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 .