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Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, MD B3-02, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov.
Abstract
The term endocrine-disrupting chemicals is used to define a structurally diverse class of synthetic and natural compounds that possess the ability to alter various components of the endocrine system and potentially induce adverse health effects in exposed individuals and populations. Research on these compounds has revealed that they use a variety of both nuclear receptor-mediated and non-receptor-mediated mechanisms to modulate different components of the endocrine system. This review will describe in vitro and in vivo studies that highlight the spectrum of unique mechanisms of action and biological effects of four endocrine-disrupting chemicals–diethylstilbestrol, genistein, di(n-butyl)phthalate, and methoxyacetic acid–to illustrate the diverse and complex nature of this class of compounds.
A VARIETY OF structurally diverse natural and synthetic chemicals, classified as endocrine-disrupting chemicals (EDCs), have been reported to interfere with the endocrine system and ultimately disturb the normal function of tissues and organs, particularly those of the reproductive tract. Given their physicochemical differences and distinct biological effects, it is not surprising that a variety of mechanisms are used by EDCs to influence the endocrine system. Advances in our understanding of these mechanisms have been aided by increased public interest in the health effects of EDCs and the development of new tools and models for studying these compounds. Diethylstilbestrol (DES), genistein (Gen), di(n-butyl) phthalate (DBP), and methoxyacetic acid (MAA) are four compounds (Fig. 1
) that are discussed here in an effort to illustrate some of the unique mechanisms of action used by EDCs to modulate endocrine system function.
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DES is a nonsteroidal synthetic estrogen that was developed by Sir Charles Dodds and colleagues in 1938 (1). Physicians began prescribing DES in the late 1940s to maintain normal placental steroid synthesis and prevent miscarriages and premature births (2). The results of the first randomized controlled clinical trials on the effectiveness of DES in preventing miscarriage and premature birth, published in 1953, showed no protective effect of DES (3). Despite these findings, DES continued to be prescribed to pregnant women until 1971, and it has been estimated that approximately 5–10 million Americans were treated with DES during pregnancy or exposed in utero from the 1940s to 1971 (4). Its use was discontinued in 1971 after a report that associated in utero DES exposure with vaginal clear cell adenocarcinoma, a rare form of reproductive tract cancer, in a small number (
0.1%) of daughters of women who had taken the drug (5, 6). Subsequent studies have reported multiple teratogenic effects attributable to prenatal DES exposure that occur more frequently than clear cell adenocarcinomas. In females exposed to DES in utero, nonneoplastic abnormalities such as anatomical malformations of the cervix, vagina, and uterus have been reported, as well as decreased fertility and less successful pregnancies (7, 8, 9, 10). Although no increased risk of cancer has been observed in DES-exposed males, several teratogenic effects have been reported in the reproductive tract, including testicular hypoplasia, cryptorchidism, and epididymal cysts (7, 8, 11).
Adverse effects resulting from DES exposure continue to be uncovered as those exposed in utero advance in age. In an effort to better predict, prevent, and understand the effects of in utero DES exposure, several rodent models have been created to elucidate the mechanisms by which DES may impart its carcinogenic and teratogenic effects on humans. One well characterized DES exposure model is the neonatal mouse, in which female and male pups are treated with DES (2 µg/d) for the first 5 d of life and aged up to 18 months. Neonatal exposure of female mice to DES results in few reproductive tract abnormalities but a high incidence of benign reproductive tract tumors (12, 13). Although rodent models have effectively reproduced several elements of human DES exposure in utero, the complex developmental and carcinogenic effects of DES have made it difficult to study the mechanisms underlying DES-mediated action (13).
A variety of receptor-mediated and non-receptor-mediated mechanisms for DES-induced toxicity have been put forward. The generation of mice lacking either estrogen receptor 
(ER) (14) or ERß (15), i.e. ER knockout (ERKO) mice, provided effective tools to incorporate into the neonatal mouse model as a means to determine the role of the estrogen receptor (ER) in mediating DES-induced effects in vivo. Additionally, because there are two ER proteins, ER and ERß, the different animal models can be used to identify the role of each receptor, if any, in DES-mediated toxicity in specific tissues.
Actions on females
Studies in our laboratory have used the neonatal mouse model to study DES effects in female wild-type (WT), ERKO, and ßERKO mice. WT females treated neonatally with DES displayed characteristic DES-induced reproductive tract lesions in the uterus, vagina, and oviduct (16) (Table 1). In the uterus, DES treatment produced atrophy, smooth muscle disorganization, hyalinization, squamous metaplasia of the luminal and glandular epithelium, and endometrial hyperplasia. In the vagina, DES treatment produced persistent epithelial cornification and vaginal adenosis in a small percentage of animals. Finally, in the oviduct, DES induced progressive proliferative lesions of the epithelium (16, 17). Similarly treated ERKO females displayed none of these characteristic DES-induced lesions, indicating that DES elicits its effects in the female reproductive tract through an ER-dependent signaling pathway.
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ERKO males were observed regardless of age (18) (Fig. 2). With respect to the prostate gland, histological analyses revealed that neonatal DES exposure in WT and ßERKO mice resulted in changes in the aged ventral and dorsolateral prostates, including increased stromal mass, epithelial hyperplasia and dysplasia, and interstitial lymphocyte infiltration, all of which are characteristic of the phenotype of an estrogenized prostate gland (19). In addition, DES-induced epithelial cell differentiation defects indicative of estrogen imprinting were observed in the dorsolateral prostate of WT and ßERKO mice, including the presence of a continuous layer of basal cells lining the epithelial ducts and acini, luminal cell hyperplasia, and loss of expression of dorsolateral prostate-2 protein, a marker of functional differentiation in the dorsolateral prostate (18). None of these effects were observed in the prostates of ERKO males treated with DES, indicating that ER is required for DES-mediated effects in the prostate. Collectively, these data suggest that DES acts through an ER-mediated mechanism in the male reproductive tract.
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ERKO female mice to determine the role of ER in mediating DES-induced down-regulation of Hox and Wnt gene expression. Expression of uterine Hoxa10 and Hoxa11 was reduced approximately 50%, and Wnt7a expression was reduced over 80% in the uteri of 5-d-old WT mice neonatally exposed to DES (16). In contrast to WT mice, DES had no effect on Hoxa10, Hoxa11, or Wnt7a expression in similarly treated ERKO mice, indicating that the DES- mediated reduction of the expression of these genes is regulated through ER (Fig. 3). These results implicate an early developmental role for ER in DES-induced teratogenic effects and suggest that disruption of Hox and Wnt gene expression may be ultimately responsible for the anatomical abnormalities in DES-exposed mice.
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Gen
Several nonsteroidal plant-derived compounds known as phytoestrogens signal through the ER and therefore may act as endocrine disruptors. The potential benefits of phytoestrogens in preventing hormone-dependent cancers and lowering cholesterol have raised interest in studying the biological effects of these compounds. One such compound is Gen, an isoflavone present at high concentrations in soy-based products, which exhibits in vitro and in vivo estrogenic activity (31, 32, 33) as well as tyrosine kinase inhibitory properties (34). Although convincing evidence for both the beneficial and detrimental effects of Gen exposure is limited, it has been reported to reduce mammary cancer in rats (35, 36) and to lower cholesterol levels in humans (37, 38). However, it has also been associated with diminished reproductive capacity in animals (39, 40) and has been shown to induce uterine adenocarcinomas in a neonatal mouse model (41) and to increase the incidence of mammary tumors in rats (42). Further interest in studying Gen has resulted from the observation that humans, particularly infants, are exposed to it through their diet. It has been estimated that adults consuming modest amounts of soy-containing foods have a total isoflavone intake of approximately 1 mg/kg·d, whereas infants fed soy formula ingest significantly higher amounts, consuming 6–9 mg/kg·d of isoflavones, approximately 65% of which is Gen (43). This dose of isoflavones in infants is six to 11 times higher than the amount reported to have hormonal effects that alter the menstrual cycle in adult women (44).
Actions on the uterus
Given the ubiquitous nature of isoflavones in the human diet and the potential for both beneficial and adverse health consequences after exposure to Gen, efforts have been made to characterize the mechanism of action of Gen and to more clearly define the health effects of Gen exposure. In an immature mouse model, Gen elicits classical estrogenic uterotropic responses including increases in uterine wet weight, epithelial cell height, gland number, and lactoferrin expression (45). Receptor-binding assays indicate that Gen preferentially binds to ERß in comparison with ER, with relative binding affinities reported from 20- to 30-fold higher for ERß (46, 47). Despite these differences in ligand binding affinity, Gen has only a slight preference for transactivation of gene expression in vitro through ERß compared with ER, suggesting that Gen may elicit effects in vivo through both ER- and ERß-mediated pathways (47). Evidence for ER-mediated actions of Gen in vivo is derived from studies showing Gen-induced effects in the mouse uterus, which predominantly expresses ER (45). In addition, Gen activates the IGF-I signaling pathway in the mouse uterus via an ER-dependent mechanism (48). Taken together, these data suggest Gen acts through an ER-mediated mechanism in the uterus.
Evidence suggesting an ER-mediated mechanism for Gen action in the uterus has prompted studies in our laboratory to definitively establish the role of ER in the uterine response to Gen. In these studies, ovariectomized WT and ERKO mice were treated with corn oil, estradiol (10 µg/kg·d), or Gen (50 µg/kg·d) for 3 d, and uterine weights were determined. Both the estradiol and Gen treatments significantly increased uterine wet weights in WT mice, whereas neither ligand increased uterine wet weights in the ERKO mice, providing direct evidence that ER is necessary for Gen-induced uterotropic effects (unpublished data).
Actions on the ovary
To further determine the contributions of ER and ERß to Gen-induced effects in tissues other than the uterus, studies using ER null mice were incorporated into the neonatal mouse model described earlier, and the effects of Gen on the ovary were determined. Initial studies in WT CD-1 mice treated with Gen for 5 d showed a dose-dependent increase in multioocyte follicles by 19 d (49) (Table 2). To prove that the mechanism was due to the estrogenic properties of Gen and not to tyrosine kinase inhibitory properties, the nonestrogenic tyrosine kinase inhibitor lavendustin was incorporated into these experiments. No multioocyte follicles were observed after treatment with lavendustin regardless of dose, indicating that the increased incidence of multioocyte follicles was due to the estrogenic properties of Gen. Similar experiments were performed in C57BL/6 mice and in both ERKO and ßERKO mice to determine whether Gen was signaling in the ovary through ER, ERß, or a non-receptor-mediated mechanism. Neonatal treatment with Gen increased the incidence of multioocyte follicles in WT C57BL/6 and ERKO mice in a dose-dependent manner. In contrast, ßERKO mice treated with Gen had a significant decrease in the incidence of multioocyte follicles, suggesting that the induction of multioocyte follicles after neonatal Gen exposure requires ERß (49) (Table 2).
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- and ERß-mediated mechanisms. Although a variety of physiological and toxicological effects of Gen exposure have been illustrated in animal studies, conflicting data reported in epidemiological studies make it difficult to correlate animal models with human exposure (50). Additional studies characterizing other possible mechanisms of Gen action, such as non-receptor mediated, epigenetic, and transgenerational effects, are needed to broaden our understanding of the potential beneficial and detrimental effects of human exposure and to clarify inconsistencies between epidemiological studies. DBP
Phthalate esters are used extensively as plasticizers and stabilizers in a variety of plastics and consumer goods. Exposure to phthalates through ingestion, inhalation, and dermal absorption occurs throughout life (51). Select phthalate esters, including DBP, adversely affect the male rat reproductive tract after either prenatal or postnatal exposure. These adverse reproductive tract effects, which include disrupted epididymal development, hypospadias, cryptorchidism, multinucleated gonocytes, and reduced fertility, are a result of the antiandrogenic effects of some phthalate esters (52). Interestingly, the reproductive tract abnormalities present in DBP-exposed rats are similar to those that occur in humans with testicular dysgenesis syndrome, which is believed to result from altered fetal development as a result of genetic mutations and/or pharmacological or environmental disruptions (53). Given the widespread use of phthalate esters, a potential role for DBP in testicular dysgenesis is plausible (51, 55). Humans are exposed to more DBP than any other phthalate ester, with maximal DBP exposure reaching 113 µg/kg·d (56, 57). Interestingly, these same studies showed that women of childbearing age have the highest estimated DBP exposures. However, these levels are considerably lower than the minimal reported dose of DBP necessary to alter male reproductive tract development of more than 50 mg/kg·d (52).
Mechanisms of action
Animal models in which rats are exposed gestationally to various phthalate esters have been used to characterize the endocrine-disrupting effects elicited by the phthalate esters. In these studies, DBP elicits its antiandrogenic effects by reducing testosterone production in the Leydig cells of the testis through several mechanisms. However, the androgen receptor (AR) antagonist flutamide, in contrast to DBP, has little effect on the developing epididymis, suggesting a complex mechanism underlying DBP-mediated reproductive tract effects that involves more than its antiandrogenic properties (52). Furthermore, neither DBP nor its major metabolite, monobutyl phthalate, physically interacts with the AR, indicating that the antiandrogenic effects of DBP occur through AR-independent mechanisms (52).
One such established mechanism is the transcriptional down-regulation of genes associated with cholesterol transport (Scarb1 and Star) and testosterone biosynthesis (Cyp11a1, Hsd3b1, and Cyp17a1) that results in decreased testosterone production by the Leydig cells of the testis (58, 59, 60). Recent studies using microarray analyses to characterize the effects of DBP treatment on global gene expression profiles in the fetal rat testis show that DBP affects the expression of nearly 400 genes in Leydig cells, Sertoli cells, and gonocytes (61). With respect to the fetal Leydig cell, DBP increased the expression of genes such as Nalp6, which is known to inhibit Leydig cell testosterone synthesis, and decreased the expression of genes such as Npc and Lhcgr that up-regulate testosterone production. Within Sertoli cells, DBP increased the expression of testin and decreased Gja1 expression, both of which are associated with gap junction signaling, suggesting altered communication between Sertoli cells and gonocytes.
The dynamic nature of DBP-mediated effects is further illustrated by time course experiments in which DBP reduces testosterone production within 1 h of treatment, before detectable decreases in gene expression associated with cholesterol transport and steroid synthesis (62). DBP rapidly increased the expression of several genes, including members of the immediate-early gene family, which may play a role in the early decrease in testosterone production.
Although the mechanisms by which phthalate esters such as DBP alter the expression of genes required for normal male reproductive tract development have not been fully characterized, DBP serves as an example of an endocrine disruptor that elicits multiple effects on the male fetal reproductive tract, including antiandrogenic effects, without physically associating with the AR or altering AR-mediated signaling. Interestingly, recent studies have shown that DBP can activate the constitutive androstane receptor (CAR), the pregnane X receptor (PXR) (63), and the peroxisome proliferator-activated receptor subtypes (PPAR, -ß, and -
) (64), all of which are nuclear receptors (NRs) concentrated in the liver that regulate several metabolic enzymes, including those involved in steroid metabolism. These findings suggest that phthalate-induced effects on the male reproductive tract may be mediated by one or more of these NRs; however, additional research, perhaps using the CAR, PXR, and PPAR knockout mice, is needed to address this possibility.
MAA
MAA is the major metabolite of ethylene glycol monomethyl ether (EGME), an industrial solvent commonly used in varnishes, paints, dyes, and fuel additives (65). Exposure to EGME and MAA results in toxic reproductive effects in both animals and humans (66, 67, 68, 69, 70, 71). Occupational exposure to both EGME and MAA has been associated with subfertility, spontaneous abortion, and reduced sperm counts (70, 72, 73, 74). The toxic effects of MAA have prompted investigations into the cellular and molecular actions of MAA that have uncovered unique actions for an EDC.
In vitro actions
In vitro studies show that MAA exerts its effects by potentiating the ligand-induced transcriptional activity of ER, ERß, thyroid hormone receptor (TRß), progesterone receptor (PR), and AR, indicating that MAA may influence the activity of a shared component of NR signaling (75, 76). However, MAA has negligible effects on NR-mediated transcriptional activity in the absence of hormone, indicating it is not functioning simply as a NR agonist. This was confirmed by receptor-binding assays, which showed that MAA does not compete with estradiol for binding to the ER (76). The potentiation effects of MAA are also not a result of the derepression of gene transcription, because microarray data show that MAA alone altered the expression of only three transcripts after a 24-h exposure. In the same experiment, addition of the PR agonist R5020 potentiated the PR-mediated expression of 16 genes and resulted in the induction of four new genes, indicating that MAA can alter NR target gene specificity.
In vivo actions
Additional experiments were performed to determine whether MAA had similar effects on NR function and gene expression in vivo. Immature CD-1 female mice were treated with the synthetic progestin R5020 in the absence and presence of MAA, and uterine levels of calcitonin mRNA expression were determined. Results showed that neither R5020 nor MAA alone had any effect on calcitonin expression, but a combination of the two produced an approximate 4-fold increase in expression, indicating MAA was capable of potentiating ligand-dependent NR-mediated transcriptional activation in vivo.
Mechanisms of action
The observation that MAA increases the transcriptional activity of multiple NRs suggests it may target a common component of NR action. One such potential target is the MAPK signaling pathway, which upon activation has been shown to potentiate the agonist-induced activity of both ER and PR (54, 77 MAA increased the levels of activated Ras and ERK1/2 in HeLa cells, and a MAPK kinase inhibitor reduced the MAA-induced potentiation of agonist-bound PR by 60% in a reporter gene assay, indicating MAA is potentiating NR signaling through a MAPK-dependent signaling pathway (76). In addition to activating MAPK, MAA may regulate NR activity by modifying chromatin structure because MAA inhibits histone deacetylase activity in vitro and in vivo, resulting in increased levels of acetylated histone H4 (76).
The results of these studies reveal a novel mechanism of action for an EDC, in which MAA potentiates the ligand-dependent transcriptional activity of multiple NRs by targeting a common pathway(s) in NR-mediated signaling. This suggests that MAA exposure may potentiate the effects of weak nuclear receptor agonists found in the environment, producing a response indicative of a full agonist. Considering this possibility and the potential health impact associated with it, additional research evaluating the effects of MAA on NR activation by weak agonists, including environmental compounds, is needed.
Conclusions
DES, Gen, DBP, and MAA are four compounds described herein that illustrate the diverse biological effects and mechanisms of action used by EDCs to modulate endocrine system function. Studies on these compounds show that EDCs can act via receptor-mediated and/or non-receptor-mediated mechanisms to influence endocrine system function. The observation that EDCs can modulate the endocrine system in a receptor-independent manner has required investigators to reassess the criteria for classifying a compound as an EDC. The varied and sometimes complex mechanisms of action of EDCs, coupled with the physical and chemical diversity among members of the EDC family, suggest there may be numerous additional mechanisms used by EDCs that have yet to be uncovered. Future progress in identifying and characterizing EDCs will require an appreciation for their diverse mechanisms of action and will likely depend on the development of new screening methods and experimental models that account for this diversity.
Footnotes
This research was supported by the intramural research program of the National Institutes of Health, National Institute of Environmental Health Sciences.
D.V.H. and K.S.K. have nothing to declare.
First Published Online May 11, 2006
Abbreviations: AR, Androgen receptor; DBP, di(n-butyl) phthalate; DES, diethylstilbestrol; EDC, endocrine-disrupting chemical; EGME, ethylene glycol monomethyl ether; ER, estrogen receptor; ERE, estrogen response element; ERKO, ER knockout; Gen, genistein; MAA, methoxyacetic acid; NR, nuclear receptor; PR, progesterone receptor; WT, wild-type.
Received August 31, 2005.
Accepted for publication November 24, 2005.
References
knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract. Dev Biol 238:224–238[CrossRef][Medline] mediates the detrimental effects of neonatal diethylstilbestrol (DES) exposure in the murine reproductive tract. Toxicology 205:55–63[CrossRef][Medline]: studies with ERKO and ßERKO mice. Cancer Res 61:6089–6097 (ER) and ERß in human cells. Toxicol Sci 80:14–25. Endocrinology 141:3430–3439 expression and multioocyte follicles in the maturing mouse ovary: evidence for ERß-mediated and nonestrogenic actions. Biol Reprod 67:1285–1296 in mediating the effects of phthalates and metabolites in the liver. Toxicology 207:149–163[CrossRef][Medline] and stimulation of the agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells. Mol Endocrinol 14:1882–1896
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