Sex specific impact of perinatal bisphenol A (BPA) exposure over a range of orally
administered doses on rat hypothalamic sexual differentiation
Katherine A. McCaffreya, Brian Jonesb, Natalie Mabreya, Bernard Weissb, Shanna H. Swanc,
Heather B. Patisaula,d,*
aDepartment of Biology, North Carolina State University, Raleigh, NC 27695, United States
bDepartment of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States
cDepartment of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
dWM Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695, United States
Numerous attempts have been made to characterize the impact
of early life bisphenol A (BPA) exposure on sexually dimorphic
brain development in rodents because of growing concern that
similar effects may occur in humans (He et al., 2012; Palanza et al.,
2008; Richter et al., 2007; Wolstenholme et al., 2011). Two
hypothalamic regions which have garnered considerable attention,
because of their well characterized estrogen-dependent structural
and functional sex differences (Simerly, 2002), are the sexually
dimorphic nucleus of the preoptic area (SDN-POA) and the
anteroventral periventricular (AVPV) nucleus. Males and females
are born with the same number of neurons in both regions, but
estradiol-mediated selective cell death mediated by ERa (Patchev
et al., 2004) during neonatal life rapidly induces morphological sex
differences (Wright et al., 2010). Remarkably, estradiol has
opposite effects on cell survival in each region such that the
SDN-POA is larger in males, and the AVPV is larger in females. The
female AVPV also contains more dopaminergic neurons than males
(Davis et al., 1996b; Simerly, 2002; Simerly et al., 1985a). Previous
studies examining BPA-related impacts on SDN-POA and AVPV
volume and composition have yielded discordant results (He et al.,
2012; Kwon et al., 2000; Nagao et al., 1999; Patisaul et al., 2006,
2007; Rubin et al., 2006). Inconsistencies in the data likely result, at
least in part, from experimental design differences including
exposure duration, dose, route of BPA administration, and critical
differences in neural structure between rats and mice (Bonthuis
et al., 2010). To improve data continuity, study design-related
guidelines for BPA research have recently been issued including
statistical control for litter effects, examination over a wide dose
NeuroToxicology 36 (2013) 55–62
A R T I C L E
I N F O
Received 24 January 2013
Accepted 6 March 2013
Available online 13 March 2013
A B S T R A C T
Bisphenol A (BPA) is a high volume production chemical used in polycarbonate plastics, epoxy resins,
thermal paper receipts, and other household products. The neural effects of early life BPA exposure,
particularly to low doses administered orally, remain unclear. Thus, to better characterize the dose range
over which BPA alters sex specific neuroanatomy, we examined the impact of perinatal BPA exposure on
two sexually dimorphic regions in the anterior hypothalamus, the sexually dimorphic nucleus of the
preoptic area (SDN-POA) and the anterioventral periventricular (AVPV) nucleus. Both are sexually
differentiated by estradiol and play a role in sex specific reproductive physiology and behavior. Long
Evans rats were prenatally exposed to 10, 100, 1000, 10,000 mg/kg bw/day BPA through daily, non-
invasive oral administration of dosed-cookies to the dams. Offspring were reared to adulthood. Their
brains were collected and immunolabeled for tyrosine hydroxylase (TH) in the AVPV and calbindin
(CALB) in the SDN-POA. We observed decreased TH-ir cell numbers in the female AVPV across all
exposure groups, an effect indicative of masculinization. In males, AVPV TH-ir cell numbers were
significantly reduced in only the BPA 10 and BPA 10,000 groups. SDN-POA endpoints were unaltered in
females but in males SDN-POA volume was significantly lower in all BPA exposure groups. CALB-ir was
significantly lower in all but the BPA 1000 group. These effects are consistent with demasculinization.
Collectively these data demonstrate that early life oral exposure to BPA at levels well below the current
No Observed Adverse Effect Level (NOAEL) of 50 mg/kg/day can alter sex specific hypothalamic
morphology in the rat.
? 2013 Elsevier Inc. All rights reserved.
* Corresponding author. Tel.: +1 919 513 7567; fax: +1 919 515 5327.
E-mail address: email@example.com (H.B. Patisaul).
Contents lists available at SciVerse ScienceDirect
0161-813X/$ – see front matter ? 2013 Elsevier Inc. All rights reserved.
range, use of oral administration, employment of concurrent
positive controls, and performing all evaluations blinded to the
exposure groups (Goodman et al., 2006; Hengstler et al., 2011;
Hunt et al., 2009; Richter et al., 2007). Here we evaluated the
impact of perinatal BPA exposure on SDN-POA and AVPV structure
in rats of both sexes using these design guidelines to enhance the
currently available data and provide results across a wider dose
range than has been done previously.
In mammals, including humans, there are numerous structural
and functional sex differences throughout the brain, particularly
within the hypothalamus and surrounding structures (Bonthuis
et al., 2010; De Vries, 2004; Simerly, 2002), which underpin
physiological and behavioral sexual dimorphisms. The SDN-POA
and AVPV are ideal regions to examine because the estrogen-
dependent mechanisms by which they sexually differentiate, both
structurally and functionally, are well understood and can be
predictably manipulated by exogenous hormone administration
(Davis et al., 1996a; Gilmore et al., 2012; Gorski et al., 1978, 1980;
Sickel and McCarthy, 2000; Simerly, 1989; Simerly et al., 1985b;
Yang et al., 2004). Thus, they are well defined targets for endocrine
disrupting chemicals (EDCs) such as BPA. The SDN-POA is
physically larger in males while the AVPV is larger in females;
morphometric differences resulting from the presence or absence
of perinatal estrogens (Bleier et al., 1982; Bloch and Gorski, 1988;
Murakami and Arai, 1989). The specific cellular mechanisms by
which estradiol can be pro-apoptotic in the AVPV but anti-
apoptotic in the SDN-POA remain to be fully characterized, but
likely involve region specific proinflammatory cytokine and
caspase signaling pathways (Wright et al., 2010).
The number of preoptic dopaminergic neurons, identified by
the presence of tyrosine hydroxylase (TH; the rate-limiting
enzyme for dopamine biosynthesis), is also sexually dimorphic
in rats (Patisaul et al., 2006; Simerly et al., 1985b). In females, the
region comprising the AVPV and the periventricular region just
caudal to it contains approximately three times as many TH
immunoreactive cells as the comparable male region (Simerly,
1989). Perinatal exposure to estradiol or an aromatizable androgen
such as testosterone propionate results in masculinization of this
region in females, and reduced numbers of immunoreactive TH
(TH-ir) cells (Simerly, 1989; Simerly et al., 1985b). In contrast, the
rat SDN-POA is 2–4 times larger in males than females (Gorski
et al., 1978, 1980); a trait that develops in the first two weeks of life
and results from a higher apoptotic rate in females (Davis et al.,
1996a; Yang et al., 2004). In males, neonatal castration lowers
circulating testosterone, from which neural estrogen is derived,
and SDN-POA volume is consequently reduced (Davis et al., 1996a;
Gorski et al., 1978, 1980; Sickel and McCarthy, 2000). Similarly,
perinatal exposure to estradiol masculinizes the female SDN-POA
resulting in increased volume (Dohler et al., 1982, 1984; Gorski
et al., 1978). Calbindin-D28 (CALB), a calcium-binding protein and
potential neuroprotectant, is a reliable marker to define the
borders of the SDN-POA (Patisaul et al., 2007; Sickel and McCarthy,
2000). Thus, CALB immunolabeling (CALB-ir) was used here to
identify both the borders of the SDN-POA and cell density within it.
These physical sex differences are reflective of functional ones.
Both the SDN and AVPV are thought to play a role in the display of
male sex behaviors (Rhees et al., 1999; Roselli et al., 2004), and lesion
studies have demonstrated the critical importance of the AVPV in
generating the preovulatory gonadatropin surge in females (Gerall
et al., 1980; Wiegand and Terasawa, 1982). Although still highly
controversial, in humans and sheep, a smaller SDN volume has been
linked to the defeminization of sexual behavior and mate choice in
males (Roselli et al., 2004). In rodents, early life BPA exposure has
been shown to impact both male reproductive behavior (Jones et al.,
2010) and female fertility (Cabaton et al., 2011); effects consistent
with AVPV and SDN perturbation.
For the present study, Long Evans pups were perinatally
exposed to a wide range of BPA doses (10, 100, 1000, 10,000 mg/kg/
day) through daily, non-invasive oral administration of dosed-
cookies to dams. This dose range spanned the current no
observable adverse effect level (NOAEL) of 50 mg/kg bw/day and
the current reference dose (tolerable daily intake) of 50 mg/kg bw/
day (Chapin et al., 2008; Geens et al., 2012; NTP, 1982). Despite
well recognized metabolic differences between rats and humans,
the lowest dose used here likely produces dam serum levels at the
top end of, or just above, the human-relevant range (Doerge et al.,
2011), although the boundaries of this range remain a point of
contention (FAO/WHO, 2011; Hengstler et al., 2011; Sathyanar-
ayana et al., 2011; Taylor et al., 2010; Teeguarden et al., 2011;
Twaddle et al., 2010). Exposure to the pups (through gestation and
lactation) is likely lower. Prior estimates (reviewed in (Chapin
et al., 2008; FAO/WHO, 2011) suggest that gestational exposure is
1.6–18.5 times lower than that of the dam, and lactational transfer
is even lower. Daily intake of BPA by humans is not well
characterized but estimated to be approximately 0.4–1.4 mg/kg/
day (Chapin et al., 2008; FAO/WHO, 2011).
2.1. Animal care and exposure
Animal care and exposure of pregnant Long Evans (LEs) dams
was conducted at the University of Rochester. All experimental
procedures were performed in accordance with Society for
Neuroscience guidelines and University of Rochester animal care
and utilization committees (UCAR).
Sixty-four timed-pregnant LE female rats (Charles River,
Raleigh, NC) were orally exposed to one of four BPA dose levels,
BPA 10 (n = 12), BPA 100 (n = 12), BPA 1000 (n = 10), and BPA
10,000 (n = 11) mg/kg bw/day, corn-oil vehicle (n = 11), or 17b-
estradiol (n = 2). This small number of estradiol-exposed dams was
included to verify the sensitivity of LE rats to estrogenic
compounds using this specific exposure paradigm. Prior studies
have clearly established that exposure to ?2 mg/kg bw/day 17b-
estradiol during early development is sufficient to masculinize the
size and CALB-ir content of the female rat SDN-POA (Dohler et al.,
1984; Gilmore et al., 2012; Gorski et al., 1978) as well as TH-ir cell
number in the female AVPV (Patisaul et al., 2006; Simerly, 1989;
Simerly et al., 1985a).
Dams arrived on gestational day (GD) 4 and were housed under
a 12-h light cycle at 74 8F and 30–70% humidity in thoroughly
washed polysulfone cages on woodchip bedding, fed Purina 5001
rodent chow (Purina Lab Diet, Richmond, IN), and provided with
filtered tap water in glass water bottles ad libitum. BPA doses and
the corn-oil vehicle were delivered daily to pregnant dams via a
quartered, Nilla1Wafer cookie from GD 12 to postnatal day (PND)
10 using procedures similar to those described previously (Patisaul
et al., 2013). Thus, developing rat pups were exposed in utero and
during lactation for a total exposure period of 21 days.
Corn-oil vehicle or corn-oil/BPA dose (?0.2 cm3adjusted for
bw) was applied daily to quartered, standard-sized (roughly 100to
1–1/400in circumference prior to quartering) Nilla1Wafers using a
fresh, sterile 1cc syringe for each dose. The corn-oil/corn-oil BPA
solution was readily absorbed by the wafer ensuring that the
animal received the entire dose. Each animal had a separate,
labeled weigh-boat in which the dosed cookie was transferred. The
cookies were placed in the cage, away from the nesting location of
the female by lifting the wire rack at a small angle (enough to
accommodate the cookie) and dropped onto the bedding. Each
dam was observed daily during this exposure regimen to ensure
complete wafer consumption. The average time for dams to fully
consume the wafer was approximately three minutes. All pups
K.A. McCaffrey et al. / NeuroToxicology 36 (2013) 55–62
were weaned on PND 21 and randomly assigned to one of four
experimental groups. Four males and four females per litter were
used to generate the data.
2.2. Tissue collection and preparation
Animals were sacrificed between PNDs 65–68. Animals were
deeply anesthetized with sodium pentobarbital and transcardially
perfused with 0.9% NaCl followed by 400 ml 4% paraformaldehyde
in 0.01 M sodium phosphate buffer (pH 7.4). Females were
sacrificed in estrous (verified by vaginal cytology (Becker et al.,
2005)) and weight was recorded for all animals at the time of
sacrifice. Brains were removed and postfixed in 30% sucrose/4%
paraformaldehyde for 3–4 h, then cryoprotected in 30% sucrose/
PBS solution for 24–72 h (Hoffman and Le, 2004). Brains were
rapidly frozen on dry ice, shipped to NCSU for processing and
stored at ?80 8C. Each brain was coronally sectioned at 50 mm
using a freezing slide microtome, divided into four series of
alternating sections comprising the SDN-POA and AVPV and stored
free-floating in a cryoprotectant antifreeze solution (30% sucrose,
30% ethylene glycol, 10% polyvinylpyrrolidone, 5% glycerol in 0.1 M
sodium phosphate buffer) at ?20 8C until staining (Hoffman and
SDN-POA sections were immunolabeled for CALB, and
sections comprising the AVPV and the periventricular region
just caudal to it were immunolabeled for TH as detailed in our
prior publications (Patisaul et al., 2006, 2007). Selected sections
were 100 mm apart in the SDN-POA, and 200 mm in the AVPV
region. Briefly, all sections were thoroughly washed at 4 8C in
endogenous peroxidase activity was quenched with a 15 min
wash in 0.5% H2O2in DK-LKPBS (2% normal donkey serum and
0.3% Triton X in 0.2 M KPBS). The sections were then washed and
incubated in DK-LKPBS overnight at 4 8C. SDN-POA sections were
incubated in a primary antibody solution directed against CALB
(Mouse Monoclonal Anti-Calbindin-D-28K (Cat # C9848, Sigma,
St. Louis, MO), 1:100,000 in DK-LKPBS; Sigma, St. Louis, MO), and
AVPV sections were incubated in a primary antibody directed
against TH (Mouse Monoclonal Anti-Tyrosine Hydroxylase (Cat #
22941, Immunostar, Hudson, WI), 1:80,000 in DK-LKPBS) for 72 h
at 4 8C. All sections were then washed and placed in a
biotinylated donkey anti-mouse immunoglobulin G (IgG) sec-
ondary antibody (1:200, Jackson ImmunoResearch Laboratories,
West Grove, PA) for 90 min at room temperature. The signal was
amplified using an avidin-biotin complex kit (Vector Labs,
Burlingame, CA) and developed using DAB chromagen (Vector
Labs, Burlingame, CA). After a final set of washes, the sections
were serially mounted onto Fisherbrand Superfrost Plus slides
(Fisher, Pittsburgh, PA) and allowed to dry overnight. The
sections were then dehydrated via washes of increasing ethanol
stringency and cleared in xylene (Fisher, Pittsburgh, PA) for two
hours. Slides were coverslipped with DPX mountant (Electron
Microscopy Services, Hatfield, PA).
2.4. Quantification of CALB-ir and SDN-POA volume
Quantification of SDN-POA CALB-ir cells and determination of
SDN-POA nuclear volume, was accomplished via unbiased
stereology (Glaser and Glaser, 2000; Schmitz and Hof, 2005) as
we have done previously (Patisaul et al., 2007). All analyses were
done using StereologerTM(Stereology Resource Center, Inc., MD) on
a Leica DM2500P scope (Leica Microsystems, Wetzlar, Germany).
The borders of the SDN-POA were clearly defined by CALB
immunolabeling and confirmed using the Adult Rat Stereotaxic
Atlas (Paxinos and Watson, 2007). For each section, SDN-POA
borders were traced at low magnification (5?) and then analyzed
at high magnification (63?). Volume was calculated using
Calviari’s Principle. CALB-ir cells were counted using the optical
fractionator. The complete nucleus was contained within 2–3
sections per animal. The final post-processing tissue thickness of
the sections was measured to be approximately 22.7 mm, therefore
the frame height was set below that threshold at 20 mm with a
guard height of 1 mm. To quantify the dense population of cells, the
frame area was set at 15 mm2(3.873 mm ? 3.873 mm), and
framing spacing was 50 mm. The volume of the CALB-ir subregion
of the SDN was quantified with a region point counting area per
point of 1000 mm2. The mean coefficient of error for CALB-ir cells
counted with the optical fractionator was 0.14 and for CALB-SDN
volume was 0.09. Images were captured using a Qimaging Retiga
2000R 12-bit color camera (QImaging, Surry, British Columbia,
Canada) mounted on a Leica DM5000B scope (Leica Microsystems,
Wetzlar, Germany) using MCID Core Image software program
(InterFocus Imaging Ltd., Cambridge, England).
2.5. Quantification of tyrosine hydroxylase (TH) immunoreactivity in
Quantification of TH-ir was done with the optical fractionator
as described above for CALB-ir cells in the SDN-POA. TH-ir cells
were contained within 3–4 sections per animal. The final post-
processing tissue thickness of the sections was measured to be
approximately 22.7 mm, therefore the frame height was set at
20 mm with a guard height of 1 mm. The frame area was
2500 mm2(50 mm ? 50 mm), and framing spacing was 50 mm.
The region point counting area per point was set at 1000 mm2.
The mean coefficient of error for TH-ir nuclei counted with
the optical fractionator was 0.09. Images were captured using
a Qimaging Retiga 2000R 12-bit color camera (QImaging,
DM5000B scope (Leica Microsystems, Wetzlar, Germany) using
MCID Core Image software program (InterFocus Imaging Ltd.,
2.6. Statistical analysis
Data analysis was performed using published guidelines
established for assessing low-dose endocrine disruptor data
(Haseman et al., 2001). Control and 17b-estradiol exposed females
were compared by a Student’s t-test (pooled variance) for each
measure to confirm the sensitivity of the animal model to the
masculinizing influence of estradiol. For each endpoint, control and
BPA exposed animals of both sexes were compared by two-way
analysis of variance (ANOVA) with exposure group and sex as
factors, and followed up with a one-way ANOVA within sex.
Significant effects were followed up by protected Fisher’s least
significant difference (LSD) post hoc analysis. Two sample t-tests
(separate variance) were performed within each exposure group to
address if sex difference was preserved between male and female
groups. All analyses were completed using SYSTAT software
(SYSTAT, Systat Software Inc., Richmond, CA) and in all cases
effects were considered significant at p ? 0.05.
3.1. Confirmation of strain sensitivity
Both regions were completely masculinized by 17b-estradiol,
observations which confirm the sensitivity of the LE rat to
oral estrogen during this critical window of development and
K.A. McCaffrey et al. / NeuroToxicology 36 (2013) 55–62
demonstrate that it is an appropriately sensitive animal model for
examining BPA effects. Estradiol exposed females (n = 2) had
significantly fewer AVPV TH-ir neurons then unexposed controls
(t(9.000) = 3.820,
p ? 0.004).
(7.000) = 7.763,
p ? 0.001)
(t(7.000) = 7.763, p ? 0.02) was significantly increased in estradiol
3.2. Impact of BPA on SDN-POA volume and CALB-ir
As anticipated (Davis et al., 1996a; Dohler et al., 1984; Gorski
et al., 1978; Patisaul et al., 2007; Sickel and McCarthy, 2000),
(t(9.310) = 6.251, p ? 0.001) and a greater number of CALB-ir
cells within it (t(9.812) = 4.201, p ? 0.002) compared to control
females. Two-way ANOVA revealed an interaction between
exposure group and sex for both SDN volume (F(4,84) = 5.719,
p ? 0.001) and CALB-ir cell numbers (F(4,81) = 3.890, p ? 0.006).
Significant effects of exposure were only present in males. There
was a main effect of BPA exposure on male SDN-POA volume
(F(4,38) = 6.557, p ? 0.001; Fig. 1), and post hoc analysis revealed
that SDN-POA volume was significantly smaller in all BPA exposed
groups compared to unexposed controls (Fig. 3). Volumetric
effects were greatest in the BPA 10 mg/kg/day (p ? 0.001) and BPA
100 mg/kg/day (p ? 0.001) groups, with mean volume decreased
45% and 39%, respectively. There was also a main effect of
exposure on the number of CALB-ir cells in the male SDN-POA
(F(4,40) = 3.9445, p ? 0.009) with numbers significantly de-
creased in all BPA exposed groups compared to the unexposed
controls (p ? 0.003 for all) except the BPA 1000 group (p = 0.07;
Fig. 2). Two sample t-tests within each group revealed that the sex
difference in SDN-POA volume was preserved in all exposure
t(13.577) = 3.174,
t(17.424) = 3.631,
p ? 0.002;
p ? 0.001; BPA 10,000: t(8.399) = 4.098, p ? 0.003). The sex
difference in CALB-ir cell density, however, was only preserved
in the BPA 1000 exposure group (t(11.552) = 2.376, p ? 0.036),
and lost in the other groups.
p ? 0.007;
t(10.013) = 7.415,
3.3. Impact of BPA on AVPV TH-ir
As expected (Davis et al., 1996b; Patisaul et al., 2006; Simerly
et al., 1985a), AVPV TH-ir was sexually dimorphic in the unexposed
controls (t(15.201) = 4.807, p ? 0.001), with females having nearly
twice as many TH-ir neurons than males (Fig. 4). Two-way ANOVA
revealed a main effect of sex (F(4,92) = 4.694, p ? 0.002) and a
main effect of exposure (F(1,92) = 42.214, p ? 0.001) but no
significant interaction between sex and exposure. Because TH-ir
is sexually dimorphic, subsequent analyses were performed within
sex. One way ANOVA within sex revealed a main effect of BPA
exposure on AVPV TH-ir cell numbers in females (F(4,52) = 3.142,
p ? 0.02) and males (F(4,39) = 4.153, p ? 0.007) (Fig. 5). TH-ir cell
numbers were significantly lower in all BPA exposed females
(p ? 0.05 for all) with the exception of the BPA 1000 group
(yp ? 0.06). In males, TH-ir cell numbers were only significantly
impacted in the BPA 10 (p ? 0.03) and BPA 10,000 (p ? 0.013)
exposure groups, with fewer TH-ir cells compared to controls. Two
sample t-tests within each exposure group revealed that the sex
difference in TH-ir cell density was preserved in all exposure
groups (BPA 10: t(19.743) 2.779, p ? 0.01; BPA 100: t(16.069) =
2.423, p ? 0.03; BPA 1000: t(16.270) = 2.670, p ? 0.02; BPA 10,000:
t(18) = 2.943, p ? 0.01).
Perinatal BPA exposure via oral exposure to the dam altered the
structure and composition of the rat SDN-POA and AVPV within a
dose range encompassing the current reference dose of 50 mg/
kg bw/day. Effects were region and sex specific with evidence of
demasculinization in the male SDN-POA, and evidence of
defeminization in the female AVPV. All effects were significant
at the lowest dose employed (10 mg/kg bw/day). Despite well
recognized metabolic differences between rats and humans, this
dose likely produces serum levels at the top end of the human-
relevant range (Chapin et al., 2008; Doerge et al., 2011), although
the bounds of this range remain unresolved (FAO/WHO, 2011;
Hengstler et al., 2011; Sathyanarayana et al., 2011; Taylor et al.,
Fig. 1. Bisphenol-A exposure on SDN-POA CALB-ir volume in adult male and female
rats. There was a main effect of BPA exposure on the volume of the SDN-POA
observed between control male and all male exposure groups (p ? 0.001). No
significant change was observed in female exposure groups. All exposure groups are
in mg/kg/day. S.E. is indicated by error bars. Control M, F (n = 8, 7); BPA 10 M, F
(n = 10, 13); BPA 100 M, F (n = 9, 13); BPA 1000 M, F (n = 9, 7); BPA 10,000 M, F
(n = 7, 11); statistically significant change within exposure groups as compared to
control animals within the same sex are as indicated: *p < 0.01–0.05, **p < 0.001–
0.01, ***p < 0.001.
Fig. 2. Bisphenol-A exposure on SDN-POA CALB-ir cell number in adult male and
female rats. There was a significant overall effect of BPA exposure (p ? 0.009) in
male exposure groups BPA 10 and BPA 100 as compared to control males. All
exposure groups are in mg/kg/day. S.E. is indicated by error bars. Control M, F (n = 7,
9); BPA 10 M, F (n = 11, 13); BPA 100 M, F (n = 12, 5); BPA 1000 M, F (n = 7, 9); BPA
10,000 M, F (n = 8, 10); statistically significant change within exposure groups as
compared to control animals within the same sex are as indicated: *p < 0.01–0.05,
**p < 0.001–0.01, ***p < 0.001,yp = 0.072.
K.A. McCaffrey et al. / NeuroToxicology 36 (2013) 55–62
2010; Teeguarden et al., 2011; Twaddle et al., 2010). Importantly,
the effects occurred at exposure levels below those needed to
increase uterine weight, suggesting that the brain may be a more
sensitive endpoint when considering the potential of BPA, and
other endocrine disruptors, of interfering with the organizational
role of estrogen on hormone sensitive structures. Why the brain is
more responsive than the uterus is unclear but differences in the
milieu of co-factors and co-repressors needed for estrogen
dependent transcription, activation of non-classical signaling
pathways, or induction of epigenetic changes which enhance
estrogen receptor activity are plausible (McCarthy et al., 2009; Yeo
et al., 2013). Collectively, these data suggest that steroid hormone
sensitive brain regions may be vulnerable to endocrine disruption
by BPA resulting in altered sex specific morphology.
SDN-POA effects were only observed in males, which is
consistent with a prior report from our research group (Patisaul
et al., 2007). An important difference, however, is the direction of
the effect. In the prior study, male Sprague Dawley (SD) rats were
subcutaneously injected with 250 mg BPA every 12 h over the first
two days of life, which is approximately equivalent to 42 mg/kg bw
per day, and 4-fold higher than the highest dose used in the present
study. At this higher dose, SDN-POA volume was unchanged but
the number of CALB-ir cells was significantly increased. Other
research groups have also observed that SDN-POA volume is
unaltered by high dose developmental BPA exposure. For example,
neonatal injections of 300,000 mg/kg bw/day (Nagao et al., 1999),
or perinatal oral exposure to 200,000–400,000 mg/kg bw/day
failed to alter male SDN-POA volume in SD rats (Kwon et al.,
2000; Takagi et al., 2004). Here, however, we found that low dose
perinatal BPA exposure, reduced, rather than increased, CALB-ir
neuron numbers, and male SDN-POA volume was also diminished.
These effects were most pronounced at the lowest doses used (10
and 100 mg/kg/day bw orally).
One possible explanation for these dose-dependent differences
on SDN-POA morphometrics across studies is that the direction of
the effect reverses across the dose curve; with demasculinization
occurring at low doses and resistance to change or subtle
hypermasculinization occurring at higher doses. Collectively, our
data suggest that the inflection point is around 100 mg/kg bw/day.
The mechanism by which this dose-specific response reverses
direction remains unclear but likely involves a dose-dependent
interaction with estrogen signaling. In the female SDN-POA, the
masculinizing effect of estradiol can be induced by selective
agonism of ERa but not ERb, demonstrating the importance of ERa
for enhancing volume (Patchev et al., 2004). BPA may inhibit
Fig. 4. Early developmental bisphenol-A exposure on AVPV-TH-ir cell number in
adult male and female rats. There was a main effect of BPA exposure on AVPV-TH-ir
cell number observed between control female and all female exposure groups
(p ? 0.022), with the exception of BPA 1000 (yp = 0.061). In male exposure groups,
BPA exposure significantly effected (p ? 0.007) AVPV-TH-ir cell number in BPA 10
and BPA 10,000 groups as compared to control males. All exposure groups are in mg/
kg/day. S.E. is indicated by error bars. Control M, F (n = 9, 10); BPA 10 M, F (n = 9, 13);
BPA 100 M, F (n = 10, 12); BPA 1000 M, F (n = 8, 11); BPA 10,000 M, F (n = 8, 11);
statistically significant change within exposure groups as compared to control
animals within the same sex are as indicated: *p < 0.01–0.05, ***p < 0.001.
Fig. 3. SDN-POA CALB-ir cells in exposed males compared to control males and females. Representative images (10?) depicting the density and distribution of CALB-ir within
the SDN-POA of vehicle treated males (control) and females (females) and males exposed to BPA (10, 100, 1000, and 10,000 mg/kg bw/day).
K.A. McCaffrey et al. / NeuroToxicology 36 (2013) 55–62
estrogen signaling at low doses, resulting in increased apoptosis,
but augment estrogen signaling at high doses, thereby enhancing
cell preservation in this region.
The dose-dependent volumetric change hypothesis posed
above is supported by all available data published to date on
the rat SDN-POA, with the exception of a recent study, which
reported evidence for hypermasculinization at 2.5 mg/kg/day bw;
a dose lower than any used in the present study (He et al., 2012).
Key design elements in that study may account for this discrepant
finding, one of which is the route of oral dosing employed. For their
study, He and colleagues (2012) used orogastric gavage (to the
dams during gestation and then directly to the pups until weaning)
which is a popular oral exposure route because it ensures precise
dosing, but can be stressful to the animals (Balcombe et al., 2004).
Significant differences in pup weight and survival between litters
born to vehicle gavaged and naı ¨ve (ungavaged) controls were
observed (Ferguson et al., 2011), suggesting that gavage-related
stress might have confounded toxicant-related effects on hormone
sensitive brain endpoints, including the SDN-POA. Subsequent
work will be needed to more clearly establish how early life stress,
such as the stress associated with gavage or other forms of
handling, interact with exposures to endocrine disruptors and
other toxicants to alter brain morphology and sex specific
For the present study, BPA was administered to the dams using
a food treat, which eliminates the potential confound of dosing
stress but relies on lactational transfer for the pups to be effectively
dosed after birth. In humans, BPA has been shown accumulate in
fetal liver tissue (Nahar et al., 2012), serum, and amniotic fluid
(Engel et al., 2006; Ikezuki et al., 2002) demonstrating the capacity
for gestational exposure. Of note, fetal BPA concentrations were
shown to have a greater variance in retention time, greater mean
retention time, and a longer terminal half-life than that of dams
(e.g., half-life from 6 to 48 h collection was approximately 37.2
times greater in fetuses than in dam blood) (Chapin et al., 2008;
FAO/WHO, 2011). In rats, however, lactational transfer of BPA
appears to be less efficient, with pup serum concentrations 300
times lower than the exposed dams (Chapin et al., 2008; Doerge
et al., 2010). It is therefore possible that, in the present study, BPA
exposure occurred primarily during gestation. If so, then these data
would suggest that the observed decreases in male SDN-POA
volume and CALB content result primarily from exposure prior to
birth. In female rats, postnatal exposure to diethylstilbestrol (DES)
is more effective at masculinizing the SDN-POA than prenatal
exposure (Tarttelin and Gorski, 1988). Subsequent work estab-
lished that the hormone-sensitive period for SDN-POA sexual
differentiation begins on GD 18 (Rhees et al., 1990a) and ends
abruptly on PND 5 (Rhees et al., 1990b), with males being more
sensitive to hormone manipulation than females. In the present
study, lower BPA exposure during postnatal life due to poor
lactational transfer could account for why no statistically
significant effects of BPA exposure were observed in the female
SDN-POA. Moreover, in the study reporting increased male SDN-
POA volume at 2.5 mg/kg bw/day (He et al., 2012), animals were
exposed during gestation but then also gavaged directly up to the
point of sacrifice at weaning. This post-natal exposure may be why
low dose BPA enhanced SDN-POA volume in their exposure
paradigm but not ours.
In contrast to the SDN-POA, effects in the AVPV were observed
in both sexes. In the male AVPV, hypermasculinization of
TH-ir numbers occurred only in the lowest and highest BPA
exposure groups (10 mg/kg/day and 10,000 mg/kg/day) suggesting
Fig. 5. AVPV TH-ir cells in males (top panels) and females (bottom panels). Representative images (10?) depicting the density and distribution of TH-ir neurons in the vehicle
controls and BPA exposed (10, 100, 1000, and 10,000 mg/kg bw/day) animals of both sexes.
K.A. McCaffrey et al. / NeuroToxicology 36 (2013) 55–62
a non-monotonic dose response. In females, evidence of masculini-
zation was observed across all BPA exposed groups, but did not
achieve statistical significance in the BPA 1000 group (yp ? 0.06).
These results are in accord with a prior study, using CD1 mice
exposed via mini-pumps to 25 or 250 ng/kg bw/BPA from GD 16 to
PND 16, which also found masculinization of the female AVPV
(Rubin et al., 2006). Similar to what was observed in the SDN-POA, at
higher doses, the sex specific outcome appears to reverse. We have
previously shown that s.c. injection of 250 mg BPA every 12 h over
the first two days of life, has no effect on total TH-ir numbers in
females, but significantly increases TH-ir levels in males (Patisaul
et al., 2006). This effect is consistent with the hypothesis that BPA
blocks estrogen action at low doses but augments it at higher doses.
Interactions with ERb may also play a role. Neonatal exposure to an
ERb selective agonist masculinizes the female AVPV (Bodo et al.,
2006; Patchev et al., 2004), and ERbKO males have been shown to
possess an abnormally high number of TH-ir neurons in the AVPV
(Bodo et al., 2006). At birth, ERb expression is higher in the male
AVPV, but this sex difference equalizes by PND 2 (Cao and Patisaul,
2011). Exposure to 50 mg/kg bw or 50 mg/kg bw/BPA by s.c.
injection from birth to PND 2 significantly eliminates AVPV ERb
expression levels in both sexes by PND 4 suggesting that altered ER
expression could underlie the morphometric changes reported here.
Prior studies exploring the impact of early life BPA exposure on
brain development and gene expression have produced inconsis-
tent and conflicting data (Palanza et al., 2008; Richter et al., 2007;
Wolstenholme et al., 2011) which has confounded risk assessment.
Although a number of study design elements, including differences
in exposure duration, dose, route of BPA administration, and
critical species differences in neural structure between rats and
mice (Bonthuis et al., 2010), likely account for the discordance in
the literature, concerns about the sensitivity of some rat strains to
gonadal steroid-derived effects have also been raised (Long et al.,
2000; Richter et al., 2007; Steinmetz et al., 1998). Thus, we exposed
a very small group of females (n = 2) to 17b-estradiol to expressly
confirm the estrogen sensitivity of the LE rat strain used for this
study. As expected based on numerous prior studies using LE rats
(Cao et al., 2012; Fader et al., 1998; Ford et al., 2004; Laws et al.,
2000), all endpoints were fully masculinized in the estradiol-
exposed females, including SDN-POA volume (Dohler et al., 1984;
Gilmore et al., 2012; Gorski et al., 1978; Patchev et al., 2004) and
decreased TH-ir cell number in the AVPV (Patisaul et al., 2006;
Simerly, 1989). Assessment of strain sensitivity by including a
concurrent positive control for the predicted effect is critical when
attempting to evaluate the impact of BPA, especially when no
significant effects are observed.
The potential for human-relevant exposure to result in adverse
health outcomes remains a subject of active debate (Beronius et al.,
2010; Hengstler et al., 2011; Vandenberg et al., 2009). It is important
to emphasize that species differences make organizational neuro-
endocrine effects in animals difficult to apply to human risk
assessment. In humans, androgen rather than estrogens is thought
to be most important for masculinizing the brain during develop-
ment (Grumbach, 2002; Wallen, 2005). This difference makes it
challenging to infer how endocrine disrupting compounds, like BPA,
might impact the sexual differentiation of the human hypothalamus
or other brain regions. Our data reveal that the rat anterior
hypothalamus is sensitive to endocrine disruption by BPA at oral
doses below the current reference dose, suggesting that neural
effects in humans are plausible. Animals were exposed perinatally, a
critical period that is entirely prenatal in humans. Prenatal exposure
to BPA has been demonstrated in humans (Braun et al., 2011, 2009;
Calafat et al., 2008) and associated with elevated anxiety and
hyperactivity in young girls. Collectively, these epidemiological data
support the possibility that developmental BPA exposure has
adverse, sex specific, neural effects in humans.
Conflict of interest
This work was supported by NIEHS 1RC2 ES018736. The
authors would like to acknowledge Jinyan Cao, Meghan Radford,
Emily Sluzas, and Sandra Losa-Ward for their invaluable contribu-
tions to tissue processing, as well as their editorial comments. We
would also like to thank the animal care staff at the University of
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