Clenbuterol and the horse revisited

The Veterinary Journal 182 (2009) 384–391

Contents lists available at ScienceDirect

The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl

Review

Clenbuterol and the horse revisited Charles F. Kearns, Kenneth H. McKeever * Equine Science Center, Department of Animal Science, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA

a r t i c l e

i n f o

Article history: Accepted 26 August 2008

Keywords: Clenbuterol b2-agonist Horse Equine

a b s t r a c t Clenbuterol is a b2-agonist and potent selective bronchodilator that is used to treat bronchospasm in the horse. The drug is normally administered to horses orally as a syrup formulation. Once absorbed into the systemic circulation, clenbuterol has the potential to cause many side effects, including a repartitioning effect and major alterations in cardiac and skeletal muscle function. Recent studies have also reported that clenbuterol can affect bone and the immune, endocrine and reproductive systems. A great deal of information has been published on the beneficial effects of short term therapeutic doses of clenbuterol on the equine respiratory system, although there is limited information about chronic administration, particularly since this has been associated with adverse physiological effects on other systems. This review summarizes the relevant understanding of clenbuterol for clinicians and horse owners who may administer this drug to pleasure and performance horses. Ó 2008 Elsevier Ltd. All rights reserved.

Introduction Clenbuterol, a b2-adrenoceptor, is a potent selective bronchodilator that is used to treat bronchospasm in the horse (Sasse and Hajer, 1978). Unfortunately, the drug also has a history of use as an anabolic agent, both in food animals and anecdotally in humans and horses. Administration of clenbuterol at 10–20 times its therapeutic respiratory dose rate has been shown to improve carcass composition (Ricks et al., 1984). Clenbuterol is particularly well known for its ability to elicit a muscle-directed protein anabolic response in young lambs (Baker et al., 1984; Claeys et al., 1989), broiler chickens (Dalrymple et al., 1984), steers (Ricks et al., 1984; Kuiper et al., 1998), rats (Maltin et al., 1987, 1989; Reeds et al., 1988; MacLennan and Edwards, 1989) and horses (Kearns et al., 2001). Specifically, clenbuterol increases muscle mass while simultaneously decreasing fat mass (MacLennan and Edwards, 1989). This repartitioning of nutrients to alter body composition has had a profound effect on the production of meat animals. Lambs fed clenbuterol demonstrated significantly improved feed conversion, reduced fat deposition and increased muscle deposition (Baker et al., 1984). Similar improvements in muscle accretion and fat reduction were reported in steers fed clenbuterol (Ricks et al., 1984). Clenbuterol is not without adverse effects, however, especially when consumed by humans eating meat from treated animals. People who have consumed clenbuterol-contaminated meat have demonstrated symptoms of drug toxicity, including skeletal muscle tremors, tachycardia, cephalalgia, myalgia, nervousness, dizzi* Corresponding author. Tel.: +1 732 932 9390; fax: +1 732 932 6996. E-mail address: [email protected] (K.H. McKeever). 1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.08.021

ness and nausea (Hahnau and Julicher, 1996). As a result, Health Authorities have prohibited the use of clenbuterol to increase weight gain in food-producing animals. However, clenbuterol is still used illegally in several countries and, while the extent of this illicit use is uncertain, it probably ranges from 0% to 7% (Kuiper et al., 1998; Barmbilla et al., 2000). Clenbuterol is a valuable therapeutic tool to treat respiratory disease in horses and may also be prescribed in a preventive fashion for both pleasure and competition animals (Sasse and Hajer, 1978). Details of the use of clenbuterol are available in documents presented to the United States Federal Drug Administration (FDA) towards approval of the use of clenbuterol (Ventipulmin, Boehringer Ingelheim) to treat respiratory problems in the horse (US FDA, 1998). However, concerns have arisen regarding the possible ergogenic effects of clenbuterol’s repartitioning abilities (Kearns and McKeever, 2002). Horses receiving clenbuterol must have the drug withdrawn prior to competition, with the suggested withdrawal time varying with the dosage and averaging between 20 and 30 days. Acute and short-term dosing studies in horses have failed to show significant alterations in any indices of aerobic performance (Rose et al., 1983; Rose and Evans, 1984; Kiely, 1985; Kiely and Jenkins, 1985; Kallings et al., 1991; Slocombe et al., 1992). However, longitudinal studies have shown that long-term administration of a mid-level approved dose rate of the drug (2.4 lg/kg) resulted in deleterious aerobic performance (Kearns and McKeever, 2002; Beekley et al. 2003). The dose rate selected for these studies was based in part on a review of the US FDA new animal drug application (NADA 140-973) that suggested that the lowest dose rate (0.8 lg/kg) had limited efficacy for the treatment of respiratory disease in horses (US FDA, 1998). Results presented in NADA

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

140-973 suggested that higher doses may be needed for a longer period of time to elicit a clinical effect in many animals. Thus, the dosing chart from the manufacturer provided a protocol for increasing the dose to a maximum of 3.2 lg/kg. The dose originally chosen for the studies in our laboratory (Kearns et al., 2001; Sleeper et al., 2002; Kearns and McKeever, 2002; Beekley et al., 2003; Plant et al., 2003; Malinowski et al., 2004; Kearns et al., 2006b) was based on the premise that an absence of adverse effects at the higher dose could be used to assume that lower doses would be safe. However, we were unable to achieve this higher dose rate, due to severe cardiovascular, nervous, and other observed side effects, and the maximum dose rate administered was 2.4 lg/kg twice daily. The duration of our study was similar in length to protocol suggested in the NADA document (US FDA, 1998) and in the product insert from the manufacturer, which permitted incremental increases in dose rate to our maximum of 2.4 lg/kg. Since this preliminary work was published (Kearns et al., 2001; Kearns and McKeever, 2002; Beekley et al., 2003), only the lowest dose (0.8 lg/kg twice daily) is recommended for the treatment of horses with respiratory disease. Furthermore, more recent studies have shown that low doses of clenbuterol and clenbuterol combined with dexamethasone are beneficial to manage airway inflammation (Abraham et al., 2002). The purpose of this review is to summarize the published information on the direct effects of clenbuterol administration, from its nutrient repartitioning to its muscle-directed alterations. The article will also address the potential of clenbuterol as an ergogenic agent in the horse and compare data in other species. Interaction between clenbuterol and exercise training Several studies have indicated that there is an interaction between clenbuterol and exercise training. Clenbuterol decreases run time to fatigue by shifting a horse’s metabolic enzyme profile away from that of a highly aerobic middle distance runner towards that of an more anaerobic athlete and by altering markers of muscle histochemistry and immunochemistry (Kearns and McKeever, 2002; Beekley et al., 2003). Many of these changes could be either ameliorated or reversed when clenbuterol administration was combined with an exercise training program, which suggested an antagonism between clenbuterol and aerobic exercise (Plant et al., 2003). Not all studies have reported a drastic interaction between clenbuterol and exercise training. For example, rats treated with 1.6 mg/kg bodyweight (BW) of clenbuterol and exercised on a treadmill for 8 weeks were not significantly different from rats who received clenbuterol alone (Ingalls et al., 1996). Rats receiving exercise training alone had significantly longer run times and work outputs compared with controls and with both clenbuterol treatment groups. Although BW and muscle protein increased significantly in the clenbuterol treatment groups in comparison with controls and with the exercising group, there were no differences in total protein or myofibrillar protein when the data were expressed per gram of muscle weight. Neither exercise nor clenbuterol treatments alter myosin light chain (MLC) composition in any muscle studied (Ingalls et al., 1996). Acute and long-term administration of clenbuterol in horses Clenbuterol was first used as a bronchodilator to relieve pulmonary distress. It is believed that clenbuterol is effective in alleviating the signs of what was then called chronic obstructive pulmonary disease (COPD) but is now referred to as inflammatory airway disease (IAD) or recurrent airway obstruction (RAO). Several investigators have studied the effect of short-term (either

385

acute or 5.5 days) clenbuterol treatment on various cardiorespiratory functions in a variety of horse breeds. The first study to examine the effect of clenbuterol on RAO in the horse was done by Sasse and Hajer (1978). They demonstrated a clinical improvement in both bronchitis and pneumonia. Clenbuterol has also been shown to increase the mucociliary transport rate in both normal horses and horses with RAO (Kiely, 1985; Kiely and Jenkins, 1985). In another study, investigators evaluated the effect of incremental doses (0.8–3.2 lg/kg twice daily) of clenbuterol in 239 heaves-affected horses (Erichsen et al., 1994). Seventy-five percent of the horses studied responded positively. It was on the basis of this study that the FDA approved clenbuterol for the management of airway obstruction in horses. Several investigators have also looked at the effects of clenbuterol on exercise performance. Rose and Evans (1984) used a crossover design to study the effect of a single clenbuterol pretreatment (0.8 lg/kg) on maximal exercise performance in Thoroughbred geldings (n = 5). Prior to the maximal exercise test, each horse was administered either a saline or clenbuterol treatment. The treatments were reversed 1 week later. In this study, clenbuterol failed to show any significant effect in any of the major cardiovascular measures, except for a reduction in tidal volume during the final stage of work. The authors concluded that although clenbuterol did not show any major effects on cardiorespiratory function in healthy horses, future research is still warranted in horses suffering from the clinical effects of RAO. Slocombe et al. (1992) investigated the effect of intravenous (IV) clenbuterol (0.8 lg/kg) pre-treatment on respiratory mechanics during a stepwise treadmill exercise test. Five healthy Standardbreds performed an exercise test designed to evaluate four different gaits (walk, slow trot, fast trot and gallop). After baseline data were collected at each stage, the horses were rested for 10 min and made to repeat the test. Prior to the second test, each horse received 0.8 lg/kg of clenbuterol IV. As was the case with the study of Rose and Evans (1984), clenbuterol failed to show any significant effects in the tested variables, but its effects on RAO during exercise are still to be determined. Kallings et al. (1991) examined the effects of short-term (5.5 days) pre-treatment with clenbuterol during a standard exercise test. Six healthy Standardbreds were dosed with 0.8 lg/kg BW per day. Horses performed a standard exercise test, both before and after drug supplementation, acting as their own controls. Arterial pH was significantly elevated following clenbuterol treatment, but there was no difference in blood lactate levels or arterial oxygen tension. Although clenbuterol has been shown to alleviate symptoms of RAO in the horse, it has not been shown to alter exercise performance in the healthy horse after short-term administration (<5 days). The long-term effects of clenbuterol have not been studied in RAO-affected horses, but the effects of chronic administration of a middle dose (as listed on the manufacturer’s dosing chart) have recently been studied (Kearns et al., 2001; Kearns and McKeever, 2002; Sleeper et al., 2002). Aerobic capacity Long-term clenbuterol administration of a therapeutic dose (2.4 lg/kg twice daily) was reported to negatively affect aerobic performance, high-intensity exercise capacity, and the ability to recover from exercise in horses (Kearns and McKeever, 2002). When compared to non-trained and trained groups that did not receive clenbuterol, the horses that were treated with a combination of clenbuterol (2.4 lg/kg BW) and exercise, and those treated with clenbuterol only, exhibited a dramatic reduction in aerobic capacity, as measured by maximum oxygen uptake (VO2max), by 10% and

386

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

3.5%, respectively. These data are consistent with findings from previous studies in other species that used much higher doses of clenbuterol (various doses ranging from 0.4 to 0.8 mg/kg up to 1.5–2 mg/kg BW) (Torgan et al., 1993, 1995; Dodd et al., 1996; Ingalls et al., 1996; Lynch et al., 1999; Duncan et al., 2000). However, the study by Kearns and McKeever (2002) was the first to measure the interaction of clenbuterol treatment and VO2max. Previous studies in rodents measured run time to volitional fatigue as a measure of fitness (Convertino et al., 1983; Delbeke et al., 1995). In addition, clenbuterol treatment reduced plasma volume in horses, and the changes in plasma volume were correlated (P = 0.01; r = 0.984) to changes in VO2max (Kearns and McKeever, 2002). The ability to endure high-intensity exercise (100% of VO2max) was significantly reduced by clenbuterol treatment. In the study by Kearns and McKeever (2002), both clenbuterol-treated groups exhibited a 21% reduction in the time to fatigue during a highintensity exercise capacity test (ECT) where the velocity of the test was set at the velocity previously shown to elicit VO2max. In contrast, the exercise-only group improved its ECT time by 32%. Furthermore, the hemodynamic recovery kinetics for heart rate (HR), pulmonary artery pressure (PAP), and right ventricular pressure (RVP) were all reduced in the clenbuterol-treated animals. These hemodynamic variables were significantly higher at 2 min after a VO2max test, which suggested that clenbuterol also affected an animal’s ability to recover from exercise. Data collected from these same horses in a concurrent study (Sleeper et al., 2002) showed elevations in ventricular and atrial chamber dimensions and cardiac output, stroke volume, and HR measured immediately (i.e., from the stop of exercise to 30–60 s post-exercise and while HR was still above 100 beats/min) following a maximal exercise test. Taken together with the observed reduction in resting plasma volume, these data may reflect both a cardiovascular and a thermoregulatory instability. Along with the reduction in VO2max, these data indicated an increased cardiac workload at any given submaximal workload and a leftward shift in the heart rate: work curve in the clenbuterol-treated horses. Reduction of cardiac function and potential cardiac myopathy There are many ways to assess cardiovascular function, including histopathology, imaging techniques, such as ultrasonography, and via a dynamic exercise test, such as the incremental exercise and simulated race tests mentioned above. Chronic clenbuterol treatment (1.5–2 mg/kg) has been shown to induce cardiac hypertrophy and increased collagen infiltration around blood vessels as well as into the wall of the left ventricle in rodents (Lynch et al., 1999; Duncan et al., 2000). In addition, clenbuterol (50 lM) has been shown to increase passive calcium (Ca2+) leak from the sarcoplasmic reticulum (SR) of single skinned mammalian skeletal muscle fibers (Bakker et al., 1998), which could lead to Ca2+ overload within the myocardial cell if similar leakage occurred from the cardiac SR. However, while Ca2+ leakage has been seen in skeletal muscle, it has not been demonstrated in cardiac myocytes from clenbuterol-treated animals although cardiac hypertrophy, collagen infiltration, and calcium leakage are all changes that have been associated with sudden cardiac death in exercising rats (Bakker et al., 1998). In mice, clenbuterol administration (50 lmol/kg) resulted in elevated serum cardiac isoenzyme creatine kinase MB (CKMB) and a-hydroxybutyrate dehydrogenase (Cubria et al., 1998; Cubría et al., 1999). The authors speculated that these changes were perhaps due to cellular myocardial damage. Recently, it has been shown that clenbuterol (various doses: 0.003–3 mmol/kg; 1 lg to 1 mg/kg for 14 days; 10 lg/kg/day) induced cardiac and skeletal

myocyte-specific necrosis in rats (Burniston et al., 2002, 2005, 2006a,b, 2007). Furthermore, cardiac changes following clenbuterol administration (2 mg/kg for 10 days) suggested that the drug may reduce oxygen supply to the heart and increase fatigability in mice (Suzuki et al., 1997). While there are no histopathological data for the horse, there has been work on cardiac function in horses following long-term clenbuterol administration (Kearns and McKeever, 2002; Sleeper et al., 2002). Following an acute exercise test, post-exertion recovery HR and RVP were elevated in horses that received clenbuterol (Kearns and McKeever, 2002). These data were supported by echocardiographic data that showed elevations in ventricular and atrial chamber dimensions as well as cardiac output, stroke volume, and HR immediately following a maximal exercise test (Sleeper et al., 2002). More troubling, however, was the increase in aortic root diameter following maximal exercise. This is of particular concern in horses because the change may increase the risk of aortic root rupture, a potential cause of death in the exercising horse (Cornelisse et al., 2000). The data are consistent with recent studies on the pharmacokinetics and tissue distribution of clenbuterol (1.6 lg/kg twice daily for 2 weeks) that have shown large (4–12-fold) increases in the concentration of the drug in both cardiac and skeletal myocytes (Soma et al., 2004a,b). When coupled with papers reporting cardiac and skeletal muscle apotosis in rats (Burniston et al., 2002, 2005, 2006a,b, 2007), the data from the above mentioned equine studies suggested a potential danger of long term clenbuterol treatment in horses. Repartitioning and body composition In simple terms, a drug that causes repartitioning is one that causes a change in body composition by causing a decrease in fat mass and an increase in fat free or muscle mass (Kearns et al., 2002). Body composition can be measured a number of ways in humans (under water weighing, skin calipers, etc.) and through post mortem methods in rodents, which are all impractical in horses. However, in livestock and horses, one can assess changes in fat mass body composition using ultrasound techniques that are based on well established relationships between rump fat thickness and total fat mass (Kearns et al., 2002). Fat free mass, which is primarily muscle, can be calculated if one measures total body mass (Kearns et al., 2002). Clenbuterol is a potent growth promoter and repartitioning agent. The action of the drug is specific to any tissue with b2-receptors, but its effects are strongest in adipose tissue and skeletal and cardiac muscle. Clenbuterol (10–200 lg/kg/day) appeared to reduce the growth of liver and kidney tissue in rats (Reeds et al., 1986). The growth data on cardiac muscle tissue are however equivocal (Emery et al., 1984; Reeds et al., 1988), although clenbuterol (200 lg/kg–0.125 mg/kg) does induce skeletal muscle enlargement through a net accretion of protein (Emery et al., 1984; Reeds et al., 1988; MacLennan and Edwards, 1989; Benson et al., 1991). It is unclear, however, and highly debated whether this net accretion in protein is the result of increased protein synthesis (Emery et al., 1984; MacLennan and Edwards, 1989) or decreased protein degradation (Reeds et al., 1988; Benson et al., 1991). As a repartitioning agent (Kearns et al., 2001; McManus and Fitzgerald, 2003), horses treated with clenbuterol at 2.4 lg/kg twice daily (one group treated with clenbuterol and exercise and one group treated with just clenbuterol), lost 20 kg and 15 kg of fat mass, respectively, after 2 weeks (Kearns et al., 2001), which is consistent with other species (poultry 1–4 ppm; rats 0.125– 2.0 mg/kg) (Ricks et al., 1984; Maltin et al., 1987; MacLennan and Edwards, 1989). Fat mass and fat percentage continued to de-

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

crease in the clenbuterol-treated horses during the remaining 8 weeks of the study, although these further reductions did not reach significance at any other time point (Kearns et al., 2001). In contrast, a different study demonstrated a continual decrease in fat percentage in horses treated with 3.2 lg/kg clenbuterol daily (McManus and Fitzgerald, 2003). The differences in the two studies may be due to the higher dose of clenbuterol used in the latter study (3.2 lg/kg versus 2.4 lg/kg) or the lower frequency of dosing per day (once versus twice daily). The differences in study design may be associated with decreased receptor downregulation in the study by McManus and Fitzgerald (2003) than is typically seen with chronic clenbuterol administration in other species, such as frog cell culture or cattle (Sibley et al., 1987a; Sibley and Lefkowitz, 1987; Re et al., 1995), and therefore allowed for the greater effect of clenbuterol. However, the exact mechanism can only be speculation as we do not know if it is the higher dose of clenbuterol or the less frequent administration. In addition to the reduction in fat mass, clenbuterol also significantly increased fat-free mass (FFM) in horses treated with the drug, with or without exercise (Kearns et al., 2001). Unlike fat mass, where the time course for change was similar between these groups of animals, FFM increased faster in the clenbuterol-only group. The increase was significant at 2 weeks, compared to the clenbuterol-plus-exercise group where it took 4 weeks. Furthermore, FFM continued to increase in the clenbuterol-only group, but not in the clenbuterol plus exercise group, which suggested that clenbuterol and exercise are synergistic in regard to fat mass, but antagonistic in regard to FFM. In addition to its repartitioning effects, clenbuterol administration altered the concentration of the adipose-specific cytokines (adipocytokines or adipokines) leptin and adiponectin in horses (Kearns et al., 2006a). It has been previously shown that both leptin and adiponectin are proportional to fat mass in horses (Kearns et al., 2006b). These cytokines are released from fat cells and serve as signals in the array of cytokine and endocrine factors, influencing appetite and the control of energy balance (Kearns et al., 2006a,b). Clenbuterol significantly increased adiponectin and simultaneously decreased leptin, which was significantly correlated with the change in fat mass. Clenbuterol therefore not only acts as a repartitioning agent, but concomitantly alters the circulating plasma concentrations of adipokines (Kearns et al., 2006a). Muscle-specific repartitioning The effect that clenbuterol has on muscle is dependent on the type of muscle (e.g., fast versus slow twitch) and the innervation status of the muscle (innervated versus denervated) (Maltin et al., 1989). Most muscles have an array of fiber types associated with the degree of aerobic versus anaerobic work performed by the muscle. Individual fibers can be classified using a variety of methods; however, in simple terms slow twitch fibers are metabolically aerobic fibers fueled by oxidative metabolism, suited to endurance activities. Fast twitch fibers fall into two general categories, type IIa fibers and type IIb (type IIx if one is classifying using myosin heavy chain) fibers. Type IIa fibers are an intermediate fiber with a mix of aerobic and anaerobic metabolic properties that are adaptable depending on the training regimen. Type IIb fibers are highly anaerobic and suited to ballistic or sprint activities. Clenbuterol treatment has been shown to produce a shift from slow to fast in muscle fiber types (Zeman et al., 1988). Furthermore, clenbuterol has been shown to have fiber type–dependent actions, with a more profound effect on fast muscle. Maltin et al. (1987) demonstrated an increase in soleus muscle wet weight that was the result of an increase in the size of fast oxidative-glycolytic (FOG) and slow oxidative (SO) fibers. However there were no

387

changes in the size of FOG fibers in the extensor digitorum longus (EDL) muscle, despite an increase in muscle wet weight. The authors concluded that the increase in EDL wet weight was due to an increased percentage of FOG fibers and a trend toward more fibers. Taken together, these data would imply an increase in the total cross-sectional area (CSA) of these muscles, which would therefore alter their contractile function. Both muscle fiber type and contractile properties were affected by clenbuterol treatment in the study by Zeman and associates (1988). Rats fed 1.3–1.6 mg/kg/day of clenbuterol in their drinking water for 12 weeks displayed a slow-to-fast twitch migration, as assessed by myosin ATPase activity. The mean CSA of soleus type II fibers increased by 40%, but there was no effect on the mean CSA of soleus type I fibers. The increase in mean CSA did not represent an increase in the range of fiber areas, but an increase in the number of larger fibers. In the EDL muscle, both type I and type II fibers increased by between 23% and 63%. This transformation from slow to fast had a significant effect on the contractile characteristics of the hindlimb muscle. Both peak twitch and maximum tetanus tension increased in the soleus of the rats. Initially, the increase in force production was due to an increase in specific tension (force normalized to CSA). However, by the 12th day of treatment, the soleus muscles demonstrated an average 25% increase in weight and a 29% increase in muscle CSA. Twitch and tetanus tension also increased in the EDL by 43– 54% and 27–31%, respectively. Increases in tension were accompanied by increases in muscle weight and CSA. The change in fiber type composition affected the speed of contraction. Clenbuterol increased both the rates of contractile tension development and relaxation in both muscles. The data of Zeman et al. (1988) were supported by the findings of a study by Dodd and associates (1996), who looked at the effects of chronic clenbuterol (2 mg/kg/day for 14 days) on muscle myosin heavy chain (MHC) characteristics, MLC characteristics, and contractile properties. Clenbuterol treatment caused a 17% increase in muscle mass of the gastrocnemius–plantaris–soleus complex of rats. A slow-to-fast fiber alteration in the MHC composition was seen in the rat soleus (an oxidative muscle), but not in MHC composition of the gastrocnemius and plantaris muscles (glycolytic muscles). None of the muscles measured demonstrated any change in MLC composition. Clenbuterol-induced muscle enlargement produced a 14% increase in absolute tension, but there was no change in specific tension due to increases in the muscular mass of the hindlimbs (Dodd et al., 1996). Maximal shortening velocity was significantly greater (+18%) in the clenbuterol-treated rats than in the control rats, while time to fatigue was significantly decreased in the clenbuterol-treated rats ( 18%). The alterations in speed of shortening and fatigability of the clenbuterol-treated rat muscles may have been the result of alterations in metabolic enzymes. This speculation was supported by the observation that citric synthase and phosphofructokinase activities were downregulated by clenbuterol treatment in those muscles. It is clear that clenbuterol administration in mammals results in skeletal muscle MHC shifts from type I ? type IIa ? type IIx (Zeman et al., 1988; Criswell et al., 1996; Dodd et al., 1996; Beekley et al., 2003). These changes are opposite of those typically seen during aerobic training in animals (type IIx ? type IIa ? type I). In commonly used therapeutic doses in horses, clenbuterol administration results in substantial changes in skeletal muscle MHC composition that are possibly detrimental to aerobic performance (Beekley et al., 2003). Specifically, Beekley et al. (2003) reported that clenbuterol caused a significant reduction in type IIA MHC percentage, with a corresponding increase in type IIx MHC percentage. These data are in agreement with previously published work on the effect of clenbuterol on MHC content (Zeman et al.,

388

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

1988; Criswell et al., 1996; Dodd et al., 1996; Plant et al., 2003). However, there was no reduction in type I MHC. This finding may have been due to the fact that all horses were severely untrained at the outset of the study and exhibited only a modest amount (10%) of type I MHC. Clenbuterol has also been shown to alter skeletal muscle contractile properties at the single-fiber level (Lynch et al., 1996). Mice administered 2 mg/kg BW per day of clenbuterol demonstrated altered Ca2+ sensitivity in fast fibers, although there were no differences in maximal force production. Mice that received both clenbuterol and exercise, however, were not significantly different from control animals in the percentage of either type I or type II fiber. Clenbuterol treatment resulted in lower Ca2+-activated contractile characteristics in fast-twitch EDL and fast-twitch soleus fibers, but no changes in the sensitivity to strontium (Sr2+). Slowtwitch soleus fibers exhibited no differences in Ca2+ sensitivity, but a reduced sensitivity to Sr2+ when compared with control fibers. In addition, based on myosin ATPase activity, clenbuterol significantly decreased the percentage of type I fibers, while increasing the percentage of type II fibers in the EDL and soleus muscles. Furthermore, differences in Ca2+ and Sr2+ sensitivity were abolished when clenbuterol-treated mice were exercised (Lynch et al., 1996). To date, there has been only one study that investigated the effects of clenbuterol on single-fiber contractility in the horse (Plant et al., 2003). Eight weeks of clenbuterol treatment (2.4 lg/kg twice daily) had no effect on isolated muscle fiber sensitivity to Ca2+and produced only a minor increase in the steepness of the force-plasma Ca2+ relationship (Plant et al., 2003). Another interesting finding from that study was that clenbuterol treatment increased the force per cross-sectional area. These results contrast with what had been seen previously in other mammalian species (Lynch et al., 1996) and may be due to the fact that horses are more sensitive to sympathomimetic drugs (McKeever, 1993; Kearns et al., 2001). Overall, increased muscle mass and a shift of fiber profile from that of a middle distance runner to one more conducive to ballistic or sprint type activities could partially explain the decline in aerobic exercise performance reported by Kearns and McKeever (2002) in Standardbred horses. Bone growth Unlike the data for skeletal muscle, which have clearly and consistently shown an anabolic effect of clenbuterol, data for bone are less clear. Some data, from humans and rats, provide evidence that ß2-receptors are located in the osteoblasts (Togari et al., 1997; Kellenberger et al., 1998) and it has been hypothesised that clenbuterol might increase bone growth through its receptor pathway. It has been shown in rats that clenbuterol reduced the net bone loss in denervated (Zeman et al., 1991) or suspended hindlimbs (Bloomfield et al., 1997). More recently, however, Kitaura et al. (2002) reported that clenbuterol inhibited longitudinal bone growth as well as bone mineral content (BMC) and area in young male rats. The authors speculated that the clenbuterol-mediated reduction in bone growth may be due to faster epiphyseal closure or accelerated calcification of the epiphysis. Another study reported that clenbuterol treatment reduced BMC, bone mineral density (BMD), and mechanical resistance in both resting and exercised rats (Cavalie et al., 2002). There was no effect on osteocalcin, but an increase in urinary deoxypyridinoline led the authors to conclude that clenbuterol decreased BMD and BMC by increasing bone resorption (Cavalie et al., 2002). While there are no reported data in horses regarding the effect of clenbuterol on bone metabolism, the implications are that clenbuterol treatment may potentially lead to orthopedic problems, especially in young, growing horses.

Interestingly, a clinical case study reported by Smith et al. (1986) noted phalangeal and navicular bone hypoplasia and hoof malformation in the hind limbs of an Appaloosa foal born to a mare that had undergone chronic treatment with clenbuterol. They acknowledged that ‘heritability and acquired defects were the primary explanation for the defects’, but they also suggested that ‘they could not rule out the possible teratogenic effects of clenbuterol’. In horses, orthopedic problems are the number one cause of poor race performance. Studies investigating the effect of clenbuterol on bone in horses are clearly warranted.

Effects on endocrine, immune, and reproductive function Systemic administration of clenbuterol has the potential to affect many additional physiological processes. However, for brevity, the major non-exercise related actions of concern to equine clinicians and horse owners are the effects on endocrine function, the immune system and its action on reproductive function. A recent study of horses documented that the combination of chronic clenbuterol administration and exercise training resulted in a suppression of the cortisol response to acute exercise (Malinowski et al., 2004). Interestingly, the authors demonstrated an enhancement of the cortisol response to acute exercise in horses that were trained without receiving clenbuterol. The increase in cortisol in response to acute exercise is a normal response that facilitates substrate release and utilization during exercise and during the post-exercise period in horses (McKeever and Gordon, 2007). This helps maintain circulating glucose concentration during exercise preventing fatigue (Malinowski et al., 2004; McKeever and Gordon, 2007). It also aids in the synthesis of muscle glycogen in the post-exercise period. The increase in cortisol may act as a damper on the inflammatory and immune response to exercise preventing a reaction to the minor breakdown in muscle that occurs with long duration and high intensity exercise in horses (Malinowski et al., 2004; McKeever and Gordon 2007). It has also been suggested that the post-exercise increase cortisol may provide a beneficial suppression of inflammation in muscle tissue. Thus, it appears that clenbuterol may block the beneficial training-induced enhancement of the cortisol response to acute exertion that may serve as a natural way to offset delayed onset muscle soreness. Mechanistically this may be due to a down regulation of ß-adrenergic receptors at the level of the adrenal (Illera et al., 1998, 2007). Clenbuterol administration may also affect immune function, either by direct action on white blood cells or through a modulation of cytokine production. Malinowski et al. (2004) also reported that while clenbuterol alone does not alter immune function, the combined effect of chronic clenbuterol administration and exercise training resulted in altered immune function with a drop in the number of natural killer cells and CD8+ cells. Interestingly, van den Hoven et al. (2006) demonstrated that clenbuterol did not affect messenger ribonucleic acid (mRNA) expression for interleukin (IL)-4 and tumor necrosis factor (TNF)-a, but did modulate the expression of the anti-inflammatory cytokine IL-10 mRNA in the peripheral white blood cells from horses with small airway disease that were exposed to lipopolysaccharide. Cytokines, such as IL-10, may affect airway inflammation. A recent paper by Laan et al. (2006) demonstrated that clenbuterol administration reduced mRNA expression for several proinflammatory cytokines in alveolar macrophages collected from horses with RAO. Recent studies have shown that while clenbuterol alone may suppress lymphocyte ß2-adrenoreceptor density and affinity, the combination of a low dose (0.8 lg/kg) of clenbuterol with dexamethasone may offer a beneficial combined therapy (Abraham et al., 2002).

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

There is limited information available on the effects of clenbuterol on reproductive function in horses, much of which is primarily focused on the mare. One of the original uses of clenbuterol in Europe was as a tocolytic to assist in obstetrical procedures in cattle (Menard, 1984), while Bostedt (1988) suggested that clenbuterol could be used to treat a variety of reproductive problems in mares. More recently, McManus and Fitzgerald (2003) reported that chronic clenbuterol administration did not affect seasonal anestrous, despite a substantial loss of body fat and an apparent decline in circulating plasma leptin concentrations associated with the repartitioning effects. Gastal et al. (1998) demonstrated that clenbuterol administration inhibited uterine tone and contractility in pregnant mares and that this could affect shape of the conceptus, in turn affecting ultrasonic imaging. While not detrimental in pregnant mares, the change in uterine tone may have a clinically important effect in non-pregnant horses. Nikolakopoulos and Watson (1999) demonstrated that a clenbuterol-induced decline in uterine tone and motility in non-pregnant mares may contribute the inability to clear uterine fluid and thus may be connected to the pathogenesis of persistent mating-induced endometritis. However, studies of the effects of clenbuterol on reproductive function in mares may be limited by their noninvasive nature. More invasive studies using rats and pigs have demonstrated that clenbuterol (20 lg/kg for 40 days) disrupted receptors affecting the normal levels of hormones controlling female reproductive cycles in non-pregnant animals (Re et al., 1995a,b), as well as hormone concentrations in pseudopregnant animals (Zsolnai et al., 1991). Even more clinically important were the results of a study on gilts that received clenbuterol mixed with feed (1 ppm/day for 40 days) and demonstrated lesions throughout the reproductive tract that had the potential to impact negatively on reproductive efficiency (Biolatti et al., 1994). The authors stated that treated animals had macroscopic lesions characterised by microcystic ovaries and uterine atrophy. Histopathological lesions included atretic degeneration of many ovarian follicles, complete absence of functional corpora lutea, a reduction in the number of endometrial glands, and a decrease in cytoplasmic volume of endometrial and glandular epithelial cells (Biolatti et al., 1994). Such effects could clearly be a substantial problem were they to occur in a mare. However, and possibly more important for breeding farms, are data obtained from other species, such as the pig, which suggest that an anabolic dose of clenbuterol (1 ppm mixed in feed for 3 months) can impair reproductive function in males (Blanco et al., 2001, 2002a,b). In these studies, it was shown that clenbuterol administration caused a decline in reproductive function due to a quantitative modification of testicular structure (Blanco et al., 2002b). More specifically, there were morphological and quantifiable effects on the Leydig cells of pigs that would certainly have the potential to impact reproductive function if similar effects were seen in stallions. Conclusions Clenbuterol is currently the only FDA approved drug used to prevent bronchospasm in horses (Erichsen et al., 1994). It has been suggested as a potential therapy to treat muscle wasting (Guldner et al., 2000) and as a potential anti-diabetic agent in humans (Castle et al., 2001). While clenbuterol does provide repartitioning effects (Kearns et al., 2001), improved glucose homeostasis in humans with insulin resistance and diabetes (Castle et al., 2001), and increased adipocytokines associated with glucose tolerance in the horse, care must be taken before considering this drug as a therapy for other disease states, such as insulin resistance, obesity

389

or metabolic syndrome. This is because clenbuterol, although formerly used in the animal production industry, has been banned in many countries (Hahnau and Julicher, 1996; Kuiper et al., 1998) due to the numerous adverse effects associated with ingesting clenbuterol-contaminated meat (Hahnau and Julicher, 1996; Kuiper et al., 1998; Smith, 1998). Furthermore, chronic administration of clenbuterol can lead to many adverse side effects. For example, clenbuterol has been repeatedly shown to decrease aerobic performance in mice, rats, and horses (Ingalls et al., 1996; Duncan et al., 2000; Kearns et al., 2002), negatively impact on measures of cardiac performance (Kearns and McKeever, 2002; Sleeper et al., 2002), induce cardiac hypertrophy (Duncan et al., 2000), increases collagen infiltration in cardiac muscle (Duncan et al., 2000), and has been linked to sudden cardiac death in rats (Bakker et al., 1998). Clenbuterol has also been shown to induce passive Ca2+ leakage from the sarcoplasmic reticulum of single skinned mammalian skeletal muscle fibers (Bakker et al., 1998) and a Ca2+/calmodulin-dependent, protein kinase-dependent pathway has been linked to skeletal muscle hypertrophy (Nairne and Picciotto, 1994). Thus, while beneficial for the short term treatment of respiratory disease, the actions of clenbuterol are extensive and may render it unsafe for long term use in horses. Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. References Abraham, G., Brodde, O.E., Ungemach, F.R., 2002. Regulation of equine lymphocyte beta-adrenoceptors under the influence of clenbuterol and dexamethasone. Equine Veterinary Journal 34, 587–593. Baker, P.K., Dalrymple, R.H., Ingle, D.L., Ricks, C.A., 1984. Use of a ß-adrenergic agonist to alter muscle and fat deposition in lambs. Journal of Animal Science 59, 1256–1261. Bakker, A.J., Head, S.I., Wareham, A.C., Stephenson, D.G., 1998. Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers. American Journal of Physiology 274, C1718–C1726. Barmbilla, G., Cenci, T., Franconi, F., Galarini, R., Macri, A., Rondoni, F., Strozzi, M., Loizzo, A., 2000. Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy. Toxicology Letters 114, 47–53. Beekley, M.D., Ideaus, J.M., Brechue, W.F., Kearns, C.F., McKeever, K.H., 2003. Chronic clenbuterol administration alters myosin heavy chain composition in Standardbred horses. The Veterinary Journal 165, 234–239. Benson, D.W., Foley-Nelson, T., Chance, W.T., Zhang, F., James, J.J., Fischer, J.E., 1991. Decreased myofibrillar protein breakdown following treatment with clenbuterol. The Journal of Surgical Research 50, 1–5. Biolatti, B., Castagnaro, M., Bollo, E., Appino, S., Re, G., 1994. Genital lesions following long-term administration of clenbuterol in female pigs. Veterinary Pathology 31, 82–92. Blanco, A., Agüera, E., Flores, R., Artacho-Pérula, E., Monterde, J.G., 2001. Morphological and quantitative study of the Leydig cells of pigs fed with anabolic doses of clenbuterol. Research in Veterinary Science 71, 85–91. Blanco, A., Artacho-Pérula, E., Flores-Acuña, R., Agüera, E., Monterde, J.G., 2002a. Quantitative modification of the testicular structure in pigs fed with anabolic doses of clenbuterol. Veterinary Research 33, 47–53. Blanco, A., Flores-Acuña, F., Roldán-Villalobos, R., Monterde, J.G., 2002b. Testicular damage from anabolic treatments with the beta(2)-adrenergic agonist clenbuterol in pigs: a light and electron microscope study. The Veterinary Journal 163, 292–298. Bloomfield, S.A., Girten, B.E., Weisbrode, S.E., 1997. Effects of vigorous exercise training and beta-agonist administration on bone response to hindlimb suspension. Journal of Applied Physiology 83, 172–178. Bostedt, H., 1988. The use of a beta 2-mimetic agent (clenbuterol) in equine pregnancy disorders and obstetrics. Tierarztliche Praxis 16, 57–59. Burniston, J.G., Ng, Y., Clark, W.A., Colyer, J., Tan, L.B., Goldspink, D.F., 2002. Myotoxic effects of clenbuterol in the rat heart and soleus muscle. Journal of Applied Physiology 93, 1824–1832. Burniston, J.G., Ellison, G.M., Clark, W.A., Goldspink, D.F., Tan, L.B., 2005. Relative toxicity of cardiotonic agents: some induce more cardiac and skeletal myocyte apoptosis and necrosis in vivo than others. Cardiovascular Toxicology 5, 355– 364. Burniston, J.G., Tan, L.B., Goldspink, D.F., 2006a. Relative myotoxic and haemodynamic effects of the beta-agonists fenoterol and clenbuterol

390

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391

measured in conscious unrestrained rats. Experimental Physiology 91, 1041– 1049. Burniston, J.G., Clark, W.A., Tan, L.B., Goldspink, D.F., 2006b. Dose-dependent separation of the hypertrophic and myotoxic effects of the beta(2)-adrenergic receptor agonist clenbuterol in rat striated muscles. Muscle and Nerve 33, 655– 663. Burniston, J.G., McLean, L., Beynon, R.J., Goldspink, D.F., 2007. Anabolic effects of a non-myotoxic dose of the beta2-adrenergic receptor agonist clenbuterol on rat plantaris muscle. Muscle and Nerve 35, 217–223. Castle, A., Yaspelkis 3rd, B.B., Kuo, C.H., Ivy, J.L., 2001. Attenuation of insulin resistance by chronic beta2-adrenergic agonist treatment possible muscle specific contributions. Life Sciences 69, 599–611. Cavalie, H., Lac, G., Lebecque, P., Chanteranne, B., Davicco, M.J., Barlet, J.P., 2002. Influence of clenbuterol on bone metabolism in exercised or sedentary rats. Journal of Applied Physiology 93, 2034–2037. Claeys, M.C., Mulvaney, D.R., McCarthy, F.D., Gore, M.T., Marple, D.N., Sartin, J.L., 1989. Skeletal muscle protein synthesis and growth hormone secretion in young lambs treated with clenbuterol. Journal of Animal Science 67, 2245– 2254. Convertino, V.A., Keil, L.C., Greenleaf, J.E., 1983. Plasma volume, renin, and vasopressin responses to graded exercise after training. Journal of Applied Physiology 54, 508–514. Cornelisse, C.J., Schott, H.C., Olivier, N.B., Mullaney, T.P., Koller, A., Wilson, D.V., Derksen, F.J., 2000. Concentration of cardiac troponin I in a horse with a ruptured aortic regurgitation jet lesion and ventricular tachycardia. Journal of the American Veterinary Medical Association 217, 231–235. Criswell, D.S., Powers, S.K., Herb, R.A., 1996. Clenbuterol-induced fiber type transition in the soleus of adult rats. European Journal of Applied Physiology 74, 391–396. Cubria, J.C., Reguera, R., Balana-Fouce, R., Ordonez, C., Ordonez, D., 1998. Polyaminemediated heart hypertrophy induced by clenbuterol in the mouse. The Journal of Pharmacy and Pharmacology 50, 91–96. Cubría, J.C., Ordóñez, C., Reguera, R.M., Tekwani, B.L., Balaña-Fouce, R., Ordóñez, D., 1999. Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart. Life Science 64, 1739–1752. Dalrymple, R.H., Baker, P.K., Gengher, P.K., Ingle, D.L., Pensack, J.M., Ricks, C.A., 1984. A repartitioning agent to improve performance and carcass composition in broilers. Poultry Science 63, 2376–2383. Delbeke, F.T., Desmet, N., Debackere, M., 1995. The abuse of doping agents in competing bodybuilders in Flanders. International Journal of Sports Medicine 16, 66–70. Dodd, S.L., Powers, S.K., Vrabis, I.S., Criswell, D., Stetson, S., Hussain, R., 1996. Effects of clenbuterol on contractile and biochemical properties of skeletal muscle. Medicine and Science in Sports and Exercise 28, 669–676. Duncan, N.D., Williams, D.A., Lynch, G.S., 2000. Deleterious effects of chronic clenbuterol treatment on endurance and sprint exercise performance in rats. Clinical Science 98, 339–347. Emery, P.W., Rothwell, N.J., Stock, M.J., Winter, P.D., 1984. Chronic effects of b2adrenergic agonist on body composition and protein synthesis in the rat. Bioscience Reports 4, 83–91. Erichsen, D.F., Aviad, A.D., Shultz, R.H., Kennedy, T.J., 1994. Clinical efficacy and safety of clenbuterol HCl when administered to effect in horses with chronic obstructive pulmonary disease (COPD). Equine Veterinary Journal 26, 331–336. Gastal, M.O., Gastal, E.L., Torres, C.A., Ginther, O.J., 1998. Effect of oxytocin, prostaglandin F2 alpha, and clenbuterol on uterine dynamics in mares. Theriogenology 50, 521–534. Guldner, N.W., Klapproth, P., Grossherr, M., Stephan, M., Rumpel, E., Noel, R., Sievers, H.H., 2000. Clenbuterol-supported dynamic training of skeletal muscle ventricles against systemic load: a key for powerful circulatory assist? Circulation 101, 2213–2219. Hahnau, S., Julicher, B., 1996. Evaluation of commercially available ELISA test kits for the detection of clenbuterol and other b2-agonist. Food Additives and Contaminants 13, 259–274. Illera, J.C., Silvan, G., Blass, A., Martinez, M.M., Illera, M., 1998. The effect of clenbuterol on adrenal function in rats. Analyst 123, 2521–2524. Illera, J.C., Pena, L., Martinez-Mateos, M.M., Camacho, L., Blass, A., Garcia-Partida, P., Illera, M.J., Silvan, G., 2007. The effect of long term exposure to combinations of growth promoters in Long Evans rats: part 2. Adrenal morphology (histopathology and immunochemical studies). Analytica Chimica Acta 586, 252–258. Ingalls, C.P., Barnes, W.S., Smith, S.B., 1996. Interaction between clenbuterol and run training: effects on exercise performance and MLC isoform content. Journal of Applied Physiology 80, 795–801. Kallings, P., Ingvast-Larsson, C., Persson, S., Appelgren, L.-E., Forster, H.J., Rominger, K.L., 1991. Clenbuterol plasma concentration after repeated oral administration and its effects on cardio-respiratory and blood lactate responses to exercise in healthy Standardbred horses. Journal of Veterinary Pharmacology and Therapeutics 14, 243–249. Kearns, C.F., McKeever, K.H., 2002. Clenbuterol diminishes aerobic performance in horses. Medicine and Science in Sports and Exercise 34, 1976–1985. Kearns, C.F., McKeever, K.H., Malinowski, K., Struck, M.B., Abe, T., 2001. Chronic administration of therapeutic levels of clenbuterol acts as a repartitioning agent. Journal of Applied Physiology 91, 2064–2070. Kearns, C.F., McKeever, K.H., Abe, T., 2002. Overview of horse body composition and muscle architecture – implications for performance. The Veterinary Journal 64, 224–234.

Kearns, C.F., McKeever, K.H., Roegner, V., Brady, S.M., Malinowski, K., 2006a. Adiponectin and leptin are related to fat mass in horses. The Veterinary Journal 172, 460–465. Kearns, C.F., McKeever, K.H., Malinowski, K., 2006b. Changes in adiponectin, leptin, and fat mass after clenbuterol treatment in horses. Medicine and Science in Sports and Exercise 38, 262–267. Kellenberger, S., Muller, K., Richener, H., Bilbe, G., 1998. Formoterol and isoproterenol induce c-fos gene expression in osteoblast-like cells by activating beta2-adrenergic receptors. Bone 22, 471–478. Kiely, R.G., 1985. Mucociliary transport rate in the horse and its modification with various pharmacologic agents. In: Proceedings of the American College of Veterinary Internal Medicine, San Diego, Calif, USA, p. 126. Kiely, R.G., Jenkins, W.L., 1985. The effect of clenbuterol chloride on the mucociliary transport rate in horses with a clinical diagnosis of chronic obstructive pulmonary disease (COPD). In: Proceedings of the American College of Veterinary Internal Medicine, San Diego, Calif, USA, p. 146. Kitaura, T., Tsunekawa, N., Kramer, W.J., 2002. Inhibited longitudinal growth of bones in young male rats by clenbuterol. Medicine and Science in Sports and Exercise 34, 267–273. Kuiper, H.A., Noordam, M.Y., van Dooren-Flipsen, M.M.H., Schilt, R., Roos, A.H., 1998. Illegal use of b-adrenergic agonist: European community. Journal of Animal Science 76, 195–207. Laan, T.T., Bull, S., Pirie, R.S., Fink-Gremmels, J., 2006. The anti-inflammatory effects of IV administered clenbuterol in horses with recurrent airway obstruction. The Veterinary Journal 171, 429–437. Lynch, G.S., Hayes, A., Campbell, S.P., Williams, D.A., 1996. Effects of b2-agonist administration and exercise on contractile activation of skeletal muscle fibers. Journal of Applied Physiology 81, 1610–1618. Lynch, G.S., Hinkle, R.T., Faulkner, J.A., 1999. Year-long clenbuterol treatment of mice increases mass, but not specific force or normalized power, of skeletal muscles. Clinical and Experimental Pharmacology and Physiology 26, 117– 120. MacLennan, P.A., Edwards, R.H.T., 1989. Effects of clenbuterol and propranolol on muscle mass: evidence that clenbuterol stimulates muscle beta-adrenoceptors to induce hypertrophy. Biochemical Journal 264, 573–579. Malinowski, K., Kearns, C.F., Guirnalda, P.D., Roegner, V., McKeever, K.H., 2004. Effect of chronic clenbuterol administration and exercise training on immune function in horses. Journal of Animal Science 82, 3500–3507. Maltin, C.A., Delday, M.I., Hay, S.M., Smith, F.G., Lobley, G.E., Reeds, P.J., 1987. The effect of the anabolic agent, clenbuterol, on overloaded rat skeletal muscle. Bioscience Reports 7, 143–149. Maltin, C.A., Hay, S.M., Delday, M.I., Lobley, G.E., Reeds, P.J., 1989. The action of bagonist clenbuterol on protein metabolism in innervated and denervated phasic muscle. Biochemical Journal 261, 965–971. McKeever, KH., 1993. Effects of sympathomimetic and sympatholytic drugs on exercise performance. Veterinary Clinics of North America: Equine Practice 9, 635–647. McKeever, K.H., Gordon, M.E., 2007. Endocrine alterations in the equine athlete. In: Hinchcliff, K.W., Kaneps, A., Geor, G. (Eds.), Equine Exercise Physiology. Elsevier, Philadelphia, pp. 278–304. McManus, C.J., Fitzgerald, B.P., 2003. Effect of daily clenbuterol and exogenous melatonin treatment on body fat, serum leptin and the expression of seasonal anestrus in the mare. Animal Reproduction Science 76, 217–230. Menard, L., 1984. Tocolytic drugs for use in veterinary obstetrics. Canadian Veterinary Journal 25, 389–393. Nairne, A.C., Picciotto, M.R., 1994. Calcium/calmodulin-dependent protein kinases. Seminars in Cancer Biology 5, 295–303. Nikolakopoulos, E., Watson, E.D., 1999. Uterine contractility is necessary for the clearance of intrauterine fluid but not bacteria after bacterial infusion in the mare. Theriogenology 52, 413–423. Plant, D.R., Kearns, C.F., McKeever, K.H., Lynch, G.S., 2003. Therapeutic clenbuterol treatment does not alter Ca2+ sensitivity of permeabilized fast muscle fibers from exercise trained or untrained horses. Journal of Muscle Research and Cell Motility 24, 471–476. Re, G., Badino, P., Novelli, A., Girardi, C., 1995a. Down-regulation of beta-adrenergic receptors and up-regulation of estrogen and progesterone receptors induced in the reproductive system of female veal calves by dietary clenbuterol. American Journal of Veterinary Research 56, 1493–1497. Re, G., Badino, P., Novelli, A., Canese, M.G., Girardi, C., 1995b. Identification of betaadrenoceptor subtypes in bovine ovarian and myometrial cell membranes. British Veterinary Journal 151, 567–578. Reeds, P.J., Hay, S.M., Dorwood, P.M., Palmer, R., 1986. Stimulation of muscle growth by clenbuterol: lack of effect on muscle protein biosynthesis. British Journal of Nutrition 56, 249–258. Reeds, P.J., Hay, S.M., Dorward, P.M., Palmer, R.M., 1988. The effect of b-agonists and antagonists on muscle growth and body composition of young rats (Rattus sp.). Comparative Biochemistry and Physiology 89C, 337–341. Ricks, C.A., Dalrymple, R.H., Baker, P.K., Ingle, D.L., 1984. Use of a b-agonist to alter fat and muscle deposition in steers. Journal of Animal Science 59, 1247–1255. Rose, R.J., Evans, D.L., 1984. Cardiorespiratory effects of clenbuterol in fit Thoroughbred horses during a maximal exercise test. In: Gillespie, J.R., Robinson, N.E. (Eds.), Equine Exercise Physiology 2. ICEEP Publications, San Diego. Calif, USA, pp. 117–131. Rose, R.J., Allen, J.R., Brock, K.A., 1983. Effects of clenbuterol hydrochloride on certain respiratory and cardiovascular parameters in horses performing treadmill exercise. Research in Veterinary Science 35, 301–305.

C.F. Kearns, K.H. McKeever / The Veterinary Journal 182 (2009) 384–391 Sasse, H.H., Hajer, R., 1978. NAB 365, a beta 2 receptor sympathomimetic agent: clinical experience in horses with lung disease. Journal of Veterinary Pharmacology and Therapeutics 1, 241–243. Sibley, D.R., Lefkowitz, R.J., 1987. Beta-adrenergic receptor-coupled adenylate cyclase. Biochemical mechanisms of regulation. Molecular Neurobiology 1, 121–154. Sibley, D.R., Benovic, J.L., Caron, M.G., Lefkowitz, R.J., 1987. Molecular mechanisms of beta-adrenergic receptor desensitization. Advances in Experimental Medical Biology 221, 253–273. Sleeper, M., McKeever, K.H., Kearns, C.F., 2002. Chronic clenbuterol administration negatively alters cardiac function in the Standardbred mare. Medicine and Science in Sports and Exercise 34, 643–650. Slocombe, R.F., Covelli, G., Bayly, W.M., 1992. Respiratory mechanics of horses during stepwise treadmill exercise tests, and the effect of clenbuterol pretreatment on them. Australian Veterinary Journal 69, 221–225. Smith, D.J., 1998. Total radioactive residues and clenbuterol residues in edible tissues, and the stereochemical composition of clenbuterol in livers of broilers after exposure to three levels of dietary [14C]clenbuterol HCl and three preslaughter withdrawal periods. Journal of Animal Science 76, 3043–3053. Smith, D.R., Leach, D.H., Bell, R.J., 1986. Phalangeal and navicular bone hypoplasia and hoof malformation in the hind limbs of a foal. Canadian Veterinary Journal 27, 28–34. Soma, L.R., Uboh, C.E., Guan, F., Luo, Y., Teleis, D., Runbo, L., Birks, E.K., Tsang, D.S., Habecker, P., 2004a. Tissue distribution of clenbuterol in the horse. Journal of Veterinary Pharmacology and Therapeutics 27, 91–98. Soma, L.R., Uboh, C.E., Guan, F., Moate, P., Luo, Y., Teleis, D., Li, R., Birks, E.K., Rudy, J.A., Tsang, D.S., 2004b. Pharmacokinetics and disposition of clenbuterol in the horse. Journal of Veterinary Pharmacology and Therapeutics 27, 71–77.

391

Suzuki, J., Gao, M., Xie, Z., 1997. Effects of the beta 2-adrenergic agonist clenbuterol on capillary geometry in cardiac and skeletal muscles in young and middleaged rats. Acta Physiologica Scandinavica 161, 317–326. Togari, A., Arai, M., Mizutani, S., Mizutani, S., Koshihara, Y., Nagatsu, T., 1997. Expression of mRNAs for neuropeptide receptors and beta-adrenergic receptors in human osteoblasts and human osteogenic sarcoma cells. Neuroscience Letters 233, 125–128. Torgan, C.E., Etgen Jr., G.J., Brozinick Jr., J.T., Wilcox, R.E., Ivy, J.L., 1993. Interaction of aerobic exercise training and clenbuterol: effects on insulin-resistant muscle. Journal of Applied Physiology 75, 1471–1476. Torgan, C.E., Etgen Jr., G.J., Kang, H.Y., Ivy, J.L., 1995. Fiber type-specific effects of clenbuterol and exercise training on insulin-resistant muscle. Journal of Applied Physiology 79, 163–167. United States Food and Drug Administration, 1998. Freedom of Information Summary of NADA 140–973. . van den Hoven, R., Duvigneau, J.C., Hartl, R.T., Gemeiner, M., 2006. Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides. Veterinary Research Communications 30, 921–928. Zeman, R.J., Ludemann, R., Easton, T.G., Etlinger, J.D., 1988. Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a b2-receptor agonist. American Journal of Physiology 254, E726–E732. Zeman, R.J., Hirschman, A., Hirschman, M.L., Guo, G., Etlinger, J.D., 1991. Clenbuterol, a beta 2-receptor agonist, reduces net bone loss in denervated hindlimbs. American Journal of Physiology 261, E285–E289. Zsolnai, B., Varga, B., Horváth, E., Stark, E., 1991. Effects of beta 2-adrenergic stimulants on the progesterone and oestradiol-17 beta serum levels in pseudopregnant rats. Acta Physiologica Hungaria 77, 7–11.