Bisphenol A prevents the synaptogenic response
to estradiol in hippocampus and prefrontal cortex
of ovariectomized nonhuman primates
Csaba Leranth*†‡, Tibor Hajszan*, Klara Szigeti-Buck*§, Jeremy Bober*, and Neil J. MacLusky¶
Departments of *Obstetrics, Gynecology, and Reproductive Sciences,†Neurobiology, and§Pharmacology, Yale University School of Medicine,
New Haven, CT 06520; and¶Department of Biomedical Sciences, Ontario Veterinary College, Guelph, ON, Canada N1G 2W1
Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved July 14, 2008 (received for review June 25, 2008)
Exposure measurements from several countries indicate that hu-
mans are routinely exposed to low levels of bisphenol A (BPA), a
synthetic xenoestrogen widely used in the production of polycar-
bonate plastics. There is considerable debate about whether this
exposure represents an environmental risk, based on reports that
BPA interferes with the development of many organs and that it
may alter cognitive functions and mood. Consistent with these
reports, we have previously demonstrated that BPA antagonizes
spine synapse formation induced by estrogens and testosterone in
limbic brain areas of gonadectomized female and male rats. An
important limitation of these studies, however, is that they were
based on rodent animal models, which may not be representative
of the effects of human BPA exposure. To address this issue, we
examined the influence of continuous BPA administration, at a
daily dose equal to the current U.S. Environmental Protection
Agency’s reference safe daily limit, on estradiol-induced spine
synapse formation in the hippocampus and prefrontal cortex of a
nonhuman primate model. Our data indicate that even at this
relatively low exposure level, BPA completely abolishes the syn-
aptogenic response to estradiol. Because remodeling of spine
synapses may play a critical role in cognition and mood, the ability
of BPA to interfere with spine synapse formation has profound
implications. This study is the first to demonstrate an adverse
effect of BPA on the brain in a nonhuman primate model and
further amplifies concerns about the widespread use of BPA in
medical equipment, and in food preparation and storage.
monkey ? stereology ? synaptic plasticity
broad range of uses, including dental prostheses and sealants (1),
the polycarbonate lining of metal cans used to preserve foods
(2), baby bottles (3) and the clear plastic cages used to house
laboratory animals (4). BPA also is used as an additive in many
Polycarbonate is less durable than commonly believed, because
the ester bond linking BPA molecules to the plastic can be
hydrolyzed. With the rate of hydrolysis increasing dramatically
under both acidic and basic conditions, and at elevated temper-
atures, BPA leaches out of polycarbonate containers into food
and beverages under normal conditions of use (3–5). As a result,
exposure measurement data from several countries, including
the United States, indicate that humans are widely exposed to
low levels of BPA on a continuous basis (6).
There is considerable debate about whether this exposure
represents an environmental risk, based mostly on the fact that
some hormonally active chemicals exhibit radically different
potencies in different bioassay systems, making it difficult to
assess their potential adverse effects (7). For example, in a
two-generation trial in rats, BPA was reported to not induce
significant reproductive abnormalities at doses up to 200 ?g/kg
(8), consistent with the relatively low affinity of BPA for the
nuclear estrogen receptors (ERs) ER? and ER?, and its weak
ince the 1950s, the synthetic xenoestrogen bisphenol A
(BPA) has been used in the manufacture of plastics with a
bioactivity in standard tests of estrogenicity (9). Nonetheless,
some reports have indicated that in mice, BPA doses of 2–100
?g/kg interfere with the development of prostate, preputial, and
mammary glands (10), and the central nervous system (11, 12).
BPA administration at doses below the U.S. Environmental
Protection Agency’s (EPA) reference safe daily limit (50 ?g/kg)
for human exposure also interferes with the development of
nonreproductive behaviors, such as play and maze learning, in
both female and male rodents (13–17). These developmental
actions of BPA are particularly worrisome, because relatively
high levels of BPA have been detected in human amniotic fluid
and placenta (18, 19). Recent experiments also have revealed
that BPA is capable of influencing other receptor mechanisms
besides the nuclear ERs that play important roles in the brain,
including the membrane ER (20), thyroid hormone receptor
(21), and androgen receptor (22–25).
Our laboratory has demonstrated that BPA antagonizes spine
synapse formation induced by estrogens and testosterone in
limbic brain areas of gonadectomized female and male rats (26,
27). Because sex steroids are widely thought to play critical roles
in higher brain activities, such as cognition and mood, through
modulating structural and functional synaptic plasticity (28–30),
our findings suggest that exposure to low-dose BPA may have
widespread effects on brain structure and function.
An important limitation of previous studies of BPA is that the
studies were based on rodent animal models. One argument
defending the safety of BPA use is that the clinical predictive
power and experimental utility of rodent studies is limited, due
to dissimilarities between rodent and human endocrine systems
observed in rodents might not occur in primates, including
humans, within the dose range expected from normal environ-
mental exposure. To address this issue, we examined the influ-
ence of continuous exposure to BPA, at a daily dose representing
spine synapse formation in hippocampus and prefrontal cortex
(PFC) of a nonhuman primate model. Our data indicate that the
primate response to estradiol is quite similar to that of rodents,
and that BPA completely abolishes the synaptogenic effect of
estradiol even at relatively low exposure levels.
Effects of Estradiol and BPA on the Number of Spine Synapses.
Experimental manipulation of hormone levels and administra-
tion of BPA resulted in major changes in the number of spine
Author contributions: C.L. and T.H. designed research; C.L., K.S.-B., and J.B. performed
research; T.H. and N.J.M. analyzed data; and C.L., T.H., and N.J.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Commentary on page 13705.
‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2008 by The National Academy of Sciences of the USA
September 16, 2008 ?
vol. 105 ?
no. 37 ?
synapses in all hippocampal and cortical areas examined (Fig. 1).
Two-way ANOVA (treatment ? sampling area) found signifi-
cant treatment (F3,32? 698.815; P ? .001) and sampling area
effects (F3,32? 583.567; P ? .001), indicating significant differ-
ences in the number of spine synapses both among treatment
groups and among analyzed areas. In addition, a significant
treatment ? sampling area interaction effect was seen (F9,32?
86.304; P ? .001), suggesting that the analyzed areas did not
respond to treatment in the same way. Compared with vehicle-
treated control animals, supplementation with estradiol benzo-
ate (EB) significantly increased the number of spine synapses, by
198.1% in the CA1 stratum radiatum (CA1sr), by 94.7% in the
CA3 stratum lucidum and radiatum (CA3sl/sr), by 134.1% in the
dentate gyrus stratum moleculare (DGsm), and by 159.9% in
the PFC. BPA alone had no effect, whereas administration of
BPA along with EB (EB?BPA) completely prevented the strong
synaptogenic response to estradiol; both the BPA- and the
EB?BPA-treated groups did not differ significantly from the
control animals (Fig. 1). These analyses had strong statistical
power; with ? ? 0.05, power was 100% for all of the treatment,
sampling area, and treatment ? sampling area interaction
There were no significant differences among the experimental
groups in terms of volumes of brain areas examined (data not
shown). For all of the experimental groups combined, mean
sampling area volume was 37.51 ? 3.2 mm3for layer II/III of the
PFC, 75.18 ? 10.2 mm3for the CA1sr, 27.04 ? 2.5 mm3for the
CA3sl/sr, and 39.39 ? 3.4 mm3for the DGsm.
Serum Estradiol Levels. A nondetectable level of circulating estra-
diol was found in the animals that were vehicle- or BPA-treated.
In all animals except one that received estradiol treatment (EB-
and EB?BPA-treated groups), the serum estradiol concentra-
tion was in the range of 80–90 pg/ml, in line with our previous
measurements (31). This estradiol level represents the early
follicular phase of the monkey menstrual cycle (32). In one
animal (EB?BPA-treated), the Silastic capsule was filled with
extracellular fluid, indicating that a small hole had developed in
the capsule during treatment, leading to a very high estradiol
level of 450 pg/ml. This animal was not removed from the study,
however, because the number of spine synapses was within the
range established for the other animals in the experimental
This study demonstrates that continuous exposure of ovariecto-
mized nonhuman primates to BPA at a daily dose of 50 ?g/kg
completely abolishes the synaptogenic effect of estradiol in all
hippocampal subregions and layer II/III of the PFC. This finding
is in line with our previous results in rat models, demonstrating
a strong negative effect of BPA on gonadal hormone–induced
spine synapse growth in both gonadectomized females and males
(26, 27). The finding of no significant alterations in the volume
of brain areas examined indicates that the changes in the number
of spine synapses are attributable to varying spine synapse
It appears that among the brain regions investigated, the
CA1sr and PFC were the most sensitive to estradiol. In both of
these regions, the number of spine synapses was about three
times higher in the estradiol-treated animals than in all other
groups. This increase may seem unusually high compared with
replacement of ovariectomized monkeys caused only an ?70%
rise in CA1 spine synapse density (31). In this earlier study,
however, the animals were maintained on a regular, phytoestro-
gen-containing diet, whereas in the present experiment, the
animals received soybean-free food. Comparing data from the
two studies shows that withdrawal of soy caused a ?50% drop
in CA1 spine synapse density, whereas synapse counts after
estradiol supplementation were approximately the same in both
studies (31). Thus, the difference in the apparent magnitude of
estradiol effects between the two studies can be explained by the
well established estrogenic action of diets containing crude soy
meal (33). However, spine synapses in the CA3sl/sr and DGsm
hippocampal areas appear to be less responsive to estradiol. In
these regions, the increase in spine synapse numbers was ap-
proximately twofold. Many earlier studies have consistently
failed to demonstrate morphological cellular plasticity in re-
sponse to estradiol in these areas (28, 34, 35); however, those
studies were based on light microscopic approaches to studying
presynaptic and/or postsynaptic elements, and presynaptic and
postsynaptic structural changes may not necessarily directly
reflect alterations in the number of synapses (36). As our
preliminary report demonstrated (37), in line with the present
results, proper electron microscopic stereology revealed consid-
erable synaptogenic responses to estradiol across the hippocam-
pus of female rats, including the CA3 and dentate gyrus. Further
work is needed to elucidate the relationship between estradiol-
dendritic structure observed in such brain regions as the CA1.
As mentioned earlier, due to an accidental leakage from a
Silastic capsule, one EB?BPA-treated monkey had a very high
serum estradiol level (450 pg/ml), five times higher than that in
any of the other estradiol-treated animals. However, even this
high level of circulating estradiol was unable to overcome the
inhibitory effect of BPA. In this estradiol-overtreated monkey,
the number of spine synapses was within the range of that of
other EB?BPA-treated animals in every brain region examined.
stratum lucidum and radiatum (CA3sl/sr), dentate gyrus stratum moleculare
(DGsm), and layer II/III of PFC of vehicle-treated (Control), estradiol benzoate-
treated (EB), BPA-treated (BPA), and EB?BPA-treated (EB?BPA) monkeys.
One-way ANOVA revealed significant treatment effects in all four brain
indicate significant differences versus the corresponding sampling area of
vehicle-treated controls (Tukey-Kramer test; P ? .05). Spine synapse numbers
The number of spine synapses in CA1 stratum radiatum (CA1sr), CA3
www.pnas.org?cgi?doi?10.1073?pnas.0806139105Leranth et al.
Functional Considerations and Relevance to Human Health. Alter-
ations in patterns of synaptogenesis appear to play critical roles
in some neurologic/neuropsychiatric disorders, including mental
retardation and developmental disabilities (38), Alzheimer’s
disease (39), schizophrenia (40, 41), and mood disorders (42–
44). The neurobiology of these disorders remains unclear, al-
though growing evidence indicates that the intricate balance of
effects from growth factors and hormones, which may be re-
quired to maintain normal synaptic plasticity, becomes derailed
in patients with these diseases. Estrogens derived both from
circulation and from local biosynthesis within the brain itself
represent an important contributory factor to these mechanisms
(45–47). Estradiol has long been known to induce a strong
synaptogenic response in limbic brain areas (31, 48, 49). Re-
modeling of dendritic spines and their synapses may play critical
roles both in learning and memory (50) and in the neurobiology
of mood disorders (29, 44). Thus, synapse formation on pyra-
midal cell dendritic spines in the hippocampus and PFC, areas
critically involved in mnemonic functions and mood regulation,
may contribute to the modulatory effect of gonadal steroids on
cognition and mood (28–30, 51, 52). On this basis, the ability of
BPA to interfere with spine synapse formation in the hippocam-
and maze learning behaviors reported in both female and male
rodents after developmental BPA exposure (13–17) may be
based, at least in part, on this mechanism. In addition, exposure
to BPA and the resulting loss of hippocampal spine synapses may
elicit depressive behavior. Although limited data are available,
two studies have demonstrated that BPA indeed promotes
helpless behavior in the learned helplessness paradigm (53) and
increases immobility in the forced swim test (54), signs of
depressive behavior in two widely accepted animal models of
The mechanism by which BPA exerts its inhibitory effect on
synaptogenesis is currently unknown. Because BPA has a rela-
tively low affinity for the ERs (9), a potential explanation could
be that BPA directly targets intracellular mechanisms, such as
the ERK and Akt pathways, that are activated by estradiol (20,
55) and are involved in the remodeling of spine synapses and
cognitive functions (56–59). For example, low concentrations of
BPA interfere with estradiol action in cerebellar granule cells,
possibly through activation of protein phosphatase 2A–like ERK
dephosphorylation (20). A similar mechanism may contribute to
the inhibition of estradiol/phospho-ERK–mediated synaptogen-
esis in the hippocampus and PFC. Moreover, it is not yet clear
whether BPA acts directly or indirectly on limbic brain areas.
Critical prefrontal and hippocampal functions, such as memory
and attention, are all modulated by subcortical cholinergic,
dopaminergic, and serotonergic systems (60). Recent studies
have demonstrated that oral administration of BPA results in
hyperactivity at 4–5 weeks of age, degeneration of mesence-
phalic dopaminergic neurons at 7 weeks of age, and decreased
gene expression levels for dopamine transporter in adult rats
(11). It also has been reported that prenatal and neonatal
exposure of mice to BPA induces memory impairment as
measured by a step-through avoidance test, associated with a
dramatic reduction in the cholinergic innervation of the hip-
pocampus at 7 weeks of age (61).
In summary, this study demonstrates for the first time an
adverse effect of BPA on spine synapse numbers in the brain of
a nonhuman primate model. The powerful inhibition of estra-
diol-induced hippocampal and PFC spine synapse formation by
BPA was observed at the low exposure level defined by the EPA
as the reference safe daily limit. Although additional studies are
needed, especially in primates, these findings further amplify
concerns about the widespread use of BPA in the production of
materials used in food preparation and storage.
Materials and Methods
Animals. Young adult female African green monkeys (Chlorocebus aethiops
sabaeus) of reproductive age were used (n ? 12; body weight, 4–5 kg).
ical Research Foundation (SKBRF). The SKBRF traps or breeds its animals and
with all applicable U.S. regulations and has provided an assurance of compli-
ance (#A3005) to the Office of Laboratory Animal Welfare. All animal proto-
cols used in this study were in compliance with the National Institutes of
Health’s Guide for the Care and Use of Laboratory Animals and were
approved by SKBRF’s Institutional Animal Care and Use Committee.
Surgery and Hormone Treatment. All monkeys were anesthetized (20 mg/kg
ketamine, i.m, followed by 20 mg/kg pentobarbital i.v.), intubated, and
ovariectomized through a median laparatomy under sterile conditions. In the
same surgical session, animals received the following treatments:
Y Vehicle-treated controls (three animals): a cholesterol-filled 4-cm long Si-
lastic capsule (Dow Corning; 3.5 mm i.d., 4.65 mm o.d.) and a vehicle-filled
minipump (Alzet 2ML4 osmotic pump, delivering fluid at a rate of 2.5 ?l/h
for 4 weeks).
and an Alzet minipump loaded with vehicle.
Y BPA-treated group (three animals): a cholesterol-filled Silastic capsule and
a BPA-filled Alzet minipump.
Y EB?BPA-treated group (three animals): a Silastic capsule containing crys-
talline EB and an Alzet minipump loaded with BPA.
We demonstrated that this type of estradiol treatment induces marked
changes in CA1 spine synapse density in female monkeys (31). The Alzet
minipumps delivering BPA were filled with 4.17 ?g/?l of BPA dissolved in
propylene glycol (Sigma) to supply BPA at a rate of 50 ?g/kg/day. The Silastic
capsules and minipumps were implanted below the skin of the back.
extracted from the pumps after the animals were euthanized. After surgery
and recovery, the animals were housed in individual cages. They were given
buprenorphine analgesia (Buprenex, 0.01 mg/kg i.m.) at the conclusion of
surgery, followed by carprofen (Remidil, 2 mg/kg by mouth) every 6 h for the
first 2 postoperative days. Water and food intake (soybean-free diet:
TD.06476; Harlan Teklad) and incision sites were monitored until complete
wound healing occurred.
Euthanasia and Tissue Processing. Twenty-eight days later, the animals were
deeply anesthetized (ketamine 20 mg/kg i.m, followed by an overdose of
pentobarbital, 100 mg/kg i.v.). After blood samples were collected, the
animals were euthanized by transcardial perfusion of heparinized saline
(1.0 liter), followed by a fixative [1.5 liters, containing 4% paraformalde-
descending aorta clamped. Brains were dissected out and postfixed over-
night in the same, but glutaraldehyde-free, fixative. The tissues were
stored and transported to Yale University in phosphate buffer containing
0.1% sodium azide.
Unbiased Quantitative Synaptology. The number of spine synapses in the
CA1sr, CA3sl/sr, DGsm, and layer II/III of Walker’s area 46 (PFC) was calcu-
lated as described in ref. 62. Serial sections (200 ?m) were cut in the coronal
then systematically sorted into 10 groups. One randomly selected group of
ethanol containing 1% uranyl acetate for 40 min, and flat-embedded in
Durcupan (Electron Microscopy Sciences) between slides and coverslips.
The volume of the sampling areas was estimated using the Cavalieri
estimator module of the Stereo Investigator system (MicroBrightField).
to the highly organized cytoarchitecture of the hippocampus. The bound-
aries of Walker’s area 46 were determined according to the method
described by Tang et al. (49).
Subsequently, 20 sampling sites for electron microscopic analysis were
localized in each sampling area using a systematic-random approach, as
modified from MacLusky et al. (62). In brief, we used a two-dimensional
coordinate system with length (L) and height (H) axes. Using the same group
of sections that previously underwent volume estimation, the length of the
(L/20), and a random number (N) between 0 and L/20 was selected. The
Leranth et al.
September 16, 2008 ?
vol. 105 ?
no. 37 ?
subsequent coordinates at L/20 ?m apart, going along the L axis (the CA1
stratum pyramidale) in the direction of the CA3 ? subiculum and from rostral
to caudal. Subsequently, the height of CA1sr was measured at each L-axis
sampling coordinate, along lines drawn perpendicular to the stratum pyra-
midale. These 20 height measurements were then combined to create the
H-axis. Sampling sites along the H axis were localized using the same method
as described for the L-axis, going along the height measurement lines in the
direction of the stratum pyramidale ? stratum lacunosum moleculare and
from rostral to caudal. This technique was repeated to localize sampling sites
in the remaining sampling areas. The CA3 stratum pyramidale, dentate gyrus
DGsm, and PFC, respectively. Finally, blocks were assembled for ultracutting
and trimmed, and then approximately four 75-nm-thick consecutive ultrasec-
tions were cut at each identified sampling site using a Reichert Ultracut E
for the physical disector using a Tecnai-12 transmission electron microscope
(FEI Company) furnished with a Hamamatsu HR/HR-B CCD camera system, at
depicting identical regions in adjacent ultrasections, with these identical
significantly between adjacent ultrasections due to their size. Before synapse
counting, the pictures were coded for blind analysis. This sampling technique
provided 20 disectors for each of the CA1sr, CA3sl/sr, DGsm, and PFC (i.e., 80
disectors total per brain).
Asymmetric spine synapses were counted according to the rules of the
disector technique (63) within an unbiased counting frame superimposed
onto each electron micrograph. Synapsing spines were identified by the
presence of postsynaptic densities, and by the absence of mitochondria,
microtubules, and synaptic vesicles. The average volumetric density (syn-
apse/?m3) of spine synapses within each sampling area was then deter-
mined by dividing the sum of spine synapses counted in all samples taken
from that particular sampling area by the disector volume. The disector
volume was calculated by multiplying the area of the unbiased counting
frame (79 ?m2) by ultrasection thickness (average 75 nm) and by the
number of disectors (i.e., 20). Thus, the average disector volume, uniformly
for each sampling area, was 237.6 ?m3. Finally, the volumetric density of
spine synapses was multiplied by the volume of the sampling area, deter-
mined earlier, to arrive at the total number of spine synapses. The number
of spine synapses was calculated independently by two different investi-
gators (C.L. and T.H.), and the results were cross-checked to preclude
systematic analytical errors.
Statistical Analysis. Spine synapse numbers obtained from individual sam-
pling areas were used to calculate means ? standard deviations for each
treatment group. Results were analyzed using Bartlett’s test for homoge-
neity of variance and two-way (treatment ? sampling area) ANOVA, to test
for significant interaction effects that might indicate that responses to
treatment were region-dependent. The data for each brain region were
then analyzed individually by one-way ANOVA, followed by the conserva-
tive Tukey-Kramer post hoc test for comparison of individual group means.
these methods, standard deviations for spine synapse numbers are ?5% of
the mean. With a standard deviation of 5% and sample sizes of n ? 3 per
group, a 15% change in mean spine synapse numbers can be detected with
? ? 0.05 and 80% power.
Hormone Assay. Blood samples were allowed to clot, then centrifuged to
separate serum. Serum samples were frozen at ?80°C until being assayed
using an IMMULITE LKE-21 estradiol chemiluminescent enzyme immunoassay
kit (Siemens Healthcare Diagnostics). According to the manufacturer’s speci-
fications, the analytical sensitivity of the kit is 15 pg/ml, with coefficients of
variance of 9.5% for intraassay precision and 9.3% for interassay precision in
the concentration range of our samples.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grants ES014893 (to C.L.) and MH074021 (to T.H.), and by a National
Alliance for Research on Schizophrenia and Depression Young Investigator
Award (to T.H.).
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