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

N-methylurea were killed on PND 180 and rats that
received 25 mg/kg bw N-nitroso-N-methylurea were
killed on PND 110 or 180. Whole-mounted mammary
glands were examined for tumors. Immunostaining was
conducted to identify cytokeratin 8 (an epithelial marker)
and p63 (a myoepithelial marker). Data were statistically
analyzed using the Mann–Whitney U-test.
Bisphenol A exposure did not affect successful
pregnancies, dam weight gain, pregnancy duration,
number of pups/litter, or percent females/litter. Anogenital
distance on PND 1 or 5 and postnatal body
weights were unaffected in pups exposed to bisphenol
A. Vaginal opening was accelerated in pups from the
bisphenol A group (mean 34 days of age compared to 39
days of age in controls). On PND 50, the BrdU/apoptosis
ratio was significantly increased and apoptosis was
significantly decreased in mammary parenchyma and
stroma of bisphenol A-exposed animals; the effects
were not observed on PND 30 or 110. Significantly
increased percentages of hyperplastic ducts, density of
stromal nuclei, and numbers of mast cells were
observed in the bisphenol A group on PND 110 and
180. Exposure to bisphenol A resulted in formation
of a dense stroma layer around mammary epithelial
structures and replacement of normal adipose tissue with
a fibroblastic stroma. In rats exposed to 25 mg/kg bw
N-nitroso-N-methylurea on PND 50, incidence of
hyperplastic lesions on PND 180 was significantly higher
in the group with prenatal bisphenol A compared to
DMSO exposure (mean incidence of 35.5% compared to
15.7% in controls). Although statistical significance was
not achieved, exposure to 25 mg/kg bw N-nitroso-Nmethylurea
resulted in tumors in 2 of 15 rats in the
prenatal bisphenol A group and 0 of 10 rats in the
prenatal vehicle control group on PND 180. Cytokeratin 8
immunostaining revealed no invasion by stromal epithelial
cells. The study authors concluded that rats prenatally
exposed to environmentally relevant doses of
bisphenol A may have an increased risk of developing
mammary tumors.
Strengths/Weaknesses: Weaknesses include route of
administration and the high single dose is a weakness as
is the use of pure DMSO.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for inclusion due to the use of
99.9% DMSO as a vehicle to administer bisphenol A via
s.c. osmotic pump.
Hong et al. (2005), sponsored by the Korea Research
Foundation, investigated the effects of acute exposures to
bisphenol A during late pregnancy on expression and
protein level of calbindin-D 9k, a putative biomarker of
estrogen activity, in the uteri of offspring and lactating
rats on PND 5. Pregnant Sprague–Dawley rats were
given free access to water and a diet of soy-free pellets in
polycarbonate caging. [Housing conditions (individual
or group) and bedding material were not indicated.] On
GD 17–19, pregnant rats were s.c. injected daily with 200,
400, or 600 mg/kg bw/day bisphenol A [purity not
provided] in corn oil (n 5 5/group). Negative and
positive control groups (n 5 10/group) were administered
corn oil or 17b-estradiol 40 mg/kg bw/day. On PND
5, lactating dams and female pups were killed and their
uteri harvested. Dose response changes in calbindin-D9k,
expression levels in uteri of lactating dams and female
offspring (3/group) were analyzed by Northern blot and
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247
RT-PCR, with appropriate housekeeping gene controls.
Protein levels and localization of calbindin-D 9k were
performed by Western blot and immunohistochemistry
for lactating dams only. Statistical analyses were performed
using the Kruskall–Wallis and Dunnett tests. [It
was not clear if dams or offspring were considered the
statistical unit.]
Northern blot analysis revealed a significant increase
[B6.4-fold] in the level of calbindin-D 9k expression in the
uteri of lactating dams exposed to 600 mg/kg bw/day
bisphenol A compared to oil controls. 17b-Estradiol
treatment produced a significant [B3.9-fold] increase
in calbindin-D9k mRNA expression in the dam uterus
that was not statistically distinct from the effect of the
high bisphenol A dose. Uteri of offspring exposed to the
highest dose level of bisphenol A also showed a
significant upregulation [B4.4-fold] in calbindin-D 9k
expression. Expression levels of ERa were unaffected in
maternal uteri exposed to bisphenol A. However, ERa
expression was increased significantly in uteri of pups
exposed to 400 and 600 mg/kg bw bisphenol A [m33%
and 66%, estimated from a graph]. Protein levels of
calbindin-D9k in lactating dam uteri were elevated
significantly at all dose points [50, 40, and 50%, for
200, 400, and 600 mg/kg bw/day, respectively]. 17b­
Estradiol-treatment was not associated with a significant
increase in calbindin-D 9k protein. The density of calbindin-D9k-immunopositive
cells was increased in uterine
sections from lactating dams exposed to all doses of
bisphenol A relative to oil controls, correlating with
Western blot results. Authors note insufficient material
or low detectability of calbindin-D9k protein in offspring
tissue, and protein analyses were not performed.
The authors suggest that calbindin-D 9k can serve as a
reliable biomarker of acute estrogenic exposure, particularly
for insight into maternal-fetal metabolic exchange,
given that calbindin-D9k is tightly regulated and rapidly
induced by 17b-estradiol, diethylstilbestrol, alkylphenols,
and now, bisphenol A. They further point out that
calbindin-D9k expression is absent in immature rat and
ovariectomized rat uteri.
Strengths/Weaknesses: This study supports the use of
calbindin-D 9k as a uterine biomarker of estrogenic effect
in the perinatal period in the rat, and provides some
dose–response information for bisphenol A induction of
an estrogenic response. Limitations are the subcutaneous
route of exposure, small sample size, high-doses and
uncertain statistical analyses of the F1 data.
Utility (Adequacy) for CERHR Evaluation Process:
While providing some dose–response information regarding
bisphenol A-induced estrogenic effects following
exposure of rats in the perinatal period, the lack of
clarity regarding whether the dam or offspring was
considered the statistical unit, route of exposure, and use
of high doses render this study inadequate for consideration
in the evaluation process.
3.2.3 Rat—oral exposure postnatally with or
without prenatal exposure.
3.2.3.1 Reproductive studies: The International Research
and Development Corporation (General Electric,
1976), sponsored by General Electric, examined the
effects of bisphenol A exposure on CD rats and their
offspring. Male and female F0 rats were housed in wire
mesh cages and fed Purina Laboratory Chow. Ten rats/
sex/group (body weights of 110–170 g for males and