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Histol Histopath (1993) 8: 449-455 Histology and Histopathology

Ultrastructural changes induced by anabolic in liver of trained rats

R. Grageral, A. saborido2, F. ~olano~,L. ~irnenezl, E. ~uiiizland A. Megias2 'Department of Biology, Faculty of Biology and 2Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutensis University, Madrid, Spain

Summary. The effects of anabolic treatment in have been shown in top-level and amateur using association with endurance training on biochemical anabolic steroids (Wilson, 1988; Hickson et al., 1989; serum parameters and liver ultrastructure have been Lamb, 1989; Craham and Kennedy, 1990). In the case of investigated in male rats. Values of serum alanine oral anabolizing , the presence of the 17a- aminotransferase, aspartate aminotransferase and alkyl group introduced to retard hepatic degradation, alkaline phosphatase were not significantly affected seems to be associated with hepatotoxic effects and, in by administration of high doses of fact, three cases of fatal liver tumours have been or methylandrostanolone. Electron microscopic reported in otherwise healthy athletes who had been examination of hepatic tissue from treated animals taking anabolic steroids for several years (Hickson et al., revealed ultrastructural alterations of hepatocytes. The 1989). Levels of and metabolites related to most prominent changes were swelling of mitochondria, hepatic function have been reported to be slightly which presented electron-lucent matrix and slightly elevated (Hakkinen and Alen, 1986; Lenders et al., defined cristae, and a marked increase in the number 1988) or not substantially modified (Alen, 1985; Ballarin of lysosomes. These changes were evident in both et al., 1986; Thompson et al., 1989) in the serum of sedentary and trained treated rats, indicating that liver athletes using different anabolic steroids. However, the cell damage is produced by anabolic-androgenic steroids possibility of liver lesions not being detected by despite the simultaneous realization of physlcal exercise. conventional exists. The alterations observed were not detected by means of It is well-known that androgens modulate several conventional biochemical liver tests. hepatic functions such as of lipoproteins, steroids, , ... (Rahwan, 1988). Likewise, Key words: Fluoxymesterone, Methylandrostanolone, morphometric analysis of rat liver after shows Hepatotoxic effects, Serum enzymes, Exercise training a significant reduction in the number of hepatocytes (Tanganelli et al., 1988). The anabolic-androgenic steroids, although exhibiting some of the characteristics Introduction and actions of the endogenous androgens, could be regarded in the high doses taken by athletes as a Anabolic-androgenic steroids, a group of synthetic xenobiotic load for the liver, probably related to compounds structurally related to , are used functional and structural alterations of this organ. On the in medical practice in status of muscle wasting or other hand, exercise is known to influence a large debilitation, to stimulate erythropoiesis in some anaemia number of physiological factors (haemodynamics, blood and in the treatment of hypogonadal status (Rahwan, pH, body temperature, etc.) which may affect the 1988). They are also taken in high doses by athletes who pharmacokinetics of numerous drugs. Thus, the wish to improve physical performance (Wilson, 1988; concurrence of training and ingestion Lamb, 1989). Although adverse effects have been could be expected to modify the potential reported in patients treated with these compounds (Ishak of these compounds. Nevertheless, to date information and Zimmerman, 1987; S0e et al., 1992), knowledge about the effects of anabolic-androgenic steroid about the side effects of suprapharmacological doses of treatment and simultaneous exercise training on liver anabolic steroids in athletes is limited. Reduced levels of structure is scarce. Pathological changes indicative of serum high density lipoproteins and testicular atrophy have been observed in the liver of motor- active mice (Mus wagneri rotans) after 4 weeks of Offprint requests to: Dra. Alicia Megias, Department of Biochemistry, methandrostenolone treatment (Stang-Voss and Appell, Faculty of Chemistry, Complutensis University, 28040 Madrid, Spain 1981), but the relevance of this model with respect to the Steroids and liver of trained rats

physical training of athletes is questionable. blood was collected by cardiac puncture. Serum was The purpose of the present work was to study the obtained by centrifugation at 3000g for 15 min, effects of a prolonged treatment with high doses of oral aliquoted and stored at -40 "C for assays. anabolizing androgens, in association with endurance Aspartate aminotransferase (AST), alanine amino- training, on rat liver ultrastructure and levels of serum transferase (ALT) and alkaline phosphatase (ALP) were parameters related to hepatic function. The results show analyzed by using commercial kits from Bohringer that anabolic-androgenic steroids induce ultrastructural (Bohringer Biochemica GmbH, Mannheim, Germany). changes in liver cells although hepatic function Total and direct were determined in fresh serum indicators in serum are not modified. Exercise training aliquots by a calorimetric procedure (Arnes, Miles does not prevent the morphological alterations observed. Italiana S.p.A., Milano, Italy).

Materials and methods Electron microscopy

Anabolic-androgenic steroids For electron microscopy, livers of the animals from the different groups were perfused with 2.5% Fluoxymesterone (1 113, 1713-dihydroxy-9a-fluoro- glutaraldehyde in Millonig's buffer, pH 7.3, and quickly 17a-methyl-4-androsten-3-one) and methyl- removed. Small tissue specimens were post-fixed in (1713-hydroxy- l7a-methyl-5a- buffered 2% osmium tetroxide, dehydrated in a graded androstan-3-one) were obtained from Sigma Chemical series of and embedded in Araldite. Ultrathin CO (St. Louis, MO. USA). sections were cut by a diamond knife with a Reichert UM-2 ultra-microtome and stained with uranyl acetate Training programme and anabolic-androgenic steroid and lead citrate. Observations were carried out on a treatment Philips EM-201 electron microscope. Male Wistar rats (initial body weight 115 + 5 g) were Statistical analysis obtained from Charles River (Barcelona, Spain). Animals had free access to laboratory chow and tap In these experiments, values of serum parameters water. They were maintained on a 12: 12 h light-dark were analyzed using two-way analysis of variance cycle and housed in an animal room where temperature (ANOVA: factor l= exercise training; factor 2 = steroid (22-24 "C) and humidity (65-75%) were controlled. treatment). If an overall significant F value was Thirty-six rats were randomly divided into sedentary obtained, a Scheffk post hoc analysis was performed. A and exercise training groups. The animals of the trained level of p < 0.05 was selected to indicate statistical group were exercised by running on a motor-driven significance. treadmill 5 dayslweek for 12 weeks. During the first 4 weeks, the speed and duration of the daily exercise Results sessions were progressively increased until the rats were capable of running continuously for 45 min at 25 mlmin. The training programme used in these experiments At the beginning of the fifth training week, when induced a significant decrease in the body weight of the maximal exercise intensity was reached, each group was exercised animals when compared to the sedentary arbitrarily subdivided into three groups: control; controls (Table 1). On the other hand, body weight of the fluoxymesterone-treated; and methylandrostanolone- rats was not affected by anabolic steroid treatment, as treated. The animals selected for anabolic-androgenic shown in Table 1. This fact suggests that abnormal steroid treatment received by gastric intubation 2 mg retention of fluids was not produced as a consequence of steroidkg body weight, as a homogeneous suspension in the anabolizing administration. 1 ml of water, 5 days per week for 8 weeks. The high The determination of metabolites and enzyme level of anabolizing androgens was chosen in an attempt activities in the serum can be of great value for the to simulate the massive doses of anabolic-androgenic detection of toxic effects on the liver. In this respect, steroids reported to be used in athletics (Wilson, 1988). mean values of serum bilirubin as well as transaminases An additional group of six sedentary rats received 5 mg and alkaline phosphatase activities remained within fluoxymesterone/kg body weight for 8 weeks, following normal range in all the groups studied (Table 1). These the same protocol described above. results suggest that neither exercise training nor the administration of anabolic-androgenic steroids induced Serum analyses modifications in the liver function, being consistent with those obtained with power athletes who self- After completion of the 12-week exercise administered very high doses of testosterone and programme, rats were not exercised for 36-44 h and anabolic steroids (Alen, 1985; Ballarin et al., 1986; received the last dose 14-18 h before they were Thompson et al., 1989). sacrificed (between 8:00 and 12:OO a.m.). Animals were The principal features of the hepatic cells in trained fasted overnight, weighed, and under ether anaesthesia control rats were similar to those of sedentary control 451 Steroids and liver of trained rats

Table 1. Effect of anabolic-androgenic steroid administration and exercise training on body weight and serum levels of transaminases, alkaline phosphatase and bilirubin.

EXPERIMENTAL GROUP BODY WEIGHT (g) AST (UII) ALT (UII) ALP (UII) BlLlRUBlN Total (pgtdl) Direct (pgldl)

SEDENTARY C F M

TRAINED C F M

AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; C: untreated control group; F: fluoxymesterone-treated group; M: methylandrostanolone-treated group. Results are meanstSD (n= 6 animals). '= Pe0.05 and "= Pc0.01 (trained group vs corresponding sedentary group). animals (Figs. 1, 2) and in accord with the well-known Another prominent change in the hepatocytes of characteristics of normal rat liver. However, significant animals treated with anabolic steroids was an increase in changes were observed in the liver ultrastructure of both the number of lysosomes dispersed throughout the the trained and untrained rats subjected to anabolic . They appeared as single-membrane-bound steroid treatment. Mitochondria were numerous and bodies with variable electron density, morphology and heterogeneous with regard to size and shape. They were size. The lysosomes contained heterogeneous material, widely distributed throughout the cytoplasm of i.e., substrates in the process of being digested and hepatocytes. The most striking change was the swelling undigested residues, which appeared as irregular and the presence of slightly defined cristae embedded in electron-dense masses in a clear matrix (Fig. 6). When a matrix of low electron-density (Figs. 3, 4). In some higher doses of fluoxymesterone (5 mdkg body weight instances, mitochondria formed part of autophagic for 8 weeks) were administered to the sedentary animals, vacuoles; they were identified as mitochondria by their different forms of lysosomes with respect to their double membrane or by remnants of cristae (Fig. 5). morphology were observed in the hepatocytes. Thus, These mitochondrial alterations were found in the liver lysosomes appeared as irregular electron-dense masses of both sedentary and trained animals and no apparent in a less dense matrix (Figs. 7A,B) and as irregular and differences in the magnitude of the changes very compact electron-dense masses, containing inside were observed between fluoxymesterone- or methyl- low electron-density vesicle-like zones (Figs, 7A, 8). androsranolone-treated rats. The latter cellular structures were observed associated

Fig. 1. Electron micrograph of a hepatic cell from a control rat tra~nedfor Fig. 2. Electron micrograph of a hepatocyte from a control sedentary rat. 12 weeks. S. sinusoid; BC, biliary canaliculi. X 14,400 S, sinusoid; BC, biliary canaliculi. X 14,400 452 Steroids and liver of trained rats

with lipid droplets and they seemed to be expelled into is known to affect a large number of physiological the sinusoids (Fig. 8). Multivesicular bodies were seen in factors, it was necessary to control the possible influence the hepatic cells of the anabolic steroid-treated rats (Fig. of training on the effects of anabolizing androgens on the 7A). They were spherical structures surrounded by a liver. Thus, in this investigation we have studied the single membrane and containing membranous vesicles in effect individually and in combination of both variables, a low electron-dense matrix. exercise training and steroid treatment. The exercise programme employed can be considered Discussion as of moderate-intensity for normal rodent standards. The 12 weeks of endurance training induced the well- The use of anabolic-androgenic steroids as ergogenic aids is accompanied by exercise training. Since exercise

Fig. 4. Mitochondria of a hepatocyte from a sedentary rat treated with methylandrostanolone (2 mglkg body weight). Mitochondria appear swollen and cristae are embedded in a matrix of low electron-density. Fig. 3. Hepat~ccell from a trained rat treated for 8 weeks with Rough endoplasmic reticulum (RER) appear surrounding mitochondria. fluoxymesterone (2 mglkg body weight). Mitochondria are swollen and N, nucleus, 84,000 present reduced matrical density. X 29.500

Fig. 6. Electron micrograph of a hepatocyte from a trained rat treated Fig. 5. Autophagic vacuole including a mitochondrion in a hepatic cell with fluoxymesterone (2 mgkg body weight). Lysosomes (LI) containing from a sedentary rat treated with fluoxymesterone (2 mglkg body heterogeneous material appear as irregular electron-dense masses in a weight). 300,000 clear matrix. 127,000 453 Steroids and liver of trained rats

Fig. 7. A, B. Hepatic cell from a sedentary rat treated for 8 weeks with fluoxymesterone (5 mglkg body weight). Lysosomes (L2) appear as irregular electron-dense masses in a less dense matrix or as irregular and very compact electron-dense masses containing low electron density vesicle-like zones (L3). ME, multivesicular bodies. A) X 85,500. B) X 94,500

showing absence of changes in liver ultrastructure even after exhaustive exercise (King and Gollnick, 1970). However, treatment with fluoxymesterone or methyl- androstanolone induced morphological alterations of the liver cells both in sedentary and trained rats. Main abnormalities observed were changes in mitochondria and lysosomal proliferation. Mitochondrial alterations similar to those described in this work have been previously observed in mouse- kidney proximal tubules after administration (Koenig et al., 1980a), in cardiac and of rats, Guinea pigs and mice following methandrostenolone and treatment (Behrendt and Boffin, 1977; Appell et al., 1983; Soares and Duarte, 1991) and in perivenous hepatocytes from rats treated with the synthetic sexual steroid (Kretzschmar et al., 1989). Besides, studies in vitro with isolated liver mitochondria have shown that some steroids exert a direct effect on mitochondrial membranes and inhibit mitochondrial respiration Fig. 8. Hepatic cell from a sedentary rat treated with fluoxymesterone (Mohan and Cleary, 1989). The effects that (5 mg/kg body weight). Electron-dense masses containing low electron- mitochondrial swelling may have had on the metabolic density vesicle-like zones (L4) appear associated with lipid droplets. capacity of the liver are unclear but it is well known that X 88,000 in isolated mitochondria, disruption of the basic structural configuration can loosen or completely characterized increase in oxidative capacity as indicated uncouple oxidative phosphorylation. Thus, it is possible by the elevation of the activity of some skeletal muscle that the functional capacity of the liver and post-exercise mitochondrial enzymes, such as carnitine palmitoyl- oxygen uptake have been adversely affected, if such loss transferase I (73% in soleus and 48% in extensor of respiratory control were to occur in vivo in the treated digitorurn longus) and succinate dehydrogenase (38% animals. and 4896, respectively) (Guzmin et al., 1991; Saborido An interesting finding in this study was the presence et al., 1991a). On the other hand, serum aspartate of numerous lysosomes in the hepatic cells after anabolic aminotransferase activity was not increased in trained steroid administration. We could observe four animals (Table 1) indicating the absence of muscle morphological types of lysosomes, which probably damage as expected for a mild endurance training. correspond to different phases of lysosomal activity. A Training was also without apparent effects on liver similar response has been observed in fibroblasts of ultrastructure since the characteristics of the hepatic cells mouse tendon after 10 weeks of treatment with the in trained rats were similar to those of sedentary animals. anabolic steroid methandrostenolone (Michna, 1989). This result is consistent with that of previous studies The characteristic changes consisted of the appearance Steroids and liver of trained rats

of numerous intracellular lysosomes with different shape, size and electron density and the emergence Acknowledgements. This research was supported by grants from of matrix vesicles, some of which were identified Universidad Complutense de Madrid (Grupos Precompetitivos) and as extracellular lysosomes. On the other hand, C.I.C.Y.T. (nQDEP90-0558). methandrostenolone in combination with exercise training has been shown to cause an increase in lysosomal hydrolytic activities in the right ventricular References wall of the dog muscle (Takala et al., 1992). Testosterone has also been reported to induce significant Alen M. (1985). Androgenic steroid effects on liver and red cells. Br. J. increases in the specific and total activities of several Med. 19, 15-20. lysosomal hydrolases in mouse kidney (Koenig et al., Appell H.J., Heller-Umpfenbach B., Feraudi M. and Weicker H. (1983). 1980a; Wilson et al., 1988), skeletal muscle (Koenig et Ultrastructural and morphometric investigations on the effects of al., 1980b), aorta (Goldstone et al., 198 l), brain (Koenig training and administration of anabolic steroids on the myocardium and I,u, 1980) and myocardium (Koenig et of Guinea pigs. Int. J. Sports Med. 4, 268-274. al., 1982). In mouse kidney and myocardium, Ballarin E., Guglielmini C., Martinelli S., Casoni I., Borsetto C., Ziglio the modification of these biochemical parameters P.G. and Conconi F. (1986). Unmodified performance in runners is associated with alterations in mechanical stability following anabolic steroid administration. Int. J. Sports Med. 7, 302- of the lysosomal membrane and i~ltrastructuralchanges 306. of the lysosomal-vacuolar system, including an increase Behrendt H. and Boffin H. (1977). Myocardial cell lesions caused by an in autophagy and accumulation of enlarged lysosomes anabolic . Cell Tissue Res. 181.423-426. filled with myeline-like membranes. These results Goldstone A.D.. Koenig H. and Lu C.Y. (1981). Androgens regulate cell suggest that an acceleration of lysosome-mediated growth, lysosomal hydrolases and mitochondrial cytochrome c degradation may be a significant feature oxidase in mouse aorta. Biochim. Biophys. Acta. 673, 170-176. of the liver response to endogenous androgens. To our Graham S. and Kennedy M. (1990). Recent developments in the knowledge, increases in lysosomal activities have not toxicology of anabolic steroids. Safety 5, 458-476. been reported as a response of the liver to anabolic- Guzman M,, Saborido A., Castro J., Molano F. and Megias A. (1991). androgenic steroid treatment and this subject is currently Treatment with anabolic steroids increases the activity of the being investigated in our laboratory. The possibility that mitochondrial outer carnitine palmitoyltransferase in rat liver and the increase in the lysosomal content observed in the fast-twitch muscle. Biochem. Pharmacol. 41, 833-835. hepatic cells of treated rats reflects, at least partly, a Hakkinen K. and Alen M. (1986). Physiological performance, serum disordered metabolism in liver because of the changes in , enzymes and lipids of an elite power during mitochondria, cannot be dismissed. training with and without androgens and during prolonged It is interesting to notice that the treadmill exercise detraining. J. Sports Med. 26, 92-100. performed by the rats does not appear to prevent Hickson R.C., Ball K.L. and Falduto M.T. (1989). Adverse effects of the effects that the anabolic steroid treatment induced anabolic steroids. Med. Toxicol. Adverse Drug Exp. 4, 254-271. on liver ultrastructure. Accordingly, we have previously lshak K.G. and Zimmerman H.J. (1987). Hepatotoxic effects of the demonstrated that the capacity of the liver to metabolize anaboliclandrogenic steroids. Sem. Liver Dis. 7, 230-236. drugs can be modified by prolonged ingestion of these King D.W. and Gollnick P.D. (1970). Ultrastructure of rat heart and liver compounds in sedentary as well as trained male and after exhaustive exercise. Am. J. Physiol. 218. 1150-1155. female rats (Saborido et al., 199 1 b, 1993). Koenig H, and Lu C.Y. (1980). Testosterone-mediated sex differences in This study also shows that serum parameters lysosomal enzymes in rodent brain. Trans. Am. Soc. Neurochem. commonly evaluated in athletes to test hepatic function 11,99. are not substantially modified by administration Koenig H., Goldstone A., Blume C. and Lu C.Y. (1980a). Testosterone- of the anabolic steroids employed. Likewise, in previous mediated sexual dimorphism of mitochondria and lysosomes in studies showing functional liver changes in anabolic mouse kidney proximal tubules. 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