Dietary bisphenol A prevents ovarian degeneration and bone lossKatsumi Toda1, Chisato Miyaura2, Teruhiko Okada3and Yutaka Shizuta1 1 Department of Medical Chemistry, Kochi Medical School
Trang 1Dietary bisphenol A prevents ovarian degeneration and bone loss
Katsumi Toda1, Chisato Miyaura2, Teruhiko Okada3and Yutaka Shizuta1
1
Department of Medical Chemistry, Kochi Medical School, Nankoku, Japan;2Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan;3Department 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
loss in a dose-dependent manner Northern blot analyses of
ovarian RNA of ArKO mice showed differences in the
expression levels of insulin-like growth factor (IGF)-I, IGF-I
receptor, growth differentiation factor 9 and bone
mor-phogenetic protein 15 as compared with those in the ovaries
of wild-type mice The differences in the expression levels were restored by dietary BPA In the ArKO uteri, expression
of progesterone receptor and vascular endothelial growth factor mRNAs was diminished, and was restored by BPA to the levels in wild-type mice In contrast, BPA had little effect
on the ovarian, uterine and skeletal structures of wild-type mice In conclusion, estrogenic effects of BPA on the reproductive tract as well as skeletal tissue were evident in adult female ArKO mice These results suggest that the ArKO mouse is an animal model suitable for studying effects
of estrogenic chemicals as well as estrogen in vivo
Keywords: ArKO mouse; bisphenol A; estrogens; IGF-I
Estrogens are synthesized from androgens by three
succes-sive hydroxylation reactions which are catalyzed by the
enzyme aromatase (CYP19) [1] In order to study the
physiological roles of estrogens in vivo, aromatase-knockout
(ArKO) mice were generated by targeted disruption of
Cyp19[2–4] These mice can be used also as a good animal
model for the postmenopausal woman Female ArKO mice
are characterized by phenotypes such as follicular depletion
and hemorrhage formation in the ovaries, underdeveloped
uteri and immature mammary glands [2–5] Female ArKO
mice also showosteopenia with increased bone turnover
[6,7] Administration of 17b-estradiol (E2) protects the
ArKO mice from ovarian degeneration and bone loss [4,7]
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 lowdoses
of BPA accelerated the onset of puberty in pups [22] However, it is still not known whether the effects of BPA
in vivoare due to its hormonal or its toxic effects Because endogenous E2 might affect the consequences of the physiological actions of BPA in vivo, ArKO is a useful animal model for characterization and evaluation of
Correspondence to: K Toda, Department of Medical Chemistry,
Kochi Medical School, Nankoku, Kochi 783-8505, Japan.
Tel.:/Fax: +81 88 880 2316, E-mail: todak@kochi-ms.ac.jp
Abbreviations: ArKO mice, aromatase knockout mice; BMD, bone
mineral density; BMP15, bone morphogenetic protein 15; BPA,
bis-phenol A; Cyp19, murine aromatase P450 gene; E2, 17b-estradiol;
FSH, follicle stimulating hormone; GAPDH, glyceraldehyde-3
phos-phate dehydrogenase; GDF9, growth differentiation factor 9; IGF-I,
insulin like-growth factor-I; OVX, ovariectomized; pQCT, periferal
quantitative tomography; UGT, uridine diphosphate-glucuronosyl
transferase; VEGF, vascular endothelial growth factor.
(Received 23 November 2001, revised 18 February 2002, accepted 12
March 2002)
Trang 2chemical compounds with putative estrogenic actions The
objective of present study was to examine the in vivo
estrogenic effects of BPA on the female reproductive tract
and bone by using ArKO mice
M A T E R I A L S A N D M E T H O D S
Materials
A standard rodent chow(NMF) was obtained from
Oriental Yeast (Tokyo, Japan) BPA and E2 were from
Sigma-Aldrich An ELISA kit for BPA was from Takeda
Pharmaceutical Co Ltd (Tokyo, Japan) All other
chem-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 22–25C and given
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 chowdiets supplemented with BPA (BPA-diet)
w ere prepared by impregnation w ith BPA, w hich w as
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 chowdiet (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 lg per 25 lL per mouse per injection) once
per week for 5 months Mice were started on each diet at
5 weeks of age and sacrificed at 5 months of age for
examination We repeated a series of the experiments and
obtained essentially same results
Preparation and analysis of RNA
Uteri and ovaries were collected from each mouse and used
for preparation of total RNA according to the method of
Mirkes [23] Northern blot analyses were performed using
15 lg of total RNA according to the method described [24]
Complementary DNA probes were prepared by PCR
amplification using oligo d(T)-primed cDNA derived from
ovarian RNA as a template with the following sets of
primers: insulin-like growth factor (IGF)-I (a 560-bp
fragment with sense primer: 5¢-GTCGTCTTCACACCTC
TTCTACCTG-3¢ and antisense primer: 5¢-CCCATCTTT
GTAATGTTATTGGACT-3¢), IGF-II (a 378-bp fragment
with sense primer: 5¢-AGCTTGTTGACACGCTTCAGT TTGT-3¢ and antisense primer: 5¢-GTAACACGATCAG GGGACGATGACG-3¢), IGF-I receptor (a 1387-bp fragment with sense primer: 5¢-GGGGCCAAACTCAA CCGTCTAAAC-3¢ and antisense primer:CGTAAGGC TGTCTCTCATCAAAACT-3¢), bone morphogenetic protein (BMP) 15 (a 1057-bp fragment with sense primer: 5¢-CCCTGGCAAGGAGATGAAGCAATGG-3¢ and antisense primer: 5¢-GGGAAACCTGAGATAGCAACA ACTT-3¢), growth differentiation factor (GDF) 9 (a 1299-bp fragment with sense primer: 5¢-GCAAGAGCAGGCA CCCAGCAACCAG-3¢ and antisense primer: 5¢-TTCCGT CACATAAAACCACAGCACT-3¢), follicle stimulating hormone (FSH) receptor (a 684-bp fragment with sense 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 Klenowfragment 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-lm thick and 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 added to untreated serum was 91.8% and the limit for detection of BPA was 2.2 ngÆmL)1under the experimental conditions used
Radiographic analysis of the femur Radiographs of femurs were taken with a soft X-ray generator (model CMB-2; SOFTEX, Tokyo, Japan) [7] The bone mineral density (BMD) of the femurs was measured using a dual X-ray absorptiometer (model DCS-600R; Aloka, Tokyo, Japan), as reported previously [7] Trabecular bone density of the femurs was measured by peripheral quantitative tomography (pQCT) using a pQCT system (model XCT Research SA+) with a version 5.4 soft-ware (Stratec Medizintechnik GMBH., Pfzheim, Germany) The position of the 500-lm slice was located 1.2 mm away from the growth plate in the distal metaphysis
Trang 3Statistical analysis
Data were expressed as means ± SD The significance of
the differences was analyzed using Student’s t-test using
INSTAT (GraphPad Software, Inc., San Diego, CA, USA)
R E S U L T S
Serum levels of BPA
We first determined the serum concentrations of BPA in
mice fed chowdiets supplemented with BPA The levels of
BPA in serum were elevated in a dose-dependent manner in
the mice of both genotypes No significant differences were
observed in the concentrations between the wild-type and
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
using cDNA probes for progesterone receptor and VEGF
While the expression of these genes in the uterus was
diminished in ArKO mice as compared with that in
wild-type mice, it was restored to the levels of the wild-wild-type mice
by dietary BPA (Fig 3) These results demonstrate that dietary BPA activates the estrogen signaling pathway in the uteri of ArKO mice, as does E2
Estrogenic effects of dietary BPA on the ovaries
of ArKO mice
To examine the effects of dietary BPA on the ovaries of ArKO mice, histological analysis was performed Depletion
of follicles and formation of hemorrhagic cysts were evident
in the ovaries of untreated ArKO mice at 5 months of age (Fig 4D) as reported previously [4] When the mice were fed
on BPA-diet, ovarian degeneration was suppressed in a dose-dependent manner With 0.1% BPA, no apparent protective effects against follicular depletion in the ovary were observed (Fig 4E) In contrast, with 1% BPA, ArKO mice were completely protected from hemorrhage forma-tion and follicular loss in the ovaries (Fig 4F) Nevertheless, typical corpus lutea were not detectable These histological observations made in the ovaries of ArKO mice fed 1% BPA are similar to what is seen in the ovaries of ArKO mice treated with E2 [4] The ovaries of wild-type mice fed BPA-diets showed no obvious structural alterations (Fig 4A–C) Estrogenic effects of BPA on the ovaries were examined
by measuring the mRNA expression of genes for IGF-I, IGF-II, IGF-I receptor, BMP15, GDF9 and FSH receptor, which have been reported to be important for ovarian function [26–34] As shown in Fig 5, the expression level of 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)
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 chowdiet 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 untreated wild-type mice in panel B, P < 0.001; c, significantly different from untreated ArKO mice in panel B, P < 0.001.
Trang 4manner (Fig 5) Recovery of the expression of these genes was also observed in the ovaries of ArKO mice treated with E2 The levels of expression of IGF-II and FSH receptor mRNAs in the ovaries were not affected by BPA (Fig 5) These results demonstrate that BPA regulates ovarian expression of the IGF-I, IGF-I receptor, BMP15, and GDF9 genes in vivo, as does E2
Estrogenic effects of dietary BPA on bone mass
in ArKO mice
It is well known that estrogen is essential for the maintenance
of bone mass in rodents and humans We reported that ArKO mice exhibit marked bone loss due to increased bone resorption, and that the treatment with E2 restored the bone mass in ArKO mice [7] To examine the effects of BPA on bone mass in ArKO mice, the femur was subjected to radiographic X-ray analysis and measurement of BMD As reported previously, the femoral BMD was markedly reduced in ArKO mice and the loss of mineralized cancellous bone was evident in the distal metaphysis of the femur in ArKO mice (Fig 6A) Dietary BPA prevented ArKO mice from bone loss in a dose-dependent manner (Fig 6A) In pQCT analysis, the distinct trabecular bone could be detected visually, seen as red and yellow, in wild-type mice, but the trabeculae disappeared and the area was occupied by bone marrow, seen as gray and black, in ArKO mice (Fig 6B) Consumption of a BPA-diet completely reversed the loss of femoral trabecular bone in ArKO mice (Fig 6B) BPA did not affect femoral bone density in wild-type mice (Fig 6)
D I S C U S S I O N
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 withBPA 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 lg total RNA from
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.
Trang 5replacement with estrogen can prevent the mutant
pheno-types of ArKO mice [4,7,8,10]
In the present study, we examined in vivo estrogenic
effects of BPA, a kind of xenoestrogen, on ovarian
degeneration and bone loss of female ArKO mice Because
these phenotypes have been reported to become evident in
aged ArKO mice [4,7], we treated the mice with
dietary-BPA for a relatively long time When ArKO mice were fed a
0.1% BPA-diet for 5 months, bone loss was significantly
prevented and uterus size was increased, but ovarian
degeneration was not protected fully With a 1% BPA-diet,
full estrogenic effects on these tissue-sites were observed
Serum concentrations of ArKO mice fed 0.1% and 1%
BPA-diets were measured as 84 ngÆmL)1and 760 ngÆmL)1,
respectively As BPA binds to estrogen receptors with
10 000-fold lower affinity than E2 in vitro [18], 84 ngÆmL)1
and 760 ngÆmL)1BPA might be, respectively, equivalent to
the concentrations of 8.4 pgÆmL)1and 76 pgÆmL)1 E2 in
terms of the binding ability to estrogen receptors in vitro
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 humans [39] It is not clear whether or not these amounts of BPA are physiologically important
In the ovaries, the intrafollicular IGF-I system is consi-dered to play important roles in follicular selection, which distinguishes follicles destined to ovulate from those destined to succumb to atresia [26,28] Furthermore, targeted disruption of the IGF-I gene was reported to cause infertility of female mice due to anovulation [27] Such studies thus demonstrate that IGF-I is essential for ovarian function Yet little is known about regulatory factors involved in the ovarian expression of the IGF-I gene In the present study, we showed that the expression of IGF-I mRNA was markedly elevated in the ovaries of ArKO mice, and that the level of this expression was attenuated by dietary BPA (Fig 5) In contrast, the expression of IGF-I receptor mRNA was suppressed in the ovaries of ArKO mice, and elevated to the same level as in wild-type mice by BPA Treatment with E2 also restored the levels of expression of IGF-I and IGF-I receptor mRNAs in
Fig 4 Histology of the ovaries of ArKO mice fed diet supplemented with BPA Wild-type and ArKO mice were fed 0% BPA-diet, 0.1% BPA-diet or 1% BPA-diet from 5 weeks of age until 5 months of age Ovaries were collected from wild-type mice fed 0% (A), 0.1% (B) and 1% (C) BPA and from ArKO mice fed 0% (D), 0.1% (E) and 1% (F) BPA and processed for histological analysis The sections were stained with hematoxylin & eosin Note that many hemorrhagic cysts (Hr) were formed in the ovary of untreated ArKO mice (D) In contrast, hemorrhage formation was suppressed and many follicles were observed in the ovaries of ArKO mouse fed the diet supplemented with 1% BPA (F), although no typical corpora lutea (CL) are observed Bar; 200 lm.
Trang 6the ovaries of ArKO mice, indicating that transcription of
IGF-I and its receptor genes are regulated by E2 in the
ovary Nevertheless it is also plausible that estrogens affect
the expression of these genes through altering the levels of
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 lg of 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.
Trang 7Fig 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 1% BPA from 5 weeks of age until 5 months of age (A) Femurs were dissected from the mice, and BMD was measured in the total area of the femur *, Significantly different from 0% BPA group, P < 0.05 The data are expressed as the mean ± SEM The upper panel shows soft X-ray radiograms of the femurs collected from animals of each group Note that there was marked bone loss in ArKO mice, and that the bone loss was prevented by dietary BPA (B) pQCT analysis of femoral distal metaphysis Scanning was performed at a site 1.2 mm from the growth plate, and the density of trabecular bone was determined visually as described in Materials and methods The value of trabecular bone density (mg per cm 3 ) is show n in each panel.
Trang 8mice are not known, and are now under investigation in our
laboratories
The dosages of BPA, 0.1% and 1%, used in the present
study are extremely high compared with the levels of BPA
found in the environment The amounts of BPA eluted from
a polycarbonate bottle by autoclaving were reported to be
10–15 nM[42] One percent BPA is thus calculated to be
approximately 5· 106-fold higher than the concentration
released from bottles Howdeshell et al [22] have shown
that exposure of pregnant mice to environmental levels of
BPA (2.4 lgÆkg body weight)1) advanced the puberty of the
offspring pups Assuming that the mean body weight of
adult ArKO mice is 30 g, and that they eat 3.5 ± 0.48 g
chowper day per mouse, then 1% BPA means 1.16 gÆkg)1
body weight This level is 5· 105-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
A C K N O W L E D G E M E N T S
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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