University of Texas, M.D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957
Received September 8, 1999; accepted December 13, 1999
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ABSTRACT |
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Key Words: uterine leiomyoma; uterine myometrium; endocrine disruptors; organochlorine pesticides; 17ß-estradiol.
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INTRODUCTION |
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Although the etiology of uterine leiomyomas is unknown, estrogen and downstream signaling from the estrogen receptor (ER) is generally believed to play a major role in the pathogenesis of these tumors in women. Leiomyomas develop during the reproductive years and decrease in size after menopause (Novak and Woodruff, 1979). Creation of a hypoestrogenic state, such as during GnRH agonist therapy, causes a reduction in tumor volume (Friedman et al., 1990
). Increased ER levels along with an elevated transcriptional response to estrogen in leiomyoma compared to normal myometrium has been reported, suggesting that leiomyomas may be hypersensitive to estrogen (Andersen et al., 1995b
; Rein et al., 1990
). In addition, several estrogen-regulated genes, such as the progesterone receptor, and the growth factors IGF-I, EGF, and their receptors have been found to have elevated expression in uterine leiomyomas (reviewed in Andersen and Barbieri, 1995a). The hormone responsive nature of the normal uterus and the increased sensitivity of leiomyomas to estrogen make them potential targets of endocrine disruption by exogenous chemicals.
Organochlorine pesticides are in widespread use and even pesticides that have been banned in western countries for more than two decades are still detectable in these regions (Blais, 1998; Simonich and Hite, 1995) and in mammalian fat stores (Stellman et al., 1998
). Many organochlorine pesticides have been associated with estrogenic activity both in vivo and in vitro. Exposure to the organochlorine pesticide kepone, previously banned in part for its estrogenic activity, produces persistent vaginal estrous and anovulation in rats treated neonatally (Gellert, 1978
). Methoxychlor, a pesticide still in common use, exhibits multiple estrogenic effects in neonatally exposed mice such as precocious vaginal opening, persistent vaginal estrous, and alterations in initiating and/or maintaining pregnancy (Eroschenko and Cooke, 1990
; Swartz and Eroschenko, 1998
). Soto et al. found that endosulfan, toxpaphene and dieldrin have estrogenic effects on breast cancer cells using the E-screen assay (Soto et al., 1994
). Significantly higher levels of DDT and its metabolites have been detected in the blood of women with leiomyoma (Khare, 1985
) and also in leiomyomatous tissue (Saxena et al., 1987
). These studies suggest the possibility of a link between the development of leiomyoma and exposure to organochlorine pesticides with estrogenic properties. However, studies to determine if these compounds have agonist activity in uterine myometrial cells are lacking.
As a first step toward addressing the possibility that organochlorine pesticides act as estrogen receptor agonists in the uterine myometrium, we tested a panel of 7 organochlorine pesticides for their ability to stimulate leiomyoma cell growth and induce an estrogenic response on the molecular level in this cell type. The cell lines used in these assays were derived from spontaneous uterine leiomyoma from the Eker rat and have been characterized previously for their estrogen responsiveness (Everitt et al., 1995; Howe et al., 1995a
,b
). Several functional assays for estrogen activity were utilized: stimulation of cell growth in vitro, a transcriptional assay utilizing the vitellogenin estrogen response element (ERE), and induction of an endogenous estrogen-responsive gene, the progesterone receptor (PR). The results presented here indicate that all the pesticides examined act as estrogen receptor agonists at the molecular level in leiomyoma cells and several are fully functional agonists that stimulate uterine myometrial cell growth. Consequently, these results suggest that exposure to these compounds could contribute to the pathogenesis of uterine leiomyoma by altering endocrine signaling via the estrogen receptor.
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MATERIALS AND METHODS |
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Chemicals
Kepone and toxaphene were purchased from Supelco (Bellefonte, PA). Methoxychlor and dieldrin were purchased from Aldrich (Milwaukee, WI). 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) was a kind gift of Dr. Leo T. Burka. The endosulfan isomers and ß were purchased from Chem Service (West Chester, PA). 17ß-Estradiol was purchased from Sigma. ICI 182,780 was a kind gift of Dr. Robin Fuchs-Young. DMSO was used as a solvent for toxaphene (TXP), kepone (KPN), methoxychlor (MXC), dieldrin (DLN), and HPTE. Ethanol was used as a solvent for 17ß-estradiol (E2), ICI 182,780, and the endosulfan isomers
and ß (Endo
, ß). According to the manufacturers, the minimum chemical purity of each of the organochlorine pesticides is: MXC-95%, HPTE-98%, KPN-96%, DLN-90%, Endo
- 99.5%, Endo ß-98%. The purity of TXP is not available due to its complex mixture. All solutions of chemicals used to treat cells were diluted 1:1000 in DF8-basal medium, unless otherwise stated.
Proliferation Assays
ELT 3 cells were plated into 24-well cell-culture dishes (Corning, Corning, NY) in DF8 medium and incubated 48 h at 37°C in 5% CO2-humidified atmosphere. The wells were aspirated and rinsed twice with 1X phosphate-buffered saline (PBS), and triplicate wells were dosed with 1 ml of DF8-basal medium containing pesticide or control solutions. At each time point, triplicate wells were rinsed with 1X PBS and cells detached with 5X trypsin-EDTA (Gibco BRL, Grand Island, NY), resuspended in DF8 medium, and counted with a Coulter counter (Coulter Electronics, Hialeah, FL).
Reporter Gene Assays
ELT 3 cells were transiently transfected in 12-well plates with the vitellogenin ERE-tk-LUC6a reporter plasmid, human ER expression plasmid pRSVT7 (both described by Tzukerman et al., 1994) and a control plasmid containing a constitutive CMV promoter-driven ß-galactosidase reporter (kind gift of Dr. Andrew Butler). Transfections were performed using the calcium phosphate method at a reporter:receptor:ß-gal ratio of 9:1:1. The following day, cells were rinsed twice with 1X PBS, and were dosed with DF8-basal medium containing DMSO, 17ß-estradiol, or test compounds. Two days later, cells were rinsed with 1X PBS and harvested and assayed for reporter-gene activity using GalactolightTM and Luciferase Assay Kit by TropixTM (Bedford, MA) according to the manufacturer's instructions. Luminescence was detected using a Dynex-MLX luminometer (Chantilly, VA), and luciferase values from triplicate wells were normalized to ß-galactosidase values for the same wells, to control for transfection efficiency. The ß-galactosidase values for 50 and 100 µM dieldrin were consistently lower than vehicle, suggesting some cytotoxicity at high doses for this compound.
Quantitative RT-PCR
The expression levels of PR, as well as of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were measured with a quantitative RT-PCR technique utilizing a competitive template. The competitor template was generated for each gene using the method of Celi (Celi et al., 1993) from rat cDNA. The primer sets for each gene included upstream (A), internal downstream (BC), and external downstream (C) primers. PR and GAPDH sequences were as follows: PR (A) 5'-CTGAATGAGCAGAGGATGAA-3', (CB) 5'-GACCACCCCTTTCTGTCTTAACCTCATCTCTTCAAACTGG-3', (C) 5'-GACCACCCCTTTCTGTCTTA-3', and GAPDH (A) 5'-AAACCCATCACCATCTTCCAG-3', (BC) 5'-AGGGGCCATCCACAGTCTTCTTCCACGATGCCAAAGTTGTCA-3' and (C) 5'-AGGGGCCATCCACAGTCTTCT-3'. Primers were designed so that amplification of both endogenous and competitor targets could be performed using a single primer pair (A and C), with the competitor product being 3638 bp shorter than the endogenous product. Each competitor template was amplified using the A and BC primers, purified by electrophoresis, quantified by spectrophotometry, serially diluted in H2O, and stored at 20°C.
ELT 3 cells were plated in DF8 medium and switched to DF8-basal medium 20 to 30 h before the addition of chemicals. The following concentrations of chemicals were used: 10 µM KPN, 10 nM MXC, 100 nM TXP, 1 µM HPTE, 1 µM ICI 182,780, 10 nM E2. These concentrations were chosen based on their ability to stimulate growth in the in vitro proliferation assay, or if no proliferation was observed, the highest dose that did not significantly inhibit proliferation was chosen. Three concentrations of DLN were used: 100 nM,1 µM and 10 µM. Cells were harvested after 48 h from log phase cultures. Total RNA was isolated from ELT 3 cells by standard methods of cesium chloride isolation. An RT reaction of 20 µg of total RNA was performed for 1 h at 37°C in a 100 µl reaction containing 50 mM KCl, 20 mM TrisHCl (pH 8.4), 2.5 mM MgCl2, 0.4 mM dNTPs, 5 pM/ml random hexamer primers, 400 U RNase inhibitor (Promega, Madison, WI), and 1000 U MMLV-reverse transcriptase (Gibco BRL, Gaithersburg, MD). The cDNA produced was amplified by PCR, using A and C primers, in a 25 µl reaction containing 2.5 µl 10X PCR buffer, 1.25 µl of 4 mM dNTPs, 1 µl of each primer, 0.25 µl Taq polymerase and 6 µl H2O. Three µl of 1:2 diluted cDNA were added along with increasing amounts of competitor template cDNA (0 to 25 ng). Amplification was performed for 25 cycles with an annealing temperature of 56°C. PCR products were visualized using electrophoresis on 6% TBE/urea gels (NOVEX, San Diego, CA), and stained with ethidium bromide. The density of the bands was determined using the Fluorimager SI and ImageQuant software, both purchased from Molecular Dynamics (Sunnyvale, CA). The ratio of band densities for competitor/endogenous versus known competitor concentrations was plotted, and linear regression was used to calculate a line with at least r2 0.95 from this figure. The concentration of endogenous target in each reaction was assumed to be equal to the concentration of competitor template, calculated by the ratio of band densities equaling 1 in the equation generated by linear regression. Quantitative PCR, using the GAPDH primer set and competitive template, was also performed to normalize for the amount of cDNA produced in each RT-PCR reaction, using the PR primers. The same reaction conditions were used in the GAPDH reaction, except that 3 µl of 1:200 diluted cDNA and increasing amounts from 0 to 5 ng of GAPDH competitive template were used, due to higher expression levels.
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RESULTS |
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DISCUSSION |
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The increased activity of metabolite HPTE over its parent compound, methoxychlor, in the proliferation assay illustrates that the metabolic products of organochlorine pesticides may be more estrogenic than the original chemical, most likely due to the absence of relevant metabolizing enzymes. Bulger et al have shown that in whole rat uterine extracts methoxychlor is a proestrogen that requires metabolic activation by hepatic microsomes to induce translocation of the ER to the nucleus (Bulger et al., 1978a,b
). In addition, certain isomers of a chemical may exhibit more estrogenic activity than other isomers as we have shown for endosulfan. Although the
and ß isomers of endosulfan were estrogenic in the transcription assay and PR induction, only the
isomer showed agonistic activity for cell proliferation. While HPTE and KPN stimulated cell proliferation, higher doses were inhibitory to proliferation, most likely due to cytotoxicity. There may be a very fine line in cell signaling whereby the estrogenic response is overcome by toxic effects. A similar dose-effect has been observed by the phytoestrogen genistein on leiomyoma cells, with higher doses of genistein exhibiting non-ER-mediated inhibition (Hunter, et al., 1999
). In addition, proliferative doses of HPTE, KPN and Endo-
are 101000 times higher than for 10nM E2, most likely due to the lower binding affinities of these compounds for ER (Bulger et al., 1978a
; Kuiper et al., 1997
).
The Eker rat model of spontaneous uterine leiomyoma closely models the human disease since they develop tumors with similar frequencies and proliferate in response to ovarian hormones. Ovariectomizing these animals results in virtual ablation of tumors (Walker et al., manuscript submitted), although the precise role of E2 and progesterone in tumor initiation and progression have not been elucidated. Uterine leiomyoma isolated from Eker rats exhibit an altered responsiveness to steroid hormones (Burroughs et al., manuscript submitted), exhibiting proliferation even in response to very low hormone levels. Uterine leiomyoma thus may be sensitive to very low levels of steroid hormones and the presence of even modest levels of xenoestrogens such as organochlorine pesticides could contribute to the growth of these tumors. These data, along with previous work by us correlating in vitro AF-2 transactivation with agonist activity in vivo (see below), indicate that the uterine myometrium, in addition to the more obvious hormone-responsive tissues, represent a potential target tissue of endocrine-active compounds. These in vitro assays can be used for not only screening potential endocrine disruptors that target the uterine myometrium, it also can be used to dissect mechanisms behind tissue-specific responses elicited by endocrine disruptors.
To consider xenoestrogens in a mass-action context in which their impact is relegated to the production of a hyperestrogenic state may be too limiting, since the ER-xenoestrogen complex may form conformations that are distinct from the ER-E2 complex. The receptor-ligand complex could potentially interact with estrogen-responsive promoter regions in a manner that may produce a dose response to xenoestrogens that differs from the dose response seen to E2 (Stancel et al., 1995). The partial ER agonist tamoxifen produces a dose-response pattern distinct from E2 for regulating estrogen-responsive genes (Nephew et al., 1993
). Furthermore, ER ligands have varying effects in hormonally responsive tissues, perhaps contributing to a unique response of the uterine myometrium to exogenous estrogens. For example, tamoxifen and its active metabolite 4-hydroxytamoxifen act as antagonists on Eker rat uterine leiomyoma-derived cells (Howe et al., 1995b
) and decreases tumor incidence in vivo in this animal model (Walker et al., manuscript submitted). Tamoxifen also acts as an antagonist in the breast (Jordan, 1992
). In contrast, tamoxifen is a partial agonist for the uterine endometrium, bone, and cardiovascular system (Sato et al., 1996
). Thus, the cellular milieu of the uterine myometrium may be an important factor in determining whether this often overlooked, hormonally-sensitive tissue will be a target for agonist activity by endocrine disruptors.
The tissue and promoter specificity of ER ligands may be partly due to the ability of ligands to activate transcription via AF-1 alone or via both AF-1 and AF-2 domains of the ER. While activation of the AF-1 domain is generally considered a constitutive function of partial and full agonists, activation of the AF-2 domain of the ER has been shown to be ligand-, promoter-, and cell-type-specific (Berry et al., 1990). The vitellogenin ERE has been shown to require AF-2 for transcriptional activity and the partial agonist tamoxifen is unable to activate transcription from this construct (Tzukerman et al., 1994
). The partial agonists tamoxifen and the raloxifene analogs LY117018 and LY317783, all antagonists for uterine leiomyoma cell growth in vivo, are unable to stimulate transcription from the vitellogenin ERE in a myometrial cell background. This is in contrast to the synthetic estrogen diethylstilbestrol (DES) which can stimulate transcription in vitro as well as proliferation in vivo (Hunter et al., 1999
). These results led us to propose previously that AF-2 activation of the ER is necessary but not sufficient for full agonist activity in this cell type. All of the organochlorine pesticides in these experiments stimulated transcription of the reporter gene containing the vitellogenin ERE, suggesting that these endocrine active compounds can activate AF-2 and have the potential to act as agonists in the uterine myometrium in vivo. Given the tissue, cell, and promoter specificities of many ER ligands, it is important to consider each target tissue as unique and correlate in vitro data with agonist activity in vivo.
In the 3 in vitro assays used to determine agonist activity, the organochlorine pesticides examined in this study exhibited differential activity profiles depending on the assay employed. All compounds were able to induce expression of an endogenous estrogen-responsive gene, the PR, whereas only HPTE, KPN, and Endo- were able to stimulate transcription from the vitellogenin ERE and induce cell proliferation. Previous in vitro and in vivo data suggest that the latter 2 assays, proliferation and transactivation, are the most predictive for agonist activity in this target tissue. We have examined other xenoestrogens, namely the pharmaceutical agents DES, tamoxifen, and two raloxifene analogs, LY117018 and LY317783, for activity in these same in vitro assays and for agonist activity in vivo. In vitro, only DES induces proliferation of ELT 3 cells and exhibits the ability to transactivate the vit-ERE; tamoxifen and the raloxifene analogs both fail to induce proliferation or vit-ERE transactivation, whereas all 3 compounds upregulate the progesterone receptor in vitro (Hunter et al., 1999, and unpublished data). In vivo, only DES induces proliferation in the uterine myometrium (Hunter et al., 1999
), while treatment with tamoxifen or LY326315 reduces the incidence of uterine leiomyoma by ~50% (Walker et al., manuscript submitted). Therefore, transactivation of the vitellogenin ERE and stimulation of cell proliferation correlate best with in vivo agonist activity in uterine myometrial cells. In contrast, the induction of the PR appears to be a promiscuous response to estrogen receptor ligands in this cell type, possibly because it may require functional activation only of AF-1 of the ER. Consequently, induction of PR does not correlate well with in vivo agonism in uterine myometrial cells.
It has been suggested that the increased incidence of cancer in hormonally responsive tissues is linked to environmental factors (Colborn et al., 1993). However, there have been several conflicting reports correlating breast cancer and organochlorine exposure. Wolff et al. reported in 1993 that there are higher levels of the DDT metabolite DDE in the serum of women with breast cancer, while more recently Hunter et al. (1997) found no association between organochlorine pesticide levels in serum and breast cancer. A recent study by Hoyer et. al. (1998) found no increase in the levels of DDT or its metabolites in the serum of breast cancer patients, but they did find a significantly higher level of dieldrin. Determining exposure levels may be more complex than just measuring serum levels, due to complex issues such as bioaccumulation and the developmental stage in which people are exposed to these compounds. They bioaccumulate in fat stores and are mobilized during lactation (Sonawane, 1995
) and fasting (Bigsby et al., 1997
). This mobilization could result in exposure levels that are several-fold higher than those originally encountered in the environment. The timing of exposure to endocrine disruptors is another important factor, since hormones play a pivotal role in many stages of the life cycle including development, puberty, pregnancy, lactation, and menopause. Exposure to xenoestrogens at a time when endogenous estrogens are low could potentially produce an inappropriate estrogenic response resulting in deleterious effects on the reproductive system. For example, neonatal treatment of rats to KPN results in persistent vaginal estrus and anovulation (Gellert, 1978
).
In the present study, although only a subset of compounds (HPTE, KPN, and Endo-) were able to stimulate cell proliferation, all the organochlorine pesticides in the panel exhibited agonistic activity on the molecular level. Even in the absence of cell proliferation, an alteration in endocrine signaling could potentially impact the growth and/or development of uterine leiomyoma by perturbing the hormonal milieu or downstream signaling from the PR and/or ER. The increase in PR message produced by these compounds in uterine leiomyoma cells is of particular concern, especially since PR is found to be increased in leiomyomas compared to the normal myometrium (Brandon et al., 1993
; Rein et al., 1990
; Viville et al., 1997
). The consequences of these molecular changes in uterine leiomyomas are unknown at present but further study of the impact of the pesticides in vivo will be necessary to determine if they constitute a novel mechanism of uterine leiomyoma pathogenesis due to altered endocrine signaling.
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ACKNOWLEDGMENTS |
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NOTES |
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