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Laboratorium of Experimental Medicine and Endocrinology (LEGENDO) (D.V., E.V.H., R.B., Onderwijs en Navorsing, Gasthuisberg, B-3000 Leuven, Belgium;, Unit of Reumatology (J.N.), University Hospital Gasthuisberg, B-3000 Leuven, Belgium; Department of Endocrinology (E.A.), Scientific Development Group, NV Organon, NL-5340 BH Oss, The Netherlands; and the Janssen Research Foundation (R.D.C.), B-2340 Beerse, Belgium
Address all correspondence and requests for reprints to: Dr. Dirk Vanderschueren, Laboratory of Experimental Medical and Endocrinology (Legendo), Onderwijs en Navorsing, Gasthuisberg, Leuven Belgium B-3000.
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Abstract |
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Rats were either Sham-operated (Sham) or Orchidectomized (Orch) and treated with or without the aromatase inhibitor VOR. One Sham-operated group was killed at Baseline (Base); the four other groups (Sham, Sham + VOR, Orch, Orch + VOR) were killed 18 weeks after surgery. As expected, all groups gained body weight, but body weight gain was significantly (-25%) lower in Orch, Orch + VOR, and Sham + VOR. Both bone formation, as assessed by serum osteocalcin, and bone resorption, as assessed by urinary (deoxy)pyridinoline, decreased significantly in all groups compared with Base. Orchidectomy resulted in a relative increase of biochemical markers of bone formation and resorption compared with Sham. Treatment with VOR, however, resulted only in a very moderate increase of (deoxy)pyridinoline compared with Sham. As expected, femoral length increased compared with Base, but orchidectomy reduced the relative growth of the femur whereas VOR did not influence femoral length.
Ex vivo, densitometric and geometric properties of the femora were evaluated by peripheral computerized quantitative tomography (pQCT) and dual-energy x-ray absorptiometry (DXA). The lumbar vertebrae were measured by DXA. At the end of the experimental period, volumetric trabecular bone mineral density (vTBMD) measured at the distal end of the femur was significantly lower not only in both Orch groups but also in Sham + VOR. The decrease of cancellous bone density in Sham + VOR was lower than in the orchidectomized animals.
A relative decrease of femoral inner and outer diameters compared with Sham and Base was observed in both Orch groups and in Sham + VOR, suggesting that both orchidectomy and VOR-treatment inhibited periosteal bone formation and endosteal bone resorption. Only orchidectomy, however, resulted in a decrease of cortical thickness. Bone area, mineral content, and density of both femora and lumbar vertebrae, measured by DXA, were decreased to a similar extent by VOR and Orch (bone mineral content of the femur was 467 ± 18 mg in Orch and 461 ± 10 mg in Sham + VOR vs. 521 ± 11 mg in Sham; P < 0.001).
In conclusion, treatment with the aromatase inhibitor VOR impairs body weight gain and skeletal modeling and decreases bone mineral density. Aromatase inhibition had similar final effects on bone mass and size as castration, but with less marked effects on bone turnover.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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The aim of this study is therefore to compare the skeletal effects of VOR with effects of castration in growing male rats.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Animals were put in metabolic cages at regular intervals (see legend of
Fig 2
) for collection of 24 h urine for measurement of the
collagen cross-links. Regularly, serum was obtained by tail bleeding
for measurement of osteocalcin and IGF-I (see legend of Figs. 1 and 2).
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All procedures were approved by the ethical committee of our university.
Assays
Serum osteocalcin (14), IGF-I (15) and testosterone (16) were
measured by an in-house RIA. Intraassay and interassay variation were
respectively 5.9 and 5.2% for osteocalcin, 10.8 and 7.6% for IGF-1,
and 3.5 and 5.1% for testosterone. Estradiol and its tetradeuterated
internal standard were extracted from serum at neutral pH using diethyl
ether. After an additional column purification, the residue was
acylated using trifluoroacetic acid anhydride (TFA) and analyzed by gas
chromatography coupled with high resolution mass spectrometry. The mass
spectrometer was set to monitor the accurate molecular mass of
estradiol-TFA and 2H4-estradiol-TFA. Urinary excretion of
collagen cross-links [deoxypyridinoline (DPD) and pyridinoline (PYD)]
was measured by HPLC as described (17). Intra- and interassay
coëfficients of variation were respectively 8.6% and 10.2% for
PYD, 12 and 12.3% for DPD.
Dual energy x-ray absorptiometry (DXA)
The right femur was scanned with the Hologic QDR-100 as
described (18). Total area and bone mineral content (BMC) were
measured; total bone mineral density (BMD) was calculated as bone
mineral content/area. Bone mineral density was also calculated for an
area containing only cortical bone at the middle femur area and an area
containing both cortical and cancellous bone at the distal femur. The
first four lumbar vertebrae were selected on the screen and their area,
mass, and density were calculated. The results are given as total area,
BMC and BMD for the combined four lumbar vertebrae.
pQCT
The defatted specimens were placed in a specially constructed
vial and mounted in the Stratec XCT 960A densitometer (Dutoit Medical,
Wommelgem, Belgium) with a special support. Two CT-slices of each 1 mm
thickness were obtained using a voxel size of 0.022 mm3.
The slice taken at 6 mm proximal from the distal end of the femur gave
data of trabecular bone density using a 0.53 cm-1
attenuation setting, whereas the second slice at 15 mm represented
cortical bone where the attenuation threshold was 0.93
cm-1. Both slices were calculated with special software
with the following settings: contour mode 1, peel mode 20 and cortical
mode 1. Femoral outer diameter, inner diameter and cortical thickness
were measured at the diaphyseal part of the femur using the pQCT
software and assuming a circular shape for the midfemur. Cortical
thickness is calculated from the difference in total bone area and
trabecular area assuming a circular ring model as described by Louis
et al., 1995 (19). The reproductivity (CV) of measurements
for trabecular BMD and for cortical thickness are 2–3% and for
cortical BMD 0.5%. Moreover, we found in a different experiment a
correlation between TBMD and histomorphometrically determined BV/TV of
r = 0.9.
In the diaphyseal region, the software also provided data of the second moment of inertia.
Statistical analysis
Statistical analysis was performed using a statistical package
(NCSS, Kaysville, UT). All data are expressed as mean ±
SEM. A Tukey’s honest significant difference multiple
comparison test was used to find out if a parameter changed with time
and if this change with time was different between groups. This test
was only used separately for those parameters that were regularly
followed during the experiment, such as body weight, serum IGF-1,
osteocalcin and urinary (deoxy)pyridinolines, as presented in the Figs. 1
and 2.
Two-way ANOVA was for all other parameters used to determine the
effects of orchidectomy, VOR, and their possible interaction at the end
of the experiment, as presented in the tables and Fig. 3. To be conservative in the application of the two-way
ANOVA, one-way ANOVA with Tukey’s test was applied when the
interaction had a P value 
0.15.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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and Table 1). Serum IGF-1 was not significantly different between
groups, except for a significant decrease of serum IGF-1 in Sham + Vor,
compared with Sham, Orch, and Orch + VOR during the experimental period
(Fig. 1b and Table 1). At the end of study, weight gain was
significantly lower, both in the Orch (-19%, P <
0.0001) and in the VOR groups (-29%, P < 0.0001). As
expected, serum testosterone was only significantly decreased in
orchidectomized animals (Table 1). Castration did not significantly
reduce estradiol levels, whereas VOR did reduce estradiol levels to the
same extent in both intact and castrated rats.
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, a–c, respectively). However, a
relative increase of both serum osteocalcin as well as urinary DPD and
PYR was observed in both orchidectomized groups (Orch and Orch + VOR)
compared with both Sham and Sham + VOR (P < 0.0001)
(Fig. 2, a–c, respectively). At the end of the experiment, however,
both DPD and PYR (but not serum osteocalcin) were also significantly
increased in Sham + VOR compared with Sham (Table 1).
DXA of the femur and four distal lumbar vertebrae
Total area, BMC, and areal density of the femur and distal lumbar
vertebrae increased, compared with Baseline (Table 2).
Total area, BMC and areal density of the femur and four distal lumbar
vertebrae were significantly reduced both by orchidectomy and treatment
with VOR, compared with Sham. Similar reductions were observed in a
region of the femur that contains only cortical bone (middle femur) as
well as in a region that contains both cortical and cancellous bone
(distal femur) (significance was only reached in the Orch group for
regional density).
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Cortical areas were smaller compared with Sham as a result of decreased
radial growth in both Orch groups and in Sham + VOR (Table 3). Therefore, lower moments of inertia were calculated
in these groups compared with Sham.
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), but the relative increase was lower
after orchidectomy. Compared with baseline, radial growth as measured
by femoral outer diameter also increased, but this increase was
significantly lower in both Orch groups and in the Sham + VOR compared
with Sham. Femoral inner diameter did not increase compared with Base
in the Sham-operated control group. The inner femoral diameter
decreased however in both Orch groups and in the Sham+VOR group,
compared with Sham and Base. Femoral cortical thickness was, however, only decreased after orchidectomy.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Surprisingly, as a result of VOR treatment, skeletal size and modeling were reduced to a similar extent as by orchidectomy. These findings were unexpected. Indeed, these data suggest that androgens need aromatization into estrogens and therefore that skeletal modeling in male rats would be estrogen dependent. This would be completely opposite to the well-established inhibitory effects of estrogens on skeletal modeling in the female rat (13). Earlier observations of reduced skeletal modeling in androgen resistant male rats with increased estrogen concentrations also do not fit with a direct stimulatory effect of estrogens on skeletal modeling in the male (20). Moreover, nonaromatizable androgens without estrogenic activity were shown to have stimulatory effects on skeletal modeling in male orchidectomized rats (13).
An alternative explanation for the unexpected decrease in skeletal modeling observed in VOR-treated rats could be the decrease of mechanical load of the skeleton as the result of the lower body weight gain. Clearly, the inhibition of conversion of androgens into estrogens leads to a decrease in body weight gain to a similar extent as obtained by orchidectomy. Such a decrease of mechanical load may also switch off skeletal modeling drifts, at the periosteal and endosteal envelopes (22). Both VOR in this and earlier experiments (11) and estrogens (6) were shown to suppress growth in male rats. This may seem contradictory. However, also androgens were shown to be stimulatory for growth at lower concentrations and inhibitory at higher concentrations in male rats (23). Moreover, the inhibitory effects of androgens on growth were attributed to stimulation of the estrogen receptor (23). Therefore, the effects of both androgens and estrogens on growth may be concentration dependent with lower concentrations being stimulatory and higher concentrations inhibitory.
The finding that VOR (and not orchidectomy) decreases serum IGF-1, as described earlier (11), may be an important observation in this respect. It has indeed been shown that administration of IGF-1 to growing rats increases bone size (24). Moreover, preliminary unpublished findings in our laboratory suggest that VOR treatment also significantly decreases lean body mass. Therefore, its effect on skeletal modeling may be indirect through its effect on lean body mass with or without its interaction with serum IGF-1 concentrations.
Not only skeletal size but also bone mass and density were equally affected by orchidectomy and aromatase inhibition in concordance with earlier observations in aged male rats (11). The decrease of bone mass observed in both castrated and VOR-treated growing male rats was explained by a decrease of (cortical) bone size that was the result of a decreased periosteal bone formation. Cortical bone density was not affected by either condition, whereas trabecular bone density was lower in both conditions.
Whereas the effects of castration and aromatase inhibition on skeletal size, mass and density were similar, their respective effects on biochemical parameters of bone turnover were different. As expected, orchidectomy increased bone turnover as assessed by both biochemical parameters of bone formation such as serum osteocalcin, and biochemical parameters of bone resorption such as urinary (deoxy)pyridinoline (17). It was also shown earlier that the increase of biochemical parameters of bone turnover in androgen-deficient male rats correlates well, not only with histomorphometric measurements of bone turnover but also with important loss of cancellous bone (6). The increase of bone turnover (certainly of serum osteocalcin) was clearly significantly lower in VOR-treated animals than in orchidectomized animals.
Therefore, it is surprising that in VOR-treated rats cancellous bone loss occurred without major changes in bone turnover. However, as measured by pQCT [which is more accurate than DXA for the measurement of cancellous bone (24)], relative cancellous bone loss was significantly lower in VOR-treated Sham operated rats, compared with the Orch groups. Moreover, VOR treatment did not significantly decrease cortical thickness in contrast with orchidectomy. These observations suggest that both androgens and estrogens may have synergistic effects on the skeleton. Recently, such a synergistic action of estrogens and androgens in the prevention of ovariectomy-induced bone loss has been observed in female rats (25). Low dose replacement therapy with 17ß-estradiol during VOR treatment could conclusively confirm our present hypothesis that estrogens may also have physiological skeletal effects in male rats, and such work is in progress. In the aged male rat model (age 12 months), however, 17ß-estradiol replacement by SILASTIC brand (Dow Corning, Midland, MI) implant (3 µg/per day for 15 weeks) could largely correct the bone loss induced by VOR treatment (unpublished personal results). Overall, the treatment with VOR combined with castration had, as expected, similar effects as castration alone, suggesting that VOR has no direct toxic effects on bone.
Finally, these data may also be relevant in humans. Similar decreases of bone size and density were reported in hypogonadal males (12). Because both bone size and bone density are believed to be important determinants of bone strength (26), hypogonadal men may be predisposed to osteoporosis later in life (27). Also, skeletal mineral gain during growth is crucial for final bone mass. Delayed puberty is associated with decreased bone density (28) and androgen treatment in hypogonadal men started after puberty does not restore bone mass and is far less effective in this respect than androgen therapy started before puberty (12). In humans, androgen action during growth may also depend on the conversion into estrogens. Both a man with estrogen receptor deficiency (9) and another man with aromatase deficiency (10) were reported to have osteopenia and delayed epiphyseal closure. As in the male rat, estrogen deficiency probably switches off modeling drifts at periosteal and endosteal sites. The bone turnover parameters were more increased in these estrogen deficient humans than in our VOR-treated animals (9, 10). However, contrary to humans, rats lack Haversian remodeling and epiphyseal closure. Cortical osteopenia and delayed epiphyseal closure will therefore complete the picture of estrogen deficiency syndrome in humans.
We conclude that treatment of growing rats with an aromatase inhibitor such as VOR impairs skeletal modeling and decreases bone size and mass to a similar extent as orchidectomy. However, the increase in bone turnover and the decrease in bone density were more pronounced after orchidectomy than after treatment with VOR.
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Acknowledgments |
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Footnotes |
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Received November 26, 1996.
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References |
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