Phytoestrogens: an example of endocrine disruptor

1. Introduction

The endocrine system is responsible for regulating the functioning of an organism so that it maintains homeostasis. In this system, hormones are responsible for transmitting information between the organs, in addition to being essential in functions such as reproduction, being involved in the normal development of organisms, and in brain function and differentiation.

Hormones will coordinate the development of each individual from a single fertilized cell, and will help that developing organism to cover the needs of each of its organs through the different stages of its life. Hormones are crucial in an individual’s life, and a deficiency or failure in the endocrine system can lead to illness or even death. That is why any substance or compound that can modify the endocrine function of an organism puts its homeostasis and survival at risk. In general, we call this type of compound endocrine disruptors (ED) (Craveadi et al., 2007).

Kavlock et al. (1996) defined the disruptors as foreign substances to the organism that induce deleterious effects on an organism or its offspring as a consequence of an endocrine alteration; this is an exogenous agent that interferes in the production, liberation, transport, metabolism, the union to a receptor or a modifier of the action or elimination of natural ligands responsible for maintaining homeostasis and the regulation and development of the organism.

The clearest example of a disruptor is diethylstilbestrol (DES), a chemical that behaves like a hormone and not only affects the health of the exposed person, but also can generate irreversible damage to their descendants since the exposure of a mother to these compounds during pregnancy can generate varied and unexpected effects on the offspring several decades after having been exposed in utero. One of these effects can be any type of cancer or infertility (Toppari et al., 1996).

Based on epidemiological studies and laboratory animals, ED hypothetically affect reproduction, thyroid function and brain differentiation. Some of the effects that have been related to ED in girls are breast cancer, endometriosis and precocious puberty (Craveadi et al., 2007). In man, reproductive health is affected; men present a decrease in sperm quality, testicular cancer and reproductive tract anomalies (Jégou et al., 1999).

2. Classification of endocrine disruptors

ED can be classified into two major groups:

1. Xenoestrogens, which are synthetic chemical compounds, and
2. Naturally occurring compounds such as phytoestrogens and estrogen compounds found in fungi.

Some examples of these compounds are:

• Pharmaceutical and veterinary contaminants: Dietilestilbestrol, growth promoters.
• Contaminants of the chemical industry: bisphenol A, phthalates, heavy metals.
• Phyto-sanitary products: herbicides, fungicides, insecticides, etc.
• Natural substances, phytoestrogens: Genistein, Daidzein, coumestrol, zearalenone, etc. (Cravedi et al., 2006).

3. Phytoestrogens

Phytoestrogens are chemical compounds found naturally in plants, in which they act as fungicides (Sukumuran and Gnanamanickam, 1980) and regulate various functions that are required for the physiological maintenance of the plant (Feet and Osman, 1982). Some factors that can generate stress in the plant, such as ultraviolet radiation, plant cut or nutrient deficiency, stimulate the phytoestrogen levels (Hanson et al., 1965; Rossiter and Bech, 1966a; Rossiter and Bech 1966b; Medina et al., 1982a; Medina et al., 1982b). These compounds, when ingested, interact with the estrogen receptors, inducing an estrogenic / antiestrogenic effect (Livingston et al., 1961; Loper and Hanson 1964; LeBars et al., 1990).

The first evidence that phytoestrogens can alter reproduction in mammals dates back to 1946 in Australia. Sheep fed with red clover presented various reproductive alterations, infertility and galactorrhea in males. All these alterations manifested an estrogenic effect and it was known as the clover disease (Bennets et al., 1940). Subsequently, similar alterations were reported in bovine cattle that were also fed with red clover (Kalla et al., 1984), which is why these compounds were considered as inducers of infertility in both species (Hughes, 1988).

Romero et al. (1997) described an estrogenic syndrome in dairy cows which could present repeated heat, abortions, ovarian cysts, false estrus, and turgid uterus in animals with 40-60 pregnancy days. All these alterations occurred as a consequence of the consumption of alfalfa with large amounts of coumestrol, induced by the contamination of alfalfa with the fungus Pseudopeziza medicaginis which increases up to 75 times the basal levels of coumestrol.

In other species such as the cheetahs, the decrease in fertility has been related to the consumption of a soy-based diet (Setchell et al., 1987) or, for example, in women with diets rich in soy (Amsterdam et al., 2005; Chandrareddy et al., 2008). Rodents whose diet contained phytoestrogens showed alterations in uterotrophic activity and decreased fertility (Thigpen et al., 1999).

In the eastern countries soy-derived foods are consumed, which, on average, equate to 1.5 mg/kg/d of isoflavones compared to the western populations consuming 0.2 mg/kg/d (Coward et al., 1993). In the eastern populations there is a lower incidence of some types of cancer (breast, prostate, rectum and stomach), and pre and postmenopausal women have few alterations related to hormonal changes (e.g. hot flashes, osteoporosis, angina). Several studies have linked the consumption of isoflavones with a series of beneficial effects on health when these compounds act as anticancers or antioxidants, providing great benefits as estrogen agonists in the cardiovascular and bone system (Song et al., 2007). In parallel, a series of negative effects on embryonic development is suggested and they interfere with normal sexual differentiation (Bar-El and Reifen, 2010; Wuttke et al., 2007).

Other phytoestrogens such as coumestrol are ingested mainly in milk, food supplements based on alfalfa, bean sprouts, alfalfa sprouts and legumes. In newborn babies, the main intake of coumestrol is suggested to be taken through breast milk during breastfeeding (Moon et al., 2009).

4. Structure, classification and mechanisms of action of phytoestrogens

Phytoestrogens are non-steroidal compounds with a structure similar to estradiol, capable of mimicking or modulating the action of endogenous estrogens by binding to the estrogen receptor; they present an in vitro estrogenic activity that is superior to some synthetic molecules (Kuiper et al., 1998). These compounds are found in plants and are synthesized from simple phenols or phenylpropanoids (Rolfe, 1998).

Currently more than 200 substances of plant origin have been found to have estrogenic activity. The majority of phytoestrogens belong to the group of flavonoids which are divided into 5 subgroups (Moutsatsou, 2007) (Figure 1).

• Isoflavones present in legumes: soybeans, lentils, peas. The main compounds are genistein, daidzein, biochanin A, formononetin.
• Lignans are found in various cereals: flax seed, bran, rye, buckwheat, millet, soy, oats and barley, as well as in fruits, some vegetables and berries. The main compounds are enterodiol and enterolactone.
• Coumestans are found mainly in alfalfa. The main compound is coumestrol.
• Flavones are found in celery, thyme, dandelion, clover flower, chamomile, green pepper. The main compound is luteolin.
• Stilbenes are found in grapes, blueberries, raspberries and blackberries. The main composite resveratrol.

Figure 1. The five groups of flavonoids compared to 17estradiol.

Estrogens and phytoestrogens have a phenolic ring, and the distance between both hydroxyls in the two groups is similar (Bojase et al., 2001; Shirataki et al., 1999) (Figure 2). Despite this similarity, phytoestrogens have the capacity to act as agonists as well as antagonists. These compounds classically bind to estrogen receptors and produce a typical estrogenic response when administered or consumed in animals or humans (Shutt and Cox, 1972). Phytoestrogens are more stable than natural estrogens, they have a longer life because they are not as rapidly metabolized as endogenous hormones are (Moutsatsou, 2007).

Figure 2. Comparison between an isoflavone and 17estradiol; both have a phenolic ring and the distance between the two hydroxyls is similar.

The potential antagonistic effect of phytoestrogens can be explained in part by the identification of two types of receptors: α and β. These compounds have greater affinity for the latter, which are expressed in different tissues in which they exert specific effects (Cassidy and Faughnan, 2000) (Table 1). At the cellular level, the effect they induce depends on various factors such as the concentration of the compound, the status of the receptors, the presence or absence of endogenous estrogens, as well as the target organ.

Table 1. Distribution of receptors (ER) and in human tissues (Cassidy and Faughnan, 2000).

Other proposed mechanisms of action for phytoestrogens are related to an antiangiogenic, antioxidant and antiproliferative effect (Setchell and Cassidy, 1999). The intracellular mechanisms of phytoestrogens are not limited to the classic genomic effects in estrogen receptors and , they can also inhibit enzymes involved in steroidogenesis (3and 17 hydroxysturoid dehydrogenase, aromatase), inhibit tyrosine kinase, modify biological responses by acting on cytochrome P450 by regulating the expression of some genes or growth factors and stimulating protein synthesis that binds sex hormones (Brown and Setchell, 2001; Duza et al., 2006; Beck et al., 2005; Magee and Rowlan, 2004; Whitten and Patisaul, 2001).

Specifically, the synthesis of proteins that bind sex hormones occurs in the liver. Phytoestrogens, like estrogens, are capable of inducing the synthesis of sex hormone binding globulin (SHBG). However, phytoestrogens have a low binding affinity to SHBG compared to estradiol, they induce an antiestrogenic effect by decreasing levels of free endogenous hormone (Nagel et al., 1998).

The relative potency of phytoestrogens depends on:

• Acute or chronic treatment.
• Stage: Prenatal, neonatal, juvenile, adult.
• Dosage.
• Route of administration: oral, subcutaneous, diet.
• Animal factors: Species, age, sex.
• Monitored response: Uterine weight, vaginal opening age, body weight, fertility etc.

5. Phytoestrogen metabolism

Most phytoestrogens are consumed as glycosylated precursors, which is why they need to be metabolized to active compounds by means of glucose cleavage (Setchell, 2000) (Figure 3). Once ingested, the compounds are metabolized in the intestine by the intestinal flora. In some cases, compounds with greater biological activity are produced, such as the transformation of daidzein to equol.

Figure 3. Formation of active isoflavones from glycosylated compounds in which the cleavage of glucose is carried out.

For a time, it was considered that phytoestrogens did not imply any risk in neonates when the metabolism of these compounds is limited since the glucose scission could not be carried out, however, an alternative mechanism was found which is carried out in the jejunum and converts the glycosylated flavonoids to active compounds by means of uridine 5 diphosphate glucuronyl transferase (Spencer et al., 1999).

6. Effect of phytoestrogens in embryonic and neonatal stages

In the last 50 years, a high incidence of reproductive alterations has been reported in men such as failure in the testicular descent (cryptorchidism), hypospadias, increased incidence of testicular cancer and poor semen quality. This set of alterations is known as Testicular dysgenesis syndrome (Skakkepack et al., 2001). This increase in the reproductive alterations, observed in humans and animals, suggests exposure to environmental factors and a lifestyle that favors exposure to endocrine disruptors, among which less attention has been given to the naturally occurring disturbances called phytoestrogens, it is considered that exposure to them is increasing (Cederroth et al., 2009).

The organs and systems of any individual are more sensitive in the stages of development than in adult life. Morphogenesis and extensive differentiation of tissues and organs important for reproduction occur in pre and postnatal stages. Many of these events depend on the signaling of steroid hormones (Young et al., 1964; Ma, 2009; Sakuma, 2009).

Endocrine disruptors, including phytoestrogens, can have a significant impact on the development and subsequently affect reproduction. These effects can be manifested in the long term, until the adult stage (Jefferson et al., 2012). In humans, phytoestrogen values have been found in the amniotic fluid in the second trimester of pregnancy, similar to those reported in adults. It is important to remember that the affinity of phytoestrogens to SHBG is negligible because the exposure of the fetus correlates with the maternal levels of these compounds (Milligan et al., 1998).

The exposure in infants in the neonatal stage to this type of compounds occurs via the mother through lactation. The levels of phytoestrogens will depend on the diet, since variations of 14.3 nM/L to 378 nM/L are reported. During lactation, lactose intolerant infants, who are fed with soy milk supplements, can reach circulating levels of isoflavones in a range of 552 μg/L to 1775 μg/L (Setchell et al., 1997). At this stage, the endogenous values of estradiol are 40-80 pg/mL, so the levels of isoflavones are 13,000 to 22,000 times higher than the endogenous levels of estradiol (Zung et al., 2001).

Those researches carried out with humans regarding neonatal exposure due to consumption of soy milk are controversial, since some of them suggest there is no relation between the ingested phytoestrogens and the reproductive alterations observed at puberty and at adulthood (Brian et al., 2001). However, as for girls, other studies report the prevalence of breast inflammation during the first two years of life (Zung et al., 2008), estrogenization of the vaginal epithelium (Bernbralum et al., 2008), and an increased risk of uterine tumors (D´Alosio et al., 2001), as for men, the presence of hypospadias (North and Golding, 2008), as well as infertility, conditions related to the estrogenic effects of these compounds.

Given the difficulty of conducting studies in humans or in some animal species, rodents have been used as animal models to investigate the effect of these compounds. In general, it has been reported that prenatal administration of genistein in rats reduces birth weight, as well as anogenital distance. At the brain level, it increases the volume of sexually dimorphic nuclei in the hypothalamic preoptic area, and specifically in females, it induces an advance of puberty as well as alterations of the vaginal cycles (Levy et al., 1995; Lewis et al., 2003). In males, it also modifies the anogenital distance and induces a reduction in testicular size. In adulthood, they may have low testosterone levels and poor copulatory behavior (Wisniewski et al., 2003).

We have observed that a single dose of coumestrol on day 10 of gestation in equimolar doses at 2 and 4 g of estradiol, and analyzed on day 18, causes a delay in development in the ovary, for scarce follicles are observed. In the case of the males, the germinal cords are observed less defined and with a fibrillar aspect, a decrease in the Sertoli and germinal cells. Our results suggest opposite effects since there is an estrogenic effect of coumestrol in males and antiestrogenic in females (Montes de Oca, 1996; Bedolla, 1996). The perinatal stage is very important since the differences in the reproductive activity between males and females are carried out during this period by the aromatization of androgens (Arai, 2001). The treatment with isoflavones during this period can cause masculinization of the sexually dimorphic nuclei in the preoptic area, and in turn suppress the response of the luteinizing hormone (LH) in the pituitary to the stimulation of GnRH (Faber and Hughes, 1991; Faber and Hughes, 1993).

It has been shown that genistein consumed by the mother during pregnancy not only crosses the placenta but also the hematocephalic barrier of the fetal brain (Doerge et al., 2001). In the preoptic area, ERβ are present, which are involved in the regulation of sexually dimorphic neurons that respond to estradiol (Tempte et al., 2001). Consequently, genistein can induce the differentiation of a male brain in the female, or females neonatally exposed to coumestrol can present anovulatory cycles in the pubertal and adult stages (Whiten et al., 1993).

The administration of phytoestrogens in neonatal females causes morphophysiological alterations in the reproductive tract, which become visible at 40 days of age and are irreversible. The alterations include persistent vaginal cornification (Burroughs, 1995), ovaries with hemorrhagic bodies and few ovulatory follicles (Burroughs et al., 1990), squamous metaplasia of the uterus (Forsberg and Kalland, 1981), and early vaginal opening (Burroughs, 1995), increased uterine weight (Medlock et al., 1995), low body weight and irregularity in vaginal cycles (Whitten and Naftolin, 1992).

The presence of anovulatory cycles is frequent due to both ovarian alterations and alterations in the pituitary response to GnRH for the release of LH, which suggests a general disturbance of the hypothalamic pituitary axis that generates alterations in ovarian function (Jefferson and Williams, 2011) presenting a pattern similar to the ovulatory delay syndrome (Whitten et al., 1993).

We have observed that the neonatal administration of coumestrol in doses of 150 μg per day during the first five days induces persistent estrus, the females have a lordosis coefficient of 100%, as well as the presence of ovarian cysts (Tarragó et al., in process).

The effects described by the neonatal administration in males are contradictory and depend both on the dose and time of administration and the phytoestrogen that was administered. The administration of coumestrol in the first five postnatal days does not modify the physiological or sperm parameters in the adult animal (Awoniyi et al., 1997). However, administration of genistein during the same period delays the process of spermatogenesis and induces a decrease in FSH levels (Atanassova et al., 2000). When administration is carried pre and postnatal, testosterone levels decrease (Klein et al., 2002).

In a study conducted in our laboratory, we administered genistein to Wistar rats in a dose equivalent to that consumed by babies in the first weeks of birth. On the sixth day, we could observe estrogen-type modifications in the seminiferous cords. We observed an increase in the area of the seminiferous duct, a decrease in the number of Sertoli cells and an increase in the number of interstitial cells (Tarragó et al., in process). In the adult stage, when assessing sexual behavior, we observed an antiestrogenic effect in the behavioral parameters, since the treatment induced a behavioral facilitation effect by increasing the number of ejaculations (Otal et al., 2016). Despite having a greater number of ejaculations, the number of pregnant females and offspring decreases, which coincides with an estrogenic effect.

7. The effect of phytoestrogens in adults

We have mentioned that the effects of the administration or consumption of phytoestrogens during the perinatal stages can manifest up to the adult stage, which makes it difficult to determine if some alterations are induced in the perinatal or adult stage. An example of this problem is related to the decrease in the number of spermatozoa that has been observed in various populations. A recent study conducted in Asia (Iwamoto et al., 2009), where the consumption of phytoestrogens is high compared to other parts of the world, observed that the average number of spermatozoa per ejaculation is similar to the lowest levels reported in some European countries; however, it cannot be determined if this is due to diet, lifestyle, genetic factors or a relation among these (Giwercman, 2011). However, since the action of phytoestrogens is not limited to interaction with estrogen receptors, it has been suggested that they can also interfere with the functioning of androgen receptors and in turn affect the last steps of spermatogenesis (Chavarro et al., 2009). Exposure to high levels of phytoestrogens over a long period in adult life or during critical stages of development could affect fertility by interfering with the spermatogenesis process.

Most of the studies have been carried out in embryonic and neonatal stages, with fewer studies carried out in the adult stage. In a study carried out on Wistar male rats, which were administered different doses of coumestrol for three days, it was found that the treatment induced a reduction of the testosterone hormone levels without modifying the gonadotropin levels, suggesting a direct effect on the biosynthesis of steroids probably inhibiting the enzyme 17 hydroxysteroid dehydrogenase. At the testicular level alterations are observed in the seminiferous tubules, mainly in stages VII and VIII of the seminiferous epithelium cycle (Figure 4). The results observed in this work do not follow a monotonic behavior because the type of response they present is not monotonic or hormonal (Tarragó et al., 2006).

Figure 4. Cross-section photomicrograph of rat testicle, in which seminiferous tubules are observed. A and C, control treatment with oil; B and D show images of the right testicle, coumestrol treatment, which induces a cell diversity decrease, as well as inhibition of some phases of the seminiferous epithelium. In addition, a clear increase in intracellular space is observed (modified from Tarragó et al., 2006).

In the case of females, there are few studies available at this stage. In humans, a unique case has been reported related to a high consumption of phytoestrogens (4 ounces of soy per day). This induced a persistent sexual arousal syndrome characterized by dysmenorrhea and menometrorrhagia, as well as a high pelvic tension. The effect of phytoestrogens was reversible and did not recur after three months of changing the diet (Amsterdam et al., 2005).

8. Conclusions

Phytoestrogens are non-steroidal compounds that can induce effects such as estradiol agonists or antagonists.

The responses they induce are of non-monotonic types, so the greater effect can be produced in both high and low doses.

The risk window for the exposure of these compounds are the embryonic and neonatal stages, sometimes the damage is detected several decades later.

The binding to estradiol receptors is not the only mechanism of action that phytoestrogens have.

The effect of these compounds depends on factors such as compound, dose, route and time of administration, stage in which it is administered and response or organ analyzed.