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NMDAR consists of one NR1 subunit and at least one NR2
subunit. The identity of the NR2 subunit determines the molec-
Environmental Toxicology and Chemistry, Vol. 29, No. 1, pp. 176–181, 2010during embryonic development. Because of the sensitivity to
estrogen, it is possible that the developing brain is particularly complexes of two different known subunits: NR1 and NR2.endocrine organs, little is known about its action on the central
nervous system (CNS). The developing brain is highly regu-
lated by endogenous hormones, and hormonally mediated
events play critical roles at early stage of the development. It
was found that ERb was expressed in the fetal brain and was
regulated by estrogen [4,5]. 17b-Estradiol increased the ratio of
neurons to glia cells in embryonic but not in adult neural stem
cells [6], suggesting an influence of estrogen on neurogenesis
N-methyl-D-aspartate (NMDA) receptor (NMDAR), a sub-
type of the glutamate receptor, is crucial for normal CNS
function, and is ubiquitously distributed in high density
throughout the brain. N-methyl-D-aspartate receptor partici-
pates in a wide variety of physiological processes. Evidence
for its involvement in synaptogenesis and synaptic plasticity,
neurogenesis and migration [9,10], long-term potentiation
(LTP), as well as in learning and memory [11], has been
presented. N-methyl-D-aspartate receptors are heteromeric
The latter consists of four subtypes: NR2A to 2D. Eachendocrine disrupter have been studied in the reproductive and* T
(xuxh6
Pub
(www.INTRODUCTION
sphenol-A (BPA), one of the most common environmental
rine disrupters with mixed estrogen agonist/antagonist
rties, is widely used in the manufacture of plastics, food
and dental sealants [1]. Bisphenol-A has weak estro-
ty, with approximately 15,000 times less potency than
stradiol estrogen receptor alpha (ERa) and beta (ERb)
addition, BPA can interfere with hormone synthesis and
nce, as well as altering hormone receptor expression and
ne activity in target tissues [3]. Although the effects of this
vulnerable to environmental endocrine-disrupting chem
such as BPA, and some of the cognitive deficits and behav
alterations may result from developmental exposure to
endocrine disrupters. Published results have demonstrated
perinatal exposure to levels of BPA below the no-obse
adverse-effect level of <50 mg/kg/d affects the neoco
histogenesis, sexual differentiation of the brain, and beha
in the offspring of rodents [7,8]. Neuronal differentia
migration was accelerated during later embryonic days i
BPA-treated brains, although the proliferation of precursorKeywords—Bisphenol-A N-methyl-D-aspartate receptor Estrogen receptor beta Aromatase cytochrome P450
HippocampusPERINATAL EXPOSURE TO BISPHENO
RECEPTOR EXPRESSION IN THE HIP
XIAO-HONG XU,*yz§ YA-MIN WANG,y JING ZHAN
yChemistry and Life Science College, Zhejiang Norm
zInstitute of Psychology, Zhejiang Normal Un
§Institute of Ecology, Zhejiang Normal Univ
(Submitted 23 January 2009; Returned for
Abstract—Bisphenol-A (BPA) is one of the most common envir
properties. The toxicity of BPA has been extensively evaluated in
toxicity, and carcinogenicity. The objective of the present study is
0.5, 5, 50, and 200 mg/kg/d affects N-methyl-D-aspartate (NMDA
(ERb), and aromatase cytochrome P450 (P450arom) protein
development. Western-blotting analyses showed that perinatal
subunits. At the lower doses of 0.05 to 50 mg/kg/d, BPA conc
However, at the higher dose (200 mg/kg/d), the effects of BPA
expression and a slighter inhibition of NR2A, 2B expression w
perinatal exposure to BPA inhibited the expression of ERb pro
dependent manner, especially during the early postnatal period
P450arom was observed at postnatal week 8. These data suggest
brain, enhancing the local biosynthesis of estrogen in the braino whom correspondence may be addressed
3@zjnu.cn).
lished online 23 September 2009 in Wiley InterScience
interscience.wiley.com).
176CHANGES N-METHYL-D-ASPARTATE
AMPUS OF MALE RAT OFFSPRING
ING-QING LUO,y YIN-PING YE,y and QIN RUANy§
iversity, Jinhua 321004, People’s Republic of China
y, Jinhua 321004, People’s Republic of China
, Jinhua 321004, People’s Republic of China
ion 4 May 2009; Accepted 26 July 2009)
ntal endocrine disrupters with mixed estrogen agonist/antagonist
ety of tests in rodents, including developmental and reproductive
aluate whether or not perinatal maternal exposure to BPA at 0.05,
ptor (NMDAR) subunits NR1, NR2A, 2B, estrogen receptor beta
sions of hippocampus in male rat offspring during postnatal
ure to BPA significantly affected the expression of NMDAR
ion dependently inhibited the expression of NMDAR subunits.
ese subunits were different, with a stronger inhibition of NR1
ompared with those at the lower dosage of BPA. In addition,
but increased P450arom protein expression in a concentration-
first 1–3 postnatal weeks). No significant influence of BPA on
nvironmental BPA exposure may affect the development of the
iting ERb and NMDAR expressions. Environ. Toxicol. Chem.
# 2009 SETAC
Printed in the USA
DOI: 10.1002/etc.18ular composition and functional properties of the receptor, and
hence, NMDAR channel diversity [11]. Expression and location
in the brain of the different NMDAR subunits varies during

Bisphenol-A changes NMDA receptor expression in hippocampus Environ. Toxicol. Chem. 29, 2010 177brain development and throughout life. In the visual cortex,
high expression of the NR2B subunit occurs soon after birth,
whereas the expression of NR2A subunit is gradually increased
postnatally [10]. The peak expression of the NR1 subunit occurs
approximately on postnatal day (PND) 10 in the hippocampus
and PND 60 in the cerebellum [10]. In the developing brain, the
number of NMDAR peaks during formation of the excitatory
synapses and declines in adulthood [10].
Endogenous estrogen affects the expression of NMDAR and
the activity of NMDAR subunits in the brain. It was reported
that endogenous estrogen was involved in the formation of the
excitatory NMDA synapses in the hippocampus [12] and
exerted profound effects on the sprouting of hippocampal
dendritic spines and the concomitant upregulation of NMDARs
[13]. N-methyl-D-aspartate receptors in the hippocampus are
involved in LTP, a cellular model for learning and memory,
which is under the influence of estrogen and other sex steroid
hormones [14]. In addition, it was reported that aromatase and
NMDAR were coexpressed in hippocampus neurons [15]. The
mice made estrogen deficient by aromatase knockout displayed
a higher expression in transcript levels of NR1, NR2A, and
NR2B in the hippocampus [16]. Based on these findings, it is
speculated that the xenoestrogen BPA may affect the expression
of NMDAR subunits of the brain. Fetal and neonatal develop-
ment is a hormonally sensitive period of life; thus, the present
study was designed to investigate the effects of perinatal
exposure to BPA on NMDAR subunits expression in the hippo-
campus during postnatal development. The expressions of
ERb and aromatase cytochrome P450 (P450arom) in the hippo-
campus were also analyzed to explore whether the expression of
ERb and local biosynthesis of estrogen were involved in BPA-
induced alteration of NMDAR subunit expression during post-
natal development.
MATERIALS AND METHODS
Animals and treatments
Male (300–350 g) and female (250–280 g) Sprague–Dawley
rats were purchased from the Experimental Animal Center,
Zhejiang Academy of Medical Science, and maintained on a
12:12 h light:dark cycle with free access to food and water. All
studies described were conducted in accordance with the Care
and Use Standard of the Laboratory Animal (China Ministry of
Health publication, 1998, unpublished data). After acclimatiza-
tion for 1 week, female rats were placed with males (two
females: one male) and vaginal smears were examined daily.
A sperm-positive smear determined gestational day (GD) 0.
After detection, pregnant dams were placed individually and
assigned to an exposure condition randomly (n¼ 8–9 per con-
dition). Dams were orally (ig) exposed to BPA (Shanghai
Chemical Reagent Research Institute) dissolved in sesame oil
(200, 50, 5, 0.5, or 0.05 mg/kg/d) or only sesame oil as a vehicle
control at between 8:00 and 9:30 per day from GD 7 through
postnatal day (PND) 21. The oral route of BPA administration
was chosen to mimic the most likely route of exposure to the
compound in humans and wildlife. The lowest dose in the
present study (0.05 mg/kg/d) was much lower than the low-
est-observed-adverse-effect level (50 mg/kg/d), and was equal
to the safe daily limit estimated by the U.S. Environmental
Protection Agency (U.S. EPA) for human exposure to BPA.After parturition (PND 0), the pups were counted, weighed, and
culled to eight pups, maintaining equivalent sex distributions if
possible. The pups were identified individually on PND 4 and
weighed on PND 4, 7, 14, 21, and 56. At weaning (PND 21), the
pups were separated into same-sex littermates and housed. The
present study reports the results of the male pups, and the data of
the females will be presented at a later time.
Tissue preparation
The male pups were sacrificed on PND 4, 7, 14, 21, and 56,
respectively. The hippocampal regions of the brain were dis-
sected and then stored at 708C until use. For membrane
fraction, the tissue was thawed and homogenized in ice-cold
0.32 M sucrose (pH 7.4) containing protease inhibitor cocktail
(Sigma Chemical) and then centrifuged at 800 g for 10 min at
48C to remove nuclei. The supernatant was then centrifuged at
12,000 g for 30 min to sediment crude membranes. The crude
membrane pellet was suspended in 50 mm Tris-HCl (pH 7.4) to
form a sample solution and stored at 208C until processed for
Western blot. For nuclear fraction (used for ERb protein
detection), the tissue was homogenized on ice with a cold
Tris/EDTA (ethylenediaminetetraacetic acid) buffer (50 mm
Tris, 1 mm EDTA, pH 7.5), and centrifuged at 10,000 g for
20 min at 48C. Supernatant was collected and stored at 208C
until processed for Western blot. The protein concentration for
each sample was estimated using Bradford dye-binding proce-
dure with bovine serum albumin as a standard.
Gel electrophoresis and immunoblotting
A sample solution including approximately 30 to 40 mg of
protein was mixed with the buffer for the sodium dodecyl
sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) and
denatured by boiling at 908C for 5 min in Laemmli sample
buffer containing 5% b-mercaptoethanol. Receptor polypepti-
des were separated using 7.5% SDS-PAGE under a reducing
condition. Two gels were run in parallel: one was used for
Coomassie brilliant blue staining and the other for immuno-
blotting. Polypeptides were transferred to a nitrocellulose mem-
brane. The membrane was washed with a blocking solution
(TBS-T) containing 20 mm Tris base, 137 mm sodium chloride,
5% (w/v) milk powder, and 0.1% (v/v) Tween 20. The blot was
then incubated with one of the following: a rabbit anti-NR1
polyclonal antibody (Santa Cruz) diluted by 1:400, a rabbit anti-
NR2A polyclonal antibody (Santa Cruz) diluted by 1:300, a
rabbit anti-NR2B polyclonal antibody (Santa Cruz) diluted by
1:300, a rabbit anti-ERb polyclonal antibody (Santa Cruz)
diluted by 1:300, or a mouse anti-P450arom polyclonal anti-
body (Beijing Biosynthesis Biotechnology) diluted by 1:200.
The blot was washed three times with TBS-T containing 0.1%
Tween 20 and then incubated with secondary antibody, that is,
peroxidase-conjugated antirabbit IgG (Wuhan Boster Biolog-
ical Technology) and peroxidase-conjugated anti-mouse IgG
(Beijing Biosynthesis Biotechnology) diluted by 1:5,000.
b-Actin was adopted as an intrareference. Adobe Photoshop1
9.0 software for Windows1 (Microsoft) was used for the
quantification of immunoblots. Enhanced chemiluminescence
(ECL) films of Western blots were scanned into Photoshop with
an optical scanner (Tsinghua Unisplendour). The images were
inverted, and the Western blots’ intensity was determined by a