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

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

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 and prostate in males.

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 evident only in presence of an additional stress factor.

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 systems.

#### **7.1 Effects on female reproductive system**

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

Soybean Phytoestrogens – Friends or Foes? 147

progressive proliferative lesions of the oviduct, cystic endometrial hyperplasia, and uterine

In addition, the reproductive performance of the neonatally-treated mice was tested during adulthood and there was a significant negative trend for the number of dams with litters. Because the effects were more pronounced in animals at 6 months of age than at 2 or 4 months of age, the authors suggested that reproductive senescence may occur earlier in these animals as a result of the neonatal G treatments (Jefferson et al., 2005). These authors explained that, although G-treated mice ovulate under exogenous hormonal influence, the ovulation rate was changed. The lower doses of G treatment enhanced ovulation rate, while the higher doses decreased this parameter. Ovulation of too many oocytes early in life may reduce the number of oocytes available for fertilization and lead to lower fertility rates later in life (McLachlan et al., 1982). The development of the ovary and ovarian follicles was altered following neonatal G treatment (Jefferson et al., 2002). Ovaries of G-treated mice contained multioocyte follicles (MOFs) at 19th postnatal day. This phenotype is a marker for altered development of the ovary, which lead to oocytes of poor quality (Jefferson et al., 2005). These oocytes are less potent, since the oocytes derived from single oocyte follicles were far more likely to be fertilized in vitro than oocytes derived from MOFs (Iguchi et al., 1990). In our laboratory, results obtained on the ovaries of immature rats treated with 50mg G/kg for three days (from 19th till 21th postnatal day) showed that G disturbed the follicular parenchyma-ovarian stroma ratio (Fig. 3), induced increase of total ovary volume

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

Data are not consistent about onset of puberty and sexual maturation in rats and mice following exposure during gestation and lactation or continuous exposure to soy diet or supplements. An earlier onset of vaginal opening was observed in mice exposed directly to G during the period of lactation (Nikaido et al., 2004.) and in rats treated by sc injection as neonates with 10 mg G/kg bw/day (Bateman & Patisaul, 2008). However, other authors

Only a very small number of studies have been published on D and its estrogenic metabolite equol, and no studies have evaluated the effects of developmental exposure to glycitein. Detection of typical estrogenic effects in these studies are controversial. Kouki et al. (2003) reported no effect on estrous cyclicity in rats treated by sc injection with ~19 mg D/kg bw/day on PND1-5. In contrast, treatment with the same dose levels of G caused the predicted estrogenic effect in all of these studies. Similar to these authors, in our laboratory (unpublished data) no uterotrophic response was detected after subcutaneous injection of immature female rats with 50mg D/kg/day (treatment lasted from 19th postnatal till 21th postnatal day), though the same treatment with G caused predicted

Isoflavones can pass from mother to fetus through placenta. However, this exposure is considerably lower than in infants fed with soy formula. Initially developed as an alternative to bovine milk formulas for babies with a milk allergy, use of soy infant formula became more popular among environmentally oriented population with vegetarian life style. A recent prospective study in human infants observed that female infants fed soybased formulas exhibit estrogenized vaginal epithelium at times when their breast fed or cow milk- based formula fed peers did not (Bernbaum et al., 2008). Patisaul and Jefferson

times as many genes as does E2, most of which were down-regulated (Barnes, 2004).

reported delay in vaginal opening (Anzalone et al., 1998).

carcinoma) relative to control females (Newbold et al., 2001).

(Medigović et al., 2009).

estrogenic response.

mechanisms of E2 action and thus interfere with both endocrine and reproductive functions of the pituitary-gonadal axis. The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Genistein administration to ovariectomized rats induced a dose-dependent uterine growth and altered expression of estrogen-regulated genes (Diel et al., 2004). As E2 does not stimulate these uterine parameters in ERα KO mice (Couse & Korach, 2001), this test is considered as the proof for estrogenic action of phytoestrogens via ERα.

The first recognized health benefit of isoflavones was their potential to alleviate climacteric complaints, namely hot flushes and night sweats in perimenopausal women (Adlercreutz, 1998). Within the short period of time, numerous isoflavone and soy products became available in a form of food supplements and remedies. They were advertised as natural alternative to hormone replacement therapy, useful in prevention of climacteric symptoms. However, majority of recent placebo-controlled clinical trials support the opinion that isoflavone preparations are not superior to placebo, as placebo effect is 30% to 50% when dealing with psychosomatic climacteric complaints (Patisaul & Jefferson, 2010). Animal studies also demonstrated that only high doses of isoflavones were able to suppress overactivation of hypothalamic gonadotropin –release hormone pulse generator induced by estrogen deprivation (the major cause of hot flushes and other climacteric symptoms (Wuttke et al., 2007). It is important to stress that exposure to high doses of soy isoflavones (150mg/kg) is similar in biological effects to classical hormone replacement therapy. Therefore, their consumption bears a risk of increased proliferation of endometrial and mammary gland tissue with so far unpredictable risk of cancer development.

Multiple human studies demonstrated that exposure of premenopausal women to soybean isoflavones have a suppressive effect on pituitary-gonadal axis; consumption of isoflavonesrich soy food suppresses serum estrogen and progesterone levels and attenuate the preovulatory surge of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (Hooper et al., 2009; Nagata et al., 1998; Schmidt et al., 2006;). However, some researchers found no impact of isoflavones on female hormone levels (Maskarinec et al., 2002). Soybean phytoestrogens may also affect the women menstrual cycle, but findings are inconsistent. It was shown that a diet with soy protein delays menstruation and prolongs the follicular phase of the menstrual cycle (Cassidy et al., 1994). Other studies demonstrated increased or unchanged follicular phase length, decreased or unchanged midcycle LH and FSH, increased, decreased or unchanged estradiol, decreased dehidroepiandrosterone sulfate, and decreased or unhanged luteal phase progesterone in relation to isoflavone ingestion (Cassidy et al., 1994; Duncan et al., 1999). Therefore, women who try to become pregnant or have menstrual cycle irregularities should be cautious with consumption of isoflavoneenriched soy products or supplements.

Animal studies in rodents produced clear evidence of adverse effects of G on the female reproductive system following treatment during development (Chen et al., 2007; Kouki et al., 2003; National Toxicilogy Program, 2008). Studies that demonstrated clear evidence of developmental toxicity for G involved treatment during the period of lactation in rodents, as well as multigenerational studies that included exposure during gestation, lactation, and post-weaning. In adulthood, the effects of neonatal exposure to 50 mg G/kg bw/day were manifested as a lower number of live pups per litter (Padilla-Banks et al., 2006), a lower number of implantation sites and corpora lutea (Jefferson et al., 2005), and a higher incidence of histomorphological changes of the reproductive tract (i.e., cystic ovaries,

mechanisms of E2 action and thus interfere with both endocrine and reproductive functions of the pituitary-gonadal axis. The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Genistein administration to ovariectomized rats induced a dose-dependent uterine growth and altered expression of estrogen-regulated genes (Diel et al., 2004). As E2 does not stimulate these uterine parameters in ERα KO mice (Couse & Korach, 2001), this test is considered as the proof for

The first recognized health benefit of isoflavones was their potential to alleviate climacteric complaints, namely hot flushes and night sweats in perimenopausal women (Adlercreutz, 1998). Within the short period of time, numerous isoflavone and soy products became available in a form of food supplements and remedies. They were advertised as natural alternative to hormone replacement therapy, useful in prevention of climacteric symptoms. However, majority of recent placebo-controlled clinical trials support the opinion that isoflavone preparations are not superior to placebo, as placebo effect is 30% to 50% when dealing with psychosomatic climacteric complaints (Patisaul & Jefferson, 2010). Animal studies also demonstrated that only high doses of isoflavones were able to suppress overactivation of hypothalamic gonadotropin –release hormone pulse generator induced by estrogen deprivation (the major cause of hot flushes and other climacteric symptoms (Wuttke et al., 2007). It is important to stress that exposure to high doses of soy isoflavones (150mg/kg) is similar in biological effects to classical hormone replacement therapy. Therefore, their consumption bears a risk of increased proliferation of endometrial and

mammary gland tissue with so far unpredictable risk of cancer development.

Multiple human studies demonstrated that exposure of premenopausal women to soybean isoflavones have a suppressive effect on pituitary-gonadal axis; consumption of isoflavonesrich soy food suppresses serum estrogen and progesterone levels and attenuate the preovulatory surge of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (Hooper et al., 2009; Nagata et al., 1998; Schmidt et al., 2006;). However, some researchers found no impact of isoflavones on female hormone levels (Maskarinec et al., 2002). Soybean phytoestrogens may also affect the women menstrual cycle, but findings are inconsistent. It was shown that a diet with soy protein delays menstruation and prolongs the follicular phase of the menstrual cycle (Cassidy et al., 1994). Other studies demonstrated increased or unchanged follicular phase length, decreased or unchanged midcycle LH and FSH, increased, decreased or unchanged estradiol, decreased dehidroepiandrosterone sulfate, and decreased or unhanged luteal phase progesterone in relation to isoflavone ingestion (Cassidy et al., 1994; Duncan et al., 1999). Therefore, women who try to become pregnant or have menstrual cycle irregularities should be cautious with consumption of isoflavone-

Animal studies in rodents produced clear evidence of adverse effects of G on the female reproductive system following treatment during development (Chen et al., 2007; Kouki et al., 2003; National Toxicilogy Program, 2008). Studies that demonstrated clear evidence of developmental toxicity for G involved treatment during the period of lactation in rodents, as well as multigenerational studies that included exposure during gestation, lactation, and post-weaning. In adulthood, the effects of neonatal exposure to 50 mg G/kg bw/day were manifested as a lower number of live pups per litter (Padilla-Banks et al., 2006), a lower number of implantation sites and corpora lutea (Jefferson et al., 2005), and a higher incidence of histomorphological changes of the reproductive tract (i.e., cystic ovaries,

estrogenic action of phytoestrogens via ERα.

enriched soy products or supplements.

progressive proliferative lesions of the oviduct, cystic endometrial hyperplasia, and uterine carcinoma) relative to control females (Newbold et al., 2001).

In addition, the reproductive performance of the neonatally-treated mice was tested during adulthood and there was a significant negative trend for the number of dams with litters. Because the effects were more pronounced in animals at 6 months of age than at 2 or 4 months of age, the authors suggested that reproductive senescence may occur earlier in these animals as a result of the neonatal G treatments (Jefferson et al., 2005). These authors explained that, although G-treated mice ovulate under exogenous hormonal influence, the ovulation rate was changed. The lower doses of G treatment enhanced ovulation rate, while the higher doses decreased this parameter. Ovulation of too many oocytes early in life may reduce the number of oocytes available for fertilization and lead to lower fertility rates later in life (McLachlan et al., 1982). The development of the ovary and ovarian follicles was altered following neonatal G treatment (Jefferson et al., 2002). Ovaries of G-treated mice contained multioocyte follicles (MOFs) at 19th postnatal day. This phenotype is a marker for altered development of the ovary, which lead to oocytes of poor quality (Jefferson et al., 2005). These oocytes are less potent, since the oocytes derived from single oocyte follicles were far more likely to be fertilized in vitro than oocytes derived from MOFs (Iguchi et al., 1990). In our laboratory, results obtained on the ovaries of immature rats treated with 50mg G/kg for three days (from 19th till 21th postnatal day) showed that G disturbed the follicular parenchyma-ovarian stroma ratio (Fig. 3), induced increase of total ovary volume (Medigović et al., 2009).

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 E2, most of which were down-regulated (Barnes, 2004).

Data are not consistent about onset of puberty and sexual maturation in rats and mice following exposure during gestation and lactation or continuous exposure to soy diet or supplements. An earlier onset of vaginal opening was observed in mice exposed directly to G during the period of lactation (Nikaido et al., 2004.) and in rats treated by sc injection as neonates with 10 mg G/kg bw/day (Bateman & Patisaul, 2008). However, other authors reported delay in vaginal opening (Anzalone et al., 1998).

Only a very small number of studies have been published on D and its estrogenic metabolite equol, and no studies have evaluated the effects of developmental exposure to glycitein. Detection of typical estrogenic effects in these studies are controversial. Kouki et al. (2003) reported no effect on estrous cyclicity in rats treated by sc injection with ~19 mg D/kg bw/day on PND1-5. In contrast, treatment with the same dose levels of G caused the predicted estrogenic effect in all of these studies. Similar to these authors, in our laboratory (unpublished data) no uterotrophic response was detected after subcutaneous injection of immature female rats with 50mg D/kg/day (treatment lasted from 19th postnatal till 21th postnatal day), though the same treatment with G caused predicted estrogenic response.

Isoflavones can pass from mother to fetus through placenta. However, this exposure is considerably lower than in infants fed with soy formula. Initially developed as an alternative to bovine milk formulas for babies with a milk allergy, use of soy infant formula became more popular among environmentally oriented population with vegetarian life style. A recent prospective study in human infants observed that female infants fed soybased formulas exhibit estrogenized vaginal epithelium at times when their breast fed or cow milk- based formula fed peers did not (Bernbaum et al., 2008). Patisaul and Jefferson

Soybean Phytoestrogens – Friends or Foes? 149

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

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,

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.,

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

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,

al., 2003; Kang et al., 2002).

2009).

2009).

failure to detect adverse functional effects.

**7.3 Effects on pituitary-thyroid axis** 

2003; Sathyapalan et al., 2011).

(2010) concluded that further determination if soy infant formula have long-term reproductive health effects should be a public health imperative.

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 method; unpublished image of Medigović et al.
