Effects of some dietary phytoestrogens in animal studies: review of a confusing landscape

A variety of effects have been attributed to phytoestrogens, which are present in variable concentrations in soy-based laboratory animal diets. The authors review the inherent complexities in evaluating the effects of soy and dietary phytoestrogens on various in vitro and in vivo systems.     

Endocrine modulation and the fragile balance of homeostasis--an overview

Endocrine modulation by natural and synthetic chemicals and the eventually resulting beneficial or adverse effects for human and animal health are controversially debated not only among scientists but particularly in the public. Most information is available on so-called environmental estrogens, however the amount of information on substances interfering with other hormonal axes steadily increases, particularly on those exhibiting (anti)androgenic activities. The aim of this paper is to summarize existing data and to give an overview on the potential pathways leading to interferences of environmental hormones with homeostasis and eventually resulting health effects. Experimental evidence suggests the hypothesis that fetal and neonatal organisms may be at risk if exposed to environmental estrogens. In contrary, it appears as if phytoestrogens, particularly those with selective estrogen receptor modulator- (SERM-)like activities have the potential to be useful in medical application, both as dietary means and as pharmaceuticals. Lacking valid information about the detailed analysis of the molecular mode of action for environmental estrogens, the possibility for an ultimate classification of environmental estrogens in "dangerous endocrine disruptors" and phytoestrogens in "useful pharmaceuticals" cannot be supported conclusively. Nevertheless both activities are likely.     

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.     

The Roles of Phytoestrogens in Primate Ecology and Evolution

Most primates depend heavily on plant foods; thus their chemical composition is key to understanding primate ecology and evolution. One class of plant compounds of strong current interest are phytoestrogens, which have the potential to alter fertility, fecundity, and survival. These plant compounds mimic the activity of vertebrate estrogens, resulting in altered physiology and behavior. Here, we review what is known about phytoestrogens from an ecological and evolutionary perspective. Much of what is known about the effects of phytoestrogens on the endocrine system comes from research on human foods, especially soybeans (Glycine max). Two opposing perspectives have resulted from this research: 1) phytoestrogens provide health benefits, such as cancer prevention, or 2) phytoestrogens act as endocrine disruptors and threaten reproductive health. Studies of wild primates have only recently begun examining the presence of estrogenic plants in the primate diet and the effects of their consumption. Evidence that a number of primate species eat plants containing phytoestrogens and research documenting behavioral and hormonal effects of estrogenic plant consumption for red colobus monkeys (Procolobus rufomitratus) augment captive and laboratory studies to suggest that these compounds promote differential survival and reproduction. Although much debate is currently taking place over the role of phytoestrogens and other endocrine disruptors in human health issues and in threatening biodiversity, we argue that an ecological and evolutionary approach is needed to reach appropriate conclusions.     

Fetal Programming of Adult Glucose Homeostasis in Mice

Background

Emerging evidence suggests that dietary soy and phytoestrogens can have beneficial effects on lipid and glucose metabolism. We have previously shown that male mice fed from conception to adulthood with a high soy-containing diet had reduced body weight, adiposity and a decrease in glucose intolerance, an early marker of insulin resistance and diabetes.

Objectives

The purpose of this study was to identify the precise periods of exposure during which phytoestrogens and dietary soy improve lipid and glucose metabolism. Since intrauterine position (IUP) has been shown to alter sensitivity to endocrine disruptors, we also investigated whether the combination of IUP and fetal exposure to dietary phytoestrogens could potentially affect adult metabolic parameters.

Methods

Male outbred mice (CD-1) were allowed ad libitum access to either a high soy-containing diet or a soy-free diet either during gestation, lactation or after weaning. Adiposity and bone mass density was assessed by dual x-ray absorptiometry. Glucose tolerance was assessed by a glucose tolerance test. Blood pressure was examined by the tail-cuff system.

Results

Here we show that metabolic improvements are dependent on precise windows of exposure during life. The beneficial effects of dietary soy and phytoestrogens on adiposity were apparent only in animals fed post-natally, while the improvements in glucose tolerance are restricted to animals with fetal exposure to soy. Interestingly, we observed that IUP influenced adult glucose tolerance, but not adiposity. Similar IUP trends were observed for other estrogen-related metabolic parameters such as blood pressure and bone mass density.

Conclusion

Our results suggest that IUP and fetal exposure to estrogenic environmental disrupting compounds, such as dietary phytoestrogens, could alter metabolic and cardiovascular parameters in adult individuals independently of adipose gain.     

Behavioral effects of endocrine-disrupting substances: phytoestrogens

A major source of endocrine-disrupting substances, usually not considered in laboratory animal experiments, is the diet used in research investigations. Soy represents the main protein source in almost all natural-ingredient commercially available formulated diets. Soy-derived isoflavones are the most abundant and in many ways the most studied phytoestrogens, and phytoestrogens (isoflavones) are known endocrine disruptors. Research is reviewed that identifies the physiological and behavioral endocrine-disrupting effects of dietary phytoestrogens (isoflavones) in animal diets, including most of the isoflavones, which are in a glycoside form and biologically inactive, and those in the gastrointestinal tract, which are biologically active. The isoflavones genistein and daidzein have similar molecular weights and structural characteristics to that of 17-beta estradiol, which may enable them to exert estrogenic and antiestrogenic properties are described and characterized. Daidzein can be further metabolized to the potent and abundant molecule equol, which in rodents is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones. Equol has the unique and important ability to specifically bind 5 alpha-dihydro-testosterone, and to act in turn to inhibit the action of this potent androgen. The specific influence of dietary soy phytoestrogens on consumptive, learning and memory, and anxiety-related behaviors is identified. Regulatory behaviors such as food and water intake, adipose deposition and leptin, and insulin levels affected by dietary isoflavones are also discussed.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.     

Liquid chromatographic-photodiode array mass spectrometric analysis of dietary phytoestrogens from human urine and blood

Dietary phytoestrogens have been implicated in the prevention of chronic diseases. However, it is uncertain whether the phytoestrogens or the foods associated with phytoestrogens account for the observed effects. We report here a new liquid chromatography photodiode array mass spectrometry (LC-PDA-MS) assay for the determination of nanomolar amounts of the most prominent dietary phytoestrogens (genistein, dihydrogenistein, daidzein, dihydrodaidzein, glycitein, O-desmethylangolensin, hesperetin, naringenin, quercetin, enterodiol, enterolactone) in human plasma or serum and urine. This assay was found to be suitable for the assessment of quercetin exposure in an onion intervention study by measuring urinary quercetin levels. Other successful applications of this assay in clinical and epidemiologic studies validated the developed method and confirmed previous results on the negative association between urinary isoflavone excretion and breast cancer risk.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.     

Synergistic chemoprotective mechanisms of dietary phytoestrogens in a select combination against prostate cancer

Combination of dietary phytoestrogens with diverse molecular mechanisms may enhance their anticancer efficacy at physiological concentrations, as evidenced in epidemiological studies. A select combination of three dietary phytoestrogens containing 8.33 μM each of genistein (G), quercetin (Q) and biochanin A (B) was found to be more potent in inhibiting the growth of androgen-responsive prostate cancer cells (LNCaP) as well as DU-145 and PC-3 prostate cancer cells in vitro than either 25 μM of G, B or Q or 12.5+12.5 μM of G+Q, Q+B or G+B. Subsequent mechanistic studies in PC-3 cells indicated that the action of phytoestrogens was mediated both through estrogen receptor (ER)-dependent and ER-independent pathways as potent estrogen antagonist ICI-182780 (ICI, 5 μM) could not completely mask the synergistic anticancer effects, which were sustained appreciably in presence of ICI. G+Q+B combination was significantly more effective than individual compounds or their double combinations in increasing ER-β, bax (mRNA expression); phospho-JNK, bax (protein levels); and in decreasing bcl-2, cyclin E, c-myc (mRNA expression); phospho-AKT, phospho-ERK, bcl-2, proliferating cell nuclear antigen (protein levels) in PC-3 cells. Phytoestrogens also synergistically stimulated caspase-3 activity. Our findings suggest that selectively combining anticancer phytoestrogens could significantly increase the efficacy of individual components resulting in improved efficacy at physiologically achievable concentrations. The combination mechanism of multiple anticancer phytochemicals may be indicative of the potential of some vegetarian diet components to elicit chemopreventive effects against prostate cancer at their physiologically achievable concentrations, in vivo.     

Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: comparison with other steroid metabolizing enzymes

Effects of phytoestrogens on human health have been reported for decades. These include not only beneficial action in cancer prevention but also endocrine disruption in males. Since then many molecular mechanisms underlying these effects have been identified. Targets of phytoestrogens comprise steroid receptors, steroid metabolising enzymes, elements of signal transduction and apoptosis pathways, and even the DNA processing machinery. Understanding the specific versus pleiotropic effects of selected phytoestrogens will be crucial for their biomedical application. This review will concentrate on the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases from a comparative perspective with other steroid metabolizing enzymes.     

Soy of dietary source plays a preventive role against the pathogenesis of prostatitis in rats.

This study examined the effects of diet on the development of prostatitis in male rats. Adult male rats were placed on either of two specially formulated diets which differed from one another by the presence or absence of soy as the protein source. A third group of rats (control) was fed standard laboratory rat chow which also includes soy as a source of protein. After 11 weeks, it was found that rats maintained on soy-free diet developed prostatitis mainly in the lateral lobe of the prostate. Increased severity and incidence of prostatitis in rats maintained on the soy-free diet coincided with a significant decrease in urinary excretion of various phytoestrogens. There was no evidence of prostatitis in rats maintained on soy-containing diets. Urinary excretion of phytoestrogens in rats maintained on soy-containing diet was also not different from controls. These results suggest that soy as a dietary source plays a protective role against the development of prostatitis in rats, and indicate that the ventral, lateral and dorsal lobes of the rat prostate have different sensitivities to alterations in dietary factors.     

Genotoxicity of phytoestrogens

Plant extracts containing phytohormones are very popular as 'alternative' medicine for many kinds of diseases. They are especially favored by women who enter menopause and are concerned about the side effects of hormone replacement therapy. However, adverse health effects of phytoestrogens have often been ignored. This review examines the literature on genotoxicity and apoptotic effects of phytohormones. Genistein, coumestrol, quercetin, zearalenone, and resveratrol exerted genotoxic effects in in vitro test systems. Other phytoestrogens such as lignans, the isoflavones daidzein and glycetein, anthocyanidins, and the flavonol fisetin exhibited only weak or no effects in vitro. However, some metabolites of daidzein showed a genotoxic activity in vitro. Practically all of the phytoestrogens exhibit pro-apoptotic effects in some cell systems. Further investigations regarding dose-response-relationships and other aspects relevant for extrapolation to human exposure seem necessary. Until then, care may be advised in taking concentrated phytohormones. Nevertheless, the intake of substantial amounts of plant-food in a normal diet constitutes an important, individual contribution to cancer prevention.     

Phytoestrogens as modulators of steroid action in target cells

Although numerous reports exist on the potential beneficial role of nutritional phytoestrogens in human health, their molecular mechanism in target cells is still not completely understood. Phytoestrogens promote estrogen and antiestrogen effects by interacting with numerous molecules, carrier proteins, enzymes and membrane and nuclear receptors, directly or indirectly involved in the transfer of estrogen signals. The hypothesis that the ER beta subtype plays a key role in antiproliferative effect of phytoestrogens, especially in breast cancer, is examined here.This review focus on the effects of phytoestrogens in developmental processes such as those linked to reproductive function, tumorigenesis and angiogenesis.     

Pharmacological activities of Genistein,an isoflavone from soy (Glycine max): part II--anti-cholesterol activity,effects on osteoporosis & menopausal symptoms

Phytoestrogens represent a family of plant compounds such as isoflavones, flavones and lignans. A wide range of commonly consumed foods contains appreciable amounts of different phytoestrogens such as isoflavones and lignans. Soy and its products are particularly good sources of isoflavones and lignans. The evidence reviewed here represents the beneficial effects of most potential and promising isoflavone, Genistein in various types of diseases such as osteoporosis, cardiovascular diseases, menopausal symptoms by accumulating evidence from molecular and cellular biology experiments, animal studies, and, to a limited extent, human clinical trials. This review suggests that phytoestrogens may potentially confer health benefits related to various diseases such as cardiovascular disorder, menopausal symptoms, and osteoporosis.     

Potential tissue selectivity of dietary phytoestrogens and estrogens

The recent discovery of a second estrogen receptor subtype, estrogen receptor-beta, may significantly advance our understanding of tissue specific effects of estrogenic compounds, both natural and synthetic. Although specific effects mediated by estrogen receptor beta in vivo remain to be elucidated, hypothetically the existence of two estrogen receptor subtypes (differing in both tissue distribution and biological activity) may help to explain the curious pharmacological behaviour of many estrogenic and antiestrogenic compounds, including the naturally occurring dietary phytoestrogens.     

Selecting the appropriate rodent diet for endocrine disruptor research and testing studies

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.     

Extraction and quantification of lignan phytoestrogens in food and human samples

Dietary phytoestrogens have a number of biological effects, including endocrine disruption, antioxidant potential, and protein tyrosine kinase inhibition. Secoisolariciresinol, matairesinol, and shonanin are lignan phytoestrogens found in foodstuffs, especially flaxseed. Normally they are glycosidically linked to carbohydrates and in the large intestine are deconjugated from the carbohydrate portion by bacteria. The aglycone lignans can be further modified to form the mammalian phytoestrogens enterodiol, enterolactone, and enterofuran, which are absorbed into the body and excreted in urine. To assess the health implications of phytoestrogens in general populations, knowledge of the quantity in the foods eaten is necessary. This article describes a simple preparative procedure for the assay of secoisolariciresinol, matairesinol, and shonanin in foodstuffs after hydrolytic removal of any conjugated carbohydrate. The difficulties in the practical application of the assay procedure are illustrated and discussed. Analytical results indicating the concentration of secoisolariciresinol, matairesinol, and shonanin in a number of foodstuffs are presented. Also, the mass spectral data of a putative mammalian phytoestrogen, called enterofuran, identified in urine are presented.