Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.
DESPITE THE WIDESPREAD USE of anabolic androgenic steroids to enhance physical performance, the mechanisms of androgen action in skeletal muscle remain poorly understood. Androgens are required to maintain normal muscle mass and strength in men, since suppression of testosterone levels reduces these parameters, and, conversely, exogenously administered androgens have anabolic effects on muscle. Testosterone and anabolic androgenic steroids, administered at supraphysiological doses, can induce hypertrophy of type I and II muscle fibers (4, 23) and are effective in increasing skeletal muscle mass and strength in eugonadal males (24). There is also experimental evidence that treatment of rodents with testosterone and anabolic androgenic steroids may improve work capacity and fatigue resistance of skeletal muscles (2, 8, 17, 44, 46), but the molecular basis underlying these effects remains unclear. It has been suggested (2) that androgens could regulate muscle function and fatigue properties through a modification of oxidative metabolism, since an increased oxidative capacity is closely related to an augmented ability to resist fatigue. In favor of this hypothesis, androgens have been reported to modify metabolic enzyme activities (16, 26), mitochondrial size (41), and muscle cell composition (7). In other studies, however, no changes in muscle oxidative capacity were detected after treatment with testosterone and anabolic androgenic steroids (6, 17, 46).
The effect of androgens on the redox status of skeletal muscle has been scarcely investigated, despite its influence on muscle function and fatigue properties. Skeletal muscle cells continuously generate reactive oxygen species (ROS), which play a critical role in the modulation of muscle contractility: low and physiological levels of ROS are required for normal force production, but high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fatigue, likely due to oxidative damage of several molecular targets (19). Exhaustive exercise induces an augmented generation of ROS within skeletal muscle (35), which alters intracellular oxidant-antioxidant balance in favor of the former and can result in oxidative damage of exercising muscles when the production of ROS overwhelms the antioxidant defense systems. Multiple potential sites for ROS generation in skeletal muscle have been identified, including mitochondria, NAD(P)H oxidase enzymes, phospholipase A[SUB]2[/SUB]-dependent processes, and xanthine oxidase (19). Mitochondria have been considered as the main ROS generator during exhaustive exercise and, at the same time, the primary target for oxidative modification, but, surprisingly, there is little evidence that mitochondria generate ROS in vivo and are under oxidative stress during exercise (20, 25, 27, 48).
Testosterone and anabolic androgenic steroids have been shown to increase antioxidant defenses in certain tissues and cell types (1, 6, 34, 43). These results support the idea that sex hormones may be involved in the redox homeostasis and suggest a mechanism by which androgens could influence muscle function and fatigue properties. In the present work, we tested the hypothesis that anabolic androgenic steroids could protect skeletal muscle mitochondria against exercise-induced oxidative modification by modulating mitochondrial ROS generation and/or scavenging. With this aim, we have isolated mitochondria from gastrocnemius muscles of sedentary and acutely exercised rats to study the effect of a treatment for 8 wk with the anabolic androgenic steroid stanozolol on: 1) the rate of mitochondrial ROS production; 2) the levels of specific markers of protein oxidation [the specific protein carbonyls glutamic (GSA) and aminoadipic semialdehydes (AASA)], glycoxidation [carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML)], and lipoxidation [malondialdehyde-lysine (MDAL)], and on the activity of the superoxide-sensitive enzyme aconitase; and 3) the antioxidant enzymatic activities. Since the lipid environment can affect membrane function, including mitochondrial electron transport chain, and, conversely, mitochondrial membranes can vary in sensitivity to oxidative damage, depending on their unsaturated fatty acid content (30), the full fatty acid composition of mitochondrial membranes was also determined.
[h=2]MATERIALS AND METHODS[/h][h=4]Steroid treatment and single bout of exhaustive exercise.[/h]Thirty-two male Wistar rats (initial body weight, 283 ± 7 g; 8 wk old) were obtained from Charles River (Barcelona, Spain). They were housed in an animal room at 22 ± 2°C and 50 ± 10% relative humidity and had free access to laboratory chow and tap water. The animals were adapted to an inverse 12:12-h light-dark cycle (dark period, 0800–2000) before the beginning of the steroid treatment period. All of the experimental procedures employed, as well as rat care and handling, were approved by the Ethics Committee of the Complutense University and complied with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.” Initially, animals were randomly divided between a control (C) group (n = 16) and a stanozolol-treated (ST) group (n = 16). The animals selected for stanozolol (17β-hydroxy-17α-methyl-5α-androstano[3,2-c]-pyrazole) (Zambon, Barcelona, Spain) treatment received, by gastric gavage, 2 mg steroid/kg body wt as a suspension in 1 ml of water 5 days/wk for 8 wk. Untreated C animals received the same amount of vehicle by the same procedure and with the same periodicity. Our laboratory has previously shown that oral administration of these doses of stanozolol reduced serum testosterone levels, indicating that the steroid was absorbed and distributed through the tissues of the rats (6). After completion of the 8-wk treatment period, eight rats from the C group (C-Ex) and eight rats from the ST group (ST-Ex) were arbitrarily selected to perform a single session of exhaustive exercise on a rodent motor-driven treadmill (Columbus Instruments, Columbus, OH) with a 15% slope. The exercise bout started with a 5-min warm-up at 15 m/min, after which the rats ran for 10 min at 20 m/min, and finally at 25 m/min until exhaustion. Animals were judged to be exhausted when they could no longer continue at the required pace or maintain upright posture on the treadmill; at this point, they were usually unable to rapidly upright themselves when placed on their back. Total running time for C-Ex and ST-Ex rats was 56 ± 5 and 54 ± 4 min, respectively. The exercised rats were killed immediately after the single session of exercise. During the 8-wk treatment period, all of the rats performed weekly an exercise session for 5 min at 15 m/min to familiarize themselves with treadmill exercise and handling.
DESPITE THE WIDESPREAD USE of anabolic androgenic steroids to enhance physical performance, the mechanisms of androgen action in skeletal muscle remain poorly understood. Androgens are required to maintain normal muscle mass and strength in men, since suppression of testosterone levels reduces these parameters, and, conversely, exogenously administered androgens have anabolic effects on muscle. Testosterone and anabolic androgenic steroids, administered at supraphysiological doses, can induce hypertrophy of type I and II muscle fibers (4, 23) and are effective in increasing skeletal muscle mass and strength in eugonadal males (24). There is also experimental evidence that treatment of rodents with testosterone and anabolic androgenic steroids may improve work capacity and fatigue resistance of skeletal muscles (2, 8, 17, 44, 46), but the molecular basis underlying these effects remains unclear. It has been suggested (2) that androgens could regulate muscle function and fatigue properties through a modification of oxidative metabolism, since an increased oxidative capacity is closely related to an augmented ability to resist fatigue. In favor of this hypothesis, androgens have been reported to modify metabolic enzyme activities (16, 26), mitochondrial size (41), and muscle cell composition (7). In other studies, however, no changes in muscle oxidative capacity were detected after treatment with testosterone and anabolic androgenic steroids (6, 17, 46).
The effect of androgens on the redox status of skeletal muscle has been scarcely investigated, despite its influence on muscle function and fatigue properties. Skeletal muscle cells continuously generate reactive oxygen species (ROS), which play a critical role in the modulation of muscle contractility: low and physiological levels of ROS are required for normal force production, but high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fatigue, likely due to oxidative damage of several molecular targets (19). Exhaustive exercise induces an augmented generation of ROS within skeletal muscle (35), which alters intracellular oxidant-antioxidant balance in favor of the former and can result in oxidative damage of exercising muscles when the production of ROS overwhelms the antioxidant defense systems. Multiple potential sites for ROS generation in skeletal muscle have been identified, including mitochondria, NAD(P)H oxidase enzymes, phospholipase A[SUB]2[/SUB]-dependent processes, and xanthine oxidase (19). Mitochondria have been considered as the main ROS generator during exhaustive exercise and, at the same time, the primary target for oxidative modification, but, surprisingly, there is little evidence that mitochondria generate ROS in vivo and are under oxidative stress during exercise (20, 25, 27, 48).
Testosterone and anabolic androgenic steroids have been shown to increase antioxidant defenses in certain tissues and cell types (1, 6, 34, 43). These results support the idea that sex hormones may be involved in the redox homeostasis and suggest a mechanism by which androgens could influence muscle function and fatigue properties. In the present work, we tested the hypothesis that anabolic androgenic steroids could protect skeletal muscle mitochondria against exercise-induced oxidative modification by modulating mitochondrial ROS generation and/or scavenging. With this aim, we have isolated mitochondria from gastrocnemius muscles of sedentary and acutely exercised rats to study the effect of a treatment for 8 wk with the anabolic androgenic steroid stanozolol on: 1) the rate of mitochondrial ROS production; 2) the levels of specific markers of protein oxidation [the specific protein carbonyls glutamic (GSA) and aminoadipic semialdehydes (AASA)], glycoxidation [carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML)], and lipoxidation [malondialdehyde-lysine (MDAL)], and on the activity of the superoxide-sensitive enzyme aconitase; and 3) the antioxidant enzymatic activities. Since the lipid environment can affect membrane function, including mitochondrial electron transport chain, and, conversely, mitochondrial membranes can vary in sensitivity to oxidative damage, depending on their unsaturated fatty acid content (30), the full fatty acid composition of mitochondrial membranes was also determined.
[h=2]MATERIALS AND METHODS[/h][h=4]Steroid treatment and single bout of exhaustive exercise.[/h]Thirty-two male Wistar rats (initial body weight, 283 ± 7 g; 8 wk old) were obtained from Charles River (Barcelona, Spain). They were housed in an animal room at 22 ± 2°C and 50 ± 10% relative humidity and had free access to laboratory chow and tap water. The animals were adapted to an inverse 12:12-h light-dark cycle (dark period, 0800–2000) before the beginning of the steroid treatment period. All of the experimental procedures employed, as well as rat care and handling, were approved by the Ethics Committee of the Complutense University and complied with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.” Initially, animals were randomly divided between a control (C) group (n = 16) and a stanozolol-treated (ST) group (n = 16). The animals selected for stanozolol (17β-hydroxy-17α-methyl-5α-androstano[3,2-c]-pyrazole) (Zambon, Barcelona, Spain) treatment received, by gastric gavage, 2 mg steroid/kg body wt as a suspension in 1 ml of water 5 days/wk for 8 wk. Untreated C animals received the same amount of vehicle by the same procedure and with the same periodicity. Our laboratory has previously shown that oral administration of these doses of stanozolol reduced serum testosterone levels, indicating that the steroid was absorbed and distributed through the tissues of the rats (6). After completion of the 8-wk treatment period, eight rats from the C group (C-Ex) and eight rats from the ST group (ST-Ex) were arbitrarily selected to perform a single session of exhaustive exercise on a rodent motor-driven treadmill (Columbus Instruments, Columbus, OH) with a 15% slope. The exercise bout started with a 5-min warm-up at 15 m/min, after which the rats ran for 10 min at 20 m/min, and finally at 25 m/min until exhaustion. Animals were judged to be exhausted when they could no longer continue at the required pace or maintain upright posture on the treadmill; at this point, they were usually unable to rapidly upright themselves when placed on their back. Total running time for C-Ex and ST-Ex rats was 56 ± 5 and 54 ± 4 min, respectively. The exercised rats were killed immediately after the single session of exercise. During the 8-wk treatment period, all of the rats performed weekly an exercise session for 5 min at 15 m/min to familiarize themselves with treadmill exercise and handling.