**3. Biological basis of soybean phytoestrogen actions**

The estrogenic effect of the isoflavones was first recognized when examining impaired fertility in grazing animals (Bennetts et al., 1946). Three decades later, Setchell et al. (1987) established that isoflavone-rich soy was a factor in reduced fertility of cheetahs in North American zoos. Isoflavones were classified as phytoestrogens following in vivo and in vitro demonstration of their binding potency of isoflavones for estrogen receptors (ER), as well as for sex-hormone binding globuline (Kuiper et al., 1998).

Testosterone actions in numerous male tissues are mediated through its conversion to estrogen catalyzed by aromatase enzymes. Specific and β ER are detected in different male and female tissues (Korach, 1994), but the ratio between ERα and ERβ is different (Rosen, 2005). This finding has finally changed the classical view of the estrogens as exclusively female hormones. ERβ is known to modulate ERα transcriptional activity acting as an activator at low concentrations of mammalian estrogen - estradiol 17β (E2) and as an

Soybean Phytoestrogens – Friends or Foes? 135

Growing evidence shows that isoflavones may also modulate the activity/expression of steroidogenic enzymes. These enzymes are present in the adrenal glands and gonads but also in many tissues that have the ability to convert circulating precursors into active hormones (i.e. brain, liver, reproductive tracts, adipose tissue, skin and breast tissue). Genistein and daidzein were reported to inhibit the activity of 3β- hydroxysteroid dehydrogenases (HSD) purified from bovine adrenal microsomes (Wong & Keung, 1999). The same isoflavones were also shown to inhibit 3β-HSD type II in mitochondrial and microsomal preparations of the human adrenocortical H295R cell line, and subsequently a similar inhibition of the conversion of dehydroepiandrosterone (DHEA) to androstenedione by these isoflavones was observed in total membrane fractions of Sf9 insect cells in which human 3β-HSD had been over-expressed (Ohno et al., 2002). However, Mesiano et al. (1999) showed that genistein and daidzein specifically inhibited the activity of 21-hydroxylase (P450c21/CYP 21) in H295 cells but had no effect on other steroidogenic enzymes, including

**4. Soybean phytoestrogens in prevention and therapy of cancer** 

The incidence of hormon-dependent cancers, namely breast and prostate, is lower in Asia than in western countries (Messina et al., 2006; Parkin, 2005). Migrants from Asia, who maintained their traditional diet, even when living in the West, had a lower risk of these diseases. However, shifting towards a more of a western diet increased the risk (Ziegler et al., 1993). Once SERM properties of soybean isoflavones were discovered, it was hipothesized that high soy dietary intake might be associated with low incidence of hormone-dependent cancers in Asian population, as well as with other putative health benefits (Setchell, 1999). That is why soyfood and its isoflavones in a form of dietary supplements or concentrated extracts have been increasingly used in the western

However, when Patisaul and Jefferson (2010) disscussed potential safety of infant soy formula, they stressed the essential difference between Asians (on a traditional "soy-reach" diet) and Caucasians (on a traditional "Western" diet) in exposure to soy over the lifespan. In Asia, soy consumption is high during entire lifespan, except for a brief breast-feeding period in early infancy. People in the West feed their babies soy infant formula, so the pattern is just the opposite - the highest intake of isoflavones occurs in the first year of life and then drop to near zero, with eventual increase later in advanced adult age. In relation to this, some authors support the opinion that lower incidence of breast cancer in Asian women is due to their continous exposure to soy from early life throuought their whole lifespan (Warri et al., 2008). Maskarinec et al. (2004) concluded that Caucasian women who ate more soy during their lifespan had denser breast tissue (a risk factor for breast cancer)

Overexposure to estrogen (early menarche, short duration of breastfeeding and low parity) is a major contributing factor in the development of breast cancer. As soybean isoflavones have a relatively high binding potency for ERs, a concern has been raised that high phytoestrogen intake may promote growth of estrogen-sensitive tumors or put breast cancer

survivors at risk of reoccurrence (Helferich et al., 2008; Messina &Loprinzi, 2001).

3β-HSD.

populations in the recent years.

than those who did not.

**4.1 Effects on breast cancer** 

inhibitor at high concentrations of E2. E2 has equal binding affinities for ERα and ERβ, while isoflavones have a higher potency for ERβ.

The molecular structures of genistein, daidzein and E2 are similar in many aspects. The intra-molecular distance between the hydroxyl groups at each end of the molecules is almost identical for both isoflavones and E2. These distances determine hydrogen bond interaction with amino acids of the ligand-binding site of the ER (Vaya and Tamir, 2004). Though molecular binding for ER between isoflavones and E2 are similar, both G and D binding potency for ERs is significantly lesser in comparison to E2. In addition, they bind with higher potency to estrogen receptor (ER) β in comparison to ERα (Kuiper et al., 1998). These features classify them as potential natural selective estrogen receptor modulators (Phyto SERMs). Thus, soybean isoflavones may exert estrogenic, antiestrogenic, or estrogen non-reactive biological actions, depending on their concentration and concentration of endogenous estrogen, tissue, and amount and type of estrogen receptors present in the tissue (Wuttke et al. 2007). Therefore, it is of importance to determine the estrogenic action of isoflavones compared with the effects of E2 (in females) and both testosterone and estradiol (in males) in each individual organ.

Phyto SERMs represent a new and very promising class of potential hormonal therapy agents. Major potential advantage of SERMs over estrogen analogue therapy is that it may demonstrate all of the favorable effects of estrogens. However, in order to declare isoflavones as safe, it needs to be demonstrated that they do not share the risks associated with estrogens used in hormone replacement therapy, osteoporosis treatment or in treatment of prostate carcinoma (Wuttke et al., 2007).

Besides their estrogenic activities, isoflavones also exhibit non-hormonal actions such as antioxidant effects. Antioxidant properties are one of the most important claims for food ingredients, dietary supplements and anticancer products. In addition, the free radical theory of aging continues to be among the most popular theories. Therefore, the antioxidant property of isoflavones offers an additional important mechanism through which they protect against age-related diseases. All soy isoflavones act as antioxidants, playing role in scavenging free radicals that can cause DNA damage and lipid peroxidation (Kruk et al., 2005) and activate antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase (Mitchell et al. 1998). The determining factors for isoflavone antioxidant activities are the absence of the 2, 3-double bond and the 4-oxo-group on the isoflavone nucleus and the position of the hydroxyl groups, with hydroxyl substitution being of utmost importance at the 4' position, of moderate importance at the 5 position, and of little significance at the 7 position. That is why G has higher antioxidant capacity than daidzein and the reason why both have stronger antioxidant activity than their glycosides (Cherdshewasart and Sutjit, 2008).

Genistein in high concentration is a potent inhibitor of Tyr kinases (Akiyama et al., 1987), DNA topoisomerases I and II, and ribosomal S6 kinase, resulting in inhibition of cell growth. Tyrosine kinases are responsible for <1% of protein phosphorylation within cells, but they appear to phosphporylate many proteins required for regulation of cell functions. Genistein has been shown to induce cell cycle arrest and apoptosis in numerous cell lines, including ER (+) and ER (-). Many of the reported beneficial effects of isoflavones and particularly those on tumor growth may be attributed to this mechanism. However, some authors critycize this by underlying that the concentrations necessary for such inhibition in the tested cell systems or organs by far exceed the serum concentrations achieved by isoflavone ingestion alone (Jiménez and Montiel 2005; Wuttke et al., 2007).

inhibitor at high concentrations of E2. E2 has equal binding affinities for ERα and ERβ,

The molecular structures of genistein, daidzein and E2 are similar in many aspects. The intra-molecular distance between the hydroxyl groups at each end of the molecules is almost identical for both isoflavones and E2. These distances determine hydrogen bond interaction with amino acids of the ligand-binding site of the ER (Vaya and Tamir, 2004). Though molecular binding for ER between isoflavones and E2 are similar, both G and D binding potency for ERs is significantly lesser in comparison to E2. In addition, they bind with higher potency to estrogen receptor (ER) β in comparison to ERα (Kuiper et al., 1998). These features classify them as potential natural selective estrogen receptor modulators (Phyto SERMs). Thus, soybean isoflavones may exert estrogenic, antiestrogenic, or estrogen non-reactive biological actions, depending on their concentration and concentration of endogenous estrogen, tissue, and amount and type of estrogen receptors present in the tissue (Wuttke et al. 2007). Therefore, it is of importance to determine the estrogenic action of isoflavones compared with the effects of E2 (in females) and both testosterone and

Phyto SERMs represent a new and very promising class of potential hormonal therapy agents. Major potential advantage of SERMs over estrogen analogue therapy is that it may demonstrate all of the favorable effects of estrogens. However, in order to declare isoflavones as safe, it needs to be demonstrated that they do not share the risks associated with estrogens used in hormone replacement therapy, osteoporosis treatment or in

Besides their estrogenic activities, isoflavones also exhibit non-hormonal actions such as antioxidant effects. Antioxidant properties are one of the most important claims for food ingredients, dietary supplements and anticancer products. In addition, the free radical theory of aging continues to be among the most popular theories. Therefore, the antioxidant property of isoflavones offers an additional important mechanism through which they protect against age-related diseases. All soy isoflavones act as antioxidants, playing role in scavenging free radicals that can cause DNA damage and lipid peroxidation (Kruk et al., 2005) and activate antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase (Mitchell et al. 1998). The determining factors for isoflavone antioxidant activities are the absence of the 2, 3-double bond and the 4-oxo-group on the isoflavone nucleus and the position of the hydroxyl groups, with hydroxyl substitution being of utmost importance at the 4' position, of moderate importance at the 5 position, and of little significance at the 7 position. That is why G has higher antioxidant capacity than daidzein and the reason why both have stronger antioxidant activity than

Genistein in high concentration is a potent inhibitor of Tyr kinases (Akiyama et al., 1987), DNA topoisomerases I and II, and ribosomal S6 kinase, resulting in inhibition of cell growth. Tyrosine kinases are responsible for <1% of protein phosphorylation within cells, but they appear to phosphporylate many proteins required for regulation of cell functions. Genistein has been shown to induce cell cycle arrest and apoptosis in numerous cell lines, including ER (+) and ER (-). Many of the reported beneficial effects of isoflavones and particularly those on tumor growth may be attributed to this mechanism. However, some authors critycize this by underlying that the concentrations necessary for such inhibition in the tested cell systems or organs by far exceed the serum concentrations achieved by

isoflavone ingestion alone (Jiménez and Montiel 2005; Wuttke et al., 2007).

while isoflavones have a higher potency for ERβ.

estradiol (in males) in each individual organ.

treatment of prostate carcinoma (Wuttke et al., 2007).

their glycosides (Cherdshewasart and Sutjit, 2008).

Growing evidence shows that isoflavones may also modulate the activity/expression of steroidogenic enzymes. These enzymes are present in the adrenal glands and gonads but also in many tissues that have the ability to convert circulating precursors into active hormones (i.e. brain, liver, reproductive tracts, adipose tissue, skin and breast tissue). Genistein and daidzein were reported to inhibit the activity of 3β- hydroxysteroid dehydrogenases (HSD) purified from bovine adrenal microsomes (Wong & Keung, 1999). The same isoflavones were also shown to inhibit 3β-HSD type II in mitochondrial and microsomal preparations of the human adrenocortical H295R cell line, and subsequently a similar inhibition of the conversion of dehydroepiandrosterone (DHEA) to androstenedione by these isoflavones was observed in total membrane fractions of Sf9 insect cells in which human 3β-HSD had been over-expressed (Ohno et al., 2002). However, Mesiano et al. (1999) showed that genistein and daidzein specifically inhibited the activity of 21-hydroxylase (P450c21/CYP 21) in H295 cells but had no effect on other steroidogenic enzymes, including 3β-HSD.
