Ultrastructural Changes Induced by Anabolic Steroids in Liver of Trained Rats

Histol Histopath (1993) 8: 449-455 Histology and Histopathology Ultrastructural changes induced by anabolic steroids in liver of trained rats R. Grageral, A. saborido2, F. ~olano~,L. ~irnenezl, E. ~uiiizland A. Megias2 'Department of Cell Biology, Faculty of Biology and 2Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutensis University, Madrid, Spain Summary. The effects of anabolic steroid treatment in have been shown in top-level and amateur athletes 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 androgens, 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 fluoxymesterone 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 enzymes 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 liver function tests 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 metabolism of lipoproteins, steroids, drugs, ... (Rahwan, 1988). Likewise, Key words: Fluoxymesterone, Methylandrostanolone, morphometric analysis of rat liver after castration 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 testosterone, 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 anabolic steroid ingestion Lamb, 1989). Although adverse effects have been could be expected to modify the potential hepatotoxicity 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 cholestasis 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 enzyme 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 bilirubin 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 androstanolone (1713-hydroxy- l7a-methyl-5a- buffered 2% osmium tetroxide, dehydrated in a graded androstan-3-one) were obtained from Sigma Chemical series of acetone 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 androgen 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

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