Anabolic Steroids: A Practical Guide - liveriga.info
The aim of this study was to assess if the dose and exposure duration of the anabolic androgenic steroids AAS boldenone BOL and stanazolol ST affected memory, anxiety, and social interaction, as well as acetylcholinesterase AChE activity and oxidative stress in the cerebral cortex CC and hippocampus HC.
Male Wistar rats 90 animals were randomly assigned to three treatment protocols: Each treatment protocol included a control group that received an olive oil injection vehicle control and AAS were administered intramuscularly a total volume of 0. BOL and ST significantly affected social interaction in all protocols. In conclusion, our findings show that the impact of BOL and ST on memory, anxiety, and social interaction depends on the dose and exposure duration of these AAS.
February 14, ; Accepted: May 1, ; Published: This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The authors have declared that no competing interests exist.
Anabolic androgenic steroids AAS form a large class of synthetic androgens that mimic the effects of male sex hormones such as testosterone and dihydrotestosterone. AAS are widely used by athletes to increase muscle mass and enhance physical performance, and by non-athletes for esthetic reasons. Both young athletes and non-athletes may take 10—fold the physiological dose of AAS [ 1 ]. Further, several studies have investigated the potentially severe side-effects of AAS abuse [ 2 , 3 ]. AAS in supraphysiological doses affect several central nervous system- CNS related behaviors such as memory, aggression, anxiety, and depression [ 2 , 4 ].
Studies investigating the mechanisms underlying AAS demonstrated that AAS influence neurotransmission in the CNS by directly affecting the cellular membrane, modulating synthesis and degradation of neurotransmitters, and altering neurotransmitter metabolism [ 5 , 6 ].
In addition, androgen receptors are expressed in brain structures such as the hippocampus HC , amygdala, and cerebral cortex CC. Further, the use of AAS interferes with important signaling and neurotransmission systems, such as glutamatergic [ 7 ], cholinergic [ 8 ], and opioid systems, that modulate animal behavior [ 9 ].
While several controlled studies have described the behavioral changes induced by AAS, these studies primarily used hormone cocktails.
Therefore, the aim of this study was to comparatively and separately assess the effects of the AAS boldenone BOL and stanazolol ST on behavioral tasks to determine if one or both altered learning, memory, anxiolytic-like behavior, and dominant or submissive social behavior. In addition, in an attempt to simulate different user groups, we analyzed the effects of three different treatment protocols that varied in dose and treatment duration to determine their impact on user outcomes. The animals had access to a standard rodent pellet diet and water ad libitum.
Both the vehicle olive oil and AAS were administered intramuscularly a total volume of 0. A representative treatment scheme is shown in Fig 1. The animals were anesthetized using halothane before being euthanized by total exsanguination. Intramuscular injection of vehicle olive oil, 0. On the first day of the evaluation of the behavioral parameters, anxiolytic-like behavior was evaluated using the elevated plus maze DAY 1 as previously described [ 12 ].
The apparatus consisted of a wooden structure raised to 50 cm from the floor, and was composed of four equally sized arms two closed arms [walls 40 cm] and two open arms. The rats were placed on the central platform of the maze facing an open arm, and were allowed 5 min to explore the apparatus.
The time spent and the number of entries in the open and closed arms were recorded. Locomotor and exploratory activities were evaluated in the apparatus used for the object recognition task as previously described [ 13 ]. The number of crossings and rearings was assessed on the first day of exposure to the apparatus habituation day, DAY 2.
During the 5-min open-field session, the number of crossings and rearings was recorded. The object recognition task makes use of the spontaneous tendency of a rat to explore its environment and does not require punishment or reward [ 14 ]. Here, it consisted of three sessions: Briefly, the rats were left to freely explore a square arena length: Twenty-four hours after habituation, the training session was conducted by placing one rat into the arena in which two identical objects objects A1 and A2 were positioned at two adjacent sides of the arena.
The rat was allowed to explore the arena and objects for 10 min. Here, during the test session performed 24 h after the training session, the rats were allowed 10 min to explore the arena in which one of the familiar objects used during the training session was replaced by a novel object object B. The objects were made of odorless plastic and were similar in size.
The rats were tested for agonistic behavior using the resident—intruder paradigm described by Salas-Ramires et al. After a 5-min acclimation period, an age- and weight-matched male intruder was placed among the rats in the vehicle, BOL, and ST home cage. The task was recorded and the recordings were analyzed. The duration and number of occurrences of dominant behavior over the intruder contact team and offensive posturing and dominant behavior over the territory number of flank marks were recorded.
Aggressive behavior was determined by the number of attacks and bites over the intruder. Submissive behavior was determined by the frequency of defensive posturing, walking with tail upward, and escape dashes.
The intruders were used for more than one behavioral test. All rats were tested during the first 4 h of the dark cycle under dimmed red light conditions to control for circadian influences on behavioral responses [ 16 ].
Aliquots of the homogenate were separated. After the addition of DCFH-DA, the medium was incubated in the dark for 1 h until fluorescence was measured excitation at nm and emission at nm, with slit widths of 1. Briefly, the supernatant was diluted 1: Subsequently, the supernatant was incubated with 10 mM DTNB in a final volume of 2 ml, and the absorbance was read at nm. A cysteine solution was used as the reference standard. AChE activity was determined as previously described [ 22 ], with a modification of the spectrophotometric method [ 23 ].
The reaction was initiated by adding 0. The protein concentration was determined beforehand in a piece of the respective brain tissue: Protein concentrations were measured by the Coomassie Blue method [ 25 ] using bovine serum albumin as the standard. The Shapiro-Wilk test was included in the statistical analysis for the determination of the normal distribution of data SPSS Statistics One-way ANOVA followed by Tukey's test was used for the analysis of behavioral results, stress parameters, and enzyme activity tests.
Fig 2 shows the anxiogenic-like behavior of rats in the elevated plus maze. Dominant behavior over territory in rats treated intramuscularly with vehicle olive oil, 0.
In the present study, we measured the physiological effects of BOL and ST using three different treatment protocols. Protocol I consisted of a dose higher than the recommended dose administered over a shorter time interval. Protocol II consisted of a moderate dose administered over an intermediate period, and Protocol III consisted of a reduced dose administered over an extended period [ 26 ].
These protocols were based on steroid user groups known to use widely ranging steroid doses and treatment durations [ 1 , 27 ].
Our research focused on comparing the behavioral changes in rats receiving BOL and ST according to the three protocols described. Locomotion parameters were unchanged in the rats receiving BOL and ST, regardless of the treatment protocol used.
In contrast, Bronson and Bronson [ 28 ] and colleagues [ 29 ] have reported that female mice treated with a combination of AAS at low and high doses for either 9 weeks or 6 months high dose only exhibited significantly reduced spontaneous activity in a running wheel relative to that observed for the controls.
Further, these authors hypothesized that female-specific effects of AAS on locomotion might reflect AAS antagonism of estrogen-induced spontaneous activity [ 28 , 29 ]. Here, we showed that the anxiety levels were significantly increased in the rats receiving BOL and ST according to Protocol I since these rats spent less time in the open arms of the elevated plus maze. Bitran and colleagues performed the first study of the effects of AAS on anxiety [ 30 ]. Another study showed that high doses of testosterone propionate induced an anxiogenic effect after 6 days of treatment; however, after 14 days of treatment the animals did not behave differently from those in the control group [ 31 ].
These results are indicative of a transitional character treatment, which might explain the fact that the relatively longer treatments at a relatively low dose used in our study did not increase anxious behavior. Nevertheless, Minkin and colleagues showed that nandrolone administered for 8 weeks increased anxious behavior, contradicting the findings of anxiolytic activity of some steroids [ 32 ].
Dominant behavior over the intruder and territorial dominance increased in all protocols tested, while submissive behavior was not affected by the BOL and ST treatments according to Protocol I and III. However, submissive behavior events decreased only when the animals were exposed to the higher dose of ST PI. The findings of Kalinine et al. Furthermore, it is plausible that functional or structural abnormalities in these regions can increase the susceptibility to impulsive aggression and violence [ 34 ].
AAS also affect aggressive behavior in rodents [ 35 ]. Single compounds such as testosterone and testosterone propionate increase dominance and aggression in healthy adult male rats and mice [ 36 , 37 ]. Further, adolescent male Syrian hamsters exposed to a cocktail of AAS for 2 or 4 weeks showed increased aggressive behavior [ 10 ].
Consequently, the choice of a specific protocol for drug administration both time and dose dependent , as well as the type of AAS used can affect the brain differently, resulting in a greater or lesser degree of dominance and aggressiveness.
The array of physiological and behavioral effects of these chemically disparate drugs is vastly compounded not only by complexity in the patterns of self-administration, but also by the heterogeneity of the subjects that take these drugs [ 38 , 39 ].
Symptoms resulting from the chronic use of supra-therapeutic doses of AAS include mania, increased anxiety, irritability, extreme mood swings, and abnormal levels of aggression, depression, and even suicide. These findings support our data that clearly show different behavioral responses of the rats treated with BOL and ST depending on the exposure duration and the dose.
However, to investigate the effect of BOL and ST on cognitive processes, additional experimental techniques can be used. Since gonadal hormones are known to play a crucial role in cognitive processes such as spatial learning performance [ 41 ] and extinction responses in a passive avoidance task [ 42 ], it is conceivable that BOL and ST also influence cognitive functions.
In particular, the current literature contains several controversial data regarding the absence of cognitive disorders in animals treated with AAS [ 43 , 44 ], while other studies have clearly shown that individuals abusing AAS have a high risk of developing cognitive disorders and important morphological changes in the amygdala, HC, and forebrain [ 45 , 46 ].
Thus, the present study evaluated memory performance of rats treated with BOL and ST in the object recognition task. Therefore, the use of BOL at a reduced dose but for a relatively long duration had a higher impact on learning and memory compared to that observed for ST treatment.
The biochemical mechanisms underlying the effects of AAS are still poorly understood. The mesocorticolimbic dopaminergic pathway is considered to play an important role in the reinforcement circuitry of the brain [ 47 ], and a connection between AAS and central dopaminergic and serotoninergic activity has been found in animal studies [ 48 , 49 ]. However, only little information is available to explain the potential role of the cholinergic system in the biochemical mechanisms underlying the CNS effects of AAS.
Such information is, however, especially important given that the cholinergic system plays an essential role in memory formation and learning [ 50 , 51 ], and perhaps also in the behavioral changes we observed according to the type, treatment duration, and dose of the AAS used.
AChE is an important enzyme that regulates the concentration of acetylcholine in the synaptic cleft. These results are supported by those of other studies that have shown that the anabolic androgenic steroid methandrostenolone changes the expression of neuronal growth factor NGF and its receptors while reducing the activity of choline acetyltransferase ChAT. Methandrostenolone also induces the synthesis of acetylcholine in the basal forebrain and impairs behavioral performance in the Morris water maze task [ 46 ].
Experimental studies in animals suggest that the redox status may be involved in AAS-induced neurotoxicity [ 54 ]. We hypothesize that both BOL and ST cause a physiological adaptation via changing of the redox status homeostasis. The main limitations of this preliminary study were that we did not explore the underlying molecular mechanisms of the effects observed, and that we did not assess the expression of superoxide dismutase and glutathione peroxidase and reductase in the brain.