Effect of daidzein, a soy isoflavone, on bone metabolism in Cd treated ovariectomized rats

Vol. 54 No. 3/2007, 641–646
on-line at: www.actabp.pl
Regular paper
Effect of daidzein, a soy isoflavone, on bone metabolism in Cd-treated
ovariectomized rats
Ae-Son Om? and Jae-Young Shim
Department of Food and Nutrition, College of Human Ecology, Hanyang University, Seoul, South Korea
Received: 08 August, 2006; revised: 28 November, 2006; accepted: 01 August, 2007
available on-line: 24 August, 2007
This study compared the ability of daidzein, a soy isoflavone, with that of 17?-estradiol to pre-
vent bone loss in cadmium (Cd)-exposed ovariectomized (OVX) rats during growth. Four week-
old female Wistar rats were randomly assigned to five treatment groups of 9 rats each, either
(1) sham-operated (SH); (2) OVX and placed on experimental diets (OVX); (3) OVX fed 50 ppm
of CdCl2 (OVX-Cd); (4) OVX fed 50 ppm of CdCl2 and 10 µg of daidzein per kg of body mass
(OVX-CD-D); or (5) OVX fed 50 ppm of CdCl2 and 10 µg of estrogen per kg of body mass (OVX-
CD-E). All rats were given free access to AIN-76 modified diet and drinking water, with or
without Cd, for 8 weeks. The OVX groups gained more (P?< 0.05) body mass than the SH group.
Femoral mass was increased by feeding daidzein and estradiol, whereas femoral length was
not (P?> 0.05) significantly different among groups. Femoral breaking force was not significantly
different among groups, however, femoral BMD was significantly lower in OVX-Cd than in the
SH and OVX groups. Morphologically proliferative cartilage and hypertrophic cells in femur
showed normal distribution in OVX-Cd-D and OVX-Cd-E groups unlike those in OVX-Cd group.
These findings suggest that Cd-OVX-induced osteopenia or osteoporosis probably results from
an increase in bone turnover.
Keywords: daidzein, soy isoflavone, bone metabolism
INTrODuCTION
can contribute to bone loss are exposure to heavy
metals such as Cd and lead (Pb), excessive mass,
Osteoporosis is known as a skeletal disease poor nutrition, and disorders such as primary hy-
mainly characterized by a reduction of bone mass perparathyroidism and hyperthyroidism. Cadmium
(Hallworth, 1998) and impairment of microarchi-
(Cd) is a very toxic contaminant that has a long
tecture (Riggs & Melton, 1986) with a consequent biological half life in both humans and animals. This
increase in bone fragility and susceptibility to frac-
toxic metal can lead to itai-itai disease (Freiberg et
ture. The main risk parameters for osteoporosis are al., 1986), kidney tubular dysfunction (Freiberg et al.,
genetically influenced low peak bone mass, estrogen 1984; 1986; Om et al., 2002) and cancer (Itokawa et al.,
deficiency, and age. Women have a higher risk than 1978; Waalkers et al., 1999). Moreover, Cd exposure
men since women have a lower peak bone mass and can influence bone tissue leading to osteomalacia
a higher bone loss than man. Especially estrogen de-
and (or) osteoporosis (Itokawa et al., 1978). Several
ficiency is regarded as a critical cause of osteoporo-
studies have shown that Cd affects the activity and
sis, which can result from naturally or surgically metabolism of bone cell directly (Iwami & Moriya-
induced menopause and endocrine disorders that ma, 1993; Wang & Bhattacharyya, 1993; Wilson &
reduce estrogen secretion in premenopausal women. Bhattacharyya, 1997). Therefore, there has been an
It influences osteoclast which enhances bone loss by increased public awareness and concern about expo-
stimulating bone resorption. Other parameters that sure of humans to Cd.
?Corresponding author: Ae-Son Om, Department of Food and Nutrition, College of Human Ecology, Hanyang Univer-
sity, Seoul 133-791, South Korea; tel: (82 2) 2220 1203; fax: (82 2) 2292 1226; e-mail: aesonom@hanyang.ac.kr
Abbreviations: AIN, American Institute of Nutrition; BMD, bone mineral density; Cd, cadmium; Ca, calcium; OC, Serum
osteocalcin; OVX, ovariectomized.

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On the other hand, Asian women have lower
Rats were maintained and utilized in accord-
rates of osteoporosis-related fractures than Western ance with Hanyang University Lab Animal Care
women (Ho et al., 1993) because they consume more Committee (HALACC) animal use protocols.
soy products such as miso and tofu that are rich in
Measurement of breaking force and bone
isoflavones, than Western women do (Adlercreutz et mineral density (BMD). Bones were kept in NaCl
al., 1995; de Kleijn et al., 2001). Daidzein is one of the (9 g/l) at 4°C until femoral breaking force was de-
main soy isoflavones along with genistein and is a termined 24 h later, using a three point bending test,
representative of a family of diphenolic compounds with a Texture Analyzer (TAXT2i, Godalming, UK)
with structural similarities to estrogen. The estro-
(Ezawa & Ogata, 1979). BMD was assessed by dual-
genic effects of daidzein as a phytoestrogen (Naim energy x-ray absorptiometry (Hologic France, Massy,
et al., 1974; Eldridge, 1982; Nogowski et al., 1993) are France) (Omi et al., 1994).
associated with prevention of bone resorption and
Femoral, fecal and urinary calcium (Ca)
augmentation of bone density (Picherit et al., 2000; measurement. Bone and feces were dried at 60 ?±?
Sugimoto & Yamaguchi, 2000; Jia et al., 2003). The 10°C for 24 h in a ceramic pot. The dry bones were
current study examined the effects of daidzein on weighed and then ashed at 550°C for 8 h to deter-
bone metabolism in ovariectomized (OVA) rats with mine the ash mass. The ashed samples were dis-
Cd exposure.
solved in 1 M HCl and diluted with 1% lanthanum
oxide (Association of Official Analytical Chemists,
1984). The Ca content was quantified with an atomic
MATErIALS AND METHODS
absorption spectrophotometer (Model 400, Perkin
Elmer, Norwalk, CT, USA). The Ca concentrations
Experimental animals and treatments. Fe-
in urine were measured by the same method as that
male Wistar rats (25 days old) were purchased of the bone samples after using trichloroacetic acid
from Samtako Inc. (Osan, Korea) and kept in a solution to remove protein (Yeager et al., 1971; Zin-
Hanyang University (Seoul, Korea) controlled terhofer, 1971).
environment animal facility at 22 ±?2°C with a
Serum osteocalcin (OC). Serum OC was de-
12-hour light/dark cycle. After 3 days of acclima-
termined by an ELSA OC kit (CIS, France) using
tion, the rats were assigned to either the sham-
a gamma counter (Cobra?, Hewlett-Packard, Pala
operated (SH) group or to groups to be operated Alto, CA, USA).
on, anesthetized with an intraperitoneal injection
Histomorphometric measurement. The femur
of ketamine hydrochloride (Yahan Inc., Seoul, was dissected and fixed immediately in 0.1 M phos-
Korea) at doses of 50 mg/kg, and either OVX or phate-buffered formalin for 24 h. Undecalcified sec-
subjected to the sham operation. One week after tions for histomorphometric analyses were obtained
survey, the OVX rats were randomly assigned to by dehydrating the bone in acetone for 36 h, fol-
the following groups (n = 9/group): OVX, OVX-Cd, lowed by immersion in xylene for 24 h. The bones
OVX-Cd treated with daidzein at 10 µg/g of body
mass (OVX-Cd-D), or OVX-Cd treated with 17?-
Table 1. Composition of soy-protein-free powdered semi-
estradiol at 10 µg/g of body mass (OVX-Cd-E). Di-
purified diet
ets were prepared by mixing powdered daidzein
Ingredient
g/kg of diet
(88.0% pure, BioSpectrum, Yongin, Korea) or 17?-
Protein
estradiol (Sigma-Aldrich Inc., Yongin, Korea) with
Casein
200
AIN-76 powdered semipurified diet (Bifido, Seoul,
Carbohydrate
Korea) (American Institute of Nutrition, 1997). The
Cornstarch
600
composition of the diet is shown in Table 1. The
Fiber
rats in the Cd-treated groups were given 50 ppm
Cellulose
50
of Cd (CdCl2, Sigma-Aldrich Inc.) in drinking wa-
Fat
ter for 8 weeks. After the experimental period,
Corn oil
100
blood samples were collected by heart puncture,
Vitamin mixturea
10
and the serum was centrifuged (3 000 r.p.m., 20
Mineral mixturea
35
min at 4°C), aliquoted, and stored at –70°C until
dl-Methionine
3
measurements were made. Femurs were cleaned
Chlorine chloride
2
from adjacent tissues and stored at –70°C until From Bifido (Seoul, Korea), containing: casein (Screma, Ciudeville,
measurements were made. Urine and feces were France), cornstarch (Daesang, Seoul, Korea), vitamin mixture (Ro-
collected in separators in metabolic cages for 16 h. che, Neuilly sur Seine, France), mineral mixture (Prolaba, Fontenay
The urine samples were acidified with 2 ml of 1 M sous Bois, France), dl-methionine (Sigma-Aldrich), and chlorine
chloride (Sigma-Aldrich). aPrepared according to AIN-76 forma-
HCl and stored at –20°C until assayed.
tion (Dyets Inc., Bethlehem, PA, USA).

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Daidzein and bone
643
Table 2. Effects of daidzein on body mass gain1
Group
Body mass gain (g)
SH
144.57 ?±? 18.07a
OVX
208.33 ?±? 26.62b
OVX-Cd
216.38 ?±? 16.92b
OVX-Cd-D
183.44 ?±? 25.04b
OVX-Cd-E
178.86 ?±? 33.23b
1Data are mean ?±?S.D. values of nine rats per group. a,bValues with
1
different letters are significantly different among groups at P < 0.05
2
by Duncan’s multiple range test.
were then embedded in methyl methacrylate. Fron-
tal sections (10 mm) were cut with a Reichert-Jung
Microtome (Model 2050, Leica, Deerfield, IL, USA),
and then stained with hematoxylin-eosin for histo-
morphometric analyses.
Statistical analysis. Means and S.D.s of all Figure 1. Effects of daidzein on serum OC and Ca con-
variables were computed for each of the groups. centrations.
Analysis of variance (ANOVA) was first performed Data are expressed as mean ± S.D. values of nine rats per
on the means to determine whether there were sig-
groups. a,b,cSignificantly different among groups at P < 0.05
nificant differences (P < 0.05). When ANOVA indicat-
by Duncan’s multiple range test.
ed statistical significance, Duncan’s multiple range
test (Duncan, 1957) was used to determine which
means were significantly different. SPSS (Chicago, mass in the OVX and OVX-Cd group was signifi-
IL, USA) software was used for all statistical analy-
cantly (P < 0.05) less than that in the SH, OVX-Cd-D,
ses.
and OVX-Cd-E groups. The femoral mass was in-
creased by feeding daidzein and estradiol, whereas
femoral lengths among the groups were not (P > 0.05)
rESuLTS
significantly different.
Body mass gain and uterine mass
Breaking force and BMD
Table 2 shows the body mass gain. The OVX,
The mean femoral breaking force was not
OVX-Cd, OVX-Cd-D, and OVX-Cd-E rats had a sig-
(P > 0.05) significantly different among groups (Ta-
nificantly (P < 0.05) increased body mass compared ble 3). The BMD was significantly (P < 0.05) lower in
with the SH groups. Uterine mass was strikingly de-
the OVX-Cd group than in the SH and OVX groups,
creased in OVX rats. Also, the OVX and OVX-Cd-D but there were no (P > 0.05) significant differences in
groups had a significantly lower uterine mass com-
femoral BMD among the OVX-Cd, OVX-Cd-D, and
pared with the OVX-CD-E group (not shown).
OVX-Cd groups.
Femoral wet mass and length
Femoral ash and Ca contents
Table 3 shows wet mass and length of the
Femoral ash was not (P > 0.05) significantly dif-
femoral bones. The femoral mass per 100 g of body ferent among groups (Table 4). Femoral Ca content
Table 3. Effects of daidzein on femoral wet mass, length, breaking force and BMD1
Group
Mass
Length
Breaking force
BMD
(mg/100 g b.m.)
(mm)
(kg)
(mg/cm2)
SH
532.67?±?75.68a
32.52?±?2.31ND
9.26?±?1.29ND
218.28?±?7.86a
OVX
399.14?±?38.17b
32.86?±?1.58
9.59?±?1.51
221.35?±?6.37a
OVX-Cd
400.42?±?32.52b
34.28?±?0.53
9.35?±?1.18
199.46?±?1.36b
OVX-Cd-D
455.01?±?59.74a
33.84?±?1.15
9.47?±?0.52
201.30?±?4.08b
OVX-Cd-E
491.58?±?57.22a
32.87?±?1.55
8.64?±?1.15
200.47?±?8.34b
1Data are mean ?±?S.D. values of nine rats per group. a,bValues with different letters are significantly different among groups at P < 0.05 by
Duncan’s multiple range test.

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Table 4. Effects of daidzein on femoral ash and calcium and cadmium1
Group
Ash/dry bone
Ca
Cd
(%)
(mg/g of dry bone)
(mg/g of dry bone)
SH
62.15 ± 0.94
401.42 ± 13.61b
1.61 ± 0.55b
OVX
65.11 ± 1.87
391.54 ± 24.49b
1.91 ± 0.73b
OVX-Cd
66.99 ± 0.82
385.48 ± 16.47b
2.17 ± 0.19a
OVX-Cd-D
66.24 ± 2.02
412.44 ± 7.35b
2.09 ± 0.52a
OVX-Cd-E
66.01 ± 1.32
458.47 ± 21.94a
2.32 ± 0.83a
1Data are mean ?±?S.D. values of nine rats per group. a,bValues with different letters are significantly different among groups at P < 0.05 by
Duncan’s multiple range test.
in the OVX-Cd-E group was significantly (P < 0.05) normal distribution characteristics in the OVX-Cd-D
higher than in the other groups.
and OVX-Cd-E groups.
Serum OC and Ca concentrations
DISCuSSION
Both serum OC and Ca concentrations were
(P < 0.05) significantly higher in the OVX-Cd group
We investigated in this study the possibil-
compared with the other groups (Fig. 1). Thus, os-
ity of a protective action of daidzein, an isoflavone,
teopenia caused by OVX or Cd probably resulted against bone loss in young OVX rats.
from an increase in bone turnover.
Body mass of all the rats increased over time.
Unlike in a study of Picherit et al. (2000) who re-
Fecal and urinary Ca concentration
ported that ovariectomy did not influence body
mass, OVX rats were significantly heavier than SH
Fecal and urinary Ca concentrations were operated rats, conforming some other previous stud-
significantly (P < 0.05) higher in the OVX-Cd group, ies (Kalu, 1991; Ishimi et al., 1999; Kim & Om, 2001;
showing that Cd may accelerate Ca excretion with Paik et al., 2003). Among the OVX groups, there
urine and feces.
were no significant differences in body mass. How-
ever, rats in the OVX-Cd-E group had a slight de-
Histomorphological change
crease in food consumption, suggesting a decrease
in appetite from the use of estradiol (Picherit et al.,
Femoral histomorphological changes in pro-
2000). Osteoblasts and adipocytes are differentiated
proliferative cartilage and hypertrophic cells are from the same mesenchymal stromal cells. Estrogen
shown in Fig. 2. Both cells were decreased by feed-
stimulates the differentiation from mesenchymal
ing Cd and an irregular arrangement was also found stromal cell to osteoblast, while it inhibits the differ-
in proliferative cells. However, both cells retained entiation to adipocytes. Hence, the body mass gain
SH
OVX
X
V
O

d
C
+
d
C
+
X
V
O
D
+
OVX+Cd+E
Figure 2. Morphological slides of femur.
Hematoxylin-eosin staining, ×100.

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Daidzein and bone
645
after OVX could be caused by the accumulation of increased serum Ca concentrations. This suggests
adipocytes due to estrogen deficiency (Grigoriadis et an increased bone resorption, thereby accelerating
al., 1988).
osteoporosis or osteromalacia. When daidzein was
The BMD was lower in OVX-Cd rats than in provided, bone turnover occurred more slowly com-
SH rats. This trend of BMD was the same as in the pared with the Cd-treated group. This may be due
study of Paik et al. (2003) who reported that the re-
to formation of insoluble compounds of daidzein,
duction of BMD could be partially attributed to a containing hydroxyl groups, with Cd leading to an
decrease in osteoblastic activity as shown by serum accelerated Cd excretion.
OC concentrations. They also reported that reduc-
In conclusion, daidzein may play an
tion of BMD in the OVX and OVX-Cd groups com-
important role in preventing Cd-induced bone loss
pared to the SH group was associated with a higher in OVX rats by inhibiting bone resorption and de-
serum OC concentration and urinary Ca excretion. creasing serum Ca. Actually, the results and conclu-
A decrease in efficiency of Ca absorption with age sion in the current study are very similar to those
and an increase in fecal and urinary Ca excretion in the previous study of genistein performed by
might result in the reduction of BMD (Avioli et al., our laboratory. However, further work is needed to
1965; Gaumet et al., 1997). Moreover, Wronski et al. compare the effects of daidzein and genistein on the
(1985) demonstrated that bone modeling in rats was Cd-induced bone loss. Furthermore, there is a neces-
accelerated after the cessation of ovarian function. sity to examine these effects in a longer study and
According to Picherit et al. (2000), only daidzein elucidate the molecular mechanisms by which daid-
prevented the loss of lumbar and femur BMD but zein inhibits Cd-induced bone disease.
geneistein did not.
OC is formed in the osteoblast and then de-
Acknowledgements
posited in bone matrix. OC is used as a biomarker of
bone loss since serum OC concentration is increased
This work was supported by the Korea Re-
after menopause. The osteoblast activity and synthe-
search Foundation Grant funded by the Korean
sis of OC increase when BMD is decreased by an Government (KRF-2003-C00149). And the authors
increase in bone resorption (Junqueira et al., 1989). thank Do Youn Kelly Kim, University of Wisconsin-
In the current study, serum OC in the OVX-Cd Madison, for reviewing the manuscript.
group was significantly higher compared with the
SH, OVX, and OVX-Cd-E groups. Furthermore, OC
as an indicator of bone formation, along with Ca
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