Address correspondence to P. Whitten, Dept. of Anthropology, Emory University, Atlanta, GA 30322 USA. Telephone: (404) 727-7594. Fax: (404) 727-2860. E-mail: antpw@emory.edu
We are grateful to S. Peymer, A. Ippel, and B. Russell for their assistance in the production of this manuscript.
Received 4 August 2000; accepted 11 October 2000.
Concerns about environmental substances with hormonal actions have generated scientific debate about the risks they pose for human and wildlife health. At issue are both the degree to which low potency may limit hormonal effects (
1,
2) and the difficulty of assigning potencies to compounds with life stage-, cell-, and gene-specific effects (
3-
6). Evaluating risk requires an accurate assessment of the relation between
in vitro and animal tests of endocrine action and human responses. This task is daunting for many environmental substances, as the timing and quantity of human exposure are often uncertain, and suspected toxicity generally precludes their use in clinical trials.
The phytoestrogens, naturally occurring substances with estrogenic actions, provide an opportunity to examine these relationships more fully. Phytoestrogens exhibit a number of actions that have the potential to alter basic reproductive and developmental processes but also exhibit many potentially beneficial actions (2,7). Phytoestrogen actions and dose-response relationships have been assessed in both in vitro and in vivo studies and can be compared to human clinical and epidemiologic studies. Phytoestrogen exposures have been quantified for human subjects in a number of studies, and some pharmacokinetic data also are available.
This review article represents a first attempt to compile animal and human data on 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.
A phytoestrogen is defined as any plant compound structurally and/or functionally similar to ovarian and placental estrogens and their active metabolites. This definition includes compounds with agonistic, partial agonistic, and antagonistic interactions with estrogen receptors (ERs) and other targets of estrogenic steroids involved in estrogen transport, synthesis, and metabolism. Functions affected by phytoestrogens include the regulation of ovarian cycles and estrus in female mammals and the promotion of growth, differentiation, and physiologic activities of the female genital tract, pituitary, breast, and many other organs and tissues in both sexes. Additional end points may include induction of RNA-protein synthesis and prolactin (PRL) secretion; prevention of bone loss; stimulation of hepatic production of sex hormone-binding globulin (SHBG), thyroid-binding globulin, plasminogen, and blood clotting factors VII-X; inhibition of antithrombin III and low-density lipoprotein (LDL) formation; and many others. Phytoestrogens differ in the combination of these actions they express and can be characterized as anticarcinogens, antioxidants, antiviral agents, and so forth. For example, genistein (GEN) can be characterized as a protein tyrosine kinase inhibitor, in contrast to its inactive chemical analogs daidzein (DAI) and chrysin. The latter phytoestrogens are equal or more potent inhibitors of steroid-metabolizing enzymes such as aromatase and 17-hydroxysteroid dehydrogenase (
8,9).
The two major classes of phytoestrogens are lignans and isoflavonoids (Table 1). Some structurally related flavonoids also exhibit estrogenic properties. Lignans are minor components of cell walls and fibers of seeds, fruits, berries, vegetables, grains, and nuts. The isoflavonoids are prominent in legumes, especially soybeans, but detectable levels occur in whole-grain products, potatoes, fruits, vegetables, and alcoholic beverages (9,10), and in cows' milk, meat, and even fish grown with soy-containing food (11). The isoflavonoids are divided into three major classes: isoflavones, isoflavans, and coumestans (Table 1). The best-known isoflavones are GEN and DAI, which are formed from the plant precursors formononetin (FRM) and biochanin A, respectively. The most significant isoflavan is equol (EQL) (Table 2), a metabolite of DAI. Coumestrol (COM) is the best-known coumestan and the isoflavonoid with the highest estrogenic potency (12). The mycoestrogens are estrogenic fungal products that are not intrinsic components of plants but are found in pasture grasses and legumes infected by the fungal genus Fusarium.
Estrogen Receptor Affinity and Activation
Estrogen receptor binding is the best-documented action of phytoestrogens. Numerous studies have shown that the isoflavonoids and lignans, as well as the fungal estrogens, compete effectively with estradiol (E2) for binding to ER-rich cells or cytosol (Table 2). Interpretation of these data is complicated by the recent discovery that ERs are divided into two distinct subtypes: the originally sequenced ER, ER
, and a newly discovered variant, ERß, with 95% homology in the DNA-binding region but only 55% homology in the ligand-binding domain (12). Binding studies using the purified receptor proteins show that the isoflavonoids COM and GEN are very active competitors for both receptor subtypes, but bind more avidly to the beta than to the alpha subtype (Table 2). COM has a 2-fold higher affinity for ERß than for ER, whereas GEN has a 30-fold higher affinity for ERß (12). A recent study using human hepatoma cells has now shown that DAI also binds to ERß with a higher affinity than ER (13). This differential affinity is likely to be of functional significance, as the two receptor subtypes have different distributions across estrogen-responsive tissues (12,14-16) and during development (17). Moreover, as none of the common screening tests for estrogens such as ligand competition assays using rodent uterine cytosol or cell proliferation assays using breast cancer cells [e.g., E-screen (18)] or recombinant cells containing ER and an estrogen-responsive reporter gene [e.g., recombinant yeast cell bioassay (18)] include significant concentrations of ERß (18,19), phytoestrogen potency may be underestimated.
Recombinant assays provide evidence that phytoestrogens not only bind to ERs but also initiate gene transcription. These assays are based on recombinant yeast cells containing an ER and one or more estrogen response elements (EREs) linked to genes for reporter proteins like ß-galactosidase (GAL) (20), chloramphenicol acetyltransferase (CAT) (20), or human placental alkaline phosphatase (21). These assays have demonstrated that isoflavones, coumestans, and mycoestrogens all induce ERE-linked reporter proteins (Table 2). Most of these assays are based on ER, but a recent study has shown that GEN activates reporter gene expression through both ER and ERß (21). However, in spite of GEN's higher affinity for ERß, it may be a more effective inducer of ER-mediated actions. Although GEN was slightly more potent via ERß-linked reporters, it was less efficacious, displaying only partial agonism via ERß but full agonism via ER (21). Phytoestrogens can also alter the expression of receptors for hormones other than estrogen, including receptors for progesterone (PR), oxytocin (OTR), and testosterone (AR). In cell culture assays GEN induces PR expression at doses 1,000-fold lower than the concentration required to act as a tyrosine kinase inhibitor (22). GEN also acts as an antiandrogen in a dose-dependent fashion in a breast cancer cell line (23).
Perhaps the most compelling data come from in vivo studies using oral doses of phytoestrogens. Oral doses of DAI too low to be uterotropic significantly reduced mRNA expression of AR and ER in the rat uterus while upregulating the expression of complement 3, a gene known to be sensitive to estrogen, in a dose-dependent fashion (24). A 0.01% COM diet also failed to significantly alter uterine weight in female rats but significantly decreased OTR expression in the hypothalamus (25). These studies suggest that phytoestrogen can have a significant impact at the molecular level at doses too low to produce gross physiologic changes.
Postreceptor Activation
Some isoflavones and flavones inhibit protein tyrosine kinases (26,27), which play important roles in cell signal transduction and the regulation of the cell cycle (Table 3). GEN is the most potent inhibitor. GEN can also inhibit DNA topoisomerases I and II, enzymes essential for DNA replication (28,29), and is the most powerful antioxidant of the phytoestrogens. It also has the ability to increase the activities of antioxidant enzymes as well as directly inhibit hydrogen peroxide production.
Hormone Synthesis
Phytoestrogens have the potential to affect steroid biosynthesis and metabolism through a number of pathways (Table 3). A variety of in vitro assays have now shown that isoflavones and lignans are inhibitors of aromatase (30,31) and 5-reductase (32). A number of phytoestrogens also inhibit 17ß-hydroxysteroid dehydrogenase Type I (33).
However, these effects may not be as pronounced in vivo. Male rats fed a diet containing mulitple phytoestrogens (224 µg/g DAI, 319 µg/g GEN, and 39.5 µg/g glycitin) for 70 days showed no change in hypothalamic aromatase activity, even though isoflavone levels were 8 times higher in the brains of these animals than in the controls (34). Similarly, male rats fed a diet containing 200 µg/g phytoestrogens for 29 days also showed no significant changes in aromatase activity in either the amygdala or the preoptic area. However, significant changes were seen in both areas in the activity of 5-reductase (35). The difference between the in vitro and in vivo data may stem from the significant role metabolism and the actions of binding proteins and second messenger systems play in the activity of phytoestrogens.
Cell Proliferation
Cell growth assays provide evidence that phytoestrogens can either enhance or suppress proliferation in estrogen-responsive cells, depending on their concentration and relative potency (RP) (Tables 4 and 5). COM, GEN, and -zearalenol (-ZEL) exhibit minimal antagonism of E2 in cancer cell or recombinant yeast cell assays and sometimes even augment its action (Table 5). The weaker phytoestrogens, such as biochanin A and enterolactone (ENL), are antagonistic at higher levels, whereas flavonoids like phloretin (PHL), narigenin, kaempferol (KMP), apigenin (APG), and ß-ZEL have been reported to display triphasic activity, inhibiting estradiol at low and high concentrations and augmenting estrogen action at intermediate levels (18).
Hormone Transport
Interactions with binding proteins can have important effects on the RP of estrogens. Although serum albumins do not affect the bioavailability and activity of serum hormones, SHBG and -fetoprotein differentially reduce sex-steroid availability, resulting in relatively greater bioavailability of estrogens that bind only weakly to these glycoproteins (36). Reports of phytoestrogen interactions with binding globulins are somewhat contradictory. An early study using dilute human serum reported phytoestrogen relative binding affinities of 14-27% (37), but affinities estimated in more recent studies using partially purifed SHBG range from 0.01 to 0.1% (38,39) (Table 6). The low affinity of isoflavonoids would be expected to result in enhanced bioavailability in the presence of SHBG, and in fact the effective free fraction of COM and GEN in human serum is 45-50%, whereas only 4% of E2 is free (40). Addition of human serum to whole-cell binding assays raises the relative binding affinity of isoflavonoids 10-fold (40), but normal serum concentrations of SHBG (0.002 mg/mL) reduce the activity of COM and GEN in the yeast estrogen screen assay by only 5-10%, which is similar to the reduction of E2 activity (5%) (41). However, pregnancy concentrations of human -fetoprotein (40-160 mg/mL) reduce the activity of E2, COM, and GEN by 50% (41).
Sequestering steroids is not the only action of binding globulins; binding globulins also contribute to hormone uptake by target cells (42,43-45). Cellular uptake of albumin- and SHBG-bound estrogens precedes their interaction with nuclear receptors and may directly affect the delivery of hormonal signals. Whereas free estrogens and the albumin-bound fraction are internalized into the cytoplasm, SHBG-estrogen complexes bind to membrane receptors, initiating nongenomic influences on cell metabolism through second messenger systems and priming effects on nuclear super-receptor complexes. ATP-binding cassette carriers within cell membranes also may expel estrogens out of cells, preventing their entry into the brain and other tissues. GEN binds to both the multidrug resistance protein (46) and P-glycoprotein (47), but only relatively high concentrations (200-400 µM) have been tested. These transport glycoproteins have low total capacity; ligands compete with one other for the binding sites, with the result dependent on the relative concentrations of binding proteins and endogenous and exogenous estrogens (9,48,49). These interactions may play a role in the selectivity of the blood-brain barrier and other tissue-blood barriers and in absorption from the intestine. Although phytoestrogen influences on these processes are not well understood, their affinity for all these binding proteins and relatively high concentrations in plasma provide opportunities for significant effects on these mechanisms of hormone uptake.
In addition to binding competition, phytoestrogens might also influence estrogen availability by raising SHBG synthesis (9). The clinical evidence for this effect is mixed, with some studies demonstrating an increase in SHBG levels in postmenopausal women on a soy-based diet (50) and other studies showing no change in SHBG levels (51-53) or even a decrease (54) in SHBG on soy-based diets.
Estimates of estrogenic potency vary across assays. Because recombinant cells often contain multiple copies of EREs, resulting in E
2 sensitivities as low as 0.1-7 pM, these assays can underestimate the
in vivo concentrations required for estrogen action (
55). Purified receptor preparations also provide unrealistic estimates of binding affinity caused by the absence of binding proteins. Cytosol and whole-cell assays provide more accurate estimates of potency, with effective concentrations in the picomolar to nanomolar range for E
2.
Generally, lower concentrations are required for ER-mediated events and for cell proliferation than for other types of responses. The range of estrogen concentrations required for stimulation of cell proliferation varies across assays, but in general, concentrations of 1-100 nM stimulate cell growth by isoflavonoids (Table 4). Relatively high concentrations, ranging from 5 to 100 µM, are generally required for suppression of proliferation (Table 5), and biphasic patterns are frequently seen, with proliferative actions occurring at nanomolar concentrations and inhibition of proliferation occurring at micromolar concentrations. There is evidence that lower concentrations of GEN are required to suppress environmental estrogen-stimulated cell proliferation if other phytoestrogens are also present. However, the concentrations required for these additive effects (25 µM) are still quite high, so it is unclear if the action would occur in vivo (56). Similarly, high micromolar levels are required for estrogen antagonism of cell proliferation, with minimal action by the most potent phytoestrogens.
Inhibition of tyrosine receptor kinases and other targets of phosphorylation occur at micromolar concentrations (Table 3). Micromolar concentrations are required for lignan and isoflavone binding to SHBG (5-50 µM) (38) and -fetoprotein (Kd: 0.5-5 µM) (57,58). Concentrations required for inhibition of most steroid metabolizing enzymes are generally in the micromolar range, with the exception 17ß-hydroxysteroid dehydrogenase type I, which is inhibited by isoflavonoids at concentrations ranging from 120 nM to 1 µM (33,59).
Dietary Sources
The highest concentrations (1-3 µmol/g) of lignans are found in flaxseed (linseed) products (60,61), which are consumed by some Europeans. Good sources of lignans in the United States and Europe are pumpkin seed (20 mg/100 g), sunflower seed, cranberry, black tea, coffee, garlic, broccoli, brans, and peanuts (62). Green tea is a rich source of lignans and a popular beverage in many Asian countries (63).
Phytoestrogenic isoflavonoids are less prevalent than lignans. The highest concentrations are found in legumes, especially in soybeans and soybean-based products, which can contain as much as 0.2-1.6 mg of isoflavones per gram dry weight (64). The isoflavone contents of the basic food types are summarized in Table 7.
Plasma Concentrations
Humans. Phytoestrogen exposures vary substantially across human populations and individuals (Table 8). Differences across populations are related to dietary variation (9,65), whereas within-population variation smay reflect individual differences in the intestinal microflora, local differences in the phytoestrogen content of foods, and dietary effects on phytoestrogen absorption and metabolism (11,66,67). Asian diets are particularly high in soy, resulting in isoflavone consumption as high as 1 mg/kg body weight (bw)/day, whereas vegetarian diets are high in whole grains, vegetables, and legumes, resulting in higher consumption of lignans. These dietary exposures produce plasma isoflavone concentrations as high as 1 µM in Japanese men and women consuming a traditional diet. In Europe and North America, plasma concentrations are generally less than 0.07 µM for omnivores, whereas vegetarians may have levels as high as 0.4 µM isoflavones and 0.8 µM lignans. A recent study of Japanese infants indicates that isoflavonoids gain access to fetal tissues, resulting in concentrations in cord blood and amniotic fluid (0.2-0.3 µM) similar to maternal plasma concentrations (0.2 µM) (68).
Feeding trials provide more detailed data on phytoestrogen bioavailability in adults (Tables 9,10). Single soy meals providing isoflavone doses of 0.06-1.2 mg/kg (4-71 mg) produce peak plasma concentrations of 0.06-2.2 µM. Peak plasma concentrations of GEN and DAI are reached within 4-8 hr, with an absorption half-life of 1-3 hr. The excretion half-life ranges from 3 to 8 hr. DAI, with estimates ranging from 16-66%, appears to be more bioavailable than GEN (5-37%).
The highest human dietary exposures occur in infants fed a soy-based formula (SBF). SBF contains high concentrations of isoflavones (100-175 µM), significantly more than breast milk before (18-56 nM) or after (10-70 nM) soy consumption (69) or in bovine based-milk formula (70,71). An infant fed only SBF consumes approximately 6-11 mg/kg/day isoflavone, which is considerably more than the average for adults, even on a soy-rich Asian diet (0.3-1.2 mg/kg/day) (72,73). These high intakes result in markedly higher plasma concentrations (2.4-6.5 µM) in infants fed a soy milk diet than in infants fed either cows' milk formula (0.03 µM) or breast milk (0.02 µM) (71,74), plasma levels that are 10-fold higher than the average levels reported for Japanese women and equal to those seen at peak plasma levels following soy challenges in adults.
Animal models. Species differences in metabolism may influence bioactive doses. For example, relatively high oral doses are required to produce micromolar concentrations in plasma in sheep, whereas similar peak concentrations can be achieved in cattle and pigs with lower doses (Table 11). However, few studies have examined the pharmacokinetics of isoflavones in rodent models of estrogen action. Estimates of bioavailability of GEN in rats and mice (10-20%) are similar to values reported for humans (Tables 10,11); however, the doses tested in rodents are substantially higher than the doses tested in humans and use free GEN rather than the GEN glycosides present in the soy protein generally used in human trials. Peak plasma levels are achieved in rats within 2 hr and in mice within 3-30 min after oral dosage (Table 11). Single or repeated oral GEN doses of 20-45 mg/kg produce peak plasma concentrations of 2-11 µM in rats. Single oral doses of 50-200 mg/kg GEN produce peak free plasma concentrations of 1-4 µM in mice. These doses are considerably higher than those reported to produce micromolar plasma levels in humans, but data on plasma concentrations resulting from lower dietary doses in rodents will be required to determine whether these dose responses represent species differences.
Tissue Concentrations
Only a few studies have examined phytoestrogen concentrations in tissues and bodily fluids other than plasma and urine (Table 12). Single-dose treatment of female mice with 40 mg/kg/day subcutaneous (sc) FRM produced peak plasma concentrations of 9 µM in 2 hr and peak mammary tissue concentrations of 7 nmol/g in 4 hr, with mean half-lives of elimination of 2 and 2.5 hr, respectively (75). In rats, plasma levels of EQL increased from 91 to 450 nM 1 hr following a single sc injection of 4 mg/kg EQL, and the corresponding uterine concentrations were 0.4 and 4 nmol/g (76). Chronic oral doses of 8-40 mg/kg GEN resulted in reproductive tissue concentrations of 0.3-1.4 nmol/g (77). On a parts per million basis, total isoflavone levels in rodent plasma and mammary gland appear to be quite similar. However, the high proportion of GEN aglycone in reproductive tissues compared to serum suggests that some accumulation occurs (77).
A very small sampling of human breast milk (HBM) suggests that isoflavone concentrations are lower in breast milk than in plasma (69,78) (Table 12). On the other hand, a large sample of prostatic fluid concentrations indicates that isoflavone and lignan concentrations are higher on average than mean plasma levels (79,80) (Table 12). Plasma and prostatic fluid concentrations in individual men were well correlated for DAI (rs = 0.7) but not for enterolactone (rs = 0.2-0.4) (80), suggesting that isoflavones are concentrated in the prostate.
The most illustrative study to date used an oral dose of [14C]GEN (4 mg/kg) in male and female rats (81). The highest concentration of radioactivity was found in the gut; significant levels of radioactivity were also found in a variety of other organs, particularly the liver and reproductive organs, but also the brain, heart, lungs, and kidneys. Interestingly, retention in the liver was sexually dimorphic, with females showing nearly 2.5 times the radioactivity as males 2 and 7 hr after the initial dose was given.
Exposure and Bioactivity
The data reviewed above indicate that total plasma levels of isoflavonoids and lignans in humans range from 10 to 400 nM, of which only about 10% (1-25 nM) is unconjugated (Table 8). Plasma concentrations as high as 2 µM total isoflavonoids may be achieved for a few hours after food consumption. Limited data for tissue concentrations indicate no more than nanomolar levels, even in tissues like the prostate where isoflavonoids may be concentrated relative to plasma. Therefore, overall it appears that average concentrations may be too low to induce most of the reported in vitro actions other than those mediated by ERs (Table 3). Moreover, current data suggest that agonism and cell proliferation are more likely than antagonism and suppression of cell proliferation. However, the latter predictions may be biased by the ER-based systems from which they were drawn, and a different picture may emerge from assays based on ERß.
A variety of
in vivo actions of phytoestrogens have been reported in animal experiments and human clinical studies. Although the majority of these actions are in the reproductive tract, there is evidence for effects on functions of the cardiovascular, skeletal, and central nervous systems.
Proliferation of Uterine and Vaginal Epithelium
Proliferation of the female reproductive tract is a classic test of estrogenicity that has been demonstrated for a number of phytoestrogens in a variety of animal species (Table 13). More variable results have been achieved using soy treatments. A soy-based diet providing 7 mg/kg/day GEN and 3 mg/kg/day DAI to ovariectomized rhesus macaques did not induce uterine growth or maturation of the vaginal epithelium, although they produced significant changes in cardiovascular risk factors (see below) (82). A similar diet also failed to alter uterine growth or vaginal cytology in ovariectomized rats (83).
More variable results have been obtained with postmenopausal women. Soy protein supplements that provided postmenopausal women with 0.7 mg/kg/day GEN and 2.1 mg/kg/day DAI over a 4-week period did not significantly alter the vaginal maturation index, although there was a trend for an increase in superficial cells (84). On the other hand, increases in the maturation index were observed in an Australian study during 6-week supplementation with a soy flour (85) estimated to have provided similar isoflavone doses (e.g., about 1-2 mg/kg/day) (84). This study did not include a control group, however, so the results are difficult to separate from the normal waning of menopausal symptoms over time. It may be that the actual dietary content was higher, as another Australian study that provided the same supplement reported much higher urinary excretion (45 µmol/day) of DAI (86) than has been reported in other feeding studies (Table 8); however, the latter study did not observe any changes in vaginal cytology. Two more recent studies did not observe changes in vaginal cytology during soy supplementation (50,87).
Ovarian Cyclicity
Phytoestrogens could influence ovarian cycles through several pathways. Phytoestrogen interaction with ERs in ovarian granulosa cells might augment or inhibit follicular development, especially in light of the high affinity of isoflavones for ERß. ERß is abundant in granulosa cells, although the high intraovarian concentrations of E2 may limit the potential for such effects. Phytoestrogen inhibition of ovarian aromatase (88-90) could reduce the availability of endogenous estrogen and limit follicular development. However, the very high concentrations required for inhibition by isoflavones (Table 3) make it unlikely that they would significantly influence estrogen production in the ovary. A more likely route for isoflavone influences on E2 bioavailability would be via inhibition of 17-hydroxysteroid dehydrogenase type I, an effect that occurs at concentrations of 0.1-1 µM (33,59). In addition, phytoestrogens could augment or inhibit estrogen negative feedback by binding to ERs in the anterior pituitary or hypothalamus and indirectly alter ovarian steroidogenesis (see below).
Natural dietary exposures to phytoestrogens have been associated with cystic ovaries, irregular estrus, and anestrus in cattle (91), and reduced breeding success in California quail (92). Compromised follicular development (91) and reductions in luteal phase plasma progesterone and E2, as well as shortened luteal phases, have been reported in cycling ewes whereas increases in estradiol and cortisol have been reported in pregnant ewes (93,94). Soy isoflavone diets providing doses of 1 mg/kg/day were associated with infertility in captive cheetahs (95).
Experimental studies of rats suggest that effects on ovarian cycles depend on the hormonal milieu. For example, a 0.01% dietary concentration of COM (a dose of 16 mg/kg bw/day) hastened the onset of ovarian cycles in immature females but suppressed ovarian cycles when provided to cycling adult females (96). Although the suppression of cycles is reversed upon cessation of treatments, prepubertal exposure may affect cyclicity later in life. Rats treated prepubertally with 500 mg/kg/day sc GEN spent more time in the estrous stage of vaginal cycles at 50 days of age (97), and rats treated prepubertally with 16 mg/kg/day oral COM exhibited more cycle irregularities at 100 days of age (96).
Several studies have examined the effects of phytoestrogen supplementation on the human menstrual cycle, with variable results (52,53,98-101) (Table 14). Isoflavone doses of 0.8-3 mg/kg/day produced significant changes in menstrual cycles, but the direct and type of effect varied considerably across studies. An increased length of the menstrual cycle was the most common outcome, but this change was accompanied by elevations as well as declines in serum E2 and testosterone. The practice of sampling hormonal levels on only a few days of the cycle becomes problematical when cycle length is altered, as the days compared no longer represent comparable stages of the cycle. A recent study reported consistent reductions in urinary estrogens but minimal changes in plasma estrogens, suggesting that urinary steroids may be more sensitive indices of changes in steroid production than plasma concentrations (100,102). Two studies have reported declines in periovulatory luteinizing hormone (LH) and follicle-stimulating hormone (52,100), a response with implications for fertility suggestive of pituitary or hypothalamic actions of phytoestrogens. However, sample size and duration of treatment limit the conclusions that can be derived from these studies. The effects observed after only a single cycle of treatment may not be representative of the longer-term consequences of phytoestrogen consumption. Larger samples of women observed over longer periods of treatment with controlled doses are needed to establish the effects of soy isoflavones on the menstrual cycle.
Proliferation of Breast Epithelium
Low rates of breast cancer in populations with high phytoestrogen exposure suggest phytoestrogens may inhibit epithelial cell proliferation (9,65,103-106). On the other hand, the biphasic effects on proliferation observed in in vitro studies suggest that both proliferative and antiproliferative effects might be observed, depending on the dose. Results from multiple in vivo studies across species have been contradictory. A variety of effects have been noted, ranging from antiproliferation through enhanced proliferation, depending on the tumor cell type, dose, timing of phytoestrogen exposure, and the phytoestrogen given. In general the isoflavones, and particularly GEN (likely due at least partially to its tyrosine kinase-inhibiting properties), produce the most significant results and are thus the most widely explored.
Results seen in vitro have been difficult to reproduce in vivo. A recent study demonstrated that GEN (20 µM) inhibits cell proliferation in estrogen-independent human breast cancer cells (MDA-MB-231) by as much as 50%. Mice with breast tumors produced by inoculation with these same cells were then placed on a GEN-rich diet (750 µg/g). No significant changes in tumor size or morphology were seen, even when the treatment diet was given before the initial innoculation with the MDA-MB-231 cells (107).
Soy protein and GEN reduce tumor multiplicity, but not incidence, in other animal models of breast cancer (Table 15). Soy protein isolate-containing diets providing oral doses of 2-8 mg/kg/day GEN and 1-3 mg/kg/day DAI reduced the number of mammary tumors in 7,12-dimethylbenz[a]anthracene (DMBA)-treated rats (108). GEN or DAI intraperitoneal (ip) injections that provided doses ranging from 8 mg/kg/day at 35 days of age to 3 mg/kg/day at 215 days of age resulted in an observable but nonsignificant reduction in the number of mammary tumors in N-methylnitrosourea (MNU)-treated rats (109). The effect of DAI was delayed compared with that of GEN. No significant changes in protein tyrosine kinase-mediated phosphorylation or topoisomerase II activity were observed in the mammary gland or mammary tumors, even with higher isoflavone doses (40 mg/kg/day), suggesting that the observed chemoprevention was mediated via other mechanisms (109). Three-day sc treatments with a much higher GEN dose (500 mg/kg/day) during the neonatal (110) or prepubertal period (97) resulted in more marked reductions (50%) in tumor number in DMBA-treated rats.
Stimulation of mammary tissue has been seen in FRM-treated mice (111), GEN-treated rats, (97,112), and in cattle grazing on phytoestrogen-rich clover (113) (Table 15). Five-day treatment with sc doses of 40 mg/kg/day FRM, resulting in peak plasma and mammary levels of 9 µM and 4 nmol/g, increased mammary gland proliferation in ovariectomized mice (111). Oral doses of 36 mg/kg/day GEN maintained lobulo-alveolar structure in ovariectomized rats without affecting ducts; no effects were seen in immature rats (112). Transitory increases in mammary gland weight also were produced with three sc doses of 500 mg/kg/day GEN in prepubertal and neonatal rats (97). Moreover, soy isoflavones or oral GEN actually increased the growth of implanted MAC-33 or MCF7 tumor cells in some recent experiments in rats (114,115). However, soy isoflavones did not increase mammary epithelium proliferation in surgically postmenopausal long-tailed macaques (116).
Early exposure to estrogen also may influence susceptibility to mammary cancer by altering the proliferation and differentiation of epithelial structures that are sensitive to transformation by carcinogens. Neonatal exposure to E2 stimulates the mammary epithelium in rodents, increasing terminal end bud proliferation and reducing the differentiation of terminal end buds during the period from 4 to 16 weeks of age (117). This effect is hypothesized to enhance susceptibility to carcinogenesis, as the terminal end bud is the site for transformation in the rodent mammary gland (118). Perinatal treatment with E2 or diethylstilbestrol (DES) increases the incidence of mammary tumors in mice carrying a mammary tumor virus (119,120). Similar effects have been observed with phytoestrogens. Neonatal treatment with 10 mg/kg/day zearalenone (ZLN) increases the incidence of spontaneous mammary tumors in Wistar rats (121). Increased numbers and reduced differentiation of terminal end buds have been observed in mice after in utero exposure to ZLN (117).
Neonatal or prepubertal exposure to GEN, however, appears to have the opposing effect of enhancing terminal duct differentiation in rats. Neonatal or prepubertal doses of 500 mg/kg/day GEN significantly reduced the number of terminal end buds and cell proliferation indices at 50 days of age (97,110). Transforming growth factor (TGF)- and epidermal growth factor (EGF) receptor were upregulated in terminal duct structures immediately after treatment, but the EGF signaling pathway was downregulated by 50 days of age (122). These data suggest that high-dose GEN enhances epithelial differentiation. However, in the absence of data on mammary structure beyond 50 days of age, it is difficult to evaluate whether the observed changes represent permanent changes in mammary organization or simply alterations in the timing of developmental events. Moreover, these high-dose treatments induced changes in the reproductive tract that resemble the alterations in sexual differentiation produced by perinatal DES or E2. Both prenatal and neonatal GEN treatments resulted in precocious vaginal opening, and neonatal treatments were associated with reductions in serum progesterone at 50 days of age and follicular abnormalities such as atretic antral follicles and fewer corpora lutea (123).
Only two studies have directly examined isoflavone influences on the breast in women (Table 15). Chinese and Japanese women had lower nipple aspirate fluid volume, lower mean levels of gross cystic disease fluid protein, fewer hyperplasic cells, and fewer atypical epithelial cells in their nipple aspirate fluid than American women, an effect that was postulated to be a consequence of consumption of soy isoflavones (53). However, supplementation of the diets of American women with soy protein, providing GEN and DAI doses of 0.6 and 0.4 mg/kg/day, respectively, stimulated breast epithelial cell proliferation and 2- to 6-fold increases in nipple aspirate fluid volume in 29% of premenopausal (PRE) women (53). Monthly plasma collections suggested that E2 also was erratically elevated. A second study examined the proliferation rate of breast epithelium in biopsy samples from women with benign or malignant breast disease treated with and without a soy supplement providing an isoflavone dose of 0.75 mg/kg/day (124). After 14 days of treatment, the proliferation rate of the lobular epithelium and PR expression were significantly increased when day of menstrual cycle and patient age were taken into account. These unexpected results illustrate the difficulties of predicting human responses and the need for careful clinical trials.
Prostate and Testes
Evidence for high expression of ERß in the prostate suggests that it could be an important target for phytoestrogen action. There is evidence for estrogenic action of isoflavones in the prostate. Isoflavonoids induce c-fos expression in the murine prostate at doses of 0.025-2.5 mg/kg/day sc (GEN) and 5 mg/kg/day (COM, DAI) (125,126). Ten days of treatment with GEN (2.5 mg/kg/day sc) induced neoplastic transformation in neonatally estrogenized mice, whereas a sc dose of 1.2 mg/kg/day had no effect (125). On the other hand, rats maintained on a soy-free diet for 11 weeks developed prostatitis in the lateral lobe of the prostate, an inflammation that can be induced by estrogen (127), suggesting that soy isoflavones might antagonize the actions of endogenous estrogens in the prostate. A recent study provided evidence for a suppressive effect of isoflavones on the hypothalamic-pituitary-gonadal axis, showing that 9 days of treatment with sc GEN at a dose of 2.5 mg/kg/day reduced testicular and serum testosterone, pituitary LH content, and prostate weight in adult male mice (126).
GEN inhibited the growth of prostate cancer tissue in 3-dimensional histoculture at concentrations of 4-37 µM (128), but dietary doses of 0.07-0.3 mg/kg/day GEN had no effect on the growth of matairesinol (MAT) LyLu prostate cancer cells implanted sc in male rats (129). Intraperitoneal doses of 0.1-0.4 mg/kg/day also had minimal effect (129). However, a diet containing a high isoflavone soy protein isolate reduced tumor incidence and prolonged the latency to tumor development in MNU-treated Lobund-Wistar rats (130).
There are no published tests of preventative actions of phytoestrogens in human prostate cancer. However, a single clinical case has been reported in which apoptosis typical of high-estrogen therapy was seen in a man who took 160 mg of phytoestrogen per day for 1 week prior to radical prostatectomy (131).
Bone Density
Estrogen is required for maintenance of normal bone density, and some phytoestrogens have been investigated as potential estrogen replacement therapy in postmenopausal women. Ipriflavone, (IPR; 7-isopropoxyisoflavone), an isoflavone derivative, has been the phytoestrogen most studied as a therapeutic agent in osteoporosis. Daily oral doses of 3-10 mg/kg/day IPR prevent bone turnover and loss in bone density in women who are postmenopausal or who have been treated with gonadotropin-releasing hormone (GnRH) agonists (132-134). These doses produce plasma concentrations (135) similar to the in vitro concentrations (>= 100 nM) required for inhibition of osteoclast differentiation (136) and bone resorption (137). Much higher oral doses (50-400 mg/kg/day) of IPR have been required to prevent bone loss in ovariectomized rats (138), doses that also produce plasma concentrations similar to the effective in vitro concentrations (139,140). COM also inhibits bone resorption in vitro (141) and is a more potent agent, preventing bone loss at oral doses of 1-2 mg/kg/day (142,143). Results with GEN, on the other hand, have been inconsistent. No effect on bone density is seen in ovariectomized rats with oral doses of 0.1-30 mg/kg/day GEN (142), whereas doses of 2 mg/kg/day (but not 6-20 mg/kg/day) preserve bone density in ovariectomized lactating rats (144). A dietary supplement providing approximately 10 mg/kg/day GEN, 17 mg/kg/day FRM, and 19 mg/kg/day biochanin A also had no effect on bone density (142). DAI, in contrast, actually stimulates bone resorption in vitro at concentrations of only 0.01-0.1 nM (145) but has not been tested in vivo. DAI is one of the metabolites of IPR, and its opposing effects may help to explain why such high doses of IPR are required to prevent bone loss.
The role of estrogen receptors in these actions is unclear. Although both ER and ERß are expressed in human (146) and rat (147) osteoblasts, there is no convincing evidence for either in osteoclasts (148), and preosteoclastic cells have not been examined. Unlike other isoflavonoids, IPR does not displace E2 binding in MCF7 cells or induce ER-dependent gene transcription (149). IPR enhances E2 binding to preosteoclasts, but its binding to the same cells (Kd = 68 nM) is not displaced by E2 (149). Moreover, IPR appears to have minimal or no estrogen action on its own, although it augments the estrogenic effect of E2 and estrone (E1) on uterine weight and thyroid calcitonin release (150).
Cardiovascular Function
Reductions in serum cholesterol were produced in ovariectomized rats by 4 days of oral treatment with -zearalanol (ZAL) (ED50 = 0.2 mg/kg/day), COM (ED50 = 0.4 mg/kg/day), and GEN (ED50 = 0.5 mg/kg) (142). One month of treatment with 100 mg/kg/day IPR also reduced serum cholesterol in ovariectomized rats (151). A soy supplement providing approximately 7 mg/kg/day GEN plus 3 mg/kg/day DAI to ovariectomized female rhesus macaques with diet-induced atherosclerosis produced free plasma concentrations as high as 20 nM GEN and 40 nM DAI (152,153). The isoflavone diet improved a number of cardiovascular risk factors such as lower total plasma cholesterol, elevated high-density lipoprotein cholesterol, lowered arterial lipid peroxidation, and enhanced dilator response of atherosclerotic arteries (152-154).
Five- to 12-week treatments with soy supplements providing 0.7-1.5 mg/kg/day isoflavones did not alter serum lipids in men and women with average serum cholestrol concentrations (86,155-157), although reductions in LDL cholesterol have been observed in hypercholesterolemic women following soy isoflavone treatment (158). However, arterial compliance, a measure of arterial elasticity, was improved in menopausal women after 10 weeks of treatment with 0.7-1.2 mg/kg/day clover isoflavones (157).
Hypothalamic and Pituitary Feedback
Several studies have examined the influence of phytoestrogens on basal LH or LH release (Table 16). In ovariectomized rats, a low GEN dose (1 ng/kg iv) enhanced GnRH-induced LH release, whereas higher doses (1-10 µg/kg iv and 0.1-10 mg/kg iv) inhibited LH release (159,160). Pretreatment with GEN 8 mg/kg sc or ZLN (0.8-8 mg/kg sc) did not inhibit tonic LH release, but these doses did block GnRH-stimulated LH release (161). However, oral doses of 0.1-10 mg/kg GEN, administered by gavage, had no effect on tonic LH (160). In rhesus macaques, ZLN suppressed LH at sc doses of 5-14 µg/kg and oral doses of 400 µg/kg (162).
There is some evidence that phytoestrogens can influence human LH secretion. Soy supplements providing 0.7-1 mg/kg/day isoflavones reduced mid-cycle LH (52,100), and suppression of the LH response to GnRH was observed in postmenopausal women with a similar supplement (163). ZLN suppressed LH secretion in postmenopausal women at similar dietary doses (0.4 mg/kg/day) (164).
Developmental Effects
The developmental actions of phytoestrogens have been studied mostly in rats and interpreted to have implications for human health. The impact of phytoestrogens on human fetal development is unclear, but the results of DES exposure have shown that early estrogen exposure can have profound effects. Although the immediately observable effects are limited in rodents, prenatal or neonatal exposure to phytoestrogens results in altered prepubertal or adult morphology and/or function in the uterus, vagina, ovary, breast, pituitary, and hypothalamus (Table 17). Reduced responsiveness to estrogen is apparent in the uterus, breast, and prostate, whereas symptoms of hyperestrogenization are apparent in the vaginal tract. Although the phytoestrogens vary in effective dose range and end points effected, COM, GEN, EQL, and ZLN all appear to exhibit some of the developmental actions that have been reported for other more potent nonsteroidal estrogens like DES. Active sc doses in neonates range from 0.005 to 50 mg/kg/day for COM and 10 to 500 mg/kg/day for GEN and ZLN. Effective maternal doses range from 0.2 to 17 mg/kg/day for both oral and sc doses. Although many of these doses are quite high, reports of effects at rather low doses, particularly in rat dams, argue for a fuller examination of dose-response relationships.
Binding studies using purified receptor proteins show that the isoflavonoid phytoestrogens are high-affinity ligands for ERs, especially ERß. In whole-cell assays, however, their RP is significantly lower, probably as a result of interactions with binding proteins and other as yet unidentified factors. As with other endocrine-active compounds, a wide variety of enzymatic actions have been reported for phytoestrogens. However, many of these
in vitro 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 vivo responses have been observed in animal and human tests for bone, breast, ovary, pituitary, vasculature, prostate, and serum lipids. These actions represent a broader range of tissues and processes, including many beneficial outcomes, than the end points generally used to assess health risks posed by exposure to endocrine-active compounds (5). Reports of ERß in many of the tissues that appear to be responsive to phytoestrogens are intriguing, but currently there are not sufficient data to assess the relative sensitivity of different end points. For some of these end points, similar responses to phytoestrogens have been reported in humans and animal models, although there are currently insufficient data to carry out detailed comparisons.
The biphasic actions of phytoestrogens complicate the process of assigning lowest observed adverse effect levels. In this case, the actions presumed to be more beneficial (e.g., antiproliferative actions) occur at higher in vitro concentrations (micromolar) than the proliferative actions presumed to be more harmful (nanomolar concentrations). This pattern would predict that higher, rather than lower, doses would be more beneficial in vivo. Moreover, human plasma and tissue concentrations are primarily in the nanomolar range, suggesting that proliferative effects should be more likely to be observed.
The in vivo situation is more complex, however. Oral soy isoflavone doses of 3-8 mg/kg/day delay the development of carcinogen-induced breast tumors in rats, whereas GEN doses reported to stimulate growth of breast and uterine epithelia range from 10 to 36 mg/kg/day. However, a recent study has shown that oral GEN doses as low as 11 mg/kg/day enhance the growth of implanted mammary tumors as well as the mammary gland. Moreover, two studies have reported proliferation of breast epithelium in women with oral soy isoflavone doses of 0.7-1 mg/kg/day. 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 lowest effective concentrations in animals have been reported for induction of c-fos in the prostate and regulation of cholesterol, testosterone, and LH. The low effective doses reported for c-fos suggest that molecular or biochemical end points may be more sensitive indices of phytoestrogen response than the morphologic or functional end points commonly tested. The low doses reported for phytoestrogen effects on hormonal secretion suggest that steroidogenesis and the hypothalamic-pituitary-gonadal axis are important loci of phytoestrogen actions. However, these inferences must be tentative because good dose-response data are not available for many end points and many of the doses may not be the lowest active doses.
The available data indicate that phytoestrogens are biologically active in humans at dietary doses of 0.4-10 mg/kg/day (Table 18). The active isoflavone doses are similar to the daily intakes estimated for adults consuming soy-rich Asian diets, which may be primarily a consequence of attempts to test the hypothesized health benefits of Asian diets. Few human studies have attempted to test a range of doses. Moreover, all of the human studies have used isoflavone mixtures present in extracts of soy or red clover, making it difficult to assess the contributions and activity of individual isoflavones. This issue is particularly important in light of the in vitro and in vivo data showing that GEN and DAI sometimes exhibit opposing effects. Moreover, most studies have relied on manufacturer's data on isoflavone content to estimate daily intake, so the actual doses used may vary even in studies using the same soy supplements.
The doses reported to be active in humans are lower than the doses generally reported to be active in rodents (10-100 mg/kg/day), although some studies have reported rodent responses at lower doses (0.005-0.2 mg/kg/day). However, the available estimates of bioavailability and peak plasma levels in rodents and humans are more similar. More studies are needed to determine whether lower doses are effective in rodents and humans and whether bioactive doses are associated with similar plasma levels in humans and animal models.
These comparisons illustrate the rich database available on phytoestrogen actions, mechanisms, and metabolism and highlight gaps and discrepancies in the dataset. These findings demonstrate some of the complexities of extrapolating across assays, species, and compounds. As dietary components, phytoestrogens generally have been treated and tested like nutrients rather than pharmacologic agents. As a result, researchers have investigated a number of beneficial properties that would be overlooked in toxicologic studies but also have failed to anticipate some potentially adverse properties. There is currently very little information on the balance of effects in individuals, data that ultimately will be required in order to assess the wisdom of phytoestrogen consumption. More detailed pharmacokinetic data are required to more accurately evaluate the reliability of extrapolating from in vitro to in vivo studies or across species. Such comparisons are likely to provide new insights into the evaluation of other exogenous estrogens.
REFERENCES AND NOTES
1. Stone R. Environmental estrogens stir debate. Science 256:308-310 (1994).
2. Safe SH. Environmental and dietary estrogens and human health: is there a problem? Environ Health Perspect 103:346-351 (1995).
3. Stancel GM, Boetter-Tong HL, Chiapetta C, Hyder SM, Kirkland JL, Murthy L, Loose-Mitchell DS. Toxicity of endogenous and environmental estrogens: what is the role of elemental interactions? Environ Health Perspect 103:29-33 (1995).
4. Rudel R. Predicting health effects of exposures to compounds with estrogenic activity: methodological issues. Environ Health Perspect 105 (suppl 3):655-663(1997).
5. Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG, Baetchke KP, Hoffmann JL, Morrow MS, Rodier DJ, Schaeffer JE, et al. Environmental endocrine disruption: an effects assessment and analysis. Environ Health Perspect 106:11-56 (1998).
6. Zacharewski T. Identification and assessment of endocrine disruptors: limitations of in vivo and in vitro assays. Environ Health Perspect 106:577-582 (1998).
7. Sheehan DM. Herbal medicines, phytoestrogens and toxicity: risk:benefit considerations. Proc Soc Exp Biol Med 217:379-385 (1998).
8. Whitten PL, Kudo S, Okubo KK. Isoflavonoids. In: Handbook of Plant and Fungal Toxicants (D'Mello JPF, ed). Boca Raton, FL:CRC Press, 1997;117-137.
9. Adlercreutz H, Mazur W. Phyto-oestrogens and western diseases. Ann Med 29:95-120 (1997).
10. Lapcik O, Hill M, Hampl R, Wahala K, Adlercreutz H. Identification of isoflavonoids in beer. Steroids 63:14-20 (1998).
11. Markiewicz L, Garey J, Adlercreutz H, Gurpide E. In vitro bioassays of non-steroidal phytoestrogens. J Steroid Biochem Mol Biol 45:399-405 (1993).
12. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870 (1997).
13. Casanova M, You L, Gaido KW, Archibeque-Engle S, Janszen DB, Heck H. Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors a and b in vitro. Toxicol Sci 51:236-244 (1999).
14. Hess RA, Gist DH, Bunick D, Lubahn DB, Farrell A, Bahr J, Cooke PS, Green GL. Estrogen receptor (alpha and beta) expression in the excurrent ducts of the adult male reproductive tract. J Androl 18:602-611 (1997).
15. Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 388:507-525 (1997).
16. Osterlund M, Kuiper GG, Gustafsson JA, Hurd YL. Differential distribution and regulation of estrogen receptor-alpha and -beta mRNA within the female rat brain. Brain Res Mol Brain Res 54:175-180 (1998).
17. Prins GS, Marmer M, Woodham C, Chang W, Kuiper G, Gustafsson JA, Birch L. Estrogen receptor-beta messenger ribonucleic acid ontogeny in the prostate of normal and neonatally estrogenized rats. Endocrinology 139:874-883 (1998).
18. Collins BM, McLachlan JA, Arnold SF. The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast. Steroids 62:363-372 (1997).
19. Couse JF, Lindzey J, Grandien K, Gustafsson J-A, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-a (ERa) and estrogen receptor-b (ERb) messenger ribonucleic acid in the wild-type and ERa-knockout mouse. Endocrinology 138:4613-4621 (1997).
20. Mayr U, Butsch A, Schneider S. Validation of two in vitro test systems for estrogenic activities with zearalenone, phytoestrogens and cereal extracts. Toxicology 74:135-149 (1992).
21. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J-A, Nilsson S. Differential response of estrogen receptor a and estrogen receptor b to partial estrogen agonists/antagonists. Mol Pharmacol 54:105-112 (1998).
22. Fitzpatrick SL, Berrodin TJ, Jenkins SF, Sindoni DM, Deecher DC, Frail DE. Effect of estrogen agonists and antagonists on induction of progesterone receptor in a rat hypothalamic cell line. Endocrinology 140:3928-3937 (1999).
23. Zand RSR, Jenkins DJA, Diamandis EP. Genistein: a potent antiandrogen. Clin Chem 46:887-888 (2000).
24. Diehl P, Schultz T, Smolnikar K, Strunck E, Vollmer G, Michna H. Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotrophic activity. J Steroid Biochem Mol Biol 733:1-10 (2000).
25. Patisaul HB, Whitten PL, Young LJ. Regulation of estrogen receptor beta mRNA in the brain: opposite effects of 17b-estradiol and the phytoestrogen, coumestrol. Mol Brain Res 67:165-171 (1999).
26. Piontek M, Hangels KJ, Porschen R, Strohmeyer G. Anti-proliferative effect of tyrosine kinase inhibitors in epidermal growth factor-stimulated growth of human gastric cancer cells. Anticancer Res 13:2119-2123 (1993).
27. Boutin JA. Minireview - Tyrosine protein kinase inhibition and cancer. Int J Biochem Cell Biol 26:1203-1226 (1994).
28. Okura A, Arakawa H, Oka H, Yoshinari T, Monden Y. Effect of genistein on topoisomerase activity and on the growth of [Val 12] Ha-ras-transformed NIH 3T3 cells. Biochem Biophys Res Commun 157:183-189 (1988).
29. Constantinou A, Kiguchi K, Huberman E. Induction of differentiation and DNA strand breakage in human HL-60 and D-562 leukemia cells by genistein. Cancer Res 50:2618-2624 (1990).
30. Wang C, Makela T, Hase T, Adlercreutz H, Kurzer MS. Lignans and flavonoids inhibit aromatase enzyme in human preadipocytes. J Steroid Biochem Mol Biol 50:205-212 (1994).
31. Kao YC, Zhou C, Sherman M, Laughton CA, Chen S. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106:85-92 (1998).
32. Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol 147:295-302 (1995).
33. Makela S, Poutanen M, Lehtimaki J, Kostian ML, Santti R, Vihko R. Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 208:51-59 (1995).
34. Lephart ED, Thompson JM, Setchell KD, Adlercreutz H, Weber KS. Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats. Brain Res 859:123-131 (2000).
35. Weber K, Jacobson N, Setchell K, Lephart E. Brain aromatase and 5-alpha-reductase, regulatory behaviors and testosterone levels in adult rats on phytoestrogen diets. Proc Soc Exp Biol Med 221:131-135 (1999).
36. Crane DA, Noriega N, Vonier PM, Arnold SF, McLachlan JA, Guilette LJJ. Cellular bioavailability of natural hormones and enviromental contaminants as a function of serum and cytosolic binding factors. Toxicol Ind Health 14:261-273 (1998).
37. Martin PM, Horwitz KB, Ryan DS, McGuire WL. Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology 103:1860-1867 (1978).
38. Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA. Interactions between phytoestrogens and human sex steroid binding protein. Life Sci 58:429-436 (1996).
39. Milligan SR, Khan O, Nash M. Competitive binding of xenobiotic oestrogens to rat alpha-fetoprotein and to sex steroid binding proteins in human and rainbow trout (Oncorhynchus mykiss) plasma. Gen Comp Endocrinol 112:89-95 (1998).
40. Nagel SC, vom Saal FS, Welshons WV. The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc Soc Exp Biol Med 217:300-309 (1998).
41. Arnold SF, Collins BM, Robinson MK, Guillette LJ Jr, McLachlan JA. Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen. Steroids 61:642-646 (1996).
42. Meyer S, Brumm C, Stegner HE, Sinnecker GH. Intracellular sex hormone-binding globulin (SHBG) in normal and neoplastic breast tissue--an additional marker for hormone dependency? Exp Clin Endocrinol Diabetes 102:334-340 (1994).
43. Germain P, Metezeau P, Hellio R, Habrioux G. Internalization and biological effects of serum albumin in the breast cancer MCF-7 and MDA-MB 231 cells. Cell Mol Biol 41:1119-1129 (1995).
44. Damassa D, Cates J. Sex hormone-binding globulin and male sexual development. Neurosci Biobehav Rev 19:165-175 (1995).
45. Germain P, Egloff M, Kiefer H, Metezeau P, Habrioux G. Use of confocal microscopy to localize the SHBG interaction with human breast cancer cell lines--a comparison with serum albumin interaction. Cell Mol Biol 43:501-508 (1997).
46. Versantvoort CHM, Broxterman HJ, Lankelma J, Feller N, Pinedo HM. Competitive inhibition by genistein and ATP dependence of daunorubicin in intact MRP overexpressing human small cell lung cancer cells. Biochem Pharmacol 48:1129-1136 (1994).
47. Castro AF, Altenbery GA. Inhibition of drug transport by genistein in multidrug-resistant cells expressing P-glycoprotein. Biochem Pharmacol 53:89-93 (1997).
48. Hobbs CJ, Jones RE, Plymate SR. The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid. J Steroid Biochem Mol Biol 42:629-635 (1992).
49. Fritsch MK, Murdoch FE. Estrogens, progestins, and contraceptives. In: Human Pharmacology (Brody TM, Larner J, Minneman KP, eds). St Louis:Mosby, 1998;499-518.
50. Brzezinski A, Adlercreutz H, Shaoul R, Rosler A, Shmueli A, Tanos v, Schenker JG. Short-term effects of phytoestrogen-rich diet on postmenopausal women. Menopause 4:89-94 (1997).
51. Shultz TD, Bonorden WR, Seaman WR. Effect of short-term flaxseed consumption on lignan and sex hormone metabolism in men. Nutr Res 11:1089-1100 (1991).
52. Cassidy A, Bingham S, Setchell KDR. Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. Am J Clin Nutr 60:333-340 (1994).
53. Petrakis NL, Barnes S, King EB, Lowenstein J, Wiencke J, Lee MM, Miike R, Kirk M, Coward L. Stimulatory influence of soy protein isolate on breast secretion in pre- and postmenopausal women. Cancer Epidemiol Biomark Prev 5:785-794 (1996).
54. Nagata C, Kabuto M, Kurisu Y, Shimizu H. Decreased serum estradiol concentration associated with high dietary intake of soy products in premenopausal Japanese women. Nutr Cancer 29:228-233 (1997).
55. Chen S, Kao Y, Laughton C. Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase. J Steroid Biochem Mol Biol 61:107-115 (1997).
56. Verma SP, Salamone E, Goldin B. Curcumin and genistein, plant products, how synergistic inhibitory effects on the growth of human breast cancer MCF-7 cells induced by estrogenic pesticides. Biochem Biophys Res Commun 233:692-696 (1997).
57. Garreau B, Vallette G, Adlercreutz H, Wahala K, Makela T, Benassayag C, Nunez EA. Phytoestrogens: new ligands for rat and human alpha-fetoprotein. Biochim Biophys Acta 1094:339-345 (1991).
58. Baker M, Medlock K, Sheehan D. Flavonoids inhibit estrogen binding to rat alpha-fetoprotein. Proc Soc Exp Biol Med 217:317-321 (1998).
59. Makela S, Poutanen M, Kostian ML, Lehtimaki N, Strauss L, Santti R, Vihko R. Inhibition of 17B-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells. Proc Soc Exp Biol Med 217:310-316 (1998).
60. Axelson M, Sjovall J, Gustafsson BE, Setchell KDR. Origin of lignans in mammals and identification of a precursor from plants. Nature 298:659-660 (1982).
61. Thompson LU, Robb P, Serraino M, Cheung F. Mammalian lignan production from various foods. Nutr Cancer 16:43-52 (1991).
62. Thompson LU, Rickard SE, Cheung F, Kenaschuk EO, Obermeyer WR. Variability in anticancer lignan levels in flaxseed. Nutr Cancer 27(1):26-30 (1997).
63. Mazur WM, Wahala K, Rasku S, Salakka A, Hase T, Adlercreutz H. Lignan and isoflavonoid concentrations in tea and coffee. Br J Nutr 79:37-45 (1998).
64. Kurzer MS, Xia X. Dietary phytoestrogens. Annu Rev Nutr 17:353-381 (1997).
65. Adlercreutz CH, Goldin BR, Gorbach SL, Hockerstedt KA, Watanabe S, Hamalainen EK, Markkanen MH, Makela TH, Wahala KT, Adlercreutz T. Soybean phytoestrogen intake and cancer risk. J Nutr 125:757S-770S (1995).
66. Hutchins AM, Slavin JL, Lampe JW. Urinary isoflavonoid phytoestrogen and lignan excretion after consumption of fermented and unfermented soy products. J Am Diet Assoc 95:545-551 (1995).
67. Rolwand I, Wiseman H, Sanders T, Adlercreutz H, Bowey E. Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora. Nutr Cancer 36:27-32 (2000).
68. Adlercreutz H, Yamada T, Wahala K, Watanabe S. Maternal and neonatal phytoestrogens in Japanese women during birth. Am J Obstet Gynecol 180:737-743 (1999).
69. Franke AA, Custer LJ. Daidzein and genistein concentrations in human milk after soy consumption. Clin Chem 42:955-964 (1996).
70. Setchell KDR, Welsh MB. High-performance liquid chromatographic analysis of phytoestrogens in soy protein preparations with ultraviolet, electrochemical and thermospray mass spectrometric detection. J Chromatogr A 368:315-323 (1987).
71. Setchell KDR, Zimmer-Nechemias L, Cai J, Heubi JE. Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 350:23-27 (1997).
72. Barnes S. Effect of genistein on in vitro and in vivo models of cancer. J Nutr 125:S777-S783 (1995).
73. Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE. Isoflavone content of infant formulas and the metabolic fate of these phytoestrogens in early life. Am J Clin Nutr 68:1453S-1461S (1998).
74. Winter JSD, Hughes IA, Reyes FI, Faiman C. Pituitary-gonadal relations in infancy: patterns of serum gonadal steroid concentrations in man from birth to two years of age. J Clin Endocrinol Metab 42:679-686 (1976).
75. Wang W. Radioimmunoassay determination of formononetin in murine plasma and mammary glandular tissue. Proc Soc Exp Biol Med 217:281-287 (1998).
76. Gamache PH, Acworth IN. Analysis of phytoestrogens and polyphenols in plasma, tissue, and urine using HPLC with coulometric array detection. Proc Soc Exp Biol Med 217:274-280 (1998).
77. Chang HC, Churchwell MI, Delclos KB, Newbold RR, Doerge DR. Mass spectrometric determination of genistein tissue distribution in diet-exposed Sprague-Dawley rats. J Nutr 130:1963-1970 (2000).
78. Franke AA, Custer LJ, Wang W, Shi CY. HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids. Proc Soc Exp Biol Med 217:263-273 (1998).
79. Morton M, Wilcox G, Wahlqvist M, Griffiths K. Determination of lignans and isoflavonoids in human female plasma following dietary supplementation. J Endocrinol 142:251-259 (1994).
80. Morton MS, Chan PS, Cheng C, Blacklock N, Matos-Ferreira A, Abranches-Monteiro L, Correia R, Lloyd S, Griffiths K. Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal, Hong Kong, and the United Kingdom. Prostate 32:122-128 (1997).
81. Coldham N, Sauer M. Pharmacokinetics of [14C]genistein in the rat: gender-related differences, potential mechanisms of biological action, and implications for human health. Toxicol Appl Pharmacol 164:206-215 (2000).
82. Cline JM, Paschold JC, OBasanjo IO, Adams MR. Effects of hormonal therapies and dietary soy phytoestrogens on vaginal cytology in surgically postmenopausal macaques. Fertil Steril 65:1031-1035 (1996).
83. Tansey G, Hughes CLJ, Cline JM, Krummer A, Walmer DK, Schmoltzer S. Effects of dietary soybean estrogens on the reproductive tract in female rats. Proc Soc Exp Biol Med 217:340-344 (1998).
84. Baird DD, Umbach DM, Lansdell L, Hughes CL, Setchell KDR, Weinberg CR, Haney AF, Wilcox AJ, McLachlan JA. Dietary intervention study to assess estrogenicity of dietary soy among postmenopausal women. J Clin Endocrinol Metab 80:1685-1690 (1995).
85. Wilcox G, Wahlqvist ML, Burger HG, Medley G. Oestrogenic effects of plant foods in postmenopausal women. Br Med J 30:905-906 (1990).
86. Murkies AL, Lombard C, Strauss BJ, Wilcox G, Burger HG, Morton MS. Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat. Maturitas 21:189-195 (1995).
87. Albertazzi P, Pansini F, Bonaccorsi G, Zanotti L, Forini E, De Aloysio D. The effect of dietary soy supplementation on hot flushes. Obstet Gynecol 91:6-11 (1998).
88. Kellis JT Jr, Vickery LE. Inhibition of human estrogen synthetase (aromatase) by flavones. Science 225:1032-1034 (1984).
89. Adlercreutz H, Bannwart G, Wahala K, Makela T, Brunow G, Hase T, Arosemena PJ, Kellis JT Jr. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 44:147-153 (1993).
90. Pelissero C, Lenczowski MJP, Chinzi D, Davail-Cuisset B, Sumpter JP, Fostier A. Effects of flavonoids and aromatase activity, an in vitro study. J Steroid Biochem Mol Biol 57:215-223 (1996).
91. Adams NR. Detection of the effects of phytoestrogens on sheep and cattle. J Anim Sci 73:1509-1515 (1995).
92. Leopold A, Erwin M, Oh J, Browning B. Phytoestrogens: adverse effects on reproduction in California quail. Science 191:98-99 (1976).
93. Obst JM, Seamark RF. Plasma progesterone concentrations during the reproductive cycle of ewes grazing Yarloop clover. J Reprod Fertil 21:545-547 (1970).
94. Newsome FE, Kitts WD. Effect of alfalfa consumption on estrogen levels in ewes. Can J Anim Sci 57:531-535 (1977).
95. Setchell KDR, Gosselin SJ, Welsh MB, Johnston JO, Balistreri WF, Kramer LW, Dresser BL, Tarr MJ. Dietary estrogens - a probable cause of infertility and liver disease in captive cheetahs. Gastroenterology 93:225-233 (1987).
96. Whitten PL, Lewis C, Russell E, Naftolin F. Phytoestrogen influences on the development of behavior and gonadotropin function. Proc Soc Exp Biol Med 208:82 (1995).
97. Murrill WB, Brown NM, Zhang J, Manzolillo PA, Barnes S, Lamartiniere CA. Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 17:1451-1457 (1997).
98. Phipps WR, Martini MC, Lampe JW, Slavin JL, Kurzer MS. Effect of flax seed ingestion on the menstrual cycle. J Clin Endocrinol Metab 77:1215-1219 (1993).
99. Lu LJ, Anderson KE, Grady JJ, Nagamani M. Effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction. Cancer Epidemiol Biomark Prev 5:63-70 (1996).
100. Duncan AM, Merz BE, Xu X, Nagel TC, Phipps WR, Kurzer MS. Soy isoflavones exert modest hormonal effects in premenopausal women. J Clin Endocrinol Metab 84:192-197 (1999).
101. Nagata C, Takasuka N, Inaba S, Kawakami N, Shimizu H. Effect of soymilk consumption on serum estrogen concentrations in premenopausal Japanese women. J Clin Endocrinol Metab 84:1830-1850 (1998).
102. Xu X, Duncan AM, Merz BE, Kurzer MS. Effects of soy isoflavones on estrogen and phytoestrogen metabolism in premenopausal women. Cancer Epidemiol Biomark Prev 7:1101-1108 (1998).
103. Carter BS, Cater HB, Isaacs JT. Epidemiologic evidence regarding predisposing factors to prostate cancer. Prostate 16:187-197 (1996).
104. Zava DT, Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 27:31-40 (1997).
105. Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet 350:990-994 (1997).
106. Morton MS, Chan PSF, Cheng C, Blacklock N, Matos-Ferreira A, Abranches-Monteiro L, Correia R, Lloyd S, Griffiths K. Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal, Hong Kong, and the United Kingdom. Prostate 32:122-128 (1997).
107. Santell R, Kieu N, Helferich W. Genistein inhibits growth of estrogen-independent human breast cancer cells in culture but not in athymic mice. J Nutr 130:1665-1669 (2000).
108. Barnes S, Peterson G, Grubbs C, Setchell K. Potential role of dietary isoflavones in the prevention of cancer. Adv Exp Med Biol 354:135-147 (1994).
109. Constantinou AI, Mehta RG, Vaughan A. Inhibition of N-methyl-N-nitrosourea-induced mammary tumors in rats by the soybean isoflavones. Anticancer Res 16:3293-3298 (1996).
110. Lamartiniere CA, Murrill WB, Manzolillo PA, Zhang J-X, Barnes S, Zhang Z, Wei H, Brown NM. Genistein alters the ontogeny of mammary gland development and protects against chemically-induced mammary cancer in rats. Proc Soc Exp Biol Med 217:358-364 (1998).
111. Wang W, Tanaka Y, Han Z, Higuchi CM. Proliferative response of mammary glandular tissue to formononetin. Nutr Cancer 23:131-140 (1995).
112. Santell RC, Chang YC, Nair MG, Helferich WG. Dietary genistein exerts estrogenic effects upon the uterus, mammary gland, and the hypothalamic/pituitary axis in rats. J Nutr 127:263-269 (1997).
113. Nwannenna AI, Madej A, Lundh TJO, Fredriksson G. Effects of oestrogenic silage on some clinical and endocrinological parameters in ovariectomized heifers. Acta Vet Scand 35:173-183 (1994).
114. Charland SL, Hui JW, Torosian MH. The effects of a soybean extract on tumor growth and metastasis. Int J Mol Med 2:225-228 (1998).
115. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen-receptive-positive breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 58:3833-3837 (1998).
116. Foth D, Cline JM. Effects of mammalian and plant estrogens on mammary glands and uteri of macaques. Am J Clin Nutr 68:1413S-1417S (1998).
117. Clarke R, Hilakivi-Clarke L, Cho E, James MR, Leonessa F. Estrogens, phytoestrogens and breast cancer. In: Dietary Phytochemicals in Cancer Prevention and Treatment (Research AIfC, ed). New York:Plenum Press, 1996;63-85.
118. Russo J, Russo IH. Biological and molecular bases of mammary carcinogenesis. Lab Investig 57:112-137 (1987).
119. Lopez J, Ogren L, Verjan R, Talamantes F. Effects of perinatal exposure to a synthetic estrogen and progestin on mammary tumorigenesis in mice. Teratology 38:129-134 (1988).
120. Walker BE. Tumors of female offspring of mice exposed prenatally to diethylstilbestrol. J Natl Cancer Inst 73:133-140 (1984).
121. Schoental R. Trichothecenes, zearalenone, and other carcinogenic metabolites of Fusarium and related microfungi. Adv Cancer Res 45:217-290 (1985).
122. Brown NM, Wang J, Cotroneo MS, Zhao YX, Lamartiniere CA. Prepubertal genistein treatment modulates TGF-alpha, EGF and EGF-receptor mRNAs and proteins in the rat mammary gland. Mol Cell Endocrinol 144:149-165 (1998).
123. Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ, Barnes S. Genistein supresses mammary cancer in rats. Carcinogenesis 16:2833-2840 (1995).
124. McMichael-Phillips DF, Harding C, Morton M, Roberts SA, Howell A, Potten CS, Bundred NJ. Effects of soy-protein supplementation on epithelial proliferation in the histologically normal breast. Am J Clin Nutr 68:1431S-1435S (1998).
125. Makela S, Santti R, Salo L, McLachlan JA. Phytoestrogens are partial estrogen agonists in the adult male mouse. Environ Health Perspect 103:123-127 (1995).
126. Strauss L, Makela S, Joshi S, Huhtaniemi I, Santti R. Genistein exerts estrogen-like effects in male mouse reproductive tract. Mol Cell Endocrinol 144:83-93 (1998).
127. Sharma OP, Adlercreutz H, Strandberg JD, Zirkin BR, Coffey DS, Ewing LL. Soy of dietary source plays a preventative role against the pathogenesis of prostatitis in rats. J Steroid Biochem Mol Biol 43:557-564 (1992).
128. Geller J, Sionit L, Partido C, Li L, Tan X, Youngkin T, Nachtsheim D, Hoffman RM. Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture. Prostate 34:75-79 (1998).
129. Naik HR, Lehr JE, Pienta KJ. An in vitro and in vivo study of antitumor effects of genistein on hormone refractory prostate cancer. Anticancer Res 14:2617-2619 (1994).
130. Pollard M, Luckert PH. Influence of isoflavones in soy protein isolates on development of induced prostate-related cancers in L-W rats. Nutr Cancer 28:41-45 (1997).
131. Stephens FO. Phytoestrogens and prostate cancer: possible preventative role. Med J Aust 167:138-140 (1997).
132. Gambacciani M, Spinetti A, Piaggesi L, Cappagli B, Taponeco F, Manetti P, Weiss C, Teti GC, Commare PI, Facchini V. Ipriflavone prevents the bone mass reduction in premenopausal women treated with gonadotropin hormone-releasing hormone agonists. Bone Miner 26:19-26 (1994).
133. Valente M, Bufalino L, Castiglione G, D'Angelo R, Mancuso A, Galoppi P, Zichella L. Effects of 1-year treatment with ipriflavone on bone in postmenopausal women with low bone mass. Calcif Tissue Int 54:377-380 (1994).
134. Adami S, Bufalino L, Cervetti R, Di Marco C, Di Munno O, Fantasia L, Isaia G, Serni U, Vecchiet L, Passeri M. Ipriflavone prevents radial bone loss in postmenopausal women with low bone mass over 2 years. Osteoporos Int 7:119-125 (1997).
135. Rondelli I, Acerbi D, Ventura P. Steady-state pharmacokinetics of ipriflavone and its metabolites in patients with renal failure. Int J Clin Pharmacol Res 11:183-192 (1991).
136. Benvenuti S, Peilli M, Frediani U, Tanini A, Fiorelli G, Bianchi S, Bernabei P, Albanese C, Brandi M. Binding and bioeffects of Ipriflavone on a human preosteoclastic cell line. Biochem Biophys Res Commun 201:1084-1089 (1994).
137. Albanese C, Cudd A, Argentino L, Zambonin-Zallone A, MacIntyre I. Ipriflavone directly inhibits osteoclastic activity. Biochem Biophys Res Commun 199(2):930-936 (1994).
138. Cecchini MG, Fleisch H, Muhlbauer RC. Ipriflavone inhibits bone resportion in intact and ovariectomized rats. Calcif Tissue Int 61:S9-S11 (1997).
139. Yoshida K, Tsukamoto T, Torii H, Doi T, Naeshiro I, Shibata K, Uemura I, Tanayama S. Disposition of ipriflavone (TC-80) in rats and dogs. Radioisotopes 34:618-623 (1985).
140. Kim SH, Lee JS, Lee MG. Determination of a isoflavone derivative, ipriflavone, and its metabolites, M1 and M5, in rat plasma, urine, and tissue homogenate by high-performance liquid chromatography. Res Commun Mol Pathol Pharmacol 98:313-324 (1997).
141. Tustsumi N. Effect of coumestrol on bone metabolism in organ culture. Biol Pharmacol Bull 18:1012-1015 (1995).
142. Dodge JA, Glasebrook AL, Magee DE, Phillips DL, Sato M, Short LL, Bryant HU. Environmental estrogens: effects on cholesterol lowering and bone in the ovariectomized rat. J Steroid Biochem Mol Biol 59:155-161 (1996).
143. Draper CR, Edel MJ, Dick IM, Randall AG, Martin GB, Prince RL. Phytoestrogens reduce bone loss and bone resorption in oophorectomized rats. J Nutr 127:1795-1799 (1997).
144. Anderson JJ, Ambrose WW, Garner SC. Biphasic effects of genistein on bone tissue in the ovariectomized, lactating rat model. Proc Soc Exp Biol Med 217:345-350 (1998).
145. Tobe H, Komiyama O, Komiyama Y, Maruyama HB. Daidzein stimulation of bone resorption in pit formation assay. Biosci Biotechnol Biochem 61:370-371 (1997).
146. Arts J, Kuiper GG, Janssen JM, Gustafsson JA, Lowik CW, Pols HA, van Leeuwen JP. Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 138:5067-5070 (1997).
147. Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T. Expression of estrogen receptor beta in rat bone. Endocrinology 138:4509-4512 (1997).
148. Collier FM, Huang WH, Holloway WR, Hodge JM, Gillespie MT, Daniels LL, Zheng MH, Nicholson GC. Osteoclasts from human giant cell tumors of bone lack estrogen receptors. Endocrinology 139:1258-1267 (1998).
149. Petilli M, Fiorelli G, Benvenuti S, Frediani U, Gori F, Brandi M. Interactions between ipriflavone and the estrogen receptor. Calcif Tissue Int 56(2):160-165 (1995).
150. Yamazaki I. Effect of ipriflavone on the response of uterus and thyroid to estrogen. Life Sci 38:757-764 (1986).
151. Arjmandi BH, Khan DA, Juma SS, Svanborg A. The ovarian hormone deficiency-induced hypercholesterolemia is reversed by soy protein and the synthetic isoflavone, ipriflavone. Nutr Res 17:885-894 (1997).
152. Anthony MS, Clarkson TB, Hughes CL Jr, Morgan TM, Burke GL. Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. J Nutr 126:43-50 (1996).
153. Honore EK, Williams JK, Anthony MS, Clarkson TB. Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques. Fertil Steril 67:148-154 (1997).
154. Wagner JD, Cefalu WT, Anthony MS, Litwak KN, Zhang L, Clarkson TB. Dietary soy protein and estrogen replacement therapy improve cardiovascular risk factors and decrease aortic cholesteryl ester content in ovariectomized cynomolgus monkeys. Metabolism 46:698-705 (1997).
155. Goodman MT, Wilkens LR, Hankin JH, Lyu LC, Wu AH, Kolonel LN. Association of soy and fiber consumption with the risk of endometrial cancer. Am J Epidemiol 146:294-306 (1997).
156. Hodgson JM, Puddey IB, Beilin LJ, Mori TA, Croft KD. Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: a randomized trial in humans. J Nutr 128:728-732 (1998).
157. Nestel PJ, Pomeroy S, Kay S, Komesaroff P, Behrsing J, Cameron JD, West L. Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal women. J Clin Endocrinol Metab 84:895-898 (1999).
158. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JWJ. Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 68:1371S-1379S (1998).
159. Hughes CLJ. Effects of phytoestrogens on GnRH-induced luteinizing hormone secretion in ovariectomized rats. Reprod Toxicol 1:179-181 (1987).
160. Hughes CL, Kaldas RS, Weisinger AS, McCants CE, Basham KB. Acute and subacute effects of naturally occurring estrogens on luteinizing hormone secretion in the ovariectomized rat: Part 1. Reprod Toxicol 5:127-132 (1991).
161. Hughes CLJ, Chakinala MM, Reece SG, Miller RN, Schomberg DW, Basham KB. Acute and subacute effects of naturally occurring estrogens on luteinizing hormone secretion in the ovariectomized rat: Part 2. Reprod Toxicol 5:133-137 (1991).
162. Hopkins W, Bailey J, Fuller GB. Hormone effects of zearalenone in nonhuman primates. J Toxicol Environ Health 3:43-57 (1977).
163. Nichols J, Lasley BL, Gold EB, Nakajima ST, Schneeman BO. Phytoestrogens in soy and changes in pituitary response to GnRH challenge tests in women. J Nutr 125:803S (1995).
164. Hidy PH, Baldwin RS, Greashan RL, Keith CL, McMullan JR. Zearalenone and some derivatives: production and biological activities. Adv Appl Microbiol 22:59-82 (1977).
165. Coldham NG, Dave M, Sivapathasundaram S, McDonnell DP, Connor C, Sauer MJ. Evaluation of a recombinant yeast cell estrogen screening assay. Environ Health Perspect 105:734-742 (1997).
166. Zava DT, Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 27:31-40 (1997).
167. Molteni A, Brizio-Molteni L, Persky V. In vitro hormonal effects of soybean isoflavones. J Nutr 125(suppl 3):751S-756S (1995).
168. Rosenblum ER, Stauber RE, Van Thiel DH, Campbell IM, Gavaler JS. Assessment of the estrogenic activity of phytoestrogens isolated from bourbon and beer. Alcohol Clin Exp Res 17:1207-1209 (1993).
169. Miksicek RJ. Estrogenic flavonoids: structural requirements for biological activity. Proc Soc Exp Biol Med:44-50 (1995).
170. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870 (1997).
171. Soto AM, Lin T-L, Justicia H, Silvia RM, Sonnenschein C. An "in culture" bioassay to assess the estrogenicity of xenobiotics (E-screen). In: Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection (Colburn T, Clement C, eds). Princeton, NJ:Princeton Scientific 1992;295-310.
172. Welshons WV, Rottinghaus GE, Nonneman DJ, Dolan-Timpe M, Ross PF. A sensitive bioassay for detection of dietary estrogens in animal feeds. J Vet Diagn Invest 2:268-273 (1990).
173. Shutt DA, Cox RI. Steroid and phyto-oestrogen binding to sheep uterine receptors in vitro. J Endocrinol 52:299-310 (1972).
174. Bickoff EM, Livingston AL, Hendrickson AP, Booth AN. Relative potencies of several estrogen-like compounds found in forages. Agric Fd Chem 10:410-412 (1962).
175. Campbell DR, Kurzer MS. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J Steroid Biochem Mol Biol 46:381-388 (1993).
176. Fotsis T, Pepper M, Adlercreutz H, Hase T, Montesano R, Schweigerer L. Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J Nutr 125:790S-797S (1995).
177. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh NM, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262:5592-5595 (1987).
178. Barnes S, Peterson TG. Biochemical targets of the isoflavone genistein in tumor cell lines. Proc Soc Exp Biol Med 208:103-108 (1995).
179. Agullo G, Gamet-Payrastre L, Manenti S, Viala C, Remesy C, Chap H, Payrastre B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol 55:1649-1657 (1997).
180. Wei H, Bowen R, Cai Q, Barnes S, Wang Y. Antioxidant and antipromotional effects of the soybean isoflavone genistein. Proc Soc Exp Biol Med 208:124-130 (1995).
181. Ruiz-Larrea MB, Mohan AR, Paganga G, Miller NJ, Bolwell GP, Rice-Evans CA. Antioxodant activity of phytoestrogenic isoflavones. Free Radic Res 26:63-70 (1997).
182. Wiseman H, O'Reilly J. The cardioprotective antioxidant activity of dietary phytoestrogens compared to oestrogen. Biochem Soc Trans 25(1):107S (1997).
183. Gaudette DC, Holub BJ. Effect of genistein a tyrosine kinase inhibitor on U46619-induced phosphoinositide phosphorylation in human platelets. Biochem Biophys Res Comm 170:288-292 (1990).
184. Dees C, Foster JS. Dietary estrogens stimulate human breast cells to enter the cell cycle. Environ Health Perspect 105:633-636 (1997).
185. Mousavi Y, Adlercreutz H. Enterolactone and estradiol inhibit each other's proliferative effect on MCF-7 breast cancer cells in culture. J Steroid Biochem Molec Biol 41:615-619 (1992).
186. Dechaud H, Ravard C, Claustrat F, Brac de la Pierre A. Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG). Steroids 64:328-334 (1999).
187. Reinli K, Block G. Phytoestrogen content of foods - a compendium of literature values. Nutr Cancer 26:123-148 (1996).
188. Kaufman PB, Duke JA, Brielman H, Boik J, Hoyt JE. A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: implications for human health and nutrition. J Altern Complement Med 3:7-12 (1997).
189. Adlercreutz H, Fotsis T, Lampe J, Wahala K, Makela T, Brunow G, Hase T. Quantitative determination of lignans and isoflavonoids in plasma of ominvorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry. Scand J Clin Lab Investig Suppl 215:5-18 (1993).
190. Gooderham MH, Adlercreutz H, Ojala ST, Wahala K, Holub BJ. A soy protein isolate rich in genistein and daidzein and its effects on plasma isoflavone concentrations, platelet aggregation, blood lipids and fatty acid composition of plasma phospholipid in normal men. J Nutr 126:2000-2006 (1996).
191. Adlercreutz H, Fotsis T, Watanabe S, Lampe J, Wahala K, Makela T, Hase T. Determination of lignans and isoflavonoids in plasma by isotope dilution gas chromatography-mass spectrometry. Cancer Detect Preven 18:259-271 (1994).
192. Arai Y, Uehara M, Sato Y, Kimura M, Eboshida A, Adlercreutz H, Watanabe S. Comparison of isoflavones among dietary intake, plasma concentratin and urinary excretion for accurate estimation of phytoestrogen intake. J Epidemiol 10:127-135 (2000).
193. Kelly GE, Nelson C, Waring MA, Joannou GE, Reeder AY, Mayr U, But
