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en
NeuroToxicology xxx (2010) xxx–xxx
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NEUTOX-1162; No. of Pages 12
Contents lists available at ScienceDirect
NeuroTox1. Introduction
It is well established that mammalian neuroendocrine devel-
opment is sensitive to changes in steroid hormone levels (Simerly,
2002; Wallen, 2005). This sex specific organization of endocrine
sensitive tissues and circuits is thought to be vulnerable to
endocrine disruption by compounds that act as ‘hormone mimics’.
Such compounds can mimic or interfere with the natural
organizational effects of hormones by interacting with hormone
receptors or disrupting hormone-dependent signaling pathways
(Gore, 2008; Welshons et al., 2006). Collectively referred to as
endocrine disrupting compounds (EDCs), they are now ubiquitous
in the human environment and their potential health effects are
only beginning to be elucidated. The present study tested the
hypothesis that exposure to the EDC bisphenol-A (BPA) during the
neonatal critical period interferes with the sex specific organiza-
tion of the female rat hypothalamus. Each of the hypothalamic
areas selected for the present study is well known to be densely
populated with estrogen receptor (ER) containing neurons and
critical for some aspect of female reproduction, including
regulation of the estrous cycle, mating or maternal behaviors.
We also assessed the impact of neonatal BPA exposure on sexual
proceptivity and body weight, two endpoints of emerging concern
(Monje et al., 2009; Ryan et al., 2010b).
Apprehension over the long-term health effects of neonatal and
childhood exposure to EDCs arises from animal data indicating that
the critical periods of fetal, infant and pubertal development are
more sensitive to low doses of hormones than adult tissues, and
thusmore vulnerable to endocrine disruption (Selevan et al., 2000;
Vom Saal and Moyer, 1985). Research focused on neonatal critical
windows of development in rodents (prior to and immediately
after birth) has shown that perinatal exposure to BPA can alter
Article history:
Received 3 June 2010
Accepted 29 July 2010
Available online xxx
Keywords:
Xenoestrogen
Endocrine disruption
Brain
Sexual differentiation
Development
Estrogen receptor
Neuroendocrine
Now under intense scrutiny, due to its endocrine disrupting properties, the potential threat the plastics
component bisphenol-A (BPA) poses to human health remains unclear. Found in a multitude of
polycarbonate plastics, food and beverage containers, and medical equipment, BPA is thought to bind to
estrogen receptors (ERs), thereby interfering with estrogen-dependent processes. Our lab has previously
shown that exposure to BPA (50 mg/kg bw or 50 mg/kg bw) during the neonatal critical period is
associated with advancement of puberty, early reproductive senescence and ovarian malformations in
female Long Evans rats. Here, using neural tissue obtained from the same animals, we explored the
impact of neonatal BPA exposure on the development of sexually dimorphic hypothalamic regions
critical for female reproductive physiology and behavior. Endpoints included quantification of oxytocin-
immunoreactive neurons (OT-ir) in the paraventricular nucleus (PVN), serotonin (5-HT-ir) fiber density
in the ventrolateral subdivision of the ventromedial nucleus (VMNvl) aswell as ERa-ir neuron number in
themedial preoptic area (MPOA), the VMNvl, and the arcuate nucleus (ARC). Both doses of BPA increased
the number of OT-ir neurons within the PVN, but no significant effects were seen on 5-HT-ir fiber density
or ERa-ir neuron number in any of the areas analyzed. In addition to hypothalamic development, we also
assessed female sex behavior and body weight. No effect of BPA on sexual receptivity or proceptive
behavior in females was observed. Females treated with BPA, however, weighed significantly more than
control females by postnatal day 99. This effect of BPA on weight is critical because alterations in
metabolism, are frequently associatedwith reproductive dysfunction. Collectively, the results of this and
our prior study indicate that the impact of neonatal BPA exposure within the female rat hypothalamus is
region specific and support the hypothesis that developmental BPA exposure may adversely affect
reproductive development in females.
 2010 Elsevier Inc. All rights reserved.
* Corresponding author at: North Carolina State University, Zoology, 127 David
Clark Labs, Raleigh, NC 27695, United States. Tel.: +1 919 513 7567;
fax: +1 919 515 5327.
E-mail address: Heather_Patisaul@ncsu.edu (H.B. Patisaul).
0161-813X/$ – see front matter  2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuro.2010.07.008The impact of neonatal bisphenol-A ex
hypothalamic nuclei in the female rat
Heather B. Adewale, Karina L. Todd, Jillian A. Mick
Department of Biology, NC State University, Raleigh, NC 27695, United States
A R T I C L E I N F O A B S T R A C TPlease cite this article in press as: Adewale HB, et al. The impact of neo
nuclei in the female rat. Neurotoxicology (2010), doi:10.1016/j.neursure on sexually dimorphic
s, Heather B. Patisaul *
icologynatal bisphenol-A exposure on sexually dimorphic hypothalamic
o.2010.07.008

H.B. Adewale et al. / NeuroToxicology xxx (2010) xxx–xxx2
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NEUTOX-1162; No. of Pages 12reproductive development and influence reproductive potential
later in life, a concept referred to as the ‘Fetal Basis of Adult Disease’
(FEBAD) hypothesis (Heindel, 2005; Heindel and Levin, 2005;
Patisaul and Adewale, 2009; Vandenberg et al., 2009). In rodents,
perinatal exposure to low, environmentally relevant doses of BPA
(ranging between 2 and 250mg/kg body weight) has been shown
to advance puberty and reproductive senescence, alter estrous
cyclicity, disrupt ovarian and mammary gland development and
has been correlated with an increased incidence of mammary
tumors (Adewale et al., 2009; Durando et al., 2007; Honma et al.,
2002; Markey et al., 2001, 2003; Susiarjo et al., 2007). All of these
reproductive and neuroendocrine endpoints are regulated by the
hypothalamic–pituitary–gonadal (HPG) axis. Thus, disruption of
hypothalamic sexual differentiation could be a unifying mecha-
nism underlying this suite of effects induced by neonatal BPA
exposure.
BPA entered commercial production in the 1950s after initially
undergoing development as a possible synthetic estrogen (Dodds
et al., 1938), and is now primarily used as a component of
polycarbonate plastics, epoxy resins and thermal paper receipts
(Biedermann et al., 2010). It can be found in a wide variety of food
and beverage containers, medical equipment and plastic tubing,
among other products (Calafat et al., 2009; Vandenberg et al., 2007,
2009;Welshons et al., 2006). The Centers for Disease Control (CDC)
has estimated that an estimated 93% of the US population has
detectable levels of BPA in their bodies (Calafat et al., 2005). Urine
analysis has detected BPA inmen andwomen across all age groups,
but children and hospitalized infants have significantly higher
circulating levels of BPA than adults, prompting concern about the
long-term health effects that might result from developmental
exposure (Calafat et al., 2009; Lakind and Naiman, 2010; Meerts
et al., 2001;Welshons et al., 2006). Exposure can occur throughout
life, including during fetal development because BPA fails to bind
to a-fetoprotein and can thus jeopardize the developing fetus
through placental transfer. Once the infant is born, further
susceptibility can occur through lactational transfer from the
mother, or by interacting with BPA-containing toys and products,
thus exposing the child during multiple critical developmental
periods (Ikezuki et al., 2002; Nagel et al., 1999; Tsutsumi, 2005).
The lowest observed adverse effect level (LOAEL) for BPA is
currently set in the US at 50 mg/kg body weight (bw) per day and
the ‘‘safe’’ or reference dose is defined as 50mg/kg bw per day.
Although evidence that BPA exposure to doses equivalent to or
lower than the LOAEL can adversely affect the development of the
female reproductive tract is relatively robust, whether or not it can
affect neuroendocrine development remains unclear. We have
previously shown, for example, that neonatal exposure to BPA can
affect adversely affect ovarian development, pubertal timing, and
the ability to maintain a regular estrous cycle in female rats
(Adewale et al., 2009; Patisaul et al., 2009b). The goal of the present
study was to determine, within this same group of animals, if
sexually dimorphic hypothalamic development had also been
disrupted. In rodents, estrogen is well recognized to masculinize
hypothalamic regions which govern sex specific physiology and
behavior, including ovarian cyclicity and sexual receptivity
(Bachman, 1995; Baum, 1979; McCarthy, 2008; Simerly, 2002).
Inmales, testosterone from the testis enters the bloodstream and is
carried to the brain where it is aromatized to estrogen. It is this
estrogen which then masculinizes the brain (Simerly, 1998, 2002).
The female brain, on the other hand, develops largely in the
absence of estradiol and is considered to be feminized. Thus,
exposure to EDCs that mimic estrogen could potentially mascu-
linize the female hypothalamus. In primates, the relative role
steroid hormones play in hypothalamic differentiation are less
well understood but androgens are now recognize to be important
for masculinization (Wallen, 2005). To further elucidate potentialPlease cite this article in press as: Adewale HB, et al. The impact of neo
nuclei in the female rat. Neurotoxicology (2010), doi:10.1016/j.neurmechanisms throughwhich BPAmight be acting within the female
rat brain, we employed the ERa-selective agonist PPT as an
additional positive control group to compare the effects of ERa
agonism with BPA exposure. Estradiol benzoate (EB) and vehicle
were also included as additional control groups (positive and
negative respectively).
BPA is hypothesized to work through an ER mediated pathway
and although the RBA of BPA for both ERa and ERb is similarly low
(Kuiper et al., 1998; Welshons et al., 2003), at least one study has
shown it to be a more effective ligand for ERb than ERa (Routledge
et al., 2000). We therefore hypothesized that low dose exposure to
BPA, during the neonatal critical window of postnatal days (PND)
0–4, would disrupt the organization of sexually dimorphic regions
of the hypothalamus important in regulating female reproductive
behavior and physiology, particularly those known to contain ERb.
It was further hypothesized that this disruption would result in a
male-like pattern of development in neonatally exposed females.
The neonatal windowwas selected as the exposure period because
it is a well recognized critical window of sensitivity in the rat
(Bachman, 1995; Baum, 1979; Simerly, 2002), approximately akin
to the 2nd trimester in humans (Bayer et al., 1993) (www.endo-
crinedisruption.com).
We focused on four ER-rich hypothalamic regions involved in
female reproduction and which exhibit sexually dimorphic
differences in volume (size or cell number) or hormone receptor
expression between males and females. The first area of interest,
the paraventricular nucleus (PVN), is the primary site of oxytocin
(OT) synthesis. OT is crucial for many aspects of maternal, social,
sexual and cognitive behaviors and in many species its release is
sensitive to circulating levels of estradiol (Amico et al., 1997; Lee
et al., 2009; Neumann, 2008; Rosenblatt et al., 1988). Because OT
neurons coexpress ERb (also referred to as ESR2), but not the ERa
(ESR1) subtype (Hrabovszky et al., 2004; Patisaul et al., 2003;
Sharon and Allan, 1997), we hypothesized that any BPA-induced
effects within this region are likely mediated via ERb and not
through ERa. The second area of interest was the ventrolateral (vl)
subdivision of the ventromedial nucleus (VMN) an area critical for
regulation of female sex behavior (McCarthy, 2008; Pfaff and
Sakuma, 1979). It receives numerous serotonergic (5-HT) projec-
tions and is sexually dimorphic, with males having a significantly
greater density of 5-HT fibers than females (Patisaul et al., 2008).
The medial preoptic area (MPOA) was selected as the third area
because it is another region critical for the regulation of the female
reproductive cycle, as well as maternal and sexual behavior
(Baskerville and Douglas, 2008; Charlton, 2008; Numan and
Stolzenberg, 2009). The MPOA expresses both ER isoforms and
exhibits sexually dimorphic expression of ERb but not ERa, with
males showing greater expression of ERb than females (Kudwa
et al., 2004; Weiser et al., 2008). The final area of interest was the
arcuate nucleus (ARC), which, while involved in feedback
regulation of the female reproductive cycle, is typically not
considered to be sexually dimorphic in volume or cell number, thus
we hypothesized that this area might be more resistant to
endocrine disruption (Walsh et al., 1982). Body weight and sexual
behavior were also assessed as part of this project. Elucidating the
mechanisms by which BPA disrupts the ontogeny and function of
the HPG axis will help establish whether or not this compound
poses a potential threat to human health.
2. Materials and methods
2.1. Animals and neonatal exposure
Animals used for this study were also used for a prior study and
detailed methods concerning housing conditions, diet, behavioral
testing and surgical procedures can be found in Adewale et al.natal bisphenol-A exposure on sexually dimorphic hypothalamic
o.2010.07.008