**7.2 Effects on male reproductive system**

Soy phytoestrogens, alone or in combination with some other EDC, may adversely affect androgen hormone production, spermatogenesis, sperm capacitation and fertility. Results of recent meta-analysis suggest that neither soy foods nor isoflavone supplements alter bioavailable T concentration in adult men (Hamilton-Reeves et al., 2010). However, Tanaka et al. (2009) reported that short-term administration of soy isoflavones decreased testosterone and dihydrotestosterone (DHT) and increased sex hormone-binding globulin levels.

Male reproductive system is particularly sensitive in prenatal stage and during early infancy, when disruption of the hormonal balance in favor of estrogens can lead to irreversible abnormalities in sex specific physiology and behavior in the adulthood (Patisaul & Jafferson, 2010).

Only few animal studies reported results on the developmental effects of exposure to soy infant formula. Their study designs were based on the same group of male marmosets treated during infancy, and assessed either as juveniles (Sharpe et al., 2002) or adults (Tan et al., 2006). The soy infant formula-fed male marmosets had significantly lower plasma testosterone levels than their cow milk formula-fed co-twins. Histopathological analysis on the testes of a subset of the co-twins revealed an increase in Leydig cell abundance per testes in the soy infant formula-fed marmosets compared to their cow milk formula–fed co-twin, in the absence of a significant change in testicular weight. A follow up study was conducted on the remaining animals when they were sexually mature (80 weeks of age or older). The males fed with soy infant formula as infants had significantly heavier testes and increased number of both Leydig and Sertoli cells per testicle compared to cow milk formula-fed controls. In addition, there was no significant onset of puberty, level of adult plasma testosterone, or fertility. The authors suggest that the increase in testes weight was likely due to an increase in testicular cell populations. Therefore, these results demonstrated permanent effects on testicular cell populations, but no obvious effects on reproductive function, namely fertility or permanent changes in testosterone levels of experimental animals.

(2010) concluded that further determination if soy infant formula have long-term

Fig. 3. Ovaries of 21 day old control (a) and genistein – treated rat; hematoxylin - eosin staining method; OS, ovarian stroma; f, follicular parenchyma; hematoxylin – eosin staining

Soy phytoestrogens, alone or in combination with some other EDC, may adversely affect androgen hormone production, spermatogenesis, sperm capacitation and fertility. Results of recent meta-analysis suggest that neither soy foods nor isoflavone supplements alter bioavailable T concentration in adult men (Hamilton-Reeves et al., 2010). However, Tanaka et al. (2009) reported that short-term administration of soy isoflavones decreased testosterone and dihydrotestosterone (DHT) and increased sex hormone-binding globulin

Male reproductive system is particularly sensitive in prenatal stage and during early infancy, when disruption of the hormonal balance in favor of estrogens can lead to irreversible abnormalities in sex specific physiology and behavior in the adulthood (Patisaul

Only few animal studies reported results on the developmental effects of exposure to soy infant formula. Their study designs were based on the same group of male marmosets treated during infancy, and assessed either as juveniles (Sharpe et al., 2002) or adults (Tan et al., 2006). The soy infant formula-fed male marmosets had significantly lower plasma testosterone levels than their cow milk formula-fed co-twins. Histopathological analysis on the testes of a subset of the co-twins revealed an increase in Leydig cell abundance per testes in the soy infant formula-fed marmosets compared to their cow milk formula–fed co-twin, in the absence of a significant change in testicular weight. A follow up study was conducted on the remaining animals when they were sexually mature (80 weeks of age or older). The males fed with soy infant formula as infants had significantly heavier testes and increased number of both Leydig and Sertoli cells per testicle compared to cow milk formula-fed controls. In addition, there was no significant onset of puberty, level of adult plasma testosterone, or fertility. The authors suggest that the increase in testes weight was likely due to an increase in testicular cell populations. Therefore, these results demonstrated permanent effects on testicular cell populations, but no obvious effects on reproductive function, namely fertility or permanent changes in testosterone levels of experimental

reproductive health effects should be a public health imperative.

method; unpublished image of Medigović et al.

**7.2 Effects on male reproductive system** 

levels.

animals.

& Jafferson, 2010).

Some studies on rats and mice demonstrated increased testicular weight when animals were treated with soy diet or isoflavone supplements during gestation and lactation or continuous exposure, similar to the effect described above in marmosets treated with soy infant formula during infancy (Akingbemi et al., 2007; McVey et al., 2004; Piotrowska et al., 2011; Ruhlen et al., 2008; Wisniewski et al., 2005). Other authors reported a decrease (Atanassova et al., 1999; Wisniewski et al., 2003) or no effect on testicular weight (Fielden et al., 2003; Kang et al., 2002).

Controversial results are found as to the effects of lifelong exposure of rodents to phytoestrogens on reproductive function, namely fertility or changes in testosterone levels. The litter size was not affected when male rats were exposed to dietary soy throughout life (Atasnassova et al., 1999). Also, chronic dietary exposure to G did not adversely affect spermatogenesis or seminal vesicle weight in rats (Delclos et al., 2001; Roberts et al., 2000). On the other hand, a few studies indicate negative effects of phytoestrogens on male reproductive success. Thus, a continuous exposure to low combined doses of G and vinclozolin affects male rats' reproductive health by inducing reproductive developmental anomalies, alterations in sperm production and quality, and fertility disorders (Eustache, 2009).

Exposure to G was found to induce hyperplasia of Leydig cells in mice (Lee et al., 2004b). The exposure to isoflavones during 5 weeks decreased the level of circulating testosterone, depending on the dose used (Weber et al., 2001). No significant differences in serum testosterone concentration was detected in rats receiving high doses of G and D from intrauterine life through sexual maturity (Piotrowska et al., 2011). In vitro investigation showed that G can promote the testosterone production of rat Leydig cells at a low concentration, but both D and G can inhibit it at a higher concentration (Zhu et al., 2009).

Effect of phytoestrogens on male reproduction system is a complex process that depends on developmental stage and time of exposure, applied dosage, and other factors. Together, these factors determine the potential risk for adverse consequences with long-lasting effects on male reproductive function. At present, the evidence is insufficient to determine whether soy products cause or do not cause adverse developmental effect on male reproductive system, due to the small number of studies, limitations in their experimental designs, and failure to detect adverse functional effects.

#### **7.3 Effects on pituitary-thyroid axis**

Goitrogenic effects of a soybean diet in animals were reported in 1933 (McCarrison, 1933). Similar to animals, goiter and hypothyroidism were reported in infants fed with adapted soy formula without adequate iodine supply (Van Wik et al., 1959). This effect was eliminated by supplementing commercial soy infant formulas with iodine, or by switching to cow milk (Chorazy et al., 1995). However, infants with congenital hypothyroidism that were fed with iodine supplemented diet still needed higher doses of L-thyroxine (Jabbar et al., 1997). In addition, the incidence rate of autoimmune thyroid disease was doubled in teenage children who consumed soy formula as infants (Fort et al., 1990). However, results of clinical studies with adults are not consistent: some authors suggest that isoflavones have a mild or no effect on thyroid function (Dillingham et al., 2007; Duncan et al., 1999), while others indicate that isoflavones suppress the thyroid function (Haselkorn et al., 2003; Ralli, 2003; Sathyapalan et al., 2011).

Soybean Phytoestrogens – Friends or Foes? 151

normal (Refetoff, 1989). In contrast, there is evidence that the role of serum binding proteins is to allow the equal distribution of hormone delivery to tissues (Mendel et al., 1987). In rats, TTR is a major serum transport protein of thyroid hormones. In humans TTR is produced in the choroid plexus and appears to be important for thyroid hormone action in the brain (Richardson et al., 2007). Thus, TTR may mediate transport of environmental chemicals into various compartments such as placenta (Meerts et al., 2002). Chemical binding to the TTR may not only decrease the availability of thyroid hormone to various tissues, it may also

Fig. 4. Thyroid gland tissue of control orchidectomized (a and b) and daidzein-treated orchidectomized (c and d) rat; hematoxylin - eosin and immuno-staining for thyroglobulin;

In order to accurately asses thyroid function it must be understood that deiodinase enzymes are essential control points of cellular thyroid activity that determine intracellular activation and deactivation of thyroid hormones. Apart from the hormone synthesis by the thyroid gland, deiodination pathways in liver and kidney are the main contributors to thyroid hormone metabolism, turnover and homeostasis. Enzyme 5′ deiodinase type I (5′DI) is the key enzyme in thyroid hormone activation and inactivation in extra thyroidal tissues. This enzyme catalyzes deiodination of the thyroid hormone precursor thyroxine (T4) to the biologically active triiodo-thyronine (T3), as well as the inactivation of T4 and T3 to "reverse" T3 and T2. It is expressed in different tissues, with

unpublished image of Šošić-Jurjević et al.

selectively target these chemicals for transport and uptake.

Rats provide a useful risk assessment model for various thyroid toxins (Choksi et al., 2003). However, compared to the human, rodent thyroid gland is more sensitive to adverse chemicals (Capen, 1997). Several investigators have reported induction of goiter in iodinedeficient rats maintained on a soybean diet (Ikeda et al., 2000; Kajiya et al., 2005; Kimura et al., 1976), although only in cases of iodine deficiency or presence of some other goitrogenic factor. Rats receiving low iodine diet that included 20% of defatted soybeans developed severe hypothyroidism, characterized by a reduction in serum thyroxin and an increase in serum TSH (Ikeda et al., 2000). In addition, a diet containing higher percentage of soy (40% of defatted soybeans) in combination with iodine deficiency induced the development of thyroid carcinoma in rats (Kimura et al., 1976).

Doerge and his associates demonstrated that genistein and daidzein inhibit the activity of thyroid peroxidase (TPO), the key enzyme in the synthesis of thyroid hormones (TH), both in vitro and in vivo (Divi et al., 1997; Chang & Doerge, 2000; Doerge et al., 2002). However, despite significant inactivation of this enzyme, serum thyroid hormone levels were unaffected by isoflavone treatments in young adult rats of both sexes. Most other authors, who performed their studies on young adult animals of both sexes, also reported that soy or isoflavones alone, in the absence of other goitrogenic stimulus, did not affect thyroid weights, histopathology and the serum levels of TSH and thyroid hormones (Chang & Doerge, 2000, Schmutzler et al., 2004). The authors suggested that soy could cause goiter, but only in animals or humans consuming diets marginally adequate in iodine, or who were predisposed to develop goiter, or exposed to additional goitrogenic compounds such as perchlorate, a potent inhibitor of the sodium-iodide-symporter (NIS) of thyrocytes.

Increasing evidence is available that set points of the HPT axis change during various life phases and tend to be less sensitive to negative feedback by thyroid hormones in aging individuals. However, the results on isoflavone effects in aged humans and rodents are scarce. In rodent models, we are the first who demonstrated that both genistein and daidzein induce micro-follicular changes in the thyroid tissue, including hypertrophy of Tgimmunopositive follicular epithelium and colloid depletion (Fig.4), and reduce the level of serum thyroid hormones in orchidectomized (Orx) middle-aged male rats, a model of andropause (Šošić-Jurjević et al., 2010). The concentration of total T4 in serum decreased more prominently than concentration of total T3 in serum in comparison to the corresponding control values. This reduction consequently led to a feedback stimulation of pituitary TSH cells, detected by the increase in cell volume and relative volume density of TSHβ-immunopositive cells per pituitary unit volume, as well as by the increased concentration of TSH in serum. Besides the TPO, there might be other molecular targets for isoflavone interference with the pituitary-thyroid axis.

Soy isoflavones may interfere with thyroid hormones at binding sites of serum distribution proteins such as transthyretin (TTR). In vitro analysis demonstrated that soy isoflavones are potent competitors for T4 binding to TTR in serum and cerebrospinal fluid (Radović et al., 2006). As an outcome of this interference, isoflavones may alter free thyroid hormone concentrations, resulting in altered availability and metabolism of thyroid hormones in target tissues (Köhrle, 2008; Radović et al., 2006). The role of serum binding proteins for thyroid hormone in thyroid homeostasis is not well understood. No single serum T4 binding protein is essential for good health or for the maintenance of euthyroid state in humans (Robbins, 2000). There are a number of clinical situations in which serum binding proteins are elevated or reduced (even completely absent) and the thyroid state remain

Rats provide a useful risk assessment model for various thyroid toxins (Choksi et al., 2003). However, compared to the human, rodent thyroid gland is more sensitive to adverse chemicals (Capen, 1997). Several investigators have reported induction of goiter in iodinedeficient rats maintained on a soybean diet (Ikeda et al., 2000; Kajiya et al., 2005; Kimura et al., 1976), although only in cases of iodine deficiency or presence of some other goitrogenic factor. Rats receiving low iodine diet that included 20% of defatted soybeans developed severe hypothyroidism, characterized by a reduction in serum thyroxin and an increase in serum TSH (Ikeda et al., 2000). In addition, a diet containing higher percentage of soy (40% of defatted soybeans) in combination with iodine deficiency induced the development of

Doerge and his associates demonstrated that genistein and daidzein inhibit the activity of thyroid peroxidase (TPO), the key enzyme in the synthesis of thyroid hormones (TH), both in vitro and in vivo (Divi et al., 1997; Chang & Doerge, 2000; Doerge et al., 2002). However, despite significant inactivation of this enzyme, serum thyroid hormone levels were unaffected by isoflavone treatments in young adult rats of both sexes. Most other authors, who performed their studies on young adult animals of both sexes, also reported that soy or isoflavones alone, in the absence of other goitrogenic stimulus, did not affect thyroid weights, histopathology and the serum levels of TSH and thyroid hormones (Chang & Doerge, 2000, Schmutzler et al., 2004). The authors suggested that soy could cause goiter, but only in animals or humans consuming diets marginally adequate in iodine, or who were predisposed to develop goiter, or exposed to additional goitrogenic compounds such as perchlorate, a potent inhibitor of the sodium-iodide-symporter (NIS)

Increasing evidence is available that set points of the HPT axis change during various life phases and tend to be less sensitive to negative feedback by thyroid hormones in aging individuals. However, the results on isoflavone effects in aged humans and rodents are scarce. In rodent models, we are the first who demonstrated that both genistein and daidzein induce micro-follicular changes in the thyroid tissue, including hypertrophy of Tgimmunopositive follicular epithelium and colloid depletion (Fig.4), and reduce the level of serum thyroid hormones in orchidectomized (Orx) middle-aged male rats, a model of andropause (Šošić-Jurjević et al., 2010). The concentration of total T4 in serum decreased more prominently than concentration of total T3 in serum in comparison to the corresponding control values. This reduction consequently led to a feedback stimulation of pituitary TSH cells, detected by the increase in cell volume and relative volume density of TSHβ-immunopositive cells per pituitary unit volume, as well as by the increased concentration of TSH in serum. Besides the TPO, there might be other molecular targets for

Soy isoflavones may interfere with thyroid hormones at binding sites of serum distribution proteins such as transthyretin (TTR). In vitro analysis demonstrated that soy isoflavones are potent competitors for T4 binding to TTR in serum and cerebrospinal fluid (Radović et al., 2006). As an outcome of this interference, isoflavones may alter free thyroid hormone concentrations, resulting in altered availability and metabolism of thyroid hormones in target tissues (Köhrle, 2008; Radović et al., 2006). The role of serum binding proteins for thyroid hormone in thyroid homeostasis is not well understood. No single serum T4 binding protein is essential for good health or for the maintenance of euthyroid state in humans (Robbins, 2000). There are a number of clinical situations in which serum binding proteins are elevated or reduced (even completely absent) and the thyroid state remain

thyroid carcinoma in rats (Kimura et al., 1976).

isoflavone interference with the pituitary-thyroid axis.

of thyrocytes.

normal (Refetoff, 1989). In contrast, there is evidence that the role of serum binding proteins is to allow the equal distribution of hormone delivery to tissues (Mendel et al., 1987). In rats, TTR is a major serum transport protein of thyroid hormones. In humans TTR is produced in the choroid plexus and appears to be important for thyroid hormone action in the brain (Richardson et al., 2007). Thus, TTR may mediate transport of environmental chemicals into various compartments such as placenta (Meerts et al., 2002). Chemical binding to the TTR may not only decrease the availability of thyroid hormone to various tissues, it may also selectively target these chemicals for transport and uptake.

Fig. 4. Thyroid gland tissue of control orchidectomized (a and b) and daidzein-treated orchidectomized (c and d) rat; hematoxylin - eosin and immuno-staining for thyroglobulin; unpublished image of Šošić-Jurjević et al.

In order to accurately asses thyroid function it must be understood that deiodinase enzymes are essential control points of cellular thyroid activity that determine intracellular activation and deactivation of thyroid hormones. Apart from the hormone synthesis by the thyroid gland, deiodination pathways in liver and kidney are the main contributors to thyroid hormone metabolism, turnover and homeostasis. Enzyme 5′ deiodinase type I (5′DI) is the key enzyme in thyroid hormone activation and inactivation in extra thyroidal tissues. This enzyme catalyzes deiodination of the thyroid hormone precursor thyroxine (T4) to the biologically active triiodo-thyronine (T3), as well as the inactivation of T4 and T3 to "reverse" T3 and T2. It is expressed in different tissues, with

Soybean Phytoestrogens – Friends or Foes? 153

demonstrated remarkable influence of isoflavones on morphology and function of adrenal cortex. The continuous administration of genistein (40mg/kg) to weanling rats resulted in greater total protein content in zona fasciculata (ZF) and zona reticularis (ZR) of adrenal cortex, and low serum corticosterone concentration (corticosterone is a major glucocorticoid hormone in rats; Ohno et al., 2003). Genistein administration to orchidectomized middleaged rats, as a model of andropause, increased zona glomerulosa (ZG), ZF (Fig. 5) and ZR cell volumes, and decreased serum aldosterone and corticosterone concentrations (p<0.05), whereas serum DHEA concentration significantly increased (Ajdžanović et al., 2009a). Genistein and daidzein increased androgen and decreased glucocorticoid production (Mesiano et al., 1999) in human adrenocortical cells in a culture. Recent study on human adrenocortical H295R cell line demonstrated that daidzein and genistein strongly inhibited

secretion of cortisol with IC50 values below 1 μM (Ohlsson et al., 2010).

Fig. 5. Zona fasciculata of adrenal cortex in control orchidectomized (a) and genisteintreated orchidectomized (b) rat; azan staining method; unpublished image of Ajdžanović

The isoflavones possess structural features similar to estradiol, which enables them to act via ERs (Lephart et al., 2004). Production of steroids in human fetal adrenocortical cells is modulated by estrogens (Fujieda et al., 1982, Mesiano & Jaffe, 1993; Voutilainen et al., 1979). It was shown that 17β-estradiol in high concentrations increased ACTH-stimulated androgen production and inhibited glucocorticoid synthesis in cultured human fetal adrenal cortical cells (Mesiano & Jaffe, 1993). Although these results indicate the influence of estrogens on the adrenocortical cells in vitro, their physiological significance is still unclear. Under physiological conditions the endogenous estrogen concentration does not reach 1 µM/L in nonpregnant adults. However, it is possible that dietary phytoestrogens, as estrogen-related compounds, could reach circulating levels high enough to exert estrogenic actions. Consuming the large amounts of soy-derived foods, for example in Japanese diet, circulating concentrations of phytoestrogens can reach higher levels (1-5 micromole/L)

Isoflavones may also affect activity or expression of steroidogenic enzymes, which seems to be the case for its action on rat adrenal cortex (Malendowicz et al., 2006; Mesiano et al., 1999; Ohno et al., 2003). Within the adrenals, steroids are produced through the action of five forms of cytochrome P450 and 3β-hydroxysteroid dehydrogenase (3βHSD) (Simpson & Waterman, 1992). Differential expression of these enzymes in the three adrenocortical zones

et al.

(Adlercreutz & Mazur, 1997).

highest expression rate found in rat liver, kidney, thyroid gland and pituitary (Bianco et al., 2002). It is regulated in a TH-dependent manner (Köhrle, 2002). In response to iodine deficiency or hypothyroidism, plasma TH values are reduced, TSH is increased and the organism tries to restore normal T3 levels by down-regulation of 5'DI in brain and liver, respectively. In addition, activity of 5'DI in different tissues seems to be sex- (Köhrle et al., 1995; Lisboa et al., 2001) and age- dependent (Corrêa da Costa et al., 2001). According to Corrêa da Costa et al. (2001) decreased serum T3 was detected only in old males, which was explained by a two-times-higher hepatic deiodination of T4 to T3, detected in aged females in comparison to males. Genistein was shown to increase hepatic 5'DI activity of about 33% in young adult female rats, but the detected increase was not statistically significant (Schmutzler et al., 2004). In our model – system (orchidectomized middle-aged rats) G significantly increased (p<0.05) 5'DI activity by 33% (unpublished data). However, neither 5'DI in thyroid nor pituitary 5'DII activity were affected by G or D treatment (unpublished data). These data indicate that although pituitary-thyroid axis in male rats is more vulnerable compared to the one in young adults, it still has great ability to compensate the adverse effects of isoflavones.

Isoflavones may also affect the thyroid function indirectly, via its estrogenic action. Estrogen receptors were located both in pituitary thyrotrophs and in thyroid follicular cells (González et al., 2008; Hampl et al., 1985). Donda et al. (1990) found that pituitary TSH cells in adult female rats have a higher density of T3 and TRH receptors than in male rats, probably due to a modulatory effect of estradiol. Males are more prone to develop goitrogenesis in response to goitrogenic stimuli, probably due to higher TSH levels in comparison to females (Capen, 1997). It seems that estradiol make the TSH cells more sensitive to the negative feedback regulation with thyroid hormones (Ahlquist et al., 1987). In orchidectomized middle-aged rats we demonstrated that pharmacological doses of testosterone and estradiol disturbed the endocrine homeostasis of pituitary-thyroid axis, but in different directions. Testosterone acted stimulatory, probably through central stimulation of pituitary TSH cells, since both serum TSH and T4 levels were increased. Estradiol acted inhibitory and, though detected structural changes corresponded to centrally induced hypothyroidism, the level of TSH in serum was not significantly altered, suggesting that estradiol may interfere with TSH action within the thyrocytes (Sekulic et al., 2010).

Estrogen was also demonstrated to inhibit activity of thyroid follicular cells in the absence of TSH both in vitro and in vivo (Furlanetto et al., 2001; Vidal et al., 2001). Our previous research of a young adult and middle-aged rat menopause models indicated that chronic estradiol treatment modulated pituitary TSH cells and thyroid structure and decreased serum levels of thyroid hormones, with no significant changes in serum TSH level (Šošić-Jurjević et al., 2005, 2006). Genistein acted as estrogen agonist in an estrogen-responsive pituitary cell line (Stahl et al., 1998).

In conclusion, though there are multiple molecular targets for interference of isoflavones with pituitary-thyroid-peripheral network, this system has considerable capacity to compensate disturbances of its feedback mechanism. If thyroid function is impaired, the risk of developing hypothyroidism increases. Elderly population and individuals with thyroid dysfunction should be aware of potential risk when use isoflavone supplements.

#### **7.4 Effects on pituitary-adrenocortical axis**

Results concerning the potential effects of the soy phytoestrogens on pituitaryadrenocortical axis in humans are very limited. The animal studies and in vitro experiments

highest expression rate found in rat liver, kidney, thyroid gland and pituitary (Bianco et al., 2002). It is regulated in a TH-dependent manner (Köhrle, 2002). In response to iodine deficiency or hypothyroidism, plasma TH values are reduced, TSH is increased and the organism tries to restore normal T3 levels by down-regulation of 5'DI in brain and liver, respectively. In addition, activity of 5'DI in different tissues seems to be sex- (Köhrle et al., 1995; Lisboa et al., 2001) and age- dependent (Corrêa da Costa et al., 2001). According to Corrêa da Costa et al. (2001) decreased serum T3 was detected only in old males, which was explained by a two-times-higher hepatic deiodination of T4 to T3, detected in aged females in comparison to males. Genistein was shown to increase hepatic 5'DI activity of about 33% in young adult female rats, but the detected increase was not statistically significant (Schmutzler et al., 2004). In our model – system (orchidectomized middle-aged rats) G significantly increased (p<0.05) 5'DI activity by 33% (unpublished data). However, neither 5'DI in thyroid nor pituitary 5'DII activity were affected by G or D treatment (unpublished data). These data indicate that although pituitary-thyroid axis in male rats is more vulnerable compared to the one in young adults, it still has great ability to

Isoflavones may also affect the thyroid function indirectly, via its estrogenic action. Estrogen receptors were located both in pituitary thyrotrophs and in thyroid follicular cells (González et al., 2008; Hampl et al., 1985). Donda et al. (1990) found that pituitary TSH cells in adult female rats have a higher density of T3 and TRH receptors than in male rats, probably due to a modulatory effect of estradiol. Males are more prone to develop goitrogenesis in response to goitrogenic stimuli, probably due to higher TSH levels in comparison to females (Capen, 1997). It seems that estradiol make the TSH cells more sensitive to the negative feedback regulation with thyroid hormones (Ahlquist et al., 1987). In orchidectomized middle-aged rats we demonstrated that pharmacological doses of testosterone and estradiol disturbed the endocrine homeostasis of pituitary-thyroid axis, but in different directions. Testosterone acted stimulatory, probably through central stimulation of pituitary TSH cells, since both serum TSH and T4 levels were increased. Estradiol acted inhibitory and, though detected structural changes corresponded to centrally induced hypothyroidism, the level of TSH in serum was not significantly altered, suggesting that estradiol may interfere with TSH action

Estrogen was also demonstrated to inhibit activity of thyroid follicular cells in the absence of TSH both in vitro and in vivo (Furlanetto et al., 2001; Vidal et al., 2001). Our previous research of a young adult and middle-aged rat menopause models indicated that chronic estradiol treatment modulated pituitary TSH cells and thyroid structure and decreased serum levels of thyroid hormones, with no significant changes in serum TSH level (Šošić-Jurjević et al., 2005, 2006). Genistein acted as estrogen agonist in an estrogen-responsive

In conclusion, though there are multiple molecular targets for interference of isoflavones with pituitary-thyroid-peripheral network, this system has considerable capacity to compensate disturbances of its feedback mechanism. If thyroid function is impaired, the risk of developing hypothyroidism increases. Elderly population and individuals with thyroid

Results concerning the potential effects of the soy phytoestrogens on pituitaryadrenocortical axis in humans are very limited. The animal studies and in vitro experiments

dysfunction should be aware of potential risk when use isoflavone supplements.

compensate the adverse effects of isoflavones.

within the thyrocytes (Sekulic et al., 2010).

pituitary cell line (Stahl et al., 1998).

**7.4 Effects on pituitary-adrenocortical axis** 

demonstrated remarkable influence of isoflavones on morphology and function of adrenal cortex. The continuous administration of genistein (40mg/kg) to weanling rats resulted in greater total protein content in zona fasciculata (ZF) and zona reticularis (ZR) of adrenal cortex, and low serum corticosterone concentration (corticosterone is a major glucocorticoid hormone in rats; Ohno et al., 2003). Genistein administration to orchidectomized middleaged rats, as a model of andropause, increased zona glomerulosa (ZG), ZF (Fig. 5) and ZR cell volumes, and decreased serum aldosterone and corticosterone concentrations (p<0.05), whereas serum DHEA concentration significantly increased (Ajdžanović et al., 2009a). Genistein and daidzein increased androgen and decreased glucocorticoid production (Mesiano et al., 1999) in human adrenocortical cells in a culture. Recent study on human adrenocortical H295R cell line demonstrated that daidzein and genistein strongly inhibited secretion of cortisol with IC50 values below 1 μM (Ohlsson et al., 2010).

Fig. 5. Zona fasciculata of adrenal cortex in control orchidectomized (a) and genisteintreated orchidectomized (b) rat; azan staining method; unpublished image of Ajdžanović et al.

The isoflavones possess structural features similar to estradiol, which enables them to act via ERs (Lephart et al., 2004). Production of steroids in human fetal adrenocortical cells is modulated by estrogens (Fujieda et al., 1982, Mesiano & Jaffe, 1993; Voutilainen et al., 1979). It was shown that 17β-estradiol in high concentrations increased ACTH-stimulated androgen production and inhibited glucocorticoid synthesis in cultured human fetal adrenal cortical cells (Mesiano & Jaffe, 1993). Although these results indicate the influence of estrogens on the adrenocortical cells in vitro, their physiological significance is still unclear. Under physiological conditions the endogenous estrogen concentration does not reach 1 µM/L in nonpregnant adults. However, it is possible that dietary phytoestrogens, as estrogen-related compounds, could reach circulating levels high enough to exert estrogenic actions. Consuming the large amounts of soy-derived foods, for example in Japanese diet, circulating concentrations of phytoestrogens can reach higher levels (1-5 micromole/L) (Adlercreutz & Mazur, 1997).

Isoflavones may also affect activity or expression of steroidogenic enzymes, which seems to be the case for its action on rat adrenal cortex (Malendowicz et al., 2006; Mesiano et al., 1999; Ohno et al., 2003). Within the adrenals, steroids are produced through the action of five forms of cytochrome P450 and 3β-hydroxysteroid dehydrogenase (3βHSD) (Simpson & Waterman, 1992). Differential expression of these enzymes in the three adrenocortical zones

Soybean Phytoestrogens – Friends or Foes? 155

So, are soy isoflavones friends or foes? The answer is complex and may ultimately depend on age, sex, health status, quantity of intake, and even the composition of an individual's intestinal micro flora. In vitro and animal research, as well as human research including both clinical and epidemiologic data, suggests that isoflavone-containing products pose a risk to estrogen-sensitive breast cancer patients and in women at high risk of developing this disease. Results of animal and human studies suggest a modest benefit in prevention of prostate cancer. Exposure to isoflavones by feeding soy infant formula bears a risk of adverse effects on the long–term development of infants. Women who tend to get pregnant or have irregularities in menstrual cycle, as well as persons who are at risk of thyroid dysfunction, should avoid soy isoflavone supplements. The usage of soy protein (with or without isoflavones) seems to have a modest beneficial effect on cardiovascular system and protective role in prevention and treatment of osteoporosis. Research of potential synergy of isoflavones and drugs, and/or other functional food could be a new promising strategy in

reducing risk of age-related diseases, improving life quality and expanding life span.

This work was supported by the Ministry of Education and Science of the Republic of Serbia, Grant No. 173009. Publishing of this chapter was in part financially supported by SOJAPROTEIN, member of Victoria Group, Soybean Processing Company – PLC, Bečej, Serbia. The authors express their sincere thanks to late Dr. Dana Brunner for her guidance and contribution, and to Mr. Kristijan Jurjevic for his valued assistance with English

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**8. Conclusion** 

**9. Acknowledgment** 

manuscript preparation.

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**10. References** 

leads to the production of specific steroids within each zone (Suzuki et al., 2000). As a precursor of steroidogenesis, the glomerulosa cells use pregnenolone which can be metabolized by either 3βHSD or 17α-hydroxylase, 17, 20-lyase. The relative expression of these enzymes influences the synthesis of aldosterone and cortisol/corticosterone in ZG and ZF, as well as adrenal androgens in ZR (Conley & Bird, 1997). The major physiological regulators of adrenal aldosterone production are angiotenzin II (Ang II) and potassium. Ang II stimulates aldosteron production through the activation of multiple intracellular signaling pathways including a number of tyrosine kinases (Berk & Corson, 1997; Ishida et al., 1995). It was showed that genistein, as a potent inhibitor of various tyrosine kinases may inhibit aldosteron production (Akiyama et al., 1987; Dhar et al., 1990). Genistein and daidzein are also potent competitive inhibitors of human adrenocortical 3βHSD and cytochrome P450 21 hydroxylase, suppressing cortisol and stimulating DHEA production in vitro (Mesiano et al., 1999). Part of the inhibition of aldosteron production may result from an increase in 17αhydroxylase, 17, 20-lyase activity, which removes the substrate from the pathway leading to aldosterone and directs it towards the synthesis of adrenal androgens (Sirianni et al., 2001). Isoflavones could also affect adrenal function indirectly, by affecting pituitary ACTH cells. It was previously reported that estrogen replacement lowered the *proopiomelanocortin* (POMC) gene mRNA level and the ACTH response to repeated stressful stimuli in ovariectomized rats (Redei et al., 1994). A certain synergism between CRH (*corticotrophin releasing hormone*) and the various cytokines, namely IL-1, IL-2 and IL-6, has been shown to exist in stimulation of the pituitary ACTH secretion (Bateman et al., 1989; Besedowsky & del Ray, 1996). Genistein may interrupt the stimulatory effects of CRH and cytokines on POMC gene transcription and reduce the level of ACTH, through inhibition of tyrosine kinase phosphorylation cascades (Katahira et al., 1998), but the biological significance of this mechanism is still unclear. We treated orchidectomized middle-aged rats with different doses of genistein or daidzein (10 and 30mg/kg body weight); (Ajdžanović et al., 2009; Ajdžanović et al., 2010, Milošević et al., 2009), and detected similar decrease in pituitary ACTH cellular volume and plasma ACTH levels. Corticosterone levels were also decreased, supporting that some other mechanism, aside from feedback regulation, is involved in effect of isoflavones on pituitary ACTH cell regulation. Keeping in mind that aging is associated with augmented activity of the pituitary-adrenal axis and higher incidence of stress-related psychiatric disorders (Hatzinger et al. 2000), this decline might be considered beneficial at some point.

On the other hand, chronic treatment of weanling rats with genistein (40mg/kg body weight) elevated ACTH level, most probably due to decreased serum corticosterone level and thus release from a negative feedback regulation (Ohno et al., 2003). This finding is of importance since glucocorticoids have important "programming" effects during development. This means that alternations in the circulating levels of glucocorticoid hormones may affect the timing and set points of other endocrine axes (Manojlovic-Stojanoski et al., 2010), as well as brain development, memory and learning capabilities in adults (de Kloet et al., 1988).

Based on animal studies and in vitro research it may be concluded that soy isoflavones interfere with the function of pituitary-adrenocortical axis. This hormonal axis plays a major role in control of stress response and regulation of numerous body processes (digestion, metabolism of carbohydrates, protein and fat, attenuation of the inflammatory response, mood, emotions and sexuality). Therefore, the biological impact of this interference is high. Potential health risks for various age groups should be further assessed.

#### **8. Conclusion**

154 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

leads to the production of specific steroids within each zone (Suzuki et al., 2000). As a precursor of steroidogenesis, the glomerulosa cells use pregnenolone which can be metabolized by either 3βHSD or 17α-hydroxylase, 17, 20-lyase. The relative expression of these enzymes influences the synthesis of aldosterone and cortisol/corticosterone in ZG and ZF, as well as adrenal androgens in ZR (Conley & Bird, 1997). The major physiological regulators of adrenal aldosterone production are angiotenzin II (Ang II) and potassium. Ang II stimulates aldosteron production through the activation of multiple intracellular signaling pathways including a number of tyrosine kinases (Berk & Corson, 1997; Ishida et al., 1995). It was showed that genistein, as a potent inhibitor of various tyrosine kinases may inhibit aldosteron production (Akiyama et al., 1987; Dhar et al., 1990). Genistein and daidzein are also potent competitive inhibitors of human adrenocortical 3βHSD and cytochrome P450 21 hydroxylase, suppressing cortisol and stimulating DHEA production in vitro (Mesiano et al., 1999). Part of the inhibition of aldosteron production may result from an increase in 17αhydroxylase, 17, 20-lyase activity, which removes the substrate from the pathway leading to aldosterone and directs it towards the synthesis of adrenal androgens (Sirianni et al., 2001). Isoflavones could also affect adrenal function indirectly, by affecting pituitary ACTH cells. It was previously reported that estrogen replacement lowered the *proopiomelanocortin* (POMC) gene mRNA level and the ACTH response to repeated stressful stimuli in ovariectomized rats (Redei et al., 1994). A certain synergism between CRH (*corticotrophin releasing hormone*) and the various cytokines, namely IL-1, IL-2 and IL-6, has been shown to exist in stimulation of the pituitary ACTH secretion (Bateman et al., 1989; Besedowsky & del Ray, 1996). Genistein may interrupt the stimulatory effects of CRH and cytokines on POMC gene transcription and reduce the level of ACTH, through inhibition of tyrosine kinase phosphorylation cascades (Katahira et al., 1998), but the biological significance of this mechanism is still unclear. We treated orchidectomized middle-aged rats with different doses of genistein or daidzein (10 and 30mg/kg body weight); (Ajdžanović et al., 2009; Ajdžanović et al., 2010, Milošević et al., 2009), and detected similar decrease in pituitary ACTH cellular volume and plasma ACTH levels. Corticosterone levels were also decreased, supporting that some other mechanism, aside from feedback regulation, is involved in effect of isoflavones on pituitary ACTH cell regulation. Keeping in mind that aging is associated with augmented activity of the pituitary-adrenal axis and higher incidence of stress-related psychiatric disorders (Hatzinger et al. 2000), this decline might be considered beneficial at

On the other hand, chronic treatment of weanling rats with genistein (40mg/kg body weight) elevated ACTH level, most probably due to decreased serum corticosterone level and thus release from a negative feedback regulation (Ohno et al., 2003). This finding is of importance since glucocorticoids have important "programming" effects during development. This means that alternations in the circulating levels of glucocorticoid hormones may affect the timing and set points of other endocrine axes (Manojlovic-Stojanoski et al., 2010), as well as brain development, memory and learning capabilities in

Based on animal studies and in vitro research it may be concluded that soy isoflavones interfere with the function of pituitary-adrenocortical axis. This hormonal axis plays a major role in control of stress response and regulation of numerous body processes (digestion, metabolism of carbohydrates, protein and fat, attenuation of the inflammatory response, mood, emotions and sexuality). Therefore, the biological impact of this interference is high.

Potential health risks for various age groups should be further assessed.

some point.

adults (de Kloet et al., 1988).

So, are soy isoflavones friends or foes? The answer is complex and may ultimately depend on age, sex, health status, quantity of intake, and even the composition of an individual's intestinal micro flora. In vitro and animal research, as well as human research including both clinical and epidemiologic data, suggests that isoflavone-containing products pose a risk to estrogen-sensitive breast cancer patients and in women at high risk of developing this disease. Results of animal and human studies suggest a modest benefit in prevention of prostate cancer. Exposure to isoflavones by feeding soy infant formula bears a risk of adverse effects on the long–term development of infants. Women who tend to get pregnant or have irregularities in menstrual cycle, as well as persons who are at risk of thyroid dysfunction, should avoid soy isoflavone supplements. The usage of soy protein (with or without isoflavones) seems to have a modest beneficial effect on cardiovascular system and protective role in prevention and treatment of osteoporosis. Research of potential synergy of isoflavones and drugs, and/or other functional food could be a new promising strategy in reducing risk of age-related diseases, improving life quality and expanding life span.

#### **9. Acknowledgment**

This work was supported by the Ministry of Education and Science of the Republic of Serbia, Grant No. 173009. Publishing of this chapter was in part financially supported by SOJAPROTEIN, member of Victoria Group, Soybean Processing Company – PLC, Bečej, Serbia. The authors express their sincere thanks to late Dr. Dana Brunner for her guidance and contribution, and to Mr. Kristijan Jurjevic for his valued assistance with English manuscript preparation.

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**8** 

*Brazil* 

**Soybeans (***Glycine max***) and Soybean Products** 

Leilane Rocha Barros Dourado1,2, Leonardo Augusto Fonseca Pascoal1,3,

Soy is a legume and has been successfully cultivated around the world. Today, the world's top producers of soy are the United States, Brazil, Argentina, China and India. According Brazilian Association of Vegetable Oil Industries (Abiove), the Brazil is responsible for some 28 percent of the world's soybean production, with the estimate of a production of 57 million tons. The Brazil is the world's second largest producer and exporter of soybeans, soybean meal and soybean oil. The soybean complex, which gathers the productive chain of soybean, soybean meal and soybean oil, is the main item in the country's Trade Balance. Other activity that involves the use of soy products (oil) is the production of biodiesel. In fact, so much in the Brazil as in most of the countries of the World, the soy represents one of the largest oilseeds of the world and to main source of vegetable protein for the poultry

**2. The nutritional composition of the soybeans and soybean products used** 

Soybeans and soybean products are now used widely in animal feeding. The crop is grown as a source of protein and oil for the human market and for the animal feed market. Soybean meal is generally regarded as the best of plant protein source in terms of its nutritional value. Also, it has a complementary relationship with cereal grains in meeting the amino acids (AA) requirements of farm animals. Consequently, it is the standard to which other

Soybeans provide an excellent source of both energy and protein for poultry and swine. As with any ingredient, their usage rate depends upon economics, although in the case of soybeans such economics relate to the relative price of soybean meal and of supplemental fats. Soybeans contain about 38% crude protein, and around 20% oil (Leeson Summers, 2008). However, soybeans contain compounds that inhibit the activity of the proteolytic

**1. Introduction** 

and swine feeding.

**in the feeding of poultry and swine** 

plant protein sources are compared (Blair, 2008).

Nilva Kazue Sakomura1,4, Fernando Guilherme Perazzo Costa1,3

**in Poultry and Swine Nutrition** 

and Daniel Biagiotti1,2 *1Department of Animal Science, 2Federal University of Piauí, 3Federal University of Paraíba and 4São Paulo State University* 

