Monograph on the Potential Human Reproductive and ... - OEHHA

processes. The study authors noted altered dopamine
transporter gene expression, which was impaired by all
chemicals tested. Bisphenol A also lowered galanin
receptor 2 expression. The study authors concluded that
intracisternal exposure to bisphenol A induced hyperactivity
in rats, possibly by regulating gene or protein
expression of G protein-coupled receptor and dopaminergic
neurotransduction systems.
Strengths/Weaknesses: Despite certain strengths, a
significant weakness is the inability to correlate the
internal exposure to bisphenol A provided by the
intracisternal route with that seen by the oral route.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process.
Patisaul et al. (2006), supported by the American
Chemistry Council, evaluated the effect of neonatal
bisphenol A on the anteroventral periventricular nucleus
of the Sprague–Dawley rat. Pregnant rats (n 5 5) were fed
a phytoestrogen-free diet (Purina 5K96) during the last
week of gestation. [No information was provided about
caging or bedding.] Dams were permitted to litter. Pups
were cross-fostered among all dams so that 4 dams
reared 6 females and 6 males and 1 dam reared 5 males.
Pups (n 5 5–8/group) were randomly assigned to receive
s.c. injections of 17b-estradiol 50 mg/pup, genistein
250 mg/pup, bisphenol A [purity not indicated] 250 mg/
pup, or sesame oil vehicle every 12 hr for 48 hr. The
authors estimated that the twice daily dosing with
250 mg/pup was approximately equivalent to 100 mg/
kg bw/day. Injections began the morning of PND 1
(delivery 5 PND 0). On PND 19, the pups were transcardially
perfused with ice-cold saline followed by paraformaldehyde.
Brains were post-fixed in 20% sucrose in
paraformaldehyde, sectioned coronally, and processed
for immunohistochemistry for ERa and tyrosine hydroxylase.
Sections were counterstained with Nissl stain.
Cells of the anteroventral periventricular nucleus positive
for ERa, tyrosine hydroxylase, or both were counted.
Statistical analysis used 2-way ANOVA with sex and
treatment as factors, followed by 1-way ANOVA and
post-hoc Fisher least significant difference test.
There was a significant, sex-related effect on tyrosine
hydroxylase-positive cells in the anteroventral periventricular
nucleus with the number in males about 29% that
of females [estimated from a graph]. The authors
concluded that neonatal treatment with bisphenol A
interfered with the normal testosterone-associated masculinization
of the anteroventral periventricular nucleus.
Because 17b-estradiol is aromatized to testosterone in the
brain, the authors interpreted this effect of bisphenol A
as anti-estrogenic. Cells staining for both ERa and
tyrosine hydroxylase are not present in rodents after
puberty, and the authors stated that these cells may play
a role in the organization of the LH-surge. They
postulated that the decrease in these cells with neonatal
exposure to bisphenol A may result in cycle disruption in
adulthood.
Strengths/Weaknesses: Strengths of this study are the
use of 17b-estradiol as a positive control and the measurement
of ERa receptors. Weaknesses are the relatively highdose
level of bisphenol A and the use of the subcutaneous
route of exposure on a per pup basis without adjustment
for body weight. Critical weakness include small sample
size (5 treated dams) and lack of adequate experimental
and statistical control for litter effects.
Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A
277
Utility (Adequacy) for CERHR Evaluation Process:
Despite certain strengths, this study is inadequate for the
evaluation process for the reasons cited above.
Patisaul et al. (2006), supported by the American
Chemistry Council, investigated the effects of an acute
neonatal exposure to bisphenol A or genistein (not
discussed here) on the SDN-POA and the anteroventral
periventricular nucleus in the adult male rat. Five
pregnant Sprague–Dawley rats were obtained and
maintained on a 12-hr light/12-hr dark cycle, with free
access to water and a soy-free, phytoestrogen-free diet
that was maintained throughout the duration of the
experiment. [Details on housing (individual or group),
type of caging, and bedding material were not
provided.] Most of the dams were cross-fostered with 6
male and 6 female pups. Starting on PND 1, all male
pups were given s.c. injections every 12 hr over 48 hr with
250 mg bisphenol A [purity not provided] or oil vehicle.
[Assuming a Sprague–Dawley pup weighs B7.5 g, this
dose would be equivalent to B66 mg/kg bw/day.] On
PND 85, males were gonadectomized. Six ovariectomized
female rats served as controls. After a recovery
period, the rats were given s.c. injections of 10 mg
estradiol benzoate, and 48 hr later, a s.c. injection of
500 mg progesterone. The authors note that this protocol
has consistently induced fos expression in GnRH neurons,
leading to LH release in females. About 8 hr later,
the animals were killed, formalin-perfused, and brains
were harvested. Regions containing the SDN-POA and
anteroventral periventricular nucleus were cryopreserved.
SDN-POA sections were serially stained with
Nissl or labeled for calbindin-d28 K. The vascular organ
of the lamina terminalis was double-immunostained for
Fos and GnRH. An automated stereomicroscope was
used to gauge the volume areas of the anteroventral
periventricular nucleus, the SDN-POA, the calbindinimmunoreactive
regions of the SDN-POA, and number
of calbindin-positive nuclei. Calbindin-positive nuclei
were also counted by independent evaluators blinded to
the treatments. Quantification analyses of GnRH and Fos
staining were evaluated visually. Statistical analysis was
performed using ANOVA, and Fisher least significant
difference test.
Acute neonatal treatment of bisphenol A did not affect
the volume of the SDN-POA. Similarly, the volumes of
the calbindin-immunoreactive regions of the SDN-POA
were roughly equivalent to SDN volumes [estimated
from a graph] with no apparent bisphenol A treatment
effect. Bisphenol A treatment induced a significant
increase [B50–60% estimated from a graph] in calbindin-positive
nuclei. Bisphenol A had no effect on the
volume of the anteroventral periventricular nucleus or
the total number of GNRH-positive nuclei, and no
induction of Fos protein was identified.
The authors noted that the long-term effect of neonatal
exposure to bisphenol A on male brain development and
reproductive behavior cannot be predicted solely on
anatomical changes in sexually dimorphic brain regions.
They concluded that the development of more precise
and predictive biomarkers is needed.
Strengths/Weaknesses: Strengths of this study are the
use of 17b-estradiol as a positive control. Weaknesses are
the relatively high-dose level of bisphenol A and the use
of the subcutaneous route of exposure on a per pup basis
without adjustment for body weight. Critical weakness