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

6.5–13.5 days post-coitum and 6.5 to 17.5 days postcoitum.
Day of vaginal plug was considered 0.5 days
post-partum. [No information was provided about the
method of oral dosing.] Another group of mice was
dosed with 5 mg/kg bw/day 17b-estradiol during the
same time periods. Twelve mice/group were killed at
14.5 and 18.5 days post-coitum, 24 hr after receiving the
final dose. Embryos were dissected to obtain cerebrum,
cerebellum, ovary, testis, and liver. RT-PCR analysis was
used to measure mRNA levels of genes. Western
immunoblotting was used to measure protein levels of
CYP1A1 and glutathione-S-transferase in liver. Data were
analyzed by ANOVA. [It was not clear if the litter or
offspring was considered the measurement or statistical
unit.]
Numerous changes in mRNA expression were observed
following bisphenol A exposure, and they varied
according to dose, sex, tissue, and exposure period. In at
least one sex and time period, exposure to 17b-estradiol
increased expression of mRNA arylhydrocarbon receptor
in all tissues, arylhydrocarbon receptor repressor in
testes and ovaries, arylhydrocarbon receptor nuclear
translocator in brain or testes, CYP1A1 in brain, and
glutathione-S-transferase in brain. Changes in protein
levels of CYP1A1 and glutathione-S-transferase in liver
were also examined in embryos at 18.5 days post-coitum
and levels of both proteins were increased with exposure
to bisphenol A at doses Z0.2 mg/kg bw/day and with
exposure to 17b-estradiol. The study authors proposed a
novel mechanism of toxicity involving upregulation of
mRNA for arylhydrocarbon receptor and other factors by
bisphenol A.
Strengths/Weaknesses: The wide dose range and the
oral route are strengths. The study has value for
understanding mechanisms of action although these
changes were not tied to any adverse findings that might
be related to these changes. Weaknesses include the lack
of specification of the method of oral dosing and lack of
clarity on sample origins and sizes for each assay. This is
not considered a critical weakness because it is known
that standard procedures for these methods require
pooling of embryos within litter.
Utility (Adequacy) for CERHR Evaluation Process:
This study is adequate but of limited utility for the
evaluation because of the mechanistic nature of the
endpoints.
Imanishi et al. (2003), supported by the Ministry of
Education, Culture, Sports, Science, and Technology of
Japan, used DNA microarrays to investigate potential
mode of action of bisphenol A on alterations in
expression of 20 nuclear hormone receptors and a few
other genes in the mouse placenta. ICR male and female
mice were housed in polycarbonate cages, given ad lib
access to tap water and CM rodent feed (Oriental Yeast),
and maintained under standard 12-hr/12-hr light/dark
cycle. Between 6.5 and 17 days post-coitum, pregnant
dams were orally administered 0 or 0.002 mg/kg bw/
day bisphenol A [purity not provided] in olive oil
[method of oral administration not given]. The dams
were killed 18.5 days post-coitum, and placentas and
fetuses were frozen at 801C. Placental RNA from male
and female embryos was separately extracted, reverse
transcribed, and hybridized to a microarray chip for 18 hr
at 421C. Images were analyzed using Atlas navigator
software, and statistical analyses were performed using
Birth Defects Research (Part B) 83:157–395, 2008
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the Pearson correlation coefficient, normalized to the
Fisher z transformation. Differentially expressed genes
were identified using paired t-test, and significant
changes were noted in percent values increased or
decreased relative to control mRNA expression values.
[The number of dams used and arrays run was not
given. It was not clear if the litter or offspring were
considered the statistical unit.]
Nuclear receptor genes that showed differential
expression in male and/or female fetuses were: neuronderived
orphan receptor 1, retinoic acid-related orphan
receptor g, estrogen receptor b, liver X receptor a,
progesterone receptor, chicken ovalbumin upstream
promoter transcription factor a, germ cell nuclear factor,
steroidogenic factor 1, and photoreceptor-specific nuclear
receptor. Nuclear receptor genes that did not show
differential expression included thyroid hormone receptor
b, peroxisome proliferators activated receptor a and g,
constitutive androstane receptor, farnesoid X receptor,
chicken ovalbumin upstream promoter transcription
factor b, testis receptor b, estrogen-related receptor g,
aryl hydrocarbon receptor, small heterodimer partner,
and dosage-sensitive sex reversal receptor. Other genes
the expression of which was both significantly altered in
pair-wise comparison with control treatment and exhibited
opposing up- or downregulation in a sexdependent
manner included fast skeletal troponin C,
probasin, RNA-specific adenosine deaminase, and
ADAM25/testase 2, a-fetoprotein, and kinesin light
chain 1. These genes were downregulated in placentas
of male fetuses and upregulated in placentas of female
fetuses. Placentas of male and female fetuses exhibited
downregulation if a-fetoprotein (k60%, male and k24%,
female) and kinesin light chain 1 (k70%, male and k10%,
female).
The authors conclude that fetal sex-based differences
in placental physiology resulting from bisphenol A
exposure may lead to subsequent sex-specific developmental
perturbance. They also indicated that important
but largely unknown effects of bisphenol A may occur
with respect to a cluster of orphan nuclear receptors,
which exhibited significant changes in gene expression.
Strengths/Weaknesses: Strengths: The evaluation of
several molecular endpoints including gene activity for
several receptors that are not commonly examined, oral
dosing, and use of a low dose represent strengths.
Weaknesses are the use of only one dose level of BPA and
absence of many critical experimental details such as the
number of litters used.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for inclusion due to lack of
reporting key experimental details.
Yoshino et al. (2004), supported by the Japanese
Ministry of Education, Science, Sports, and Culture and
the Japan Private School Promotion Foundation, examined
the effect of prenatal bisphenol A exposure on
immune response in mice. [No information was provided
about feed or caging and bedding materials.]
DBA/l J mice were fed bisphenol A [purity not
indicated] at doses of 0 (ethanol/corn oil vehicle),
0.003, 0.030, 0.300, or 3 mg/kg bw/day for 18 days
[stated to be 17 days in the Methods section but 18 days
in other parts of the report], beginning on the day of a
24-hr mating period (Day 0). Twelve mice/group were
treated and 7–9/group became pregnant. [The specific