Phytoestrogen are of biological interest because they exhibit both in vivo and in vitro weak estrogenic and anti-estrogenic actions through their binding to ERs . The dual action of phytoestrogens as agonists and antagonists has resulted in a general confusion in the literature regarding the role these compounds may play in disease progression and/or protection. In common with many other weak estrogens, isoflavones have been shown to be anti-estrogens in model systems, competing for E2 at the receptor complex, yet failing to stimulate a full estrogenic response after binding to the nucleus . This raises the possibility that they may be protective in hormone-related diseases, such as breast cancer . In vitro studies have established that phytoestrogens are weakly estrogenic, since they have the ability to bind to mammalian ERs to a low degree. The relative potencies, determined by human cell culture bioassays (compared with E2, to which an arbitrary value of 100 was given) are: coumestrol 0.202, genistein 0.084, equol 0.061, daidzein 0.013 and formononetin 0.0006 . The biological potency of these compounds, in animal and in vitro models, varies considerably and may depend on many factors including target tissue, functional state of the target tissue, species, age of the subject, route of delivery, dose, length of exposure, and metabolism. Furthermore, it is important to note that these wide ranges are derived from studies of cell-culture systems or animal models, rather than from direct effects in humans.
The human, rat and mouse ER exists as two subtypes, ERa and ERfi, which differ in the C-terminal ligand-binding domain and in the ^-terminal transac-tivation domain. Kuiper et al.  investigated the estrogenic activity of phytoestrogens in competition binding assays with ERa or ERb protein and in a transient gene expression assay. In most instances, the relative binding affinities (RBA) of phytoestrogens are at least 1000-fold lower than that of E2. Some phy-toestrogens, such as coumestrol and genistein, compete more strongly with E2 for binding to ERb than to ERa. Phytoestrogens can stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the trans-activation assay is different; that is E2>coumestrol>genistein>daidzein>bio-chanin A>formononetin = ipriflavone for ERa and E2>genistein = coumestrol > daidzein > biochanin A > formononetin = ipriflavone for ERb The binding affinity of coumestrol to ERb is 7-fold higher in comparison to ERa, whereas genistein has a 20- to 30-fold higher binding affinity for ERb The exact position and number of the hydroxy substituents on the isoflavone molecule seems to determine the ER binding affinity. For example, the isoflavone genistein has a particular high binding affinity for ERb,but elimination of one hydroxy group (e.g., daidzein, biochanin A) or two hydroxy groups (e.g., formononetin) causes a great loss in binding affinity (Fig. 1). Anti-estrogenic activity of the phytoestrogens could not be detected in this particular study. However, the estrogenic potency of phytoestrogens in this study is significant, especially for ERb, and they may trigger many of the biological responses that are evoked by the physiological estrogens. Interestingly, ERb shows a different anatomical distribution from ERa, being expressed more prominently in tissues such as the brain, ovary, uterus, prostate, lung and urinary tract . ERb is also expressed in breast cells, although apparently weakly .
In vitro research has played an important role in determining the biological potencies of dietary estrogens. In cultured cells, both proliferative and antiproliferative effects have been ascribed to genistein [74-77]. Wang et al.  observed that genistein stimulated estrogen-responsive pS2 mRNA expression, and this effect could be inhibited by tamoxifen. Thus, the estrogenic effect of genistein would appear to be a result of an interaction with the ER. At lower concentrations (10-8 to 10-6 M), genistein stimulated growth of ER-positive cells, but at higher concentrations (>10-5 M), genistein inhibited growth. Genistein failed to stimulate the proliferation of ER-negative cells at low concentrations. The biphasic effects of genistein on growth at lower concentrations appeared to be via the ER pathway, while the effects at higher concentrations were independent of the ER. These effects might appear to be involved in the mechanism by which genistein contributes to the decreased risk in hormone-dependent cancers associated with ingestion of soy products. However, this may be unlikely since the human circulating level of genistein does not exceed 2.5x10-5 M . Even with increased intake of soy products, circulating levels of genistein normally do not exceed 10-6 M. It is more likely that the effects of dietary genistein on tumorigenesis are mediated through the ER, for which it has a relatively high affinity. Taken together, the results support the idea that genistein, at physiologically achievable concentrations, may interfere with the action of estrogen through direct competition for binding to ER and by reducing ER expression. These effects could be considered anti-estrogenic, consistent with the proposed cancer-preventive effects of soy diet. In addition, prolonged experimental exposure to phytoestrogens may result in the reduction of ER expression and hence lead to decreased responsiveness to endogenous estrogens since prolonged exposure to genistein resulted in a decrease in ER mRNA level, as well as a decrease in response to stimulation by E2 .
Although there have been many interesting studies on the effects of isoflavones on biochemical targets in tissue culture experiments, the concentrations used by investigators have exceeded 10 |M in most cases. Based on simple pharmacokinetic calculations involving daily intakes of isoflavones, absorption from the gut, distribution to peripheral tissues, and excretion, it is unlikely that blood isoflavone concentrations, even in high soy consumers, could be greater than 1-5 |M . Although the plasma genistein levels achievable with soy food feedings are unlikely to be sufficient to inhibit the growth of mature, established breast cancer cells by chemotherapeutic-like mechanisms, these levels are sufficient to regulate the proliferation of epithelial cells in the breast and thereby may cause a chemopreventive effect. For breast cancer and prostate cancer, delivery of genistein via the blood supply is the only route available physiologically. The effective concentration of genistein in blood will therefore depend on plasma protein binding. Physiological steroids are 98% bound to plasma proteins, thereby leaving less than 2 % available for uptake into tissues.
There are several other anti-cancer effects of isoflavones which do not involve ER mechanisms. Genistein is known to inhibit protein tyrosine kinases (PTKs), which are responsible for phosphorylating proteins required for the regulation of cell function, including cell division . These enzymes appear to be necessary for epidermal growth factor function and the action of other growth factors, which indicate the anti-proliferative potential of this iso-flavonoid. Since many of the protein products of human oncogenes were found to take part in growth factor signaling through PTKs, a rationale was developed for the role of genistein in cancer prevention . Genistein can inhibit cell cycle progression in tumor cells independent of action at the ER [80-82]. Genistein has also been shown to inhibit the DNA repair enzyme topoisomerase , and to act as an antioxidant, thus potentially preventing apoptosis, or programmed cell death, a protective mechanism induced in cells that have been damaged in order to prevent the proliferation of harmful mutations and possible cancer . Alternative mechanisms of action of genistein have also been postulated. They include the effects of reactive oxygen species , and the expression of DNA transcription factors c-fos and c-jun . Recently, genistein's regulation of the level of transforming growth factor b (TGFb), an inhibitor of the G1/S phase of cell cycle control, has been identified . It has also been shown to inhibit ras gene expression in a rat pheochromocytoma cell line . In addition, genistein has been shown to inhibit angiogenesis, the formation of new blood vessels, which is an abnormal event that occurs as a part of the growth and expansion of malignant tumors . It has been pointed out that many of these effects have also been shown with very high concentrations, and not in cells treated with the levels likely to be achieved in plasma of human subjects eating foods containing phytoestrogens . For example, 100 |M concentrations were needed for a significant suppression of angiogenesis, although proliferation was inhibited at 5 |M levels . This compares with plasma levels of about 0.4 |M in Japanese men and women fed on dietary supplements of isoflavones [40, 78, 89] and peak levels of about 4 |M in women receiving 126 mg isoflavones as soy milk, rising to 12 |M when receiving 480 mg isoflavones per day [22,90].
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