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Bisphenol-A, an Environmental Contaminant that Acts as a Thyroid Hormone Receptor Antagonist in Vitro, Increases Serum Thyroxine, and Alters RC3/Neurogranin Expression in the Developing Rat Brain

Department of Biology and Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003

Address all correspondence and requests for reprints to: R. Thomas Zoeller, Biology Department, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003. E-mail: tzoeller{at}bio.umass.edu.

	Abstract

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Considering the importance of thyroid hormone (TH) in brain development, it is of potential concern that a wide variety of environmental chemicals can interfere with thyroid function or, perhaps of greater concern, with TH action at its receptor (TR). Recently bisphenol-A (BPA, 4,4' isopropylidenediphenol) was reported to bind to the rat TR and act as an antagonist in vitro. BPA is a high production volume chemical, with more than 800 million kg of BPA produced annually in the United States alone. It is detectable in serum of pregnant women and cord serum taken at birth; is 5-fold higher in amniotic fluid at 15–18 wk gestation, compared with maternal serum; and was found in concentrations of up to 100 ng/g in placenta. Thus, the human population is widely exposed to BPA and it appears to accumulate in the fetus. We now report that dietary exposure to BPA of Sprague Dawley rats during pregnancy and lactation causes an increase in serum total T₄ in pups on postnatal d 15, but serum TSH was not different from controls. The expression of the TH-responsive gene RC3/neurogranin, measured by in situ hybridization, was significantly up-regulated by BPA in the dentate gyrus. These findings suggest that BPA acts as a TH antagonist on the ß-TR, which mediates the negative feedback effect of TH on the pituitary gland, but that BPA is less effective at antagonizing TH on the -TR, leaving TR-mediated events to respond to elevated T₄.

	Introduction

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THYROID HORMONE (TH) is essential for normal brain development in both humans (1, 2) and animals (3, 4, 5). Moreover, it is becoming increasingly clear that mild and transient TH insufficiency can affect cognitive outcome in humans (6), and that the developmental timing of transient TH insufficiency produces different cognitive deficits (7). An important implication of these studies is that environmental chemicals that produce TH insufficiency or interfere with TH signaling during development may alter important developmental events. Moreover, if an environmental chemical alters TH signaling by selectively interfering with subset(s) of TH receptors (TRs), the consequences to brain development may be a mosaic of effects on the nervous system because different TRs mediate different actions of TH during development (8, 9, 10, 11).

Although several authors have speculated that specific environmental chemicals might bind to TRs and alter TH signaling (12, 13, 14, 15, 16), we (17) and others (18) have failed to find convincing evidence that suspected thyroid toxicants can bind with high affinity to the TR. Therefore, it was surprising that Moriyama et al. (19) recently reported that the estrogenic compound bisphenol-A (BPA, 4,4' isopropylidenediphenol) binds to the TR. Best characterized as a weak estrogen (20), binding to the estrogen receptor with an inhibitory constant of approximately 10^–5 M (21, 22), BPA binds to and antagonizes T₃ activation of the TR (19, 23). The inhibitory constant for BPA inhibition of T₃ binding to rat TR is approximately 10^–4 M, but as little as 10^–6 M BPA significantly inhibits TR-mediated gene activation (19). Moreover, Moriyama et al. found that BPA reduced T₃-mediated gene expression in culture by enhancing the interaction with the nuclear receptor corepressor (N-CoR) (19).

Thus, BPA is the first environmental chemical known to bind to the TR and affect TH signaling in vitro. BPA is produced at a rate of more than 800 million kg annually in the United States alone (24) and is used primarily in the manufacture of plastics including polycarbonate plastics and epoxy resins that coat food cans and in dental sealants (25, 26). Howe et al. (25) estimated human consumption of BPA from food cans alone to be about 6.6 µg/person·d. BPA has been reported in concentrations of 1–10 ng/ml in serum of pregnant women and cord serum taken at birth (27, 28). Importantly, BPA was found to be 5-fold higher in amniotic fluid at 15–18 wk gestation, compared with maternal serum (29), and was found in concentrations of up to 100 ng/g in placenta (27). Thus, the human population is widely exposed to BPA and concentrations appear to accumulate in the fetus.

Despite these observations, there is no direct information about its effects on TH signaling in vivo, especially in brain development. Iwamuro et al. (29) reported that BPA has an antimetamorphic effect on Xenopus laevis, blocking T₃-induced tail resorption and T₃-induction of TR in tail tissue. In addition, Seiwa et al. (30) showed that BPA antagonizes the ability of TH to affect oligodendrocyte differentiation in vitro. We now report that maternal exposure to BPA in rats can elevate serum T₄ in nursing rat pups but that the effect on TH signaling in the developing brain is consistent with the elevated levels of T₄, producing a profile that is reminiscent of thyroid resistance syndrome (31).

	Materials and Methods

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Animals
All animal procedures followed the National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the University of Massachusetts-Amherst Institutional Animal Care and Use Committee. Timed-pregnant female Sprague Dawley rats (n = 36, 314 ± 53 g; Zivic Miller Laboratories, Inc., Pittsburgh, PA) arrived in our facility on gestational d (G) 2. Animals were individually housed in plastic cages with food and water provided continuously and maintained on a 12-h light, 12-h dark cycle (0600–1800 h). Beginning on the day of arrival, each dam was weighed in the morning and provided with a single untreated wafer (Keebler miniwafers; Keebler, Elmhurst, IL) 1 h before lights off. This initial period (G2-G6) trained the animals to consume the wafer quickly during the experimental procedure. Beginning on G6 and continuing throughout the experiment, dams were weighed in the morning and provided daily with a wafer dosed with 1 µl/g body weight of a solution calibrated to deliver specific doses of BPA. To accomplish this, BPA was dissolved in contaminant-free methanol at one of four concentrations: 0, 1, 10, and 50 µg/µl and pipetted (1 µl/g body weight of the dam; final dose of 0, 1, 10, and 50 mg/kg) onto a wafer and allowed to dry under a fume hood throughout the day before feeding. Administration of BPA on a wafer that the animals voluntarily consume has the advantage of mimicking the route of exposure in humans (oral) and avoiding the variability in exposure by mixing BPA in the feed or water or causing the stress associated with gavage.

Control wafers (0 mg/kg) were dosed with methanol alone and allowed to dry. The dose range of BPA was chosen to overlap with that of Tyl et al. (32), who chose these nominal doses to bracket the effects of BPA on mouse prostate and testis weight (33, 34, 35). Pups were weighed and killed on postnatal day (P) 4, P8, P15, and P35. Trunk blood was collected for serum at all time points; the brain was collected, labeled, and stored at –80 C.

In situ hybridization
Frozen brain tissues were sectioned in coronal plane at 12 µm in a cryostat (Reichert-Jung Frigocut 2800N, Leica Corp., Deerfield, IL). Coronal sections were made through the rostral hippocampus of P15 males approximately corresponding to Figs. 29–33 of Paxinos and Watson (36). Two adjacent sections were thaw mounted onto each twice gelatin-coated microscope slides and stored at –80 C until hybridization. Prehybridization treatments, hybridization, and posthybridization washes were carried out for RC3/neurogranin as described earlier (17, 37). Two slides from each brain (four sections) were hybridized, arranged in x-ray cassettes with ¹⁴C standards (American Radiolabeled Chemicals, St. Louis, MO), and apposed to BioMax film (Eastman Kodak Co., Rochester, NY).

The RC3 probes (complementary or sense-strand) were generated in vitro from an RC3 cDNA [kindly provided by Dr. Juan Bernal (plasmid PRC/cytomegalovirus-RC3) (38), Madrid, Spain]. The transcription reaction was performed in a final volume of 10 µl. RNA was synthesized in the presence of 1 µg DNA template (linearized plasmid); 500 µM each of GTP, ATP, and CTP; and 12 µM uridine 5-triphosphate (UTP) (UTP to ³³P-UTP at a molar ratio of 1:1). For cRNA production, pPRC/CMV-RC3 was linearized with HindIII and transcribed in the presence of SP6 RNA polymerase. The DNA template was then removed by deoxyribonuclease digestion, and the probe was purified by phenol/chloroform extraction followed by two ethanol precipitations.

Autoradiography and signal quantitation
To analyze the hybridization signal, a 5-fold magnified image of the signal over the hippocampus was captured using a Scion AG-5 capture board interfaced with the public domain NIH-Image 1.61/ppc (W. Rasband, National Institute of Mental Health, Bethesda, MD) run on a Macintosh (Cupertino, CA) G4. The optical system consisted of a Dage-72 (Michigan City, IN) series video camera equipped with a Nikon (Melville, NY) macrolens mounted onto a bellows system over a light box. Film density was measured over the dentate gyrus (upper and lower leaflet) or cortex of P15 brains as an index of the relative levels of mRNA expression. Resulting values were average over the four sections for each brain, with one brain per litter and nine litters per treatment. Differences in sample sizes reflect both sample loss during processing and differences in litter sizes among individual dams.

RIA
Total T₄ was measured in 5 µl of rat serum as we have described previously (17). Briefly, each assay tube contained 100 µl barbital buffer [0.11 M barbital (pH 8.6), 0.1% wt/vol 8-anilino-1-napthalene-sulfonic acid ammonium salt, 15% bovine -globulin Cohn fraction II, and 0.1% gelatin], 100 µl anti-T₄ (rabbit, Sigma, St. Louis, MO) diluted to provide a final concentration of 1:30,000, and 100 µl ¹²⁵I-labeled T₄ (Perkin-Elmer/NEN Life Science Products, Boston, MA). Standards were prepared from T₄ (Sigma) measured using a Cahn electrobalance; standards were run in triplicate and calibrated to measure serum T₄ from 0.4 to 25.6 µg/dl. Tubes were incubated at 37 C for 30 min and then chilled on wet ice for 30 min. Bound counts were precipitated by adding 300 µl ice-cold polyethylene glycol 8000 (20% wt/vol; Sigma). Tubes were centrifuged at 1800 x g for 20 min at 4 C, and the supernatant was aspirated and counted in a -counter (CobraII, Packard, Meriden, CT).

Statistical analysis
A two-way ANOVA was performed on body weight of the dams, followed by two single one-way ANOVAs on body weight gain (pregnancy vs. lactation). Serum total T₄ was initially analyzed using a three-way ANOVA (age x treatment x gender as main factors); because there was no interaction between treatment and gender, two-way ANOVAs were then performed separately on data derived from males and females. One-way ANOVAs were used to analyze all other data. Post hoc tests, where appropriate, were performed by Bonferroni’s t test, where the mean square error term in the ANOVA table was used as the point estimate of the pooled variance (SuperAnova Software, Abacus Concepts, Inc., Berkeley, CA).

	Results

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Body weight
Two-way ANOVA of maternal body weight revealed a significant effect of treatment (F_3,396 = 9.005; P = 0.0001), day (of pregnancy/lactation) (F_12,396 = 32.607; P = 0.0001), and a significant interaction (F_36,396 = 1.458; P = 0.0464) (Fig. 1A). However, further evaluation indicated that maternal body weight gain during pregnancy was significantly lower in dams treated with 50 mg/kg BPA, compared with controls (Fig. 1B; F_3,27 = 2.908; P = 0.0089) but that body weight gain during lactation was not affected by BPA treatment. Despite these effects on maternal body weight during pregnancy, there were no observed effects of BPA exposure on litter size (data not shown) or pup weights taken at P4, P8, or P15 (F_3,121 = 2.033; P = 0.113) (Fig. 2).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Effect of BPA treatment on maternal body weight (A) and body weight gain (B) in Sprague Dawley dams (n = 9/group). Although ANOVA revealed that, overall, BPA caused a reduction in body weight (F3,365 = 13.734; P = 0.0001), the extent to which dams gained weight during pregnancy was significantly affected by all doses of BPA (B). Note that maternal body weight gain during lactation was not significantly affected by BPA exposure. Data points (A) or bars (B) represent mean ± SEM. *, Significantly different from body weight gain in control animals (P > 0.001) using Bonferroni-Dunn’s post hoc test.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effect of BPA treatment on body weight of male (A) and female (B) pups on P4, P8, and P15. No effects of treatment were observed. Bars represent mean ± SEM. Group size is listed in the bar.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Effect of BPA treatment on serum total T4 in pups on P4, P8, P15 and P35. Bars represent mean ± SEM (n). *, Significantly different from control (P > 001).

View larger version (22K): [in this window] [in a new window]   FIG. 4. Effect of BPA on serum TSH in male pups at P15. No significant differences were observed among the groups. Bars represent mean ± SEM (n).

View larger version (57K): [in this window] [in a new window]   FIG. 5. Effect of BPA on RC3/neurogranin expression in the P15 male brain. A, Pseudocolor image of a film autoradiogram of the signal resulting from in situ hybridization RC3 in coronal sections containing the dentate gyrus. Note the difference in intensity of the signal in the image of the dentate taken from a BPA-treated animal (50 mg/kg). B, Graphical representation of the results of the RC3/neurogranin densitometry. Bars represent mean ± SEM (n). DG, Dentate gyrus; CA1, subfield of Ammon’s horn; Cx, somatosensory cortex; upper, upper leaflet of the dentate gyrus; lower, lower leaflet of the dentate gyrus. *, Significantly different from controls (P > 0.01).

	Discussion

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The flat dose response of BPA on serum T₄ levels may be attributable to the mechanism by which BPA antagonizes T₃-induced TR activation. Specifically, BPA causes the TR to recruit a corepressor (N-CoR) (19), which is likely to be in limited abundance. Thus, the antagonism will saturate in direct proportion to N-CoR abundance. This type of dose response may be characteristic of this type of antagonism. For example, RU 486, an indirect antagonist of the progestin receptor, exerts it antiprogestin activity (40, 41) in the same way that BPA affects the TR. Like the effect of BPA on serum T₄, RU 486 produces a flat dose response on progestin receptor-regulated gene expression (40, 41). Thus, the shape of this dose response (i.e. flat) does not abrogate its therapeutic efficacy. Likewise, the flat dose response of BPA on serum T₄ may be no less important.

Control pups exhibited a postnatal rise in serum T₄ levels from P4 (1.5 µg/dl) to P15 (6 µg/dl); this postnatal rise is well documented (e.g. Refs.37 , 42 , 43) and is not related to gender. However, BPA had no effect on serum T₄ on P4 (Fig. 2), perhaps because the negative feedback action of TH on the hypothalamic-pituitary axis does not mature until around P7 in the rat (44). Therefore, failure of BPA to elevate serum T₄ on P4 is consistent with the hypothesis that BPA inhibits TH-negative feedback. In addition, BPA did not affect serum T₄ in animals on P35, perhaps because these animals had not received BPA since they were weaned on P21 and BPA body burden would have diminished due to metabolic clearance. BPA is rapidly metabolized in rats and humans; the predominant metabolite of BPA is BPA-monoglucuronide (45, 46, 47), which is devoid of estrogenic activity (48). However, BPA-glucuronide has not been tested for its ability to bind to the TR.

BPA bioavailability is dependent on the route of administration. BPA is rapidly metabolized to BPA-glucuronide within an hour after oral administration in adult rats, but injection (ip or sc) results in a prolonged (8–12 h) presence of parent BPA in rat serum (48). In contrast, the serum half-life of parent BPA in pups after direct oral administration is about 7–8 h (48). However, the transfer of BPA to pups from the dam is quite low. Snyder et al. (45) reported that milk contains only 0.0008% of the maternal dose (100 mg/kg) 24 h after oral administration and that P14 pups receive about 0.006% of the dose given orally to the dam. Therefore, we can estimate that pups in this experiment received about 0.06, 0.6, and 3.0 µg BPA when the dams were treated with 1, 10, or 50 mg/kg, respectively. However, our findings indicate that as low as 10 mg/kg oral dose to the dam (0.6 µg/pup) caused a significant increase in serum T₄.

We focused our additional work on P15 males because P15 is a time during postnatal development that RC3/neurogranin is particularly sensitive to TH (49) and there appear to be no gender differences in TH regulation of RC3/neurogranin on P15. Serum TSH levels were not significantly different in BPA-treated pups on P15, despite elevated levels of T₄. Because of the duration of BPA treatment (from G6), the TSH levels likely represent a balance between the antagonistic effect of BPA on the pituitary, which would tend to increase TSH release by inhibiting negative feedback, and the elevated T₄ levels in serum, which would tend to suppress TSH release. Thus, in the presence of BPA, T₄ levels increase to maintain serum TSH levels similar to those of control animals

Considering the effect of BPA on serum T₄ and TSH, we expected that measures of TH action in the developing brain would follow the same pattern as that of TSH (i.e. balanced antagonism of BPA and elevated T₄, producing no difference among treatment groups). In contrast, BPA significantly increased RC3/neurogranin expression in the dentate gyrus but not the somatosensory cortex. This spatial pattern of RC3 regulation by BPA is identical with the spatial pattern of RC3 regulation by TH (37, 38), suggesting that BPA is not affecting RC3 expression independently of the TR. Therefore, the parsimonious interpretation is that the observed increase in RC3 expression after BPA exposure is due to increased circulating levels of TH. Because RC3 expression is likely to be regulated by the TR in the dentate (38, 50), these data indicate that BPA may not interfere with TH action on TR as it does on the TRß, leaving the TR unopposed to respond to elevated levels of T₄.

The BPA-induced reduction in maternal body weight and body weight gain during pregnancy is similar to that observed by others (32, 51). In contrast, BPA exposure did not affect body weight gain during lactation in the current study (Fig. 1). The effect of BPA on maternal body weight may well be related to the estrogenic action of BPA inasmuch as ethinyl estradiol exposure causes a similar decrease in maternal body weight gain during pregnancy (52). In contrast, the increased serum T₄ in the offspring of BPA-treated dams cannot be explained by an estrogenic action of BPA because, unlike humans in which serum T₄ is elevated by estrogen (53, 54), estrogen treatment in rats decreases serum T₄ (55). Although it is theoretically possible that the BPA-induced decrease in maternal weight gain during pregnancy may have affected pup RC3 expression independent of TH, this seems unlikely considering that the effect of BPA on RC3 expression was observed only 2 wk after birth and at a time when RC3 expression is known to be most sensitive to TH. Thus, at the developmental time that RC3 expression is known to be sensitive to TH, it was associated with BPA exposure.

The results of this study indicate that BPA can exert a selective TH antagonism independent of estrogenic effects. Specifically, BPA appears to exert a selective antagonism on the TRß, causing serum levels of T₄ to rise, which in turn produces a local hyperthyroidism on the TR, causing RC3 mRNA to rise. Considering that BPA is an indirect antagonist, its effects on TH action in vivo will likely be dependent on the composition and relative abundance of cofactors available in the cell. This could lead to a mosaic of effects in the developing brain, producing affects on different brain regions that reflect TH antagonism or potentially TH agonism. Likewise in humans, BPA could produce effects that are not consistent with global TH insufficiency or excess. In addition, BPA is commercially halogenated (brominated or chlorinated) for use as flame retardants; tetrabromobisphenol-A is the most commonly used with more than 60,000 tons produced annually (56, 57). Thomsen et al. (58) recently reported that brominated flame retardants, including tetrabromobisphenol-A, have increased in human serum from 1977 to 1999, with concentrations in adults ranging from 0.4 to 3.3 ng/g serum lipids. However, infants (0–4 yr) exhibited serum concentrations that ranged from 1.6 to 3.5 times higher (58). Considering that the halogenated BPAs exhibit a much higher affinity than parent BPA for the TR (23), this class of environmental chemicals will be important to evaluate for their ability to interfere with TH action in development.

	Footnotes

 
This work was supported in part by National Institutes of Health Grant ES10026 (to R.T.Z.).

First Published Online October 21, 2004

Abbreviations: BPA, Bisphenol-A; G, gestational day; N-CoR, nuclear receptor corepressor; P, postnatal day; TH, thyroid hormone; TR, TH receptor; UTP, uridine 5-triphosphate.

Received August 4, 2004.

Accepted for publication October 13, 2004.

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