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Métabolisme Minéral, Laboratoire des Maladies Métaboliques et Micronutriments (U3M), I.N.R.A. Clermont-Ferrand/Theix, 63122 Saint Genès Champanelle, France;
*
ENITA de Bordeaux, 33175 Gradignan cedex, France, LBRP, Université Bordeaux I, 33405 Talence cedex, France; and
LPA, UFR Biosciences, Université de Cocody, Abidjan 22, Côte d’Ivoire
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethinylestradiol, to prevent
bone loss in ovariectomized rats, a model for postmenopausal
osteoporosis. Female Wistar rats (n = 65; 12 mo
old) were either sham-operated (SH; n = 13) or
ovariectomized (OVX; n = 52). On d 0, OVX rats were
randomly assigned to groups as follows: 13 received genistein [G; 10
µg/(g body weight · d)], 13 were treated with
daidzein [D; 10 µg/(g body weight · d)], 13
received 17-ethinylestradiol [E2; 30
µg/kg body weight · d)] and 13 were untreated
(OVX). Compounds were mixed with a soy protein–free powdered
semipurified diet and given orally for 3 mo. On d 90, the bone mineral
density (BMD) in lumbar vertebrae, femur and its metaphyseal and
diaphyseal zones (rich in cancellous and cortical bone, respectively)
was lower in OVX than in SH (P < 0.01). In D or
E2, the four BMD were not different from SH, whereas in G,
only the diaphyseal BMD was not different from SH. Image analysis
performed in the distal femur metaphysis revealed that the cancellous
bone area was lower in OVX than in SH (P < 0.01).
Only the area in D was not different from that in SH. Finally, the bone
turnover, which was higher in OVX than in SH (P < 0.005 and P < 0.05 for plasma osteocalcin
concentration and urinary deoxypyridinoline excretion, respectively),
was not different in G, D or E2 compared with SH.
Therefore, consumption of 17-ethinylestradiol or daidzein was more
efficient than genistein in preventing ovariectomy-induced bone
loss in rats.
KEY WORDS: • genistein • daidzein • prevention • bone • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and also constitutes a
bone microarchitectural impairment (Riggs and Melton 1986
). Hypoestrogenemia after menopause is an important cause
of osteoporosis. For this reason, hormone replacement therapy
(HRT)2
is often recommended for osteoporosis prevention and treatment.
However, HRT may be associated with side effects. Thus, increased
research into alternatives to estrogen for postmenopausal women is of
clinical, scientific and health policy importance (Kessel 1998
).
Broadly defined, phytoestrogens include isoflavones, coumestans and
lignans, found mainly in soybeans, clover or alfalfa sprouts, and
oilseeds such as flaxseed, respectively (Kurzer and Xu 1997
). Although few foods containing coumestrol (the major
coumestan) are consumed by humans, both lignans and isoflavones were
identified in many human physiological fluids after the consumption of
ordinary diets. Because soybean consumption in postmenopausal women
could be associated with potential health benefits, such as prevention
of atherosclerosis progression, lowering of cancer risks, positive
effects on hot flushes, vaginal symptoms, cognitive function or
dementia, and bone preservation, isoflavones are under intensive
investigation. Furthermore, although phytoestrogens are 1000- to
10,000-fold less estrogenic than 17ß-estradiol, their plasma
concentrations were found to be high in Japanese men eating a
traditional Asian diet (Adlercreutz et al. 1993b
), and
even up to 1000-fold higher than the highest levels of circulating
17ß-estradiol in premenopausal women (Anderson and Garner 1997
). Moreover, phytoestrogens were shown to interact with sex
hormone production, metabolism or action at the cellular level
(Murkies et al. 1998
). In effect, they inhibit enzymes
involved in steroid metabolism, such as 5-reductase (Evans et al. 1995
) or aromatase (Adlercreutz et al. 1993a
), whereas they stimulate sex hormone binding globulin
(SHBG) production, reducing the proportion of free estrogens
circulating in plasma. On the other hand, because of a hydroxyl group
aligned in a stereochemical position analogous to estradiol, they can
bind to the estrogen receptor (ER). Consequently, they could appear as
selective estrogen receptor modulators (SERM) and act either as
antiestrogens or as weak estrogens because their occupancy time or
affinity for the receptor is reduced significantly compared with
17ß-estradiol (Miksicek 1994
). Moreover, understanding
their mechanism(s) of action has become even more complex since the
discovery of a new ER subtype (ERß) in rats
(Kuiper et al. 1996
) and humans (Mosselman et al. 1996
). Furthermore, in ER-expressing cells and as
endogenous estrogens, phytoestrogens could also act through nongenomic
effects mediated by membrane-bound ER or other cellular proteins
(Anderson et al. 1999
). In this way, the isoflavone
genistein could inhibit phosphorylation of cytoplasm proteins via its
anti-tyrosine kinase properties (Akiyama et al. 1987
).
Thus, isoflavones appear to have potential promise for maintaining or
modestly improving bone mass of human subjects when consumed at optimal
dosages (Anderson and Garner 1997
). Moreover, in
ovariectomized rats used classically as an animal model for
postmenopausal osteoporosis (Kalu 1991
), dietary soybean
proteins were found to prevent bone loss (Arjmandi et al. 1996
), and this bone-sparing effect was mediated by the
isoflavone content of soybean (Arjmandi et al. 1998
).
Therefore, this study investigated the effects of the two major soybean
isoflavones (genistein and daidzein), given orally and separately at
equal dose, on ovariectomy-induced bone loss in adult rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in accordance with current legislation on
animal experiments in France. Female Wistar rats (n
= 75; 11 mo old; ~ 375 g) were purchased from I.N.R.A.
(Clermont-Ferrand/Theix, France) and housed individually in metallic
cages that allowed separation and collection of urine, at 21°C, with
a 12-h light:dark cycle. Rats were fed a soy protein–free powdered
semipurified diet (I.N.R.A., Jouy en Josas, France) for 1 mo. The
composition of the diet is given in Table 1
. After the adaptation period, 10 rats designated as initial controls
(IC) were killed; the remaining 65 rats were either sham-operated
(SH: controls; n = 13) or surgically ovariectomized
(OVX; n = 52), under anesthesia using chloral hydrate
(80 g/L in saline solution; 0.4 mL/100 g body weight,
intraperitoneally). In the sham procedure, the ovaries were
exteriorized and replaced to create a stress similar to that obtained
with bilateral ovariectomy. On d 1 after surgery (designated as d 0),
the OVX rats were randomly assigned to groups as follows: 1)
treated with genistein at 10 µg/(g body weight · d)
(n = 13; G); 2) received daidzein at 10
µg/(g body weight · d) (n = 13; D);
3) treated with 17-ethinylestradiol at 30
µg/(kg body weight·d) (n = 13;
E2); or 4) untreated (n
= 13; OVX: ovariectomized controls). During the 3-mo experimental
period, all compounds were given orally. Diets were prepared by mixing
the powdered genistein (Sigma, L’Isle d’Abeau, France), daidzein
(Sigma) or 17-ethinylestradiol (Sigma) with the soy protein–free
powdered semipurified diet. SH and OVX rats were fed the soy
protein–free powdered semipurified diet without any additional
compound. To prevent ovariectomy-induced hyperphagia, the daily
diet quantity distributed to each rat was adjusted to the mean level
consumed by SH the previous day. Food was humidified (1 mL/g), and each
rat had free access to water. Every week, rats were weighed to adjust
the genistein, daidzein or 17-ethinylestradiol doses to body weight.
On d 89, a 24-h urine sample was collected to measure urinary excretion
of deoxypyridinoline, a marker of bone resorption (Robins 1994
). On d 90, at 0900 h, rats were killed by cervical
dislocation. Blood samples were collected into ice-cooled
heparinized plastic tubes containing 200 peptidase inhibitory units of
aprotinin (Iniprol, Choay, Paris, France) per milliliter blood, and
centrifuged immediately (3500 g for 5 min at 4°C).
Then, plasma was frozen at -20°C until measurements of
phytoestrogens and osteocalcin, a marker of osteoblastic activity, were
made. Uterine horns were removed from each rat and immediately weighed.
Femurs and lumbar vertebrae were cleaned from adjacent tissues and used
for physical and chemical measurements. Successes of ovariectomy and
phytoestrogen treatment were confirmed by uterine weight and plasma
genistein or daidzein concentrations, respectively.
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BMD was assessed by dual-energy X-ray absorptiometry (DEXA), with
the Hologic QDR-4500 A X-ray bone densitometer (Hologic, Massy,
France). The total right femur BMD (T-BMD), as well as the BMD of two
subregions, one corresponding to the distal femur metaphyseal zone
(M-BMD), rich in cancellous bone, and the other to the diaphyseal zone
(D-BMD), rich in cortical bone, were determined (Pastoureau et al. 1995
). The second, third, fourth and fifth lumbar vertebrae
(mainly cancellous bone) were also scanned and the mean BMD measured.
Femoral calcium content.
Femoral Ca was determined with an atomic absorption spectrophotometer (Perkin Elmer 400, Norwalk, CT), in ashed femurs (dissolved in HCl and diluted with 1 g/L lanthanum oxide).
Femoral mechanical testing.
Immediately after collection, the length of the left femur and the mean
diameter of the femoral diaphysis were measured with a precision
caliper (Mitutoyo, Shropshire, UK). Bones were kept in NaCl (9 g/L) at
4°C, and femoral failure load was determined 24 h later, using a
3-point bending test (Turner and Burr 1993
), with a
Universal Testing Machine (Instron 4501, Instron, Canton, MA).
Image analysis.
To measure cancellous bone area in the distal femur metaphyseal zone,
frontal sections were cut with a saw (Isomet 2000, Buehler,Krautkramer,Champagne-Mont d’Or,France), ground to
80-µm sections (Metaserv 2000 polisher,Buehler), and
stained with Von Kossa’s reagent (AgNO3, Sigma). The
underlying zone to growth plate was then analyzed with an automated
microscope image-analysis system, as previously described
(Rose et al. 1996
).
Marker of osteoblastic activity.
Osteoclacin (OC) in plasma was measured by RIA, using rat 125I-labeled OC, goat anti-rat OC antibody and donkey anti-goat second antibody (Biochemical Technologies, Stoughton, MA). The sensitivity was 0.01 nmol/L. The intra- and interassay precisions were 6.8 and 8.9%, respectively.
Marker of bone resorption.
Deoxypyridinoline (DPD) in urine was determined by competitive RIA,
using rat monoclonal anti-DPD antibody coated to the inner surface
of a polystyrene tube and 125I-labeled DPD (Pyrilinks-D RIA
kit, Metra Biosystems, Mountain View, CA). The sensitivity was 2
nmol/L. The intra- and interassay precisions were 4 and 6%,
respectively. Results were expressed as nmol of DPD per mmol of
creatinine (Robins 1994
). The creatinine assay was based
on a modified Jaffés’s method in which picric acid forms a
colored solution in the presence of creatinine (Cook 1975
).
Plasma phytoestrogen concentrations.
Genistein and daidzein in plasma were measured by ELISA, according to a
recently described method (Bennetau-Pelissero et al. 2000
, Le Houérou et al. 2000
). The
sensitivity was 0.45 and 0.12 nmol/L for genistein and daidzein,
respectively. The intra-assay precision was 18 and 14% for
genistein and daidzein, respectively, whereas the interassay precision
was 19 and 17% for genistein and daidzein, respectively.
Statistical methods.
Results were expressed as means ± SEM. All data were analyzed using the GraphPad InStat software (Microsoft, San Diego, CA). ANOVA was first performed to test for any significant differences among groups. When significant (P < 0.05), the Student-Newman-Keuls multiple comparisons test was used to determine the specific differences between means. Parametric ANOVA was performed when data were sampled from populations with equal variance. If not, nonparametric methods were selected. Thus, a Kruskall-Wallis test was first performed. If it indicated a significant difference among groups (P < 0.05), the Mann-Whitney U test was used to determine specific differences. The level of significance was set at P < 0.05 for all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the experimental period, body weight increased compared with d 0
(P < 0.005), except in E2
(Fig. 1
). As a result, although no significant difference was observed among
SH, OVX, G and D, rats in the E2 group were
lighter than the others (P < 0.01). Uterine weight,
higher in IC than in SH (P < 0.05), was lower in OVX
than in SH on d 90 (Table 2
; P < 0.01). It was higher in E2
than in OVX, but lower than in SH (P < 0.01). Uterine
weights in G and D were not different from OVX.
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No significant difference in plasma phytoestrogen concentration was
observed among IC and SH, or SH, OVX and E2
(Table 3
). On the contrary, plasma concentrations of genistein and daidzein on d
90 were higher in G (P < 0.005) and D (P
< 0.01), respectively, compared with all other groups.
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The mean BMD of lumbar vertebrae was not different between IC and SH
(Fig. 2A
). On d 90, the value in OVX was lower than in SH (P < 0.01). This effect of OVX was prevented by daidzein and
17-ethinylestradiol because no difference was observed between SH
and D or E2. By contrast, there was no difference
between G and OVX. The highest values of T-, M- and D-BMD were
observed in IC, and they were greater than those in SH (P
< 0.05) (Fig. 2B, C, D
). The
three variables were lower in OVX than in SH (P < 0.05). This effect of OVX was prevented by daidzein (P <
0.01) and 17-ethinylestradiol because no difference was observed
between SH and D or E2. By contrast, no
significant difference was observed between G and OVX for the three
variables. However, the D-BMD in the G group also was not different
from SH. A positive linear relationship was found between the total
femur mineral content (measured by DEXA) and the femoral calcium
content (assessed by atomic absorption spectrophotometry) (r
= +0.87; P < 0.01).
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No significant difference among groups was demonstrated for femoral length (mean: 33.95 ± 0.12 mm) or diaphyseal diameter (mean: 3.76 ± 0.02 mm). Similarly, the femoral failure load was not different among groups (mean value: 110 ± 2 N).
Image analysis.
The cancellous bone area in the distal femur metaphysis was not
different between IC and SH (Fig. 3
). This variable, lower in OVX than in SH on d 90 (P < 0.01), was not different among OVX, G, D and E2.
However, the D group also was not different from SH. Photomicrographs
of histological slides used for image analysis are shown in
Figure 4
.
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Plasma OC concentration was higher in IC than in SH (P
< 0.01) (Fig. 5A
). On d 90, it was also higher in OVX than in SH (P < 0.005) and the effect of OVX was prevented by daidzein because no
difference was observed between SH and D. Concentrations in G and
E2 were not different from OVX or SH. The urinary
DPD excretion did not differ between IC and SH (Fig. 5B
). On
d 90, it was significantly higher in OVX than in SH (P
< 0.05) and this effect was prevented by genistein and daidzein
because no difference was observed between SH and G or D. Excretion in
E2 was not different from OVX or SH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Miller et al. 1995
, Mosekilde 1995
, Wronski and Yen 1991
). Furthermore, they may provide a useful model for
investigating the biological effects of soy isoflavones because of the
similarity in plasma genistein concentrations attainable in rats
(King et al. 1996
) and humans (Xu et al. 1994
). Isoflavones are degraded by gut microflora, which
profoundly influences their bioavailability (Xu et al. 1995
). In rats, genistein is highly bioavailable; not only is
it well absorbed from the intestines, but it is also extracted
efficiently from the portal blood into the liver and excreted into bile
(Sfakianos et al. 1997
). This enterohepatic cycle leads
to a new circulation of genistein in the general circulation. However,
differences in bioavailability occur when we consider the conjugate or
glycoside forms of genistein and daidzein (genistin and daidzin,
respectively). These forms occur naturally in vegetables and are then
metabolized into genistein and daidzein, respectively, by gut bacteria
glycosidases). Daidzein was reported to be more bioavailable than
genistein in rats (King 1998
) and humans (Xu et al. 1994
). Because dietary soybean proteins (Arjmandi et al. 1996
), their isoflavones (Arjmandi et al. 1998
), and genistin and daidzin (Ishida et al. 1998
) prevent bone loss in young ovariectomized rats, this
study investigated the potential preventive effects of genistein and
daidzein, given orally and separately at equal dose, and compared these
effects with those of orally administered 17-ethinylestradiol on
ovariectomy-induced bone loss in adult rats.
The T-, M- and D-BMD were lower in 15- than in 12-mo-old female
rats. This could be attributed in part to a decrease in osteoblastic
activity, as shown by plasma OC concentrations. In addition, an
increase in fecal and urinary calcium excretions, as well as a decrease
in calcium absorption efficiency with age, might contribute to the
reduction of BMD (Avioli et al. 1965
, Gaumet et al. 1997
). For cancellous bone, the BMD reduction in the distal
femur metaphysis was not sufficient, however, to reduce the
corresponding cancellous bone area. Again, the BMD decrease in the
cortical bone was not sufficient to affect its mechanical properties.
At both the cancellous and cortical sites, ovariectomy greatly reduced
BMD resulting from increased bone turnover as indicated by the higher
plasma OC concentration and urinary DPD excretion in the OVX group
compared with the SH group. These results are in agreement with those
of Wronski et al. (1985)
, which demonstrated that bone
remodeling in rats is accelerated after the cessation of ovarian
function. Moreover, related to the M-BMD reduction, ovariectomy
also decreased cancellous bone area, probably by lowering trabeculae
number rather than by thinning them. In cortical bone, however, the BMD
decrease was less pronounced than that in cancellous bone and was not
sufficient to impair its mechanical properties. In contrast, ingestion
of 17-ethinylestradiol prevented the BMD reduction at both the
cancellous and cortical sites in rats, by suppressing the increase in
bone turnover. In effect, in ovariectomized rats, estradiol prevents
bone loss by depressing bone turnover (Wronski et al. 1988
). However, under our experimental conditions, the decrease
in cancellous bone area of the distal femur metaphysis was not
prevented by ingestion of 17-ethinylestradiol.
As reported previously in cortical bone (Ishida et al. 1998
), daidzein, like estrogen, prevented the
ovariectomy-induced BMD reduction at both the cancellous and
cortical sites by suppressing the bone turnover increase. Moreover,
daidzein consumption also resulted in the preservation of cancellous
bone area in the distal femur metaphysis. By contrast, genistein did
not prevent cancellous bone loss; however, as previously reported
(Ishida et al. 1998
), it did prevent
ovariectomy-induced cortical bone loss. Our results obtained in
cancellous bone are consistent with those of Anderson et al. (1998)
demonstrating that, in ovariectomized lactating rats,
orally administered genistein induced a cancellous bone tissue
retention at a low dose [1.5 µg/(g body weight · d)],
whereas there were no effects at a higher dose [from 5 to 15
µg/(g body weight · d)]. In this study, because the
exchange surface with plasma was higher in cancellous than in cortical
bone, it is possible that cancellous bone was overexposed to genistein,
thus inducing potential mechanisms of ER saturation or impairments of
cellular activity (such as protein phosphorylation). Genistein was
found to suppress osteoclastic activity through tyrosine-kinase
inhibition (Blair et al. 1996
, Williams et al. 1998
). However, the hypothesis based on the inefficiency of a
high genistein dose is unlikely because injections of 5 (Fanti et al. 1998
) or 20 µg/(g body weight · d)
(Ishimi et al. 1999
), which likely provided higher
plasma genistein concentrations than oral administration of 5 or 15
µg/(g body weight · d) (Anderson et al. 1998
), respectively, induced cancellous bone-sparing
effects. Moreover, as reported by Ishimi et al. (1999)
,
but contrary to results from Fanti et al. (1998) and
Ishida et al. (1998)
, the bone loss preventive effects
of genistein in this study resulted from a suppression in bone turnover
increase; thus, these effects could be due to a mechanism similar to
that of estrogen. Nevertheless, our results do not explain why
genistein and daidzein exhibited different effects on cancellous bone
and similar effects on cortical bone, whereas both molecules reduced
the increase in bone turnover. It is possible that the difference in
cancellous and cortical bone responsiveness to phytoestrogens depends
on the ER subtype. Indeed, some differences in both the binding
affinity of phytoestrogens to ER or
ERß and the ER or
ERß content in cancellous and cortical bones
could be involved. Thus, genistein possesses a higher affinity for
ERß than for ER
(Kuiper et al. 1997
), and although both
ER and ERß mRNAs are
expressed in osteoblasts, the expression of ERß
mRNA is higher in cancellous bone of the rat distal femoral metaphysis
and lumbar vertebrae than in cortical bone of the femoral diaphysis
(Onoe et al. 1997
). Again, recent studies also showed
that ERß mRNA was expressed predominantly in
rat osteoblasts covering the metaphyseal bone trabecular surface
(Windahl et al. 2000
), and neither
ERß or ER mRNA was
detected in rat cortical bone (Lim et al. 1999
).
Moreover, an ERß-like immunoreactivity was
demonstrated not only in the nuclei of human and murine osteoblasts,
but also in the osteoclast cytoplasm (Vidal et al. 1999
). Thus, the response of target tissues to phytoestrogens
could be modulated by the
ER/ERß ratio in each
tissue. Further studies are required to determine whether
phytoestrogens act via ER-dependant mechanisms.
Because a nutritional approach was used to meet the goals of this
experiment, all treatments were given orally. Because 17ß-estradiol
is a weak oral estrogen (Barnes 1998
), we used
17-ethinylestradiol, which is at least 200-fold more active than
17ß-estradiol when equivalent doses are given orally (Messina et al. 1994
). We chose the dose of 30 µg/(kg body
weight · d) because it prevents bone loss in ovariectomized rats
(Ke et al. 1997
). The dose choice for genistein and
daidzein was based on data computed from studies by Anderson et al. (1998)
, Fanti et al. (1998)
, and Ishimi et al. (1999)
. In this experiment, oral administration
of 10 µg/(g body weight·d) for 3 mo induced high plasma
genistein and daidzein concentrations in the G and D groups,
respectively. These concentrations were ~1000- to 10,000-fold greater
than plasma estradiol levels usually observed in rats (basal and peak
estradiol concentrations during the estrous cycle are 7–17 and 50–88
pg/L, respectively) (Butcher et al. 1974
). On the other
hand, the presence of both daidzein and genistein in the G and D groups
could be explained by an unspecific cross-reaction between
genistein and antidaidzein antibody, and vice versa
(Bennetau-Pelissero et al. 2000
). Moreover, because
equol is obtained naturally by a gut microbial transformation of
daidzein (Axelson et al. 1984
, Braden 1967
) in ruminants, monogastrics and therefore humans, it is
absorbed in the gut, conjugated in the liver and excreted in urine
(Axelson et al. 1984
). Because orally administered
daidzin in rats can be metabolized to equol (Yasuda and Oshawa 1998
), we also measured plasma equol concentrations in the D
group. The mean level obtained with a very specific antibody
(Bennetau-Pelissero et al. 2000
) was 281 ± 55
nmol/L, indicating that equol was produced in D. Moreover, in various
estrogenic tests in many species, the estrogenicity of isoflavone and
isoflavane compounds can be ordered as follows: daidzein < genistein < equol. Indeed, the last-mentioned is at least 10-
to 100-fold more estrogenic than daidzein and at least 10-fold more
estrogenic than genistein in fish (Pelissero et al. 1991
). It is possible, then, that the bone estrogenic effects
observed in D could be due to equol rather than daidzein, or to both
equol and daidzein. However, it would be also reasonable to consider
that the degree of estrogenic activity of genistein, daidzein and equol
is not universal, and there could be tissue differences in the effects
of these compounds.
Ovariectomized rats may exhibit some protection against bone loss by
obesity (Kalu 1991
). However, under our experimental
conditions, ovariectomy did not influence the body weight evolution,
indicating that pair-feeding to SH avoided the
ovariectomy-induced hyperphagia. On the contrary, rats in the
E2 group were significantly lighter than the
others because of a reduced food consumption (daily mean food intake
was 75% of the daily SH diet consumption), which was likely due to the
palatability of 17-ethinylestradiol. Uterine weight was
significantly decreased by ovariectomy, confirming the effects of the
surgical intervention. In these rats, the 17-ethinylestradiol intake
[22–23 rather than 30 µg/(kg body weight · d)]
induced an uterotrophic activity. Furthermore, when results are
expressed as g/100 g body weight rather than as grams, values in
E2 were not different from SH (data not shown).
On the contrary, genistein and daidzein did not exhibit any
uterotrophic activity, confirming results obtained in previous studies
(Anderson et al. 1998
, Arjmandi et al. 1996
and 1998
, Ishimi et al. 1999
, Tansey et al. 1998
). Effectively, these phytoestrogens are weakly estrogenic
in the rat uterus. In the study of Fanti et al. (1998)
,
a high genistein dose [25 µg/(g body weight · d),
subcutaneously] was required to cause an increase in uterine mass. In
the same way, in the study of Ishida et al. (1998)
, only
the highest orally administered daidzin dose [50 µg/(g
body weight·d)] increased uterine weight in ovariectomized rats.
However, when genistin was administered at the same dose, no
uterotrophic activity was demonstrated (Ishida et al. 1998
). Again, it is possible that ER subtype–dependant
mechanisms could be involved because both ER
and ERß are present in the rat uterus
(Hiroi et al. 1999
).
In conclusion, we demonstrated that both cancellous and cortical bone loss or only cortical bone loss was prevented by orally administrated daidzein or genistein, respectively, in the ovariectomized rat model of postmenopausal osteoporosis. Like estradiol, these phytoestrogens suppressed the ovariectomy-induced increase in bone turnover. Moreover, neither genistein nor daidzein exhibited estrogenic activity on the uterus, demonstrating the usefulness of a soybean isoflavone to prevent postmenopausal bone loss without any adverse effects on the uterus.
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FOOTNOTES |
|---|
-ethinylestradiol; ER,
estrogen receptor; G, ovariectomized rats given genistein; HRT, hormone
replacement therapy; IC, initial control rats; OC, osteocalcin; OVX,
ovariectomized rats; SERM, selective estrogen receptor modulators; SH,
sham-operated rats; SHBG, sex hormone binding globulin. 
Manuscript received January 3, 2000. Initial review completed February 11, 2000. Revision accepted March 9, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), ovariectomized (OVX: 
),
ovariectomized + genistein (G: 
), ovariectomized + daidzein (D: •)
and ovariectomized + 17
