Physiological plasma levels of androgens reduce bone loss in
the ovariectomized rat
C. K.
Lea and
A. M.
Flanagan
Department of Histopathology, Imperial College School of Medicine at
St. Mary's, London W2 1PG, United Kingdom
 |
ABSTRACT |
The effect of androstenedione (ADIONE)
slow-release pellets on cancellous bone volume (BV/TV) at the tibial
metaphysis was investigated in ovariectomized (OVX) rats at various
times from 21 to 180 days. Plasma levels of ADIONE and testosterone (T)
in OVX rats were significantly reduced at 21 days and were restored close to levels in the sham rats with the 1.5-mg ADIONE pellet. OVX
animals with and without ADIONE pellets resulted in close to a 50%
reduction in BV/TV by day 21. By
day 180, OVX rats had only ~5%
BV/TV, whereas that in ADIONE-treated OVX rats was significantly greater at ~12%. The reduced BV/TV was associated with increased bone resorption and formation. In a separate 90-day experiment, we
found that the antiandrogen, Casodex, abrogated the ADIONE-induced skeletal-protective effect in OVX rats, whereas the antiaromatase, Arimidex, had no effect. This provides evidence that ADIONE protects against the development of osteopenia in the estrogen-deficient rat and
mediates its effect through androgens and not estrogens.
bone resorption; bone formation; estrogen; androstenedione; Arimidex
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INTRODUCTION |
THE FALL IN PLASMA LEVELS of estrogen that occurs at
menopause has been implicated in the increased bone turnover and
associated loss of bone mass that occur in postmenopausal women (3).
Although all women lose estrogen and bone mass during the menopause,
not all women develop osteoporosis. This may be partially explained on
the basis that women with a high peak bone mass are protected against
osteoporosis (see Ref. 18). However, other factors may also be
involved, since the rate of bone loss is variable in the menopausal
years (4, 17). It is now believed that a high bone turnover state can
exist late into the eight and ninth decade, and furthermore, it has
been found that women with bone turnover above a certain level are at a
greater risk of sustaining a fracture (10), but the reason for the
variability in bone turnover in postmenopausal women has not been
established. Our hypothesis is that ovarian androgens protect against
bone loss, particularly in estrogen-deficient states. If this were the
case, low levels of these hormones in postmenopausal women might, at
least partially, account for the variability in their bone turnover.
This opinion is based on previous reports, including data that show a
positive correlation between sera levels of androgens and bone mass in pre- and postmenopausal women (1, 6), and the report that treatment of
postmenopausal women with anabolic androgenic steroids is associated
with increased bone mass (2). Furthermore, it has also been shown that
ovariectomy results in loss of testosterone (T) and androstenedione
(ADIONE) (11), and finally there is evidence indicating that ADIONE and
T plasma levels are reduced in peri- and postmenopausal women compared
with those in premenopausal women (11).
The sex steroid precursor, dehydroepiandrosterone, has been shown to
protect the ovariectomized (OVX) rat skeleton against bone loss (24),
but this study did not test whether androgens or estrogens mediated the
effect. We have recently reported that ADIONE and T are significantly
reduced in OVX rats and that ADIONE slow-release pellets
protect against cancellous bone loss in a dose-responsive manner in the
OVX rat (15). This was achieved using static and dynamic
histomorphometric analyses of the tibial metaphyses. However, only
supraphysiological plasma levels of ADIONE showed a convincing
skeletal-protective effect in the previous 21-day experiment. We also
found, in this study, that the ADIONE-mediated effects in response to
pharmacological levels of ADIONE (100-mg pellets) were abrogated by
Arimidex, the aromatase inhibitor (21).
In this study, we elected to use the mature OVX rat model (13) to
determine whether physiological plasma levels of ADIONE could
unequivocally reduce bone loss if the experiment was continued beyond
21 days for a further 159 days. Once we established that this was the
case, we tested whether the skeletal-protective effect of ADIONE was
mediated by androgens or estrogens in the aged rat model. In these
experiments, OVX animals were treated with ADIONE with and without an
androgen antagonist, Casodex (7), and an aromatase inhibitor, Arimidex.
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MATERIALS AND METHODS |
Animal Experimentation and Histomorphometry
Female Sprague-Dawley rats were purchased from Harlan Olac (Bicester,
Oxon, UK), housed at 21°C with a 12:12-h light-dark cycle, and fed
rat laboratory diet (Lillico, Betchworth, Surrey, UK) and water. The
animals were pair fed. All pellets were purchased from Innovative
Research of America (Toledo, OH).
Experiment I.
Thirteen-week-old animals, with an average weight of 216 g (range
196-234 g), were subjected to ovariectomy or a sham operation under halothane anesthesia using a dorsal approach on
day 1. The animals were divided into
six treatment groups and analyzed at five time points. At each time
point, each of the six groups contained either six or eight animals (a
total of 210 animals in experiment). Group
1 (n = 8) was
subjected to a sham operation. Group 2 (n = 6) had a bilateral ovariectomy.
Groups 3 (n = 8) and
5 (n = 8) had a bilateral ovariectomy and ADIONE slow-release pellets of 1.5 and 5 mg, respectively, inserted at the back of the neck at the time of
ovariectomy. Groups 4 (n = 6) and
6 (n = 6) had bilateral ovariectomy and placebo pellets of 1.5 and 5 mg,
respectively, inserted subcutaneously.
Experiment II.
Six-month-old animals, with an average weight of 270 g (range
252-290 g), were subjected to ovariectomy or a sham operation as
described above. At this age, the animals exhibit imperceptible growth
(13). Ten animals were included in each of the following groups:
group 1, sham OVX;
group 2, OVX plus placebo pellet;
group 3, OVX plus ADIONE (1.5-mg
slow-release pellet); group 4, OVX plus Casodex (kindly provided by Dr. B. M. Vose, Zeneca
Pharmaceuticals, Macclesfield, UK); group
5, OVX plus ADIONE (1.5-mg pellet) and Casodex;
group 6, OVX plus Arimidex (kindly
provided by Dr. Vose); and group 7,
OVX plus ADIONE (1.5-mg pellet) and Arimidex. Casodex (5 mg · kg
1 · day
1)
and Arimidex (0.1 mg · kg
1 · day
1)
were both dissolved in water and administered orally.
Calcein (30 mg/kg, Sigma Chemicals, Dorset, UK) and tetracycline
hydrochloride (25 mg/kg, Lederle Laboratory, Gosport, Hants, UK) were
injected intraperitoneally 14 and 7 days before each group of animals
was killed. Cardiac puncture was performed under anesthesia, and plasma
samples were stored at
70°C until required. The animals were
then killed by cervical dislocation after periods of 21, 60, 90, 120, and 180 days in experiment
I and after 90 days in experiment
II.
The uteri were removed and weighed, and ovariectomy was confirmed by
the absence of ovarian tissue. The tibiae were cleaned of soft tissue,
fixed in 70% alcohol for 24 h, dehydrated through graded alcohols, and
embedded without decalcification in London Resin (London Resin,
Basingstoke, Hants, UK). Longitudinal sections of the proximal
metaphysis were cut using a Reichart-Jung microtome (Leica, Germany).
Sections (5 µm) were stained with toluidine blue, and 12-µm
unstained sections were cut for fluorescent microscopy. Bone
histomorphometry was performed using transmitted and epifluorescent microscopy linked to a computer-assisted image analyzer (Seescan, Cambs, UK). Bone volume and surface parameters were measured by tracing
the relevant features with a cursor on the video screen. Cancellous
bone volume (BV/TV) measurements were performed at ×40
magnification, and the surface parameters were measured at ×400
magnification. All sections were analyzed without knowledge of the
group from which they came.
BV/TV at the proximal metaphysial cancellous bone from animals killed
on days 21 and
60 was measured on two nonconsecutive sections, and four nonconsecutive sections were analyzed from animals
killed on days 90,
120, and
180. The latter was done because of
the relative lack of bone spicules in OVX rats. A standard area of 2 mm2 (at least 2 mm from growth
plate to exclude primary spongiosa) was measured. Trabecular number and
thickness were calculated as previously described (20). Static
parameters were measured in the same way as that described for BV/TV
and included osteoblast surface, osteoclast surface, and osteoclast
number. Longitudinal growth rate (LGR) was derived by measuring the
distance between the tetracycline and calcein fluorescent bands that
parallel the growth plate at four equally placed sites per section and
dividing by the time interval between the two injections. The bone
formation rate (BFR; tissue level, total surface referent) was
calculated from the product of the percentage of the trabecular bone
surface with a double fluorochrome label and the mineral apposition
rate (MAR): the former was obtained by measuring the percentage of the
trabecular bone surface, covered by two fluorochrome labels, and the
latter by dividing the interlabel distance by the time interval between
the injections of the labels in the corresponding area. The BFR values
were not corrected for label escape errors. MAR values were not
corrected for the obliquity of the plane of section of cancellous bone.
Radioimmunoassays for Plasma Hormone Levels
Plasma levels of ADIONE, estrone
(E1), and T (Diagnostics Systems
Laboratories, TX) and estradiol
(E2; IncStar, Berks, UK) were
measured in the animals from experiment
I, which were killed on day
21, and in animals from experiment
II on day 90 by
radioimmunoassay (RIA) as instructed in the manufacturer's guidelines.
The RIAs had an intra-assay coefficient of variation of <5% and an
interassay coefficient of variation of <6%. Each assay was validated
by spiking serum samples with known amounts of the four hormones being
tested.
Statistics
The results were analyzed using Fisher's least significant difference
method for multiple comparisons in a one-way analysis of variance and
expressed as means ± SE. Significance was considered when
P < 0.05. Statview 4.0 (Abacus
Concepts, Cupertino, CA) was used to analyze the results.
 |
RESULTS |
Experiment I
OVX rats were significantly heavier than sham-operated rats at all time
points. There was no difference in the weight gained by any of the OVX
groups at any time point. All the animals were significantly heavier by
day 21 compared with
day 1. They continued to increase
their weight until day 180 (day 180: sham, 299.80 ± 5.30 vs.
OVX, 336.56 ± 4.53; P < 0.05 vs.
relevant day 0). The uteri of OVX
animals were markedly atrophic compared with the sham animals; the
average weight of the former on day 21 was 107 mg (range 96-110 mg) and on day
180 was 102 mg (range: 89-108 mg). The average
weight of the sham-operated animals on day
21 was 444 mg (range 349-588 mg) and on
day 180 was 672 mg (range 579-822
mg), and that of ADIONE-treated OVX rats on day
21 was 116 mg (range 111-124 mg) and on
day 180 was 120 mg (range 113-129 mg).
Ovariectomy caused the expected reduction in the plasma levels of
E2 and also resulted in a
significant reduction in plasma levels of ADIONE, T, and
E1. The 1.5- and 5-mg ADIONE
pellets raised the plasma concentrations of ADIONE and T significantly above OVX levels. The androgens levels, however, remained below sham
levels in the presence of the 1.5-mg pellets, whereas they were raised
close to male levels in response to the 5-mg pellet. The
E2 level in the ADIONE-treated
rats was only marginally increased above those in OVX rats. The level
of E1 in ADIONE-treated rats was
elevated above sham levels; the reason for this was not clear. No
effect on hormone levels were seen in placebo-treated animals (Fig.
1).

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Fig. 1.
Plasma levels of testosterone (T), androstenedione (ADIONE), estradiol
(E2), and estrone
(E1) in ADIONE-treated rats
after ovariectomy of 13-wk-old rats on day
21. T: * P < 0.01 vs. sham; P < 0.001 vs. ovariectomized (OVX). ADIONE:
* P < 0.001 vs. sham;
P < 0.01 vs.
OVX. E2:
* P < 0.0001 vs.
sham; P < 0.0001 vs. OVX. E1:
* P < 0.01 vs. sham;
P < 0.0001 vs.
OVX.
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The LGR of OVX rats was found to be significantly increased above that
of the sham rats until day 90 but
returned to control levels at subsequent times. The increased LGR in
OVX animals on day 21 partially
returned to sham levels after administration of the 1.5-mg ADIONE
pellet, and the larger 5-mg pellet completely reverted the measurements
to those in the sham-operated animals. After day
21, the LGR of the 1.5- and 5-mg ADIONE-treated rats was similar to that of the sham animals (Fig.
2).

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Fig. 2.
Effect of ADIONE (A) and placebo (P) pellets on longitudinal growth
rate (LGR) after ovariectomy of 13-wk-old rats.
* P < 0.0001 vs. sham and 5-mg
ADIONE-treated rats.
P < 0.0001 vs.
1.5-mg ADIONE-treated rats. ** P < 0.0001, OVX (36.43 ± 1.58 µm/day, mean ± SE) and placebo
groups (35.13 ± 0.84 µm/day) vs. sham (26.45 ± 1.19 µm/day)
and ADIONE-treated groups (27.18 ± 1.05 µm/day).
P < 0.0001, OVX (16.41 ± 1.16 µm/day) and placebo groups (17.65 ± 1.31 µm/day) vs. sham (11.31 ± 1.41 µm/day) and ADIONE-treated
groups (11.98 ± 1.15 µm/day).
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Cancellous bone volume was largely maintained throughout the experiment
in the sham animals, whereas it was reduced by ~50% in OVX rats as
early as day 21 (Fig.
3). This was followed by a slower decline
in bone loss over the next 100 days, resulting in a further ~25%
loss of cancellous bone. After this time, the bone volume appeared to
be maintained at ~5%. There was a minor but significant difference
in the values of OVX and ADIONE-treated rats as early as
day 21, but the difference became more
pronounced with time. In fact, there was no further reduction in the
cancellous bone volume in ADIONE-treated animals after
day 21. Cancellous bone loss in
ADIONE-treated OVX rats was reduced by ~35%, measured 180 days
post-OVX. By the end of the experiment, ADIONE-treated animals had
130% greater cancellous bone volume than did OVX rats (Fig. 3). This
greater cancellous bone volume was largely the result of an increase in
the thickness of the metaphysial trabeculae (Table
1), whereas there was no increase in
trabecular number (data not shown).

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Fig. 3.
Effect of ADIONE and placebo pellets on percentage of cancellous bone
volume (BV/TV) at proximal tibial metaphysis after ovariectomy of
13-wk-old rats. * P < 0.0001 vs. sham. P < 0.05 vs. 1.5 and 5 mg ADIONE.
P < 0.001 vs.
1.5 and 5 mg ADIONE.
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Table 1.
Effect of ovariectomy and ADIONE pellets on trabecular thickness of
13-wk-old rats at various time points after surgery
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The loss in cancellous bone in OVX rats was associated with an increase
in osteoclast number that was most marked on day
21 but remained significantly different from the
controls even at day 180. A reduction
in osteoclast numbers and the bone surface covered with osteoclasts
were significantly reduced in ADIONE-treated animals compared with OVX
animals, and by day 180, both of these parameters were similar to those in the sham-operated animals (Fig.
4).

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Fig. 4.
Effect of ADIONE and placebo pellets after ovariectomy of 13-wk-old
rats on percentage of bone surface covered with osteoclasts
(OcS/BS). * P < 0.0001 vs. sham. P < 0.001 vs. 1.5 and 5 mg ADIONE.
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BFR was increased in OVX animals compared with controls. Again, the
greatest difference was seen early in the experiment on days 21 and
60. Thereafter it declined gradually
but always remained significantly different from the controls (Fig. 5).
The increase in BFR was the result of an increase in the double-labeled
bone surface and in the MAR. ADIONE reduced the increase in the
fluorochrome-based indexes of bone turnover to values midway between
those of sham and OVX animals (data not
shown). Likewise, osteoblast numbers were
significantly increased in OVX animals compared with controls and this
parameter was reduced in the presence of ADIONE. The effects brought
about by administration of ADIONE occurred in the presence of both
sizes of pellets, and although the larger pellet was always found to
exert a marginally greater effect, there was no significant difference
between the results(Fig. 5). The results obtained from the
placebo-treated animals were not significantly different from those
from OVX animals (Figs. 2-5).

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Fig. 5.
Effect of ADIONE and placebo pellets on histomorphometric indexes of
cancellous bone formation (BFR) after ovariectomy of 13-wk-old rats.
BFR (top):
* P < 0.0001 vs. sham.
P < 0.005 vs.
1.5 and 5 mg ADIONE. Osteoblast surface (ObS/BS;
bottom):
* P < 0.0001 vs. sham.
P < 0.001 vs.
1.5 and 5 mg ADIONE.
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Experiment II
Both sham and OVX rats gained weight in the 90-day experiment. There
was no significant difference in the weight gained by any of the OVX
groups. The sham animals gained 22.70 ± 2.17 g, and the latter
gained 70.46 ± 3.86 g.
To determine whether the results in experiment
I were accounted for by conversion of ADIONE to
androgens and/or estrogens, 1.5-mg ADIONE pellets were
administered in the presence and absence of Casodex and Arimidex. As
shown in experiment I, the 1.5-mg ADIONE pellet protected cancellous bone loss significantly in OVX rats
(Table 2). As in
experiment I, this was achieved by reducing bone turnover (Figs. 6 and
7). The ADIONE-induced protective effect
was completely abrogated by antiandrogen therapy, whereas it was
maintained in the presence of Arimidex (Figs. 6 and 7). The finding
that neither the antiandrogen nor the aromatase inhibitor treatment in OVX animals reduced parameters below the OVX placebo group
is consistent with previous reports (8, 15) (Figs. 6 and 7, Table
2).
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Table 2.
Effect of ADIONE, placebo pellets, Casodex, and Arimidex on uterine
weight, cancellous bone volume, and LGR of 6-mo-old rats 90 days
postovariectomy
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Fig. 6.
Effect of ADIONE, placebo, Casodex (Cas) and Arimidex (Ar) on
percentage of bone surface covered with osteoclast (OcS/BS;
top) and number of osteoclasts per
mm of bone surface (NOc/BS; bottom).
P < 0.0001 vs.
all groups. * P < 0.0001 vs.
all groups except each other.
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Fig. 7.
Dynamic histomophometric indexes of bone formation of 6-mo-old rats,
after treatment with 1.5 mg ADIONE, placebo pellets, Casodex, and
Arimidex for 90 days. BFR: * P < 0.0001 vs. all groups. ** P < 0.0001 vs. all groups except each other. ObS/BS:
* P < 0.001 vs. all groups.
P < 0.001 vs.
all groups except sham.
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The hormone plasma levels in the animals provide supportive evidence
that the skeletal protective effect of ADIONE was mediated through
androgens and not estrogens (Fig. 8). The
finding that T plasma level was increased in ADIONE-Arimidex-treated
OVX rats above OVX ADIONE-treated rats is probably the result of a
greater proportion of the ADIONE being converted into T, since it was prevented from being converted into
E1 by Arimidex. The increase in
the uterine weight that occurred in response to ADIONE was abrogated by
Arimidex, indicating that the effect was mediated by estrogens and not
androgens (Table 2). Likewise, the ADIONE-induced reduction in the LGR
was reversed by Arimidex, demonstrating that the effect was mediated by
estrogens and not androgens (Table 2).

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Fig. 8.
Plasma levels of T, ADIONE, E2,
and E1 in 6-mo-old rats
after treatment with 1.5 mg ADIONE with and without Casodex or Arimidex
for 90 days. ADIONE and T:
*, P < 0.0001 vs. all groups except each other.
E2:
* P < 0.0001 vs. all groups
except each other;
P < 0.005 vs.
all groups except each other. E1:
*, P < 0.0001 vs. all groups except each other.
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DISCUSSION |
The role of ovarian-derived estrogen in the maintenance of the skeleton
is well characterized (12, 16, 22, 28, 29), whereas the effect of
ovarian androgens is less clear. We now report that cancellous bone
loss in the OVX rat can be significantly reduced by restoring plasma
levels of ADIONE and T to those found in sham-operated animals. Similar
results were found in 13-wk- and 6-mo-old animals. The 6-mo-old animals
were used in experiment II, since
older rats are generally considered to provide a better model of
postmenopausal bone loss in humans (12). However, similar results were
observed in both experiments. Our results, in terms of static and
dynamic bone histomorphometry in sham-operated and OVX rats of the
serial histomorphometric analysis, are very similar to the effects seen
in OVX rats in response to estrogen (28). Hence the skeletal-protective
effect of estrogens (28) and androgens in OVX rats are both brought
about by suppressing bone turnover.
ADIONE is a precursor of estrogens and T, either or both of which could
potentially protect the skeleton against bone loss. The results of
experiment II show that the protective
effect of ADIONE was maintained in the presence of Arimidex, when
E1 and E2 plasma levels were returned to
levels in OVX placebo-treated animals. This excludes the possibility
that estrogens mediated the effect of ADIONE. The finding that there
was complete abrogation of the ADIONE-induced protective effect in the
presence of the antiandrogen demonstrates that androgens account for
our findings. Androgens have previously been shown to exert an anabolic
effect on the female rat skeleton at physiological levels (9, 14, 23),
but on this occasion, we were unable to detect such an effect. However,
the anabolic effect of androgens may be masked by the
resorption-induced changes in formation, i.e., where suppression of
bone resorption suppresses bone formation.
It is not surprising that the ADIONE-induced reduction in the LGR of
OVX rats was exerted by estrogens, because longitudinal bone growth
increases as a result of ovariectomy (28) and is reversed by estrogen
(25), but it is interesting that this occurred in the presence of very
low levels of circulating E2,
which were found to be without effect on the cancellous bone volume in
OVX rats. It is therefore possible that these circulating levels of E2 do not account for the
reduction in LGR in OVX rats. The alternative explanation is that
localized peripheral synthesis of
E2 is responsible for the
compartmentalized estrogenic effect that we have observed at the growth
plate. If the appropriate enzymes were present at this site, conversion
from T to E2 and ADIONE to
E1 could be catalyzed by aromatase
cytochrome P-450, and conversion of
the latter to E2 could be
catalyzed by 17
-hydroxysteroid dehydrogenase.
Our results indicate that androgens inhibit bone resorption in the
female, just as they do in the male (26, 27). Previous reports have
shown that treatment of female rats with antiandrogens do not affect
osteoclast parameters, but these experiments were carried out on
estrogen-replete rats (9, 14). Our results are at odds with those
showing that nonaromatizable dihydrotestosterone only suppresses
histomorphometric indexes of resorption in OVX rats at nonphysiological
levels (23). The reason for our different findings is not clear.
However, the experimental designs were not the same.
Our data suggest that the antiresorptive effect of androgens in the
female rat can only be detected in estrogen-deficient states, such as
menopause. If this were the case, it might explain why ADIONE treatment
in our study does not protect bone loss from day
1. There was only a minor difference between the bone
lost in OVX and ADIONE-treated OVX animals on day
21, after which ADIONE prevented any further bone loss.
This suggests that, after exposure to estrogen, the androgen-sensitive
cells in the skeleton involved in the process of inhibiting bone
resorption are resistant to the effects of androgens. The results also
suggest that, after a period of estrogen depletion, the cellular target
for androgens in bone becomes responsive to this hormone. At present,
it is unclear whether androgens inhibit bone resorption by exerting their effect directly on the osteoclast or indirectly through other
cells, including osteoblasts, since receptors have been identified on
both (5, 19).
The absence of a significant difference in the results of the
histomorphometric data between the 1.5- and 5-mg pellets, particularly in the presence of such a large difference in the plasma levels of
ADIONE and T, suggests that a maximal inhibitory effect on the
parameters of bone resorption is being exerted by the androgens in
response to the 1.5-mg pellet. However, this is not the case, since, in
a separate series of experiments, supraphysiological levels of T were
able to further reduce the resorptive parameters toward control levels
(15).
Our findings that restoration of ADIONE and T plasma levels to those in
sham-operated animals protects against cancellous bone loss and that
this effect is exerted by androgens have not previously been reported.
The results suggest that ovarian androgens may be important in
protecting against bone loss, particularly in the absence of estrogens.
It will now be important to discover whether equivalent plasma levels
of androgens exert a similar effect on the human skeleton. If this
proves to be the case, screening postmenopausal women to identify those
with low levels of androgens may be a useful means of identifying
individuals at risk of developing osteoporosis. Furthermore,
administration of androgens to postmenopausal women who are deficient
in these hormones should reduce bone turnover and consequently reduce
bone loss. Because our findings indicate that physiological plasma
levels of androgens protect against bone loss, the adverse effects
generally associated with high levels of androgens should not be seen.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Arthritis and Rheumatism Council,
UK.
 |
FOOTNOTES |
Address for reprint requests: A. M. Flanagan, Dept. of Histopathology,
Imperial College School of Medicine at St. Mary's, Norfolk Place,
London W2 1PG, UK.
Received 27 March 1997; accepted in final form 31 October 1997.
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