INTRODUCTION
Anabolic Androgenic Steroids (AASs) abuse is a prevalent social problem and
no longer limited to professional athletes. Adolescents abuse AAS to increase
muscular development, improve physical fitness and tolerance to high intensity
training (Hartgens and Kuipers, 2004; Talih
et al., 2007; Matrisciano et al., 2010).
More recently, the negative psychiatric side effects of AASs use have received
attention (Lumia and McGinnis, 2010). AASs use has
been associated with a propensity for indiscriminate and unprovoked aggression
and violence in humans. This gratuitous display of aggression and violence has
been referred as "roid rage" (Trenton and Currier, 2005).
Today, violence and aggression are the most serious problems facing society
(Wood et al., 2012).
There is an increased awareness of the fact that specific nutritional substances
are thought to influence physiological functions in the body to improve performance
(Maughan and Shirreffs, 2012; Tokaev
et al., 2011). Among these, amino acids have proven to be effective
in improving performance, reducing fatigue and maintaining a favourable physical
condition (Bishop, 2010; Colombani
and Mettler, 2011). Indeed, the use of amino acids supplements is gaining
popularity in sport community with a commonly held view that they are safe and
free of toxic effects. While several studies reported side effects of amino
acids mixtures such as impairment of hepatic and renal function, little is known
about possible effects of amino acids mixture on behaviour.
However, less attention has been paid to the effects of stacking combinations of anabolic steroids and amino acids on behaviour and neural circuits that underlie these behavioural effects.
In this context, the present study aimed to assess how AASs and amino acids
interaction affect behavioural and physiological responses in rats since amino
acids have both direct and indirect effects on neurotransmitters. The studied
agents were administered either separately or in combination to accurately reflect
human abuse paradigm.
EXPERIMENTAL PROCEDURES
Animals: The experiments were carried out using adult male albino rats, weighing 120-140 g. Animals were obtained from the animal house (National Research Centre, Giza, Egypt). All animals were housed under conventional laboratory conditions throughout the period of experimentation and fed standard laboratory pellets (20% proteins, 5% fats, 1% multivitamins) and allowed free access to tap water. Animals were allowed at least one week of acclimatization before using them. Experimental protocols were approved by the Research Ethical Committee of the National Research Centre (Giza, Egypt).
Drugs: Nandrolone Decanoate (Nile Pharmaceutical Company, Egypt) was
dissolved in arachis oil and injected intramuscularly in a dose of 10 mg kg-1,
once per week (Kurling et al., 2005). Amino acids
mixture (MEPACO Pharmaceutical Company, Egypt) was dissolved in 1% tween 80
in water and used in a dose of 0.8 g kg-1, p.o., once daily, 5 days
week-1 (Gibala, 2002).
Animal treatment: Rats were randomly assigned to one of six treatment groups; each consisting of 10-12 rats that were treated according to the following scheme: Group 1: received arachis oil, i.m. and served as nandrolone decanoate control, Group 2: received nandrolone decanoate (10 mg kg-1, i.m.), Group 3: received 1% tween 80, p.o. and served as amino acids control, Group 4: received amino acids mixture (0.8 g kg-1, p.o.), Group 5: received arachis oil and 1% tween 80 and served as combination control and Group 6: received nandrolone decanoate (10 mg kg-1), i.m. and amino acids mixture (0.8 g kg-1, p.o.). All groups were injected with test agents for 8 weeks.
Basal counts of open field and hot plate tests were recorded at zero time before injection. Behavioural tests were measured at the end of experiments, 24 h after last treatment in the following order: open field, defensive aggression and hot plate tests. All behavioural tests were carried out between 8 am and 1 pm to avoid the effect of diurnal variation.
At the end of experiments, animals were weighed and sacrificed by decapitation 48 h following the last treatment. Brains were rapidly excised, transferred to a dry ice-cold glass plate and dissected into different brain regions (cerebral cortex, striatum, hippocampus and hypothalamus). Brain samples were stored at -80°C till analysis of the following neurotransmitters: 5-HT, DA, NE and glutamate.
Behavioral testing
Open field test: Animals were tested in an open field arena using square
wooden arena (80x80x40 cm high) divided into 16 equal squares (Pruus
et al., 2002). Each rat was placed at the same corner square and
observed during 10 min. Parameters measured were: Ambulation frequency (e.g.,
number of squares crossed by the animal, rearing frequency (e.g., number of
times the animal stood stretched on its hind limbs with or without forelimb
support) and grooming frequency (e.g., number of face scratching, washing with
the hind limbs and licking of the forelimbs).
Defensive aggression test: The rat was lifted by its tail and placed
in a plexiglas cage (60x31x41 cm high) and allowed to habituate for 30 sec.
The rats’ reaction to five different stimuli was then assessed according
to Johansson et al. (2000).
The hot plate test: Each animal was gently placed onto a 52±0.1°C
hot plate to perform the test. Latency to exhibit nociceptive responses, such
as licking paws or jumping off the hot plate was determined at zero time (pre-treatment)
and 24 h after last injection of test drugs or their corresponding controls
(Woolfe and McDonald, 1944).
Neurochemical analyses: The following endogenous compounds in various
brain regions were determined by the use of High Performance Liquid Chromatography
(HPLC) according to the method of Pagel et al. (2000):
NE, DA and 5-HT. Prior to analysis, brains were homogenized in 1/5 wt/v of 75%
aqueous methanol to obtain 20% homogenate. Each homogenate was centrifuged at
3000 rpm (4°C) for 10 min. The supernatants were injected onto AQUA column
C18 (150x4.6 mm I.D., 5 μm) purchased from Phenomenex, USA. Glutamate concentration
was determined by mass spectrometry/electron spray ionization technique (MS/ESI)
technique according to Zoppa et al. (2006).
Statistical analysis: Results were expressed as Means±S.E. Comparisons between means were carried out using one-way ANOVA test followed by Tukey-HSD post hoc analysis. The level of significance was set at p<0.05. Graph pad Prism software (version 5) was used to carry out all statistical tests.
RESULTS
Behavioral tests
Defensive aggression: Administration of nandrolone decanoate or amino
acids mixture increased defensive aggression scores to about 100% as compared
to control group. Meanwhile, the combination of nandrolone decanoate and amino
acids mixture increased defensive aggression by 126.7% as compared to their
respective control (Fig. 1).
Open field test
Ambulation frequency: Administration of nandrolone decanoate did
not alter the ambulation frequency, while administration of amino acids mixture
markedly depressed the ambulation frequency to about 63% as compared to control
(38% vs. 104%, p<0.001). Administration of both nandrolone decanoate and
amino acid mixture significantly decreased ambulation frequency to about 60%
as compared to control (Fig. 2a).
Rearing frequency: Nandrolone decanoate depressed the rearing frequency
to nearly 45% of the control value (58.7% vs. 108.2%, p<0.001), while oral
administration of amino acids mixture showed marked increase in rearing frequency
reaching 146.2% of the control values (265.9% vs. 108.23, p<0.001).
|
| Fig. 1: |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on defensive aggression, Data are Mean±SE,
*Significantly different from corresponding control group at p<0.05 |
|
| Fig. 2(a-c): |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on, (a) Ambulation, (b) Rearing and (c) Grooming
frequencies in open field test, Data are Mean±SE, *Significantly
different from corresponding control group at p<0.05 |
Administration of both drugs did not affect rearing frequency as compared to
sedentary control group (Fig. 2b).
|
| Fig. 3: |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on reaction time in hot plate test, Data are
Man±SE |
Grooming frequency: Grooming frequency was not altered in rats receiving nandrolone decanoate at a dose of 10 mg kg-1 for 8 weeks. On the other hand, rats receiving amino acid mixture exhibited marked elevation in grooming frequency reaching 70% as compared to control group (211.7% vs. 124%, p<0.001). Administration of both nandrolone decanoate and amino acid mixture did not affect grooming frequency (Fig. 2c).
Hot plate test: None of the studied drugs at the given doses, significantly altered reaction time in hot plate test as compared to their respective sedentary control group (Fig. 3).
Neurochemical assays
Serotonin: Rats receiving nandrolone decanoate showed decreased serotonin
content in striatum (0.6 vs. 9.4, p<0.001) and hippocampus (0.3 vs. 1.5,
p<0.01), while no significant change occurred in both cerebral cortex and
hypothalamus as compared to control group. Oral administration of amino acids
mixture resulted in a decrease in serotonin content in striatum (2.7 vs. 9.4,
p<0.001) and hypothalamus (0.04 vs. 0.18, p<0.01). On the other hand,
administration of combination of nandrolone decanoate and amino acids significantly
decreased serotonin content in all studied regions as compared to the control
values (Fig. 4a-d).
Dopamine: Treatment with nandrolone decanoate resulted in increased
dopamine content in hypothalamus (0.9 vs. 0.4, p<0.01), as compared to control
while treatment with amino acids increased dopamine content in hippocampus only
(2.2 vs. 1.3, p<0.05). However, a decrease in dopamine content in cortex
(9.05 vs. 12.4, p<0.05) and striatum (2.2 vs. 4.04, p<0.01) was found
after treatment with combination of nandrolone decanoate and amino acids for
8 weeks as compared to control group (Fig. 5a-d).
Norepinephrine: Intramuscular injection of nandrolone decreased norepinephrine
contents in hippocampus (0.3 vs. 0.6, p<0.05) only, as compared to control
rats. Oral administration of amino acids mixture increased norepinephrine contents
in striatum (1.14 vs. 0.65, p<0.01) and hypothalamus (0.34 vs. 0.09, p<0.01),
as compared to control.
|
| Fig. 4(a-d): |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on serotonin content in, (a) Crebral cortex,
(b) Sriatum, (c) Hippocampus and (d) Hypothalamus, Data are Mean±SE,
*Significantly different from corresponding control group at p<0.05,
@Significantly different from nandrolone-treated group at p<0.05, #Significantly
different from amino acids-treated group at p<0.05 |
However, combination of both nandrolone and amino acids mixture resulted in
a significant reduction in norepinephrine content in hippocampus (0.3 vs. 0.6,
p<0.05) as compared to control rats (Fig. 6a-d).
Glutamate: Nandrolone decanoate did not alter glutamate content in cerebral
cortex, while it increased significantly in hippocampus as compared to control
group (50 vs. 25, p<0.05). Oral administration of amino acids mixture did
not affect glutamate content in either cortex or hippocampus as compared to
control group. Combination of both nandrolone decanoate and amino acids mixture
significantly increased glutamate content in cortex (17 vs. 8.7, p<0.01)
and hippocampus (64.3 vs. 25, p<0.05) as compared to control group (Fig.
7a, b).
DISCUSSION
To date, possible effects of combined administration of nandrolone decanoate
and amino acids mixture on behavior have never been explored in animal model.
The major finding in the present study is that both nandrolone decanoate and
amino acids increased defensive aggression scores and their combination further
elevated this response. These effects correlated with decrease in 5-HT content
in rats receiving either nandrolone decanoate, amino acids or both of them.
Moreover, nandrolone affects dopaminergic system. In particular DA content increased
in cortex and hypothalamus.
|
| Fig. 5(a-d): |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on dopamine content in, (a) Cerebral cortex,
(b) Striatum, (c) Hippocampus and (d) Hypothalamus, Data are Mean±SE,
*Significantly different from corresponding control group at p<0.05,
@Significantly different from nandrolone-treated group at p<0.05, #Significantly
different from amino acids-treated group at p<0.05 |
Chronic treatment with supratherapeutic doses of nandrolone decanoate mainly
reflects a stimulatory influence on the mesolimbic DA system rather than the
nigro-striatal DA system. Stimulation of the mesolimbic DA system is known to
be related to reinforcement of behavior so enhanced hyperactivity of the DA
system might account for some positive effects as euphoria, increased self-esteem
and confidence that frequently appear as early effects following the administration
of AASs in humans (Kurling et al., 2005).
Oral administration of amino acids mixture increased defensive aggression.
This can be explained by the fact that ingestion of tryptophan (TRP)-free amino
acids mixtures in laboratory animals leads to extremely rapid changes in plasma
TRP and brain serotonin (5-HT) content with maximal reductions of brain 5-HT
occurring within 2 h. It also alters behavioral indices of 5-HT function (increasing
pain sensitivity, acoustic startle, motor activity and aggression) (Bell
et al., 2001).
Administration of amino acids mixture decreased 5-HT, increased DA in hippocampus
only and increased NE in striatum and hypothalamus. The synthesis of neurotransmitters
in mammalian brain responds rapidly to changes in precursor availability. 5-HT
synthesis depends largely on the brain concentrations of L-TRP, its precursor
amino acid. Similarly, the synthesis of catecholamines (e.g., DA and NE) in
the brain varies with the availability of the precursor amino acid L-tyrosine
(Fernstrom, 1977).
|
| Fig. 6(a-d): |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on norepinephrine content in, (a) Cerebral cortex,
(b) Striatum, (c) Hippocampus and (d) Hypothalamus, Data are Mean±SE,
*Significantly different from corresponding control group at p<0.05,
#Significantly different from amino acids-treated group at p<0.05 |
|
| Fig. 7(a-b): |
Effect of 8 weeks treatment with nandrolone decanoate, amino
acids and their combination on glutamate content in, (a) Cerebral cortex
and (b) Hippocampus, Data are Mean±SE, *Significantly different from
corresponding control group at p<0.05, @Significantly different from
nandrolone-treated group at p<0.05, #Significantly different
from amino acids-treated group at p<0.05 |
Oral administration of amino acids mixture did not affect glutamate levels in either cortex or hippocampus. The absence of effect on glutamate content suggests that glutamate does not play a key role in amino acids-induced aggression.
Combination of both nandrolone decanoate and amino acids increased aggression. While there exists a large literature on aggression that describes the behavioral and neurochemical sequelae that accompany AASs cocktail administration, no studies have demonstrated that AASs and nutritional supplements administration increases aggressive behavior. In the current investigation, the combination of both nandrolone decanoate and amino acids decreased 5-HT in striatum, hippocampus and hypothalamus, reduced NE content in hippocampus and increased glutamate in cortex and hippocampus. The finding that in nandrolone-amino acids animals there is an increase in glutamate content strengthens the notion that glutamate plays a key role in AAS-amino acid-induced aggression. Since it has been hypothesized that serotonergic neurons play an important role in some types of depressive disorders and also a reduction of noradrenergic transmission is central in depressive disorders, our results suggest that chronic treatment with nandrolone and amino acids seems to reproduce the neurochemical substrate seen in a depressive state.
Open field test was used to gain a general overview of the behavioural characters.
Ambulation is an indicator of motor activity. Rearing, on the other hand, is
an indicator of exploratory behavior, whereas grooming is considered as a measure
of emotional response (Inone et al., 1996; Tamasidze,
2006).
There is a negative correlation between increased levels of 5-HT and locomotor
activity (Plaznik et al., 1983). Strombom
(1975) proved that norepinephrine is important for exploratory behavior
and consequently rearing frequency. Grooming activity was attributed to dopaminergic
system (Carneiro et al., 2005).
In the current study, injection of nandrolone decanoate decreased rearing frequency. This was correlated with the observed decrease in norepinephrine content. Amino acids-treated animals showed decreased ambulation frequency, while exhibited marked increase in rearing and grooming frequencies. Decreased locomotion in the open field is viewed as generally indicative of behavioral inhibition (e.g., less freezing). Previous studies have shown that locomotion by the rat in open field is inversely associated with anxiety ratings, depending upon whether the locomotion appears purposeful and whether the animal exhibits other exploratory behaviors such as rearing.
Pain sensitivity in hot plate test was assessed since altered pain sensitivity
could contribute to altered responding in aggression testing. Nandrolone decanoate
-treated rats did not differ from controls in reaction time in hot plate test.
These results are consistent with findings showing that nandrolone decanoate
did not affect acute nociceptive thresholds on the hot plate, tail withdrawal
and paw pressure tests (Tsutsui et al., 2011).
In the present study, no noticeable effect on reaction time in the hot plate test was observed after oral administration of amino acids mixture. At present, specific studies on possible analgesic effects of amino acids supplements are not available.
In conclusion, the present data indicate that treatment with nandrolone decanoate, amino acids mixture and their combination may induce neurochemical alterations in brain regions regulating aggressive behavior. This finding is of relevant interest considering that often athletes combine many drugs that interfere with 5-HT and DA function.
ACKNOWLEDGMENTS
This work was supported by a research grant (7/10/2) from National Research Center.
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