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Looking for Organ Damages Due to Anabolic-androgenic Steroids (AAS): is Oxidative Stress the Culprit? Daniela Cerretani1, Margherita Neri2, Santina Cantatore2, Costantino Ciallella3, Irene Riezzo2, Emanuela Turillazzi2, Vittorio Fineschi2* 1

Department of Medical, Surgical Science and Neuroscience, University of Siena, Siena, Italy; 2Department of Forensic Pathology, University of Foggia, Foggia, Italy; 3Department of Anatomical, Histological, Forensic & Orthopedic Sciences, Sapienza University of Rome Abstract: The aim of this review is to discuss the evidence on the potential role of oxidative stress in the mechanisms of AAS-induced toxicity. The adverse effects of chronic consumption of supraphysiological doses of AAS include endocrine, behavioral, hepatic, renal and cardiovascular abnormalities; this is of considerable importance because of the wellknown AAS abuse by adolescent bodybuilders and athletes and the emergence of adverse side effects including a number of cardiac and cardiovascular complications leading eventually to death in some cases. Accumulating evidence indicate that abuse of AAS may cause cardiovascular adverse side-effects including elevated blood pressure, alteration of the structure of the heart, congestive heart failure, stroke, sudden cardiac death and endothelial dysfunction. Under normal physiological conditions a major source of ROS in liver is mitochondria, additional sources in generating ROS are peroxisomes, xanthine oxidase, NADPH oxidase, acyl-CoA oxidase and cytochrome P-450. Poor information are available on the effects of AAS treatment on hepatic antioxidant capacity. Evidence of side effects affecting kidney and the renal function are sporadically emerging from clinical reports of renal disorders among AASs users, especially with elevated and prolonged use. Experimental evidence suggests that both nandrolone administration and strenuous exercise increase the extent of renal damage in response to renal toxic injury. From the data presented, we can realize that to date considerable research has led to the identification of a growing number of AAS-adverse effects due to abuse of these substances.

Keywords: Anabolic androgenic steroids, cardiac pathology, kidney alterations, liver damage, oxidative stress. INTRODUCTION Anabolic-androgenic steroids (AAS) are synthetic testosterone derivatives that exhibit a greater anabolic activity and reduced androgenic activity. AAS are used in medical practice in status of muscle wasting and to treat a variety of other conditions [1]. In addiction AAS are employed in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance; the abuse of AAS to enhance physical performance is widespread in sport communities despite their reported side effects and is becoming a public health issue. AAS therapy is associated with various adverse effects that are generally dose related; therefore, illicit use of the high doses taken by sportsmen carries substantial risks for health. The adverse effects of chronic consumption of supraphysiological doses of AAS include endocrine, behavioral, hepatic, renal and cardiovascular abnormalities; this is of considerable importance because of the well-known AAS abuse by adolescent bodybuilders and athletes and the emergence of adverse side effects including a number of cardiac and cardiovascular complications leading eventually to death in some cases [2, 3]. Another side effect of 17-alkylated AAS use is hepatotoxicity, including elevated levels of liver *Address correspondence to this Author at the Department of Forensic Pathology, Ospedale Colonnello D’Avanzo, Viale degli Aviatori 1, 71100 Foggia, Italy; Tel: +39 0881 733193; Fax: +39 0881 736903; E-mail: [email protected] 1570-193X/13 $58.00+.00

enzymes, cholestatic jaundice, peliosis hepatis, and various neoplastic lesions [1, 4]. Some authors point out to the role of oxidative stress in the mechanisms of AAS- induced toxicity in various organs such as liver, cardiovascular system and kidney; the potential hepatotoxicity of these compounds cannot be excluded because chronic treatments with stanozolol caused histopatological changes and oxidative stress in rat liver [5]. Vascular oxidative stress with an increased production of reactive oxygen species is described; oxidative stress is considered deleterious for endothelial function and is considered a significant risk factor in cardiovascular disease [6]. In this review, we will focus the evidence on the potential role of oxidative stress in the mechanisms of AASinduced toxicity. OXIDATIVE STRESS It is very well established that there is a relationship between exercise training and oxidative stress [7, 8]. Several lines of evidence suggest that exercise induces adaptations in many tissues against an oxidative insult, due mainly to a mild oxidative stress, which would upregulate antioxidant enzymes gene expression through redox-regulated transcription factors [9-11]. However, any situation in which the consumption of oxygen is increased, as during physical exercise, could result in an acute state of oxidative stress. Primary reactive oxygen species (ROS) generation in response to acute exercise can occur via several pathways. These include © 2013 Bentham Science Publishers

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Mini-Reviews in Organic Chemistry, 2013, Vol. 10, No. 4

mitochondrial respiration (electron leakage from electron transport chain and subsequent production of the superoxide radical), prostanoid metabolism, the autooxidation of catecholoamines, and oxidase enzymatic activity (NAD(P)H oxidase, xanthine oxidase) [12]. The initial increase in ROS during exercise, as well as following cessation of the work bout can lead to additional secondary generation of prooxidants via phagocytic respiratory burst, a loss of calcium homeostasis and/or the destruction of iron-containing proteins [12]. ROS are by-products of aerobic cellular metabolism and antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), play a crucial role in order to circumvent their deleterious effects. The imbalance between ROS generation and the intracellular levels of antioxidant defenses leads to oxidative stress, a condition that has been associated with apoptosis, neurodegenerative diseases and ischemia–reperfusion injury [13]. Several lines of evidence indicate a massive production of ROS upon ischemia– reperfusion events in different tissues such as brain, vascular endothelial cells and heart [14]. Liver is a key organ actively involved in numerous metabolic and detoxifying functions, it is continuously exposed to high levels of endogenous and exogenous oxidants that are by-products of many biochemical pathways and, in fact, it has been demonstrated that intracellular oxidant production is more active in liver than in other rat tissues [15, 16] like the increase of inflammatory cytokines, apoptosis and the inhibitors of apoptosis NF- B and Heat Shock Proteins [17]. AAS, CARDIOVASCULAR SYSTEM AND OXIDATIVE STRESS Accumulating evidence indicate that abuse of AAS may cause cardiovascular adverse side-effects including elevated blood pressure, alteration of the structure of the heart, congestive heart failure, stroke, sudden cardiac death [18] and endothelial dysfunction [19, 20]. One of the most crucial vasoactive substances released from endothelial cells is nitric oxide (NO). Endothelial NO has been shown to have many anti-atherogenic properties and vasodilatory effect. NO also inhibits inflammation, platelet aggregation and smooth muscle proliferation [21].Vascular oxidative stress with an increased production of ROS contributes to mechanisms of vascular dysfunction. Oxidative stress is mainly caused by an imbalance between the activity of endogenous prooxidative enzymes (such as NADPH oxidase, xanthine oxidase, or the mitochondrial respiratory chain) and antioxidative enzymes (such as SOD, glutathione peroxidase (GPX), heme oxygenase, thioredoxin peroxidase/peroxiredoxin, CAT, and paraoxonase) in favor of the former. Also, small molecular weight antioxidants may play a role in the defense against oxidative stress. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite. Peroxynitrite-in turncan "uncouple" endothelial NO synthase (eNOS) to become a dysfunctional superoxide-generating enzyme that contributes to vascular oxidative stress. Oxidative stress and endothelial dysfunction can promote atherogenesis [22]. In a recent study of Skogastierna et al. [23] was investigated the effect of supraphysiological doses of testosterone enanthate (500 mg) in healthy volunteers on endothelial NO release and the impact of testosterone on the oxidative stress status

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measured as total antioxidative capacity in urine prior and after testosterone administration. The authors show that supraphysiological doses of testosterone inhibit the urinary excretion of NO in healthy volunteers 48 hours after the testosterone administration. Since urinary NO is a biomarker for endothelial function, these results indicate that even a single dose may induce a decrease in NO formation in endothelial cells. The authors also show, by study in vitro in two endothelial cell lines, that testosterone down-regulates the gene expression of eNOS after 48 hours from treatment. This effect was partly abolished by addiction of seleno-Lmethionine indicating that oxidative stress may be involved in the down-regulation of eNOS. Further studies with supraphysiological doses of testosterone revealed an inhibition of the antioxidative capacity in vivo probably by generation of ROS and/or inhibition of the antioxidative activity. This observation were in line with the results obtained in vitro where several defense enzymes genes (CAT, SOD-1 and GPX-4) were down-regulated by supraphysiological doses of testosterone after 8 h exposure, indicating that oxidative stress may by acutely induced by testosterone The authors point out to limitation of them study because the few subjects observed and after only a single testosterone dose. It is possible that chronic adaptations develop to counterbalance the increased level of oxidative stress or the acute reduction of eNOS mRNA and NO production [23]. The beneficial effects of exercise in reducing the incidence of cardiovascular diseases are well known and the abuse of anabolic androgenic steroids (AAS) has been associated to cardiovascular disorders. Previous studies showed that heart protection to ischemic events would be mediated by increasing the antioxidant enzyme activities. Chaves et al. [24] have investigated the impact of exercise and high doses of the AAS nandrolone decanoate (DECA), 10 mg kg1 body weight during 8 weeks, in cardiac tolerance to ischemic events as well as on the activity of antioxidant enzymes in rats. After a global ischemic event, hearts of control trained group recovered about 70% of left ventricular developed pressure, whereas DECA trained, control sedentary and DECA sedentary animals recovered only about 20%. Similarly, heart infarct size was significantly lower in the control group compared to animals of the three other groups. The activities of the antioxidant enzymes SOD, GPX, and glutathione reductase (GR), were significantly higher in control animals than in the other three groups, whereas CAT activity was not affected in any group. The data presented in this work suggest that the cascade of events that lead to cardioprotection is impaired by DECA at least the one involved in cellular ability to detoxify ROS. Although the mechanisms by which DECA promote the deleterious effects in rat heart are not known, this drug might be acting in one of the events involved in the improved cardiac protection against ischemic/reperfusion injury triggered by exercise. The main outcome would be that DECA treated animals do not show the adaptive response of the exercise-induced increase of antioxidant enzymes activities establishing a chronic oxidative stress condition, which would explain the cardiac injuries frequently found in AAS users [25]. In a recent study of Sadowska-Krepa [26] was evaluated the changes in activities of selected antioxidant enzymes (SOD, CAT, GPX, and GR) and contents of key non-enzymatic antioxidants (glutathione (GSH), protein thiol groups, and a- and c-tocopherols) in the left heart ventricle of

Anabolic-androgenic Steroids and Oxidative Stress

young male Wistar rats subjected to endurance training (treadmill running, 1 h daily, 5 days a week, for 6 weeks) or/and testosterone propionate treatment (8 or 80 mg/kg body weight, intramuscularly, once a week, for 6 weeks) during adolescence. The training alone increased the activities of key antioxidant enzymes, but lowered the pool of nonenzymatic antioxidants and enhanced myocardial oxidative stress as evidenced by elevation of the lipid peroxidation biomarker malondialdehyde. The lower-dose testosterone treatment showed mixed effects on the individual components of the antioxidant defense system, but markedly enhanced lipid peroxidation. The higher-dose testosterone treatment decreased the activities of the antioxidant enzymes, lowered the contents of the non-enzymatic antioxidants, except for that of c-tocopherol, reversed the effect of endurance training on the antioxidant enzymes activities, and enhanced lipid peroxidation more than the lower-dose treatment. These data demonstrate the potential risk to cardiac health from exogenous androgen use, either alone or in combination with endurance training, in adolescents [26]. Recently, it has been confirmed that high doses of AAS hindered the cardioprotection provided by exercise by blocking its positive effects on antioxidant enzymes activities [27]. AAS, LIVER AND OXIDATIVE STRESS Under normal physiological conditions a major source of ROS in liver is mitochondria, additional sources in generating ROS are peroxisomes, xanthine oxidase, NADPH oxidase, acyl-CoA oxidase and cytochrome P-450 [15, 28]. Poor information are available on the effects of AAS treatment on hepatic antioxidant capacity. Pey et al. [4] have designed a study to investigate in sedentary and trained rats whether a prolonged treatment with high doses of stanozolol, a 17-alkylated derivative of testosterone, modified oxidative stress biomarker levels, redox status of glutathione and activities of the antioxidant enzymes in liver. In addition, the expression of the heat shock protein HSP72, stress proteins that may represent an important mechanism of protection against oxidative damage, was determined. Since prolonged administration of stanozolol provokes dysfunction of mitochondrial respiratory chain complexes and mono-oxygenase systems [29, 30], it would be possible that these alterations were accompanied by an increased ROS generation; an enhancement in ROS production exceeding the antioxidant defenses and repair capacity could lead to oxidative stress and cell damage. This kind of risk should be higher if the consumption of O2 were increased as occurring during exercise [4]. The main finding of the study of Pey et al. [4], is that prolonged (8 wks) ingestion of stanozolol induced a significant increase in the content of Thiobarbituric acidreactive substances (TBARS, as index of lipoperoxidation) and in the activities of the antioxidant enzymes total SOD, CAT and GPX and did not modify the expression levels of HSP72 in rat liver. Simultaneous realization of exercise training did not alter the effects of stanozolol administration. The increase of total SOD, CAT and GPX activities detected in liver from both sedentary and trained stanozolol-treated animals is consistent with the fact that oxidative stress occurred in some extent. The authors found augmented levels of the lipid peroxidation marker TBARS in liver homogenates from treated animals 48 hr after receiving the last steroid


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dose. Thus, the continuous and prolonged ingestion of stanozolol appears to provoke a local and sustained oxidative stress state in liver that could lead to increased expression of antioxidant enzymes through free radical-mediated induction of redox sensitive signal cascades. In this contest, the end product of oxidized fatty acid metabolism has been reported to activate signal transduction mechanisms and to modulate the expression of various genes [31]. How oral stanozolol treatment could be involved in excessive free radical production is unknown, but experimental evidence let suppose the involvement of the mitochondrial electron transport chain and/or the cytochrome P450 oxidase systems [29, 32]. In a review of Neri et al. [33] where the AAS abuse and liver toxicity is debated, oxidative stress is indicated as another mechanism of liver toxicity [34] resulting e.g. in the impairment of the canalicular bile salt export [35]. A previous study conducted by Welder et al. underlined an oxidative stress-induced damage showing the GSH depletion in cultured hepatocytes of rats treated with 17- alkylated AAS Those cultures exposed to the 17α-alkylated AAS, methyltestosterone and stanozolol for 24 hr and the 17 α alkylated AAS, oxymetholone, for 4 and 24 hr showed significant increase in Lactate dehydrogenase release while there were no significant differences with the nonalkylated steroids. GSH depletion was evaluated in cultures treated with methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr. Cultures exposed to oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested. These data indicate that the 17α-alkylated steroids (methyltestosterone, oxymetholone, and stanozolol) are directly toxic to hepatocytes [36]. An experimental study demonstrated that the prolonged AAS administration provokes dysfunction of mitochondrial respiratory chain complexes and mono-oxygenase systems [29] leading to an increased ROS generation. Afterwards, when ROS production exceeds the high levels of enzymatic and non-enzymatic antioxidant defenses and liver repair capacity, the oxidative stress induced liver damage appears [34]. In a work of Vieira et al. [37] the hepatic function and structure after 5 wk of nandrolone decanoate administration at three different doses in adult male Wistar rats were determined. The results obtained suggest that subchronic treatment with nandrolone decanoate, mainly administered at higher-than-clinical doses, are potentially deleterious to the liver, leading to incipient fibrosis probably correlated to increase in the number of Kupffer cells that when activated they produce many harmful by products such as TGF-B1, ROS, NF-kB, TNF-a, and IL-1B, which directly are able to stimulate the liver fibrosis process [37] (Fig. 1). AAS, KIDNEY AND OXIDATIVE STRESS Evidence of side effects affecting kidney and the renal function are sporadically emerging from clinical reports of renal disorders among AASs users, especially with elevated and prolonged use [38-43]. Experimental evidence suggests that both nandrolone administration and strenuous exercise increase the extent of renal damage in response to renal toxic injury. Misuse of boldenone undecylenate, an anabolic steroid developed for

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Fig. (1). proposed mechanisms of oxidative stress related liver damage due to anabolic androgenic steroids.

veterinary use, may determine the occurrence of a chronic renal injury, such as glomerulus mass reduction, that may lead to a progressive renal failure. Recently, has been proposed an experimental mechanism related to the expression of CD34 in renal specimens, a marker for endothelial cells deterioration, due to boldenone undecylenate abuse [44]. One possible mechanism for the latter would be the depletion of reduced glutathione (GSH), which has been suggested in some studies to sensitize to tumor necrosis factor (TNF-)-induced cell death (Fig. 2) [45]. Evidence of oxidative damage in kidneys of mice treated with nandrolone decanoate is showed by the increase of MDA level (marker of lipid peroxidation) and by the reduction of antioxidant enzymes GR and GPx activity resulting in the decreased ability of the kidney to scavenge toxic hydrogen peroxide and lipid peroxides [46]. GPx metabolizes hydrogen peroxide to water by using reduced glutathione as a hydrogen donor, resulting in the formation of oxidized glutathione [47]. GR subsequently regenerates reduced glutathione from oxidized glutathione. The reduction of activity of these enzymes may result in the involvement of deleterious oxidative changes due to the accumulation of toxic products. The results of experimental study indicated that a long term administration of nandrolone, an anabolic androgenic steroid, promotes

oxidative injury [46]. One possible mechanism of this effect could be related to 17--estradiol conversion of testosterone derivatives. The testosterone derivatives could become aromatic and convert to 17--estradiol which is a potent toxic, genotoxic, and cancer-forming compound [48]. The genotoxic activity of steroids is also due to an indirect process that takes place in the redox cycle, as well as the production of oxygen reactive types [49]. In this way, the metabolic activation of testosterone derivatives leads to the formation of free radicals and consequently to induction of oxidative stress in the kidney. CONCLUSION AAS use is associated with various adverse effects that are generally dose related; therefore, illicit use of the high doses taken by sportsmen carries substantial risks for health [50-52].Various adverse effects of supraphysiological dose of AAS are described; however, special attention has been paid to AAS induced adverse effects on liver and cardiovascular system. Many factors are involved in the induction of toxicity; in particular a significant alteration of antioxidant cellular defense system such as antioxidant enzymes and GSH can produce an increase in ROS and induce oxidative



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Fig. (2). pathogenic mechanisms about nandrolone decanoate generates kidney alterations and focal segmental glomerulosclerosis.

stress and lipid peroxidation in cells. It was demonstrated that the higher-dose AAS treatment decreased the activities of the antioxidant enzymes, lowered the contents of the nonenzymatic antioxidants, reversed the effect of endurance training on the antioxidant enzymes activities, and enhanced lipid peroxidation. These data demonstrate the potential risk to cardiac health from exogenous androgen use [26]. Furthermore a prolonged AAS administration cause a decrease in some components of the hepatic microsomal drugmetabolizing system and in the activity of the mitochondrial respiratory chain complexes leading to an increased ROS generation. When ROS production exceeds the high levels of enzymatic and non-enzymatic antioxidant defenses and liver repair capacity, the oxidative stress induced liver damage appears [34]. Finally vascular oxidative stress with an increased production of ROS contributes to mechanisms of vascular dysfunction. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite that contributes to vascular oxidative stress. Oxidative stress and endothelial dysfunction can promote atherogenesis [22]. Again, the metabolic activation of testosterone derivatives leads to the formation of free radicals and consequently to induction of oxidative stress in the kidney too. From the data presented, we can realize that to date considerable research has led to the identification of a growing number of AAS-adverse effects due to abuse of these substances. The role of oxidative stress in the mechanisms of AAS-induced toxicity in various organs such as liver, cardiovascular system and kidney is an important field of research related to AAS-related organ damage and could explain the pathogenetic basis of a significant social problem that affects mainly the youth community and players do not just amateurs [53].

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1]

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Accepted: ??????? ??, 2013