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J Appl Physiol 102: 1502-1509, 2007. First published December 21, 2006; doi:10.1152/japplphysiol.00815.2006
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Doping dose of salbutamol and exercise: deleterious effect on cancellous and cortical bones in adult rats

N. Bonnet,1 C. L. Benhamou,1 H. Beaupied,1 N. Laroche,2 L. Vico,2 E. Dolleans,1 and D. Courteix1

1Caracterisation du Tissu Osseux par Imagerie, Techniques et Applications and Architecture du Tissu Osseux-Exercise Physiology, Institut National de la Santé et de la Recherche Médicale U658, School of Sports Sciences and Physical Education, Orleans Regional Hospital and University of Orleans, Orleans, and 2Laboratoire de Biologie du Tissu Osseux, Institut National de la Santé et de la Recherche Médicale E366, University of St. Etienne, France

Submitted 25 July 2006 ; accepted in final form 18 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal studies suggest that bone remodeling is under beta-adrenergic control via the sympathetic nervous system. To our knowledge, the impact of beta-agonist substances, at doping doses, has not been studied in adult rats. The purpose of this study was to examine the effects of salbutamol injections with or without treadmill exercise on trabecular and cortical bone in adult rats. Adult (36 wk of age) female Wistar rats (n = 56) were treated with salbutamol (3 mg·kg–1·day–1 sc, 5 days/wk) or vehicle (sham) with or without subsequent treadmill exercise (13 m/min, 60 min/day, 5 days/wk) for 10 wk. Tibial and femoral bone mineral density was analyzed by dual-energy X-ray absorptiometry. Metaphysic trabecular bone structure was analyzed by micro-CT at the time of the animals' death. Bone cell activities were assessed histomorphometrically. After 10 wk, the increase in bone mineral density was less in salbutamol-treated than in sham rats (+3.3% vs. +12.4%, P < 0.05), and trabecular parameters were altered and bone resorption was increased in salbutamol-treated rats compared with controls. The negative effect on bone architecture in salbutamol-treated rats persisted, even with treadmill exercise. These results confirm the deleterious effect of beta2-agonists on bone mass during chronic treatment and describe its effects on bone mechanical properties in adult rats. Bone loss occurred independently of a salbutamol-induced anabolic effect on muscle mass and was equally severe in sedentary and exercising rats, despite a beneficial effect of exercise on the extrinsic and intrinsic energy to ultimate strain. These bone effects may have important consequences in athletes who use salbutamol as a doping substance.

beta-agonist; architecture; histomorphometry; doping

THE WIDESPREAD ABUSE OF DOPING agents that increase lean body mass in female athletes can be responsible for menstrual irregularities and low bone mineral density (BMD) (16). Salbutamol, a beta2-adrenergic agonist, is the most commonly prescribed medication for bronchospasm and exercise-induced asthma, which affect ~10–20% of athletes (40). Systemic administration of salbutamol is prohibited by the International Olympic Committee because of its potential for performance enhancement. Most published studies dealing with the effects of salbutamol have failed to demonstrate its anabolic effect on human muscle. However, the therapeutic doses used in these studies (0.2 mg·kg–1·day–1) did not reflect the actual doses used by athletes, which can be 10- to 100-fold higher. Animal experiments using beta2-agonists at 10–20 times therapeutic doses showed an anabolic effect on muscle (23). Libretto (23) reported that high doses of salbutamol can induce several toxicological effects, such as an increased heart weight associated with inflammation, hypertrophy of muscle fibers, focal myocardial necrosis, and fibrosis.

An increase of muscle strength, possibly associated with a higher muscle mass, usually induces an anabolic effect on bone tissue. This effect is mediated by the so-called mechanostat system regulating bone mass (14). Therefore, according to the theory of muscle-bone interaction of Frost et al. (15), an augmentation of muscle mass by beta2-agonists would be expected to result in a gain of bone tissue.

Clenbuterol, another powerful beta2-agonist, has been shown to reduce BMD loss in denervated (42) or tail-suspended rats (1). Albuterol, together with resistance exercise, reduces bone loss induced by immobilization (7). Preliminary data published by Pataki et al. (30) demonstrated that salbutamol may increase the trabecular bone volume [i.e., bone volume-to-trabecular volume ratio (BV/TV)] in ovariectomized rats.

A previous study carried out in our laboratory demonstrated a deleterious effect of salbutamol on the vertebral trabecular architecture in young rats evaluated by micro-CT (6). Our data showed a decrease in BV/TV (–19.7% vs. placebo), and biomechanical tests revealed a lower ultimate force (–15.5% vs. placebo) (6). Kitaura et al. (21) found that clenbuterol inhibited longitudinal bone growth and decreased bone mineral content (BMC) in growing rats. Takeda et al. (36) showed that isoproterenol, a nonspecific beta-agonist, decreased bone mass in mice. They showed a decrease of BV/TV (–34.3% vs. placebo) due to a lower bone formation rate (–23.6%) and osteoblast number (–41.7%) (36). No study has clearly demonstrated whether beta2-agonists can improve bone mass and muscle mass. Arai et al. (2) and Togari (37) also reported that beta2-receptors are expressed in osteoblasts and osteoclasts. beta2-Agonists can therefore act directly on bone and via their effects on muscle, and these two mechanisms would account for the contradictory results reported in the literature.

To our knowledge, no study has investigated the effects of salbutamol at doping doses on treadmill-exercising rats, which is the actual condition of use of this substance by human athletes. Few studies have evaluated the effects of salbutamol on the skeleton, especially regarding bone metabolism and bone trabecular microarchitecture in adult rats (6, 30).

The aim of the present study was to evaluate the effect of salbutamol on mature bone with or without exercise in female rats. We hypothesized that salbutamol might alter bone status in adult rats and that mechanical loading combined with an anabolic effect on muscle mass could reduce the net bone loss induced by salbutamol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals and Treatment

Female Wistar rats (n = 56, 34 wk old; Animal Production Center, Olivet, France) were acclimatized for 2 wk before the experiment and maintained under constant temperature (21 ± 2°C) and a 12:12-h light-dark cycle during the experiment. The rats were housed in groups of three in standard cages and fed a commercial standard diet. The whole body BMD of rats was determined by dual-energy X-ray absorptiometry (DXA; model QDR-1000W, Hologic) to match all groups. At 36 wk of age, 12 SHAM rats, selected at random, were killed for evaluation of histomorphometry and microarchitecture (baseline). Twenty SHAM rats were constrained to perform exercise and were immediately treated with saline (n = 10 SHAM EXE) or salbutamol (n = 10 SHAM EXE SAB). The remaining untrained rats were randomized to receive treatment with saline (n = 12 SHAM) or salbutamol (n = 12 SHAM SAB; Fig. 1). Two salbutamol dose regimens were used: 16 µg/kg (therapeutic) and >3 mg/kg (doping). Dose and treatment protocols were based on those described by Yang and McElligott (41). Treated rats were injected with salbutamol (3 mg/kg sc, 5 days/wk for 10 wk; Sigma-Aldrich Chimie, St. Quentin Fallavier, France). The dose used in the salbutamol study was therefore equivalent to a doping dose. The control (SHAM and SHAM-EXE) groups were treated with sterile saline with a dose regimen identical to that used for the salbutamol-treated animals (5 days/wk for 10 wk). Treadmill exercise and salbutamol and saline treatment started at 36 wk of age.


Figure 1
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  		Fig. 1. Changes in body weight and muscle and fat masses during 10 wk of salbutamol treatment and treadmill exercise. Because fat and muscle mass were different at baseline, results are expressed as percent change from baseline. SAB, salbutamol; EXE, exercise; SH, sham. Values are means ± SE. aSignificantly different from SH; bsignificantly different from SHSAB; csignificantly different from SHEXE; dsignificantly different from SHEXESAB (P < 0.05).

 
For evaluation of histomorphometric parameters, bone labeling was performed by an intraperitoneal injection of tetracycline (30 mg/kg body wt) 14 and 4 days before death. At the end of the study, all groups were killed by a pentobarbital sodium overdose. Immediately after death, the hindlimb muscles (soleus, gastrocnemius, and extensor digitorum longus), uterus, and heart were weighed. Femurs and tibias of all rats were excised and cleared of fat and connective tissues. The right tibias were immediately fixed in 10% formaldehyde for 48 h at 4°C. The other bones were placed in plastic tubes and frozen at –20°C for subsequent microarchitectural and biomechanical tests. The procedure for the care and killing of animals was in accordance with the European Community standards on the care and use of laboratory animals (Ministere de l'Agriculture, France, Authorization Institut National de la Santé et de la Recherche Médicale 45-001 IACUC).

Exercise Protocol

SHAM EXE and SHAM EXE SAB rats were trained 5 days/wk for 10 wk. During the 1st wk, the rats were familiarized with treadmill locomotion by gradual increases in the speed and duration of each running session from 8 m/min for 15 min to 13 m/min for 60 min. The rats were then constrained to run at 13 m/min for 60 min/day for 9 wk. This protocol corresponded to moderate exercise for the age range of the rats (19). Untrained (SHAM and SHAM SAB) rats were handled twice daily at 1-h intervals to mimic the stress induced by handling before and after running.

Body Mass, Fat Mass, and Lean Mass

Body mass was recorded at weekly intervals throughout the study. Lean and fat masses were measured at –1, 3, 6, and 9 wk by DXA using specific rat body composition software (1.5-mm line spacing, 0.7-mm resolution). Inasmuch as muscle mass represents 94–96% of lean mass, extrapolation of muscle mass from lean mass is generally accepted. The coefficients of variation (CV = standard deviation ÷ mean) were determined for these parameters from seven repeated measurements with repositioning on an animal cadaver. The CVs were 4.76% and 1.64% for fat and muscle masses, respectively.

BMC and BMD Measurements

In vivo BMC and BMD of the left tibia and femur were measured at baseline and at 3, 6, and 9 wk by DXA using a Hologic QDR-1000 apparatus adapted to small animals. An ultra-high-resolution mode (0.254-mm line spacing, 0.127-mm resolution) was used with a 0.9-mm-diameter collimator.

Morphological and Topological Characteristics of Trabecular Bone

Microarchitecture of the femoral and tibial trabecular bone was investigated using a micro-CT. The characteristics and methods have been described elsewhere (5, 25). The X-ray source was set at 75 kV and 100 µA, with a pixel size of 11 µm. Four hundred projections were acquired over an angular range of 180° (angular step of 0.45°). The image slices were reconstructed using cone-beam reconstruction software (version 2.6) based on the Feldkamp algorithm. The registered data sets were segmented into binary images. Because of low noise and relatively good resolution of the data sets, we used simple global thresholding methods (26, 27). On the femur, 250 slices were selected from the distal growth plate to the shaft proximally. On the tibia, 250 slices were selected from the proximal growth plate to the shaft distally.

For extraction of the trabecular bone, ellipsoid contours were drawn with CT Analyzer software (Skyscan, Aartselaar, Belgium). BV/TV and trabecular number were calculated by the mean intercept length method. Trabecular thickness and trabecular separation were calculated according to the method of Hildebrand and Ruegsegger (17). The trabecular bone pattern factor (TBPf) was measured for the prevalence of platelike or rodlike trabecular structures: the higher the TBPf, the more trabecular bone is organized in the form of rodlike structures.

Morphological Characteristics of Cortical Bone

Cortical bone of the femoral and tibial middiaphyses was analyzed by micro-CT. The characteristics and methods have been described elsewhere (24). The acquisition characteristics are the same as those described for trabecular bone. After reconstruction, polygon contours were drawn with CT Analyzer software for extraction of the cortical bone. Before inverting the image, we applied simple global threshold methods, and the algorithms developed for trabecular bone analysis were used to characterize the porosity network. The pore number was measured by the mean intercept length method. The pore diameter and pore spacing were derived from the method of Hildebrand and Ruegsegger (17), and pore surface on volume was derived from the triangulation method.

For analysis of the femoral cortex, 100 slices were selected, starting 12 mm from the distal growth plate on the shaft proximally, corresponding to the distal diaphysis region.

For analysis of the tibial cortex, 100 slices were selected, starting 12 mm from the proximal growth plate on the shaft distally, corresponding to the proximal diaphysis region.

Diameters and cortical width of the middiaphysis (equal to 50% of the femur or tibia length) were measured by micro-CT software. Tissue area, cortical bone area, and bone marrow area were measured at the middiaphysis using CT Analyser software.

Bone Histomorphometry

After 48 h of fixation, the right tibia was dehydrated in absolute acetone and embedded in methylmethacrylate at low temperature according to the method developed by Chappard et al. (9). The central plane of the proximal part of the tibia was sliced frontally with a microtome (Reichert-Jung Polycut, Heidelberg, Germany). Five 8-µm-thick sections were stained with Goldner's trichrome and used for measurement of the following parameters in secondary spongiosa according to the American Society for Bone and Mineral Research histomorphometry nomenclature (29) using an automatic image analyzer (BIOCOM): BV/TV; trabecular thickness, number, and separation; and osteoid surface and thickness. Five 8-µm-thick sections were stained for tartrate-resistant acid phosphatase activity to measure active osteoclastic surfaces and osteoclast number. Histodynamic parameters were determined on five unstained, 12-µm-thick sections under UV light: mineral apposition rate (MAR), single-labeled surface (sLS/BS), and double-labeled surface (dLS/BS). Mineralizing surface per bone surface (MS/BS) was calculated as follows: dLS/BS + 1/2(sLS/BS). Bone formation rate was calculated as follows: MS/BS x MAR.

All bone remodeling parameters were measured using a semiautomatic analyzer consisting of a digitizing table (Summasketch-Summagraphics) connected to a personal computer and a microscope equipped with a drawing system (Camera Lucida, Reichert-Jung Polyvar).

Bone Mechanical Tests

Mechanical properties of the left femur were assessed by three-point bending tests. At 4 h before mechanical testing, the bones were thawed at room temperature. Each bone was secured on the two lower supports of the anvil of the testing machine (Instron 4501, Instron, Canton, MA). The diameter of the supports is 4 mm, and the distance between the two supports is 20 mm. The cross-head speed for all tests was 1 mm/min. Load-displacement curves were recorded using specialized software (Instron 4501). Biomechanical properties were calculated from the following curves: ultimate force (i.e., the maximum force supported by the bone before fracture, characterized by a drop of the load-displacement curve), total energy (i.e., work energy required to fracture the bone), and stiffness (i.e., extrinsic rigidity of the femur). Because of the irregular shape of the femoral diaphysis, the femoral diameter used in the calculation was the mean of mediolateral and anteroposterior femoral middiaphysis diameters. Stress and Young's modulus (i.e., modulus of elasticity) were determined by the stress-strain curve. To ensure comparable testing sites at middiaphysis, the femur was always mounted so that the cross-head could be applied in the middle of the bone, as previously described by Turner and Burr (38).

The distal metaphysis of the right femur was tested in compression by the same material testing system used for the bending test. For extraction of specimens at the same relative position of each femur, the distal metaphysis was cut to a length of 2.5 mm using a standardized procedure: the location of each specimen was standardized from image analysis of the micro-CT for extraction of the same region of interest used to evaluate bone microarchitecture. The location of the point at which the primary spongiosa below the epiphyseal growth plate transitioned to fully cancellous bone was determined from the image. From the distance measured between this point and the distal end of the femur, we calculated the ratio of this distance to the total length of the femur for each bone. These data were then averaged for all bones. The overall average ratio was multiplied by the length of each femur to define the point of the first cut for each bone. The more proximal cut was then made to produce ~2.5-mm-long specimens. Both cuts were made perpendicular to the long axis of the bone using a low-speed diamond-blade wafering saw under continuous irrigation (Buehler Isomet 4000). One limitation of this method is the difficulty to discriminate between primary and secondary spongiosa, for which there is not a clear boundary.

The specimens were loaded between flat parallel plates by compression. A steel disk (5 cm) was used to apply the load in the craniocaudal direction at a deformation rate of 0.5 mm/min (18, 28). Load-displacement curves were recorded during testing. We directly measured extrinsic parameters from the force-displacement curve: ultimate force, displacement at ultimate force, energy to failure, and stiffness. Extrinsic properties reflected the combined effects of bone size and shape and tissue material properties. Intrinsic properties referred to the tissue-level material behavior and were derived by adjustment of the extrinsic properties to the size and shape of the specimen with use of appropriate engineering analysis and assumptions. In the present study of the metaphysis, intrinsic properties represented combined contributions from cancellous and cortical bone. Therefore, the area used to calculate intrinsic parameters was the total cross-sectional area determined from micro-CT images (which corresponds to trabecular area + cortical area). The following intrinsic properties were calculated with the assumption of purely uniaxial loading: ultimate stress, ultimate strain, intrinsic energy to ultimate strain, and Young's modulus.

Biochemical Analyses

Osteocalcin (a marker of bone formation) and COOH-terminal collagen cross-links (CTx, a marker of bone resorption) were assayed in duplicate by enzyme-linked immunosorbent assay (Nordic Bioscience Diagnostics, Herlev Hovedgade, Denmark). The within- and between-assay CVs were <10% in our laboratory.

Statistical Analysis

Values are means ± SE. Body composition, BMD, geometric data, architectural parameters, biochemical analyses, and femoral mechanical properties were analyzed by a two-way (exercise and treatment) ANOVA at baseline and a two-way ANOVA with repeated measurements (baseline and end of treatment). When necessary, post hoc differences were determined with the Newman-Keuls test.

Because no significant difference in trabecular microarchitecture, histomorphometric, and bone marker parameters was observed between groups at baseline, the data are not mentioned in RESULTS. However, baseline body composition and BMD data are given where significant differences were observed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Body Mass and Muscle Mass

For normalization of data in relation to baseline body mass, fat mass and muscle mass are expressed in Fig. 1 as percent changes from baseline. At the end of the protocol, no difference in body mass gains was observed between SHAM (+10.6%), SHAM SAB (+8.9%), SHAM EXE (+7.2%), and SHAM EXE SAB (+10.9%) rats.

Gain in muscle mass was greater in SHAM SAB (+15.8%, P < 0.05) and SHAM EXE SAB (+15.2%) than in SHAM (+10.8%, P < 0.05) and SHAM EXE (+10.6%, P < 0.05) rats.

Fat mass gain was lower in SHAM EXE (–52.9%) and SHAM EXE SAB (–46.1%) than in SHAM (+1.6%, P < 0.01) and SHAM SAB (–10.9%, P < 0.01) rats (Fig. 1).

Gastrocnemius and soleus muscle mass was greater in salbutamol-treated than in SHAM groups, but no significant differences were observed between SHAM and SHAM EXE groups (Table 1).


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  		Table 1. Uterine and skeletal muscle mass in salbutamol-treated and untreated exercising and nonexercising

 
Bone Mineral Density

Longitudinal analyses of tibia BMD indicated less BMD gain in SHAM SAB (+3.0%) than in SHAM EXE (+5.1%, P < 0.01) and SHAM EXE SAB (+7.2%, P < 0.01) rats (Fig. 2).


Figure 2
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  		Fig. 2. Time course of bone mineral density (BMD) changes in long bone. Values are means ± SE. aSignificantly different from SH; bsignificantly different from SHSAB; csignificantly different from SHEXE; dsignificantly different from SHEXESAB (P < 0.05).

 
Analysis of the femur showed less BMD gain in SHAM SAB (+3.3%) and SHAM EXE SAB (+5.6%) than in SHAM (+12.4%, P < 0.05) and SHAM EXE (+11.7%, P < 0.05) rats.

Trabecular Bone Microarchitecture

Distal femur.   Salbutamol induced significant alterations of femoral trabecular microarchitecture in SHAM animals. Trabecular thickness (–6.7%, P < 0.001) and BV/TV (–13.6%, P < 0.05) were lower in SHAM SAB rats than in controls (Table 2). No significant difference in trabecular parameters was observed between SHAM and SHAM EXE rats. Trabecular thickness was lower in SHAM EXE SAB (–9.7%, P < 0.01) than in SHAM EXE rats.


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  		Table 2. Long bone trabecular microarchitecture in salbutamol-treated and untreated exercising and nonexercising

 
Proximal tibia.   BV/TV was lower (–14.3%, P < 0.001), trabecular thickness was lower (–6.1%, P < 0.001), and TBPf was higher (+96.6%, P < 0.001) in salbutamol-treated than in control animals (Table 2), indicating a severe loss of architectural integrity of trabecular bone. As in the tibia, no difference was observed between SHAM and SHAM EXE groups. No significant differences in BV/TV or trabecular thickness were observed between SHAM EXE SAB and SHAM EXE groups, but TBPf was higher in the SHAM EXE SAB (+94.9%, P < 0.01) than in the SHAM EXE group.

The results obtained by TBPf were confirmed by the structural model index (r = 0.93, P < 0.0001).

Cortical Investigation

Analysis of cortical architectural parameters showed lower cortical width of the tibial middiaphysis in SHAM EXE SAB (522 ± 24.3 µm, P < 0.05) than in SHAM (575 ± 15.8 µm) and SHAM EXE (564.2 ± 17.9 µm) rats. Cortical width of the SHAM SAB group (541 ± 11.2 µm) was not significantly different from the other groups. The same trends were observed in the femur.

Cortical porosity was significantly higher in the SHAM SAB group than in controls in the tibia (+52%, P < 0.001) and the femur (–32.9%, P < 0.05; Table 2).

Cellular Activity

No effect of salbutamol alone on bone formation parameters was observed (Fig. 3). Higher bone formation parameters were observed in SHAM EXE than in SHAM rats, whereas no differences were seen between SHAM and SHAM EXE SAB rats. Salbutamol appeared to eliminate the beneficial effect of exercise on bone formation.


Figure 3
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  		Fig. 3. Histomorphometric parameters of proximal tibia after 10 wk of salbutamol treatment or no treatment with and without treadmill exercise. BFR, bone formation rate; BS, bone surface; Oc.S, osteoclastic surface; MAR, mineral apposition rate; MS, mineralizing surface. Values are means ± SE. aSignificantly different from SHAM; bsignificantly different from SHAM SAB; csignificantly different from SHAM EXE; dsignificantly different from SHAM EXE SAB (P < 0.05).

 
Osteoclast surface on bone surface was higher in the SHAM SAB group than in the SHAM (+30.4%, P < 0.01), SHAM EXE, and SHAM EXE SAB groups.

Biomechanical Parameters

Bending test revealed a significantly lower ultimate force, cross-sectional area, and moment of inertia in the SHAM SAB than in the SHAM group (Table 3) but no difference in ultimate stress and energy to failure. Furthermore, no difference in intrinsic parameters expressed by intrinsic energy and Young's modulus was detected between SHAM SAB and SHAM groups. Stress was significantly higher in the SHAM EXE than in the SHAM group. Energy to ultimate force and Young's modulus were higher in SHAM EXE (+21.2% and +34.3%, respectively, P < 0.05) than in SHAM rats. Moment of inertia was lower in SHAM EXE SAB than in SHAM EXE animals, but no significant difference was observed between groups for the other parameters (Table 3).


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  		Table 3. Influence of exercise on biomechanical properties of the femur in bending test.

 
The extrinsic and intrinsic biomechanical properties of the metaphysis assessed by the compression test were significantly lower in the SHAM SAB than in the SHAM group (Table 4). Salbutamol appeared to have a negative impact on ultimate force and energy to failure (–21% and –34%, respectively). In addition, the toughness of the femur was altered, with stress, intrinsic energy, and Young's modulus decreased by –16.5%, –23%, and –25.5%, respectively. No significant differences in extrinsic and intrinsic parameters were observed between SHAM and SHAM EXE groups.


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  		Table 4. Influence of salbutamol on biomechanical properties of the distal femur metaphysis in compression test

 
Analysis of extrinsic properties showed a lower ultimate force and stiffness in the SHAM EXE SAB than in the SHAM EXE group (–25.0% and –45.9%, respectively). No significant difference in the other extrinsic parameters was observed between SHAM EXE and SHAM EXE SAB groups. Among the intrinsic properties, Young's modulus was lower in the SHAM EXE SAB than in the SHAM EXE group (–24.2%, P < 0.05).

Bone Turnover

At the end of the experiment, the osteocalcin level was not significantly different between SHAM (136.25 ± 22.4 ng/ml), SHAM SAB (106.51 ± 19.2 ng/ml), SHAM EXE (153.6 ± 17.5 ng/ml), and SHAM EXE SAB (132.54 ± 18.7 ng/ml) groups.

CTx was lower in the SHAM EXE group (12.66 ± 2.3 ng/ml) than in the SHAM (19.76 ± 2.2 ng/ml, P < 0.05), SHAM SAB (25.22 ± 2.6 ng/ml), and SHAM EXE SAB (17.35 ± 3.7 ng/ml) groups and significantly higher in the SHAM SAB group than in the SHAM and SHAM EXE SAB groups.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The present study demonstrates that salbutamol is associated with an alteration of bone tissue (trabecular bone microarchitecture and biomechanical properties of cortical and trabecular bone) due to an increase in bone resorption. For the first time, this study displays deleterious effects of a doping substance on bone in adult sedentary and trained rats, despite an increase in muscle mass due to the substance and the training activity. These findings suggest consequences of the metabolism of an adrenergic substance in bone that are different from those in muscle.

We observed an anabolic effect of salbutamol on the muscle mass of the soleus and gastrocnemius. However, no anabolic effect was shown on total body muscle mass. BMD gain of the femur and tibia was significantly altered in the sedentary salbutamol-treated group. Furthermore, the ex vivo analysis of BMD indicated much more alteration of the cortical bone represented by the midshaft analysis (data not reported).

Salbutamol induced a deleterious effect on tibial trabecular microarchitecture parameters that is supported by lower trabecular bone proportion (–14.3%), trabecular number (–14.7%), and trabecular thickness (–6.1%) and higher proportion of rod-shaped trabeculae (+96.6%). Less effect was observed in the femur, with a lower trabecular bone proportion (–13.6%) and trabecular thickness (–6.7%). In these animals, deleterious effects of salbutamol were observed in the tibia and femur, despite an increase in muscle mass, suggesting that the deleterious effects of salbutamol outweigh the beneficial effects of muscle mass on bone. The increased muscle mass would not be effective to increase bone mass without enhanced physical activity in sham-operated rats (35, 39). Consistent with this argument, we failed to demonstrate a stimulant effect of salbutamol on the animal's overall activity in the open-field test (data not reported).

Cortical width (tibia and femur) was decreased by salbutamol. As previously reported by de Souza et al. (12), our data also suggest that sensitivity to the beta2-agonist (i.e., salbutamol) differs between the cortical and trabecular compartments, with greater sensitivity of the cortical bone. The cortical porosity of the middiaphysis of long bones demonstrated a higher pore number in the SHAM SAB than in the SHAM group, suggesting a deleterious effect of salbutamol not only on cortical width but also on cortical porosity.

The bending test indicated a lower femoral ultimate force and moment of inertia in sedentary salbutamol-treated rats than in sedentary placebo-treated rats, but we did not observe any difference in Young's modulus, indicating that the lower mechanical properties were mainly due to extrinsic bone properties (bone size and shape). These results are consistent with the study by Currey (11), who demonstrated that cortical porosity is not the main parameter accounting for the intrinsic mechanical properties in bending. However, the compression test indicated that salbutamol alters the intrinsic properties of the bone metaphysis: salbutamol-treated groups displayed lower stress and intrinsic energy than sham groups. The absence of a salbutamol effect on intrinsic parameters in the bending test indicates that salbutamol has a greater effect on cortical sahpe than on bone material properties. However, the effect of salbutamol on the intrinsic parameters in the compression test suggests that salbutamol preferentially alters the tissue material properties of trabecular bones, rather than the shape.

Kellenberger et al. (20) and Togari (37) observed stimulation of osteoclastogenesis after treatment in vitro of MC3T3-E1 osteoblast cells with epinephrine or isoproterenol; we observed a higher CTx level in the salbutamol-treated group. The histomorphometric data confirm an increase of bone resorption as shown by Kondo and Togari (22) and Takeda et al. (36) with isoproterenol. Compared with our first study in young rats (6), we demonstrated that a longer treatment induced a deleterious effect on cortical bone and that the deleterious effect of salbutamol on the trabecular bone cannot only be attributed to an inhibition of the bone growth, as also suggested by Kitaura et al. (21).

One of our objectives was to assess whether training, combined with treatment, would attenuate the deleterious effect of the beta-agonist (8).

In the present study, treadmill exercise did not positively influence BMD of long bones (tibia and femur) or their trabecular microarchitecture. However, we observed higher biomechanical properties in the SHAM EXE group (+21% of energy to ultimate strain and +25% of Young modulus vs. SHAM in the bending test). The lack of exercise effect on bone quantity in our SHAM group is consistent with the findings of Barengolts et al. (3, 4), who demonstrated a greater influence of exercise on BMD and microarchitecture in ovariectomized than in sham rats. Peng et al. (31) suggested that the higher bone turnover in ovariectomized rats might have increased the sensitivity of bone cells to treadmill exercise; Robling et al. (33) suggested more pronounced effects of exercise on bone quality than on bone quantity; Rutherford (34) reported that the bone tissue response to exercise depends on the initial bone status and that exercise is more effective when bone is more fragile.

To provide a better understanding of the influence of salbutamol and physical training on bone tissue, we studied the efficiency of the combination of these two interventions. We observed that salbutamol had a less deleterious effect on the femoral and tibial BMD in the training rats than in the sedentary group. Furthermore, we observed a lower effect of salbutamol on the trabecular bone microarchitecture in the training group, with no difference in trabecular bone proportion, trabecular number, and trabecular thickness between the training and SHAM groups. However, we still observed a lower cortical width of the tibia in the SHAM EXE SAB group, suggesting a severe deleterious effect of salbutamol on the cortical bone. These results are consistent with those of Cavalie et al. (8), who demonstrated a deleterious effect of clenbuterol, despite exercise, on the cortical bone. However, our study demonstrated a slight beneficial effect of exercise on the trabecular bone of salbutamol-treated rats.

Interestingly, bending tests revealed a lower moment of inertia in the SHAM EXE SAB than in the SHAM EXE group but a higher moment of inertia in the SHAM EXE SAB than in the SHAM SAB group. We observed a higher energy to ultimate strain with the combination of salbutamol and exercise than with salbutamol alone. Exercise had a beneficial effect on the bone mechanical properties; however, it was not sufficient to prevent totally the effect of salbutamol. Particularly, it is suggested that exercise did not change the effect of salbutamol on the bone shape (or had a slight effect), whereas it had a beneficial effect on the intrinsic properties of the cortical bone. This bone mechanical investigation completes the information given by Cavalie et al. (8), who described only a lower ultimate load in the group treated with clenbuterol vs. placebo.

In compression tests, we observed the same half effect of EXE observed in bending tests: SHAM EXE SAB groups displayed a higher extrinsic and intrinsic energy to ultimate strain than the SHAM SAB group, but ultimate force, stiffness, and Young's modulus were lower than with exercise alone. This suggests that, despite training in sham rats, some of the bone mechanical properties of salbutamol-treated sedentary and exercising animals did not differ.

Histomorphometric data showed that the bone formation rate difference between SHAM and SHAM EXE rats disappeared when we treated the SHAM EXE group with salbutamol but demonstrated a lower osteoclast surface on bone surface with exercise than with salbutamol treatment alone.

Serum bone markers indicate a tendency to increase osteocalcin level in salbutamol-treated exercising rats. On the contrary, salbutamol increased the CTx level in exercising rats. Our results concerning the effects of salbutamol on bone marker levels are consistent with those reported by Cavalie et al. (8), who showed an increase in urinary deoxypyridinoline excretion due to increased bone resorption in a sedentary group (+8.72% in sedentary clenbuterol group vs. placebo) and an exercising group (+8.97% in exercising clenbuterol group vs. exercising placebo group) (8).

As reported by Prather et al. (32), the doses delivered in animal experiments are larger than those prescribed for patients for clinical treatment of asthma or induction of womb dilatation during delivery. However, the dose we administered to our animals is close to that taken by young athletes on a milligram-per-kilogram basis (10, 16). One limitation of our study is that the doping dose for humans and rats might be different on a milligram-per-kilogram basis. It would be of interest to compare the serum level of salbutamol between rats and humans, inasmuch as salbutamol metabolism in rats cannot be considered exactly the same as in humans.

Recently, De Vries et al. (13) demonstrated a higher risk of hip/femur fracture in patients using higher doses of beta2-agonists (odds ratio = 1.94, 95% confidence interval = 1.41–2.66). Unfortunately, the effects of these substances at doping doses have not been tested in sedentary humans or in athletes. It would be of interest to know whether the effect of beta2-adrenergic agonists on bone are attenuated or potentiated by an estrogen deficiency observed in overtrained female athletes.

In the present study, a harmful effect of salbutamol on the skeleton of adult rats was observed in trabecular and cortical compartments. In addition, despite muscle hypertrophy induced by salbutamol, this substance alters the mechanical properties of long bones. Finally, this study demonstrated that moderate exercise is not sufficient to counterbalance the deleterious effect of salbutamol.

Great attention should be paid to the side effects of salbutamol used at doping doses on the skeleton. The influence of salbutamol on bone tissue must also be considered in asthma patients using this drug, although via a different route of administration and at lower doses.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by the World Anti-Doping Agency.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We are very grateful to Prof. Eddie Van Praagh for assistance with English grammar.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Bonnet, Inserm U658, CHR Orleans, 1, rue porte Madeleine, 45000 Orleans, France (e-mail: nicolas.bonnet15{at}wanadoo.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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N. Bonnet, N. Laroche, H. Beaupied, L. Vico, E. Dolleans, C. L. Benhamou, and D. Courteix
Doping dose of salbutamol and exercise training: impact on the skeleton of ovariectomized rats
J Appl Physiol, August 1, 2007; 103(2): 524 - 533.
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