Anabolic androgenic steroids (AAS) are synthetic compounds derived by selective chemical manipulations of the 19-carbon testosterone molecule. These modifications affect the pharmacokinetics of the resulting molecule (e.g., orally active compounds), as well as the ratio of the anabolic/androgenic effect of the parental molecule. Although some of these agents are useful to treat profound body wasting in the elderly or in HIV patients and to counteract pathological conditions characterized by low amount of testosterone (e.g., delayed puberty or some type of impotence), they are often abused by bodybuilders and athletes of both sexes seeking to enhance their performance. AAS abuse has been coupled with several medical complications, such as sterility, gynecomastia, and increased risk of cardiovascular and hepatic disease (for a review, see the study of Hartgens and Kuipers ). In spite of these well characterized metabolic and cardiovascular adverse effects, few studies have examined the potential toxic effects of AAS on the CNS in nonclinical setting. In particular, investigations of the neuropsychiatric effects of AAS have been hampered by several methodological issues such as the lack of placebo control, concomitant drug coadministration, as well as that many steroid users use multiple steroids simultaneously, a practice known as “stacking.” The first published placebo-controlled prospective study demonstrated in male volunteers a significant increase in symptom scores during methyltestosterone administration compared with baseline in positive mood, negative mood, and cognitive impairment (36). Subsequent studies have confirmed the emergence of cognitive symptoms after AAS administration (9). Recent experimental studies have shown that AAS is neurotoxic on their own (4) and amplify excitotoxic neuronal death (28), a mechanism that is largely implicated in the pathophysiology of acute and chronic neurodegenerative disorders. Nerve growth factor (NGF) is a member of the neurotrophin family that promotes the differentiation, growth, and survival of specific neuronal populations during development and in the adulthood. NGF exerts its actions through the activation of two cell surface receptors: (i) the TrkA receptor, which is endowed of intrinsic tyrosine kinase activity and (ii) the low affinity p75 neurotrophin receptor (p75NTR), which is also activated by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (32). Activation of TrkA supports neuronal survival and promotes neuronal repair via the activation of the phosphatidylinositol-3-kinase/Akt pathway and other mechanisms (27,35). There are only a few studies examining the effect of ASS treatment on the neurotrophin system in the brain. Tirassa et al. (38) have found that chronic treatment with high doses of testosterone or 19-nortestosterone (nandrolone) causes an increase in NGF levels in the hippocampus and septum of adult rats. Testosterone and nandrolone had opposite effect on the expression of p75NTR (expression of TrkA was not examined) (38). Using a well-characterized treatment protocol (2,5,7,22) that uses an administration regimen and doses equivalent to those usually taken by AAS abusers (13,29), we have found that nandrolone and stanozolol reduced BDNF expression in the hippocampus and prefrontal cortex, and induced depressive-like behavior in the forced swim test (24). Two specific questions were raised in this study. First, what are the effects of exposure to supraphysiological doses to AAS on NGF status? Second, are cognitive impairments associated with AAS long-term use? Thus, we decided to examine the effects of nandrolone and stanozolol on the expression of NGF and its receptors, and because NGF appears to be uniquely important for the development and maintenance of the basal forebrain cholinergic neurons, we extended the analysis to the specific cholinergic marker, choline acetyltransferase (ChAT), and to spatial learning and memory using the Morris water maze.
Nandrolone and stanozolol were purchased from Sigma-Aldrich (Italy). Nandrolone (5.0 mg·kg−1) and stanozolol (5.0 mg·kg−1) were dissolved in sesame oil and injected s.c. in a volume of 1.0 mL·kg−1. The doses of nandrolone and stanozolol we have used (5.0 mg·kg−1·d−1) are considered equivalent to those usually taken by AAS abusers on a milligram per kilogram of body weight basis (2,7).
Experimental design and drug treatments
Adult male Wistar rats (250–275 g, body weight; Charles River, Italy) were housed two to three per cage (42 × 27 × 16 cm) under controlled conditions (temperature, 24°C ± 2°C), on a 12-h light/dark cycle (light on at 7:00 a.m.) with food (standard 4RF21 diet, Charles River) and water ad libitum. Experiments were performed between 9:00 a.m. and 12:00 a.m. and were carried out according to the European (86/609/EEC) and Italian (D.Lgs 116/92) guidelines on animal care. All efforts were made to minimize animal suffering, to reduce the number of animal used, and to use alternatives to in vivo techniques. In a first series of experiments, three groups of rats (n = 6 per group) were injected s.c. once a day for 28 d with vehicle (sesame oil), nandrolone or stanozolol. These groups of animals were used for measurements of NGF levels in the hippocampus and basal forebrain. Three additional groups of rats (n = 6 per group) were treated once a day for 28 d as described previously. These animals were used for measurements of TrkA, p75NTR, and ChAT protein levels in the hippocampus (p75NTR) and basal forebrain (TrkA, p75NTR, and ChAT). For these biochemical studies, rats were euthanized 24 h after the last injection. Additional groups of rats (n = 10 per group) were treated with AAS as mentioned and used to assess spatial learning and memory using the Morris water maze 24 h after the last injection.
Measurements of NGF levels
NGF levels were measured in the hippocampus and basal forebrain. The basal forebrain region was dissected from the coronal brain slice that spanned approximately 0.20–0.70 mm relative to bregma. The basal forebrain, including the medial septum and diagonal band, was dissected bilaterally around the midline up to the borders of the ventral pallidum and the shell of the nucleus accumbens. Subsequently, the brain stem and cerebellum were removed with a spatula and forceps to reveal the ventral surface of the hippocampus. A spatula was inserted underneath the hippocampus, and the septal and temporal ends were severed to gently remove the whole hippocampus. Tissues were stored at −80°C. NGF protein levels were measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (NGF EmaxTM Immunoassay System; Promega, Italy). In brief, tissue was sonicated in a lysis buffer (100 mM Tris–HCl, 400 mM NaCl, 2% BSA, 0.05% NaN, 1 mM PMSF, 7 μg·mL−1 aprotinin (6000 kIU·mg−1, ICN), 4 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0). Homogenates were centrifuged at 12,000g at 4°C for 30 min, and supernatants were diluted with a buffer supplied by the manufacturer. ELISA was performed in 96-well plates (MaxiSorp, Nunc, Denmark). The antibodies used have negligible cross-reactivity (<3%) with other growth factors including BDNF, NT-3, and NT-4/5. All samples were assayed in duplicate. Colorimetric detection of peroxidase activity was achieved by adding TMB solution and peroxidase substrate, after a 10-min incubation at room temperature, according to the manufacturer. The enzymatic reaction was stopped with HCl (1.0 M), and the optical density of each well was measured at 450 nm using a Bio-Rad Model 550 Microplate Reader (Bio-Rad, Hercules, CA). Protein levels were measured as described by Bradford (1).
Western blot analyses
p75NTR, TrkA, and ChAT protein levels were determined by Western blot analysis. Tissues were homogenized at 4°C in Triton X-100 lysis buffer containing 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg·mL−1 leupeptin, and 10 μg·mL−1 aprotinin. Homogenates were centrifuged at 12,000g at 4°C for 30 min, and the soluble protein fraction levels in the supernatants were measured as described by Bradford (1). Aliquots of the supernatants containing 70 μg of proteins were resuspended in sodium dodecyl sulfate (SDS)–bromophenol blue buffer containing 40 mM dithiothreitol. Samples were then separated by electrophoresis on 8% SDS polyacrylamide gels (Protean II xi, Bio-Rad). Proteins were transferred to nitrocellulose membranes (Amersham Bioscience, Italy) in 35 mM Tris, 192 mM glycine, and 20% methanol at 450 mA for 4 h. Membranes were then incubated for 1 h in a blocking solution containing 0.5% (w/v) Tris-buffered saline, 1% (w/v) Tween-20, nonfat milk, and 1% (w/v) bovine serum albumin. Afterward, blots were incubated overnight at 4°C in blocking solution with a rabbit polyclonal anti-TrkA antibody (1:500, Millipore, Billerica, MA), with a rabbit polyclonal anti-p75NTR antibody (1:500, Millipore) or with a mouse monoclonal anti-ChAT antibody (1:500; Chemicon, Temecula, CA). Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:5000, Amersham Bioscience) for TrkA and p75NTR, and goat–anti-mouse IgG peroxidase conjugated (1:2000, Chemicon) for ChAT. Expression of β-actin was detected by a primary monoclonal antibody (Sigma-Aldrich). Immunoreactivity was assessed by an enhanced chemiluminescence system (Amersham Biosciences). Digitized images of immunoreactive bands were acquired and measured using the Image J software (National Institute of Health, Bethesda, MD).
Morris water maze
Spatial learning was investigated using the behavioral approach described by Morris (25) with minor modifications. A gray circular pool (diameter = 120 cm, height = 60 cm) was filled with water maintained at 26°C ± 0.5°C; the water level in the pool was 40 cm. A Plexiglas transparent platform (11 × 11 cm) was submerged 2 cm below the surface of water. The maze was in a room (3 × 3 m) exclusively used for these behavioral studies with a constant environment and visual cues on curtain walls around the pool. Above the pool, a video camera was mounted to record the animal behavior and to allow thorough analysis of each trial. Each day, four training trials were conducted for five consecutive days. At the beginning of each trial, an animal was started at one of four different starting points, in random order (designated north, east, south, and west). The rat was carefully placed in the water and positioned to face the wall of the pool; it was allowed to locate the hidden platform, which was situated in the southeast quadrant during all training trials. A maximum time of 120 s was allowed for each trial, and when the rat did not locate the platform within the maximum time, it was guided to the platform and allowed to remain on the platform for 30 s. Eight to 10 d later, the platform was removed and the animals underwent a 60-s probe trial to determine the extent to which they had learned the location of the platform. The amount of time the rats spent in each of the four quadrants was recorded.
Data were analyzed using one-way ANOVA (with repeated measures when indicated) after Bartlett test for the homogeneity of variances and Kolmogorov–Smirnov test for the Gaussian distribution followed by the Dunnett multiple comparisons test. Data from water maze were subjected to a mixed-model ANOVA with one between-subject factor (treatment at three levels) and one repeated-measures factor (time at five or three levels). Bonferroni correction was used for multiple comparison. P < 0.05 was used as the criterion for statistical significance.
AAS treatment did not affect body weight gain
Chronic AAS treatment did not influence body weight gain (data not shown). All rats progressively gained weight over the 28-d period of observation (day effect, F 4,89 = 599.3, P < 0.001), with no difference between control and AAS-treated rats (P > 0.05) as previously reported for nandrolone (23). AAS-treated rats did not show gross behavioral abnormalities in their home cage.
Effects of chronic AAS treatment on NGF levels
The effects of AAS treatment on NGF levels in the hippocampus and basal forebrain are shown in Figure 1. In control rats, NGF levels detected by ELISA were 0.74 ± 0.04 ng·mg−1 prot. in the hippocampus and 2.57 ± 0.24 ng·mg−1 prot. in the basal forebrain (mean ± SEM, n = 6). Treatment with either nandrolone or stanozolol significantly increased NGF levels in the hippocampus and reduced NGF levels in the basal forebrain.
Effects of chronic AAS treatment on NGF receptor expression
The expression of p75NTR and TrkA proteins were determined by Western blot analysis in the hippocampus and basal forebrain. Immunoblot analysis with p75NTR and TrkA antibodies showed a major band at the expected molecular size (75 and 140 kDa, respectively). Treatment with nandrolone or stanozolol reduced p75NTR expression in the hippocampus with no changes in the basal forebrain (Fig. 2). TrkA expression is known to be very low in hippocampal neurons (21). Hence, we limited the analysis of TrkA to the basal forebrain. Neither nandrolone nor stanozolol changed the expression of TrkA receptor in the basal forebrain (Fig. 3).
Effects of chronic AAS treatment on ChAT expression
Western blot analysis was used for the detection of ChAT in the basal forebrain. Immunoblot showed a single immunoreactive signal at 67 kDa corresponding to the molecular size of ChAT. A significant decrease in ChAT expression was seen in rats treated with either nandrolone or stanozolol for 28 d (Fig. 4).
Effects of chronic AAS treatment on spatial learning
Results obtained in the Morris water maze are shown in Figure 5. During the training phase, control and AAS-treated rats showed a similar robust decrease in the latency to reach the platform across test sessions. A main effect of trial (F 4,28 = 126.8, P < 0.001) was found, whereas treatments did not change the performance (F 2,28 = 0.54, not significant). In contrast, a significant effect of treatments was found in the probe trial (F 2,28 = 20.18, P < 0.001). Multiple comparisons showed a significant difference between vehicle and nandrolone-treated animals on the second and the last probe day (P < 0.001). Stanozolol also influenced learning capacity because stanozolol-treated rats spent significantly less time in the quadrant in which the platform had previously been positioned as compared with nandrolone or vehicle-treated rats. This effect was observed from the first probe day (P < 0.001) and was still present the second (P < 0.01) and the last probe day (P < 0.001). No differences were recorded in swimming speed and the average distance swum among the experimental groups (data not show).
Here, we have demonstrated that nandrolone and stanozolol, two of the most frequently abused AAS, caused divergent effects on NGF and p75NTR levels in the hippocampus and basal forebrain, without changing the expression of the high-affinity NGF receptor, TrkA, in the basal forebrain. Remarkably, nandrolone and stanozolol were injected for 28 d at doses that are considered to be equivalent to dose usually taken by AAS abusers. Our data on hippocampal NGF levels are consistent with those obtained by Tirassa et al. (38) using a 6-wk treatment regimen with high doses of testosterone and nandrolone. Thus, the hippocampal NGF system appears to be particularly vulnerable to the effects of AAS. NGF is known to be retrogradely transported by cholinergic terminals from the hippocampus to the basal forebrain (septum and diagonal band), where the cell bodies of cholinergic neurons are localized (16,34). As demonstrated by in vivo and in vitro approaches, NGF improves neuronal survival, increases sprouting of cholinergic neurons (11,15), and rescues cholinergic cells from lesion-induced atrophy (6). The progressing memory deficits associated with aging have been related to degenerative changes of cholinergic neurons of the basal forebrain, resulting in cholinergic hypofunction (reviewed by Schliebs and Arendt (33)) according to the cholinergic hypothesis of memory dysfunction in senescence and Alzheimer diseases (35). Our finding that NGF levels were reduced in the basal forebrain of rats treated with nandrolone or stanozolol suggests that AAS caused an impairment of the retrograde transport of NGF from the hippocampus to the basal forebrain. This hypothesis fits nicely with the reduction of ChAT expression (a specific marker of cholinergic neurons) found in the basal forebrain of AAS-treated rats. ChAT is the specific enzyme forming acetylcholine from acetyl-S-coenzyme-A and choline, and its reduced expression may reflect either a dysfunction or a loss of cholinergic neurons. Studies combining measurements of acetylcholine release by microdialysis and stereologic counting of cholinergic neurons will shed further light onto the precise effect of AAS treatment on the septohippocampal cholinergic system. Both TrkA and p75NTR are involved in the retrograde transport of NGF. p75NTR interacts with TrkA by increasing the affinity and specificity of TrkA for NGF and by enhancing TrkA autophosphorylation in response to NGF (39). We speculate that a defect in p75NTR expression in hippocampal cholinergic nerve terminals underlies the impaired transport of NGF to the basal forebrain.
The cholinergic neurons projecting from the basal forebrain to the hippocampus have an established role in spatial learning (10,26). Transgenic mice expressing a neutralizing anti-NGF antibody, which are used as a model for Alzheimer disease, show an early cholinergic deficit in the basal forebrain and an impairment of spatial memory in the eight-arm radial maze (3). Our rats treated with AAS showed a specific defect in retention of spatial memory, as reflected by an accelerated memory extinction in the Morris water maze. In particular, stanozolol-treated rats exhibited, already during the first extinction session, a reduced time spent in the quadrant where the platform was positioned, whereas the same effect was observed in nandrolone-treated rats from the second day of tests. Nandrolone and stanozolol have comparable myotropic and androgenic activities (20); thus, at present, it is difficult to explain this time-dependent difference in the onset of memory extinction. It has been demonstrated that the characteristic of androgen receptor (AR) binding, as measured by ligand-binding assay, does not fully explain the biological activity of AAS (12). For example, it has been reported in transactivation studies of androgen-responsive luciferase reporter gene that there is a remarkable difference between equimolar doses of nandrolone and stanozolol on induction of MMTV-luc reporter gene (17). Thus, the difference between nandrolone and stanozolol on the extinction of spatial memory in the Morris test may not be related to the binding capacity of both compounds to AR but may rely on additional mechanisms that remain to be identified. Nevertheless, our behavioral findings are consistent with those found by Magnusson et al. (23), who also found a deficit in spatial memory in rats after nandrolone exposure. The following scenario might have occurred in our AAS-treated rats (Fig. 6): (i) a reduced expression of p75NRT in the hippocampus might have impaired the retrograde transport of NGF, as reflected by the increased NGF levels in the hippocampus and the reduced NGF levels in the basal forebrain; (ii) the reduced availability of NGF in the basal forebrain might have caused a specific impairment of cholinergic neurons in the basal forebrain; and (iii) this might have caused the deficit in spatial memory observed in these animals. Interestingly, a similar scenario has been proposed in mutant mice modeling Down syndrome (30), which show an impairment of spatial memory in the Morris water maze (18). In these mice, NGF levels are increased in the hippocampus and reduced in the septum, suggesting a disconnection between NGF production and its retrograde transport (8).
The identification of the precise mechanism underlying changes in NGF and neurotrophin receptors expression in response to administrations is beyond the scope of the present study. We have previously reported that nandrolone, at suprapharmacologic doses, is toxic to cultured cortical neurons (4) and causes abnormalities in BDNF expression (24). Both these effects were prevented by the AR blocker flutamide. Because AR has been identified in the hippocampus and in the basal forebrain (31), both brain areas are potentially candidates responsible for the biochemical and behavioral consequence of AAS exposure. Hence, it is reasonable to hypothesize that the effect induced by AAS observed in the present study is related to overactivation of AR. However, this hypothesis remains to be tested. In the male rat, AAS treatment markedly increased serum estradiol-17 beta, an effect that is probably associated with the peripheral and central conversion of AAS to estrogens (37). A relationship between NGF and estrogen has been established. In particular, it has been reported that estrogen potentiates hippocampal NGF expression in the inflamed temporomandibular joint model (40) and that estrogen deficiency can lead to a decrease of NGF expression in this limbic structure (19). Thus, the possibility of a participation of estrogen in the AAS effects observed in the present study should not be overlooked.
In conclusion, our data strongly suggest that treatment with AAS under conditions that recapitulate AAS long-term use in humans causes a defective retention of spatial memory that may result from a dysregulation of the NGF system with an ensuing impairment of the septohippocampal cholinergic pathway. Whether these defects are transient or permanent remains to be determined. Our data raise the concern that AAS long-term use in humans may cause neuropathological consequences reminiscent of those seen in Alzheimer disease or other forms of dementia.
This study was supported by a grant from Ministero della Salute.
All the authors have declared that there is no conflict of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:©2013The American College of Sports Medicine
NERVE GROWTH FACTOR; ANABOLIC ANDROGENIC STEROIDS; HIPPOCAMPUS; BASAL FOREBRAIN; NEUROTROPHIC UNBALANCE; NGF RECEPTORS