Keywords
anabolic steroids - left ventricle - mice - morphometric analysis
Introduction
The use of anabolic androgenic steroids (AAS) has grown into a worldwide substance
abuse problem over the last decades. Today, the majority of AAS users are not competitive
athletes, but, instead, they are typically young to middle-aged men who use them primarily
for personal appearance.[1] The use of AAS is a public health concern for adolescent boys who have suffered
bullying by being labeled gay/bisexual.[2]
The doses taken by these users are usually 10 to 100 times higher than the therapeutic
ones, bringing forth hyperandrogenism. Although these doses promote increased strength
and muscle development, they concomitantly develop hormonal disorders that lead to
a variety of harmful consequences.[3]
Among the most striking AAS side effects are the increase in hematocrit and coagulation,
causing thromboembolism, intracardiac thrombosis and stroke, as well as other cardiac
disturbances including arrhythmias, cardiomyopathies, and, possibly, sudden death,
adenomas and carcinomas.[4]
The use of anabolic agents causes adverse effects on the musculoskeletal system, increasing
the risk of tendon rupture; this is due to the increase of strength and muscle mass.[5]
The anabolic activity of testosterone and its derivatives manifests primarily by its
myotrophic action, which results in increased muscle mass by rising protein synthesis
in the muscle.[6]
Krieg et al[7] analyzed cardiac changes by echocardiogram and observed an increase in the ventricular
mass index and in the interventricular septum thickness in AAS users compared with
non-users, and also a loss of diastolic function associated with a reduction in peak
velocity during the initial phase initial of diastolic filling.
Studies performed with powered athletes by means of echocardiogram examination demonstrated
that the cardiac remodeling that occurs as an effect of the use of anabolic steroids
is irreversible.[8]
This paper intends to analyze the possible morphometric changes in the left ventricular
diameter of male and female mice submitted to swimming that received supraphysiological
doses of two types of AAS.
Material and Methods
In this work, we used 60 Swiss mice (30 males and 30 females) from the Universidade
Federal de Alfenas (UNIFAL-MG) bioterium, housed in boxes with 10 animals each, treated
with commercial ration and water “ad libitum” (at will) and kept in a light-dark cycle
of 12 hours. The present experiment was analyzed and approved by the Ethics Committee
for Research and Animal Experimentation (ECRAE) of the University (protocol n° 414/2012).
The treatment with AAS consisted of intraperitoneal injections of two types of AAS,
as follows: group 1 (10 male and 10 female animals) received a dose of 0.8 mg/kg of
Deposteron (EMS, São Bernardo do Campo, SP, Brazil); group 2 (10 male and 10 female
animals) received a dose of 1.8 mg/kg of Winstrol (Stanozolol Depot, Landerlan, Lambaré,
Paraguay), and group 3 (10 male and 10 female animals) received 1.8 mg/kg saline solution.
The animals were treated for 2 months, with the doses being administered twice a week
at 2-day intervals. On each of these interposed days, all mice were submitted to swimming
for 10 minutes.
After euthanasia by inhalation of isoflurane, the chests of the mice were opened,
and the hearts were entirely removed. Finally, they were stored in glass containers
immersed in a buffered paraformaldehyde solution (pH 7.4) and remained in this fixative
solution for 24 hours. Thus, the specimens were processed following the standardized
sequence for a conventional histological procedure: alcohol dehydration, xylol diaphanization,
and paraffin inclusion. Each heart was put in a paraffin block and cut into 7 μm-thickness
sections in Jinhua YIDI Medical Appliance CO., LTD (Jinhua City, Zhejiang Province,
China) microtome and stained with hematoxylin and eosin. For the morphometric analysis,
6 distinct sections were selected and measured using an optical microscope with 40-fold
magnification, and for the morphometric analysis of the ventricular cavity we used
the Axiovision Rel. 4.8.2 (Carl Zeiss Microscopy LLC, Peabody, MA, USA) and Axiovision
4 Module Interactive Measurement (Carl Zeiss Microscopy LLC) software.[9]
To evaluate the mean values of the left ventricle areas, according to mice's gender
and treatment imposed (research groups), the variance analysis of variance was used.
When a significant difference (p < 0.01) was observed among the groups while comparing different variables, the Tukey
test was used to discriminate differences and/or similarities among the evaluated
means.[9]
Results
According to the graph and photomicrographs ([Fig. 1A] and [2E]), it can be observed that in the male mice treated with Deposteron there was a significant
increase (p < 0.001) in the diameter of the left ventricle in relation to the control group ([Fig. 2D]) and the group treated with Winstrol (p < 0.01) ([Fig. 2F]).
Fig. 1 Comparative graphs of left ventricular diameter in male and female animals, respectively.
(A) * Statistically significant differences of control group animals compared with Deposteron
animals (p < 0.001) and ** Statistically significant differences of Winstrol animals compared
with Deposteron animals (p < 0.01). (B) Statistically significant differences of control group animals in relation to Winstrol
animals (p < 0.01).
Fig. 2 Photomicrography of the cross-sections of the left ventricle of mice in both sexes
and in different groups studied. A: Female control group; B: Deposteron female; C:
Winstrol female; D: Male control group; E: Deposteron male, and F: Winstrol male.
In the females, the results show that the group treated with Winstrol ([Figs. 1B] and [2C]) presented a significant decrease in ventricular diameter (p < 0.01) in relation to the control ([Fig. 2A]) and the Deposteron ([Fig. 2B]) groups.
Discussion
Differences between genders are often ignored and underestimated when studying the
cardiovascular system, and these cause biases and losses in the performed researches.[10] However, previous clinical and epidemiological studies have corroborated and acknowledged
gender differences in cardiovascular function and disease.[11] The causal link between the use of AAS and the occurrence of cardiovascular diseases
has been increasingly evidenced through researches, which demonstrate the frequent
use of these substances associated with the rise in the occurrence of death due to
cardiac arrest among the users.[12]
[13]
[14] Such studies corroborate the findings in the present study, which show that AAS
use may cause morphological changes in the left ventricle and that they can lead to
the appearance of cardiovascular diseases.
Cardiac changes in women and men may have been influenced by both dose and time period
of administration of the drugs used, and these factors, isolated or together, may
have led to different effects in both sexes. However, there are limitations and scarcity
of data in the literature regarding the relevance and peculiarities of the different
types of AAS.
Neto et al[15] suggest that the AAS dose is directly related to the aromatization of the testosterone;
that is, the higher the dose, the greater the aromatization and the greater cardiac
compromise. Pirompol et al[16] concluded that cardiac hypertrophy is not related to the dose, but to exposure to
the induction of maladaptative heart responses. Therefore, although it was not possible
to measure the interference of the dosage, period of use and active principle of the
AAS used, the results presented here may induce and contribute to the interest and
awakening for future researches.
A second hypothesis consists in the association of AAS action and the activation and
increase of the sympathetic autonomic nervous system action. When present in the bloodstream,
AAS reach the hypothalamus through the vascular organ of the terminal lamina or through
the subfornical organ, structures that do not present a blood-brain barrier, facilitating
the absorption and interaction by specific cellular groups of neurons acting on the
control of viscera, blood osmolarity, angiotensin II levels and blood pressure. These
negative influences of AAS use on the sympathetic modulation have already been evidenced
and recorded by Neto et al,[15] whose study contributes to and strengthens the results observed here.
The consequences of non-therapeutic and abusive use of testosterone (AAS and its derivatives)
are associated with an increase in blood pressure and induction of left ventricular
changes, with consequent cardiac hypertrophy, as shown in some studies already performed.[17]
[18]
[19]
[20]
Initially, it was expected that there would be no change in the ventricular diameter
in females because they have a greater amount of estrogen as a differential characteristic.
This hormone is a protective factor for the cardiovascular system, and such concept
has already been evidenced in some previously published papers.[21]
[22]
[23] Nonetheless, some authors suggested that the decrease in the diameter of the left
ventricle in female animals under supraphysiological doses of the AAS could be due
to an increase in the left ventricular wall, leading to a decrease in the ventricular
chamber volume with consequent hypertension, resulting in heart failure and left ventricular
hypertrophy.[24]
[25]
Another hypothesis that would lead to an increase of muscle mass in females would
be based on the same reason observed by Hayward et al,[26] who administered AAS to women and consequently observed an increase in cardiac muscle
mass. This could explain the findings of increased muscle mass with a consequent decrease
in diameter in females, in this study, in addition to the fact that the drugs themselves
lead to an increase in ventricular mass. Thus, although females have to modulate hormonal
protection (estrogen) in the cardiovascular system, such hormone would not have been
able to prevent ventricular changes nor would it attenuate the androgenic actions
of supraphysiological doses of AAS.
In male animals, it is suggested that the reason for finding the opposite result to
that observed in female subjects was due to the drugs investigated in the present
experiment, for they increased the left ventricular lumen because of an atrophy of
the cardiac muscle and supposed decrease of left myocardial thickness. These effects
can lead to chronic ischemia, which drives to fibrosis, reducing heart fiber nutrition,
exactly as demonstrated in other studies.[27]
[28]
Other studies demonstrate that another reason that could lead to an increase in left
ventricular diameter in male animals, as found in this study, is that AAS would induce
dilated cardiomyopathy, primary heart muscle disease with dilation and change in the
contractile function of the left ventricle, which is more prevalent in men than in
women.[29]
[30]
Another factor that could contribute to the increase in left ventricular diameter
is that, physiologically, males have a greater amount of endogenous testosterone when
compared with females; this factor, added to supraphysiological doses, could lead
to toxicity of the cardiac muscle tissue by inducing pro-oxidative actions on the
cardiovascular system.[10]
This wide variety of hypotheses may be a reflection of the limitations pertinent to
AAS studies and of the morphological consequences to the cardiovascular system caused
by the use of AAS. This is due to several reasons, such as the manifestation of the
effects, that do not appear in a short-term period and make it difficult to provide
an early diagnosis, as well as the intrinsic factors of the AAS (dose, duration of
treatment and active principle) that interfere in their action.[1]
[31]
[32]
[33]
Conclusion
Thus, one can conclude that the use of supraphysiological doses of the administered
AAS significantly and differently alters the ventricular diameter in male and female
animals. Such findings may contribute to elucidate the possible effects and consequences
of the indiscriminate use of these drugs concerning the cardiovascular system.
