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Department of Cell Biology, University of Cincinnati College of Medicine (N.A.M., D.L.A., N.B.J.), Cincinnati, Ohio 45267; and the Department of Obstetrics and Gynecology, Indiana University School of Medicine (R.S., A.G., R.M.B.), Indianapolis, Indiana 46202
Address all correspondence and requests for reprints to: Dr. Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati Medical School, 231 Bethesda Ave, Cincinnati, Ohio 45267-0521.
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Abstract |
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Treatment with single high doses of BPA induced cell proliferation in
the uterus and vagina of ovariectomized F344 rats, as determined by
bromodeoxyuridine immunostaining. This proliferation was dose dependent
(from 37.5–150 mg/kg) and followed a time course similar to that of
estradiol (E2). Quantitative RT-PCR revealed that both BPA
and E2 increased c-fos messenger RNA levels
in the uterus 14- to 16-fold within 2 h, which returned to basal
levels after 6 h. In the vagina, BPA-induced c-fos
expression remained elevated for up to 6 h, compared with the
transient increase caused by E2. Treatment of F344 rats for
3 days with continuous release capsules that supplied a much lower dose
of BPA (
0.3 mg/kg·day) resulted in hypertrophy, hyperplasia, and
mucus secretion in the uterus and hyperplasia and cornification of the
vaginal epithelium. The reproductive tract of SD rats did not respond
to this treatment paradigm with BPA.
These studies demonstrate that 1) the molecular and morphological alterations induced by BPA in the uterus and vagina are nearly identical to those induced by estradiol; 2) the vagina appears to be especially sensitive to the estrogenic actions of BPA; 3) the reproductive tract of the inbred F344 rat appears more sensitive to BPA than that of the outbred SD rat; and 4) continuous exposure to microgram levels of BPA is sufficient for exerting estrogenic actions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently reported that BPA increased PRL gene expression and release upon in vitro incubation with pituitary cells. Moreover, in vivo administration of BPA to ovariectomized (OVEX) rats induced hyperprolactinemia and a marked increase in the expression of a putative PRL-regulating factor produced by the posterior pituitary (8). Interestingly, the PRL response to BPA was evident in Fischer 344 (F344) rats, a strain known for its hypersensitivity to exogenous estrogens (9, 10), whereas Sprague-Dawley (SD) rats, which responded to estradiol (E2) with a moderate increase in PRL secretion, were unresponsive to BPA.
The rodent reproductive tract is an excellent model for studying the effects of estrogens on cellular growth and differentiation. Administration of E2 to adult OVEX rats evokes a cascade of events. In the uterus, early events occur within minutes to a few hours after exposure to estrogen and include increases in vascular permeability, water imbibition, increases in organ wet weight, and induction of protooncogenes (11, 12) as well as genes for growth factors (13, 14, 15) and their receptors (16, 17). Delayed events occur within 20–24 h after estrogen treatment and involve DNA synthesis (18) followed by mitosis and cellular differentiation (19). The vagina is also very sensitive to ovarian steroids. Cellular proliferation and keratinization are hallmarks of the estrogenic response in the vaginal epithelium, but the sequence of the cellular and molecular events involved is not as well delineated as that in the uterus (20, 21).
The overall objective of this investigation was to examine the in vivo effects of BPA on the growth and differentiation of the reproductive tract in the female rat. Specifically, we examined whether BPA mimicked E2 in inducing the early response gene c-fos and in stimulating DNA synthesis in the uterus and vagina of the F344 rat. We also compared the responses of F344 and SD rats to delayed effects of BPA on uterine weight and on differentiation of the epithelia in the uterus and vagina.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-Bromodeoxyuridine (BrdU) immunohistochemistry for cell
proliferation
F344 rats were injected with 0, 18.75, 37.5, 75, 150, or 200
mg/kg BPA or 10 µg/kg E2. Uteri and vaginas
were removed after 20 h. One hour before death, rats were injected
ip with 100 mg/kg BrdU (Sigma), a thymidine analog. Tissues were fixed
in ethanol-chloroform-glacial acetic acid (60:30:10, vol/vol/vol),
embedded in paraffin, and sectioned. Cells in the S phase of the cell
cycle were identified by immunohistochemistry for BrdU as previously
described (22). Briefly, tissue sections were treated with
H2O2 for 30 min, 1 N HCl for 8 min,
and borate buffer for 15 min. The sections were incubated overnight at
4 C with a mouse monoclonal anti-BrdU antibody (Becton Dickinson, San
Jose, CA). Nuclear staining for BrdU was developed using the Vectastain
ABC kit (Vector Laboratories, Burlingame, CA), with diaminobenzidine as
the chromogen. The BrdU-labeled epithelial cells per microscopic field
were counted, and the length of the basement membrane underlying the
epithelium in each field was determined using image analysis software
(IPLab Spectrum, Signal Analytics, Vienna, VA). Results are expressed
as the number of labeled cells per 1000 µm luminal epithelium, using
several tissue sections from each animal.
Analysis of c-fos expression by RT-PCR
F344 rats were injected ip with 50 mg/kg BPA or 10 µg/kg
E2 and were killed after 2, 6, and 24 h.
Uteri and vaginas were removed, total RNA was isolated using
Tri-Reagent (Molecular Research Center, Cincinnati, OH), and 5 µg
were reverse transcribed using Superscript II reverse transcriptase and
random hexamers (Life Technologies, Gaithersburg, MD). Primer sequences
and the expected product size were as follows: 1) c-fos
sense primer 5'-CCAACTTTATCCCCACGGTGAC-3' and antisense
5'-TGGCAATCTCGGTCTGCAAC-3' with expected product size of 381 bp; and 2)
RPL19 (ribosomal protein L19), used as an internal standard, sense
primer 5'-AGTAGTCTTAGGCTACAGAAG-3', and antisense primer
5'-TTCCTTGGTCTTAGACCTGCG-3', with expected product size of 500 bp.
Optimal PCR conditions for quantitative analysis were first determined by varying the number of hybridization cycles or the RNA amounts from estrogen-treated uteri. PCR was then performed using 100 ng of the RT reaction products for 28 cycles (94 C for 30 sec, 60 C for 30 sec, and 74 C for 45 sec). Products were separated on a 1% agarose gel stained with ethidium bromide and analyzed by scanning (Scion Image Software, Frederick, MD). The density ratio of c-fos/RPL19 was calculated, and the results were expressed as a percentage of the values obtained from control tissues (time zero).
In situ hybridization for c-fos expression
Uteri and vaginas were removed from F344 rats injected with
either oil (control) or 50 mg/kg BPA. Tissues were cryosectioned, fixed
in 4% paraformaldehyde, and processed for in situ
hybridization as previously described (23). Sense and antisense
35S-labeled riboprobes for c-fos were
synthesized using a Promega riboprobe transcription kit (Promega,
Madison, WI), and the sections were hybridized to the probes for
18 h at 55 C and extensively washed. After dipping in Kodak NTB2
photographic emulsion (Eastman Kodak, Rochester, NY), slides were
stored at 4 C and developed after 7 days.
In vitro release of BPA and E2 from SILASTIC brand
capsules
SILASTIC brand capsules containing crystalline BPA or
E2 were prepared as previously described (8). The
capsules (length, 1 cm; id, 0.062 in.; od, 0.125 in.) were weighed
before and after filling to determine the amount of compounds contained
in each and were then incubated in 1 ml PBS at 37 C for 7 days. Media
were removed daily and analyzed by HPLC to determine the amount of
E2 or BPA released each day. Briefly, media were
isocratically fractionated at 0.8 ml/min on an analytical reverse phase
C18 column using 40% acetonitrile and 0.1%
trifluoroacetic acid. The elution profiles of BPA and
E2 were monitored at 254 and 230 nm,
respectively. The amounts of BPA or E2 in the
fractionated media were determined based on peak heights after
calibration with known amounts of each compound.
Tissue weights and morphometric analysis of reproductive tract
tissues
F344 and SD rats were implanted with capsules containing
crystalline BPA or E2. After 3 days, rats were
killed, and one uterine horn from each animal was blotted and weighed.
The second horn and the vagina were fixed in 10% formalin, embedded in
paraffin, sectioned at 4 µm, stained with hematoxylin and eosin, and
photographed. Uterine sections were viewed under the microscope, and
the heights of the luminal epithelial cells were measured using IPLab
Spectrum software; 75–100 cells/tissue section·animal were
analyzed.
Data analysis
Unless otherwise stated, results are expressed as the mean
± SEM. Statistical differences between treatments were
analyzed using ANOVA (Statmost 3.0, DataMost Corp., Salt Lake City,
UT), followed by Duncan’s or Scheffe’s post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The photomicrographs
shown in Fig. 1 are from a preliminary time-course study that showed
that, similar to stimulation by E2 (18, 19), both
uterine and vaginal epithelia were maximally labeled 20 h after a
single dose of 200 mg/kg BPA (data not shown). When rats were implanted
with BPA-containing capsules for 3 days, BrdU-labeled cells were also
seen in all uterine tissue compartments (myometrium, stroma, and
epithelium) as well as in both vaginal epithelium and stroma (data not
shown).
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, 18.75
mg/kg BPA had no effect, but the 37.5 mg/kg dose caused a significant
increase (P < 0.05) in the number of labeled
epithelial cells in both uterus and vagina. Maximal labeling in the
uterine epithelium was achieved at doses of 75 and 150 mg/kg (Fig. 2).
A secondary increase in vaginal epithelial labeling was seen when 200
and 300 mg/kg BPA were injected, but such high doses appear to cause
some toxicity to the animals (data not shown). Based on these results,
the next set of experiments was performed using 50 mg/kg BPA injected
ip.
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, left
panel, the optical density for both RPL19 (used as an internal
standard) and c-fos in the uterus was linear from 25–35
cycles. The amount of template RNA yielding a linear relationship
ranged from 50 to at least 400 ng (Fig. 3, right panel).
Based on these results, the next experiment was performed using 100 ng
RNA and 28 PCR cycles.
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shows induction of
c-fos gene expression by E2 and BPA in
the uterus (upper panel) and vagina (lower panel)
of representative animals 0, 2, 6, and 24 h after injections.
Combined results from four or five rats at each time point are shown in
Fig. 5. Within 2 h after treatment
with either BPA or E2, uterine c-fos
messenger RNA (mRNA) levels increased 14- to 17-fold above control
values (time zero, expressed as 100%). By 6 h, c-fos
mRNA levels in the uterus of BPA-treated animals had dropped sharply to
near control levels. A different pattern emerged in the vagina (Fig. 5, right panel). Both E2 and BPA
increased c-fos mRNA levels 7- to 9-fold above control
values within 2 h. However, after 6 h, c-fos mRNA
levels in the BPA-treated rats remained 6- to 7-fold above control
values (P < 0.01), whereas those in the
E2-treated rats were less than 3-fold greater
than control values (P < 0.01). After 24 h,
c-fos expression in the vagina of rats treated with either
E2 or BPA did not differ from control levels. As
shown by in situ hybridization (Fig. 6), the induction of c-fos
expression by BPA was confined almost exclusively to the luminal
epithelium of both tissues.
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, approximately 50 µg BPA,
compared with 1 µg E2, were released from the
capsules each day. This rate of release remained stable for at least 7
days. From this we estimate that administration of BPA via implanted
capsules produces a dose of approximately 0.3 mg/kg·day.
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, upper panel). In contrast,
BPA did not alter uterine cell height in SD rats, whereas
E2 stimulated more than a 2-fold increase
(P < 0.001). Uterine wet weight in response to
E2 rose 3- to 3.5-fold (P <
0.01) in both F344 and SD rats (Fig. 7, lower panel). BPA,
on the other hand, increased uterine weight less than 2-fold in F344
rats (P < 0.01) and was ineffective in SD rats.
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shows morphological changes
induced by a 3-day treatment with BPA or E2 in
the uterus and vagina of F344 rats. Similar to
E2, BPA increased the cell height of the luminal
epithelium in the uterus and stimulated mucus secretion
(arrowheads, upper panels). In the vagina, BPA increased the
thickness of the epithelium from the two or three cell layers seen in
control rats to six to eight cell layers (Fig. 8, lower
panel). Additionally, BPA stimulated keratinization of the vaginal
epithelium and sloughing of surface cells, similar to the effects of
E2 (open arrows, lower panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A rapid and transient rise in the expression of the protooncogene c-fos characterizes one of the early events of estrogenic action in the rodent reproductive tract (11). The induction of c-fos expression in the uterus by E2 is restricted to epithelial cells (24, 25, 26), even though estrogen receptors are also expressed in the stroma and myometrium (27). By itself, c-fos induction is not sufficient to promote DNA synthesis and subsequent mitosis (28). Additionally, a single injection of E2 is sufficient for stimulating uterine cell proliferation, whereas multiple doses of shorter acting estrogens, e.g. estriol and 16-E2, are required to promote cell progression from the G0 to the S phase of the cell cycle (18). Our results clearly demonstrate that a single injection of BPA can sustain DNA synthesis by responsive cells in the uterus and vagina.
Both the magnitude and the time course of E2-induced increases in uterine c-fos expression, determined by RT-PCR, were similar to those measured by ribonuclease protection assay in our previous report (23). Unlike that in the uterus, c-fos expression in the vagina remained elevated for 6 h after treatment with BPA, whereas its induction by E2 subsided after 2 h. It is now recognized that the pattern of c-fos induction by estrogens differs, depending upon the ligand and the target tissue. For example, the antiestrogen tamoxifen, which acts as a partial estrogen agonist in the uterus (29, 30), elicits a sustained induction of uterine c-fos expression (31). In contrast to the uterus, the E2-induced c-fos rise in the pituitary is low, delayed, and prolonged (23). The mechanism underlying the sustained c-fos elevation in the vagina in response to BPA is unknown. It could involve a slow dissociation of BPA from local estrogen receptors, mediation by coactivators that are specifically associated with receptor activation by BPA but not by E2, or different pharmacokinetics of BPA and E2.
Within 20–24 h of E2 administration to adult OVEX rats, the epithelial cells of the uterine lumen and glands undergo pronounced proliferation (18, 19). Our data reveal that BPA stimulates cell division in the uterus at a time course similar to that reported for E2 and that its effects are dose dependent. Notably, prolonged exposure to microgram levels of BPA released from capsules is sufficient to promote cellular proliferation in both the uterus and vagina. Similar to the well established action of E2, these relatively low levels of BPA also induce strong differentiation and cornification of the vaginal epithelium.
There is a general consensus that BPA is a weak estrogen, requiring 2000- to 5000-fold higher concentrations than E2 to stimulate MCF-7 cell proliferation (32), PRL release from GH3 cells (8), or binding to the estrogen receptor (33). Our data reveal that animals exposed to approximately 0.3–0.5 mg/kg·day BPA via the capsules (as judged by our in vitro data) responded similarly to those given 50–100 mg/kg BPA by a single injection. The reasons for the higher than expected efficacy of slowly released BPA could be as follows. First, maximal steady state levels can be achieved by a continuous presence of BPA in target tissues. As yet, there are no data on the half-life of BPA in vivo. Second, BPA could be converted to a more potent metabolite(s) than the parent compound. Indeed, it has been reported that BPA is metabolized to 5-hydroxybisphenol and further converted to 4,5,bisphenol-O-quinone, a compound capable of forming covalent DNA adducts (34, 35). This may be analogous to tamoxifen (36) and the pesticide methoxychlor (37), both of which are converted in vivo to significantly more active compounds. Third, the actions of BPA could be enhanced by binding to the sex hormone-binding protein (38) or by activating signaling pathways that synergize with the estrogen receptor (38, 39). All of these possibilities merit further investigation.
The neuroendocrine axis of F344 rats is extremely sensitive to exogenous estrogens. When chronically treated with E2 (10, 40) or diethylstilbestrol (9, 41), F344 rats develop severe hyperprolactinemia and pituitary tumors composed of lactotrophs. This is in contrast to SD rats, which respond to estrogen treatment with a modest increase in PRL release and no prolactinoma formation. It was also suggested that the estrogen sensitivity of the F344 rat is confined to the pituitary, as the increase in uterine weight in response to E2 did not differ between F344 and Holtzman rats (9). We have recently found that BPA administered for 3 days via SILASTIC brand capsules increased PRL release 7- to 8-fold in F344 rats, but did not alter PRL release in SD rats (8). The present results suggest that the reproductive tract of F344 rats is also more sensitive to BPA than that of the SD rat. Although BPA only marginally increased uterine wet weight in F344 rats, it induced profound cellular alterations in their reproductive tracts. The underlying cause(s) of the difference in strain sensitivity to BPA is currently under investigation.
Based on the estimated daily release of BPA from in vitro incubated capsules, we propose that BPA is active in vivo at microgram amounts. Notably, humans could be exposed to microgram amounts of BPA from a variety of sources. For example, BPA can be liberated from polycarbonate plastics subjected to high temperature (32) or from incompletely polymerized epoxy resins (42). Indeed, significant quantities of BPA have been detected in liquids from canned vegetables (20 µg/can) that are exposed to high temperature during autoclaving (6) and in the saliva (20–30 µg/ml) of dental patients fitted with restorative material (7). Determination of BPA in human plasma or tissues awaits the development of specific and sensitive analytical assays.
Finally, there is a striking resemblance between BPA and diethylstilbestrol, a known human teratogen and carcinogen (43, 44). This includes a similar chemical structure, activation of estrogen-responsive genes, metabolism to quinones, and formation of DNA adducts (35, 45). Thus, despite a marked difference in potency between the two compounds, the question remains whether chronic exposure to low levels of BPA constitutes health hazards to humans and, if so, which physiological functions might be affected. We do not know the answer to these questions. We raise the possibility, however, that certain human subpopulations, e.g. developing fetuses, prepubertal children, and postmenopausal women, all of whom have low endogenous estrogen levels, may be vulnerable to the undesirable effects of BPA and other xenoestrogens. Further, the potential effects of BPA on the male reproductive tract should also be examined.
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Footnotes |
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Received September 26, 1997.
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References |
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/2-hydroxyestrone: a
biologic marker of breast cancer risk. Environ Health Perspect 103:147–150
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