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Departments of Orthopedics and Biochemistry and Molecular Biology (R.T.T., G.L.E.), Mayo Graduate School of Medicine, Rochester, Minnesota 55905; Department of Endocrine Research (J.P.S., M.D.A., H.U.B., M.S.), Lilly Corporate Center, Indianapolis, Indiana 46285; and Department of Orthopedic Surgery (C.H.T.), Indiana University Medical Center, Indianapolis, Indiana 46202
Address all correspondence and requests for reprints to: Russell T. Turner, Ph.D., Orthopedic Research, Room 3–69 Medical Science Building, Mayo Clinic, 200 First Street Southwest, Rochester, Minnesota 55905.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen replacement therapy is effective in reducing postmenopausal bone loss, and it decreases fracture risk (13, 14, 15). Unfortunately, estrogen replacement is associated with many detrimental side effects, the majority of which are caused by hormonal stimulation of reproductive tissues (15, 16). As a consequence of their tissue-selective pharmacology, the substituted triphenylethylene and benzothiophene compounds described above have generated considerable interest as potential alternatives to estrogen for hormone replacement therapy. Tamoxifen, raloxifene, and CLO act as estrogen agonists by preserving bone mineral density (BMD) (9, 17, 18, 19). Also, they have similar (but not identical) effects on bone histomorphometry (5, 8, 11, 12, 20). Differences in the activities of the tissue-selective estrogen agonists may be very important in optimizing the efficacy and minimizing the incidence of detrimental side effects when these agents are applied clinically for postmenopausal hormone replacement (21, 22).
CLO differed from estrogen and other tissue selective estrogen agonists in that it uniquely increased trabecular thickness (Tb.Th) in ovariectomized (OVX’d) rats (11). CLO contains equal molar amounts of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC). The two isomers have distinctly different effects on reproductive tissues and tumor cells; ENC is a potent estrogen antagonist, whereas ZUC is an estrogen agonist (23, 24). It is uncertain whether the estrogen agonistic activity of CLO on nonreproductive tissues is conferred by ZUC only or by both isomers. To answer this question, we determined the dose-dependent effects of ENC and ZUC on selected estrogen target tissues in OVX’d rats. We then compared the results with the response of these same tissues to an optimal dose of CLO for preventing cancellous osteopenia in OVX’d rats (11).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-month-old, virgin Sprague-Dawley female rats (Harlan,
Indianapolis, IN), weighing about 270 g, were maintained on a 12-h
light/dark cycle at 22 C with ad libitum access to
food (TD 89222 with 0.5% calcium, 0.4% phosphorous, and 1000 IU of
vitamin D/kg diet; Teklad, Madison, WI) and water. Bilateral
ovariectomies were performed, except for sham-operated (SHAM) controls,
and maintained without treatment for 30 days. One group of 8
ovary-intact rats was killed on the day of surgery to provide initial
(baseline) measurements. Pretreatment groups of 8 SHAM and 8 OVX’d
rats were killed before treatment (30 days after the surgeries) to
assess the magnitude of osteopenia induced by gonadal hormone
deficiency. The remaining rats were divided into treatment groups of
n = 8 and orally dosed daily for 90 days (from days 30–120 post
surgery). The 10 treatment groups consisted of: 1) SHAM; 2) OVX’d
control (OVX); 3) OVX treated orally with 17
-ethynyl estradiol (EE2,
Sigma, St. Louis, MO) at 0.1 mg/kg·day; 4) OVX treated orally with
CLO (Eli Lilly, Indianapolis, IN) at 3 mg/kg·day; 5–7) OVX treated
orally with ENC (Lilly) at 0.03, 1, and 3 mg/kg·day; or 8–10) OVX
treated orally with ZUC (Lilly) at 0.03, 1, and 3 mg/kg·day. SHAM and
OVX control rats were administered by gavage the carrier only
which consisted of 100 µl/100 g BW of 20% wt/vol
ß-hydroxypropyl-cyclodextrin (Aldrich Chemical Co., Milwaukee, WI).
The rats received calcein (10 mg/kg·day) by ip injection 15, 14, 4,
and 3 days before death.
Estrogenic stimulation of eosinophil infiltration of the uterus of OVX’d rats was evaluated in a separate experiment by quantitating the peroxidase activity of uterine eosinophils, as described (19, 25, 26). For this assay, 6-month-old rats were OVX’d (except for SHAM controls) and maintained for 1 month before treatment. Rats were orally dosed daily from days 30–37 post surgery with carrier or test compounds. The treatments consisted of: 1) SHAM control; 2) OVX’d control; (3) OVX treated with ENC at 1 or 10 mg/kg; 4) OVX treated with ZUC at 1 or 10 mg/kg; and 5) OVX treated with ENC and ZUC at the following ratios: 10 mg/kg:1 mg/kg, 1 mg/kg:1 mg/kg, 1 mg/kg:10 mg/kg.
Tissue collection, cholesterol analysis, and densitometry
Twenty-four hours after the last dose of treatment, rats were
anesthetized with ketamine HCL (120 mg/kg): xylazine HCl (24 mg/kg),
and blood was collected by cardiac puncture. The animals were then
asphyxiated by CO2 inhalation. Uteri were removed rapidly,
and wet weights were determined on a Mettler balance to evaluate
ovariectomy or efficacy of treatments. Uteri for histomorphometry were
then fixed in 10% formalin and processed for paraffin embedding.
Tibiae for histomorphometry were removed and fixed in 50%
ethanol/saline. Femora for mechanical testing were removed and frozen.
Blood samples for cholesterol analysis were allowed to clot at 4 C for
2 h before centrifugation at 2,000 x g for 10
min. Serum were collected and stored at -70 C before analysis. Serum
cholesterol was determined using a high-performance cholesterol assay
(Boehringer Mannheim Biochemicals, Indianapolis, IN), as recommended by
the manufacturer. Absorbance at 450 nm was monitored using a
thermoregulating microplate absorbance spectrophotometer (Thermomax
Molecular Devices, Menlo Park, CA).
A 960A peripheral quantitative computed tomography (pQCT) (Norland/Stratec, Ft. Atkinson, WI) was used to analyze a 1.2-mm cross-section of the proximal tibial metaphysis, using Dichte software version 5.1 and voxel dimensions of 0.148 x 0.148 x 1.2 mm, as described (19). Measured parameters included cross-sectional area, volume, bone mineral content (BMC), and BMD.
Bone histomorphometry
Histomorphometric measurements were performed with the
OsteoMeasure Analysis System (OsteoMetrics, Atlanta, GA), which
consisted of a Pentium 1133 computer coupled to a photomicroscope and
image analysis system. This image system consisted of a high-resolution
color video camera (Sony DXC-970 MD, Ichinomiya, Japan) that
records the image specimen through the microscope (Olympus BH-2, New
Hyde Park, NY) and displays the image on a view sonic video monitor
that registers the movement of a digitizing pen on a graphics tablet
(OsteoTablet, OsteoMetrics). The region of interest is traced, and the
line lengths and area bounded by lines are calculated
automatically.
Cortical bone measurements
Ground transverse sections were used for histomorphometric
analysis of cortical bone, as described (5). The samples were
microscopically examined under UV light to visualize fluorochrome
labels. The following measurements were performed: 1) cross-sectional
area, defined as the area of bone and marrow cavity bounded by the
periosteal surface of the specimen; 2) medullary area, defined as the
area delineated by the endocortical surface of the specimen; and 3)
cortical bone area, calculated as the difference between
cross-sectional and medullary areas.
Cancellous bone measurements
The metaphysis was dehydrated, embedded without demineralization
to retain the fluorochrome labels, and sectioned at a thickness of 5
µm, as described (11). A standard sampling site was established in
the secondary spongiosa of the metaphyseal region of the proximal
tibia, as described (11).
Cancellous bone volume was calculated as the volume of total cancellous bone per mm3 metaphyseal volume within the sampling site and expressed as a percentage. Cancellous bone surface was calculated as the surface of cancellous bone per mm3 metaphyseal area. The bone formation rate (bone volume referent) was calculated as BFR/BV = double labeled-surface (dlS) plus 1/2 single-labeled surface per mm3 cancellous bone volume x the mineral apposition rate (MAR). The results are expressed as percent BV per year. The BFR (tissue referent) was calculated as BFR/TV = the dlS plus 1/2 single-labeled surface per mm3 metaphyseal volume x the MAR. The results are expressed as percent TV per year. The BFR (perimeter referent) was calculated as BFR/BS = dlS plus 1/2 single-labeled perimeter per mm cancellous bone perimeter x MAR. The MAR was the mean distance between the two calcein labels, divided by the labeling interval of 11 days.
Tb.Th, number, and separation were calculated as described (27). Node and free-ends were measured as described (28) and were normalized to cancellous bone surface. Osteoblast and osteoclast surfaces were measured in toluidine blue stained sections, as described (11), and were expressed as percentages.
Uterine histology
After fixation in 10% buffered formalin, uteri were embedded in
paraffin and sectioned. The slides were stained with Carrazi
hematoxylin and eosin. Sections of uteri for analysis were located on a
Nikon Optiphot microscope using the 40x objection. Images were then
captured with a COHU high-performance CCD camera and quantitated using
the Scion Image (version 1.57) software package. Two separate fields
were measured for each uterine section. Quantitation of epithelial cell
height for each field was accomplished by constructing a region of
interest (approximately 2.5 cm in length) around the epithelial layer.
Once selected, a grid was placed over the region of interest, which
produced 10–15 measurements of epithelial height from the lengths of
the grid lines. The median epithelial height for each of the 2
fields/uterus was calculated, and the average of the 2 median values
was recorded as the final mean epithelial cell height for the section.
Thus, the final epithelial cell height determined for each animal
resulted from 20–30 separate measurements of cell height.
Biomechanical analyses
Bone strength was measured on intact femora using a three-point
bending test (29). Load was applied midway between two supports that
were 15 mm apart. The femora were positioned so that the loading point
was 7.5 mm proximal from the distal popliteal space, and bending
occurred about the medial-lateral axis. Specimens were tested in a
saline bath at 37 C. Each specimen was submerged in the saline bath for
3 min before testing to allow equilibration of temperature.
Load-displacement curves were recorded at a cross-head speed of 1
mm/sec using a servo-hydraulic materials testing machine (MTS Corp.,
Minneapolis, MN) and an x-y recorder (Hewlett Packard 7090A, Palo Alto,
CA).
Ultimate force (Fu) was calculated as the maximum load from
the load-displacement curves. Ultimate stress (
u) was
calculated using the following equation:
 | (1) |
The value for moment of inertia used in stress analysis was calculated
under the assumption that the femoral cross-sections were elliptically
shaped (30) using the following equation:
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The bending stiffness (S) of the bone was calculated as the maximum
slope of the force-displacement curve. The Young’s modulus (E) was
calculated as:
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 | (4) |
Femoral neck strength was measured by mounting the proximal half of the femur vertically in a chuck and applying downward force at a rate of 1 mm/sec on the femoral head until the neck failed. The ultimate load was calculated as the maximum force sustained by the femoral neck. Femoral neck tests were performed at room temperature using the MTS system.
Statistics
The pretreatment sham and OVX’d groups, as well as the
treatment groups, were compared with the posttreatment sham and OVX’d
groups. These comparisons are shown in the Figures and Tables. When
possible, the treatment groups were also compared with the pretreatment
OVX’d group. These additional comparisons were performed to establish
whether treatment reversed the effects of OVX, and they are described
in Results.
Data are presented as means and SE. Group differences were assessed by ANOVA, and pair-wise contrasts were examined using Fisher’s protected least significant difference (PLSD), where the significance level for the overall ANOVA was P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and decreased uterine weight (Fig. 2). There were no differences in body
weight or uterine weight between the pre- and posttreatment OVX’d
groups, indicating that the effects of OVX on these measurements were
fully established by the end of the pretreatment interval. In contrast,
serum cholesterol increased (P < 0.05) during the
treatment interval. Treatment of OVX’d rats with EE2 restored body
weight to a value that did not differ from the posttreatment Sham
group, while reducing serum cholesterol levels to below the
posttreatment Sham group. EE2 increased uterine wet weight, but the
value was significantly less than the posttreatment SHAM group. CLO had
effects similar to those of EE2 on body weight and cholesterol. In
contrast to EE2, CLO had only a small stimulatory effect on uterine
weight (Fig. 2). ENC resulted in a dose-dependent reduction in body
weight to Sham levels. ENC also reduced serum cholesterol, to a similar
extent, at all three doses and had little effect on uterine weight. ZUC
lowered body weight at the higher doses and led to a dose-dependent
reduction in serum cholesterol. ZUC resulted in a dose-dependent
increase in uterine weight to values similar to EE2 treatment (Fig. 2).
Additionally, the treatment groups were compared with the pretreatment
OVX’d group. The significant differences in Figs. 1 and 2 were
identical to the comparison with the posttreatment control groups.
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). CLO and ENC increased epithelial cell height to values which
were intermediate between OVX and Sham and not statistically
distinguishable from either of those groups. EE2 resulted in a
significant increase in cell height to 127% of Sham, which was further
exceeded by ZUC treatment (195% of Sham).
The effects of age, OVX, and treatment on long bones were evaluated by
pQCT and histomorphometry. pQCT analysis showed an age-related 23%
(P < 0.05) reduction in the proximal tibia BMC of
10-month-old SHAM rats, compared with baselines (Table 1). In contrast, age had no significant
effect on BMD. OVX reduced BMD and BMC by 16%, compared with Sham, and
this bone loss was established during the pretreatment interval
(P < 0.05). EE2 resulted in BMD and BMC that were
intermediate between posttreatment OVX and Sham controls. BMD and BMC
of tibiae of CLO-treated rats were greater than posttreatment OVX and
not different from posttreatment Sham controls. ENC and ZUC showed
dose-dependent effects on the proximal tibia with BMD and BMC
approaching posttreatment Sham levels (Table 1).
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Histomorphometric analyses of cortical bone and cancellous bone are
summarized in Tables 2–4 and Fig. 3. Significant age-related changes in
cortical bone measurements were not observed for the tibial diaphysis,
and minimal fluorochrome labeling was detected on the periosteal and
endocortical bone surfaces, indicating that the rats had essentially
ceased radial bone growth (Table 2).
Neither OVX nor treatment with the test compounds had significant
effects on either cross-sectional area or cortical bone area. However,
there was a strong tendency for CLO to increase cortical bone area. OVX
increased medullary area, compared with the posttreatment sham control.
With the exception of the 0.03 ENC-treated rats (which increased,
compared with the posttreatment sham group), none of the treatment
groups differed in medullary area from the pre- and posttreatment OVX
and posttreatment sham groups.
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During the initial month after surgery, OVX resulted in decreases in
BV/TV, BS/TV, trabecular number (Tb.N), and trabecular thickness
(Tb.Th), and an increase in trabecular separation (Tb.Sp) (Table 3). There were no age-related changes in
the bone measurements in the SHAM rats during the 3-month treatment
interval, but there was further bone loss in the OVX’d rats
(P < 0.05). EE2 treatment stabilized bone measurements
at values which did not differ significantly from the pretreatment
OVX’d controls. CLO increased BV/TV, BS/TV, Tb.N, and Tb.Th, and
decreased Tb.Sp, compared with the pretreatment (P <
0.05) and posttreatment OVX’d controls. Interestingly, BV/TV, Tb.Th,
and Tb.Sp of CLO-treated rats did not differ from the posttreatment
sham groups. ENC and ZUC had effects similar to EE2, to stabilize
cancellous bone volume at pretreatment OVX values. OVX resulted in a
loss of trabecular connectivity, as indicated by the decrease in nodes
and increase in free-ends. None of the treatments increased the number
of nodes; but EE2, CLO, all doses of ENC, and the highest dose of ZUC
(3 mg) decreased the number of free ends.
OVX increased osteoblast and osteoclast surfaces during the pretreatment interval; there were no further changes in these measurements during the treatment interval. EE2, CLO, the two highest doses of ENC, and all doses of ZUC reduced osteoblast and osteoclast surfaces to values that did not differ from the posttreatment sham group.
During the initial month after surgery, OVX resulted in increases in
dls/BS, BFR/BV, BFR/TV, BFR/BS, and MAR (Table 4). dlS/BS, BFR/BV, and BFR/BS were
similarly increased in OVX’d rats at the end of the treatment period,
whereas BFR/TV declined to a value that did not differ from the SHAM
controls. EE2 and CLO each decreased the dynamic bone measurements in
OVX’d rats to values similar to the SHAM animals. ENC and ZUC each
resulted in decreases in the dynamic bone measurements in OVX’d rats
to the SHAM control values; the maximum response occurred at 0.3 and 3
mg/kg·day for ENC and ZUC, respectively.
The functional consequences of ovariectomy and treatment on bone
quality were evaluated by biomechanical analyses. Fu,
midshaft strength, and t were evaluated for the femur diaphysis, as
shown in Table 5. No significant
differences between groups were observed for Fu. However,
OVX decreased midshaft femoral strength (u) and this
change was prevented by treatment with EE2. The values for
u in CLO-treated rats were intermediate between OVX and
Sham. ENC resulted in dose-dependent increases in u to
values between OVX and Sham, whereas the values for ZUC-treated rats
were not different from Sham for the three doses (0.03–3 mg/kg).
Examination of t showed that CLO increased thickness, compared with
OVX, as did the high dose of ENC.
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To examine the possibility that ENC is able to antagonize the uterine
stimulatory effects of ZUC, differing ratios of ENC to ZUC were
administered to osteopenic rats for 1 week (Fig. 4). Examination of
uterine wet weights showed that ENC-treated rats had uterine weights
that were less than ZUC, and all combinations of ENC and ZUC were
intermediate between sham and OVX (Fig. 3A). However, uterine weights
for rats treated with ENC at 1 and 10 mg/kg were significantly lower
than those for rats treated with equivalent doses of ZUC. Additionally,
uterine weights for all combinations of ENC and ZUC were significantly
lower than those for ZUC only at 1 and 10 mg/kg.
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, ENC increased
Ut EPO activity above OVX levels; but at 10 mg/kg, the stimulation was
not significant. ZUC markedly increased Ut EPO activity to
significantly above OVX and ENC; and at 10 mg/kg, the activity exceeded
SHAM levels. Combination treatments showed that Ut EPO activity for ENC
(10 mg) with ZUC (2 mg/kg) and ENC (1 mg/kg) with ZUC (1 mg/kg) were
below that of ENC (1 mg/kg) with ZUC (10 mg/kg). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results are in agreement with studies reporting the differential effects of ENC and ZUC on reproductive tissues whereby ZUC was found to be the much more potent estrogen agonist of the two isomers and ENC was the more potent estrogen antagonist (23, 24). These earlier studies did not investigate the effects of the individual isomers on serum cholesterol, bone mass, bone histomorphometry, bone turnover, and bone strength.
The well-characterized changes in cancellous bone histomorphometry induced by OVX were observed in the present study and consisted of osteopenia, loss of connectivity, reduced strength, and increases in dynamic and cell measurements related to bone turnover (26, 31, 32). As expected, estrogen replacement decreased the overall rate of bone turnover, stabilized bone volume and architecture at the pretreatment values (33, 34), and improved bone strength. As has been demonstrated in numerous studies, estrogen was not capable of fully restoring cancellous bone to the osteopenic skeleton. However, the hormone was shown to increase modestly cancellous bone volume in moderately osteopenic rats in this and a previous study (35). Our finding that CLO largely restored cancellous bone volume in OVX’d rats with established bone loss was not entirely unexpected because the increase in Tb.Th was also observed in a previous study (11). CLO increased Tb.N, but did not restore node number, suggesting that there was no improvement in connectivity. Taken together, these observations suggest that the likely mechanism of action of CLO involves the net addition of bone onto existing trabecular surfaces.
There is no evidence that the difference between CLO and EE2 was caused by an overall difference in the rate of bone remodeling, because both agents decreased osteoblast and osteoclast surfaces, as well as indices of bone formation, including double-labeled perimeter and calculated BFRs, to similar extents. The present studies were not designed to measure quantitative differences in bone resorption, although it is not clear how a change in resorption could lead to an increase in bone volume of the observed magnitude. It is possible that prior transient differences in bone turnover, which would not have been detected by the fluorochrome labeling schedule, were responsible. This possibility is unlikely because CLO inhibited bone turnover in short-term studies (11). The present results could be explained if CLO inhibited the initiation of new bone remodeling units but, in addition, resulted in a strongly positive remodeling balance. In this regard, EE2 was recently shown to have independent effects on the rates of bone remodeling and the balance between bone formation and bone resorption during the bone remodeling cycle (35). Thus, although currently unknown, the precise mechanism for the increase in bone volume in CLO-treated rats with established osteopenia deserves continued study.
All doses of ENC and ZUC were effective in preventing further cancellous bone loss in OVX’d rats. Interestingly, the dynamic bone measurements in these rats decreased with dose, from values that did not differ from untreated OVX’d rats, to much lower values that were nearly identical to SHAM controls. This observation provides further evidence that high bone turnover does not, in or of itself, result in bone loss (35). The dose of estrogen and estrogen agonists sufficient to reestablish bone balance in OVX’d rats seems to be much lower than that required to inhibit the rate of bone turnover. This observation may be relevant to humans, where establishing the minimum effective dose is of great interest, because of the concern for undesirable side effects. Serum and urine markers for bone turnover are often used as indices of efficacy in patients (36) but may underestimate the skeletal effectiveness of estrogens and estrogen agonists in postmenopausal women.
In summary, ENC is able to antagonize the uterine stimulatory effects of ZUC on wet weight and Ut EPO activity. This finding is relevant to the pharmacological effects of CLO on estrogen target tissues because ENC and ZUC are found approximately at a 1:1 molar ratio in CLO. In contrast, the beneficial effects of CLO on nonreproductive estrogen target tissues in OVX’d rats are conferred by the combination of ZUC and ENC. Finally, the combination of the two isomers seems to provide a more desirable pharmacological profile on estrogen target tissues than either isomer given alone or EE2.
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Acknowledgments |
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Footnotes |
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Received December 23, 1997.
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