Introduction

Top Abstract Introduction Environmental Estrogens Phytoestrogens and Estrogen... Conclusion References

Estrogens influence growth, differentiation, maturation, and function of many different target tissues including the cells of the central and peripheral nervous system. The endogenous gonadal steroid 17-estradiol (estrogen; E₂) regulates gene expression in target tissues and dramatically influences diverse physiological processes through its cognate receptors estrogen receptor (ER) and ER. These receptors are members of the steroid/thyroid superfamily of transcription-factor receptors, and they share common modular domains whose structures and functions are conserved. Overall, the ER and ER proteins are about 47% identical and are expressed from different genes that are located on separate chromosomes (Enmark et al., 1997). Each receptor binds E₂ with high affinity (K_d = 0.1 and 0.4 nM, respectively), can interact as homo- or heterodimers with estrogen-responsive elements (ERE) or associate with the AP1 transcription factors c-jun or c-fos to influence transcription of responsive genes (Kuiper et al., 1996, 1997; Paech et al., 1997).

Numerous studies have also demonstrated that E₂, as well as other steroid hormones, can rapidly (within seconds to a few minutes) influence cellular physiology in many different cell types of reproductive and nonreproductive tissues through the activation of a diverse array of intracellular signaling mechanisms. For example, E₂ has been shown to rapidly activate adenylate cyclase, increase intracellular [Ca²⁺], activate phospholipase C to generate inositol 1,4,5-trisphosphate and diacylglycerol, stimulate nitric-oxide synthase to generate nitric oxide, and activate the extracellular regulated kinases 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway (Falkenstein et al., 2000). While the molecular mechanisms of nongenomic E₂ action are probably diverse and therefore not well understood, it is known that some rapid E₂ effects are initiated by the binding of E₂ at membrane-associated ERs that are closely related to the "classical" intracellular receptors (Levin, 1999; Norfleet et al., 1999; Razandi et al., 1999; Watson et al., 1999). However, there is additional evidence indicating that some rapid actions of E₂ are independent of the intracellular ER (Falkenstein et al., 2000; Schmidt et al., 2000).

How the diverse mechanisms through which estrogens mediate their resulting physiological effects are integrated is not known; however, it seems reasonable that the total physiological effects of E₂ are the consequence of both rapid nongenomic mechanisms and longer-duration genomic mechanisms. In this regard, little is known concerning the relative contribution of genomic and nongenomic mechanisms to the integrated physiological effects of estrogens. In this brief review, we will focus our attention on the role of estrogens in the central nervous system by briefly discussing what is known concerning rapid intracellular signaling mechanism(s) through which estrogen's effects are mediated, discuss the potential of estrogenic compounds present in the environment to influence the actions of endogenous estrogens, and suggest that these compounds may potentially modify the activities of endogenous estrogens in the nervous system.

Rapid Mechanisms of Estrogen Action in the Nervous System. In the brain, E₂ is well known as a fundamental regulator of the physiology and behaviors required for reproduction. Along with its role in regulating neuroendocrine functions and sexual behaviors, E₂ also plays a significant role during normal development and in genderization of the mammalian central nervous system (CNS), and it has important neurotrophic and neuroprotective functions in the brain (Beyer, 1999; Toran-Allerand et al., 1999; Wise et al., 2001).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the rapid mechanisms activated by estrogen in the brain. E2 acts at estrogen receptor ER binding sites either near or associated with the plasma membrane to modulate the activity of multiple signal transduction cascades. The MAPK pathway, which is normally activated by binding of neurotrophins (NT) at their cognate receptor tyrosine kinases (Trk), can also be activated by E2, resulting in the subsequent phosphorylation and activation of the MAPK kinase kinase B-Raf, the MAPK kinase MEK1/2, and the ERK1/2. Activated ERK1/2 can alter gene expression by phosphorylation of a specific set of transcription factors or by the phosphorylation of the 90-kDa ribosomal S6 kinase (p90RSK). E2 also modulates the Akt/protein kinase B (AKT/PKB) pathway through activation of phosphatidylinositol-3 kinase (PI3K) that converts phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to PtdIns(3,4,5)P3 leading to activation of the phosphoinositide-dependent kinase (PDK1/2) that subsequently activates AKT/PKB, which results in increased cell survival. Additionally, E2 can activate phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2 to generate Ins(1,4,5)P3 (IP3) and diacylglycerol, leading to the release of Ca2+ from intracellular stores and the activation of protein kinase C (PKC). The ER mediates activation of adenylate cyclase (AC) and results in the cAMP-dependent activation of PKA, leading to phosphorylation and activation of the CREB, which then alters gene expression by binding at CRE. The AC and PLC pathways can also alter neuronal excitability by PKA or PKC phosphorylating specific ion channels to modulate their function. The actions of these pathways are also likely to interact with and modulate other rapid E2-activated signaling pathways. AKT, cellular homolog of the v-akt oncogene; GRB2, growth factor receptor-bound protein 2; Shc, SH2-containing collagen-related proteins; mSos, mammalian homolog of Son of sevenless guanine nucleotide exchange factor.

Rapid Activation of MAPK Signaling in the Brain. The interaction between estrogenic activity and growth factor signaling is well established in the brain, and much evidence indicates that neurotrophic growth factors and estrogen may act in concert as well as reciprocally to regulate differentiation of their target neurons (Toran-Allerand, 1996). Because of the apparently critical role played by E₂ in modulating growth, differentiation, and viability of both neurons and glia, the mechanisms through which E₂ modulates growth factor-dependent MAPK signaling in developing brain cells is currently the subject of intensive research efforts. In neurons, the MAPK pathway is activated by the binding of neurotrophins (e.g., brain-derived neurotrophic factor, nerve growth factor) at their cognate receptor tyrosine kinases and initiates the sequential p21Ras-mediated phosphorylation and activation of downstream effector kinases (Fig. 1). Recently, the ERK1/2 MAPK signaling pathway was shown to be rapidly activated by E₂ in rat cortical neurons in culture (Singer et al., 1999), and a heteromultimeric complex containing ER and components of the MAPK cascade was described in primary cultured cortical explants (Singh et al., 1999). The results of the immunoprecipitation experiments in this latter study suggest that in these cortical cells there is a direct interaction between components of the neurotrophin-activated and E₂-mediated MAPK signaling pathways at the level of B-Raf (Toran-Allerand et al., 1999) (see Fig. 1).

	Environmental Estrogens

Top Abstract Introduction Environmental Estrogens Phytoestrogens and Estrogen... Conclusion References

Environmental estrogens are a large and structurally diverse group of compounds that can mimic and in some cases antagonize the effects of endogenous estrogens, and they are therefore often referred to as "endocrine disruptors". As a result of their estrogen-like activities and the potential for some to block the normal actions of endogenous E₂, there is currently much debate within the scientific community, and also considerable interest within the general public regarding the relative benefits or threats associated with exposure to environmental estrogens.

Compounds characterized as having estrogenic properties are typically divided into two general categories, the xenoestrogens and the phytoestrogens. Xenoestrogens are a diverse group of synthetic compounds that include pesticides such as 1,1,1-trichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane; the widespread industrial pollutants poly-chlorinated biphenyls; bisphenol-A, which is present in canned foods and dental sealants; the synthetic estrogen, diethylstilbesterol; and many others. As a result of their negative actions on reproductive tissues, xenoestrogens are a potential threat to wildlife and human populations and are therefore the subject of much active research. Because numerous studies and recent reviews have focused on various aspects of xenoestrogen action (Tyler et al., 1998; Nilsson, 2000; Rosselli et al., 2000), these compounds are addressed here only in regard to their potential as estrogenic compounds to mediate effects in the brain similar to those of phytoestrogens. It should however be pointed out that while the long-term estrogenic effects of both phytoestrogens and xenoestrogens have been extensively studied, there is a similar lack of experimental data concerning the influence of both natural and synthetic estrogenic compounds on rapid E₂-mediated mechanisms. The ability of these compounds to influence rapid actions of E₂ in the brain and how such effects may impact the normal development and physiological properties of cells in the brain is currently unknown.

The phytoestrogens are a group of naturally occurring compounds with estrogenic activity that are present in plants or that arise from bacterial or fungal metabolism of plant precursor compounds. To varying degrees, phytoestrogens can also act as agonists or antagonists of the normal actions of E₂, and in adults they may have protective effects against certain forms of cancer, cardiovascular disease, and osteoporosis and may also prevent undesirable menopausal symptoms (Bingham et al., 1998). As a result of these potentially beneficial effects, phytoestrogens, especially soy isoflavones, have increasingly gained widespread acceptance as safe and beneficial dietary components and as a "natural" alternative to estrogen-based hormone replacement therapies. This increased use of phytoestrogens has occurred even though their mechanisms of action and their effects (either positive or negative) on the developing and mature brain are not well understood. It is also of interest to note that the ready acceptance of the safety and the benefits associated with exposures to increased concentrations of the natural estrogenic compounds by the general public and the medical community is in sharp contrast to the common (and potentially accurate) perception that the actions of xenoestrogens are a threat to the health and well-being of human and wildlife populations.

In regard to human dietary exposure, there are three major classes of phytoestrogens: the isoflavonoids, the coumestans, and the lignans (Fig. 2). The most significant and therefore most well studied are the isoflavones genistein and daidzein. These phytoestrogens are regularly consumed in soy-containing food products that include infant formula andas mentioned aboveare increasingly used in the form of over-the-counter dietary supplements as an alternative to hormone-replacement therapies to relieve menopausal symptoms. Because infants that are fed soy-based formula have especially high plasma concentrations of daidzein and genistein during critical periods of brain development (Setchell et al., 1998), understanding the normal actions of E₂ during perinatal development of the brain, the way phytoestrogens may influence these actions, and whether they exert long-term effects on neuronal function is extremely important.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Comparison of the chemical structures of selected members from each major class of phytoestrogen with that of 17-estradiol. Shown are the isoflavones genistein and daidzein, the lignans enterolactone and enterodiol, the coumestan coumestrol, and the mycoestrogen zearalenone.

Lower but potentially significant concentrations of genistein and daidzein are also present in a wide variety of fruits and nuts (Liggins et al., 2000). Along with isoflavones, the coumestan coumestrol (Fig. 2), which is present in sprouts of soybeans, clover, and alfalfa, is another significant phytoestrogen regularly consumed by humans. Lignans, while receiving less research attention, are present in a wide variety of normally consumed foods and therefore represent a potentially significant source of dietary phytoestrogens. The two major mammalian lignans, enterolactone and enterodiol (Fig. 2), are absorbed as the fermentation products of gut bacterial metabolism of the precursor plant lignans matairesinol and secoisolariciresinol, respectively (Bingham et al., 1998). An additional group of natural estrogenic compounds, the mycoestrogens (e.g., zearalenone; Fig. 2), are generated by metabolic actions of molds belonging to the genus Fusarium that frequently infest pasture grasses and legumes.

Some phytoestrogens have obvious structural similarity with E₂ and are typically considered to act as E₂ mimetics; however, many compounds characterized as having estrogen-like properties have few obvious structural similarities to E₂ (this is especially true in regard to xenoestrogens). It is also likely that some estrogenic compounds act through mechanisms unrelated to those through which E₂ mediates its effects. For example, numerous studies indicate that changes in the expression or activity of E₂-metabolizing enzymes or changes in the levels of E₂-binding serum proteins can influence the rate of E₂ metabolism resulting in alterations in the availability of free-E₂ at target tissues (Rosselli et al., 2000). This structural and mechanistic diversity of different estrogenic compounds suggests that E₂ normally influences multiple mechanisms (i.e., genomic and rapid nongenomic signaling mechanisms) whose sum effects result in a cell-type specific E₂ responsive phenotype.

	Phytoestrogens and Estrogen Receptors

Top Abstract Introduction Environmental Estrogens Phytoestrogens and Estrogen... Conclusion References

In cells of reproductive tissues, many studies have shown that environmental estrogens typically bind ERs with low affinity and can mimic or block the actions of endogenous E₂. In some cases, the estrogenic effects of these compounds are thought to be protective against certain cancers and in other instances have been causatively linked with some hormone-responsive cancers (Bingham et al., 1998; Kuiper et al., 1998; Arcaro et al., 1999; Rosselli et al., 2000).

In contrast to the ability of phytoestrogens to influence reproductive tissues, much less is known concerning the mechanistic effects of these environmental estrogens on the developing nervous system, with especially little known about rapid nongenomic mechanisms of action in brain regions outside of the neuroendocrine axis. However, recent studies in smooth muscle and in a pituitary tumor cell line have demonstrated that xenoestrogens can activate rapid nongenomic mechanisms that are also activated by E₂ (Ruehlmann et al., 1998). Although the available evidence is currently limited, it seems probable that in other nonreproductive cell typesincluding those of the nervous systemxenoestrogens and phytoestrogens may have similar effects (either agonistic or antagonistic) on rapid-signaling mechanisms that are normally regulated by endogenous estrogen. Thus, it is anticipated that phytoestrogens may modify the normal activities of endogenous estrogens during critical periods in the developing brain that may influence the function of the mature adult brain.

At the ER, the binding affinity and transactivational properties of different phytoestrogens appear dependent on the model system/cell type used for analysis and also vary for different orthologs of an ER in the same experimental system (Miksicek, 1994; Kuiper et al., 1997, 1998; Casanova et al., 1999; Matthews et al., 2000). As a result, it is difficult to directly compare the absolute binding affinities and transactivational potencies reported for different compounds across different systems. However, comparison of relative binding affinities from various studies indicates that some phytoestrogens appear to have a higher affinity for ER than for ER and therefore suggests that the ER-mediated effects of phytoestrogens may be mediated through ER (Table 1). Because of the differential expression of ER and ER in different tissues and cell types, this preferential binding of phytoestrogens at ER may in part explain the observed tissue-specific variability of phytoestrogen action.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of relative binding affinity and transactivational activity of phytoestrogens at human ER and ER

As can also be seen from the results presented in Table 1, for all classes of phytoestrogens the binding affinity of a given phytoestrogen may not be predictive of its transactivational potency. In the case of the isoflavones, the relative binding affinity of daidzein at ER and ER is 1000- and 200-fold lower than E₂, respectively. However, the transactivational potency of daidzein (1000 nM) at an ERE was nearly equivalent to that observed for the same concentration of E₂ (Kuiper et al., 1998). In the case of genistein, its relative binding affinity is 25 times lower than that of E₂ at ER, whereas at ER the binding affinities of genistein and E₂ are similar. In contrast to the observed differences in binding affinities, the transactivational potency of 1000 nM genistein at ER and ER is about 2-fold greater than a similarly high concentration of E₂ (Table 1) (Kuiper et al., 1998). The physiological relevance of the differences in ER-mediated transactivational potency at EREs that were detected at these supraphysiological concentrations of E₂ and phytoestrogens is unclear.

In the nervous system, prenatal and postnatal exposures to some environmental estrogens have been reported to result in long-term effects on neuroendocrine function and reproductive behavior (Palanza et al., 1999; Ferguson et al., 2000). Because of the lack of in vitro or in vivo data regarding the ability of phytoestrogens to influence rapid actions of E₂ in the nervous system, the potential for these compounds to influence rapid E₂-mediated mechanisms in the developing CNS is uncertain. However, it seems fairly likely that these compounds may also act as estrogen mimetics to influence some rapid E₂-activated mechanisms, and based on the varying abilities of these compounds to bind ERs and to act as agonists of transactivation, it also seems plausible that phytoestrogens may influence rapid E₂-mediated mechanism in a similarly variable and unpredictable fashion.

	Conclusion

Top Abstract Introduction Environmental Estrogens Phytoestrogens and Estrogen... Conclusion References

Recently, much new insight has accumulated concerning the mechanisms of estrogen action in reproductive and nonreproductive tissues. As a result, there has been increased understanding of, and appreciation for, the significance of both ER-dependent and -independent functions of estrogen in the CNS. Because both long-term genomic and rapid nongenomic E₂-mediated mechanisms are likely to be important for normal development and function of the brain, there is a potential for environmental estrogens to impact brain functions and behavior by influencing these E₂-mediated processes. In humans the available data do not confirm any risks associated with the exposure to normal dietary levels of phytoestrogens; however, much remains unknown concerning the risks associated with exposures to high concentrations of phytoestrogens. As a result, the influence of high concentrations of phytoestrogens during critical periods of neuronal development cannot be discounted completely. The uncertainties surrounding the actions of phytoestrogens on the CNS center around an incomplete understanding of the pleiotropic role of E₂ and its receptors in the brain, the variable ability of phytoestrogens to bind at and to act through ERs, and a nearly complete lack of experimental studies assessing the influence of environmental estrogens on the rapid actions of E₂. The apparent importance of E₂-mediated mechanisms during development and function of the CNS underscores the importance for detailed understanding of the mechanisms of E₂ action in the brain. With a recent trend toward increased human exposures to higher concentrations of phytoestrogens, whether in diet or as an alternative to estrogen replacement therapy, additional research is needed to determine the effects that phytoestrogens may have on the developing and mature nervous system.