The Environmental Estrogen Bisphenol A Stimulates 

Prolactin Release in Vitro and in Vivo^*

Endocrinology V.138, N.5 1780-1786 May97

Rosemary Steinmetz, Nancy G. Brown, Donald L. Allen, Robert M. Bigsby and Nira Ben-Jonathan

[Description of prolactin]

ABSTRACT

Environmental estrogens (xenoestrogens) are a diverse group of chemicals that mimic estrogenic actions. Bisphenol A (BPA), a monomer of plastics used in many consumer products, has estrogenic activity in vitro. The pituitary lactotroph is a well established estrogen-responsive cell. The overall objective was to examine the effects of BPA on PRL release and explore its mechanism of action. The specific aims were to: 1) compare the potency of estradiol and BPA in stimulating PRL gene expression and release in vitro; 2) determine whether BPA increases PRL release in vivo; 3) examine if the in vivo estrogenic effects are mediated by PRL regulating factor from the posterior pituitary; and 4) examine if BPA regulates transcription through the estrogen response element (ERE).

BPA increased PRL gene expression, release, and cell proliferation in anterior pituitary cells albeit at a 1000- to 5000-fold lower potency than estradiol. On the other hand, BPA had similar efficacy to estradiol in inducing hyperprolactinemia in estrogen-sensitive Fischer 344 (F344) rats; Sprague Dawley (SD) rats did not respond to BPA. Posterior pituitary cells from estradiol- or BPA-treated F344 rats strongly increased PRL gene expression upon coculture with GH₃ cells stably transfected with a reporter gene. Similar to estradiol, BPA induced ERE activation in transiently transfected anterior and posterior pituitary cells.

We conclude that: a) BPA mimics estradiol in inducing hyperprolactinemia in genetically predisposed rats; b) the in. vivo action of estradiol and BPA in F344 rats is mediated, at least in part, by increasing PRL regulating factor activity in the posterior pituitary; c) BPA appears to regulate transcription through an ERE, suggesting that it binds to estrogen receptors in both the anterior and posterior pituitaries. The possibility that BPA and other xenoestrogens have adverse effects on the neuroendocrine axis in susceptible human subpopulations is discussed. (Endocrinology 138: 1780-1786, 1997)

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Received November 11, 1996. Address all correspondence and requests for reprints to: Dr. Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati Medical School, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0521.

* This work was supported by NSF Grant IBN94-09133, US Army Grant DAMD17-94-J-4452, NIH Grant NS13243, and Center for Environmental Genetics Grant P30 ES06096.

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ENVIRONMENTAL estrogens (Xenoestrogens) are a diverse group of chemicals that bind to estrogen receptors, mimic estrogenic actions, and may have adverse effects on human health (1, 2). One such compound is bisphenol A (BPA), a monomer of polycarbonate plastics. As shown in Fig. 1, BPA has two unsaturated phenol rings, but otherwise it has little structural homology with estradiol. It should be noted, however, that BPA and diethylstilbestrol (DES), a synthetic substance with potent estrogenic activity, are structurally similar. Polycarbonates are produced by condensing BPA to form the carbonate linkages of the polymer (3). Because of superior stability, toughness, and pliability, BPA-based epoxy resins are used in many consumer products, including inner coating of food cans, dental composites, and drug delivery systems. Although normally resilient, the carbonate linkages can hydrolyze at high temperatures and release BPA. BPA can also be liberated from incompletely polymerized epoxy resins (3).

Being lipophilic, many xenoestrogens can access the human body by ingestion or absorption through the skin and mucosal membranes. Of particular public health concern are two recent reports on significant amounts of BPA in foodstuff and human saliva. The first study (4) detected BPA in liquid from canned vegetables (10-20 µg/can, or 50-100 nM). The second study (5) found 2030 µg/ml of BPA in saliva collected from subjects treated with composite dental sealants.

The estrogenic activity of BPA was accidentally discovered. After reporting that yeast produced estrogens (6), the authors realized that the estrogenic substance in the conditioned media had leached from polycarbonate flasks during autoclaving of water (7). The substance was purified by HPLC and identified by mass spectrometry as BPA. When incubated with the estrogen-responsive MCF-7 breast cancer cells, BPA induced progesterone receptors, competed with tamoxifen in binding to the estrogen receptor and promoted cell proliferation (7). However, the potency of BPA was 3-4 orders of magnitude lower than that of estradiol.

Most studies to date focused on putative carcinogenic effects of Xenoestrogens, using primarily hr vitro systems, while neglecting their potential impact on the neuroendocrine axis. The pituitary lactotroph is a well characterized estrogen-responsive cell (8, 9). Estrogens can affect PRL release by acting directly on the lactotrophs (10, 11), or indirectly via hypothalamo-pituitary factors that regulate the lactotrophs. These include dopamine, the primary PRL inhibiting factor (12, 13), and PRL regulating factor (PRF) from the posterior pituitary (14). PRF, the structure of which is yet unknown, is produced by a subset of intermediate lobe cells (15) and is the most potent inducer of PRL gene expression (16). PRF-producing cells are likely targeted by estrogens because an intact posterior pituitary is necessary for mediating estrogen-induced surges of PRL (17, 18).

FIG. 1. Chemical structure of estradiol and BPA.

The overall objective of these studies was to examine the effects of BPA on PRL release in vitro and in vivo. For the in vitro system, we used both primary rat anterior pituitary cells and GH₃ cells, a somatomammotroph cell line. For the in vivo system, we used two strains of rats: Fischer 344 (F344) and Sprague Dawley (SD). The inbred F344 rat is exquisitively sensitive to exogenous estrogens that rapidly induce hyperprolactinemia and formation of prolactinomas (19, 20). The SD rat, like other rat strains, responds to estrogens with a moderate rise in plasma PRL levels and does not readily form prolactinomas (21, 22).

The specific objectives were to: a) compare the potency of estradiol and BPA in increasing PRL gene expression and release in vitro; b) determine whether BPA stimulates PRL release in vivo; c) examine whether the stimulation of PRL release by estrogens is mediated, in part, by increasing PRF activity in the posterior pituitary; and d) investigate whether BPA regulates transcription in either anterior or posterior pituitary cells through the estrogen response element (ERE).

Materials and Methods

All animal studies were performed under an institutionally approved protocol according to the USPHS Guide for the Care and Use of Laboratory Animals. Ovariectomized (OVEX) F344 and SD rats (8-10 weeks old) were maintained on a 12-h light, 12-h dark schedule (lights on at 0700 h). SILASTIC brand (Dow Corning, Midland, MI) capsules (1 cm long) made from medical grade SILASTIC tubing (id 0.062", od 0.125") were filled with crystalline 17(3 estradiol (E2; Sigma Chemical Co. St. Louis, MO) or BPA (Aldrich, Milwaukee, WI). The capsules were incubated for a few hours in PBS at 37 C before being inserted sc into the rats. Control rats received empty capsules. After 3 days, rats were decapitated, trunk blood was collected, and the serum was analyzed in duplicate for PRL by RIA, using NIDDK rat PRL kit with RP-3 as a reference preparation. The pituitary glands were removed, the anterior pituitaries weighed, and the posterior pituitaries dispersed with trypsin for use in the coculture experiment.

Because there is no established assay for measuring BPA in body fluids, we compared the release rates of estradiol and BPA under simulated in vitro conditions. SILASTIC capsules filled with crystalline estradiol or BPA were incubated in PBS for 3 days at 37 C. Daily aliquots were fractionated on reversed phase HPLC, eluted isocratically with acetonitrile-water (40:60) and monitored at 254 nm, as described (7). Quantitation was based on peak height. The results showed that BPA and estradiol diffused from the capsules at the approximate rates of 40-45 jig/day and 1.2-1.5 µg/day, respectively.

GH₃ cells were maintained in F-10 media supplemented with 15% horse serum and 2.5% FBS (Life Technologies, Grand Island, NY) and were plated in protamine precoated 96well plates (NUNC, Copenhagen, Denmark) at 2.5 x 10 cells/well as described (23). The cells were first incubated for 48 h in phenol red-free, serum-free media (SFM) composed of DMEM/F-10 (50/50; vol /vol) and supplemented with 1% ITS + Premix (Collaborative Research, Bedford, MA) and penicillin/ streptomycin and then incubated with the test substances for 7 days. Stock solutions of BPA, E2 or testosterone (T; Sigma) were made in ethanol and serially diluted in SFM; final ethanol concentration was 0.001% or less. Media aliquots were analyzed in duplicate for PRL by RIA. At different times during culture, cell number in parallel plates was estimated using the MTT optical density method (15). Anterior pituitaries, removed from OVEX F344 rats, were trypsinized and the cells plated as above at 2.5 x 104 cells/ well. After 4 days in SFM, the cells were washed and incubated with different concentrations of E2 or BPA for 3 days. Media aliquots were analyzed in duplicate for PRL.

GH₃ cells were transfected by electroporation with 5 µg PRL/luciferase plasmid containing 2.5 kb of the 5' flanking region of the rat PRL gene placed upstream of the luciferase coding sequence (a gift from Dr. R. Maurer, Oregon Health Sciences University) and 0.5 jig pcDNA3 neomycin expression vector (Invitrogen, San Diego, CA). Positive clones were selected using 300 jig/ ml geneticin (G418; Promega, Madison, WI), and the resulting stably transfected cells were maintained in 50 Ag/ ml of 6418. The GH₃11UC cells were plated at 2.5 x 104 cells/well and preincubated in SFM for 48 h. The cells were then incubated with E2 (1 pm), BPA (1 nM) or TRH (1 nM) for 8 or 24 h. Luciferase activity (designating induction of the PRL promoter) was determined in cell lysate by luminometry (16).

PRF activity was determined by a bioassay that measures the ability of posterior pituitary cells to increase PRL gene expression when cocultured with the GH₃/luc cells. Posterior pituitaries (neurointermediate lobes) were removed from OVEX F344 and SD rats pretreated for 3 days with E2 or BPA as described above. The cells were dispersed with trypsin, plated at 1 x 10⁴ cells/well and incubated for 4 days in SFM. The GH₃ /luc cells, preincubated for 48 h in SFM, were then added at 2 x 10⁴ cells/ well, and cocultured with the posterior pituitary cells for 24 h. Luciferase activity, determined in cell lysate by luminometry as above, was normalized for cell density that was determined in parallel plates using the MTT assay.

Anterior and posterior pituitaries were pooled from 2-3 OVEX F344 and SD rats. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH), and 5 µg were reverse transcribed using SuperSript II reverse transcriptase (Life Technologies, Grand Island, NY) and random hexamers. For the PCR reaction, 10% of the RT products were used. The samples contained intron-spanning primers for either the ligand binding domain of the estrogen receptor gene (ER-1 5'-GCTCCTAACTTGCTCTTGGACA-3' and ER-2  5'-ATCTCCAGCAGCAGGTCATAGA-3'), or for the POMC gene (MP-2 5'-TCCTGCTTCAGACCTCCATAGA-3' and MP-3 5'-GGAAGTGACCCATGACGTACTT-3'), a marker for intermediate lobe melanotrophs. All PCR reactions also had primers for ribosomal protein L19 (RPL19-1 5'-AGTATGCTTAGGCTACAGAAG-3' and RPL19-2 5'TTCCTTGGTCTTAGACCTGCG-3'), a housekeeping gene serving as an internal standard. Expected product sizes are 500, 415, and 209 by for RPL19, ER, and POMC, respectively. PCR reactions were denatured at 94 C for 30 sec, annealed at 57 C for 30 sec, and extended at 72 C for 30 sec for 25 cycles. Products were separated on a 1.5°/ agarose gel containing ethidium bromide, and the photograph was scanned and analyzed using Scion Image software. The number of cycles and annealing and extension temperatures were optimized, resulting in a linear relationship between band density and RNA amounts (data not shown). Band densities for ER and POMC were corrected for those for RPL19.

Transient transfection of anterior and posterior pituitary cells with ERE/ luciferase reporter gene

Anterior and posterior pituitary cells from OVEX F344 rats were plated in 24 well plates at 6-7 x 10⁴ cells/well, with 3-4 wells per treatment, and cultured for 4 days in SFM. Using calcium phosphate precipitation (Life Technologies) the cells were cotransfected with 5 µg ERE/luciferase plasmid, containing a single Xenopus vitellogenin A2 ERE sequence (GGTCACAGTGACC) placed 5' to a minimal TK promoter driving the expression of the luciferase gene (a gift from Dr. E. Holler, Regnsburg, Germany), and 0.5 µg CMV-ß-galactosidase plasmid. After 18 h, media were changed and the cells incubated with E₂ (10 nM) or BPA (1 µM) for 24 h. Luciferase activity was normalized for ß-gal activity, determined using Galacto-Lite (Tropix, Bedford, MA).

Data analysis

Data were analyzed by analysis of variance, followed by Dunnett's test.

Results 

BPA stimulates PRL release from primary anterior pituitary cells

We first tested whether BPA has estrogenic action in vitro using primary anterior pituitary cells harvested from untreated OVEX F344 rats. The cells were first cultured for 4 days to enable cell attachment and recovery and then incubated with different concentrations of estradiol and BPA for 3 days. As shown in Fig. 2, as little as 1 pm estradiol stimulated PRL release, exhibiting dose dependency up to 1 rim. The magnitude of release in response to estradiol was not large, however, and even at 10 nM (data not shown) the rise in PRL did not exceed 2.5-fold. Like estradiol, BPA increased PRL release in a dose-dependent manner, but at a 1000- to 5000-fold lower potency.

BPA increases PRL release and cell proliferation in GH₃ cells

GH₃ cells, plated in SFM, were incubated with 10 nM estradiol, 1 gm BPA, or 10 nM testosterone for 7 days. Both estradiol and BPA increased PRL release 2- to 3-fold in a time-dependent manner while testosterone was inactive (Fig. 3, left panel). The effect of BPA on cell proliferation was examined next. Within 3-5 days, cell number increased 50 - 60% in response to estradiol or BPA whereas testosterone was ineffective (Fig. 3, right panel). Because GH₃ cells are quiescent without serum, the mitogenic effects of both compounds is significant, albeit small.

A low dose of BPA induces PRL gene expression

The efficacy and time dependency of estradiol and BPA in stimulating PRL gene transcription were compared. GH₃1 'UC cells were incubated with estradiol (1 pM), BPA (1 nM) or TRH (1 nM), and luciferase activity was determined after 8 and 24 h. As shown in Fig. 4, luciferase activity increased 1.5to 2.5fold in response to either estradiol or BPA; higher doses of either compound did not further increase PRL promoter activity (data not shown). Note that 1 nM TRH, a well characterized inducer of the PRL gene, caused a 6- to 8-fold rise in PRL gene expression, whereas estradiol induced only 2-fold elevation. This suggests that the overall effect of estrogenic compounds may involve both direct and indirect effects on PRL synthesis and release.

The objective was 2-fold: a) to determine if BPA increases PRL release in vivo, and b) to compare the effects of BPA and estradiol on PRL release and anterior pituitary weight in F344 and SD rats. To minimize potential negative feedback effects of elevated PRL, a short term exposure of 3 days was chosen. As shown in Fig. 5, basal serum PRL levels were 40 and 25 ng/ml in F344 and SD rats, respectively. Within 3 days, estradiol increased serum PRL levels 10-fold in F344 rats (P < 0.05) but only 3-fold in SD rats (P < 0.5). BPA increased serum PRL levels 7- to 8-fold over controls in F344 rats (P < 0.05) and was without effect in SD rats. As evident in Fig. 6, estradiol doubled the anterior pituitary weight in F344 rats within 3 days of treatment (P < 0.05) but caused no significant increase in pituitary weight of SD rats. Unlike its marked effect on PRL release in F344 rats, BPA did not alter anterior pituitary weight in either F344 or SD rats.

One mechanism by which estrogens might stimulate PRL release in vivo is by increasing PRF activity. Therefore, we examined whether either compound increases PRF activity, using a coculture approach. Posterior pituitary cells from the above rats were incubated for 4 days in SFM and then cocultured with the stably transfected GH₃/luc cells for 24 h. Posterior pituitary cells from untreated rats of either strain increased luciferase activity 3- to 5-fold, indicating basal PRF activity (Fig. 7). The stimulation by cells harvested from estradiol- or BPA-pretreated F344 rats was 75- to 17-fold (P < 0.05), whereas PRF activity in posterior pituitary cells from SD rats was unchanged by the estrogens. Note that the estrogen-induced PRF activity was retained by the cells after several days in culture.

The difference in estrogen responsiveness between the two rat strains could be due to dissimilar expression of pituitary estrogen receptors. The minute size of the rat pituitary, especially the posterior pituitary, hinders receptor characterization by classical methods. Analysis by RT-PCR revealed no significant difference between F344 and SD rats in the level of expression of estrogen receptors in either the posterior or anterior pituitaries (Fig. 8). The estrogen receptors were strongly expressed in the anterior pituitary with a significantly lower expression in the posterior pituitary, possibly due to the scarcity of PRF-producing cells. As expected, POMC expression was much higher in the posterior than anterior pituitaries.

BPA activates ERE in both anterior and posterior pituitary cells

We next examined whether BPA regulates transcription through an ERE. Anterior and posterior pituitary cells harvested from OVER F344 rats were transiently transfected with ERE/luciferase reporter gene and incubated with estradiol (10 nM) or BPA (1 µM) for 24h. Luciferase activity was expressed as relative light units (RLU) after correction for ß-gal. As shown in Fig. 9, like estradiol, BPA stimulated ERE-dependent gene expression, suggesting its binding to estrogen receptors in both tissues.

Discussion

We are reporting that BPA, a monomer of plastics that is abundant in the environment, mimics estradiol in stimulating PRL secretion both in vitro and in vivo. Like estradiol, BPA induced hyperprolactinemia in an estrogen-sensitive rat, but had only weak estrogenic activity in vitro. The in vivo actions of estradiol and BPA in F344 rats were mediated in part by increasing PRF activity in the posterior pituitary. BPA appears to regulate transcription through an ERE, suggesting that it binds to estrogen receptors in both the anterior and posterior pituitaries.

Our data show that BPA mimicked estradiol in inducing PRL gene expression, release, and cell proliferation in both primary anterior pituitary cells and GH₃ cells. Similar to its action on MCF-7 cells (4, 5, 7), the potency of BPA in vitro was 1000- to 5000-fold lower than that of estradiol. In contrast, BPA was rather effective in stimulating PRL release in vivo, albeit only in F344 rats. The discrepancy between the efficacy of BPA in vitro and in vivo could be due to a combination of factors. First, under simulated in vitro conditions, BPA diffused from the capsules 30-35 times faster than estradiol; this alone, however, cannot explain its increased efficacy in vivo. Second, the pharmacokinetics of BPA may differ from that of estradiol because of higher resistance to degradation, lesser binding to sex-hormone binding proteins, or retention in fat tissues. All of these possibilities should be examined. Third, BPA in vivo may form metabolites, e.g. 5-hydroxy bisphenol and bisphenol o-quinone (24), that are either more active than BPA or synergize with it. As reported recently, combinations of two weak xenoestrogens can be 100 to 1000 times as potent in activating estrogen receptors as each substance alone (25).

We also explored the mechanism underlying the estrogen-sensitivity of F344. Previous reports suggested that the genetic susceptibility of F344 rats to estrogens resides in the pituitary because uterine growth in response to estrogen is normal (19). Furthermore, only pituitaries from F344, but not other strains, increased in size when grafted to the kidney capsule of estrogen-treated recipients (19). Other reports suggested increased neovascularization in the pituitary gland (26) and elevated production of basic fibroblast growth factor in F344 rats in response to estrogens (21). The present data confirmed rapid induction of hyperprolactinemia in F344, but not SD, rats by estrogens. This could be due either to altered estrogen receptors and/or estrogen-responsive gene(s) that affect the lactotrophs in F344 rats. Our results suggest that while functional estrogen receptors are present in both the anterior and posterior pituitaries (Fig. 9), there were no apparent differences in their expression between F344 and SD rats (Fig. 8). Still, the difference between the rat strains could be attributed to the presence of estrogen receptor splice variants (27) or estrogen receptor ß (28). We have preliminary evidence that both the anterior and posterior pituitaries express a truncated estrogen receptor product (TERP) as well as estrogen receptor ß. These findings are presently being confirmed and expanded. Alternatively, the difference between the rat strains could reside in factors downstream of the receptor, e.g. coactivators, repressors, or sequence and binding affinity of ERE on target genes. It would be of interest to further investigate these possibilities.

The coculture data clearly show that estrogens increase PRF activity in F344 rats. We previously reported that PRF is produced by a subpopulation of intermediate lobe cells (15, 29), is distinct from other PRL secretagogues (30, 31), and is a strong inducer of the PRL gene (16, 32). Further, we suspected that PRF-producing cells are targeted by estrogens because an intact posterior pituitary is necessary for mediating the acute estradiol-induced rise in PRL (17) and for generating the full pattern of the PRL surge on proestrus (18). This notion was supported by Frawley et al., reporting that estrogen induced a mammotropic factor (presumably MSH) that rapidly recruited additional PRL secretors into the secretory pool (33). Further, the posterior pituitary expresses estrogen receptors (34 and Fig. 8), and like the uterus, estrogen induces c- fos expression in this tissue (35).

Although basal PRF activity was similar in both rat strains (Fig. 7), pretreatment with estradiol or BPA increased PRF activity only in posterior pituitary cells from F344 rats. This suggests that the estrogen sensitivity of F344 rats is attributed, at least in part, to increased responsiveness of PRF-producing cells to estrogens. Because the structure of PRF is yet unknown, identification of PRF cells awaits the sequencing of PRF and generation of cellular and molecular probes. Of interest, BPA stimulated PRL release but did not increase the pituitary weight in F344 rats (Fig. 6). This suggests that BPA does not mimic all of the in vivo actions of estradiol. Indeed, tissue-selective estrogenic activity has been reported for several estrogenic compounds (36).

Because humans are exposed to significant amounts of BPA through canned food and dental devices (4, 5), the present findings may have implications to human hyperprolactinemia. Although oral contraceptives do not normally induce hyperprolactinemia (37), women who used oral contraceptives for menstrual irregularities rather than for prevention of pregnancy, have a 7- to 8-fold higher incidence of prolactinomas (38). This suggests that exogenous estrogens may stimulate incipient prolactinomas to grow or are more mitogenic in women with reproductive disorders. Whether this is related to the expression of multiple splice variants of the estrogen receptor by human prolactinomas (39) remains to be determined. 

In conclusion, we demonstrated estrogen-mimicking activity of BPA both in vitro and in an animal model. BPA and other Xenoestrogens constitute an unsuspected source of compounds capable of altering the natural hormonal balance. Perhaps there is a human homolog to the F344 rat, i.e. only individuals with altered estrogen receptors and/or estrogen responsive genes are predisposed to the effects of Xenoestrogens. To better evaluate potential hazards posed by such compounds to human health, more information is needed on their exposure, pharmacokinetics, synergistic interactions, and ability to activate a variety of estrogen-responsive genes.

We thank NIDDK, Hormone Distribution Program, for the rat PRL RIA kit, and Drs. R. Maurer and E. Holler for providing us with the reporter constructs. We thank Eric Waits and Natasha Mitchner for their excellent technical assistance.

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