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Department of Food Science (Y.I., M.O., X.W., S.I.), The National Institute of Health and Nutrition, Tokyo 162, Japan; Department of Biochemistry (C.M., Y.O., T.Su.), School of Dentistry, Showa University, Tokyo 142, Japan; Exploratory Research Laboratories III (T.Sa., Y.U.), Daiichi Pharmaceutical Company Ltd., Tokyo 134, Japan; Department of Radiology (M.I.), School of Medicine, Nagasaki University, Nagasaki 852, Japan; and Department of Biochemistry (C.M.), School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji 192, Japan
Address all correspondence and requests for reprints to: Yoshiko Ishimi, Department of Food Science, The National Institute of Health and Nutrition. 1–23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan. E-mail ishimi{at}nih.go.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidemiological studies suggest that the low incidence of osteoporosis and heart diseases caused by estrogen deficiency in Asian women is attributable to their high intake of soyfoods, compared with American and Finnish women (3, 4, 5). It is reported that dietary soybean proteins prevent bone loss in ovariectomized (OVX) rats (6). Possible candidates for the beneficial substances present in soybeans are isoflavones, such as genistein and daidzein, because these isoflavones show a structural similarity to estradiol (E2) and are known as phytoestrogens. Although these isoflavones have been reported to bind to estrogen receptors and protect cell growth in breast cancer cells (7, 8, 9, 10), it is less known whether these isoflavones affect bone metabolism. Furthermore, it has been reported that genistein acts as an inhibitor of tyrosine kinase (11).
We have reported that an estrogen deficiency caused by OVX selectively
stimulates B-lymphopoiesis, resulting in an accumulation of pre-B cells
in mouse bone marrow (12). Both increased B-lymphopoiesis and bone loss
in OVX mice were restored by treatment with estrogen (12). It was
assumed that the increased B-lymphopoiesis caused by estrogen
deficiency was involved in stimulating bone resorption. This is because
the increased B-lymphopoiesis, induced by the administration of
interleukin (IL)-7, resulted in marked bone loss caused by stimulated
osteoclastic bone resorption in mice with intact ovarian function (13).
Recent studies suggest that bone-resorbing cytokines, such as IL-1,
IL-6, and tumor necrosis factor 
, may be involved in bone loss
caused by estrogen deficiency (14, 15, 16, 17, 18). We found that pre-B cells
proliferate on bone marrow stromal cells and make them produce
bone-resorbing cytokines, such as IL-6. Therefore, B-lymphopoiesis in
bone marrow seems to be closely related to bone metabolism, which is
regulated by estrogen. Thus, it is important to clarify whether soybean
isoflavones have estrogenic action in bone and bone marrow.
In this study, we examined the effects of genistein, a typical soybean isoflavone, on B-lymphopoiesis and bone mass in OVX mice to see whether genistein shows an estrogenic property in bone and bone marrow. Genistein did, in fact, exhibit estrogenic properties in bone and bone marrow without exhibiting estrogenic action in the uterus.
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Materials and Methods |
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).
Control mice were treated with a vehicle solution. The mice were fed a
normal diet, containing 1.12% calcium and 1.07% phosphorus (Nippon
Clea, Tokyo, Japan), for 2–4 weeks after the surgery, under specific
pathogen-free conditions. In another series of experiments, 8-week-old
female ddy mice were administered with genistein or
E2 sc, using a mini-osmotic pump, for 2 weeks under the
same conditions. In each experiment, the uterine weight was measured,
and the right and left tibiae were removed to prepare bone marrow
cells. For the measurement of bone mineral density (BMD), femora were
dissected 4 weeks after surgery. All procedures were in accordance with
The National Institute of Health and Nutrition Guidelines for the Care
and Use of Laboratory Animals.
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Radiographic analysis of the femur
Radiographic analysis of the femora was performed using a soft
x-ray system. The BMD of the femur was measured by dual x-ray
absorptiometry (model DCS-600R; Aloka, Tokyo, Japan). The bone mineral
content of the mouse femur was closely correlated with the ash weight
(r = 0.978) (13). BMD was calculated by bone mineral content of
the measured area. The scanned area of mouse femur was equally divided
into three parts (5.3 mm each): proximal femur, mid shaft, and distal
femur.
Three-dimensional analysis of trabecular microarchitecture by
microcomputed tomography (µCT)
The femoral cancellous bone of the distal metaphysis was
analyzed three-dimensionally by the µCT system (µCT-20; Scanco
Medical, Zurich, Switzerland), as reported by Ruegsegger et
al. (19). The mean tissue volume (TV) of the scanned area was 0.44
mm3 in the trabecular bone of the femoral distal
metaphysis, which did not include any cortical bone. Using 200
two-dimensional computed tomography images of 8-µm thickness, a
three-dimensional microstructural image was reconstructed to calculate
morphometric indices, such as bone volume (BV) fraction (BV/TV),
trabecular thickness [Tb.Th = 2 x BV/bone surface (BS)],
and trabecular separation [Tb.Sp = (1/Tb.N) - Tb.Th], where
Tb.N is trabecular number. These parameters were calculated using the
parallel plate model developed by Parfitt et al. (20). The
Tb. N was defined as the number of intersections between bone and
nonbone components per total length of test lines applied to a specimen
(21).
Histomorphometry
Undecalcified 5-µm sections were prepared from femora and were
stained for tartrate-resistant acid phosphatase (TRAP).
Histomorphometry was performed with the semiautomatic image analyzing
system (System Supply, Nagano, Japan) linked to a light microscope.
Using the sections of distal femora, histomorphometric parameters were
quantified in cancellous bone tissue at secondary spongiosa. The region
in the trabecular bone within one cortical width from the endosteal
surface was excluded from the measurements. Trabecular BV [bone area
(BA)/tissue area (TA)] and the number of osteoclasts [osteoclast
number (N.Oc)/BS, mm-1] were measured.
Statistical analysis
Data were expressed as means ± SEM. The
significance of the differences was determined by ANOVA and Fisher’s
protected least-significant-difference test (Stat view 4.0, Abacus
Concepts, Calabasas, CA). Differences were considered significant at
the level of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the time course of
change in body weight, uterine weight, and the number of nucleated
cells in the bone marrow after OVX. It also shows the effects of the
treatment with genistein (0.5 mg/day) and E2 (0.01
µg/day) on those parameters in OVX mice. Four weeks after surgery,
OVX mice exhibited a significant increase in body weight, compared with
sham-operated mice; and treatment with genistein or E2
restored them to the sham level (Fig. 2A). Uterine weight strikingly
decreased in OVX mice, 2 weeks after surgery, indicating that the mice
were estrogen deficient (Fig. 2B). As reported previously,
E2 restored the decreased uterine weight in OVX mice to the
same level as in the sham mice, after 2–4 weeks (12, Fig. 2B). In
contrast, treatment with genistein (0.5 mg/day) for 4 weeks did not
affect the uterine weight at all in OVX mice. A histological study
indicated that the atrophy of uterine epithelial cells in OVX mice was
not restored by the treatment with genistein (data not shown). As
reported previously (12), OVX caused a significant increase in the
number of nucleated cells in bone marrow, and the increased hemopoiesis
was corrected with estrogen treatment, to the sham level (Fig. 2C).
Genistein also restored the OVX-induced stimulation of bone marrow
hemopoiesis to the same level as that of sham mice (Fig. 2C). These
results indicate that genistein regulates bone marrow hemopoiesis
without exhibiting estrogenic action in the uterus, at least at this
dose level.
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shows the expression pattern of B220-positive B cells in sham-operated
mice, OVX mice, and OVX mice treated with genistein or E2.
As reported previously (12), the percentage of B220-positive B cells
was markedly enhanced in OVX mice, 2 weeks after surgery (Fig. 3). When
OVX mice were treated with 0.5 mg/day of genistein, the level of
B220-positive cells returned to the sham level, and its effect was
similar to that of 0.1 µg/day E2 (Fig. 3). Table 1 shows the dose response effect of
genistein on the percentages of B220-positive B cells and Gr-1-positive
granulocytes of bone marrow cells in OVX mice. In contrast to the
increase in B220-positive cells, the percentage of Gr-1-positive cells
decreased in OVX mice, as reported previously (12, Table 1). Because
the total number of bone marrow cells increased, the absolute number of
granulocytes was unchanged in OVX mice (12). When OVX mice were treated
with genistein, at 0.5–0.7 mg/day, the percentages of B220-positive
cells and Gr-1-positive cells returned to the sham levels (Table 1).
The effects of genistein on bone marrow hemopoiesis were similar to
those of E2. Estrogen replacement completely restored
uterine atrophy in OVX mice, but genistein (at 0.1–0.7 mg/day) did not
affect the uterine weight at all.
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). In
contrast, treatment with estrogen resulted in a significant increase in
the uterine weight and a decrease in the percentage of B cells in
normal female mice. When normal mice were administered with genistein
(0.7 mg/day) and E2 (0.1 µg/day) concurrently, genistein
did not alter the uterotrophic and suppressive effects on
B-lymphopoiesis induced by E2 (data not shown). These
results indicate that genistein, at 0.1–0.7 mg/day, does not exhibit
estrogenic action in uterine wet weight in mice with intact ovarian
function.
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). Treatment with genistein (at 0.7
mg/day) or E2 (at 0.01 µg/day) markedly prevented the
bone loss from the distal metaphysis in OVX mice (Fig. 4). Effects of
genistein on bone mass in OVX mice were further determined by measuring
BMD at proximal, middle, and distal regions of the femur, 4 weeks after
surgery. The BMD was significantly reduced by OVX, especially at the
distal metaphysis, and the decreased BMD was significantly restored by
the administration of 0.7 mg/day genistein (Table 3). Estrogen, at 0.01 µg/day,
completely restored the decreased BMD to the same level as that of
sham-operated mice.
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, left). In OVX mice,
however, the plate-like structure was markedly destroyed, and many of
the connecting rods were missing (Fig. 5A, middle).
Treatment with genistein significantly prevented trabecular bone loss
in OVX mice, and the three-dimensional trabecular bone architecture was
reconstructed, becoming similar to that of the sham-operated mice (Fig. 5A, right). In OVX mice, the BV density (BV/TV) and the
Tb.Th were significantly reduced, whereas the Tb.Sp increased, compared
with that of sham-operated mice (Fig. 5B). The increase in Tb.Sp
indicates that the osteoclastic bone resorption was stimulated,
resulting in enhanced intertrabecular space. Treatment with genistein
completely prevented this decrease in BV/TV and Tb.Th and restored the
increase in Tb.Sp.
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). The number of
TRAP-positive multinucleated osteoclasts was significantly increased in
trabecular bone obtained from OVX mice, and the treatment with
genistein restored it to the sham level (Fig. 6B). Bone volume (BA/TA)
of trabecular bone was markedly suppressed by OVX, and the treatment
with genistein significantly recovered it (Fig. 6). These effects of
genistein on bone turnover were similar to those of estrogen. These
results indicate that genistein prevents bone loss by suppressing the
increased bone resorption caused by estrogen deficiency.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genistein completely prevented bone loss caused by estrogen deficiency,
in µCT and histological analyses (Figs. 5
and 6), but BMD was
recovered only partially by genistein in OVX mice (Table 3). The
discrepancy can be explained, because BMD was measured for both
cortical and trabecular bone, whereas µCT analysis was performed only
for the trabecular bone. Therefore, it is possible to speculate that
the effect of genistein in cortical bone is relatively less than that
in trabecular bone.
Genistein has a structural similarity to E2 (Fig. 1) but
binds weakly to estrogen receptors prepared from rat uterine cytosol
with 1/100th affinity, compared with E2 (7). Recently, a
novel estrogen receptor, termed estrogen receptor-ß (ERß), has been
cloned from rat prostate and human testis complementary DNA libraries
(24, 25), and the classical estrogen receptor is now called estrogen
receptor- (ER). Kuiper et al. (26) compared the
binding affinity of various estrogenic compounds for ER and ERß.
Genistein possessed a higher affinity for ERß than that for ER
(26). Tong et al. (27) also reported genistein’s higher
affinity for ERß using the computed molecular field analysis for
structure-activity relationship. Recently, Smithson et al.
(28) have suggested that the effect of E2 on
B-lymphopoiesis in bone marrow is mediated by an ERß-dependent
mechanism, because no significant increase in B-lymphopoiesis was found
in ER knockout mice. In addition, we have reported that osteoblasts
express both ER and ERß (29). Taken together, it is possible to
speculate that genistein acts on bone and bone marrow by a mechanism
involving ERß. Further studies are needed to define the mechanism of
action of genistein in bone and bone marrow.
It is known that genistein acts as a tyrosine kinase inhibitor (11). Recent studies indicated that signaling, mediated by Src tyrosine kinase, plays an important role in osteoclast function (30). Therefore, we cannot rule out the possibility that genistein directly inhibits osteoclastic bone resorption and therefore protects against bone loss caused by estrogen deficiency. To clarify this point, we examined the effect of genistein on osteoclast formation in a co-culture of mouse bone marrow cells and primary osteoblasts, and on an organ culture of fetal mouse calvaria for bone-resorbing assay. Genistein, at 0.4–10 µM, suppressed neither osteoclast formation induced by 2 ng/ml of IL-1 (mean ± SEM in N.Oc/well; control, 0; IL-1, 96.0 ± 6.4; IL-1 + 10 µM genistein, 95.6 ± 6.2) nor bone-resorbing activity in mouse calvarial cultures [mean ± SEM in released Ca into medium (mg/dl); control, 0.84 ± 0.12; IL-1, 7.7 ± 0.1; IL-1 + 10 µM genistein, 7.6 ± 0.1]. Therefore, it is unlikely that genistein acts as a tyrosine kinase inhibitor in bone to prevent increased bone resorption caused by estrogen deficiency. Further studies are necessary to define the mechanism of action of genistein in bone.
It has been shown that the intake of extremely large amounts of
isoflavone causes impaired reproductive function in a variety of animal
species, because of its estrogenic property (31, 32, 33). In this study, sc
administration of 0.7 mg/day genistein for 4 weeks did not affect
uterine weight, either in OVX or normal mice (Table 1). Santell
et al. (7) reported that the administration of large amounts
of dietary genistein (0.375 and 0.75 mg/g diet) increased uterine
weight in mature OVX rats but not in immature intact rats.
Administration of dietary genistein, however, did not affect the
uterotrophic effect of concurrently administered E2 in OVX
rats (7). Bioavailability of genistein may be influenced by gender,
age, or animal species; and the response to genistein may vary with the
levels of ERs (ER and ERß) in each target tissue.
Epidemiological studies have suggested that the much lower incidence of diseases caused by estrogen deficiency, such as osteoporosis and heart diseases, in Asian women is attributed to their high intake of isoflavone-rich soyfoods (3, 4, 5). Indeed, the concentrations of isoflavone in serum and urine in Japanese are higher than those in subjects following Western diets (3, 34, 35). The daily intake of isoflavones in Asians is estimated at 25–200 mg (36, 37), and this may account for high levels (10–100 nM) of active isoflavones in their serum (34). The serum level of genistein is 100- to 1,000-fold higher than that of endogenous estrogen in premenopausal women (38). Therefore, genistein may be effective in improving the various symptoms of estrogen deficiency, though it shows only about 1/100th the affinity of E2 for its receptors.
In this study, we found evidence, for the first time, that genistein significantly reduces bone loss caused by estrogen deficiency, by a mechanism similar to that of estrogen. Arjmandi et al. (6) reported that the administration of soybean protein(s), instead of casein, in the diet prevented bone loss in OVX rats. They speculated that the effects of soybean proteins on bone are attributable to isoflavonoids present in soybeans. Furthermore, Draper et al. (39) demonstrated that phytoestrogen, coumesterol, and zearalanol significantly prevented bone loss in OVX rats. These findings clearly indicate the protective effects of phytoestrogen on bone in a state of estrogen deficiency. In this study, 0.7 mg/day genistein prevented bone loss caused by estrogen deficiency in mice. Further nutritional studies are needed to determine to what extent isoflavone is effective for improving bone metabolism in humans.
In conclusion, an appropriate dosage of genistein restored the increased B-lymphopoiesis and bone loss caused by estrogen deficiency, without exhibiting a substantial effect on the uterus, in OVX mice. Recent studies have shown that synthetic estrogen-related compounds, such as raloxifene, selectively act on bone and on the cardiovascular system without exhibiting estrogenic action in the uterus (2, 40, 41). In OVX mice, raloxifene exhibited estrogenic actions in bone and bone marrow, preventing bone loss and regulating B-lymphopoiesis, without exhibiting estrogenic action in the uterus (42). Furthermore, it has been reported that soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system in rhesus monkeys (43). Therefore, it is likely to speculate that the tissue-selective effects of genistein are similar to that of raloxifene. Intake of soybean products may be useful in preventing bone loss caused by estrogen deficiency.
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Acknowledgments |
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Footnotes |
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Received July 14, 1998.
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), OVX (•), and OVX mice
treated with 0.5 mg/day genistein (
) or 0.01 µg/day E2
(
). C, Number of bone marrow cells: the number of bone marrow cells
collected from the right and left tibiae of sham-operated (
