Influence of exogenous estrogen receptor ligands on uterine leiomyoma: evidence from an in vitro/in vivo animal model for uterine fibroids.

The remarkable frequency of uterine leiomyoma in the human population calls into question the potential for the participation of environmental factors in tumor etiology. Having been implicated in the dramatic rise in hormone-related cancers in recent years, endocrine disruptors are salient suspects in this pathogenesis, although the mechanism by which they might participate is unclear. Investigations using the Eker rat model show that uterine leiomyoma may have an enhanced sensitivity to modulation via the estrogen receptor. This sensitivity could make these tumors a target for disruption by exogenous estrogen receptor ligands. Direct evidence for a pathogenic role of exogenous compounds in leiomyomas is lacking; however, it can be demonstrated that such diverse agents as organochlorine pesticides, dietary flavonoids, botanical extracts, and therapeutic antiestrogens have either estrogen agonist or antagonist function in myometrial tissues. The use of this model will help define the impact of exogenous estrogen receptor modulators on uterine leiomyoma and will permit the evaluation of strategies for therapeutic intervention.

Estrogen receptor activation via activation function 2 predicts agonism of xenoestrogens in normal and neoplastic cells of the uterine myometrium.

The possible contribution of endocrine disrupters to human disease, particularly those compounds that modulate the estrogen receptor (ER), has recently drawn considerable attention. The tissue specificity of effects mediated by the ER is well recognized, although the mechanism of this specificity is not understood sufficiently to predict the effects of a particular ligand in different target tissues. Although the divergence of ER-mediated effects in the breast, bone, and uterine endometrium has been described, a frequently overlooked site of estrogen action is the smooth muscle of the uterus. The uterine myometrium is the tissue of origin of an extremely common hormone-responsive tumor, uterine leiomyoma, a tumor with a significant impact on women's health and a possible environmental influence. This report describes an in vitro/in vivo system for identifying the effects of ER ligands in the myometrium and elucidating their mechanism of action. Several natural and synthetic xenoestrogens were evaluated at the cellular and molecular level for their ability to mimic estrogen action in uterine myometrial tissues. Diethylstilbestrol, coumestrol, genistein, naringenin, and endosulfan were able to activate the AF2 function of the ER in vitro and demonstrated agonist activity in estrogen-responsive myometrial cells, as determined by induction of proliferation and increased message levels of progesterone receptor. Compounds that could not activate AF2 function (4-hydroxy-tamoxifen, LY117018, and LY317783) did not act as estrogen agonists. For agonists, rank order of potency was predicted by receptor affinity; however, endosulfan displayed a surprising degree of activity, despite negligible receptor binding. Additionally, diethylstilbestrol and tamoxifen demonstrated prototypical agonist and antagonist effects, respectively, in the intact myometrium of sexually mature rats. The results presented here suggest that some exogenous ER ligands may mimic the effects of endogenous estrogens on uterine leiomyoma and may contribute to a complex hormonal milieu that impacts both normal and neoplastic myometrium.

Environmental signaling: a biological context for endocrine disruption.

Endogenous and exogenous chemical signals have evolved as a means for organisms to respond to physical or biological stimuli in the environment. Sensitivity to these signals can make organisms vulnerable to inadvertent signals from xenobiotics. In this review we discuss how various chemicals can interact with steroid-like signaling pathways, especially estrogen. Numerous compounds have estrogenic activity, including steroids, phytoestrogens, and synthetic chemicals. We compare bioavailability, metabolism, interaction with receptors, and interaction with cell-signaling pathways among these three structurally diverse groups in order to understand how these chemicals influence physiological responses. Based on their mechanisms of action, chemical steroid mimics could plausibly be associated with recent adverse health trends in humans and animals.

Cellular bioavailability of natural hormones and environmental contaminants as a function of serum and cytosolic binding factors.

Environmental contaminants have been reported to function as hormone mimics in various wildlife species. To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals. Hormone binding proteins from oviductal cytosol of the American alligator (Alligator mississippiensis) and yellow-bellied turtle (Trachemys scripta) were used in competitive binding assays with [3H] 17 beta-estradiol. Most of the environmental contaminants, and the potent, synthetic estrogen diethylstilbestrol (DES), did not interact with the cytosolic binding proteins. Among the compounds tested, o,p'-DDT and toxaphene exhibited the greatest affinity for the binding proteins. The functional consequence of the apparent lack of interaction of most contaminants with binding proteins was studied in a strain of yeast containing the human estrogen receptor (YES assay). The activation of YES with estradiol was reduced 30% in the presence of a physiological concentration (0.01 mg/mL) of human sex hormone binding globulin (SHBG), a hormone binding protein found in the blood. In contrast, the activity of DES was not inhibited by 0.01 mg/mL SHBG. Interestingly, ethinyl estradiol, a major component of contraceptives, did not appear to appreciably interact with SHBG in the YES system. Together, these data suggest that cytosolic and circulating binding proteins bind many environmental contaminants with much less affinity than native steroids. Therefore, such contaminants may be more hormonally active than previously hypothesized.

Synergistic responses of steroidal estrogens in vitro (yeast) and in vivo (turtles).

Many environmental agents exert estrogenic activity. Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994) Environ. Hlth Perspect. 102, 780-782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996) Science 272, 1489-1492). Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens. The activity of combinations of estrone, estradiol-17beta or estradiol-17alpha in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds. However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast. Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor. The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activity in vivo. In this instance, a combination of estriol and estradiol-17beta was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner. Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.

o,p'-DDT and its metabolites inhibit progesterone-dependent responses in yeast and human cells.

Using a combination of in vitro assays we have evaluated whether DDT metabolites can interact with the progesterone receptor pathway in yeast expressing human progesterone receptor (hPR) and in T47D human breast cancer cells which express endogenous hPR. In transactivation assays using both yeast and T47D cells, o,p'-DDT and the metabolites p,p'-DDT, o,p'-DDD, p,p'-DDD, o,p'-DDE, p,p'-DDE, p,p'-DDA, and DDOH inhibited progesterone-induced reporter gene activity in a dose-dependent manner. None of the DDT metabolites functioned as hPR agonists. Whole cell competition binding assays using T47D cells indicated that the inhibitory effects of DDT metabolites on progesterone-dependent activites may occur through both hPR-dependent and hPR-independent pathways. Our results and previous reports of DDT metabolites interacting with estrogen and androgen receptors suggests that this class of environmental chemicals may interact with numerous hormone receptor signaling pathways.

In vitro synergistic interaction of alligator and human estrogen receptors with combinations of environmental chemicals.

The effect of mixtures of environmental chemicals with hormonal activity has not been well studied. To investigate this phenomenon, the estrogen receptor (ER) from the American alligator (aER) or human (hER) was incubated with [3H]17beta-estradiol in the presence of selected environmental chemicals individually or in combination. The environmental chemicals included the insecticide chlordane, which has no estrogenic activity, and the pesticides dieldrin and toxaphene, which have very weak estrogenic activity. Chlordane, dieldrin, and toxaphene individually demonstrated no appreciable displacement of [3H]17beta-estradiol from aER and hER at the concentration tested. A combination of these chemicals inhibited the binding of [3H]17beta-estradiol by 20 to 40%. Alachlor, a chemical recently discovered to have weak estrogenic activity, also displaced [3H]17beta-estradiol more effectively in combination with dieldrin than alone. These results indicate that combinations of some environmental chemicals inhibit [3H]17beta-estradiol binding in a synergistic manner. This suggests that the ER may contain more than one site for binding environmental chemicals. The possibility that the ER binds multiple environmental chemicals adds another level of complexity to the interaction between the environment and the endocrine system.

Identification and characterization of estrogen and progesterone receptors from the oviduct of the American alligator (Alligator mississippiensis).

A protein extract prepared from the egg shell fiber-secreting region of the oviduct of Alligator mississippiensis was assayed for the presence of receptors for estrogen and progesterone. In the presence of [3H]-estradiol-17 beta, the extract contained an estrogen-binding activity which reached equilibrium at 25 degrees C in 1 h. Scatchard analysis demonstrated that a single estrogen-binding activity was present in the extract with a Kd of 0.5 nM for [3H]estradiol-17 beta. A steroid specificity competition assay showed the estrogen binding activity strongly recognized estradiol-17 beta and diethylstilbestrol (DES) and weakly interacted with estrone, estriol, estradiol-17 alpha, and dihydrotestosterone (DHT). The estrogen binding activity did not recognize testosterone, dexamethasone or progesterone. The extract exhibited a DNA-binding activity that recognized an estrogen response element in a gel mobility shift assay. We have also identified a high affinity binding activity in the extract that specifically recognized the synthetic progestin R5020 with a Kd of 0.9 nM. This binding activity recognized 17 alpha-estradiol, dexamethasone, testosterone, and estriol. This activity did not recognize DHT, DES, or estradiol-17 beta. These data suggest the presence of estrogen and progesterone receptors in the oviduct of the alligator.

Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator.

Reports of reproductive abnormalities in the American alligator from Lake Apopka, Florida, have been linked to a spill of DDT and other pesticides suspected of having hormonelike activity. To determine whether environmental chemicals had the potential to function as exogenous hormones in the American alligator, we examined the ability of chemicals to bind the estrogen receptor (aER) and progesterone receptor (aPR) in a protein extract prepared from the oviduct of the alligator. In competition binding assays with [3H]17 beta-estradiol, some DDT metabolites showed inhibition of [3H]17 beta-estradiol binding to aER. A combination of DDTs demonstrated an additive decrease in [3H]17 beta-estradiol binding to aER. Modern-use chemicals such as alachlor, trans-nonachlor, endosulfan, and atrazine also competed with [3H]17 beta-estradiol for binding to the aER. To test the effect of chemicals identified in alligator eggs from Lake Apopka on [3H]17 beta-estradiol binding, we mixed these chemicals at concentrations measured in eggs in the competition binding assay. 2,2-bis(4-chlorophenyl)-N-(methoxymethyl)acetamide (p,p'-DDD) and trans-nonachlor, both found in Lake Apopka, interacted with aER, whereas others such as chlordane and toxaphene did not. Surprisingly, combinations of these chemicals decreased [3H]17 beta-estradiol binding in a greater than additive manner. To assess the ability of chemicals to interact with aPR, we performed commpetition binding assays with the synthetic progestin [3H]R5020. Most of the chemicals tested did not reduce [3H]R5020 binding to aPR, whereas endosulfan, alachlor, and kepone inhibited binding. These results provide the first evidence that environmental chemicals bind the aER and aPR from the American alligator, supporting the hypothesis that the reported reproductive abnormalities may be related to the modulation of endocrine-related responses. The findings that combinations of chemicals demonstrated a greater than additive interaction with the aER and some chemicals bind to the aPR in the competition binding assay are novel. This suggests that interactions of these chemicals with the endocrine system are complex.

Synergistic activation of estrogen receptor with combinations of environmental chemicals.

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.