Interest in possible health benefits of isoflavonoids has increased due to a variety of bioprotective effects, including antioxidant, antimutagenic, anticarcinogenic, antiproliferative activities, mostly assessed in vitro (Birt et al., 2001; Miadokova et al., 2002; Ryan-Borchers et al., 2006; Iwasaki et al., 2008; Scarpato et al., 2008). Isoflavonoids have been classically defined as dietary antioxidants, i.e. compounds that may protect against oxidative stress linked to inflammation and the risk of macromolecule damage by free radicals and by related oxygen and nitrogen-based oxidizing agents (Reiter et al., 2008). They may protect the body from hormone-related cancers, like breast, endometrial (uterine) and prostatic. Despite the wide spectrum of health protective abilities that have been attributed to them, e.g. immunomodulation, risk reduction of chronic diseases including cardiovascular diseases, diabetes, cancer, osteoporosis and obesity (Bezek et al., 2008), as well as relief of menopausal symptoms (Birt et al., 2001; Ryan-Borchers et al., 2006; Ørgaard and Jensen, 2008; Sabudak and Guler, 2009), there is growing evidence from human dietary and epidemiologic studies that the role of isoflavonoids in human health is questionable. Overview Of The Biological Activities Of Isoflavones Essay. Some studies speak in favor of a protective effect of isoflavonoid consumption as dietary supplements and as a natural alternative to estrogen replacement therapy, other studies failed to demonstrate favorable effects. The aim of this article is not to present a comprehensive review of the literature but to provide a critical review of the arguments centered on a variety of effects and mechanisms of isoflavonoid actions.

Natural sources, bioavailability and metabolism

Isoflavonoids, as plant secondary metabolites, possess a 3-phenylchroman skeleton that is biogenetically derived from 2-phenylchroman skeleton of flavonoids. Isoflavonoids are particularly prevalent in Papilonoidae, subfamily of Leguminosae (Dixon and Summer, 2003). Soy (Glycine max) principle isoflavones genistein, daidzein and glycitein are the aglycones with three possible glycoside forms: β-D-glycoside, 6″-O-malonyl-glycoside and a 6″-O-acetyl-glycoside. However, primarily they are naturally present in their β-D-glycoside form as genistin, daidzin, glycitin. The aglycone form of isoflavones is biologically active. In addition to soy, isoflavone daidzein and genistein, their precursors formononetin, biochanin A, their glycosides, glycosides malonates and acetyl glycosides were determined in red clover (Trifolium pratense) extracts using chromatographic and spectrometric methods (Sabudak and Guler, 2009). Murata et al. (2006) isolated from leaves of Millettia taiwaniana (Leguminosae) two new isoflavonoids, millewanin-F and furowanin-A, together with previously known five isoflavonoids. The bioavailability of isoflavones has been shown to be influenced by their chemical form in foods (generally glycoside conjugates), susceptibility to degradation, the microbial flora of the consumer, and by the food matrix. Bioavailability of isoflavonoids may depend to some extent upon interaction with other dietary components (Birt et al., 2001).

The metabolism of isoflavones in animals and humans is complex and is a combination of mammalian and gut microbial processes. Moreover, there is a great deal of individual variability in the metabolism of isoflavonoids. Individual differences in gut microflora, intestinal transit time, and genetic polymorphisms, all are likely to contribute to this great variability (Duffy et al., 2007). During digestive and absorptive processes, isoflavonoids often undergo metabolic transformations. In some individuals, daidzein can be converted by the intestinal microflora to the metabolite equol or to O-desmethylangolensin, and genistein to p-ethyl phenol. Overview Of The Biological Activities Of Isoflavones Essay.

The degree of absorption of bioflavonoids has been the subject of frequent debates among scientists. Absorption and bioavailability of isoflavonoids is much higher than originally believed. Cesarone et al. (2009) compared absorption of an isotonic isoflavonoid solution vs. tablet form with the equivalent amount of fluid and revealed a dramatically accelerated bioavailability of isoflavonoids delivered in an isotonic formulation.

Under certain nutritional conditions total isoflavone concentrations can be measured in urine, plasma and even in breast fluid. Assessment of isoflavonoids in nipple aspirate fluid enables to elucidate their effect directly on breast tissue (Maskarinec et al, 2008a).

The hypothesis that isoflavones are absorbed more efficiently from fermented than from non-fermented soy foods was re-examined and then rejected (Maskarinec et al., 2008b). Therefore recommendations favoring fermented soy foods are not justified when the intestinal microflora is capable of hydrolyzing the isoflavone glycosides from non-fermented soy foods.

Estrogenic activity of isoflavonoids

Isoflavones are diphenolic compounds that are structurally or functionally similar to endogenous estrogens and display agonistic and antagonistic interactions with estrogen receptors (Wang et al., 2008). Their biological activity is partly ascribed to the structural similarities with the primary physiologically relevant estrogen – 17β-estradiol (E2). They bind to and activate intracellular estrogen receptors: ERα and ERβ and, mimicking the effects of estrogen, are commonly referred to as phytoestrogens. In most systems, the relative binding affinities of genistein and daidzein are greater for ERβ than for ERα, while E2 binds to both receptors with approximately equal affinities (Messina et al., 2006). The estrogenic activities of soy isoflavones are thought to play an important role by their health-enhancing properties in menopausal symptoms and in treating osteoporosis (Lockwood, 2008).Overview Of The Biological Activities Of Isoflavones Essay.  Though isoflavonoids have exhibited estrogen-like properties they are bound more weakly to estrogen receptors than E2. Arguments have been presented for considering soy isoflavones as natural selective estrogen modulators.

Controversal data on estrogen-like effects

Since overexposure to estrogen is a major contributing factor in the development of breast cancer, the relationship between soyfoods and breast cancer has become controversial. A concern has been raised that soy-derived isoflavones, which exhibit estrogen-like properties under certain experimental conditions, may stimulate the growth of existing estrogen-sensitive tumors. This concern exists because of evidence showing that isoflavones bind and transactivate estrogen receptors and elicit estrogenic effects in rodent reproductive tissues. Limited human data directly address the tumor-promoting effects of isoflavonoids (Messina et al., 2006)

The fact that genistein at low physiologically relevant levels may stimulate estrogen receptor positive tumors can be attributed to its estrogenic properties, while at higher levels, anticancer actions of isoflavonoids may predominate (Duffy et al., 2007).

Estrogen-like effects have raised concern regarding soy/isoflavone consumption particularly in the case of postmenopausal women at high risk of breast cancer. Overview Of The Biological Activities Of Isoflavones Essay. Currently there is little evidence to suggest that any potential weak estrogenic effects of dietary isoflavones have a clinically relevant impact on the breast tissue in healthy women or breast cancer survivors (Messina and Wood, 2008).

Epidemiologic evidence shows that higher soy intake in Asian women is associated with a nearly one-third reduction in breast cancer risk and that Japanese breast cancer patients, in comparison to Western women, exhibit better survival rates even after controlling stage of diagnosis. Evaluation of the short-term effects of high-dose soy isoflavonoid supplements on reproductive tissues in a postmenopausal primate model showed that high doses of dietary isoflavonoids had minimal uterotrophic or mammotrophic effects (Wood et al., 2006). These findings suggest that comparable high-dose isoflavonoid supplements, in particular those containing equol or taken by equol producers, would have minimal negative estrogenic effects on human reproductive tissues and are not likely to contribute to increased uterine or breast cancer risks.

Since endogenous estrogens are important determinants of breast cancer risk in postmenopausal women, Wood et al. (2007) evaluated the effects of dietary soy isoflavonoids on endogenous metabolism in a postmenopausal primate model. They revealed that isoflavonoid treatment did not significantly alter gene markers of estrogen metabolism or estrogen receptor agonist activity in breast tissue.

On the other hand, there are data supporting the idea that a high dietary isoflavone intake improves the prognosis of breast cancer patients. For example, among postmenopausal U.S. breast cancer patients, mortality was reduced due to isoflavonoid diet (Fink et al., 2007). Available data for estrogen therapy effects on breast cancer recurrence and mortality provide some assurance for breast cancer patients that isoflavone supplements, when taken at dietary levels, do not contribute to recurrence rates. Nevertheless more data are needed to better address this issue (Messina and Wood, 2008). Overview Of The Biological Activities Of Isoflavones Essay.

Phytoestrogens and effects on gene expression

Isoflavones can exert hormonal and nonhormonal properties in many ways. Isoflavones have been shown to inhibit the activity of enzymes involved in estrogen metabolism. Estrogens have diverse effects throughout the body, attributable in part to their ability to modulate transcription of target genes in a variety of organs. Rice et al. (2006) found that phytoestrogens and their low dose combinations inhibited mRNA expression and activity of aromatase in human granulosa-luteal cells. Due to the established association between estrogen levels and breast cancer risk, the inhibitory effect of biochanin A on enzyme aromatase, the protein product of CYP 19 gene, was studied. Investigation of the effect on gene regulation and enzyme activity of aromatase showed that this isoflavonoid inhibited CYP19 expression and aromatase activity and hampered the growth of MCF-7 breast carcinoma cells attributed to the enzyme activity (Wang et al., 2008).

At nutritionally relevant doses, phytoestrogens may selectively interact with ERβ and thus affect only expression of a subset of estrogen-responsive genes. Different regulation of estrogen-responsive genes by isoflavonoids may also depend upon the relative ratio of ERα and ERβ present within tissue types. To test the hypothesis that soy isoflavones selectively trigger Erβ-dependent gene expression, which may be particular to the cell type and dependent on relative expression of ERα and Erβ, Chrzan and Bradford (2007) studied whether genistein and daidzein could affect reporter gene transcription via the estrogen receptors ERα and ERβ1. Overview Of The Biological Activities Of Isoflavones Essay. They documented that both phytoestrogens, genistein and daidzein, increased the expression of estrogen-responsive genes in human MCF-7 breast carcinoma cells.

Data from experiments using DNA microarray analysis for examining the effects of genistein in the developing rat uterus indicate that genistein alters the expression of 6–8 times as many genes as does physiological estrogen E2. Genistein affected 227 genes, the majority of which were down regulated (Barnes, 2004).

Emerging research using genomics and proteomics enabled to understand the participation of enzymes included in the isoflavonoid metabolic pathway regulation. Ralston et al., (2005) cloned and functionally characterized soybean chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analyses revealed that the gene expression for soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other isoflavonoid-specific genes, while the legume specific type II CHIs, which uses a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Comparison of putative soybean CHI with CHI ortologs from other species showed that their amino acid sequences fell into four different subfamilies. CHIs in the first subfamily (CHI1) were found only in legumes and were similar at least in 70% to those in Plant Genome Database (Ralston et al., 2005). Overview Of The Biological Activities Of Isoflavones Essay.

Inconsistency between phytoestrogen intake and risk for colorectal cancer

Some experimental studies reported that anticarcinogenic properties of dietary soy isoflavonoids play an important role in preventing colorectal cancer. However, few epidemiologic studies have examined this association in general populations and their findings have been inconsistent (Akhter et al., 2008).

Investigation of the association between dietary isoflavone intake and incidence of colorectal cancer in a prospective cohort study of 83,063 Japanese men and women documented that the intake of isoflavones was not associated with the risk of distal colorectal and rectal cancers in either men or women. Moreover, the risk of proximal colon cancer in men decreased with increasing consumption of isoflavones, miso soup and soy food (Akhter et al., 2008). These findings are in accordance with those aimed at soy isoflavones modulation of rat colon carcinogenesis because pre- and postnatal exposure to dietary soy isoflavones suppressed the growth of colon tumors in male rats (Raju et al., 2009). Overview Of The Biological Activities Of Isoflavones Essay.

Inconsistency between phytoestrogen intake and risk for breast cancer

Data regarding the role of isoflavonoids in breast cancer prevention are conflicting. Nevertheless, they suggest that early exposure in childhood or early adolescence may be protective (Ju et al., 2006; Duffy et al., 2007). There has been some evidence suggesting that the menopausal status of women may modulate the effect of isoflavonoids. Case-control studies examining phytoestrogens and breast cancer incidence have generally found more evidence for a protective role in premenopausal women versus postmenopausal ones because isoflavonoid effects are dependent on the hormonal status of the woman, with stimulatory effects in low-estrogen environments, while in high-estrogen states they may block the effects of estrogen.

There are in vivo animal data suggesting that genistein may interfere with inhibitory effects of tamoxifen on breast cancer cell growth (Liu et al., 2005). Several studies using urine serum concentrations of phytoestrogens and their metabolites as biomarkers of their intake reported reduced risk for breast cancer among breast cancer survivors, yet other studies have failed to show an effect of phytoestrogens on breast cancer risk or on the menopausal syndrome (Duffy et al., 2007). Overview Of The Biological Activities Of Isoflavones Essay. Recently Goodman et al. (2009) examined the association of urinary phytoestrogens with the risk of postmenopausal breast cancer and results of their multiethnic cohort study revealed that a diet rich in isoflavones from soy products reduced the risk of postmenopausal breast cancer, particularly in populations with comparatively high excretion of phytoestrogens. Iwasaki et al., (2008) also found a statistically significant inverse association between plasma genistein and the risk of breast cancer.

Some differences in the results presented could be related to other abettors, such as genetic factors which may modulate the effect of phytoestrogens (Piller et al., 2006). Functional polymorphism in genes that encode for enzymes involved in the estrogen biosynthesis and metabolism and in genes that encode hormone receptors may have been associated with the risk for breast cancer (Atkinson et al., 2004). But no association between a polymorphism in genes CYP17 (5′untranslated MspA1 polymorphism), CYP19 (generated by a G → T substitution in intron 6), involved in the sex hormone biosynthesis pathway, and Pvull (lipoprotein lipase) polymorphism (generated by a C → T substitution in intron 1) in the estrogen receptor ESR1 gene and the risk of breast cancer was demonstrated (Chen et al., 2008). Piller et al. (2006) examined dietary genistein intake, CYP17 5′untranslated MspA1 genetic polymorphism and breast cancer risk in premenopausal breast cancer patients. The risk-reducing effect of genistein consumption was not modified by CYP 17 genotype. On the other hand, examination of two single nucleotide polymorphisms in Cyp 19 gene (aromatase), rs1008805 (A/G) and rs730154 (C/T) revealed that premenopausal women carrying at least one G alelle at Cyp 19 locus was associated with an increased breast cancer risk (Talbot et al., 2008). Furthermore, Cyp17 variant C alelle may increase the breast cancer risk in conjugation with long-term hormone replacemet treatment (HRT) and a high body mass index (BMI) in postmenopausal women (Chen et al., 2008). These findings support the potential role of variation in estrogen biosynthesis genes in premenopausal and postmenopausal breast cancer risks.

Additionally, results obtained by Boccia et al. (2005), proved that SULTA1Arg213Hisgene (encoding sulfotransferase) polymorphism is a potential marker for identifying gastric cancer individuals at a high-risk, and useful in defining who may benefit from specific chemopreventive interventions. Overview Of The Biological Activities Of Isoflavones Essay.

Proposed mechanisms for cancer prevention

Isoflavonoids have been shown to possess many biological properties that may account for cancer prevention. Isoflavones exert their effects through numerous pathways and with respect to cancer prevention, they use mechanisms of action which appear to be various, complementary and/or overlapping.

Some mechanisms of action have been identified for isoflavone/flavone prevention of cancer, including estrogenic/antiestrogenic activity, antiproliferation, induction of cell cycle arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regulation of host immune system and changes in cellular signaling (Birt at al., 2001; Murata et al., 2006; Chrzan and Bradford, 2007). It is expected that also combinations of these mechanisms may contribute to cancer prevention.

Dysregulated proliferation appears to be a hallmark of an incerased susceptibility to neoplasia. Cancer prevention is generally associated with inhibition, reversion or retardation of cellular hyperproliferation. It is known that dietary isoflavonoids may behave as general cell inhibitors. Although most isoflavonoids appear to be non-toxic to humans and animals, they have been shown to inhibit proliferation, the cell cycle or induce apoptosis in many kinds of cultured cancer cell lines. It was thus found that check points at both G1/S and G2/M of the cell cycle were perturbed by isoflavonoids (Birt at al., 2001).

Soy-protein-derived isoflavonoids have antiproliferative and proapoptic effects on prostatic epithelial cells in vitro and on the prostate gland of macaques, preventing prostate cancer development and/or progression in animal populations consuming soy protein (Hedlund et al., 2005; Perry et al., 2007). Overview Of The Biological Activities Of Isoflavones Essay. Activation of Erβ is supposed to result in decreased proliferation due to the receptor expression loss because Erβ probably functions as tumor suppressor within the prostate gland. The Erβ expression loss in prostatic adenocarcinoma was shown to result from an epigenetic effect – gene silencing caused by promoter methylation (Perry et al., 2007). Gene silencing due to promoter methylation provides an opportunity for clinical intervention, as gene-re-expression can be induced by a variety of DNA demethylating agents.

Apoptosis, i.e. natural programed cell death, is a physiological phenomenon indispensable for normal functioning of the organisms. The signal to apoptosis can be started practically in any cell. Disturbance in apoptosis regulation determines the essential link in the pathogenesis of many diseases, including cancer. Recent studies have shown, that genistein has multiple effects because it induces apoptosis also by mechanisms that do not involve ERs, as demonstrated in HepG2 cells, which do not express ERs (Chodon et al., 2007; Ørgaard and Jensen, 2008).

On trying to explain molecular mechanisms of apoptosis induction in human leukemia HL-60 cells by isoflavonoids from the leaves of Milettia taiwaniana, Murata et al. (2006) and Ito et al. (2006) found that isoflavonoids induced apoptosis through activation of the caspase-9/caspase-3 pathway which is triggered by mitochondrial dysfunction.

The biological mechanism by which phytoestrogens, particularly in the Western diet, can protect against hormone-dependent breast cancer is likely through their competitive effects on the generation, transport and removal of endogenous steroid hormones (Piller et al., 2006). By competing for estrogen receptors, phytoestrogens possibly inhibit binding of the more potent endogenous estrogens and decrease their potential effects on breast cancer risk (Verheus et al., 2007). They may inhibit also proliferation of hormone-independent breast cell lines via a number of mechanisms, including inhibition and down-regulation of protein tyrosine kinases, which are involved in growth signaling pathways. Genistein was shown to inhibit tyrosine kinase, particularly the autophosphorylation and activation of epidermal growth factor receptor, which is important in regulating apoptosis and cell proliferation. Genistein inhibited protein tyrosine kinase-dependent transcription of c-fos and subcellular proliferation in estrogen receptor negative human breast cancer cell lines. Key enzymes implicated in cancer invasion are also affected by genistein (Kousidou et al., 2006). Overview Of The Biological Activities Of Isoflavones Essay.

Changes in cell signaling were demonstrated in the HC11 mouse mammary epithelial cell line. Soy isoflavone genistein upregulated epithelial adhesion molecule E-cadherin expression and attenuated β-catenin signaling in mammary epithelial cells. In addition, it diminished basal and Wnt-1-(wingless)-induced cell proliferation and attenuated Wnt-1 targets c-Myc and cyclin D1 expression (Su and Simmen, 2008).

Mammographic density as a biomarker for breast cancer risk

Isoflavonoids have been increasingly advocated as potential natural alternatives to hormone replacement therapy. There are however only limited data on the effect of isoflavones on breast density, despite the fact that isoflavonoids were proved to be present in breast fluids and could directly act on breast tissue (Maskarinec et al., 2008a).

Although isoflavones have been suggested to protect against breast cancer, it is still not clear whether they act as estrogens or antiestrogens in breast tissue. Mammographic breast density, representing the amount of stromal and glandural tissue within the breast, has consistently been associated with a risk for breast cancer and a high density is often used as a predictive biomarker for breast cancer risk (Maskarinec et al., 2003). The mechanism underlying this relationship has not been fully explained, but it has been proposed that breast cancer density provides an index of current and past hormonal and reproductive events that modulate the risk for breast cancer. A high mammographic density has been associated with a 3- to 6-fold increase in breast cancer risk. Only few intervention studies with isoflavones and mammographic density have been published. In studies performed in premenopausal and postmenopausal women no effect of isoflavones from soy or other sources on mammographic density was shown (Maskarinec et al., 2003; Maskarinec et al., 2009). Overview Of The Biological Activities Of Isoflavones Essay. Neither did intervention studies with phytoestrogens from red clover or black cohosh show any statistically significant effects on mammographic density in postmenapausal women (Hirschberg et al., 2007). The findings in a 1-year double-blind, randomized, placebo-controlled trial did not support any effect (beneficial or adverse) of a large quantity of soy proteins containing isoflavones on mammographic density in postmenopausal women. The results of this trial do not support the hypothesis that a diet high in soy protein would decrease mammographic density in postmenopausal women (Verheus et al., 2008).

Although it has been hypothesized that breast density reflexes cumulative exposure to estrogens, in postmenopausal women the relationship between phytoestrogens and mammographic density may be different due to the decline of circulating levels of endogenous hormones after menopause. Yet examination of the relation between circulating sex hormones and mammographic density showed that in postmenapausal women mammographic density was not dependent on circulating sex hormone levels (Tamini et al., 2005; Elliassen et al., 2008).

Atkinson et al. (2004) did not observe a significant effect of clover-derived isoflavone supplement, taken daily for 1 year, on mammographic density in women aged 49–65 years, unlike conventional estrogen replacement therapies. Furhermore, they did not find any effect of the isoflavone supplement on estradiol, follicle-stimulating hormone or luteinizing hormone in postmenopausal women, or on hot flushes or other menopausal symptoms. A study aimed at soy isoflavonoid effects on endogenous estrogen metabolism in postmenopausal female monkeys showed that long-term exposure to soy isoflavonoids did not significantly alter gene markers of estrogen metabolism or estrogen receptor agonists in breast tissue. This long-term exposure may facilitate endogenous estrogen clearance and catabolism to more benign 2-hydroxylated metabolites (Wood et al., 2007).

Combined hormone replacement therapy may be associated with an increased risk for breast cancer and an increase in mammographic breast density. Breast cancer density increases when a woman starts on hormone replacement therapy and decreases when she discontinues it. Breast density is also reduced by antiestrogenic effect of tamoxifen, a selective estrogen receptor modulator.

Overall, recent epidemiologic and clinical data showed either a modest protective role or no effect of isoflavone intake on breast tissue density in pre- or postmenopausal women and on breast proliferation in postmenopausal women with or without a history of breast cancer (Messina and Wood, 2008). Overview Of The Biological Activities Of Isoflavones Essay.

Modulation of immune function

The isoflavone daidzein administered orally stimulated murine nonspecific immunity, activated hormoral immunity, enhanced cell-mediated immunity, and at physiologically relevant concentrations potentiated lymphocyte activation, suggesting that its immunostimulatory effects may be involved in cancer prevention (Birt et al., 2001).

Equol is known to protect against solar-simulated UV radiation induced inflammation and immunosuppression. Two protective mechanisms are responsible: antioxidant actions and phytoestrogenicity. It is supposed that both might be functionally linked (Widyarini et al., 2006).

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The role of the host immune function has become increasingly important in our understanding of the mechanisms involved in cancer prevention. The human immune system encompasses an array of defenses that help to guard against the development of age-related diseases, e.g. cancer. However, its function can be adversely affected by hormonal changes and oxidative damage (Kralova et al., 2008). The immune system may be compromised mainly after menopause because of the effects of aging and diminishing concentration of estrogen, an immune-modulation hormone. The human immune system may benefit from various biological properties of isoflavonoids. Since postmenopausal women are in particular susceptible to chronic disease associated with aging and to major shifts in hormonal status, isoflavones with estrogenic and antioxidant properties may offer immunologic benefits to women during this stage of life. Ryan-Borchers et al. (2006) reported that soy isoflavones stimulated an increase of important markers of immunity – B cells in healthy postmenopausal women.

Although soy isoflavones have been suggested to have both immune-enhancing and immune-suppressive effects, their effects on allergic disorders are less clear. Nagata et al. (2008) showed that a high intake of soy isoflavones was associated with decreased risk of cedar pollinosis, the most common seasonal allergic rhinitis. Overview Of The Biological Activities Of Isoflavones Essay.