**6. Soybean phytoestrogens in bone protection**

Bone remodeling is a continuous process of bone resorption and bone formation for the purpose of maintaining normal bone mass. As a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissues, osteoporosis is usually caused by a chronic imbalance in the bone remodeling cycle. This skeletal disorder occurs as part of the natural aging process and is associated with the rapid decline in ovarian function and subsequent reduction of circulating estrogen in women after the menopause and declining testosterone in middle-aged and older men. However, in contrast to postmenopausal osteoporosis in women, the age related bone loss in men is less well-defined. Observational studies have indicated that estrogen administration is important in bone remodeling. Thus, hormone replacement therapy (HRT) administered in a dose-dependent manner, not only significantly reduces bone loss, but also lowers the incidence of hip and vertebral fractures (Lindsay et al., 1976, 1984; Michaelsson et al., 1998*).* In the other hand, although HRT has a protective effect on bone tissue, it can increase the risk of breast, endometrial ovarian or prostate cancer developing (Davison & Davis, 2003; Loughlin & Richie, 1997; Nelson et al., 2002). For this reason, much attention has been paid to the examination of alternative therapeutic compounds that may have protective effects on bone, without adverse effects on other tissues. Epidemiological studies have demonstrated a low incidence of postmenopausal fractures and high bone mineral density (BMD) in Asian populations with a particularly soy-rich diet (Cooper et al., 1992; Lauderdale et al., 1997; Somekawa et al., 2002). Thus, phytoestrogens have been proposed as an alternative to conventional hormone therapy for preventing osteoporosis and have shown beneficial effects on bone health (Barnes, 2003; Morin, 2004).

Bone remodeling is regulated by the activity of two different cell lines. Osteoblasts stimulate bone formation and calcification, while osteoclasts promote bone resorption. It has been shown that isoflavones affect osteoblastic bone formation and osteoclastic bone resorption in vitro. The anabolic effects of genistein and daidzein on bone metabolism have been investigated in culture using femoral trabecular and cortical bone tissues obtained from elderly female rats (Gao & Yamaguchi, 1999; Yamaguchi & Gao, 1997, 1998). Genistein induced a significant increase in calcium content, alkaline phosphatase activity as a marker of osteoblasts, as well as DNA content, which is an index of bone cell numbers in bone tissues (Yamaguchi & Gao, 1997). In bone tissue culture medium daidzein significantly elevated bone components (Gao & Yamaguchi, 1999). Both genistein and daidzein increased newly synthesized protein content, alkaline phosphatase activity and DNA content in cultures of osteoblastic MC3T3-E1 cells (Sugumoto & Yamaguchi, 2000, 2000a; Yamaguchi & Sugumoto, 2000).

NO production causes relaxation of artherial myocites (Mahn et al., 2005). Research on the effect of genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium-dependent vasodilatation in postmenopausal women revealed that genistein therapy improved flow-mediated endothelium-dependent vasodilatation in healthy postmenopausal women. This improvement is probably mediated by a direct effect of genistein on vascular function and could be the result of an increased ratio of nitric oxide to

In conclusion, despite the fact that dietary soy products and isoflavones are heavily advertised for their hypolipidemic effect, their therapeutic potential is lesser than was previously hoped and depend on many factors related to inter-individual differences.

Bone remodeling is a continuous process of bone resorption and bone formation for the purpose of maintaining normal bone mass. As a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissues, osteoporosis is usually caused by a chronic imbalance in the bone remodeling cycle. This skeletal disorder occurs as part of the natural aging process and is associated with the rapid decline in ovarian function and subsequent reduction of circulating estrogen in women after the menopause and declining testosterone in middle-aged and older men. However, in contrast to postmenopausal osteoporosis in women, the age related bone loss in men is less well-defined. Observational studies have indicated that estrogen administration is important in bone remodeling. Thus, hormone replacement therapy (HRT) administered in a dose-dependent manner, not only significantly reduces bone loss, but also lowers the incidence of hip and vertebral fractures (Lindsay et al., 1976, 1984; Michaelsson et al., 1998*).* In the other hand, although HRT has a protective effect on bone tissue, it can increase the risk of breast, endometrial ovarian or prostate cancer developing (Davison & Davis, 2003; Loughlin & Richie, 1997; Nelson et al., 2002). For this reason, much attention has been paid to the examination of alternative therapeutic compounds that may have protective effects on bone, without adverse effects on other tissues. Epidemiological studies have demonstrated a low incidence of postmenopausal fractures and high bone mineral density (BMD) in Asian populations with a particularly soy-rich diet (Cooper et al., 1992; Lauderdale et al., 1997; Somekawa et al., 2002). Thus, phytoestrogens have been proposed as an alternative to conventional hormone therapy for preventing osteoporosis and have shown beneficial effects on bone health

Bone remodeling is regulated by the activity of two different cell lines. Osteoblasts stimulate bone formation and calcification, while osteoclasts promote bone resorption. It has been shown that isoflavones affect osteoblastic bone formation and osteoclastic bone resorption in vitro. The anabolic effects of genistein and daidzein on bone metabolism have been investigated in culture using femoral trabecular and cortical bone tissues obtained from elderly female rats (Gao & Yamaguchi, 1999; Yamaguchi & Gao, 1997, 1998). Genistein induced a significant increase in calcium content, alkaline phosphatase activity as a marker of osteoblasts, as well as DNA content, which is an index of bone cell numbers in bone tissues (Yamaguchi & Gao, 1997). In bone tissue culture medium daidzein significantly elevated bone components (Gao & Yamaguchi, 1999). Both genistein and daidzein increased newly synthesized protein content, alkaline phosphatase activity and DNA content in cultures of osteoblastic MC3T3-E1 cells (Sugumoto & Yamaguchi, 2000, 2000a; Yamaguchi &

endothelin (Squadrito et al., 2002).

(Barnes, 2003; Morin, 2004).

Sugumoto, 2000).

**6. Soybean phytoestrogens in bone protection** 

In addition to effects on osteoblasts, many authors have reported that isoflavones are efficacious in suppressing osteoclast activity in vitro. Genistein completely inhibited bone resorption and osteoclast-like multinucleated cells in culture with bone-resorbing factors (Gao & Yamaguchi, 1999a; Yamaguchi & Gao, 1998a). Also, daidzein inhibited the development of osteoclasts from cultures of porcine bone marrow and reduced bone resorption (Rassi et al., 2002).

While in vitro studies reveal possible actions of isoflavones on individual bone cells, in vivo studies provide insight into the effects of isoflavones on the intact system and coupling effects between osteoblasts and osteoclasts. Most of the animal bone studies investigating isoflavone action have been performed in rodents. Aged ovariectomized female and orchidectomized male rats represent a suitable model for simulating osteoporosis due to estrogen or androgen deficiency (Comelekoglu et al., 2007; Filipović et al., 2007; Pantelić et al., 2010; Turner, 2001; Vanderschueren et al., 1992). Using this animal model, supplementation with isoflavones has been shown to prevent bone loss (Fig. 2) induced by gonadal hormone deficiency (Filipović et al., 2010; Khalil et al., 2005; Lee et al., 2004; Om & Shim, 2007; Ren et al., 2007; Soung et al., 2006). In a randomized placebo controlled trial with estrogen and phytoestrogen on ovariectomized nonhuman primates, Ham et al. (2004) failed to show any efficacy of soy phytoestrogens in decreasing all indices of bone turnover as estrogen does, but soy phytoestrogens were able to increase bone volume, trabecular number and decrease trabecular separation, stressing the importance of phytoestrogens in postmenopausal osteoporosis prevention.

Fig. 2. Trabecular bone microarchitecture of the proximal tibia in control orchidectomized (a) and daidzein-treated orchidectomized (b) rat; аzan staining method; unpublished image of Filipovic et al.

Phytoestrogens may elicit a bone sparing effect by both genomic and nongenomic mechanisms. They are able to interact with enzymes and receptors and, their stable structure and low molecular weight enables them to pass through cell membranes (Adlercreutz et al., 1998). The structural similarity of phytoestrogens to mammalian estrogens and their ability to bind to estrogen receptors (Setchell et al., 1999) suggests that the actions of phytoestrogens are mediated via estrogen receptors. ERα and ERβ have been detected in bone (Arts et al., 1997; Onoe et al., 1997). The relative binding affinity of phytoestrogens for ERβ is greater than that for ERα, and the protective effect of phytoestrogens on bone is probably produced through binding to estrogen receptors, particularly ERβ (Kuiper et al., 1998). In addition, phytoestrogens such as coumestrol, genistein and daidzein increase alkaline phosphatase activity in osteoblast-like cells (Kanno et al., 2004). Daidzein stimulates

Soybean Phytoestrogens – Friends or Foes? 145

slowly over time, in short-term intervention studies this change may represent a transient remodeling rather than a long-term steady-state. In addition, a favorable effect on the spine BMD was achieved with large doses of isoflavones (≥ 80 mg/day, median 99 mg/day), but not with lower doses (< 80 mg/day, median 60 mg/day). Thus, the potential of soy isoflavones to prevent bone loss can be achieved by a dosage of 80 mg/day (Huang et al.,

Finally, a wealth of supporting data from many in vitro mechanistic studies on bone cell lines and in vivo investigations using models of osteoporosis shows bone-sparing effects from phytoestrogens. These studies indicate that positive effects of phytoestrogens, as a SERM, may be achieved through estrogen receptors or other mechanisms. However, the results of clinical studies are more inconsistent. The different efficacy of phytoestrogen treatments, in studies involving either animal or human subjects, depended on dose, route and duration of administration. The data are, however, rather tantalizing because it is possible that soy isoflavones may offer the maximum benefit for prevention of osteoporosis. Therefore, it is necessary to perform large-scale clinical dietary intervention studies with

Endocrine systems of vertebrates have essential role in regulation of growth (including bone growth/remodeling), reproduction, stress, lactation, metabolism, energy balance, osmoregulation, and all other processes involved in maintaining homeostasis. Disruption in function of any endocrine system, involving either increased or decreased hormone secretion, result inevitably in disease, the effects of which may extend to many different

An endocrine-disrupting compound (EDC) is defined by the U.S. Environmental Protection Agency (EPA) as "an exogenous agent that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental process." All hormone-sensitive physiological systems are vulnerable to EDCs, including brain and hypothalamic neuroendocrine systems; pituitary; thyroid; adrenal gland; cardiovascular system; mammary gland; adipose tissue; pancreas; ovary and uterus in females; and testes

The exposure to such chemicals does not necessarily mean that disturbance of the relevant endocrine system will occur, as much depends on the level, duration and timing of exposure. However, even subtle changes, however small, in combination and/or under different conditions and/or in later generations might reduce the ability of humans (animals) to adapt. It may also happen that the magnitude of the disruption becomes

It is beyond the scope of this chapter to discuss the potential interference of soy isoflavones with all endocrine organs; instead, the focus will be on three major endocrine axes that are affected by soybean phytoestrogens: pituitary – gonadal, -thyroid and - adrenocortical

Soybean isoflavones are ligands for both ERα and ERβ, despite the fact that their estrogenic potency is much lower than that of E2. Therefore, they can mimic and/or antagonize the

phytoestrogens to determine their effects on bone tissue in humans.

organs and functions, and may even be life-threatening.

evident only in presence of an additional stress factor.

**7.1 Effects on female reproductive system** 

and prostate in males.

systems.

**7. Soybean phytoestrogens as potential endocrine disruptors** 

2006).

osteoblast differentiation, induces changes in the action of the cytoskeleton responsible for cell adhesion and motility and activates transcription factors associated with cell proliferation and differentiation (de Wilde et al., 2004, Ge et al., 2006; Jia et al., 2003). Also, isoflavones promote insulin-like growth factor-I (IGF-I) production which enhances osteoblastic activity (Ajrmandi et al., 2000)

Isoflavones inhibit bone resorption, via direct targeting of osteoclasts. They can decrease differentiation and increase apoptosis of osteoclasts or interfere with signaling pathways such as intracellular calcium, cAMP or protein kinase and protein tyrosine phosphatase (Gao & Yamaguchi, 2000; Sliwinski et al, 2005). Furthermore, osteoblasts are essential for in vitro osteoclastogenesis through cell-to-cell interactions of cytokines. Isoflavones regulate the expression and osteoblastic production of osteoclastogenesis-regulatory cytokines, such as interleukin-6 (IL-6), which stimulates osteoclast formation, and osteoprotegerin (OPG), which is identical to osteo-clastogenesis inhibitory factor, and the receptor activator of NFκB ligand (Chen et al., 2002).

In addition to ERs, it has been shown that PPAR are new targets of phytoestrogens. PPAR directly influences osteogenesis and adipogenesis in a divergent way (Dang & Lowik, 2005). These authors suggested that biphasic dose-dependent effects of phytoestrogens are the result of concurrent activation of ERs and PPARs. Dominant ER-mediated effects that increase osteogenesis and decrease adipogenesis can only be seen at low concentrations of phytoestrogens, whereas dominant PPAR-mediated effects that decrease osteogenesis and increase adipogenesis are only evident at high concentrations.

Calcitonin (CT), a hormone secreted from thyroid C cells is known to inhibit osteoclast activity directly through its receptors (Nicholson et al., 1986). It was shown that synthesis and release of CT from thyroid C cells decreased after ovariectomy in rats, due to lack of estrogens (Filipović et al., 2002; Sakai et al., 2000). On the other hand, estrogen treatment had a stimulatory effect on CT secretion in ovariectomized rats (Filipović et al., 2003; Grauer et al., 1993). However, chronic Ca treatment of ovariectomized rats positively affected CT release without any significant changes in morphometric parameters of the C cells, suggesting an important role for estrogen in the regulation of CT synthesis (Filipović et al., 2005). Exogenous CT administration was reported to inhibit CT secretion in rats and therefore CT treatment probably suppresses C cell function due to a negative feedback (Sekulić et al., 2005). Recently, daidzein was found to stimulate CT secreting thyroid C cell activity in addition to increasing trabecular bone mass and decreasing bone turnover (Filipović et al., 2010). These results suggest that, besides direct action, daidzein may affect bone structure indirectly through enhancement of thyroid C cell activity.

Although animal studies demonstrate a clear skeletal benefit of phytoestrogens, clinical trials have given different results. Soy isoflavones were observed to retard bone loss in some (Huang et al., 2006; Newton et al., 2006), but not in other studies (Arjmandi et al., 2005; Brink et al., 2008). To date, only one study indicated that supplementation of intact soy protein providing 83 mg isoflavones daily might increase both hip and spine BMD in men (Newton et al., 2006). Also, the results of meta-analyses of soy foods and isoflavones extracted from soy protein have given conflicting results concerning the prevention of bone loss (Liu et al., 2009; Ma et al., 2008). The large heterogeneity in these conclusions might have arisen because many results were pooled from different individual studies, involving different treatment durations, different doses of soy isoflavone and study quality (Liu et al., 2009). These authors suggested that, because changes in bone mineral density (BMD) occur

osteoblast differentiation, induces changes in the action of the cytoskeleton responsible for cell adhesion and motility and activates transcription factors associated with cell proliferation and differentiation (de Wilde et al., 2004, Ge et al., 2006; Jia et al., 2003). Also, isoflavones promote insulin-like growth factor-I (IGF-I) production which enhances

Isoflavones inhibit bone resorption, via direct targeting of osteoclasts. They can decrease differentiation and increase apoptosis of osteoclasts or interfere with signaling pathways such as intracellular calcium, cAMP or protein kinase and protein tyrosine phosphatase (Gao & Yamaguchi, 2000; Sliwinski et al, 2005). Furthermore, osteoblasts are essential for in vitro osteoclastogenesis through cell-to-cell interactions of cytokines. Isoflavones regulate the expression and osteoblastic production of osteoclastogenesis-regulatory cytokines, such as interleukin-6 (IL-6), which stimulates osteoclast formation, and osteoprotegerin (OPG), which is identical to osteo-clastogenesis inhibitory factor, and the receptor activator of NF-

In addition to ERs, it has been shown that PPAR are new targets of phytoestrogens. PPAR directly influences osteogenesis and adipogenesis in a divergent way (Dang & Lowik, 2005). These authors suggested that biphasic dose-dependent effects of phytoestrogens are the result of concurrent activation of ERs and PPARs. Dominant ER-mediated effects that increase osteogenesis and decrease adipogenesis can only be seen at low concentrations of phytoestrogens, whereas dominant PPAR-mediated effects that decrease osteogenesis and

Calcitonin (CT), a hormone secreted from thyroid C cells is known to inhibit osteoclast activity directly through its receptors (Nicholson et al., 1986). It was shown that synthesis and release of CT from thyroid C cells decreased after ovariectomy in rats, due to lack of estrogens (Filipović et al., 2002; Sakai et al., 2000). On the other hand, estrogen treatment had a stimulatory effect on CT secretion in ovariectomized rats (Filipović et al., 2003; Grauer et al., 1993). However, chronic Ca treatment of ovariectomized rats positively affected CT release without any significant changes in morphometric parameters of the C cells, suggesting an important role for estrogen in the regulation of CT synthesis (Filipović et al., 2005). Exogenous CT administration was reported to inhibit CT secretion in rats and therefore CT treatment probably suppresses C cell function due to a negative feedback (Sekulić et al., 2005). Recently, daidzein was found to stimulate CT secreting thyroid C cell activity in addition to increasing trabecular bone mass and decreasing bone turnover (Filipović et al., 2010). These results suggest that, besides direct action, daidzein may affect

Although animal studies demonstrate a clear skeletal benefit of phytoestrogens, clinical trials have given different results. Soy isoflavones were observed to retard bone loss in some (Huang et al., 2006; Newton et al., 2006), but not in other studies (Arjmandi et al., 2005; Brink et al., 2008). To date, only one study indicated that supplementation of intact soy protein providing 83 mg isoflavones daily might increase both hip and spine BMD in men (Newton et al., 2006). Also, the results of meta-analyses of soy foods and isoflavones extracted from soy protein have given conflicting results concerning the prevention of bone loss (Liu et al., 2009; Ma et al., 2008). The large heterogeneity in these conclusions might have arisen because many results were pooled from different individual studies, involving different treatment durations, different doses of soy isoflavone and study quality (Liu et al., 2009). These authors suggested that, because changes in bone mineral density (BMD) occur

increase adipogenesis are only evident at high concentrations.

bone structure indirectly through enhancement of thyroid C cell activity.

osteoblastic activity (Ajrmandi et al., 2000)

κB ligand (Chen et al., 2002).

slowly over time, in short-term intervention studies this change may represent a transient remodeling rather than a long-term steady-state. In addition, a favorable effect on the spine BMD was achieved with large doses of isoflavones (≥ 80 mg/day, median 99 mg/day), but not with lower doses (< 80 mg/day, median 60 mg/day). Thus, the potential of soy isoflavones to prevent bone loss can be achieved by a dosage of 80 mg/day (Huang et al., 2006).

Finally, a wealth of supporting data from many in vitro mechanistic studies on bone cell lines and in vivo investigations using models of osteoporosis shows bone-sparing effects from phytoestrogens. These studies indicate that positive effects of phytoestrogens, as a SERM, may be achieved through estrogen receptors or other mechanisms. However, the results of clinical studies are more inconsistent. The different efficacy of phytoestrogen treatments, in studies involving either animal or human subjects, depended on dose, route and duration of administration. The data are, however, rather tantalizing because it is possible that soy isoflavones may offer the maximum benefit for prevention of osteoporosis. Therefore, it is necessary to perform large-scale clinical dietary intervention studies with phytoestrogens to determine their effects on bone tissue in humans.
