Bisphenol-A, an Environmental Contaminant that Acts as a Thyroid Hormone Receptor Antagonist in Vitro, Increases Serum Thyroxine, and Alters
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内分泌学杂志 2005年第2期
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@bio.umass.edu.
Abstract
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 ?-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
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
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
Body weight
Two-way ANOVA of maternal body weight revealed a significant effect of treatment (F3,396 = 9.005; P = 0.0001), day (of pregnancy/lactation) (F12,396 = 32.607; P = 0.0001), and a significant interaction (F36,396 = 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; F3,27 = 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 (F3,121 = 2.033; P = 0.113) (Fig. 2).
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.
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.
Serum hormone levels
A three-way ANOVA (age x treatment x gender) of T4 levels in pups revealed significant effects of age (F3,213 = 217.97; P = 0.0001) and treatment (F3,213 = 3.75; P = 0.0118) but not gender (F1,213 = 0.003; P = 0.953). In addition, there was a significant interaction between age and treatment (F9,213 = 2.43; P = 0.012), but no other significant interactions were present (age x gender, treatment x gender, or age x treatment x gender). Further inspection of these data indicated that maternal BPA exposure significantly increased serum total T4 in both male and female pups (Fig. 3). Pups derived from BPA-treated dams exhibited significantly higher levels of T4 on P15 (F3,55 = 3.704; P = 0.0169) (Fig. 3).
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).
Because P15 is a critical time for TH action in the rat brain (39) and there appeared to be no substantive gender differences in the effects of BPA on the hypothalamic-pituitary-thyroid axis, we chose to focus on the ability of BPA to alter TH signaling in P15 males. First, we determined whether the BPA-induced increase in serum T4 in male pups on P15 is associated with an increase in serum TSH; however, we observed no significant differences in mean TSH levels among P15 males born to dams being treated with various BPA concentrations (F3,23 = 0.802; P = 0.505) (Fig. 4). However, despite the finding that TSH levels were not different among groups, there were significant treatment effects of BPA on RC3/neurogranin expression in the upper (F3,22 = 6.48; P = 0.01) and lower leaflet of the dentate gyrus (F3,22 = 6.58; P = 0.0024) but not in the cortex (F3,22 = 1.514; NS) (Fig. 5). RC3/neurogranin mRNA levels in both leaflets of the dentate gyrus were significantly higher in all BPA-treated animals, compared with controls.
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).
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
The present findings strongly suggest that BPA acts as a thyroid hormone antagonist in vivo. Maternal exposure to BPA caused an increase in serum T4 of both male and female pups but simultaneously increased the expression of RC3/neurogranin in the hippocampus. The simplest explanation for these findings is that BPA acts as an antagonist of TH action on the ?-TR, inhibiting TH-negative feedback but leaving the -TR unopposed in responding to elevated T4 in the hippocampus and presumably elsewhere. This is the first report of the in vivo effects of an environmental chemical that can exert direct effects on TH signaling in vitro, and the findings suggest the possibility that incidental exposure to BPA could influence TH signaling in the developing brain.
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 TR?, 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 TR?, 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.
References
Song S, Daneman D, Rovet J 2001 The influence of etiology and treatment factors on intellectual outcome in congenital hypothyroidism. J Dev Behav Pediatr 22:376–384
Rovet J 1999 Congenital hypothyroidism: long-term outcome. Thyroid 9:741–748
Bernal J 2002 Action of thyroid hormone in brain. J Endocrinol Invest 25:268–288
Zoeller RT, Simonyi A, Butnariu O, Fletcher DL, Rudeen PK, McCrone S, Petersen SL 1995 Effects of acute ethanol administration and cold exposure on the hypothalamic-pituitary-thyroid axis. Endocrine 3:39–47
Zoeller RT, Dowling ALS, Herzig CTA, Iannacone EA, Gauger KJ, Bansal R 2002 Thyroid hormone, brain development, and the environment. Environ Health Perspect 110(Suppl 3):355–361
Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon J, O’Heir CE, Mitchell ML, Hermos RJ, Waisbren SE, Faix JD, Klein RZ 1999 Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 341:549–555
Zoeller RT, Rovet J 2004 Timing of thyroid hormone action in the developing brain-clinical observations and experimental findings. J Neuroendocrinol 16:809–818
Morte B, Manzano J, Scanlan T, Vennstrom B, Bernal J 2002 Deletion of the thyroid hormone receptor 1 prevents the structural alterations of the cerebellum induced by hypothyroidism. Proc Natl Acad Sci USA 99:3985–3989
Bernal J, Guadano-Ferraz A, Morte B 2003 Perspectives in the study of thyroid hormone action on brain development and function. Thyroid 13:1005–1012
Manzano J, Morte B, Scanlan TS, Bernal J 2003 Differential effects of triiodothyronine and the thyroid hormone receptor ?-specific agonist GC-1 on thyroid hormone target genes in the brain. Endocrinology 144:5480–5487
Morte B, Manzano J, Scanlan TS, Vennstrom B, Bernal J 2004 Aberrant maturation of astrocytes in thyroid hormone receptor 1 knockout mice reveals an interplay between thyroid hormone receptor isoforms. Endocrinology 145:1386–1391
McKinney JD, Waller CL 1994 Polychlorinated biphenyls as hormonally active structural analogues. Environ Health Perspect 102:290–297
Porterfield SP 1994 Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system. Environ Health Perspect 102(Suppl 2):125–130
McKinney JD, Waller CL 1998 Molecular determinants of hormone mimicry: halogenated aromatic hydrocarbon environmental agents. J Toxicol Environ Health B Crit Rev 1:27–58
Porterfield SP, Hendry LB 1998 Impact of PCBs on thyroid hormone directed brain development. Toxicol Ind Health 14:103–120
Porterfield SP 2000 Thyroidal dysfunction and environmental chemicals—potential impact on brain development. Environ Health Perspect 108(Suppl 3):433–438
Gauger KJ, Kato Y, Haraguchi K, Lehmler HJ, Robertson LW, Bansal R, Zoeller RT 2004 Polychlorinated biphenyls (PCBs) exert thyroid hormone-like effects in the fetal rat brain but do not bind to thyroid hormone receptors. Environ Health Perspect 112:516–523
Cheek AO, Kow K, Chen J, McLachlan JA 1999 Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107:273–278
Moriyama K, Tagami T, Akamizu T, Usui T, Saijo M, Kanamoto N, Hataya Y, Shimatsu A, Kuzuya H, Nakao K 2002 Thyroid hormone action is disrupted by bisphenol A as an antagonist. J Clin Endocrinol Metab 87:5185–5190
Staples CA, Dorn PB, Klecka GM, O’Block ST, Harris LR 1998 A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere 36:2149–2173
Krishnan AV, Stathis P, Permuth SF, Tokes L, Feidman D 1993 Bisphenol A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132:2279–2286
Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, Mcdonell DP 1997 Evaluation of chemicals with endocrine modulating activity in yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol 143:205–212
Kitamura S, Jinno N, Ohta S, Kuroki H, Fujimoto N 2002 Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A. Biochem Biophys Res Commun 293:554–559
Reporter CM 1999 ChemExpo chemical profile: Bisphenol-A. New York: Schnell Publishing Co.
Howe SR, Borodinsky L, Lyon RS 1998 Potential exposure to bisphenol A from food-contact use of epoxy coated cans. J Coatings Technol 70:69–74
Lewis JB, Rueggeberg FA, Lapp CA, Ergle JW, Schuster GS 1999 Identification and characterization of estrogen-like components in commercial resin-based dental restorative materials. Clin Oral Investig 3:107–113
Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I 2002 Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ Health Perspect 110:A703–A707
Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y 2002 Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod 17:2839–2841
Iwamuro S, Sakakibara M, Terao M, Ozawa A, Kurobe C, Shigeura T, Kato M, Kikuyama S 2003 Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larval Xenopus laevis. Gen Comp Endocrinol 133:189–198
Seiwa C, Nakahara J, Komiyama T, Katsu Y, Iguchi T, Asou H 2004 Bisphenol A exerts thyroid-hormone-like effects on mouse oligodendrocyte precursor cells. Neuroendocrinology 80:21–30
Refetoff S, Weiss RE, Usala SJ, Hayashi Y 1994 The syndromes of resistance to thyroid hormone: update 1994. In: Braverman LE, Refetoff S, eds. Clinical and molecular aspects of diseases of the thyroid. Bethesda, MD: The Endocrine Society; 336–343
Tyl RW, Myers CB, Marr MC, Thomas BF, Keimowitz AR, Brine DR, Veselica MM, Fail PA, Chang TY, Seely JC, Joiner RL, Butala JH, Dimond SS, Cagen SZ, Shiotsuka RN, Stropp GD, Waechter JM 2002 Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley Rats. Toxicol Sci 68:121–146
Sharpe RM, Rivas A, Walker M, McKinnell C, Fisher JS 2003 Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens, or with a GnRH antagonist, on Leydig cell development and function through puberty into adulthood. Int J Androl 26:26–36
Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV 1997 Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70–76
vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer KA, Nagel SC, Parmigiani S, Welshons WV 1998 A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol Ind Health 14:239–260
Paxinos G, Watson C 1986 The rat brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press
Zoeller RT, Dowling AL, Vas AA 2000 Developmental exposure to polychlorinated biphenyls exerts thyroid hormone-like effects on the expression of RC3/neurogranin and myelin basic protein messenger ribonucleic acids in the developing rat brain. Endocrinology 141:181–189
Iniguez MA, DeLecea L, Guadano-Ferraz A, Morte B, Gerendasy D, Sutcliffe JG, Bernal J 1996 Cell-specific effects of thyroid hormone on RC3/neurogranin expression in rat brain. Endocrinology 137:1032–1041
Ibarrola N, Rodriguez-Pena A 1997 Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development. Brain Res 752:285–293
Liu Z, Auboeuf D, Wong J, Chen JD, Tsai SY, Tsai MJ, O’Malley BW 2002 Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486. Proc Natl Acad Sci USA 99:7940–7944
Schulz M, Eggert M, Baniahmad A, Dostert A, Heinzel T, Renkawitz R 2002 RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by corepressor binding. J Biol Chem 277:26238–26243
Goldey ES, Kehn LS, Rehnberg GL, Crofton KM 1995 Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol Appl Pharmacol 135:67–76
Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM 1995 Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol Appl Pharmacol 135:77–88
Taylor T, Gyves P, Burgunder JM 1990 Thyroid hormone regulation of TRH mRNA levels in rat paraventricular nucleus of the hypothalamus changes during ontogeny. Neuroendocrinology 52:262–267
Snyder RW, Maness SC, Gaido KW, Welsch F, Sumner SC, Fennell TR 2000 Metabolism and disposition of bisphenol A in female rats. Toxicol Appl Pharmacol 168:225–234
Pottenger LH, Domoradzki JY, Markham DA, Hansen SC, Cagen SZ, Waechter Jr JM 2000 The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. Toxicol Sci 54:3–18
Zalko D, Soto AM, Dolo L, Dorio C, Rathahao E, Debrauwer L, Faure R, Cravedi JP 2003 Biotransformations of bisphenol A in a mammalian model: answers and new questions raised by low-dose metabolic fate studies in pregnant CD1 mice. Environ Health Perspect 111:309–319
Domoradzki JY, Thornton CM, Pottenger LH, Hansen SC, Card TL, Markham DA, Dryzga MD, Shiotsuka RN, Waechter Jr JM 2004 Age and dose dependency of the pharmacokinetics and metabolism of bisphenol a in neonatal Sprague-Dawley rats following oral administration. Toxicol Sci 77:230–242
Iniguez M, Rodriguez-Pena A, Ibarrola N, Aguilera M, Morreale de Escobar G, Bernal J 1993 Thyroid hormone regulation of RC3, a brain-specific gene encoding a protein kinase-C substrate. Endocrinology 133:467–473
Bradley DJ, Towle HC, Young WS 1992 Spatial and temporal expression of - and ?-thyroid hormone receptor mRNAs, including the ?-2 subtype, in the developing mammalian nervous system. J Neurosci 12:2288–2302
Kim JC, Shin HC, Cha SW, Koh WS, Chung MK, Han SS 2001 Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy. Life Sci 69:2611–2625
Ferguson SA, Delclos KB, Newbold RR, Flynn KM 2003 Dietary ethinyl estradiol exposure during development causes increased voluntary sodium intake and mild maternal and offspring toxicity in rats. Neurotoxicol Teratol 25:491–501
Ain KB, Mori Y, Refetoff S 1987 Reduced clearance rate of thyroxine-binding globulin (TBG) with increased sialylation: a mechanism for estrogen-induced elevation of serum TBG concentration. J Clin Endocrinol Metab 65:689–696
Ain KB, Refetoff S, Sarne DH, Murata Y 1988 Effect of estrogen on the synthesis and secretion of thyroxine-binding globulin by a human hepatoma cell line, Hep G2. Mol Endocrinol 2:313–323
Emerson CH, Cohen 3rd JH, Young RA, Alex S, Fang SL 1990 Gender-related differences of serum thyroxine-binding proteins in the rat. Acta Endocrinol (Copenh) 123:72–78
World Health Organization Environmental Health Committee 1995 Tetrabromobisphenol A and derivatives. Geneva: World Health Organization
World Health Organization Environmental Health Committee 1997 Flame-retardants: a general introduction. Geneva: World Health Organization
Thomsen C, Lundanes E, Becher G 2002 Brominated flame retardants in archived serum samples from Norway: a study on temporal trends and the role of age. Environ Sci Technol 36:1414–1418(R. Thomas Zoeller, Ruby B)
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@bio.umass.edu.
Abstract
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 ?-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
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
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
Body weight
Two-way ANOVA of maternal body weight revealed a significant effect of treatment (F3,396 = 9.005; P = 0.0001), day (of pregnancy/lactation) (F12,396 = 32.607; P = 0.0001), and a significant interaction (F36,396 = 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; F3,27 = 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 (F3,121 = 2.033; P = 0.113) (Fig. 2).
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.
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.
Serum hormone levels
A three-way ANOVA (age x treatment x gender) of T4 levels in pups revealed significant effects of age (F3,213 = 217.97; P = 0.0001) and treatment (F3,213 = 3.75; P = 0.0118) but not gender (F1,213 = 0.003; P = 0.953). In addition, there was a significant interaction between age and treatment (F9,213 = 2.43; P = 0.012), but no other significant interactions were present (age x gender, treatment x gender, or age x treatment x gender). Further inspection of these data indicated that maternal BPA exposure significantly increased serum total T4 in both male and female pups (Fig. 3). Pups derived from BPA-treated dams exhibited significantly higher levels of T4 on P15 (F3,55 = 3.704; P = 0.0169) (Fig. 3).
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).
Because P15 is a critical time for TH action in the rat brain (39) and there appeared to be no substantive gender differences in the effects of BPA on the hypothalamic-pituitary-thyroid axis, we chose to focus on the ability of BPA to alter TH signaling in P15 males. First, we determined whether the BPA-induced increase in serum T4 in male pups on P15 is associated with an increase in serum TSH; however, we observed no significant differences in mean TSH levels among P15 males born to dams being treated with various BPA concentrations (F3,23 = 0.802; P = 0.505) (Fig. 4). However, despite the finding that TSH levels were not different among groups, there were significant treatment effects of BPA on RC3/neurogranin expression in the upper (F3,22 = 6.48; P = 0.01) and lower leaflet of the dentate gyrus (F3,22 = 6.58; P = 0.0024) but not in the cortex (F3,22 = 1.514; NS) (Fig. 5). RC3/neurogranin mRNA levels in both leaflets of the dentate gyrus were significantly higher in all BPA-treated animals, compared with controls.
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).
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
The present findings strongly suggest that BPA acts as a thyroid hormone antagonist in vivo. Maternal exposure to BPA caused an increase in serum T4 of both male and female pups but simultaneously increased the expression of RC3/neurogranin in the hippocampus. The simplest explanation for these findings is that BPA acts as an antagonist of TH action on the ?-TR, inhibiting TH-negative feedback but leaving the -TR unopposed in responding to elevated T4 in the hippocampus and presumably elsewhere. This is the first report of the in vivo effects of an environmental chemical that can exert direct effects on TH signaling in vitro, and the findings suggest the possibility that incidental exposure to BPA could influence TH signaling in the developing brain.
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 TR?, 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 TR?, 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.
References
Song S, Daneman D, Rovet J 2001 The influence of etiology and treatment factors on intellectual outcome in congenital hypothyroidism. J Dev Behav Pediatr 22:376–384
Rovet J 1999 Congenital hypothyroidism: long-term outcome. Thyroid 9:741–748
Bernal J 2002 Action of thyroid hormone in brain. J Endocrinol Invest 25:268–288
Zoeller RT, Simonyi A, Butnariu O, Fletcher DL, Rudeen PK, McCrone S, Petersen SL 1995 Effects of acute ethanol administration and cold exposure on the hypothalamic-pituitary-thyroid axis. Endocrine 3:39–47
Zoeller RT, Dowling ALS, Herzig CTA, Iannacone EA, Gauger KJ, Bansal R 2002 Thyroid hormone, brain development, and the environment. Environ Health Perspect 110(Suppl 3):355–361
Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon J, O’Heir CE, Mitchell ML, Hermos RJ, Waisbren SE, Faix JD, Klein RZ 1999 Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 341:549–555
Zoeller RT, Rovet J 2004 Timing of thyroid hormone action in the developing brain-clinical observations and experimental findings. J Neuroendocrinol 16:809–818
Morte B, Manzano J, Scanlan T, Vennstrom B, Bernal J 2002 Deletion of the thyroid hormone receptor 1 prevents the structural alterations of the cerebellum induced by hypothyroidism. Proc Natl Acad Sci USA 99:3985–3989
Bernal J, Guadano-Ferraz A, Morte B 2003 Perspectives in the study of thyroid hormone action on brain development and function. Thyroid 13:1005–1012
Manzano J, Morte B, Scanlan TS, Bernal J 2003 Differential effects of triiodothyronine and the thyroid hormone receptor ?-specific agonist GC-1 on thyroid hormone target genes in the brain. Endocrinology 144:5480–5487
Morte B, Manzano J, Scanlan TS, Vennstrom B, Bernal J 2004 Aberrant maturation of astrocytes in thyroid hormone receptor 1 knockout mice reveals an interplay between thyroid hormone receptor isoforms. Endocrinology 145:1386–1391
McKinney JD, Waller CL 1994 Polychlorinated biphenyls as hormonally active structural analogues. Environ Health Perspect 102:290–297
Porterfield SP 1994 Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system. Environ Health Perspect 102(Suppl 2):125–130
McKinney JD, Waller CL 1998 Molecular determinants of hormone mimicry: halogenated aromatic hydrocarbon environmental agents. J Toxicol Environ Health B Crit Rev 1:27–58
Porterfield SP, Hendry LB 1998 Impact of PCBs on thyroid hormone directed brain development. Toxicol Ind Health 14:103–120
Porterfield SP 2000 Thyroidal dysfunction and environmental chemicals—potential impact on brain development. Environ Health Perspect 108(Suppl 3):433–438
Gauger KJ, Kato Y, Haraguchi K, Lehmler HJ, Robertson LW, Bansal R, Zoeller RT 2004 Polychlorinated biphenyls (PCBs) exert thyroid hormone-like effects in the fetal rat brain but do not bind to thyroid hormone receptors. Environ Health Perspect 112:516–523
Cheek AO, Kow K, Chen J, McLachlan JA 1999 Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107:273–278
Moriyama K, Tagami T, Akamizu T, Usui T, Saijo M, Kanamoto N, Hataya Y, Shimatsu A, Kuzuya H, Nakao K 2002 Thyroid hormone action is disrupted by bisphenol A as an antagonist. J Clin Endocrinol Metab 87:5185–5190
Staples CA, Dorn PB, Klecka GM, O’Block ST, Harris LR 1998 A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere 36:2149–2173
Krishnan AV, Stathis P, Permuth SF, Tokes L, Feidman D 1993 Bisphenol A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132:2279–2286
Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, Mcdonell DP 1997 Evaluation of chemicals with endocrine modulating activity in yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol 143:205–212
Kitamura S, Jinno N, Ohta S, Kuroki H, Fujimoto N 2002 Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A. Biochem Biophys Res Commun 293:554–559
Reporter CM 1999 ChemExpo chemical profile: Bisphenol-A. New York: Schnell Publishing Co.
Howe SR, Borodinsky L, Lyon RS 1998 Potential exposure to bisphenol A from food-contact use of epoxy coated cans. J Coatings Technol 70:69–74
Lewis JB, Rueggeberg FA, Lapp CA, Ergle JW, Schuster GS 1999 Identification and characterization of estrogen-like components in commercial resin-based dental restorative materials. Clin Oral Investig 3:107–113
Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I 2002 Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ Health Perspect 110:A703–A707
Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y 2002 Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod 17:2839–2841
Iwamuro S, Sakakibara M, Terao M, Ozawa A, Kurobe C, Shigeura T, Kato M, Kikuyama S 2003 Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larval Xenopus laevis. Gen Comp Endocrinol 133:189–198
Seiwa C, Nakahara J, Komiyama T, Katsu Y, Iguchi T, Asou H 2004 Bisphenol A exerts thyroid-hormone-like effects on mouse oligodendrocyte precursor cells. Neuroendocrinology 80:21–30
Refetoff S, Weiss RE, Usala SJ, Hayashi Y 1994 The syndromes of resistance to thyroid hormone: update 1994. In: Braverman LE, Refetoff S, eds. Clinical and molecular aspects of diseases of the thyroid. Bethesda, MD: The Endocrine Society; 336–343
Tyl RW, Myers CB, Marr MC, Thomas BF, Keimowitz AR, Brine DR, Veselica MM, Fail PA, Chang TY, Seely JC, Joiner RL, Butala JH, Dimond SS, Cagen SZ, Shiotsuka RN, Stropp GD, Waechter JM 2002 Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley Rats. Toxicol Sci 68:121–146
Sharpe RM, Rivas A, Walker M, McKinnell C, Fisher JS 2003 Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens, or with a GnRH antagonist, on Leydig cell development and function through puberty into adulthood. Int J Androl 26:26–36
Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV 1997 Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70–76
vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer KA, Nagel SC, Parmigiani S, Welshons WV 1998 A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol Ind Health 14:239–260
Paxinos G, Watson C 1986 The rat brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press
Zoeller RT, Dowling AL, Vas AA 2000 Developmental exposure to polychlorinated biphenyls exerts thyroid hormone-like effects on the expression of RC3/neurogranin and myelin basic protein messenger ribonucleic acids in the developing rat brain. Endocrinology 141:181–189
Iniguez MA, DeLecea L, Guadano-Ferraz A, Morte B, Gerendasy D, Sutcliffe JG, Bernal J 1996 Cell-specific effects of thyroid hormone on RC3/neurogranin expression in rat brain. Endocrinology 137:1032–1041
Ibarrola N, Rodriguez-Pena A 1997 Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development. Brain Res 752:285–293
Liu Z, Auboeuf D, Wong J, Chen JD, Tsai SY, Tsai MJ, O’Malley BW 2002 Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486. Proc Natl Acad Sci USA 99:7940–7944
Schulz M, Eggert M, Baniahmad A, Dostert A, Heinzel T, Renkawitz R 2002 RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by corepressor binding. J Biol Chem 277:26238–26243
Goldey ES, Kehn LS, Rehnberg GL, Crofton KM 1995 Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol Appl Pharmacol 135:67–76
Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM 1995 Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol Appl Pharmacol 135:77–88
Taylor T, Gyves P, Burgunder JM 1990 Thyroid hormone regulation of TRH mRNA levels in rat paraventricular nucleus of the hypothalamus changes during ontogeny. Neuroendocrinology 52:262–267
Snyder RW, Maness SC, Gaido KW, Welsch F, Sumner SC, Fennell TR 2000 Metabolism and disposition of bisphenol A in female rats. Toxicol Appl Pharmacol 168:225–234
Pottenger LH, Domoradzki JY, Markham DA, Hansen SC, Cagen SZ, Waechter Jr JM 2000 The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. Toxicol Sci 54:3–18
Zalko D, Soto AM, Dolo L, Dorio C, Rathahao E, Debrauwer L, Faure R, Cravedi JP 2003 Biotransformations of bisphenol A in a mammalian model: answers and new questions raised by low-dose metabolic fate studies in pregnant CD1 mice. Environ Health Perspect 111:309–319
Domoradzki JY, Thornton CM, Pottenger LH, Hansen SC, Card TL, Markham DA, Dryzga MD, Shiotsuka RN, Waechter Jr JM 2004 Age and dose dependency of the pharmacokinetics and metabolism of bisphenol a in neonatal Sprague-Dawley rats following oral administration. Toxicol Sci 77:230–242
Iniguez M, Rodriguez-Pena A, Ibarrola N, Aguilera M, Morreale de Escobar G, Bernal J 1993 Thyroid hormone regulation of RC3, a brain-specific gene encoding a protein kinase-C substrate. Endocrinology 133:467–473
Bradley DJ, Towle HC, Young WS 1992 Spatial and temporal expression of - and ?-thyroid hormone receptor mRNAs, including the ?-2 subtype, in the developing mammalian nervous system. J Neurosci 12:2288–2302
Kim JC, Shin HC, Cha SW, Koh WS, Chung MK, Han SS 2001 Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy. Life Sci 69:2611–2625
Ferguson SA, Delclos KB, Newbold RR, Flynn KM 2003 Dietary ethinyl estradiol exposure during development causes increased voluntary sodium intake and mild maternal and offspring toxicity in rats. Neurotoxicol Teratol 25:491–501
Ain KB, Mori Y, Refetoff S 1987 Reduced clearance rate of thyroxine-binding globulin (TBG) with increased sialylation: a mechanism for estrogen-induced elevation of serum TBG concentration. J Clin Endocrinol Metab 65:689–696
Ain KB, Refetoff S, Sarne DH, Murata Y 1988 Effect of estrogen on the synthesis and secretion of thyroxine-binding globulin by a human hepatoma cell line, Hep G2. Mol Endocrinol 2:313–323
Emerson CH, Cohen 3rd JH, Young RA, Alex S, Fang SL 1990 Gender-related differences of serum thyroxine-binding proteins in the rat. Acta Endocrinol (Copenh) 123:72–78
World Health Organization Environmental Health Committee 1995 Tetrabromobisphenol A and derivatives. Geneva: World Health Organization
World Health Organization Environmental Health Committee 1997 Flame-retardants: a general introduction. Geneva: World Health Organization
Thomsen C, Lundanes E, Becher G 2002 Brominated flame retardants in archived serum samples from Norway: a study on temporal trends and the role of age. Environ Sci Technol 36:1414–1418(R. Thomas Zoeller, Ruby B)