testosterone is the main androgen hormone and exerts its physiological effects through a genomic mechanism that is mediated by the androgen receptor .
these non - genomic effects are considered to be rapid , in contrast to the genomic effects , and several possible pathways have been identified .
the activation of myocardial inflammation signaling enzymes of the mapk family , mediated by the androgen receptor , has been documented , but interestingly this activation is not related to the receptor s transcriptional activity .
testosterone and its related androgens has significant anabolic and androgenic effects . since its molecular identification , testosterone has been used as a therapy for hormone replacement , infertility and libido dysfunction , as well as an anti - aging agent .
the abuse of androgens for improvement of performance has become a common , yet serious , condition that may have detrimental effects on the athletes .
testosterone abuse has been linked to an increase of cardiovascular adverse events such as significant cardiac hypertrophy and myocardial infarction , virilization of women , prostate hyperplasia in men , liver hypertrophy and hepatocellular adenomas or carcinomas [ 812 ] , apoptotic death of myocardial   and neuronal cells . on the other hand ,
recent investigations have shown that testosterone might have anti - ischemic effects   and possibly inhibits plaque development .
the aim of this study was to examine the effect of testosterone administration at the ultrastructural level of the rat myocardium , as well as to investigate whether testosterone abuse can induce apoptosis .
we used high doses of testosterone enanthate for a long period of time mimicking androgen abuse .
all procedures for animal use were in accordance with the guidelines of the greek government and the bioethics committee of the medical school of the aristotle university of thessaloniki .
we used adult male wistar rats ( weighing between 350400 g ) separated into 2 groups .
group a ( experimental ) consisted of 6 rats that received 10 mg testosterone enanthate ( which equals 7 .
5 mg testosterone ) intramuscularly for 20 consecutive days , while group b ( control group ) included 6 rats that were given normal saline via the same route .
having completed the administration period , all rats were anaesthetized and sacrificed and their hearts were removed .
sections from the hearts were placed in 10% formalin for light microscopy and glutaraldehyde 2.5% for electron microscopy .
myocardial tissue samples designated for light microscopy study were dehydrated through a series of increasing ethanol concentrations ( 25% , 50% , 70% , 80% , 96% and absolute ) and finally cleared with xylene .
then , the samples were embedded in paraffin wax and sections of 5 m were cut and stained with hematoxylin and eosin .
the myocardial tissue samples for electron microscopy were sectioned into small ( < 1 cm ) pieces and were placed into glutaraldehyde 2.5% for 2 hours and then into osmium tetraoxide 1% for 1 hour .
this was followed by staining with uranyl acetate 1% for 16 hours , dehydration with advancing ethanol concentrations and intubation into epon resin .
subsequently , ultra - thin sections ( 600900  ) were taken and stained with lead citrate ( reynolds s stain ) and studied using a joel transition electron microscope .
we used both qualitative and quantitative analyses which were conducted by 3 of the authors , blinded to the experimental groups . in order to examine the possible activation of apoptosis
mouse monoclonal antibodies ( ncl - cpp32 , novocastra ) against caspase-3 ( ccp32 ) were incubated with sections of myocardial tissue after appropriate processing .
the paraffin sections were dewaxed , rehydrated and placed in 0.5% hydrogen peroxide / methanol . after washing with tap water
, the sections were placed into high temperature and high pressure unmasking solution ( 0.01 m citrate buffer , ph 6.0 ) for 1 minute . the sections subsequently were placed into a bath of tap water , washed with tbs buffer and placed in diluted normal serum . then , the sections were incubated with primary ( mouse anti - rat - antibody ) and secondary biotinylated antibody .
the final steps included incubation in abc reagent and in dab , counter - staining with hematoxylin and dehydration .
the quantitative evaluation was made from photomicrographs of the myocardial tissue samples from each group by 1 of the authors ( v.p . )
the area of fibrosis was quantified using imagej software ( national institutes of health , bethesda , md , usa ) , and the fibrosis ratio was calculated by dividing the area of fibrosis by the total myocardial area . the dimensions and areas of mitochondria were measured with the use of imagej and were expressed as mean  se for each animal group .
the mann - whitney u - test was used for comparisons between the 2 groups .
data was analyzed using pasw statistics 18 , release version 18.0.0 , 2009 ( spss , inc . ,
finally , a quantitative measurement of the immunohistochemical staining for caspase-3 was performed using the imagej software .
the quantitative evaluation was made from photomicrographs of the myocardial tissue samples from each group by 1 of the authors ( v.p . )
the area of fibrosis was quantified using imagej software ( national institutes of health , bethesda , md , usa ) , and the fibrosis ratio was calculated by dividing the area of fibrosis by the total myocardial area . the dimensions and areas of mitochondria were measured with the use of imagej and were expressed as mean  se for each animal group .
the mann - whitney u - test was used for comparisons between the 2 groups .
data was analyzed using pasw statistics 18 , release version 18.0.0 , 2009 ( spss , inc .
finally , a quantitative measurement of the immunohistochemical staining for caspase-3 was performed using the imagej software .
we observed significant myocardial hypertrophy of rats that received testosterone ( figure 1 ) , whereas the myocardium of control rats appeared normal , without signs of hypertrophy ( figure 2 ) .
significant alterations were observed in the myocardial cells and the endothelial cells of the heart capillaries .
we found edematous mitochondria in the myocardial cells of the experimental group along with some degree of intracellular edema ( figure 3 ) . on the other hand
, the mitochondria of the control group appeared to be normal ( figure 4 ) . in order to evaluate any differences quantitatively , we measured and analyzed the shape and size characteristics of mitochondria of length exceeding 1.0 m . the maximum length found was 7.96 m .
we measured and compared the area ( a ) , the length ( l ) , the width ( w ) of the equivalent ( same area ) ellipse w = a/(l ) and the elongation ( l / w ) of 219 mitochondria in the experimental group and 23 mitochondria in the control group .
the results presented in table 1 , show that the area and the width of the mitochondria were larger for the experimental group , whereas the length and the elongation were larger for the control group .
it is indicative that the top 5 values of the area of the mitochondria in the experimental group ( 9.6830.39 m ) were all higher than the highest value of the corresponding area in the control group ( 7.67 m ) ; however , the difference was non - significant ( p=0.93 ) , as were the differences in length ( p=0.30 ) and width ( p=0.42 ) . on the other hand , the difference in elongation between the 2 groups was statistically significant ( p=0.04 ) .
there was also disarrangement of the sarcomeres , with disorganization of the myofibrils and the z discus ( figure 5 ) . between the capillaries and the myocardial cells we observed collagen fibers , which are an indication of myocardial fibrosis ( figures 6 , 7 ) .
a large number of pinocytoplasmic cysts inside the endothelial cytoplasm was observed ( figures 5,8 ) .
the appearance of the myofibrils and the mitochondria was normal in the control rats ( figure 4 ) .
myocardial immunohistochemical staining for caspase-3 was negative for the rats in group b ( control group ) ( figure 9 ) . on the other hand , the myocardial tissue of the rats which received testosterone ( group a ) exhibited significantly positive staining for caspase-3 , which indicates the detection of apoptosis ( figures 10 , 11 ) .
the differences are presented in figure 12 , in the form of rgb color histograms , determined by the use of imagej software . to quantify the differences , we calculated the delta - e ( e76 ) , as defined by the international commission on illumination ( cie ) , after conversion of the mean rgb values of each image into l * , a * and b * values in the l*a*b * color space .
the values of e76 between the control and experimental groups were found to be 63.0 and 74.3 , for figures 9 , 10 and 911 , respectively , and only 13.7 between figures 10 , 11 , which correspond to experimental group images .
a value of e76 approximately equal to 2.3 corresponds to a jnd ( just noticeable difference ) .
masson trichrome staining showed traces of local collagen fibrils ( connective tissue ) among the cardiac cells in the control group .
figure 13a shows masson s trichrome staining of a representative heart tissue section from a rat of the control group .
the amount of collagen fibrils in the experimental group that received testosterone was higher compared to the control group . in sections of cardiac veins , thickening of the muscular tunic with many foamy cells was observed as well as many collagen fibrils in the outer vascular tunica , surrounded by thick adipose tissue ( figure 13d ) .
the presence of adipose tissue was evident around the arterioles wall as well . within the adipose tissue
we observed significant myocardial hypertrophy of rats that received testosterone ( figure 1 ) , whereas the myocardium of control rats appeared normal , without signs of hypertrophy ( figure 2 ) .
significant alterations were observed in the myocardial cells and the endothelial cells of the heart capillaries .
we found edematous mitochondria in the myocardial cells of the experimental group along with some degree of intracellular edema ( figure 3 ) . on the other hand
, the mitochondria of the control group appeared to be normal ( figure 4 ) . in order to evaluate any differences quantitatively , we measured and analyzed the shape and size characteristics of mitochondria of length exceeding 1.0 m . the maximum length found was 7.96 m .
we measured and compared the area ( a ) , the length ( l ) , the width ( w ) of the equivalent ( same area ) ellipse w = a/(l ) and the elongation ( l / w ) of 219 mitochondria in the experimental group and 23 mitochondria in the control group .
the results presented in table 1 , show that the area and the width of the mitochondria were larger for the experimental group , whereas the length and the elongation were larger for the control group .
it is indicative that the top 5 values of the area of the mitochondria in the experimental group ( 9.6830.39 m ) were all higher than the highest value of the corresponding area in the control group ( 7.67 m ) ; however , the difference was non - significant ( p=0.93 ) , as were the differences in length ( p=0.30 ) and width ( p=0.42 ) . on the other hand , the difference in elongation between the 2 groups was statistically significant ( p=0.04 ) .
there was also disarrangement of the sarcomeres , with disorganization of the myofibrils and the z discus ( figure 5 ) . between the capillaries and the myocardial cells we observed collagen fibers , which are an indication of myocardial fibrosis ( figures 6 , 7 ) .
a large number of pinocytoplasmic cysts inside the endothelial cytoplasm was observed ( figures 5,8 ) .
the appearance of the myofibrils and the mitochondria was normal in the control rats ( figure 4 ) .
myocardial immunohistochemical staining for caspase-3 was negative for the rats in group b ( control group ) ( figure 9 ) .
on the other hand , the myocardial tissue of the rats which received testosterone ( group a ) exhibited significantly positive staining for caspase-3 , which indicates the detection of apoptosis ( figures 10 , 11 ) .
the differences are presented in figure 12 , in the form of rgb color histograms , determined by the use of imagej software . to quantify the differences , we calculated the delta - e ( e76 ) , as defined by the international commission on illumination ( cie ) , after conversion of the mean rgb values of each image into l * , a * and b * values in the l*a*b * color space .
the values of e76 between the control and experimental groups were found to be 63.0 and 74.3 , for figures 9 , 10 and 911 , respectively , and only 13.7 between figures 10 , 11 , which correspond to experimental group images .
a value of e76 approximately equal to 2.3 corresponds to a jnd ( just noticeable difference ) .
masson trichrome staining showed traces of local collagen fibrils ( connective tissue ) among the cardiac cells in the control group .
figure 13a shows masson s trichrome staining of a representative heart tissue section from a rat of the control group .
the amount of collagen fibrils in the experimental group that received testosterone was higher compared to the control group . in sections of cardiac veins , thickening of the muscular tunic with many foamy cells was observed as well as many collagen fibrils in the outer vascular tunica , surrounded by thick adipose tissue ( figure 13d ) .
the presence of adipose tissue was evident around the arterioles wall as well . within the adipose tissue
cardiac arrhythmias , qt dispersion , atrial fibrillation , myocardial infarction , heart failure and atherogenesis have all been linked to androgen abuse by athletes .
melchert and welder proposed 4 hypothetical models of anabolic - induced adverse cardiovascular effects : an 
atherogenic model involving the effects of androgen - anabolic steroids ( aass ) on lipoprotein concentrations ; a 
vasospasm model involving the effects of aass on the vascular nitric oxide system ; a  direct myocardial injury model involving the effects of aass on myocardial cells .
our study shows that the administration of testosterone in high doses exerts toxic effects on the myocardial cells .
this probably correlates with the direct myocardial injury model of the effects of androgen - anabolic steroids on the myocardial cells .
the morphometric approach showed that the mitochondria of the experimental group were larger and more rounded .
the contractile apparatus showed signs of deterioration , with disorganization of the sarcomeres and the z discus .
these observations have been reported earlier and have been characterized as typical for early heart failure .
another interesting finding of this study , using the masson s trichrome staining , was the presence of collagen fibrils inside the myocardium and particularly between the capillaries and the myocardium cells .
this perivascular myocardial fibrosis , together with the hypertrophy that was also induced , may be the cause of myocardial ischemia   as well as a substrate for arrhythmias .
reported that acute exposure of hearts to testosterone significantly reduces the dp / dt ( a measure of cardiac compliance ) .
the myocardial hypertrophy observed in the present study and also in our previous experiments , as well as by other researchers , has a significant role in the testosterone - induced reduction of myocardial compliance .
the hypertrophy of the myocardium correlates with the enhanced expression of the androgen receptor after testosterone administration .
abnormalities of the circulating levels of other hormones may induce adverse cardiovascular effects . increased levels of the protein hormone leptin
have been found to induce cardiac hypertrophy although diastolic dysfunction was not associated with leptin levels .
another set of hormones , the thyroid hormones , do have an impact on the myocardial diastolic properties among other various cardiovascular effects .
an unusually large number of micropinocytic vesicles was observed in the cytoplasm of the endothelial cells .
although it is common for these vesicles to occur in capillary endothelial cells , especially in the striated muscles , the abundance of such vesicles is a sign of heightened pinocytic activity that permits the cell to receive substances through the cell membrane .
apoptosis is the programmed cell death and is mediated by 2 pathways : the extrinsic death receptor signaling pathway and the intrinsic mitochondrial control pathway .
caspase-3 ( cpp 32 ) is a member of the interleukin-1 beta - converting enzyme ( ice ) family of mammalian proteases that specifically cleaves substrates at the c - terminal side of aspartic residues .
members of this family have been implicated in apoptosis , and caspase-3 acts as a control mediator of programmed cell death in mammalian cells .
caspase-3 is synthesized as an inactive 32kd proenzyme and is processed during apoptosis to its active form , which is responsible for the cleavage of poly ( adp - ribose ) polymerase ( parp ) , actin and sterol regulatory element binding protein ( sredp ) [ 3840 ] . in the present study , testosterone overdosing significantly activated apoptosis , as was clearly seen by immunohistochemistry .
although it has been reported that apoptosis activated by testosterone enanthate is due to the ester , experimental exposure of myocardial cells to enanthate alone did not activate apoptosis .
the abuse of androgen anabolic substances has been causally linked with sudden cardiac death , myocardial infarction , ventricular remodeling and cardiomyopathy .
myocardial death without coronary vessel disease or atherosclerosis has also been attributed to the activation of apoptosis by aass .
this study showed that testosterone abuse produces significant myocardial hypertrophy and fibrosis as well as of the myofibrils , the mitochondria and the capillaries .
the activation of apoptosis was a significant finding that indicates the direct myocardial injury caused by testosterone .
as testosterone is now used for a variety of possible indications such as hormone replacement in the elderly or as an antianginal in cardiac patients , more research is required to clarify the exact biochemical route of action as well as the correct dose to prevent adverse effects .
 summarybackgroundandrogen abuse is an increasing problem amongst professional and amateur athletes . 
 moreover , testosterone , apart from its widely accepted indications , is used for a variety of other indications such as aging and ischemia . 
 its actions are mainly attributed to a specific genomic mechanism through the androgen receptor , but emerging evidence reveals non - genomic effects as well . 
 the use of androgens has been linked with several adverse effects . 
 the purpose of this study was to examine the effects of testosterone on the morphology and the ultrastructure of the myocardium and to investigate the possible role of apoptosis.material/methodswe used 12 adult male wistar rats , separated into 2 groups . 
 group a consisted of 6 rats that were administered high doses of testosterone enanthate , while group b consisted of 6 male wistar rats that received placebo ( normal saline ) intramuscularly . 
 after the last day of treatment , all rats were anesthetized and sacrificed , and the hearts were removed and processed for optical and electron microscopy and immunohistochemical detection of caspase-3 , an apoptosis marker.resultswe found significant myocardial hypertrophy along with abundant ultrastructural alterations . 
 the immunohistochemical staining of the myocardial cells for caspase-3 was positive in group a ( experimental group ) , which is interpreted as an activation of apoptosis by testosterone treatment.conclusionstestosterone abuse has serious adverse effects , including myocardial hypertrophy , myocardial fibrosis and activation of apoptosis . 
 these findings need to be taken into account whenever androgens are prescribed to improve performance or as hormone therapy . 
Background
Material and Methods
Statistical analysis
Results
Optical study
Electron microscopy
Immunohistochemical study
Massons trichrome staining for collagen fibers
Discussion
Conclusions