites of 17 beta-HSOR Type 1 and estrogen binding sites of the ER. The effects of these compounds in vivo cannot be predicted on the basis of these results. Inhibition of 17 beta-HSOR Type 1 enzyme could lead to a decrease in the availability of the highly active endogenous estrogen. However, these compounds are estrogenic per se, and they may thus replace endogenous estrogens. Additional studies are needed to further understand the role of these plant estrogens in the etiology of hormone-dependent cancers. It is not easily conceivable how the chemopreventive action of Asian diets, possibly mediated by phytoestrogens in soya products, can be based on the inhibition of estrone reduction at the target cells by phytoestrogens or related compounds, unless they are "incomplete estrogens" (i.e., unable to induce all effects typical of endogenous estrogens).

Tumor tissue, including breast cancer, thyroid carcinoma, uterine carcinoma and granulosal thecal cell ovarian tumor, from twelve different patients, was analysed for sterol content by thin-layer and gas-liquid chromatography. Cholesterol was present in large amounts in all. A sterol with the retention time of desmosterol was present in 11; one with the retention time of stigmasterol in 3; of which, 2 also contained a sterol with the retention time of campesterol, and 1 of B-sitosterol. Thus, the finding of osteolytic sterols in breast cancer by Gordan and associates is partially confirmed and extended.

Quotes

The present study tends to confirm, in part, reports by Gordan and associates of the presence of osteolytic phytosterols in breast cancers, postulated by them to account for the frequent serious degree of hypercalcemia seen in these patients. We have demonstrated evidence for their presence in one of eight breast cancers as well as in a follicular carcinoma of the thyroid metastatic to the lung, and in a malignant mixed Mullerian tumor of the uterus.

We found no evidence of the presence of these sterols in normal tissue

Quotes

Hypercalcemia is a serious, life-threatening emergency. The most common cause of hypercalcemia is malignancy, and the one tumor accounting for two-thirds of all cases of the hypercalcemia of malignancy is breast cancer. In most cases, the hypercalcemia of breast cancer is associated with osteolytic metastases. As with other malignancies, however, hypercalcemia occasionally occurs in the absence of osseous lesions.

Hypercalcemia is a frequent cause of death and disability in breast cancer, occasionally even in the absence of osseous metastases

It is not caused by parathyroid hormone or vitamin D

A series of plant sterols has been identified in breast cancer extracts and in the plasma of patients with breast cancer. Some of these, campesterol, stigmasterol and sitosterol, are also found in normal plasma. Breast cancer is characterised by an abnormality of sterol metabolism leading to the accumulation of sterol esters, especially the osteolytic, short chained fatty acid acid esters of stigmasterol and 7-dehydrositosterol. Either or both was found in the plasma of all 25 cases - 19 disseminated and 6 local. They were not found in normal non-lactating women.

It is suggested that the esters of stigmasterol and of 7-dehydrositosterol may play a role in the hypercalcemia of breast cancer.

Quotes

Plant sterols and their esters have been isolated from plasma and breast extracts of a number of patients with breast cancer in concentrations much higher than is found in normal persons. This has been confirmed in patients with thyroid or renal carcinoma. Indeed, the esters of certain plant sterols have been shown to possess high osteolytic activity

Quotes

Plant sterols are structurally similar to cholesterol, all having a cyclopentenophenanthrene ring with 3-beta hydroxy substitution and a 5-6 double bond. Beta-sitosterol is proportionately the predominant plant sterol in dible oils and grains, with smaller amounts of campesterol and stigmasterol. In adult diets, plant sterols are estimated to account for about one tenth of 1 per cent of total calories and 20 per cent of total sterols. Studies in adults using labelled beta-sitosterol suggest that less than 5 per cent of orally administered sitosterols are absorbed, representing about one thenth of the amount of cholesterol absorbed under similare conditions

There is however an apparent increase in absorption of dietary phytosterols in infancy and childhood. In infants and children fed diets rich in phytosterols, plasma phytosterols rose from less than 2 to 9 mg. per 100 mL. The implications of long-term three- to fivefold elevations of the plasma phytosterols in infancy and childhood are unknown.

In adults with mature atheromatous lesions, plant sterols are present in appreciable amounts. The implications of these findings are unknown and their relationship to deposition of cholesterol in atheromatous and in normal aortic tissues remains to be elucidated.

Although balance studies of phytosterol absorption have not been done in human infants, it appears that phytosterol-rich diets induce elevations of plasma phytosterol five- to 15-fold above that observed in adults. Once abserbed, phytosterols are catabolised, esterified, and handled in a fashion similar to, but not exactly the same as cholesterol. ...there is no endogenous synthesis of beta-stiosterol

...the correlation between cholesterol and phytosterols (excluding campesterol) suggests parallel accumulation of these sterols.

...it is speculated that phytosteols may accumulate in infants faster than cholesterol.

In contrast, the ratio of plasma cholesterol to plant sterol in vegetable-oil formula-fed infants was considerably lower than relative tissue ratios. The early increments of aortic tissue phytosterol in infants on phytosterol-rich diets might be speculatively related to relatively low cholesterol and relatively high plasma phytosterol.

In the mature adult atheromatous lesion, where large amounts of cholesterol were present, there were notable increases in plant sterols.

Soy is known to produce estrogenic isoflavones. Here, we briefly review the evidence for binding of isoflavones to the estrogen receptor, in vivo estrogenicity and developmental toxicity, and estrogen developmental carcinogenesis in rats. Genistein, the major soy isoflavone, also has a frank estrogenic effect in women. We then focus on evidence from animal and human studies suggesting a link between soy consumption and goiter, an activity independent of estrogenicity. Iodine deficiency greatly increases soy antithyroid effects, whereas iodine supplementation is protective. Thus, soy effects on the thyroid involve the critical relationship between iodine status and thyroid function. In rats consuming genistein-fortified diets, genistein was measured in the thyroid at levels that produced dose-dependent and significant inactivation of rat and human thyroid peroxidase (TPO) in vitro. Furthermore, rat TPO activity was dose-dependently reduced by up to 80%. Although these effects are clear and reproducible, other measures of thyroid function in vivo (serum levels of triiodothyronine, thyroxine, and thyroid-stimulating hormone; thyroid weight; and thyroid histopathology) were all normal. Additional factors appear necessary for soy to cause overt thyroid toxicity. These clearly include iodine deficiency but may also include additional soy components, other defects of hormone synthesis, or additional goitrogenic dietary factors. Although safety testing of natural products, including soy products, is not required, the possibility that widely consumed soy products may cause harm in the human population via either or both estrogenic and goitrogenic activities is of concern. Rigorous, high-quality experimental and human research into soy toxicity is the best way to address these concerns. Similar studies in wildlife populations are also appropriate.

To the Editor:

The results of Van Patten et al confirmed previous findings that soy phytoestrogens have minimal efficacy for menopausal symptoms in breast cancer patients. However, I am concerned that patients in neither study were apparently informed of the potential stimulatory effects of phytoestrogens on breast tumor. Similar omission would have raised ethical concerns if pharmaceutical drugs were involved.

At concentrations below 10 µmol/L, phytoestrogens can stimulate breast tumor growth and antagonize the antitumor effects of tamoxifen, particularly in an environment of low endogenous estrogen. In contrast, phytoestrogens inhibit breast tumor growth and enhance the antitumor effects of tamoxifen at concentrations above 10 µmol/L. In humans, serum phytoestrogen concentrations attained after acute or chronic intake of phytoestrogens were much lower than 10 µmol/L.

Without long-term human data, cancer risk assessments need to be cautious and assume that substances that promote tumor growth in preclinical studies may pose similar risks clinically. Hence, to weigh the potential risks versus benefits before using phytoestrogens for unproven indications, breast cancer patients should be informed that phytoestrogens have the potential to stimulate tumor growth.

Mário de Lemos

OBJECTIVE: To determine whether genistein and daidzein, the major phytoestrogens in soy, can stimulate breast cancer growth. DATA SYNTHESIS: Systematic search through primary English-language literature on MEDLINE (1966-January 2001), EMBASE (1982-January 2001) and Current Contents (1998-January 2001). DATA SOURCES: Genistein and daidzein at low concentrations were found to stimulate breast tumor growth in in vitro and in vivo animal studies, and antagonize the antitumor effect of tamoxifen in vitro. At high concentrations, genistein inhibited tumor growth and enhanced the effect of tamoxifen in vitro. CONCLUSIONS: Genistein and daidzein may stimulate existing breast tumor growth and antagonize the effects of tamoxifen. Women with current or past breast cancer should be aware of the risks of potential tumor growth when taking soy products.

Environmental oestrogens have been implicated in the pathogenesis of hormonally treated cancers (such as breast and prostate cancer), male infertility, and abnormalities of the male and female reproductive tracts. They may be derived from plants (phytoestrogens), pharmaceuticals, or other synthetic compounds not originally intended to have oestrogenic activity (including soy based infant formulas). This review will discuss the evidence from both animal studies and humans for an effect of these ubiquitous compounds on the development of the human female genital tract, in addition to prolonging the menstrual cycle, alleviating symptoms of the menopause, and protecting against the development of endometrial carcinoma.

Some Take Home Messages

Environmental oestrogens have been implicated in the pathogenesis of hormonally treated cancers (such as breast and prostate cancer), male infertility, and abnormalities of the male and female reproductive tracts.

Environmental oestrogens may be derived from plants (phytoestrogens), pharmaceuticals, or other synthetic compounds not originally intended to have oestrogenic activity.

Exposure to these compounds results in structural and functional abnormalities in the female genital tract of fish, rodents, and livestock.

The age at first exposure and the duration of exposure are important, neonatal exposure having the potential to produce lasting morphological abnormalities and a persistent (gonad independent) oestrous state.

The human diet is rich in phytoestrogens, and such compounds are also present in soy based infant formulas, which may be a cause for concern.

To date, there is little evidence that such compounds affect human female genital tract development or fertility, probably because of the ubiquitous nature of such compounds in the environment and a lack of investigation, rather than the absence of a correlation.

The estrogenic soy isoflavone, 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 the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate 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 estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogen-dependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.

We have demonstrated that the isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo (C. Y. Hsieh et al., Cancer Res., 58: 3833-3838, 1998). The isoflavones are a group of phytoestrogens that are present in high concentrations in soy. Whether consumption of genistein from soy protein will have similar effects on estrogen-dependent tumor growth as pure genistein has not been investigated in the athymic mouse tumor implant model. Depending on processing, soy protein isolates vary widely in concentrations of genistein. We hypothesize that soy isolates containing different concentrations of genistein will stimulate the growth of estrogen-dependent cells in vivo in a dose-dependent manner. To test this hypothesis we conducted experiments in which these soy protein isolates were fed to athymic mice implanted s.c. with estrogen-dependent tumors. Genistein content (aglycone equivalent) of the soy isolate diets were 15, 150, or 300 ppm. Positive (with 17beta-estradiol pellet implant) and negative (no 17beta-estradiol) control groups received casein-based (isoflavone-free) diets. Tumor size was measured weekly. At completion of the study animals were killed and tumors collected for evaluation of cellular proliferation and estrogen-dependent gene expression. Incorporation of bromodeoxyuridine into cellular DNA was used as an indicator of cell proliferation, and pS2 mRNA was used as an estrogen-responsive gene. Soy protein diets containing varying amounts of genistein increased estrogen-dependent tumor growth in a dose-dependent manner. Cell proliferation was greatest in tumors of animals given estrogen or dietary genistein (150 and 300 ppm). Expression of pS2 was increased in tumors from animals consuming dietary genistein (150 and 300 ppm). Here we present new information that soy protein isolates containing increasing concentrations of genistein stimulate the growth of estrogen-dependent breast cancer cells in vivo in a dose-dependent manner.

There is widespread concern that fetal exposure to hormonally active chemicals may adversely affect development of the reproductive tract. Therefore, the present study was performed to develop the necessary analytical methods and test the hypothesis that dietary phytoestrogens can be quantified in second trimester human amniotic fluid. Amniotic fluid samples (n=59) from women (n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (+/- standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44 +/- 1.34 and 1.69 +/- 1.48 ng/ml with maximum levels of 5.52 and 6.54 ng/ml, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure.

BACKGROUND: Phytoestrogens, including genistein and other inhibitors of tyrosine kinases (TKs), inhibit specific steroidogenic enzymes. This study was designed to compare the effects of genistein, with two other TK inhibitors, on steroid synthesis in human granulosa luteal (GL) cells and to identify which steroidogenic enzymes they may affect. METHODS: GL cells, obtained from women undergoing IVF procedures, were cultured for various periods of time with and without substrates for progesterone and estradiol synthesis, in the presence or absence of the TK inhibitors. RESULTS: The TK inhibitors significantly suppressed progesterone and estradiol synthesis in a dose-dependent manner over a 48 h culture period. Progesterone production in the presence of 10(-7) mol/l pregnenolone during a 4 h period was inhibited by both acute (4 h) and chronic (24 h) exposure of GL cells to 50 micromol/l genistein (P < 0.05) whilst no significant effects of 50 micromol/l tyrphostin A23 were observed. Genistein (4 and 24 h exposure) inhibited the production of estradiol using 10(-7) mol/l estrone as a substrate, but inhibition of estradiol synthesis using androstenedione or testosterone as substrates was only observed after a 24 h exposure. In contrast, tyrphostin acutely stimulated estradiol synthesis when androstenedione and testosterone were used as substrates (P < 0.05) but not estrone. CONCLUSIONS: Genistein directly inhibits 3 and 17beta-hydroxysteroid dehydrogenase activity, whilst tyrphostin has an acute stimulatory effect on aromatase activity. Over a longer time (24 and/or 48 h period), both TK inhibitors suppress steroid synthesis.

Plasma testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulfate, androsterone and epiandrosterone sulfates, cortisol and sex hormone binding globulin were measured in six adult men before and during daily isoflavone extract ingestion (40 mg) in the form of Trinovin tablets. Although modest plasma genistein levels were achieved following three weeks of Trinovin ingestion (106-356 nmol/l) there were no significant changes in most of the analytes tested. However plasma levels of dihydrotestosterone showed an increase that reached significance when combined basal levels were compared to levels following Trinovin treatment. The results suggest that the daily ingestion of isoflavones in the form of Trinovin (1 tablet/day), over a short term, does not alter most plasma steroid levels. We therefore question the value of Trinovin, at the recommended dosage, as offering protective effects against prostate disease by mechanisms involving either significant modulation of plasma steroid or SHBG levels. In contrast the increase in dihydrotestosterone plasma levels could be seen as possibly detrimental.

The association of estrogens with benign prostatic hyperplasia and prostatic cancer has been widely studied, but no conclusive evidence exists for a role of estrogens in prostatic disease. This paper reviews the literature and describes studies which have sought to show a correlation of estrogens and alterations in the prostates of humans and experimental animal models. Using the developmentally estrogenized mouse model, we propose an alternative role for estrogens as a predisposing factor for prostatic diseases: estrogen exposure during development may initiate cellular changes in the prostate which would require estrogens and/or androgens later in life for promotion to hyperplasia or neoplasia. Thus, the critical time for estrogen action would be during the development of the prostatic tissue. We further suggest that estrogen-sensitive cells may remain in the prostate and be more responsive to estrogens later in life or less responsive to the normal controlling mechanisms of prostatic growth

OBJECTIVE: To examine the relationships between dietary intake of soy products and hot flushes and other menopausal symptoms. METHODS: Subjects were 284 women aged 40-59 years who attended a health check-up program provided by a general hospital in Gifu, Japan. They completed a health questionnaire including the Kupperman test of menopausal distress. Diet was assessed by a semiquantitative food frequency questionnaire. RESULTS: Fermented soy product intake but not total soy product intake was significantly negatively correlated with hot flush severity (r = -0.16, p = 0.01) after controlling for age and menopausal status. Neither total soy product intake nor fermented soy product intake was significantly correlated with menopausal index score. Estimated isoflavone intake from total and fermented soy products was significantly lower by 15% (p = 0.02) and 19% (p = 0.01), respectively, in women with hot flushes, compared to those without hot flushes after controlling for covariates. CONCLUSION: The data support a hypothesis that intake of fermented soy products alleviates the severity of hot flushes.

Caudal epididymal spermatozoa were used to study the influence of genistein isoflavone and dexamethasone (dxm) on the functional characteristics of spermatozoa. The effects of genistein alone and in combination with dxm on sperm motility, sperm morphology, spontaneous acrosome reaction (AcR), and ionophore A23187-induced AcR were investigated. The FITC-PSA/Hoechst 33258 staining procedure was used to assess sperm cell viability and AcR status and thus to differentiate between true AcR and acrosome degeneration. The overall results indicated that (1) lower doses of genistein alone, or in combination with dxm, did not significantly influence sperm motility or sperm morphology; (2) ionophore A23187 induced AcR in rat spermatozoa; (3) there appeared to be no direct correlation between sperm motility and AcR, (4) higher doses of genistein, alone or in combination with dxm, significantly interfered with percentage sperm motility and caused significant detachment of sperm heads but did not cause morphological defects; and (5) higher doses of genistein caused significant decrease in sperm acrosome reactivity with long duration of exposure. In view of the fact that sperm capacitation and AcR are physiological prerequisites for successful fertilization of oocytes, the findings suggest that chronic exposure of spermatozoa to high doses of genistein could be associated with infertility problems through suppression/inhibition of AcR and sperm motility. Dexamethasone did not appear to influence the effect of genistein on the functionality of postspermatogenic spermatozoa

The effects of genistein (Gn), sodium azide (naz), and dexamethasone (dxm) on testicular cells TM3, TM4 and GC-1 spg were studied in vitro. First, a series of experiments were performed to assess the response of the cells to the exposure of Gn, naz, dxm, a combination of Gn with naz and Gn with dxm. Trypan blue exclusion assay was used to determine the percentage of viability, and LDH-cytotoxicity test was used to assess the degree of treatment-induced cytotoxicity on each cell type. A second series of experiments were performed to study cytomorphology and determine the type and percentage of treatment-induced cell death (apoptosis and necrosis) on each cell line, using fluorescent dye technique to detect apoptotic and necrotic cells, and tunnel assay to confirm apoptosis. The results from the data obtained demonstrated: i) that incubation of testis cells with each of the agents (Gn, dxm, naz) alone and in two combinations (Gn-dxm, and Gn-naz) induced significant testicular cell death; ii) that both genistein and dexamethasone mostly and significantly induced apoptotic cell death while sodium azide induced necrotic cell death; iii) that addition of dexamethasone to genistein demonstrated synergism in apoptosis on testis cells; and iv) that combination of naz with Gn demonstrated synergism in necrosis on testis cells even though Gn alone did not induce significant necrosis. It is concluded that the synergistic actions of genistein and dxm, and of genistein + sodium azide in induction of apoptosis and/or necrosis may be of clinical and pathophysiological research interest considering the chemopreventive and chemotherapeutic potential of genistein; and the clinico-pharmacological application of dexamethasone and sodium azide.

The effects of genistein on testicular cells, TM3, TM4, and GC-1 spg, were studied in vitro. First, each cell line was cultured with pre-determined concentrations of genistein for a maximum of 72 h to assess the effects of genistein on in vitro growth of the test cells. A second series of experiments were performed to determine the degree of genistein-induced apoptosis in these cells, using Apop-Tag kit reagents, to detect apoptotic cells in situ by specific end labeling, and detection of DNA fragments produced by the apoptotic process. The results obtained indicate that: i) genistein inhibits the growth and proliferation of testicular cells; ii) growth inhibition and proliferation is dose- and exposure-time dependent; iii) there is significant difference in sensitivity of the different testicular cells to genistein; iv) genistein induces apoptosis in testicular cells in a concentration-dependent manner. Genistein-induced apoptosis identifies genistein as a potential diagnostic and therapeutic tool in testicular pathophysiological research.

Recent reports have demonstrated a decline in human male reproductive health: high and probably increasing prevalence of cryptorchidism and hypospadias, low and probably decreasing semen quality, a rising incidence of testicular cancer and a growing demand for assisted reproduction. These changes seem to be interrelated and may be symptoms of a common underlying entity, the testicular dysgenesis syndrome, with foundations in fetal life due to adverse environmental influences. Wildlife experience and animal studies have provided evidence that fetal or perinatal exposure to endocrine disrupters results in disturbed sexual differentiation and urogenital malformations followed by decreased reproductive health in adult life. This chapter reviews existing evidence for a connection between (i) exposure to endocrine disrupters in fetal life and childhood and (ii) adult reproductive health in humans. This topic is not only relevant to basic scientists but also to clinical endocrinologists, who should also be encouraged to participate in research concerning this problem.

The association between soyfood consumption and subsequent bladder cancer risk was investigated in a population-based cohort study, the Singapore Chinese Health Study. As of December 31, 2000, 329,848 person-years of follow-up were accrued. Sixty-one histologically confirmed incident bladder cancer cases were identified. Information on soyfood consumption at baseline was obtained through in-person interviews using a validated dietary questionnaire. Relative risks and 95% confidence intervals were calculated using the Cox proportional hazard regression method. High intake of soyfood was statistically significantly related to an elevated risk of bladder cancer. Relative to the lowest quartile of energy-adjusted total soy intake (<36.9 g/1000 Kcal), the highest quartile of total soy intake (> or =92.5 g/1000 Kcal) was associated with a 2.3-fold increase in bladder cancer risk (95% confidence interval = 1.1-5.1) after adjustment for cigarette smoking and level of education. Similar results were obtained for intakes of soy protein and soy isoflavones. The soyfood-bladder cancer risk association did not differ significantly between men and women and was not explained by other dietary factors. The soy-cancer relationship became stronger when the analysis was restricted to subjects with longer (> or =3 years) duration of follow-up. To our knowledge, this is the first epidemiological report on the effect of dietary soy on bladder cancer risk.

CONTEXT: Clinical trials demonstrating increased risk of cardiovascular disease and breast cancer among women randomized to hormone replacement therapy have increased interest in other therapies for menopausal symptoms. Dietary supplements containing isoflavones are widely used as alternatives to hormonal therapies for hot flashes, but there is a paucity of data supporting their efficacy. OBJECTIVE: To compare the efficacy and safety of 2 dietary supplements derived from red clover with placebo in symptomatic menopausal women. DESIGN, SETTING, AND PARTICIPANTS: Randomized, double-blind, placebo-controlled trial of menopausal women, aged 45 to 60 years, who were experiencing at least 35 hot flashes per week. The study was conducted between November 1999 and March 2001 at 3 US medical centers and included women who were recently postmenopausal (mean [SD], 3.3 [4.5] years since menopause) experiencing 8.1 hot flashes per day. Women were excluded if they were vegetarians, consumed soy products more than once per week, or took medications affecting isoflavone absorption. INTERVENTION: After a 2-week placebo run-in, 252 participants were randomly assigned to Promensil (82 mg of total isoflavones per day), Rimostil (57 mg of total isoflavones per day), or an identical placebo, and followed-up for 12 weeks. MAIN OUTCOME MEASURE: The primary outcome measure was the change in frequency of hot flashes measured by participant daily diaries. Secondary outcome measures included changes in quality of life and adverse events. RESULTS: Of 252 participants, 246 (98%) completed the 12-week protocol. The reductions in mean daily hot flash count at 12 weeks were similar for the Promensil (5.1), Rimostil (5.4), and placebo (5.0) groups. In comparison with the placebo group, participants in the Promensil group (41%; 95% confidence interval [CI], 29%-51%; P =.03), but not in the Rimostil group (34%; 95% CI, 22%-46%; P =.74) reduced hot flashes more rapidly. Quality-of-life improvements and adverse events were comparable in the 3 groups. CONCLUSION: Although the study provides some evidence for a biological effect of Promensil, neither supplement had a clinically important effect on hot flashes or other symptoms of menopause.

DNA topoisomerases are nuclear enzymes inducing transient breaks in the DNA allowing DNA strands or double helices to pass through each other. The clinically used DNA topoisomerase II-poison etoposide is known to induce DNA double strand breaks leading to chromosomal aberrations and leukemias. Recently, some alarming studies have been published, suggesting that maternal exposure to low doses of dietary topoisomerase II poisons, including bioflavonoids such as genistein or quercetin, may contribute to the development of infant leukemia: approximately 80% of infants with acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) have chromosome translocations involving the MLL (mixed lineage leukemia) gene. It has been shown that antineoplastic chemotherapy with the leukemogenic topoisomerase II-poison etoposide induced identical chromosomal aberrations involving the MLL gene compared to children with infant leukemia. Interestingly, the MLL cleavage sites induced by etoposide colocalized with the cleavage sites observed in infant leukemia. In addition, an almost 10-fold higher risk of infant AML has been reported for mothers consuming relatively high levels of topoisomerase II-poison containing foods. These observations are relevant, since many foods contain topoisomerase IIpoisons, predominantly soy and soy products, but also coffee, wine, tea, cocoa, as well as some fruits and vegetables. Further studies on the role of dietary topoisomerase II-poisons are urgently required. If the causal relationship between dietary exposure to topoisomerase IIpoisons and infant leukemia will be confirmed, care should be taken to reduce exposure to critical foods during pregnancy. Full Paper Available Here

To determine the effect of soy-derived isoflavones on hot flushes, endometrial thickness, and the vascular reactivity of uterine and cerebral arteries. Double-blind, randomized, placebo-controlled trial.Healthy volunteers in an academic research environment.Sixty-two postmenopausal women aged 45-60 years attending the Outpatient Menopause Clinic of our gynecological departments. The patients were administered 72 mg of soy-derived isoflavones or placebo under double-blind conditions. The daily number of hot flushes was recorded in a diary. Endometrial thickness was measured by means of transvaginal ultrasound; the uterine, internal carotid, and middle cerebral arteries were evaluated using Doppler ultrasound. The daily number of hot flushes, endometrial thickness, and arterial pulsatility index (PI). Both treatments led to a 40% reduction in the number of hot flushes. Soy-derived isoflavones had no effect on endometrial thickness or the PI of the uterine and cerebral arteries. The daily administration of 72 mg of soy-derived isoflavones is no more effective than placebo in reducing hot flushes in postmenopausal women. It also has no effect on endometrial thickness or the PI of the uterine and cerebral arteries.

OBJECTIVE:: We examined the effects on serum sex steroids, lipids and markers of oxidative stress of supplementing the diets of healthy male volunteers with scones made with soya flour. DESIGN:: A randomized placebo controlled cross-over trial. SETTING:: University Hospital of Wales. SUBJECTS:: Twenty volunteers recruited by advertisement. INTERVENTIONS:: Male volunteers ate three scones a day in addition to their normal diet for a period of 6 weeks. The scones were made with either wheat or soya flour (containing 120 mg/day of isoflavones). Blood was analysed for sex steroids (testosterone, dihydro-testosterone, oestradiol, oestrone, sex hormone binding globulin, albumin and the concentration of non-protein bound sex steroids were calculated), lipid profile (total cholesterol, high density lipoprotein cholesterol and triglycerides) and measures of oxidative stress (hydroperoxides, susceptibility of LDL to oxidation with copper and myeloperoxidase). RESULTS:: The volunteers' mean age was 35.6 (s.d. 11.2) y. Total serum testosterone fell in volunteers taking the soya scones (19.3-18.2 nmol/l; 95% CI 1.01, 1.12; P=0.03). No significant changes were seen in the concentrations of the other serum sex steroids, albumin or sex hormone binding globulin throughout the study. Significant improvements in two of the three markers of oxidative stress were seen in volunteers taking soya scones. Lag time for myeloperoxidase rose from 55.0 to 68.0 min (95% CI -16.0, -3.5; P=0.009) and the presence of hydroperoxides decreased from 2.69 to 2.34 micro mol/l (95% CI 0.12, 0.71; P=0.009). There were no changes seen in serum triglycerides or cholesterol. CONCLUSIONS:: We have shown that soya supplements reduce serum testosterone and improve markers of oxidative stress. These findings provide a putative mechanism by which soya supplements could protect against prostatic disease and atherosclerosis. Further dietary studies with clinical end points are warranted. SPONSORSHIP:: The Mason medical research foundation.

Nearly 80 percent of infant leukemias present with an abnormality involving the MLL gene at 11q23. Moreover, secondary acute myeloid leukemias (AML) that occur as the result of chemotherapy agents, which are known to inhibit DNA topoisomerase II, often manifest the same MLL abnormalities. It has been hypothesized that de novo infant leukemias may occur as a result of maternal exposure to agents in diet and medications that inhibit DNA topoisomerase II. Three epidemiologic studies of childhood leukemia with similar methodologies were conducted in the United States and Canada over the past 10 years by the Children's Cancer Group (CCG). Of the total 771 mothers of infants diagnosed at one year of age or less (< 12.5 months) who originally were interviewed (303 infant cases and 468 matched controls) across the three studies, follow-up questionnaire data on maternal exposure to potential DNA topoisomerase II inhibitors during pregnancy were available on 84 cases and 97 matched controls in the US. For maternal diet, a composite variable was created that consisted of 10 foods identified alpha priori as containing DNA topoisomerase II inhibitors. There were no significant trends with increasing maternal consumption for either the overall group, or the acute lymphoblastic leukemia (ALL) stratum. However, within the AML stratum, there was a statistically significant positive association (P trend = 0.04) with increasing consumption of DNA topoisomerase II-inhibitor containing foods (odds ratio [OR] = 9.8, 95 percent confidence interval [CI] = 1.1-84.8; OR = 10.2, CI = 1.1-96.4; for medium and high consumption, respectively). Other potential topoisomerase II inhibitors were explored; no significant findings were found. Results of this preliminary study, in combination with molecular data, should be used in future investigations of childhood leukemia (particularly, infant) to justify the incorporation of a detailed dietary history.

The clastogenic potential of the phytoestrogens coumoestrol (COUM), genistein (GEN) and daidzein (DAI) has been studied in human peripheral blood lymphocytes in vitro. After exposure of the cultured lymphocytes to 50 to 75 microM COUM or 25 microM GEN for 6 h, a clear induction of structural chromosomal aberrations was observed by cytogenetic analysis. The major alterations were chromatid breaks, gaps and interchanges. In contrast, DAI did not induce chromosome aberrations even at 100 microM. These results, together with previously published reports on the induction of micronuclei and DNA strand breaks in cultured Chinese hamster V79 cells by COUM and GEN, but not DAI, suggest that some but not all phytoestrogens have the potential for genetic toxicity.

OBJECTIVE: This study was designed to assess endometrial histology in postmenopausal women not taking hormone replacement therapy, to evaluate side effects and efficacy of phytoestrogens in treating menopause-associated symptoms, and to determine whether 6 months of phytoestrogen supplementation altered endometrial histology. METHODS: We performed a prospective, double-blinded, randomized, placebo-controlled trial comparing the effects of 6 months of dietary phytoestrogen supplementation versus placebo in postmenopausal women. Baseline endometrial biopsies were performed and, if adequate, nonhyperplastic, noncancerous, and nonovulatory, subjects were randomly assigned to receive daily placebo or soy cereal supplementation for 6 months. Study subjects completed baseline and weekly dietary, symptom, and side effect logs. Repeat endometrial biopsies were obtained at 6 months. RESULTS: Subjects were recruited from January 1998 through June 2000. Twenty-seven subjects were randomized, and 19 completed the study. One (3.7%) baseline endometrial sample was weakly proliferative. All other baseline and final biopsies were consistent with atrophic, inactive endometrium. The maximum risk of endometrial stimulation with phytoestrogens is 35%. Hot flushes, night sweats, and vaginal dryness were significantly less severe at the final week of the study compared with baseline in the placebo group. Insomnia was more common in the treated group. There were no other statistically significant differences in symptoms or side effects. CONCLUSION: Phytoestrogens did not cause stimulation of the endometrium. Insomnia was more frequent over the 6-month study in the soy group, whereas hot flushes, night sweats, and vaginal dryness improved from baseline in the placebo group but not in the soy group.

Alzheimer's disease may be due to a deficiency in neurotrophin protein or receptor expression. Consistent with this hypothesis, a reduction in BDNF mRNA expression has been observed in human post-mortem Alzheimer's disease hippocampi. To further investigate this observation, we examined whether the alteration in BDNF expression also occurred at the protein level in human post-mortem Alzheimer's disease hippocampi and temporal cortices using immunohistochemical techniques. We observed a reduction in the intensity and number of BDNF-immunoreactive cell bodies within both the Alzheimer's disease hippocampus and temporal cortex when compared to normal tissue. These results support and extend previous findings that BDNF mRNA is reduced in the human Alzheimer's disease hippocampus and temporal cortex, and suggest that a loss of BDNF may contribute to the progressive atrophy of neurons in Alzheimer's disease.

Objective: To examine associations of midlife tofu consumption with brain function and structural changes in late life.

Methods: The design utilized surviving participants of a longitudinal study established in 1965 for research on heart disease, stroke, and cancer. Information on consumption of selected foods was available from standardized interviews conducted 1965–1967 and 1971–1974. A 4-level composite intake index defined "low-low" consumption as fewer than two servings of tofu per week in 1965 and no tofu in the prior week in 1971. Men who reported two or more servings per week at both interviews were defined as "high-high" consumers. Intermediate or less consistent "low" and "high" consumption levels were also defined. Cognitive functioning was tested at the 1991–1993 examination, when participants were aged 71 to 93 years (n=3734). Brain atrophy was assessed using neuroimage (n=574) and autopsy (n=290) information. Cognitive function data were also analyzed for wives of a sample of study participants (n=502) who had been living with the participants at the time of their dietary interviews.

Results: Poor cognitive test performance, enlargement of ventricles and low brain weight were each significantly and independently associated with higher midlife tofu consumption. A similar association of midlife tofu intake with poor late life cognitive test scores was also observed among wives of cohort members, using the husband’s answers to food frequency questions as proxy for the wife’s consumption. Statistically significant associations were consistently demonstrated in linear and logistic multivariate regression models. Odds ratios comparing endpoints among "high-high" with "low-low" consumers were mostly in the range of 1.6 to 2.0.

Conclusions: In this population, higher midlife tofu consumption was independently associated with indicators of cognitive impairment and brain atrophy in late life.

Aluminum (Al) impairment of bone matrix formation and mineralization may be mediated by its direct effect on bone cells or indirectly by its effect on parathyroid hormone and calcium metabolism. Its toxic effects are proportional to tissue Al load. Al contamination of nutrients depends on the amount of Al present naturally in chemicals or from the manufacturing process. Intravenous calcium, phosphorus, and albumin solutions have high Al (greater than 500 micrograms/L), whereas crystalline amino acid, sterile water, and dextrose water have low Al (less than 50 micrograms/L) content. Enteral nutrients including human and whole cow milk have low Al, whereas highly processed infant formulas with multiple additives, such as soy formula, preterm infant formula, and formulas for specific disorders are heavily contaminated with Al. Healthy adults are in zero balance for Al. The gastrointestinal tract excludes greater than 95% of dietary Al, and kidney is the dominant organ for Al excretion. However, even with normal renal function, only 30-60% of an Al load from parenteral nutrition is excreted in the urine, resulting in tissue accumulation of Al. The risk for Al toxicity is greatest in infants with chronic renal insufficiency, recipients of long term parenteral nutrition, i.e., no gut barrier to Al loading, and preterm infants with low Al binding capacity. The rapid growth of the infant would theoretically potentiate Al toxicity in all infants, although the critical level of Al loading causing bone disorders is not known. To minimize tissue burden, Al content of infant nutrients should be similar to "background" levels, i.e., similar to whole milk (less than 50 micrograms/L).

It is widely appreciated that health food beverages are not appropriate for infants. Because of continued growth, children beyond infancy remain susceptible to nutritional disorders. We report on 2 cases of severe nutritional deficiency caused by consumption of health food beverages. In both cases, the parents were well-educated, appeared conscientious, and their children received regular medical care. Diagnoses were delayed by a low index of suspicion. In addition, nutritional deficiencies are uncommon in the United States and as a result, US physicians may be unfamiliar with their clinical features. Case 1, a 22-month-old male child, was admitted with severe kwashiorkor. He was breastfed until 13 months of age. Because of a history of chronic eczema and perceived milk intolerance, he was started on a rice beverage after weaning. On average, he consumed 1.5 L of this drink daily. Intake of solid foods was very poor. As this rice beverage, which was fallaciously referred to as rice milk, is extremely low in protein content, the resulting daily protein intake of 0.3 g/kg/day was only 25% of the recommended dietary allowance. In contrast, caloric intake was 72% of the recommended energy intake, so the dietary protein to energy ratio was very low. A photograph of the patient after admission illustrates the typical features of kwashiorkor: generalized edema, hyperpigmented and hypopigmented skin lesions, abdominal distention, irritability, and thin, sparse hair. Because of fluid retention, the weight was on the 10th percentile and he had a rotund sugar baby appearance. Laboratory evaluation was remarkable for a serum albumin of 1.0 g/dL (10 g/L), urea nitrogen <0.5 mg/dL (<0.2 mmol/L), and a normocytic anemia with marked anisocytosis. Evaluation for other causes of hypoalbuminemia was negative. Therapy for kwashiorkor was instituted, including gradual refeeding, initially via a nasogastric tube because of severe anorexia. Supplements of potassium, phosphorus, multivitamins, zinc, and folic acid were provided. The patient responded dramatically to refeeding with a rising serum albumin and total resolution of the edema within 3 weeks. At follow-up 1 year later he continued to do well on a regular diet supplemented with a milk-based pediatric nutritional supplement. The mortality of kwashiorkor remains high, because of complications such as infection (kwashiorkor impairs cellular immune defenses) and electrolyte imbalances with ongoing diarrhea. Children in industrialized countries have developed kwashiorkor resulting from the use of a nondairy creamer as a milk alternative, but we were unable to find previous reports of kwashiorkor caused by a health food milk alternative. We suspect that cases have been overlooked. Case 2, a 17-month-old black male, was diagnosed with rickets. He was full-term at birth and was breastfed until 10 months of age, when he was weaned to a soy health food beverage, which was not fortified with vitamin D or calcium. Intake of solid foods was good, but included no animal products. Total daily caloric intake was 114% of the recommended dietary allowance. Dietary vitamin D intake was essentially absent because of the lack of vitamin D-fortified milk. The patient lived in a sunny, warm climate, but because of parental career demands, he had limited sun exposure. His dark complexion further reduced ultraviolet light-induced endogenous skin synthesis of vitamin D. The patient grew and developed normally until after his 9-month check-up, when he had an almost complete growth arrest of both height and weight. The parents reported regression in gross motor milestones. On admission the patient was unable to crawl or roll over. He could maintain a sitting position precariously when so placed. Conversely, his language, fine motor-adaptive, and personal-social skills were well-preserved. Generalized hypotonia, weakness, and decreased muscle bulk were present. Clinical features of rickets present on examination included: frontal bossing, an obvious rachitic rosary (photographed), genu varus, flaring of the wrists, and lumbar kyphoscoliosis. The serum alkaline phosphatase was markedly elevated (1879 U/L), phosphorus was low (1.7 mg/dL), and calcium was low normal (8.9 mg/dL). The 25-hydroxy-vitamin D level was low (7.7 pg/mL) and the parathyroid hormone level was markedly elevated (114 pg/mL). The published radiographs are diagnostic of advanced rickets, showing diffuse osteopenia, frayed metaphyses, widened epiphyseal plates, and a pathologic fracture of the ulna. The patient was treated with ergocalciferol and calcium supplements. The published growth chart demonstrates the dramatic response to therapy. Gross motor milestones were fully regained within 6 months. The prominent neuromuscular manifestations shown by this patient serve as a reminder that rickets should be considered in the differential diagnosis of motor delay.

A 14 weeks old infant was admitted to the intensive care unit with life-threatening hypocalcemic-hyperphosphatemic spasms. Hypocalcemia-hyperphosphatemia was found to have been caused by feeding a high phosphate/ low calcium soy milk. The daily uptake of calcium was calculated to have been 3.3-6 mmol that of phosphate 30 mmol. The parents strongly believed that soy milk formulas were equivalent to breast milk and cow's milk formulas and lived on a strictly vegetarian diet. Therapy with calcium (at an initial dose of 2.25 mmol/kg/day) and 1.25 OH vitamin D3 (Rocaltrol, 0.25 microgram/day) normalized Ca, PO4, vitamin D and parathyroid hormone levels rapidly. Vegetarian feeding had led to life-threatening hypocalcemic hyperphosphatemic spasms in the infant. We conclude that malnutrition and false nutritional beliefs have to be included as a potential cause of early hypocalcemia in infants.

BACKGROUND:. In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit higher performance levels compared to females (a difference attributed to sex steroid hormonal influences). Based upon the results from our companion investigation, this study examined the influence of prenatal sex steroid hormone manipulations on VSM in adulthood, as assessed in the radial arm maze. Additionally, the influence of dietary soy phytoestrogens (i.e., the presence of high or low estrogen-like compounds present in the animal's diet) on VSM was examined in combination with the prenatal hormonal manipulations. RESULTS:. Radial arm maze performance on a phytoestrogen-rich diet: 1) females treated prenatally with testosterone were masculinized and acquired/performed in a manner similar to control or oil-treated males and 2) males treated prenatally with an androgen receptor blocker (flutamide) were feminized and acquired/performed in a fashion typical of control or flutamide-treated females. When a diet change was initiated in adulthood, control phytoestrogen-rich fed females outperformed control females switched to a phytoestrogen-free diet. Whereas, in control males the opposite diet effect was identified. Furthermore, flutamide-treated males fed a phytoestrogen-rich diet outperformed flutamide-treated males switched to a phytoestrogen-free diet. CONCLUSIONS:. These results suggest that prenatal hormonal manipulations significantly sex-reverse the normal sexually dimorphic expression of VSM. Specifically, VSM was enhanced in females treated with testosterone and inhibited in males treated with flutamide. Finally, dietary soy phytoestrogens set a bias on learning and memory in these hormonally manipulated animals in a predictable manner and these data confirm and extend the findings in our companion paper.

. In patients with the inherited disease of phytosterolemia, elevated concentrations of plant sterols (eg, campesterol and sitosterol) have been implicated as a risk factor for premature atherosclerosis. Whether plasma concentrations of campesterol and sitosterol are risk factors for coronary heart disease (CHD) in nonphytosterolemia subjects has not been established. Therefore, the present study examined the role of plant sterols in patients admitted for elective artery coronary bypass graft (ACBG). Serum concentrations of campesterol and sitosterol, as well as lathosterol, desmosterol, cholestanol, and lipoproteins were analyzed in 42 men and 11 women without lipid-lowering treatment during the past. Twenty-six patients reported a positive family history in their first-degree relatives for CHD. Lipid profile and other risk factors were comparable in both groups. Patients with a positive family history for CHD had significant higher plasma levels of campesterol (.50 +/-.17 v.38 +/-.16 mg/dL; P =.011), sitosterol (.40 +/-.11 v.31 +/-.11 mg/dL; P =.004) and their ratios to cholesterol. Lathosterol, desmosterol, cholestanol, and their ratios to cholesterol were not significantly different. Analysis of covariance (ANCOVA) analysis showed no influence of sex, age, triglycerides, total-, low-density lipoprotein (LDL)-, and high-density lipoprotein (HDL)-cholesterol on the results, but confirmed a strong influence of plant sterols. These findings support the hypothesis that plant sterols might be an additional risk factor for CHD.

Soy infant formula contains high levels of the isoflavones, genistein and daidzein, which are commonly referred to as phytoestrogens. These are non-steroidal chemicals with structural similarities to estrogen. Infants consuming soy formula have high levels of circulating isoflavones. These are an order of magnitude greater than the levels of isoflavones which have been shown to produce physiological effects in adult women consuming a high soy diet. There is conflicting evidence about the risks and benefits of soy phytoestrogens, with research presenting a contradictory picture. Some reviewers suggest that early exposure to soy may prevent cancer and heart disease. However, there is very little research on the effects of consumption of soy phytoestrogens by human neonates. Against this generally positive view there is an increasing number of recent reports that suggest that in experimental animals, phytoestrogens have adverse effects with respect to carcinogenesis, reproductive function, immune function, and thyroid disease. Despite the absence of adequate scientific research that quantifies the level of risk to infants, most would argue for a precautionary approach to be taken in situations where there are potential developmental effects from the consumption of pharmacologically active compounds in infancy and childhood.

Aims: To test the hypothesis that feeding soy formula to infants with congenital hypothyroidism (CH) leads to prolonged increase of thyroid stimulating hormone (TSH).

Methods: The study was a review of 78 patients seen during their first year of life between 1990 and 1998. Data regarding clinical diagnosis, date of treatment initiation, TSH, levothyroxine dose, weight, length, and diet information from each visit were collected from the charts.

Results: There were eight patients in the soy diet group and 70 in the non-soy diet group. There was no significant difference between the two groups in the starting dose of levothyroxine or the change in this dose over one year. There was a significant difference between the two groups in the following areas: time to TSH normalisation, first TSH on treatment, percentage with increased TSH at 4 months of age, percentage with increased TSH throughout the first year of life, and in the overall trend of TSH at each visit.

Conclusions: Infants fed soy formula had prolonged increase of TSH when compared to infants fed non-soy formula. These infants need close monitoring of free thyroxine and TSH measurements, and they may need increased levothyroxine doses to achieve normal thyroid function tests.

Breast feeding should be strongly encouraged as providing the safest, most nutritionally adequate form of feeding for most infants. Dietitians should discourage the use of soya protein in children with atopy or cow's milk allergy in the first six months of life to avoid sensitisation to soya protein and exposure to phytoestrogens while organ systems remain at their most vulnerable. This would include soy infant formula and soya products such as desserts etc. When a soy-based infant formula is used, parents should be informed of current findings relating to phytoestrogens and health and on the clinical need for soy formula. Any parent choosing to refuse soya for their infant should be supported in their decision. More research into the long-term effects of phytoestrogen exposure in infants is needed and into whether any adverse effects are dose related. This position statement will be updated as further evidence becomes available

Paediatric dietitian Judy More discusses several recent directives and guidelines on infant feeding in the first year of life. These include recommendations that babies should be fed exclusively on breast milk for their first six months and that soy formulae should normally be avoided for babies under six months old. There is also specific new advice on restricting salt intake for infants.

OBJECTIVE: To determine the effects of 5 years of treatment with soy phytoestrogens on histological characteristics of endometrium in postmenopausal women. DESIGN: Randomized, double-blind, placebo-controlled study. SETTING: Centre of Perinatal and Reproductive Medicine, Department of Gynecological, Obstetrical, and Pediatric Sciences, University of Perugia, Italy. PATIENT(S): Three hundred seventy-six postmenopausal healthy women, all with intact uterus. INTERVENTION(S): Women were distributed in two different groups using randomized criteria: group A (n = 179) patients received soy tablets (150 mg of isoflavones per day) for 5 years; group B (n = 197) patients received identical appearing placebo tablets for 5 years. MAIN OUTCOME MEASURE(S): Results of endometrial histology from biopsies obtained at baseline, 30 months, and 5 years after the beginning of the treatment. RESULT(S): Two hundred ninety-eight women completed the 5-year treatment. No cases of malignancy were detected during biopsy. Seventy percent of women undergoing treatment with soy phytoestrogens had an endometrium classified as atrophic or nonassessable versus 81% receiving placebo. The occurrence of endometrial hyperplasia was significantly higher in group A (3.37% vs. 0%). CONCLUSION(S): Long-term treatment (up to 5 years) with soy phytoestrogens was associated with an increased occurrence of endometrial hyperplasia. These findings call into question the long-term safety of phytoestrogens with regard to the endometrium.

CONTEXT: Postmenopausal estrogen therapy has been posited to have some beneficial effects on aging processes, but its use has risks. Isoflavones, estrogenlike compounds naturally occurring in plant foods, might confer positive effects with fewer adverse effects. OBJECTIVE: To investigate whether soy protein with isoflavones improves cognitive function, bone mineral density, and plasma lipids in postmenopausal women. DESIGN, SETTING, AND PARTICIPANTS: Double-blind, randomized, placebo-controlled trial of 202 healthy postmenopausal women aged 60 to 75 years, recruited from a population-based sample in the Netherlands, conducted between April 2000 and September 2001. INTERVENTION: Participants were randomly assigned to receive 25.6 g of soy protein containing 99 mg of isoflavones (52 mg genistein, 41 mg daidzein, and 6 mg glycetein or total milk protein as a powder on a daily basis for 12 months. MAIN OUTCOME MEASURES: Cognitive function was assessed using the following instruments: dementia, Mini-Mental State Examination; memory, Rey Auditory Verbal Learning Test, immediate recall, delayed recall, and recognition, the Digit Span forward and reversed, and the Doors test; complex attention tasks, Digit Symbol Substitution and Trailmaking, A1, A2, and B; and verbal skills, Verbal Fluency A and N, animals and occupations, and the Boston Naming Task. Bone mineral density of the hip and lumbar spine was assessed using dual-energy x-ray absorptiometry scanning. Lipid assessment included lipoprotein(a), total cholesterol, low-density lipoprotein, high-density lipoprotein, and triglycerides. RESULTS: A total of 175 women completed the baseline and at least 1 postintervention analysis and were included in the modified intent-to-treat analysis. Adherence was good (median plasma genistein levels, 17.2 and 615.1 nmol/L for placebo and soy group, respectively). Cognitive function, bone mineral density, or plasma lipids did not differ significantly between the groups after a year. CONCLUSION: This double-blind randomized trial does not support the hypothesis that the use of soy protein supplement containing isoflavones improves cognitive function, bone mineral density, or plasma lipids in healthy postmenopausal women when started at the age of 60 years or later.

OBJECTIVE: This double-blind, 15-month pilot study was designed to investigate the effect of soy protein isolate with varying concentrations of isoflavones on early postmenopausal bone loss and lipids. DESIGN: A total of 65 women, with a mean age of 55 years and 7.5 years since menopause, were randomized to one of three groups; soy protein with 96 mg isoflavones/day, soy with 52 mg isoflavones/day, or soy without isoflavones (< 4 mg isoflavones/day). Soy was given for 9 months and then discontinued; participants were followed for an additional 6 months. Bone mineral density (BMD) and blood lipids were measured during this time. RESULTS: Measurement of serum isoflavones at 3 months showed dose-related increases in the three groups. There was no significant effect of the soy supplements on BMD of the spine or femoral neck in any of the three groups. BMD increased significantly in the trochanter at 9 months (P = 0.0219) and at 15 months (P < 0.05) in the group given isoflavone-free soy compared with the other two groups. There was no significant effect of soy on lipid metabolism at the end of the intervention. CONCLUSION: The present study did not find a significant positive effect of soy protein isolate supplemented with isoflavones on BMD and the serum lipid profile in early postmenopausal women.

Fifteen percent of all U.S. infants are fed soy formulas containing up to 47 mg/L of isoflavones (>65% as genistin + genistein); thus, these infants' intestines are exposed to a high dose of genistein, a phytoestrogen and tyrosine kinase inhibitor. Little attention has been focused on genistein's impact on the developing intestine. We hypothesized that a high dose of genistein would inhibit intestinal cell growth. Caco-2BBe human intestinal cells were exposed to 0, 3.7, and 111 micro mol/L (0, 1, and 30 mg/L) genistein in DMEM + 0.5% fetal bovine serum for 24-48 h. Cell number, thymidine incorporation, apoptosis, and cell cycle analyses were performed. The low genistein concentration increased intestinal cell proliferation by 28% (P = 0.001), but did not affect cell number or caspase-3 activity compared to the control. Furthermore, the addition of ICI, an estrogen receptor antagonist, negated the proliferative effect of the low genistein. In contrast, the high genistein concentration reduced cell number by 40%, proliferation by 94%, and caspase-3 activity by 50% compared to the control (P < 0.05). Cell cycle analysis after 48 h exposure to high genistein revealed 37% of cells in G0/G1 and 35% in G2/M vs. 71% in G0/G1 and 17% in G2/M for the control and low genistein groups. Thus, a biphasic effect of genistein was seen with a low dose stimulating intestinal cell proliferation through the estrogen receptor, whereas a high dose of genistein inhibited intestinal cell proliferation and altered cell cycle dynamics. A high dose of genistein may potentially compromise intestinal growth.

Total femur zinc of young rats was used to evaluate the biological availability of zinc in milk and soy protein-based infant formulas. A zinc deficient diet (0.8 mug Zn/g) containing egg white protein was supplemented with graded levels of zinc from zinc sulfate, milk and soy protein-based infant formulas. A plot of total femur zinc (log) after feeding the diet for 3 weeks versus the zinc added to the diet gave a linear relationship over the range of 0, 3, 6, 9 and 12 mug/g added zinc. By using a slope-ratio bioassay model, the relative biological availability of endogenous and added zinc in milk-based formula was estimated to be 0.86 and that of soy-based formula 0.67 (zinc sulphate = 1.00) with corresponding 95% fiducial limits being 0.82 to 0.91 and 0.62 to 0.71. Thus, to provide equivalent amounts of available zinc, the total zinc content of the soy protein-based formula would need to be at least 20% higher than that of the formula containing milk protein.

Hormonally active chemicals in the human diet, such as man-made estrogenic chemicals or plant-derived compounds (phytoestrogens), have become a matter of public concern. A significant part of human exposure to phytoestrogens is attributable to soy isoflavones. Besides their estrogenic properties, soy isoflavones also exert genotoxic actions. In this paper, the micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. Genistein caused a clear dose-related induction of MN within the range of 5-25 microM; MN rates were declining at higher genistein concentrations. This was probably due to cytotoxicity of genistein since reduced neutral red uptake and MTT formation with an IC(50) of about 75 microM occurred. Daidzein induced a comparatively shallow increase in the number of MN between 25 and 100 microM. In contrast, the daidzein metabolite equol caused an increase in the number of MN up to 25 microM with no further increase at higher concentrations. Additional staining with anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST(-) micronuclei, i.e. MN with chromosomal fragments, thus indicative of a clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-), whilst equol induced mostly CREST(+) micronuclei indicative of an aneugenic action. These results point to a differential genotoxicity of phytoestrogens.

To verify our previous finding of a positive association between dietary soy and bladder cancer risk, we examined the association in a second, geographically distinct prospective cohort of Chinese subjects, the Shanghai Cohort Study. Briefly, 18,244 men aged 45-64 years were recruited between January 1986 and September 1989. As of December 31, 2002, 61 incident bladder cancer cases were identified. Information on soy consumption was obtained through in-person interviews at baseline using a food frequency questionnaire. Cox proportional hazard regression methods were used to estimate relative risks (RR) and their corresponding 95% confidence intervals (CI), with adjustment for age (years) at baseline interview, level of education and other potential confounders. Compared to men consuming soy less than once a week, the RR (95% CI) for those who consumed soy 1-<3 times per week, 3-<7 times a week and daily were 2.05 (0.80-5.29), 2.45 (0.89-6.76) and 4.61 (1.57-13.51), respectively (p for trend = 0.004), after adjustment for age, cigarette smoking and level of education. The soy-bladder cancer risk associations in smokers and non-smokers were comparable. The soy-bladder cancer relationship became stronger when the analysis was restricted to subjects with 2 or more years of follow-up.