Báo cáo Y học: Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene (Cyp19 ) pptx

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 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 show osteopenia 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 low doses of BPA accelerated the onset of puberty in pups [22]. However, it is still not known whether the effects of BPA in vivo are due to its hormonal or its toxic effects. Because endogenous E2 might affect the consequences of the physiological actions of BPA in vivo,ArKOisauseful 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) Eur. J. Biochem. 269, 2214–2222 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02879.x chemical 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. MATERIALS AND METHODS 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 22–25 °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 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 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 added to untreated serum was 91.8% and the limit for detection of BPA was 2.2 ngÆmL )1 under 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. Ó FEBS 2002 Effects of bisphenol A on ArKO females (Eur. J. Biochem. 269) 2215 Statistical 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). RESULTS Serum levels of BPA We first determined the serum concentrations of BPA in mice fed chow diets 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-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 ) 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 untreated wild-type mice in panel B, P < 0.001; c, significantly different from untreated ArKO mice in panel B, P <0.001. 2216 K. Toda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 manner (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 distincttrabecular 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). 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 replacement 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 )1 and 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 )1 BPA might be, respectively, equivalent to the concentrations of 8.4 pgÆmL )1 and 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. 2218 K. Toda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the 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 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 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 shownineachpanel. 2220 K. Toda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 mice 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 n M [42]. One percent BPA is thus calculated to be approximately 5 · 10 6 -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 chow per day per mouse, then 1% BPA means 1.16 gÆkg )1 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. REFERENCES 1. Simpson, E.R., Mahendroo, M.S., Means, G.D., Kilgore, M.W., Hinshelwood, M.M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C.R., Michael, D., Mendelson, C.R. & Bulun, S.E. (1994) Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrinol. Rev. 15, 342–355. 2. Fisher, C.R., Graves, K.H., Parlow, A.F. & Simpson, E.R. (1998) Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc. Natl Acad. Sci. USA 95, 6965–6870. 3. Honda. S., Harada, N., Ito, S., Takagi, Y. & Maeda, S. (1998) Disruption of sexual behavior in male aromatase-deficient mice lackingexons1and2ofthecyp 19 gene. Biochem. Biophys. Res. Commun. 252, 445–449. 4. Toda, K., Takeda, K., Okada, T., Akira, S., Saibara, T., Kaname, T., Yamamura, K., Onishi, S. & Shizuta, Y. (2001) Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17b-oestradiol. J. Endocrinol. 170, 99–111. 5. Britt, K.L., Drummond, A.E., Cox, V.A., Dyson, M., Wreford, N.G., Jones, M.E.E., Simpson, E.R. & Findlay, J.K. (2000) An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene. Endocrinology 141, 2614–2623. 6. Oz, O.K., Zerwekh, J.E., Fisher, C., Graves, K., Nanu, L., Mill- saps, R. & Simpson, E.R. (2000) Bone has a sexually dimorphic response to aromatase deficiency. J. Bone Miner. Res. 15, 507– 514. 7. Miyaura,C.,Toda,K.,Inada,M.,Ohshiba,T.,Matsumoto,C., Okada, T., Ito, M., Shizuta, Y. & Ito, A. (2001) Sex- and age-related response to aromatase-deficiency in bone. Biochem. Biophys. Res. Commun. 280, 1062–1068. 8. Toda,K.,Okada,T.,Takeda,K.,Akira,S.,Saibara,T.,Shiraishi, M., Onishi, S. & Shizuta, Y. (2001) Oestrogen at the neonatal stage is critical for reproductive ability of male mice as revealed by supplementation with 17b-oestradiol to aromatase gene (Cyp19) knockout mice. J. Endocrinol. 168, 455–463. 9. Robertson, K.M., O’Donnell, L., Jones, M.E.E., Meachem., S.J., Boon, W.C., Fisher, C.R., Graves, K.H., McLachlan, R.I. & Simpson, E.R. (1999) Impairment of spermatogenesis in mice lacking a functional aromatase (cyp19) gene. Proc. Natl Acad. Sci. USA 96, 7986–7991. 10. Toda, K., Okada, T., Saibara, T., Onishi, S. & Shizuta, Y. (2001) A loss of aggressive behaviour and its reinstatement by estrogen in mice lacking the aromatase gene (Cyp19). J. Endocrinol. 168, 217–220. 11. Nemoto, Y., Toda, K., Ono, M., Fujikawa-Adachi, K., Saibara, T., Onishi, S., Enzan, H., Okada, T. & Shizuta, Y. (2000) Altered expression of fatty acid-metabolizing enzymes in aromatase-defi- cient mice. J. Clin. Invest. 105, 1819–1825. 12. Jones,M.E.E.,Thorburn,A.W.,Britt,K.L.,Hewitt,K.N.,Wre- ford, N.G., Proietto, J., Oz, O.K., Leury, B.J., Robertson, K.M., Yao, S. & Simpson, E.R. (2000) Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc. Natl Acad. Sci. USA 97, 12735–12740. 13. Korach, K.S. (1993) Editorial: surprising places of estrogenic activity. Endocrinology 132, 2277–2278. 14. Ashby, J. & Tinwell, H. (1998) Uterotrophic activity of bisphenol A in the immature rat. Environ. Health Perspect. 106, 719–720. 15. Odum, J., Lefevre, P.A., Tittensor, S., Paton, D., Routledge, E.J., Beresford, N.A., Sumpter, J.P. & Ashby, J. (1997) The rodent uterotropic assay: critical protocol features, studies with nonyl phenols, comparison with a yeast estrogenicity assay. Regul. Toxicol. Pharmacol. 25, 176–188. 16. Jensen, T.K., Toppari, J., Keiding, N. & Skakkebaek, N.E. (1995) Do environmental estrogens contribute to the decline in male reproductive health? Clin. Chem. 41, 1896–1901. 17. Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedrazza, V. & Olea, N. (1995) Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect. 103, 608–612. 18. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B. & Gustafsson, J.A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 139, 4252–4263. 19. Shilling, A.D. & Williams, D.E. (2000) Determining relative estrogenicity by quantifying vitellogenin induction in rainbow trout liver slices. Toxicol. Appl. Pharmacol. 164, 330–335. 20. Schafer, T.E., Lapp, C.A., Hanes, C.M., Lewis, J.B., Wataha, J.C. & Schuster, G.S. (1999) Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro. J. Biomed. Mater. Res. 45, 192–197. 21. Rehmann, K., Schramm, K.W. & Kettrup, A.A. (1999) Applicability of a yeast oestrogen screen for the detection of oes- trogen-like activities in environmental samples. Chemosphere 38, 3303–3312. 22. Howdeshell, K.L., Hotchkiss, A.K., Thayer, K.A., Vandenbergh, J.G. & vom Saal, F.S. (1999) Exposure to bisphenol A advances puberty. Nature 401, 763–764. 23. Mirkes, P.E. (1985) Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients. Anal. Biochem. 148, 376–383. Ó FEBS 2002 Effects of bisphenol A on ArKO females (Eur. J. Biochem. 269) 2221 24. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY. 25. Hogan, B., Beddington, R., Costantini, F. & Lacy, E. (1994) Tail bleeding. In Manipulating the Mouse Embryo: A Laboratory Manual,2ndedn.(Sambrook,J.,Fritsch,E.F.&Maniatis,T., eds), p. 323. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 26. Monget, P. & Bondy, C. (2000) Importance of the IGF system in early folliculogenesis. Mol. Cell Endocrinol. 163, 89–93. 27. Baker,J.,Hardy,M.P.,Zhou,J.,Bondy,C.,Lupu,F.,Bellve, A.R. & Efstratiadis, A. (1996) Effects of an igf1 gene mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. 28. Adashi, E.Y., Resnick, C.E., Payne, D.W., Rosenfeld, R.G., Matsumoto, T., Hunter, M.K., Gargosky, S.E., Zhou, J. & Bondy, C.A. (1997) The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm. Endocrinology 138, 3881–3890. 29. Galloway, S.M., McNatty, K.P., Cambridge, L.M., Laitinen, M.P.,Juengel,J.L.,Jokiranta,T.S.,McLaren,R.J.,Luiro,K., Dodds,K.G.,Montgomery,G.W.,Beattie,A.E.,Davis,G.H.& Ritvos, O. (2000) Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nature Genet. 25, 279–283. 30. Yan, Y., Wang, P., DeMayo, J., DeMayo, F.J., Elvin, J.A., Carino, C., Prasad, S.V., Skinner, S.S., Dunbar, B.S., Dube, J.L., Celeste, A.J. & Matzuk, M. (2001) Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol. Endocrinol. 15, 854–866. 31. Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R., Lu, N. & Matuk, M.M. (1996) Growth differentiation factor 9 is required during early ovarian folliculogenesis. Nature 383, 531–535. 32. Elvin, J.A., Yan, C., Wang, P., Nishimori, K. & Matzuk, M.M. (1999) Molecular charactrization of the follicle defects in the growth differentiation factor-9-deficient ovary. Mol. Endocrinol. 13, 1018–1034. 33. Dierich, A., Sairam, M.R., Monaco, L., Fimia, G.M., Gansmul- ler, A., LeMeur, M. & Sassone-Corsi, P. (1998) Impairing follicle- stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hor- monal imbalance. Proc. Natl Acad. Sci. USA 95, 13612–13617. 34. Abel, M.H., Wootton, A.N., Wilkins, V., Huhtaniemi, I., Knight, P.G. & Charlton, H.M. (2000) The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduc- tion. Endocrinology 141, 1795–1803. 35. Nagel, S.C., vom Saal, F.S., Thayer, K.A., Dhar, M.G., Boechler, M. & Welshons, W.V. (1997) Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ. Health Perspect. 105, 70–76. 36. Elsby, R., Ashby, J., Sumpter, J.P., Brooks, A.N., Pennie, W.D., Maggs, J.L., Lefevre, P.A., Odum, J., Beresford, N., Paton, D. & Park, B.K. (2000) Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor. Biochem. Pharmacol. 60, 1519–1530. 37. Yokota, H., Iwano, H., Endo, M., Kobayashi, T., Inoue, H., Ikushiro, S. & Yuasa, A. (1999) Glucuronidation of the environ- mental oestrogen bisphenol A by an isoform of UDP- glucuronosyltransferase, UGT2B1, in the rat liver. Biochem. J. 340, 405–409. 38. Guillemette, C., Levesque, E., Beaulieu, M., Turgeon, D., Hum, D.W. & Belanger, A. (1997) Differential regulation of two uridine diphospho-glucuronosyltransferases, UGT2B15 and UGT2B17, in human prostate LNCaP cells. Endocrinology 138, 2998–3005. 39. Takeuchi, T. & Tsutsumi, O. (2002) Serum bisphenol A con- centrations showed gender differences, possibly linked to andro- gen levels. Biochem. Biophys. Res. Commun. 291, 76–78. 40. Elvin, J.A., Yan, C. & Matzuk, M.M. (2000) Oocyte-expressed TGF-b superfamily members in female fertility. Mol. Cell Endocrinol. 159,1–5. 41. Ishimi, Y., Miyaura, C., Ohmura, M., Onoe, Y., Sato, T., Uchiyama, Y., Ito, M., Wang, X., Suda, T. & Ikegami, S. (1999) Selective effects of genistein, a soybean isoflavone, on B-lympho- poiesis and bone loss caused by estrogen deficiency. Endocrinology 140, 1893–1900. 42. Krishnan, A.V., Stathis, P., Permuth, S.F., Tokes, L. & Feldman, D. (1993) Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132, 2279–2286. 43. Cagen, S.Z., Waechter, J.M. Jr, Dimond, S.S., Breslin, W.J., Butala, J.H., Jekat, F.W., Joiner, R.L., Shiotsuka, R.N., Veenstra, G.E. & Harris, L.R. (1999) Normal reproductive organ develop- ment in CF-1 mice following prenatal exposure to bisphenol A. Toxicol. Sci. 50, 36–44. 44. Ashby, J., Tinwell, H. & Haseman, J. (1999) Lack of effects for low dose levels of bisphenol A and diethylstilbestrol on the prostate gland of CF1 mice exposed in utero. Regul. Toxicol. Pharmacol. 30, 156–166. 2222 K. Toda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene ( Cyp19 ) Katsumi Toda 1 , Chisato Miyaura 2 ,. 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