Dietary bisphenol A prevents ovarian degeneration and bone loss
in female mice lacking the aromatase gene (
Cyp19
)
Katsumi Toda
1
, Chisato Miyaura
2
, Teruhiko Okada
3
and Yutaka Shizuta
1
1
Department of Medical Chemistry, Kochi Medical School, Nankoku, Japan;
2
Department of Biochemistry, School of Pharmacy,
Tokyo University of Pharmacy and Life Science, Japan;
3
Department of Anatomy and Cell Biology, Kochi Medical School, Nankoku,
Japan
We previously generated mice lacking aromatase activity by
targeted disruption of Cyp19 (ArKO mice), and reported
phenotypes of the female mice, showing hemorrhage for-
mation and follicular depletion in the ovary, diminution in
uterine size, and bone loss. In the present study, we examined
the influence of dietary bisphenol A (BPA), a monomer used
for the production of polycarbonate and known to have
estrogenic activity, on these phenotypes of the ArKO mice.
When ArKO mice were fed chow diets supplemented with
0.1% or 1% (w/w) BPA for 5 months, they were protected
from ovarian degeneration, uterine diminution and bone

ArKO mice were also used to study the roles of estrogens in
male mice, and the results demonstrated that estrogens are
critical for male reproductive ability and the development of
the potential for adult inter-male aggression [4,8–10].
Moreover, studies of ArKO mice strongly support the
notion that estrogens play important roles in lipid and
glucose metabolism [11,12].
Xenoestrogens, chemically synthesized nonsteroidal com-
pounds, have been reported to enter the body by ingestion
or adsorption and to exert estrogenic effects [13]. The effects
of these compounds are evaluated by determining the
responses of rodent uteri or testicular function [14–16].
Because estrogen plays important roles in the development
of uterine and breast cancer, exposure to xenoestrogens may
be a risk factor that affects cancer development in addition
to disturbing reproductive functions.
Bisphenol A (4,4¢-isopropylidenediphenol; BPA) is a class
of monomer widely used in the production of polycarbonate
plastic products. The level of human exposure to BPA is not
insignificant, as microgram amounts of BPA were reported
to be detectable in liquid from canned vegetables [17]. BPA
is considered as a xenoestrogen because it binds to estrogen
receptors with approximately 10 000 times less affinity than
E2 [18] and it exhibits estrogenic properties when studied in
in vitro assay systems. For instance, it stimulated the
production of vitellogenin in cultured trout hepatocytes [19]
and the growth of an MCF-7 human breast cancer cell line
[20]. BPA has also been shown to induce estrogen-depend-
ent b-galactosidase activity in an assay system using yeast
cells [21]. In vivo, the exposure of pregnant mice to low doses

icals were of analytical grade.
Animals
Animal care and experiments were carried out in accord-
ance with institutional animal regulations. All animals were
maintained on a 12-h light/dark cycle at 22–25 °Candgiven
water and rodent chow diet with or without BPA ad libitum.
The aromatase P450 gene (Cyp19) was disrupted by
homologous recombination [4]. In brief, an 87-base pair
(bp) fragment located within exon 9 of Cyp19 (the
nucleotide sequence position between +1124 and +1210
relative to the translational start site) was replaced with a
neomycin resistance gene derived from pMC1-neo. The
replacement caused a complete loss of aromatase activity as
shown by an in vitro expression study [4].
The chow diets supplemented with BPA (BPA-diet)
werepreparedbyimpregnationwithBPA,whichwas
dissolved in acetone. For example, 1 g BPA was dissolved
in 10 mL acetone and impregnated into 100 g rodent chow
to yield the chow diet supplemented with 1% (w/w) BPA.
Female wild-type and ArKO mice at 5 weeks of age were
divided into four diet groups: the first group was fed a
normal chow diet (0% BPA-diet; wild-type mice, n ¼ 4;
ArKO mice, n ¼ 5), the second group was fed a chow
diet supplemented with 0.1% BPA (0.1% BPA-diet; wild-
type mice, n ¼ 4; ArKO mice, n ¼ 5), the third group
was fed a chow diet supplemented with 1% BPA (1%
BPA-diet; wild-type mice, n ¼ 4; ArKO, mice n ¼ 4)
and the mice in the fourth group (ArKO mice n ¼ 5)
were given subcutaneous injections of E2 dissolved in
sesame oil (15 lgper25lL per mouse per injection) once

primer: 5¢-TAGATGATGAACCCAGTTATGGAA-3¢
and antisense primer: 5¢-CCACAAAGGCCAGGGCGTT
GAGTA-3¢), progesterone receptor (a 723-bp fragment with
sense primer: 5¢-TGAACCACGCACTCCT-3¢ and anti-
sense primer: 5¢-GAATCAAAGCCATACTGT-3¢), and
vascular endothelial growth factor (VEGF) (a 612-bp
fragment with sense primer: 5¢-TCAAGCCGTCCTGTG
TGCCGCTGATGC-3¢ and antisense primer: 5¢-AGAAA
ATGGCGAATCCAGTCCCACGAG-3¢). The amplified
products were cloned into the EcoRV site of pBluescript
SKII(–) (Stratagene) and verified to be the expected products
by nucleotide sequence analysis. The inserted fragments
were radiolabeled by the random primer labeling procedure
using the Klenow fragment and used as hybridization
probes. The signals were quantified by using a Bioimage
Analyzer BAS2000 (Fuji) to determine relative intensity.
Histological examination
Ovaries and uteri were removed from the mice, fixed in 10%
phosphate-buffered formalin (pH 7.4) for 24 h, dehydrated,
and embedded in paraffin. Sections were cut 3-lmthickand
stained with hematoxylin & eosin.
Serum concentration of BPA
The concentration of BPA in serum was measured using an
ELISA kit for BPA according to the manufacturer’s
instructions. Blood ( 500 lL) was collected from the tail
of each mouse according to the method described [25] and
200 lL of serum was used for the determination of BPA
concentration. The rate of recovery of 50 ngÆmL
)1
BPA

ArKO mice (Table 1). These data indicate that endogenous
estrogen does not influence the intake or the rate of
degradation of BPA.
Estrogenic effects of dietary BPA on the uteri of ArKO
mice
We reported previously that the body weights of female
ArKO mice increased significantly compared with those of
their wild-type littermates after 12 weeks of age [4,11]. The
body weights of ArKO mice fed the 1% BPA-diet were
significantly decreased as compared with those of untreated
ArKO mice, but the 0.1% BPA-diet did not influence the
body weights of ArKO mice (Fig. 1A).
Diminution of uterine size is one of the typical pheno-
types observed in aromatase-deficient mice [2–4]. When
ArKO mice were fed BPA-diets, the uterine weight
increased significantly in a dose-dependent manner
(Fig. 1B). The uterine weight of ArKO mice fed 0.1% and
1% BPA-diets increased approximately 2.5-fold and five-
fold over that of the untreated ArKO mice, respectively. The
uterine weight of the ArKO mice fed the 1% BPA-diet was
comparable to that of the wild-type mice. In contrast, the
BPA-containing diets did not cause any alterations of the
uterine weight in the wild-type mice. Histological examina-
tions showed that the uteri of ArKO mice exhibited atrophy
with suppressed proliferation of endometrium cells (Fig. 2)
[4]. Consumption of a BPA-diet resulted in proliferation of
the uterine endometrial as well as myometrial cells in ArKO
mice in a dose-dependent manner (Fig. 2). To examine the
effects of BPA on the expression of estrogen-responsive
genes in the uterus, Northern blot analysis was performed

the IGF-I gene was markedly elevated in the ArKO ovaries
(6.5-fold over the wild-type level). When the ArKO mice
were fed on BPA-diet, the expression was suppressed in a
dose-dependent manner. The expression of the IGF-I gene
was normalized in response to the treatment with E2 in
ArKO mice. BPA did not influence the expression of the
IGF-I gene in the ovaries of wild-type mice (Fig. 5). In
contrast, the levels of mRNA expression of the IGF-I
receptor, GDF9 and BMP15 were suppressed in the ovaries
of ArKO mice as compared with those of the wild-type mice
(relative intensities were 0.55 ± 0.06, 0.65 ± 0.02 and
0.86 ± 0.06, respectively). These expression levels were
increased by treatment with BPA in a dose-dependent
Table 1. Serum concentration of BPA. The concentration of BPA was
determined using 0.2 mL of serum of each mouse. Data are presented
as mean ± SD (n ¼ 4–5). No significant differences were observed
between wild-type and ArKO mice in each group.
Genotype
Concentration of BPA added to diet (ngÆmL
)1
)
0% 0.1% 1.0%
Wild-type 4.6 ± 1.7 166.1 ± 94.7 508.3 ± 104
ArKO 3.2 ± 1.9 84.3 ± 8.7 768.7 ± 204
Fig. 1. Effects of dietary BPA on body weight and uterine weight in wild-
type and ArKO mice. Body weight (A) and uterine wet weight (B) were
measured at 5 months of age. Wild-type and ArKO mice were fed
chow diet supplemented with 0%, 0.1% or 1% BPA. The data are
expressed as the mean ± SD. a, Significantly different from untreated
ArKO mice in panel A, P < 0.02; b, significantly different from

not affect femoral bone density in wild-type mice (Fig. 6).
DISCUSSION
Xenoestrogens are thought to interact with endogenous
estrogen through binding to estrogen receptors in target
tissues in vivo. ArKO mice appear to be a useful animal
model to study in vivo estrogenic actions of xenoestrogens,
because endogenous estrogen is absent in these mice, and
Fig. 2. Histology of the uteri of ArKO mice fed
diets supplemented with BPA. The uteri of
ArKO mice fed the 0% BPA-diet (A), 0.1%
BPA-diet (B), or 1% BPA-diet (C) and the
uterus of an untreated wild-type mouse (D)
were fixed and stained with hematoxylin &
eosin for histological analysis. Decreases in the
thickness of the endometrial and myometrial
cell layers in ArKO mice were prevented by
the diet supplemented with BPA in a dose-
dependent manner. Bar, 500 lm.
Fig. 3. Alterations in expression of progesterone receptor and VEGF
mRNAs in the uteri of ArKO mice fed diets supplemented with BPA.
Expression of progesterone receptor (A) VEGF (B) and glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) (C) mRNAs was ana-
lyzed by Northern blot hybridization using 15 lgtotalRNAfrom
uteri of wild-type and ArKO mice fed 0% BPA-diet, 0.1% BPA-diet or
1% BPA-diet. Signals of progesterone receptor and VEGF mRNAs
were analyzed using a radioactive image analyzer (BAS 2000) and
normalized relative to GAPDH mRNA levels to calculate the relative
intensity. The experiment was repeated at least twice for quantification
of the signals.
Ó FEBS 2002 Effects of bisphenol A on ArKO females (Eur. J. Biochem. 269) 2217

Additionally, Nagel et al. reported that estrogenic activity
of BPA was potentiated in the presence of serum [35]. Thus
these observations strongly indicate that the estrogenic
potency of BPA is strictly paralleled with the serum
concentration of BPA in ArKO mice. The present study
also demonstrated that dietary BPA showed little influence
on reproductive organs and bone in female wild-type mice.
Metabolism of BPA apparently plays an important role in
modulating estrogenic activity in vivo [36]. The major
pathway for the metabolism of BPA is glucuronidation in
the liver, where the reaction is catalyzed by an isoform of
uridine diphosphate-glucuronosyl transferase (UGT) [37].
Thus the little influence observed in the wild-type mice
might be attributable to enhanced enzymatic activity of
UGT. Indeed, the levels of the activity and transcripts of a
certain isoform of UGT were reported to be down-regulated
by androgens [38], of which serum concentration in ArKO
females is about 10-fold higher than that in the wild-type
mice [4]. However, it is also plausible that endogenous
estrogens are a more dominant factor than BPA in the
target tissues of wild-type mice in vivo.
It was of interest that we detected low amounts of BPA in
serum of mice fed control diet (about 5 ngÆmL
)1
), which is
almost the limit of detection of the experimental conditions
used. Recently, similar amounts of BPA (between 0.6 and
1.5 ngÆmL
)1
) were detected by ELISA in serum of normal

testosterone or pituitary hormones in vivo. BMP15 and
GDF9, members of transforming growth factor b gene
superfamily, were reported to regulate the development and
maturation of ovarian follicles [40]. In the present study, we
showed suppression of the levels of expression of both
BMP15 and GDF9 mRNAs and elevation of the levels by
BPA as well as E2 in the ovaries of ArKO mice (Fig. 5).
These findings indicate that the levels of expression of
BMP15 and GDF9 in addition to IGF-I and its receptor
might be sensitive molecular markers to evaluate the
estrogenic effects of xenoestrogens in the ovaries of ArKO
mice in vivo.
Estrogen plays an important role not only in the
reproductive system but also in the regulation of bone
metabolism to maintain bone mass. In the present study,
dietary BPA was shown to prevent bone loss in ArKO mice
as does estrogen (Fig. 6). Ishimi et al. [41] have reported
that genistein, a typical phytoestrogen, acted like estrogen
and reversed the bone loss in ovariectomized (OVX) mice,
suggesting the beneficial effects of phytoestrogen for the
prevention of postmenopausal osteoporosis due to estrogen
deficiency. The effects of BPA on bone metabolism in OVX
Fig. 5. Alterations in gene expression in the ovaries of ArKO mice fed diets supplemented with BPA. The expression of IGF-I (A), IGF-II (B), FSH
receptor (C), IGF-I receptor (D), BMP15 (E), GDF9 (F) and GAPDH (G) mRNAs was analyzed by Northern blot hybridization using 15 lgof
total RNA from the ovaries of wild-type or ArKO mice. Mice were fed chow diet supplemented with 0%, 0.1%, or 1% BPA from 5 weeks of age
until 5 months of age. Signals of the respective mRNAs were analyzed using a radioactive image analyzer (BAS 2000) and normalized relative to
GAPDH mRNA levels to calculate the relative intensity. The total RNA of the ovaries from the ArKO mice supplemented with E2 was also
analyzed (E2). The experiment was repeated at least twice for quantification of the signals.
Ó FEBS 2002 Effects of bisphenol A on ArKO females (Eur. J. Biochem. 269) 2219
Fig. 6. Effects of dietary BPA on bone mass in wild-type and ArKO mice. Wild-type and ArKO mice were fed diets supplemented with 0%, 0.1% or

body weight. This level is 5 · 10
5
-fold higher than the
environmental level of BPA reported by Howdeshell et al.
Therefore, 1% BPA, the dosage required to exert full
estrogenic effects in adult ArKO mice, seems to be extremely
high for an endocrine disrupter.
In summary, while the in vivo estrogenic effects of BPA
are still a subject prolific of controversy, especially at low
doses [35,43,44], our present in vivo study employing ArKO
female mice established that BPA acts as a nonsteroidal
estrogen without apparent toxic effects, but only at high
doses. This finding might imply that the enzyme activity of
aromatase is required to visualise the low-dose effects of
BPA in vivo. Furthrmore, our present study demonstrated
that the ArKO mouse is a useful animal model for studying
estrogenic effects of various compounds including xeno-
estrogens, phytoestrogens and nonsteroidal drugs in vivo.
ACKNOWLEDGEMENTS
We thank Y. Okada (Institute for Laboratory Animals at Kochi
Medical School) for technical assistance. This work was partially
supported by the grant-in Aid (13672305 for C. Miyaura) from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan and (13670145 for K. Toda) from Japan society for the
promotion of science. This work was conducted as a part of research
projects of Japan Food Industrial Center.
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