Keywords
heart - morphometric analysis - left ventricle - steroids
Introduction
Androgenic anabolic steroids (AAS) are hormones that function in the differentiation,
growth, and development of the male reproductive tract, as well as in the development
and maintenance of secondary sexual characteristics. However, they also have anabolic
effects, stimulating body growth and increasing muscle mass.[1] AAS are synthetic compounds derived from this hormone, which mimic the anabolic
effects of testosterone, while at the same time minimizing androgenic effects.[2]
In the 1950s, the first reports of abuse in the administration of AAS for non-therapeutic
purposes by Russian athletes appeared in the literature.[3] Since then, AAS hormones have been used usually by people with good health, in order
to increase muscle mass and potency, as well as to provide improvement in their physical
condition and appearance.[4]
Illicit long-term and supraphysiologic use of AAS may cause adverse cardiovascular
effects, such as pathological hypertrophy, as well as ventricular and atrial arrhythmic
events, among which atrial fibrillation is the most frequently observed in bodybuilders.
Additionally, several reports of atrial fibrillation suggest a causal link in the
use of AAS in athletes.[5]
The compounds selected for the present study are synthetic, commonly used for aesthetic
purposes, with testosterone cypionate being one of the most widely used AAS in the
market.[6]
One of the mechanisms described in the literature regarding the use of supraphysiological
doses is associated with the temporary nitrogen retention of ingested proteins, as
well as with the competition of receptors of glucocorticoids released during intense
exercise, leading to the inhibition of protein catabolism.[7]
Increased erythropoiesis is also a risk factor for erythropoietin stimulation and
hepcidin suppression. Erythropoietin is the erythrocyte stimulating hormone, whereas
hepcidin is a protein that regulates iron absorption, so the erythropoietin produced
is more active due to the higher concentration of serum iron.[8]
Understanding the ease of obtaining anabolic agents, the indiscriminate use of these
drugs and their possible side effects, it was proposed to evaluate in a specific and
controlled way the effects of supraphysiological doses of these AAS in sedentary mice
for comparison with a group of animals treated with the same drugs, but performing
physical exercise, in order to obtain accurate information and, with this, to raise
awareness, mainly to the generation of young adolescents, about the possible causes
and effects of the indiscriminate use of these substances.
Materials and Methods
For the present experiment, 60 Swiss mice were used (30 males and 30 females) from
the Universidade Federal de Alfenas (UNIFAL, in the Portuguese acronym) (Committee
for Ethics in Animal Experimentation [CEEA], Universidade Federal de Alfenas, registry
number 505/2013). These were housed in boxes containing 5 animals each, and were treated
with commercial feed and ad libitum water and kept in a light-dark 12-hour cycle.
The treatment consisted of the intraperitoneal application of two AAS, as follows:
Group 1: testosterone cypionate (Deposteron [EMS, São Bernardo do Campo, SP, Brazil])
(0.8 mg/kg); group 2: stanozolol ([Winstrol-Stanozolol Depot, Landerlan, Lambaré,
Paraguay]) (1.8 mg/kg), and group 3: physiological solution (1 ml/kg). The animals
were treated for 2 months, with doses applied twice a week at 2-day intervals.
After the euthanasia of the animals by means of the halothane anesthetic and the identification
of each animal, the thoraxes were opened and the hearts were entirely removed. Finally,
the organs were stored in glass containers containing paraformaldehyde (pH 7.4). They
remained immersed in this fixative solution for 24 hours.
The hearts were processed following the standardized histological procedures. The
paraffin-embedded samples were cut in a thickness of 7μm in a Yidi microtome ([Jinhua
YIDI Medical Appliance CO., LTD, Jinhua City, Zhejiang province, China]) and stained
with hematoxylin and eosin (H&E).
For the morphometric analysis, 10 distinct sections of the apex, of the middle region,
and of the base of the heart were selected ([Fig. 1]). To measure ventricular cavity diameter, was selected the entire area of ventricular
cavity by means of one of the tools of the software Axiovision Version. 4.8.2 (Carl
Zeiss Microscopy LLC, Peabody, MA, USA). To measure the myocardical thickness, three
areas (anterior, posterior and lateral) were selected and was used the same software
for analysis [Axiovision Version. 4.8.2 (Carl Zeiss Microscopy LLC, Peabody, MA, USA)].
The values were measure in µm2.
Fig. 1 Heart of a normal mouse demonstrating the regions to be analyzed (white trace): the
apex, the middle region, and the base. Abbreviations: LA, left atrium; LV, left ventricle;
RA, right atrium; RV, right ventricle.
The analysis was performed using an optical microscope model Axion Scope A1 (Carl
Zeiss Microscopy LLC, Peabody, MA, USA) coupled to Axiovision Version. 4.8.2 (Carl
Zeiss Microscopy LLC, Peabody, MA, USA) and to the Axiovision 4 Module Interactive
Measurement software (Carl Zeiss Microscopy LLC). To capture the selected images,
a 2.5x objective lens (Carl Zeiss Microscopy LLC, Peabody, MA, USA) was used ([Fig. 1]).
To evaluate the mean values of the left ventricle areas, according to the gender of
the mice and to the treatment imposed (research groups), the analysis of variance
(ANOVA) was used. When a significant difference (p < 0.01) was observed among the groups when comparing different variables, the Tukey
test was used to discriminate differences and/or similarities among the evaluated
means.[9]
Results
As shown in the graphics, no significant result was observed in the diameter of the
left ventricle cavity of the hearts of female mice in the control group in relation
to the treated groups ([Fig. 2A]).
Fig. 2 (A) The graphics demonstrate the nonsignificant difference in the left ventricular cavity
diameter of female mice between the experimental groups.(B) Shows the significant difference in the left ventricular myocardial thickness observed
between the control group in relation to the group treated with stanozolol (Winstrol
Depot) (**), and the group treated with testosterone cypionate (Deposteron) in relation
to the Stanozolol (Winstrol) group (*).
However, when analyzing the myocardial thickness, a significant difference was observed,
showing a decrease in the myocardial thickness of the stanozolol group in relation
to the control group (p < 0.00001), as well as in the group treated with testosterone cypionate in relation
to the group treated with stanozolol (p < 0.0001). However, when the testosterone cypionate group was compared with the control
group, no statistically significant result was obtained ([Fig. 2B]).
When comparing the diameter of the left ventricle in male mice, significant differences
were observed, showing that both of the AAS evaluated increased the diameter of the
left ventricular cavity, both in the testosterone cypionate group in relation to the
control group (p < 0.0001), and in the stanozolol group in relation to the testosterone cypionate
group (p = 0.0009) ([Fig. 3A]).
Fig. 3 The graphics show the statistically significant difference found in the left ventricular
cavity diameter (A) and in the left ventricular myocardial thickness (B) between male mice in the control group and in the testosterone cypionate (Deposteron)
group (*), between the control group and the Stanozolol (Winstrol) group (**), and
between the two groups treated with the different steroids (***).
Regarding the analysis of the thickness of the left myocardial ventricle, the male
mice presented significant differences between the control group and the 2 treated
groups: testosterone cypionate (p < 0.0001) and stanozolol (p = 0.0008). A statistically significant difference was also observed between the stanozolol
group and the testosterone cypionate group (p = 0.0004) ([Fig. 3B]).
[Fig. 4] represents histological section of the left ventricules of some of the male and
female mice studied, stained in H&E, with a 2.5x magnification, of all in the groups
studied for comparation.
Fig. 4 Histological section of the left ventricles of some of the mice studied, (A) male mouse treated with testosterone cypionate, (B) stanozolol-treated male mouse, (C) male mouse in the control group, (D) testosterone cypionate-treated female mouse, (E) stanozolol-treated female mouse, and (F) female mouse in the control group, Abbreviations: LVL: left ventricular cavity;
VMT, ventricular myocardial thickness.
Discussion
Left ventricular hypertrophy is directly related to increased blood pressure and systemic
by compensatory mechanism. High blood pressure is associated with a three to four
fold increase in the risk of stroke, fibrillation, and ventricular arrhythmia.[10]
[11] This study corroborates the results found Seara et al.[15], showing a significant increase in myocardial thickness and ventricular cavity diameter,
in male mices.
According to literature, the use of nandrolone, another AAS widely used, promotes
changes in endothelium of blood vessels, both at the sedentary and exercised female
mice, indicating that physical exercise does not decrease the effects caused by AAS,
and that sedentary users also suffer significant damage.[12] Other damages are also described by some authors, which demonstrated increased levels
of LDL, HDL, NADPH oxidase in association with ventricular hypertrophy in rats treated
with nandrolone, doing or not doing exercises.[13]
[14]
For Seara et al,[15] this cardiac hypertrophy is due to the thickening of the posterior wall of the left
ventricle and of the interventricular septum in addition to fibrosis, even with discontinuation
of the AAS treatment, thus reporting irreversible cardiac damage. And this can occur
through several mechanisms, one of which is the increase in cardiac angiotensin due
to the stimulus of the activity of the converting enzyme, and angiotensin II has been
related to cardiac remodeling by cellular hypertrophy and collagen deposition.
The studies of Nascimento et al[16] showed that the deposition of collagen in cardiac cells can lead to a loss of the
contractile function, with increased rigidity; moreover, an increase in the left ventricular
systolic pressure has been reported, whereas a decrease in the diastolic function
was observed, causing left ventricular isovolumic relaxation failure in rats treated
with AAS. This failure in relaxation is one of the major etiological factors of ischemia
and heart failure.
The possible increase in the erythrocytes count due to the stimulation caused by these
hormones can also be seen as a risk factor because it leads to an increase in blood
volume. Consequently, the heart would need more force to pump the blood to the tissues,
causing hypertrophy.[17]
The results obtained in this study did not significantly alter the diameter of the
left ventricular cavity of female mice, which can be explained by a cardiac and vascular
protective effect of the endogenous estrogen, contributing to the maintenance of caliber
and myocardial diameter, suggesting a possible compensation mechanism.[12]
[18]
According to Barp,[19] the cardiovascular protective effect of estrogen occurs in several ways, such as
the stimulation of nitric oxide (NO), which is a vasodilatation gas that prevents
platelet aggregation, endothelial leukocyte adhesion, endothelin production, and control
of the vascular tone. Another protective factor is that estrogen can decrease cell
proliferation in the tunica intima and in the tunica adventitia in the vessel wall,
inhibiting protein kinase, which is in line with the results obtained in the present
study.
Another probable hypothesis for the results found in females is the same as reported
by Clark,[20] who states that, unlike testosterone cypionate, stanozolol has the ability to interfere
with the production of estrogen by central action, as well as the potential to inhibit
the hypothalamus-pituitary-gonadal axis, thus decreasing the production of estrogen,
which explains the fact that the myocardial thickness of female mice changes once
they no longer have the protective action of the hormone.
In patients with deficiency in testosterone levels, a deficiency of myocardial contraction
is noted. In contrast, studies indicate that, at high levels, testosterone can be
harmful because it acts directly on androgen receptors, which in turn act on genes
that make the transcription of alpha-myosin heavy chain (α-MHC) and of transforming
growth factor beta (TGF-β) proteins, which act respectively on atrial contraction
and on the control of cell proliferation and differentiation.[21]
The results found by Pirampol et al.[21] may explain the results found in this study, which present a difference between
the studied genders, showing that, in female mice, the use of AAS, such as stanozolol,
may decrease the thickness of the left ventricular myocardium, while the results were
the opposite in male mice.
The use of AAS causes changes in normal physiological adaptations that occur after
exercise, from compen- sated to uncompensated hypertrophy and is related to several
pathological processes according to Nascimento and Medei[22], such as heart failure, reduced tolerance to ischemia, and ventricular dilatation,
which can also be demonstrated by the results found in the present study.
The use of AAS causes hypertrophy of cardiomyocytes, and a decrease in capillary density
and increased capillary spacing was observed. It is presumed that these alterations
affect the supply of oxygen to the myocardium by subjecting it to ischemia.[23]
Some researchers report the development of stress and cellular damage caused by the
chronic use of AAS. In biochemical tests, it is known that troponin isoenzymes (cTnI)
and creatine kinase MB (CK-MB) are specific indicators of cell lysis and are elevated
in users of anabolic steroids.[24]
[25]
The heart rate also decreases after exercise, according to dos Santos,[26] who also reports that this may be an effect of changes in the parasympathetic nervous
system, suggesting that these effects may also occur at rest in the case of sedentary
mice, due to the changes already mentioned. The decrease in heart rate is a predisposing
factor to cardiovascular pathologies.
Conclusion
The alterations found in the left ventricular cavity and thickness of the left ventricular
myocardium allow to conclude that innumerable pathologies, such as vascular and cardiac
hypertension, arrhythmias, acute myocardial infarction, and sudden death, may be associated
with the findings described in the present study and that, beyond these alterations,
it is also suggested that the indiscriminate use of AAS may bring serious risks to
the user in relation to general health.
It is possible to see the great importance of studies related to AAS due to the increasing
use of these substances in supraphysiological doses for non-medical purposes also
by sedentary individuals who believe in the increase of muscular mass without physical
effort.
