Nandrolone Decanoate Enhances Hypothalamic Biogenic Amines in Rats

1 1 2 2 TETSURO TAMAKI , TAKEMASA SHIRAISHI , HIROSHI TAKEDA , TERUHIKO MATSUMIYA , 3 3,4 ROLAND R. ROY , and V. REGGIE EDGERTON 1Department of Physiology, Division of Human Structure and Function, and 2Department of Pharmacology, Tokyo Medical College, Tokyo, JAPAN; and 3Brain Research Institute and 4Department of Physiological Science, University of California, Los Angeles, CA

ABSTRACT TAMAKI, T., T. SHIRAISHI, H. TAKEDA, T. MATSUMIYA, R. R. ROY, and V. R. EDGERTON. Nandrolone Decanoate Enhances Hypothalamic Biogenic Amines in Rats. Med. Sci. Sports Exerc., Vol. 35, No. 1, pp. 32–38, 2003. Purpose: To identify possible mechanisms for an anabolic-androgenic steroid induced increase in aggressive behavior and work capacity, the levels of some biogenic amines considered to be closely related to a systemic hyper-adrenergic state were measured in selected regions of the brain. Methods: Wistar male rats were divided randomly into five groups: nontreated (control), oil-vehicle-treated (vehicle) or one of three (therapeutic dose and 10- or 100-fold higher dose) anabolic-androgenic steroid-treated (steroid-1, -2, -3) groups. Rats in the steroid and vehicle groups were given a single dose of nandrolone decanoate or oil vehicle, respectively, one week before tissue sampling. The levels of norepinephrine (NE) and its metabolite, 4-hydroxy-3-methoxyphenylglycol (MHPG), serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) were measured in the cerebral cortex, hypothalamus and cerebellum by high-performance liquid chromatography. Immunostaining for c-fos was performed as a confirmation of increased neural activity. Results: The levels of NE and MHPG were increased by ~2- and ~7-fold in the hypothalamus of the steroid-2 compared with the control and vehicle groups. The levels of 5-HT and 5-HIAA were ~40 and ~50% higher in the steroid-2 compared with the control and vehicle groups. A significantly higher number of c-fos expressing neurons were observed in the periventricular region of the steroid-2 than the control and vehicle groups, indicating enhanced neuronal activity after nandrolone decanoate treatment. Conclusions: The present results, combined with previously reported findings of physical performance enhancement after anabolic-androgenic steroid treatment, are consistent with the interpretation that elevated levels of adrenergic and serotonergic amines in the hypothalamus could contribute to aggressive behaviors as well as improved physical performance. Key Words: NANDROLONE-DECANOATE, HYPOTHALAMUS, NOREPINEPHRINE, SEROTONIN, C-FOS

nabolic-androgenic steroids (AAS) are widely drolone decanoate in adult male rats, we have reported an abused by athletes and bodybuilders to improve enhanced work volume during an exhaustive weight-lifting Aphysical performance (ergogenic effects) and en- session and a greater fatigue resistance of the plantaris hance overall physical strength and muscle mass (38). For muscle tested in situ (35). these ergogenic effects, one possibility is that there is a Another possibility is that they could be operating direct effect on the work capacity of the skeletal muscula- through some neurally mediated mechanism, which en- ture, i.e., there is evidence that AAS treatment may directly hances the motivation to train harder, thus improving work improve the endurance capacity of skeletal muscles. For tolerance (1). In human subjects, several psychological ef- example, an elevated submaximal running capacity of rats fects such as a greater tolerance to pain and irritability and (36) and an improved fatigue resistance of the rat extensor aggression after AAS treatment have been reported, and it digitorum longus muscle tested via electrical stimulation (8) has been suggested that these behavioral changes may be have been observed after 5–6 wk of AAS treatment. Sim- related to an enhancement of performance (4,30). A rela- ilarly, 1 wk after a single intramuscular injection of nan- tionship between AAS loading and biochemical changes in the brain reward system also has been observed. For example, AAS treatment for 14 d increased ␤-endorphin levels in Address for correspondence: Tetsuro Tamaki, Ph.D., Department of Phys- the ventral tegmental area (18), increased dynorphin B and 6 iology, Division of Human Structure and Function, Tokai University met-enkephalin-Arg Phe (7)-ir activity in the hypothala- School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193 Japan; E- mus, striatum, and periaqueductal gray region (16), and mail: [email protected]. decreased N-methyl-D-aspartate receptor subunit NR2A Submitted for publication December 2001. mRNA expression in the hypothalamus and hippocampus of Accepted for publication September 2002. the brain (23). In addition, the hypothalamus and hippocam- 0195-9131/03/3501-0032/$3.00/0 pus are thought to play important roles in the expression of

MEDICINE & SCIENCE IN SPORTS & EXERCISE® aggressive behavior (33), a consistent behavioral sequel of Copyright © 2003 by the American College of Sports Medicine AAS abuse (30). Indeed, a primary effect of AAS use on DOI: 10.1249/01.MSS.0000043287.12926.12 athletic and/or exercise performance could be neurally me- 32 diated and manifested as greater motivation and/or aggres- (34). This procedure was performed by trained personnel sion (9). Furthermore, possible mechanisms of central ner- and approved by the local animal use committee. The brains vous system fatigue during exercise have been suggested were removed rapidly and dissected into discrete regions on that implicate the neurotransmitters serotonin, dopamine, a dry ice-cooled aluminum plate according to the method of and acetylcholine, as well as known neuromodulators like Glowinski and Iversen (10). The sampled regions included ammonia and various cytokines (6). the cerebral cortex, hypothalamus, and cerebellum. Each Therefore, the primary purpose of the present study was sample was placed immediately in liquid nitrogen and to determine how AAS treatment affects the levels of spe- stored at Ϫ80°C until analysis. Norepinephrine (NE), sero- cific biogenic amines and their metabolites in regions of the tonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), and CNS associated with aggressive behavior and improved 4-hydroxy-3 methoxyphenylglycol (MHPG) were measured motor performance (30,33). In addition, because immuno- using a coulometric high-performance liquid chromatogra- histochemical mapping of the inducible transcription fac- phy system based on the procedures of Takeda et al. (34). tors, such as the members of the Jun family (c-Jun, JunB, Briefly, each tissue sample was weighed and initially mixed and JunD) and the Fos family (c-fos, FosB, and the fos- with a solution of 200 ng each of deoxyepinephrine related antigenes Fra-1 and Fra-2) often has been used to hydrochloride and 5-hydroxy-N ␻-methyltryptamine investigate the spatial and temporal distribution of brain oxalate·mLϪ1 of 0.1 M perchloric acid, 30 ␮Lof0.1M functional activity (19,31), we used c-fos immunohisto- ethylenediamine-tetraacetic acid, and 30 ␮Lof1Mso- chemistry to determine whether inducible transcription fac- dium hydrogen sulfite as internal standards. The mixture tors were activated in the brain by AAS loading. then was homogenized with an ultrasonic cell disrupter (Ultrasonicator, OHTAKE WORKS, Tokyo, Japan) at METHODS 0°C for 30 s and centrifuged at 20,000 g for 15 min at 0°C. The supernatant layer was filtered through a 0.45 Experimental design. Specific pathogen and virus an- ␮m millipore filter (Nihon Millipore, Yonezawa, Japan) tigen free Wistar male rats (~17 wk old; 400-412 g body to separate the insoluble residue. A portion of the super- weight; N ϭ 50) were divided randomly into five groups: a natant was injected into a reversed-phase high-perfor- nontreated control (control; N ϭ 11), a placebo oil-vehicle- mance liquid chromatography system with the redox- treated (vehicle; N ϭ 10), or one of three AAS-treated reductive screen detection mode by using a series of three (steroid-1, -2, or -3; N ϭ 7, 14, and 7, respectively) groups. coulometric working electrodes for simultaneous deter- A single dose of nandrolone decanoate (Deca-Durabolin, mination of NE, MHPG, 5-HT, and 5-HIAA. Organon, Oss, Netherlands) was administered in the gluteus Immunohistochemical staining. To confirm neural muscles of steroid-treated rats 1 wk before the terminal activity within the CNS, we performed c-fos immunohisto- experiment. This drug is a long-acting steroid ester, which is chemical staining in the control, vehicle, and steroid-2 slowly hydrolyzed to give a constant tissue level of steroid groups. One week after the administration of AAS or oil for more than 4 wk (7). The recommended therapeutic use vehicle, three rats in each group (total N ϭ 9) were over- of nandrolone decanoate is about 0.4 mg·kgϪ1 body weight dosed with sodium pentobarbital (60 mg·kgϪ1, i.p.) and (i.m.) in human subjects. It is highly likely that athletes perfused with warmed (~37°C) 0.01 M phosphate-buffered consume 10–100 times the recommended therapeutic dose saline (PBS; pH. 7.4) for 15 min. Then the rats were per- (26). Therefore, we selected the following dosage: 0.375 fused with 4% paraformaldehyde/0.05 M phosphate buffer mg·kgϪ1 body weight of nandrolone decanoate for the ste- (pH 7.4) for 20 min at room temperature. The entire brain roid-1, 3.75 for the steroid-2, and 37.5 for the steroid-3 was removed and immersed in the same fixative overnight groups, respectively. In the vehicle group, the same amount at 4°C. After fixation, the brains were cut serially in 40-␮m of oil vehicle (same solvent used with the Deca-Durabolin) thickness (coronal sections) by using a microslicer (DTK- was administered in the same manner. The animals were 1500, Do-han EM Co., Kyoto, Japan). Thirty sections from housed in standard cages and provided food (CE-2, CLEA, each rat were processed by free-floating staining for immu- Tokyo, Japan) and water ad libitum. The room temperature nohistochemistry. Rabbit polyclonal antiserum for c-fos was kept at 23 Ϯ 1°C, and a 12:12-h light-dark cycle was (Ab-5, Oncogene Research Products, Darmstadt, Germany) maintained throughout the experiment. All experimental was used as the primary antibody (1:20,000). Free-floating procedures were conducted in accordance with the Japanese sections were incubated with 0.3% H2O2 in PBS for 30 min, Physiological Society Guide for the Care and Use of Lab- washed with PBS, and then incubated in a blocking solution oratory Animals as approved by the Tokai University (PBS/5% horse serum/0.25% Triton X-100) for2hatroom School of Medicine Committee on Animal Care and Use temperature. The sections were incubated with primary an- and followed the policy statement of the American College tibody for 48 h at 4°C. The primary antibody was removed, of Sports Medicine on research with experimental animals. and then the sections were washed in PBS and incubated Biochemical analyses. Forty-one rats (control, N ϭ 8; with a secondary biotinated antirabbit (1:400, Amersham vehicle, N ϭ 8; and steroid-1, -2, -3, N ϭ 7, 11, and 7, Pharmacia Biotech, Buckinghamshire, UK) for2hatroom respectively) were used for these analyses. One week after temperature. Sections were washed well in PBS and sub- the administration of AAS or oil vehicle, the animals were jected to an avidin-biotin-peroxidase reaction (Vectastain, sacrificed by decapitation for brain monoamine analysis Vector Laboratories, Burlingame, CA) for 1 h and visual-

ANABOLIC-ANDROGENIC STEROID AND HYPOTHALAMUS Medicine & Science in Sports & Exerciseா 33 ized with 0.02% 3,3-diaminobenzidine (Wako Pure Chem- ical, Osaka, Japan)/0.05 M Tris HCl buffer, pH 7.4, con- taining 0.005% H2O2 for 4 to 6 min. The sections then were transferred to slide-glass, air dried, dehydrated in a graded ethanol series, and mounted. A digital photograph was taken from the periventricular region of the brain of each rat. The number of c-fos positive neurons were counted using image analysis computer software (NIH Image) and expressed as mean Ϯ SE/unit area (1.85 ϫ 1.2 mm squares as shown in Fig. 4). Statistical analyses. All data are reported as mean Ϯ SE. An ANOVA was used to determine overall differences and Duncan’s post hoc analyses were used to identify indi- vidual group differences. Differences were considered sta- tistically significant at a P Յ 0.05 level.

RESULTS Biochemical data. The final body weight of the five groups were similar: 417 Ϯ 15, 412 Ϯ 12, 424 Ϯ 19, 423 Ϯ 17, and 419 Ϯ 21 g for the control, vehicle, and steroid-1, -2, and -3 groups, respectively. The simultaneous quantification of NE and MHPG, a metabolite of NE, in the cerebral cortex, hypothalamus, and cerebellum are shown in Figure 1, A and B, respectively.

FIGURE 2—Levels of serotonin (5-HT) (A) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the cerebral cortex, hypothalamus, and cerebellum of rats in the control, vehicle, and steroid-1, -2, .and -3 groups. Values are mean ؎ SE. Letters, same as in Figure 1

The levels of NE and MHPG in the cerebral cortex and cerebellum were similar among the five groups. In the hypothalamus, however, the NE levels in the steroid-2 and -3 (Fig. 1A) and the MHPG levels in all three steroid groups (Fig. 1B) were higher than in the control and vehicle groups, except that the NE level was not higher in the steroid 3 versus control. In addition, the level of MHPG was higher in the steroid-2 than in the steroid-1 and -3 groups (Fig. 1B). The levels of 5-HT were not significantly different among the five groups in any of the brain regions studied (Fig. 2A). However, there was a tendency for the steroid-2 group to have higher levels in the cerebral cortex (~30%, P ϭ 0.06) and hypothalamus (~40%, P ϭ 0.06) than in the control and vehicle groups. The levels of 5-HIAA, a metabolite of 5-HT, were higher in the hypothalamus of the steroid-2 group than in all other groups except control (Fig. 2B). Furthermore, the 5-HIAA/5-HT ratio was higher in the hypothalamus of the steroid-2 than all other groups. A higher 5-HIAA/5-HT ratio also was observed in the cerebellum of the steroid-2 and -3 compared with the other three groups (except for the steroid-3 vs control comparison, which was not significant) FIGURE 1—Levels of norepinephrine (NE) (A) and its metabolite (Fig. 3). There were no differences between the control and 4-hydroxy-3 methoxyphenylglycol (MHPG) (B) in the cerebral cortex, vehicle groups for any measurement. Overall, the most hypothalamus, and cerebellum of rats in the control, vehicle, and prominent changes were observed in the steroid-2 group ؎ steroid-1, -2, and -3 groups. Values are mean SE. Letters indicate a Ϫ significant difference from that group at P < 0.05. (3.75 mg·kg 1 dose) (Figs. 1–3).

34 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org FIGURE 3—Percent of 5-hydroxyindole-3-acetic acid/serotonin (5- HIAA/5-HT) in the cerebral cortex, hypothalamus, and cerebellum of rats in the control, vehicle, and steroid-1, -2, and -3 groups. Values are .mean ؎ SE. Letters, same as in Figure 1

Immunohistochemistry. To confirm the biochemical results, we performed c-fos immunohistochemical staining in brain sections of the control, vehicle, and steroid-2 groups, i.e., the AAS-treated group showing the largest biochemical responses. A significantly higher number of c-fos-expressing neurons were observed in the periventricular region of the steroid-2 group (Table 1). Basal expression levels of c-fos, i.e., comparable levels to those shown for the control and vehicle groups in Table 1, were observed in the cerebral cortex, septum, amygdala, and corpus striatum in all three groups (data not shown). A representative photograph of the distribution of c-fos expressing neurons in the periventricular region of the brain is shown in Figure 4. A much denser distribution of c-fos expressing neurons was observed throughout the periventricular region in the rats of the steroid-2 group (Fig. 4C) compared with either the control (Fig. 4A) or vehicle (Fig. 4B) groups.

DISCUSSION An AAS influence on brain reward systems has been suggested previously. For example, increased aggressiveness is one of the typical side effects after AAS treatment in humans (4). Increased aggressiveness also has been observed in AAS-treated animals based on the resident-in- truder paradigm test (3,28). Similarly, AAS-induced defen- FIGURE 4—Photomicrographs (normal contrast, ؋54) of coronal sive aggression has been reported in male rats (17). It has sections of the rat brain showing the distribution of c-fos expressing been reported that the hypothalamus plays an important role neurons in the periventricular region of the hypothalamus of a control (A), vehicle (B), and steroid-2 (C) rat. Note that the highest density of in the expression of aggressive behavior (33) and that the c-fos expressing neurons is in the steroid-2 treated rat (C). Square part N-methyl-D-aspartate receptor is involved in the induction (drawn by dotted line) in D shows the corresponding area in the brain of some types of aggressive response (25). The N-methyl- region taken by the photograph. Scale bar in A, 100 ␮m. D-aspartate receptor subunit mRNA expression levels are significantly reduced after 14 d of AAS treatment (23). The Nandrolone decanoate administration also has been shown androgen action is linked to its ability to bind and activate to increase the immunoreactivity of substance P, a pepti- specific androgen receptors, and the highest density is in the dergic factors associated with enhanced aggression, in sev- hypothalamus of the brain (13). This distribution also is eral brain areas, to include the hypothalamus, amygdala consistent with androgenic influences on the regulation of striatum and periaqueductal gray region (12). All of these gonadotropin secretion and reproductive behavior (13). results are consistent with the possibility that the hypothal-

ANABOLIC-ANDROGENIC STEROID AND HYPOTHALAMUS Medicine & Science in Sports & Exerciseா 35 Ϫ Ϫ TABLE 1. Number of c-fos positive neurons in the periventricular region of mg·kg 1 body weight) or higher (37.5 mg·kg 1 body the hypothalamus. weight) dose. Whether the dose used in the present study is Control (N ϭ 3) 17 Ϯ 4 the optimal dose is unknown. However, it is likely that the Vehicle (N ϭ 3) 35 Ϯ 8 Steroid-2 (N ϭ 3) 239 Ϯ 27* bell-shaped dose-response curve, as observed in the present Values are mean Ϯ SE; * P Ͻ 0.05. study, has also been reported on the brain reward system Number of c-fos positive neurons were counted and expressed mean Ϯ SE/unit using other type of agents such as substance P (11) and area (1.85 ϫ 1.2 mm2). Ϫ neuropsin (22). The administration of 0.375 mg·kg 1 body weight corresponds to the recommended therapeutic dose amus is affected by AAS treatment and with the hypothesis for nandrolone decanoate in humans. However, for the pur- that AAS loading could influence the level of aggressive- pose of increasing muscle mass or strength, it is highly ness (3,12,28,30). The interrelationships among AAS treat- likely that athletes may consume doses that are 10–100 ment, aggressive behavior and elevated NE metabolism in times this therapeutic dose (26). The rats injected with a the hypothalamus (Fig. 1, A and B) are unknown. However, single dose of 3.75 mg·kgϪ1 body weight AAS in the same functional interactions between dopamine, 5-HT, and NE manner as in present study also can perform a significantly levels in the hypothalamus and aggressive behavior in higher work volume during an exhaustive weight-lifting DSP-4 (noradrenergic neurotoxin) treated rats have been session, and their plantaris muscles have a greater fatigue suggested (39). Further study will be needed to clarify these resistance when tested in situ than age-matched untreated interrelationships. rats (35). Elevated NE metabolism in the hypothalamus has been Any relationship between the increase in the metabo- associated with a systemic adrenergic state (15). In the lism of NE and 5-HT observed in the present study and present study, the levels and metabolic rate of 5-HT and its the suggested enhancement of work capacity (8,36) after metabolite 5-HIAA (Fig. 2, A and B, and Fig. 3) were AAS treatment remains unclear. One possibility is that elevated in the hypothalamus after a single dose of AAS, cardiac output and muscle blood flow are increased via suggesting that AAS loading has a strong influence on the sympathetic vasodilator and beta-adrenergic receptors in hypothalamic 5-HT system. A systemic hyper-adrenergic a hyper-adrenergic state. Electrical stimulation of the state also is observed under stress conditions. For example, posterior hypothalamus activates the sympathetic nervous the enhancement of brain 5-HT neurotransmission, i.e., an system, which, in turn, results in an increase in mean augmentation of postsynaptic 5-HT1A receptor function and arterial pressure (MAP), an increase in muscle blood flow 5-HT turnover, contributes to the adaptation to stressful via beta-adrenergic receptors and a decrease in mesen- conditions (5,20). Thus, the observed increases in the 5-HT teric artery blood flow mediated by alpha-adrenorecep- level and metabolic rate in the hypothalamus in the present tors (24). In addition, microinjection of NE in the para- study may reflect a counter action after AAS induced sys- ventricular nucleus of the hypothalamus produces a dose- temic hyper-adrenergic state. Similarly, AAS treatment di- dependent increase both in the systolic and diastolic rectly modulates GABA-A receptor function (27), and both blood pressure (14). Consequently, a hyper-adrenergic 5-HT and GABA can modulate hypothalamically evoked state induced by AAS loading may have profound effects aggression (32,37), thus contributing to a systemic hyper- on a blood flow of skeletal muscle, as well as other adrenergic state. supporting tissues. Another possibility is that an involve- An enhanced state of activation of the hypothalamus after ment of brain serotonergic system. Generally, fatigue of AAS treatment was further suggested by the immunohisto- voluntary muscular effort has been considered on factors chemical staining patterns of c-fos expression (Figure 4, that peripheral fatigue (result in dysfunction of the con- A–C, and Table 1). The number of c-fos expressing neurons traction process within the muscle itself) and CNS fatigue was significantly higher in the periventricular region of the (implication the neurotransmitters, especially 5-HT) (6). hypothalamus of rats in the steroid-2 than in the control and It is likely that an increase in 5-HT synthesis and/or vehicle groups, suggesting a state of hyper-neuronal activity concentrations in various regions of the CNS reduced the (Table 1). Although early Fos expression does not simply endurance capacity during prolonged exercise, related to reflect neuronal activation (29), the enhanced c-fos immu- an increase in plasma free tryptophan (TRP). In the nostaining observed in the present study is consistent with present study, the levels and metabolic rate of 5-HT and the biochemical data suggesting a systemic hyper-adrener- its metabolite 5-HIAA (Fig. 2, A and B, and Fig. 3) in the gic state after a single dose of AAS. Enhanced c-fos ex- resting state were elevated in the hypothalamus after a pression has been observed previously in limbic brain re- single dose of AAS. It is not clear that how this increase gions, areas involved in stress, and behavioral and reward in serotonergic system during resting state affects and/or systems, in the guinea pig after nandrolone decanoate ad- contributes to 5-HT metabolism during intermittent in- ministration (21). These results provide further support for tense exercise and can result on its increased work vol- a role of AAS treatment to aggressive behavior. ume (35). However, it is possible that increased synthesis The present data suggest that there is an optimal dose of of 5-HT in the brain closely related to the plasma TRP AAS to produce these effects, i.e., the influences of AAS metabolism and its transport to brain across blood-brain treatment at a dose of 3.75 mg·kgϪ1 body weight were less barrier. This transport occurs via specific receptors that apparent after the administration of a much lower (0.375 TRP shares with other large neutral amino acids, most

36 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org notably the branched-chain amino acids (BCAA) such as or ammonia levels in the present study. However, it leucine, isoleucine, and valine. We have reported that the seems that AAS treatment can enhance the adrenergic and AAS treatment increased leucine uptake in the muscular serotonergic state in the hypothalamus, which, in turn, component (35), and it may possible that this enhanced can contribute to a highly integrated and complex re- uptake of BCAA means decrease in plasma BCAA con- sponse that includes an increase in aggression and an centrations, and this maybe contribute to greater fatigue augmentation in work capacity. Further studies are resistance. Elevated blood ammonia, which accumulates needed to clarify a possible relations among AAS load- quickly in the brain, is found during brief intense exercise ing, brain neurochemistry, and fatigue (and/or enhanced (2), and increased ammonia can alter CNS function and work volume). be considered to be related to the CNS fatigue. It may also be possible that the AAS-induced enhanced uptake This work was supported by a Grant-in-Aid for Scientific Re- of BCAA also diminishes an increase in plasma ammonia search (B-12480012) from the Ministry of Education, Science, level. Unfortunately, we have no data for plasma BCAA Sports and Culture of Japan.

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