spleen morphology after androgenic anabolic steroid

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Sendo assim, o objetivo do presente estudo foi anal- isar os efeitos de diferentes doses de decanoato nandrolona (DN), associado ao exercício físico de alta ...
EISSN 1676-5133

SPLEEN

MORPHOLOGY AFTER ANDROGENIC-

ANABOLIC STEROID ADMINISTRATION ASSOCIATED TO EXERCISE Vagner José da Silva1 [email protected] Jonato Prestes2 [email protected] Luis Felipe Milano Teixeira1 [email protected] Guilherme Borges Pereira2 [email protected] Richard Diego Leite2 [email protected] Cláudia Kiyomi Minazaki3 [email protected] Mauricio Cavacchini da Silveira1 [email protected] Lea Sílvia Horii1 [email protected] Sílvia Cristina Crepaldi Alves1 [email protected] Cláudia Regina Cavaglieri1 [email protected] Estela Bevilacqua3 [email protected] Rozangela Verlengia1 [email protected] doi:10.3900/fpj.8.1.32.e

Silva VJ, Prestes J, Teixeira LFM, Pereira GB, Leite RD, Minazaki CK, et al. Spleen morphology after androgenic-anabolic steroid administration associated to exercise. Fit Perf J. 2009 Jan-Feb;8(1):32-9.

ABSTRACT Introduction: Spleen represents one of the larger lymphoid organs in humans and animals. Therefore, the objective of the present study was to analyze the effects of different doses of nandrolone decanoate (ND), associated to high intensity physical exercise on spleen morphology. Materials and Methods: 20 Wistar male rats with two months and body mass of 280±12g, divided in four experimental groups: G0 - control group with propilenoglycol (0.2mL.kg-1); GI - exercised + 1.0mL.kg-1 of ND; GII - exercised + 5.0mL.kg-1 of ND; GIII - exercised + 20.0mL.kg-1 of ND. The five weeks training consisted of four sets of 10 water jumps and rest interval of 30s between sets. In the 1st and 2nd weeks the load was 50%; in 3rd and 4th, 60%; and in 5th, 70% of the body mass. Results: There were higher erythrocytes levels in GII group compared with G0 group (p=0.039) and also in GIII group compared to G0 (p=0.004) and GI (p=0.018). There was no significant modification in spleenic histological structure in the comparison between the experimental groups. Discussion: DN administration associated to exercise promotes no qualitative alterations in the spleen histological structure and induces erythropoiesis.

KEYWORDS Spleen, Physical Activity, Steroids. 1

Universidade Metodista de Piracicaba - UNIMEP - Faculdade de Ciências da Saúde - Mestrado em Educação Física - Piracicaba - Brazil Universidade Federal de São Carlos - UFSCar - Departamento de Ciências Fisiológicas - Laboratório de Fisiologia do Exercício - São Carlos Brazil 3 Universidade de São Paulo - USP - Instituto de Ciências Biomédicas - Departamento de Histologia e Embriologia - São Paulo - Brazil 2

Copyright© 2009 por Colégio Brasileiro de Atividade Física, Saúde e Esporte Fit Perf J | Rio de Janeiro | 8 | 1 | 32-39 | Jan/Feb 2009

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Fit Perf J. 2009 Jan-Feb;8(1):32-9.

A NDROGENIC-ANABOLIC STEROID ASSOCIATED TO EXERCISE

MORFOLOGIA

DO BAÇO APÓS ADMINISTRAÇÃO DE ESTERÓIDE ANABÓLICO-ANDROGÊNICO ASSOCIADO AO EXERCÍCIO

RESUMO Introdução: O baço representa um dos maiores órgãos linfóides em humanos e animais. Sendo assim, o objetivo do presente estudo foi analisar os efeitos de diferentes doses de decanoato nandrolona (DN), associado ao exercício físico de alta intensidade, sobre a morfologia do baço. Materiais e Métodos: Foram utilizados 20 animais machos da espécie Wistar, com dois meses, peso de 280±12g, divididos em quatro grupos experimentais: G0 - grupo controle com propilenoglicol (0,2mL.kg-1); GI - exercitado + 1,0mL.kg-1de DN; GII - exercitado + 5,0mL.kg-1 de DN; GIII - exercitado + 20,0mL.kg-1 de DN. O treinamento de 5 semanas consistiu de quatro séries de 10 saltos na água, com intervalos de 30s entre as séries. Na 1ª e 2ª semana a sobrecarga foi de 50%; na 3ª e 4ª de 60%; e na 5ª de 70% do peso corporal. Resultados: Foram observadas maiores concentrações de eritrócitos no grupo GII comparado com grupo G0 (p=0,039), e também no grupo GIII comparado com G0 (p=0,004) e GI (p=0,018). Não houve modificação na estrutura histológica esplênica na comparação entre os grupos experimentais. Discussão: A administração de DN associada ao exercício não promove alterações qualitativas na estrutura histológica do baço e promove aumento da eritropoiese.

PALAVRAS-CHAVE Baço, Atividade Física, Esteróides.

MORFOLOGÍA

DEL BAZO TRAS ADMINISTRACIÓN DE ESTEROIDE ANABÓLICO-ANDRÓGENO ASOCIADO AL EJERCICIO

RESUMEN Introducción: El bazo es uno de los principales órganos linfoides en los seres humanos y animales. Por lo tanto, el objetivo de este estudio fue analizar los efectos de diferentes dosis de decanoato de nandrolona (DN), asociadas al ejercicio físico de alta intensidad sobre la morfología del bazo. Materiales y Métodos: Se utilizaron 20 animales masculinos de la especie Wistar de dos meses y peso de 280±12, divididos en cuatro grupos experimentales: G0 - grupo de control con el propilenoglicol (0,2mL.kg-1); GI - con ejercicio + 1,0mL.kg-1 de DN; GII - con ejercicio + 5,0mL.kg1 de DN; GIII - con ejercicio + 20,0mL.kg-1 de DN. El entrenamiento de 5 semanas consistió en cuatro series de 10 saltos en el agua a intervalo de 30s entre las series. En las 1 ª y 2 ª semanas, la carga fue de 50%, en la 3ª y 4ª de 60%, y la 5ª de 70% del peso corporal. Resultados: Existieron concentraciones más altas de glóbulos rojos en el grupo GII en comparación con el G0 (p=0,039) y también en el grupo GIII en comparación con el G0 (p=0,004) y GI (p=0,018). No hubo cambios en la estructura histológica del bazo en la comparación entre los grupos experimentales. Discusión: La administración de la DN asociada al ejercicio no ha promovido cambios cualitativos en la estructura histológica del bazo y promueve el aumento de la eritropoyesis.

PALABRAS CLAVE Bazo, Actividad Física, Esteroides.

INTRODUCTION Testosterone, together with other androgens, is responsible for the development of the secondary sexual characteristics (androgenic effect), stimulation of the muscular proteic synthesis and hypertrophy1. Due to the diverse effects propitiated by testosterone, derived synthetic from sexual hormones steroids were used for decades in researches, for recreational sports practitioners and athletes, mainly objectifying the increase of the muscular mass, force, sportive performance and aesthetical aspects2. Alén3 demonstrated increase in the concentration of serum hemoglobin, hematocrit and in the white cells’ number, after six months of administration of raised doses of anabolic androgenic steroids (EAA). Hartgens et al.4 demonstrated increase of the plaques’ content after eight weeks of EAA use,

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while all the other hematological parameters remained unchanged. Furthermore, the medical community has been each time more interested for the therapeutical use of the EAA in the treatment of hypogonadism, sarcopenia, anemia, cancer and patients with acquired immunodeficiency syndrome3. On the other hand, Ferrández et al.1 demonstrated that the administration of supraphysiological doses (above 10mg.kg-1.week-1) of EAA associated with the high intensity exercise can lead to the reduction of the proliferation and mobility of lymphocytes in vitro, evidencing negative aspects related to the inadequate use of EAA. Another important aspect to be considered is the effect of the different doses of EAA on hematological and tissuey parameters, as the spleen.

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S ILVA , P RESTES, TEIXEIR A , P EREIR A , L EITE, M INA ZAKI,

ET AL .

In this direction, the spleen represents one of the biggest lymphoid organs in human beings and animals. Its morphologic aspect allows the visualization of different structures, as the white pulp, which is characterized by the abundant presence of lymphocytes, and the red pulp, constituted of the great erythrocytes’ content5. Stewart et al.6 showed that, after 5min, 10min and 15min of acute exercise in cycle ergometer 60% of the VO2max, the blood volume and spleen size are reduced. Chronically, after 30, 45 and 60 days of exercise an increase occur in sympathetic innervation in young rats’ spleen occurs7. Until the present moment, the inquiries regarding the effects of different doses of the EAA associates to the exercise remain insufficient, on the splenic structural alterations. The objective of this study was to analyze the effect in the spleen morphology of different doses of nandrolone decanoate (ND) associated to the high intensity physical exercise. The ND was chosen for this study for being one of the most used anabolic androgenic in clinical application and between athletes3,8.

MATERIALS AND METHODS Animals Twenty male animals of the Wistar (Rattus norvegicus albinus) species were selected, with two months and weight of 280±12g. The animals were separated in four collective cages (five animals in each) and received ration and water ad libitum. The temperature of the room was controlled in 23±2°C, clear-dark cycle of 12h/12h, with lights on starting from 6h. Before initiating the experimental period, the animals remained for 48h in adaptation to the cage conditions. All the experiment was lead in accordance with the politics for research with experimental animals of the American College of Sports Medicine and Brazilian College of Experimentation. The present study was approved by the Ethics Commission in Animal Experimentation of the Methodist University of Piracicaba. Experimental groups The animals were randomized and distributed in four experimental groups, in the following order: G0 - control group with propilenoglicol (0.2mL.kg-1); GI - exercised group with administration of 1.0mL.kg-1 of ND; GII - exercised group with administration of 5.0mL. kg-1 of ND; GIII - exercised group with administration of 20.0mL.kg-1 of ND. The animals were submitted to a program of jumps in the water in a plastic pipe of 20cm of diameter, containing an amount of water approximately equivalent to the double of the length of the rat and temperature of 30±2ºC. The high intensity

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training protocol was performed five days a week during five weeks. Administration of anabolic-androgenic steroid The anabolic-androgenic steroid was injected intramuscular way in the quadriceps’ muscles of the thigh in alternated form, three times a week after the training. The administration of the vehicle (propilenoglicol) and nandrolone® decanoate (Organon, New Jersey, USA) initiated in the first week of training, totalizing five weeks. The G0 group received only injections from propilenoglicol (0.2mL.kg-1). The animals dealt with nandrolone decanoate received the doses diluted in propilenoglicol, totalizing a final concentration of 50mg.mL-1. Training protocol The animals were adapted to the exercise protocol during two weeks, to reduce stress. The adaptation was composed by sessions of jumps in the water with weight (50% of the body weight), once a day, during five days. The additional weight was fixed in the animal’s chest by means of a vest that allows the execution of the exercise. The number of series (2-4) and jumps (5-10) was adjusted daily and increased gradually, with a period of 30s of recovery between the series. All the adaptation sessions were performed in the morning (8h to 12h). After the adaptation period, the animals were submitted to the experimental protocol of high intensity training, which consisted of jumps in the water with the adjusted weight in accordance to the animal’s weight, previously described9. All the training sessions were composed by four series of 10 jumps and recovery interval of 30s between the series. In the first and the second week the overcharge was of 50%; in third and fourth, 60%; and in the fifth week was of 70% of the body weight. The animals were weighed three times a week. Hematocrit’s determination After three days of the experimental period, the animals were sacrificed and the blood collected in hair pipes without anticoagulant. After that, the hair pipes were filled with a volume of blood equal to 70% and centrifuged at 5000rpm during 5min. After the centrifugation, the erythrocytes’ percentage was inferred through a table of the standard reading card (Fanem, Brazil). Tissue preparation Immediately after the sacrifice, the spleen was removed. The samples were fixated in paraformaldehyde 4% in PBS 0.1M, pH 7.4, for 24h. After that, the samples were washed, dehydrated in increasing concentrations (70%, 80%, 90% and 95%), bathed during 40min with xylol and fixated in liquid paraplast at 45°C.

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A NDROGENIC-ANABOLIC STEROID ASSOCIATED TO EXERCISE

Histological evaluation The tissue was cut in sections of 6µm and submitted to different procedures for identification of the splenic structures. The procedures were: coloration with hematoxiline and eosine (H&E) (Carlo Erba and Mallinckrod, Sigma®) for analysis of the general structure of the tissuey components; coloration by the Mallory’s trichrome (TM) (Carlo Erba, Sigma®) to evidence the red blood cells; histochemical reaction with peroxidase (DAB-tablets, Sigma®) to evidence the peroxidasic activity; e histochemical reaction with phosphatase (Fast Red, Sigma®) to determine the activity of phosphatase acid. Later, the samples were examined in the microscope and the analysis performed individually without the knowledge of the groups and samples. All the spleen cuts were entirely examined and the most representative area of each experimental group was photographed. Statistical treatment The quantitative data (hematocrit’s concentrations) was expressed by the average ± standard deviation. The statistical analysis was made initially by the normality test of Shapiro-Wilk and the test of homocedasticity (Bartlett’s criterion). All the analyzed variables presented normal distribution and homocedasticity, using, in such a way, the Anova Two Way for repeated measures. In all the calculations it was fixated a critical level of 5% (p≤0.05). The used software in all the statistical tests was Statistica® 6.1.

RESULTS The values of erythrocytes’ concentrations (mL.kg-1) in the different studied groups that received different doses from ND were presented in the format of average ± standard deviation. Group GII presented serum concentrations of statistical superior erythrocytes, compared with the G0 group (p=0.039). Superior concentrations of erythrocytes in group GIII, compared had been observed G0 (p=0.004) and GI (p=0.018) (Image 1). In relation to the body mass, significant variations of none of the experimental groups during the five weeks were not observed. All the described qualitative histological observations were consistently found in all the samples for each group. It was observed, in all the experimental groups, the white pulp formed by a typical condensation of lymphocytes, macrophages and plasmocites. The periarterial case, that persists around of arterioles and some of these nodules, presents a little colored central region by the hematoxylin, being able to indicate a lesser cellular accumulation, characterizing the germinative centers (Image 2A). It was also observed the red pulp consisting by a net of veinal anastomosed seins and the presence of red blood cells (Image

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Image 1 - Mensuration of the hematocrit blood concentrations (ml.kg-1)

Values = average + standard deviation. G0 Group (vehicle), GI (1.0 mg.kg-1), GII (5.0 mg.kg-1) and GIII (20.0 mg.kg-1) *Statistically significant difference in relation to G0 # Statistically significant difference in relation to G1 p < 0.05

2B). The presence of erythrocytes marked in chestnut (E) by the chemical reactions of peroxidase (positive) and the presence of lymphatic nodules (N) can be visualized (Image 2C). The samples marked positively with the acid phosphatase allowed the identification of the presence of macrophages (M) (Image 2D). In general and qualitative form, there was no modification in the splenic histological structure in the comparison between experimental groups GI, GII and GIII in relation to G0 (control). In all groups, the splenic tissuey components were present and the organization and the architecture of the organ preserved in chemical preparations colored by the hematoxylin and heosine (Images 2E, I and N) and Mallory’s trichrome (Images 2F, J and O). The positive reactions of phosphatase acid (Images 2H, M and Q) and peroxidase (Images 2G, L and P) evidenced the presence of macrophages and erythrocytes, respectively. Moreover, periarterial cases, lymphoid nodules with central arteries and the presence of germinative centers, margin zone delimited and evident red pulp, with a possible alteration in the incidence of colored erythrocytes in chestnut. This alteration can indicate an increase in the presence of the erythrocytes inside of the medullar zone, suggesting that the ND associated with the exercise can increase erythropoiesis and, consequently, greater marking by peroxidase (Images 2L and P). To confirm that there was no histological differences in the transversal cuts of the spleen, the morph metrical measures of the lymphoid nodules’ area. The analyses confirmed the absence of difference in the splenic structure between the studied experimental groups (results not presented).

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S ILVA , P RESTES, TEIXEIR A , P EREIR A , L EITE, M INA ZAKI,

ET AL .

Image 2 - Histological transversal cuts

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A NDROGENIC-ANABOLIC STEROID ASSOCIATED TO EXERCISE

G0 Group (A, B, C and D), GI (E, F, G and H), GII (I, J, L and M) and GIII (N, O, P and Q). The cuts A, E, I and N represent the coloration with hematoxylin and eosin and magnifying of 80X. The cuts B, F, J and O represent the coloration by the Mallory’s trichrome and magnifying of 80X. The cuts C, G, L and P represent the coloration by peroxidase and magnifying of 400X. The cuts F, D, H, M and Q were colored with acid phosphatase and magnified at 800X. White pulp (PB), red pulp (PV), medullar zone (ZM) and the arrow indicates arteriole, lymphatic nodules (N), erythrocytes (E), periarterial folds (BP) and macrophages (M).

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S ILVA , P RESTES, TEIXEIR A , P EREIR A , L EITE, M INA ZAKI,

ET AL .

DISCUSSION Until the present moment, this was the first study to qualitatively evaluate the morphologic structures of spleen of rats submitted to the high intensity training, associated to the administration of different doses of ND. The spleen is an important responsible agency for diverse functions, such as: antibody formation; production of lymphocytes and monocytes; filtration; phagocytosis and destruction of senescent erythrocytes; supply of ions iron and viable sanguineous cells5. The splenic morphologic adaptations, decurrent from the physical exercise associated to the ND, need bigger clarifications. It is important to point out the necessity of studies in this area, since recent estimates indicate that more than three million people manage EAA in United States10. The results of the qualitative evaluations of this study did not show differences in the spleen morphology between the exercised groups that received different doses from ND, when compared with the control. In the present study, different dosages of ND were managed in three groups. According to Hartgens & Kuipers11, only the administration of group GIII was supraphysiological (above 10 mg.kg-1.week-1), what, probably, can have contributed for absence of structural alterations. Furthermore, frequently the athletes manage many EAA at the same time, with different doses11, resulting in doses 10 to 100 times bigger than the recommended for the reposition therapies3. The prolonged use of EAA in supraphysiological doses can lead to diverse effects that vary according to different tissues, types of enzymes and concentration of androgenic receptors11. Recent studies demonstrated that the main targets that suffer from the EAA the effects are the muscles, conjunctive and boney tissue12,13,14. Additionally, when combined to the exercise, it may also occur significant alterations in cardiovascular, immunological, endocrine, reproductive, psychological systems, liver and tendons, mediated by diverse mechanisms not yet totally understood11,12,13. Another coherent justification for our results can be the insufficient time of ND administration (five weeks), that did not allow the adaptation and alteration in the morphology of the evaluated fabric. In healthful young men, supraphysiological doses of EAA induced increase in the body mass, musculature size and muscular force, associated or not to exercises, after 10 to 20 weeks14. Marqueti et al.15,16 evaluated the effects of the steroids’ administration associated to the high intensity exercise, during six weeks, on the tendons’ morphology. The authors demonstrated that the association between exercise and EAA modifies the tissuey morphology and can harm the tendon’s remodeling, potentially increasing the injury risk. In another study, Fernández et al.1 analyzed the effect of supraphysiological doses of EAA associates to the

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exercise, on mobility and proliferative reply in culture of lymphocytes derived from spleen and thymus of rats. After 12 weeks, the authors concluded that the high intensity training reduces mobility and the proliferation in vitro and, when associated to the treatment with EAA, can harm the immune response even more. EAA, including the ND, are synthetic hormones derived from the masculine hormone testosterone, developed with properties to maximize the anabolic effect and to minimize the androgenic effects11. The testosterone regulates many physiological processes in the organism, including muscular proteinic metabolism, sexual and cognitive functions, erythropoiesis, lipidic profile and boney metabolism18. In our study, the administration of different doses of ND associated with the high intensity exercise, promoted the expected increase of the circulating hematocrit and bigger positive marking of peroxidase in the medullar zone of the histological spleen cuts. Among the induced cardiovascular adverse effects for the EAA, we can cite hypertension, left ventricular hypertrophy, harmed diastolic period, arrhythmia, thrombosis and erythrocytosis18. The mechanisms by which the EAA stimulate erythropoiesis are not totally clarified. It has been postulate that the synthetic testosterone and its derivatives induce erythropoiesis by the stimulation of the erythropoietin production and direct action on the brown bone, increasing the number of responsive-erythropoietin cell19,20. These findings suggest that the administration of ND associated with the exercise does not promote qualitative alterations in the histological structure of the spleen and promotes increase of erythropoiesis. However, new similar inquiries are necessary, manipulating the combination of different types of EAA and supraphysiological dosages with periods of duration above 10 weeks. Another interesting aspect to be investigated is the responsivity (receptors/ enzymes) from the spleen to the EAA.

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14. Pärssinen M, Karila T, Kovanen V, Seppälä T. The effect of supraphysiological doses of anabolic androgenic steroids on collagen metabolism. Int J Sports Med. 2000;21(6):406-11. 15. Marqueti RC, Parizotto NA, Chriguer RS, Perez SEA, Selistre-de-Araujo HS. Androgenic-anabolic steroids associated with mechanical loading inhibit matrix metallopeptidase activity and affect the remodeling of the achilles tendon in rats. Am J Sports Med. 2006;34:1274-80. 16. Marqueti RC, Prestes J, Paschoal M, Ramos OH, Perez SE, Carvalho HF, et al. Matrix metallopeptidase 2 activity in tendon regions: effects of mechanical loading exercise associated to anabolic-androgenic steroids. Eur J Appl Physiol. 2008;104(6):1087-93.

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17. Huber DM, Bendixen AC, Pathrose P, Srivastava S, Diengerkm, Shevde NK, et al. Androgens suppress osteoclast formation induced by RANKL and macrophage-colony stimulating factor. Endocrinology. 2001;142(9):3800-8.

11. Hartgens F, Kuipers H. Effects of androgenic-anabolic steroids in athletes. Sports Med. 2004;34(8):513-54.

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12. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, et al. Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab. 2001;281(6):1172-81.

19. Blazevich AJ, Giorgi A. Effect of testosterone administration and weight training on muscle architecture. Med Sci Sports Exerc. 2001;33:1688-93.

13. Sinha-Hikim I, Artaza J, Woodhouse L, Gonzalez-Cadavid N, Singh AB, Lee MI, et al. Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. Am J Physiol Endocrinol Metab. 2002;283(1):154-64.

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20. Boyadjiev NP, Georgieva KN, Massaldjieva RI, Gueorguiev SI. Reversible hypogonadism and azoospermia as a result of anabolic-androgenic steroid use in a bodybuilder with personality disorder. A case report. J Sports Med Phys Fitness. 2000;40(3):271-4. Submitted: 09/23/2008 - Accepted: 12/12/2008

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