Boldenone undecylenate is on of anabolic steroids, (synthetic androgen hormone) that derived from testosterone (Yesalis et al., 1993; Sullivan et al., 1998; Cannizzo et al., 2007; Soma et al., 2007; Tousson et al., 2012, 2013). Boldenone is well known under the trade names Equipoise, Ganabol, Equigan and Ultragan. They were developed mainly for veterinary use, mostly for the horse treatment (Yesalis et al., 2000; Soma et al., 2007). Recently, it used by bodybuilders in both off-season and pre-contest where it is well known for increasing vascularity while preparing for a bodybuilding contest. Boldenone has a very long half-life and can show up on a steroid test for up to 1.5 years. Trace amounts of the drug can be easily detected for months after discontinued use (Hoffmann, 2002; Brookhouse, 2007). Groot and Biolatti (2004) study the histopathological effects of boldenone in adult male cattle and reported that boldenone causes degeneration of the germinal epithelium of the testis and hypersecretion and cyst formation in the prostate. Boldenone has dual effects on humans, both directly and indirectly; directly as injection to build muscles and indirectly as through consuming meat of animals that where treated with boldenone.

Oxidative stress has been defined as a disturbance in the balance between antioxidants and prooxidants (free radicals and other reactive species) with increased levels of prooxidants leading to potential damage. This imbalance can be an effect of depletion of endogenous antioxidants, low dietary intake of antioxidants and/or increased formation of free radicals and other reactive species (Halliwell, 1997). In human tissues, a condition of oxidative stress can be revealed through searching for specific biomarkers of oxidative damage to lipids, proteins and nucleic acids (Zhang and Li, 2006). El-Moghazy et al. (2012) reported that; intramuscular injection of rabbits with boldenone increased total protein, total lipid, Nitric oxide (NO), superoxide dismutase activity (SOD), glutathione (GSH) and malondialdehyde (MDA) in liver and kidney tissues comparing with the control group. Upon the previous information, the aim of the present study was to investigate the possible effect of growth promoter boldenone undecylenate on total antioxidant capacity and oxidative stress on muscular tissues (skeletal, cardiac and smooth muscles).

MATERIALS AND METHODS

The experiment adhered to the guidelines of the ethical committee of the National Research Center, Egypt. The present study was conducted at a rabbit private farm in Dakhahleia governorate and Zoology Department, Faculty of Science, Tanta University, Egypt, during winter 2011.

Animals: The experiment was performed on 40 adult New Zealand rabbits weighing (3.65±0.35 kg) and of 9 months age. The animals were fed ad libitum pellets standard rabbit ration and free access to water. Animals were divided into four groups (10 animals each). Control group (G₁) includes animals that injected intramuscularly with olive oil. Groups 2, 3 and 4 include animals that receive one, two and three intramuscular injections of 5 mg kg^-1 body weight boldenone undecylenate, respectively. Boldenone undecylenate was dissolved in the olive oil. The animals were dissected after 3, 6 and 9 weeks, respectively (El-Moghazy et al., 2012; Tousson et al., 2013). At the end of the experiment, the rabbits were fasted for 10 h then euthanized with intravenous injection with sodium pentobarbital and subjected to a complete necropsy.

Muscles (Cardiac, skeletal and smooth) homogenate (10%; w/v) was prepared in ice-cold 0.067 M phosphate buffer (pH = 7) then, the homogenate was centrifuged at 3000 rpm for 10 min at 4°C. The resulting supernatant was used to determine the total protein by comassie blue according to Bradford (1976); Nitric Oxide (NO) according to Vodovotz (1996); total lipids according to Esher et al. (1973); superoxide dismutase activity (SOD) according to Oyanagui (1984); glutathione (GSH) according to Beutler et al. (1963) and lipid peroxidation (malondialdehyde; MDA) according to Lahouel et al. (2004).

Statistical analysis: Data were expressed as mean values±SE and statistical analysis was performed using one way ANOVA to assess significant differences among treatment groups. The criterion for statistical significance was set at p<0.05 for the biochemical data. All statistical analyses were performed using SPSS statistical version 16 software package (SPSS^® Inc., USA).

RESULTS

The animals from practice appeared healthy and did not show clinical signs of disease and no rabbit was dead during experiment. As represented in Table 1, there was a significant increase in cardiac total protein, total lipid, superoxide dismutase (SOD), malondialdehyde (MDA), Nitric oxide (NO) and glutathione (GSH) in boldenone treated rabbit (G₂-G₃) as compared to control (G₁). These changes in total antioxidant capacity and the oxidative stress parameters were increased with the increase the boldenone dose injection from one dose to three doses (G₂ and G₄), respectively. On the other hand there was a significant increase in cardiac SOD, MDA and nitric oxide and a non significant change in cardiac total protein, total lipid and GSH as compared to G₂. In G₄, there was a significant increase in cardiac total protein, total lipid, SOD, MDA, NO and GSH as compared to control and G₂. On the other hand there was a significant increase in cardiac total lipid, MDA and GSH and a non significant change in cardiac total protein, SOD and NO as compared to G₃.

Table 2 showed that, a significant increase in smooth total protein, SOD, MDA, NO and GSH in one dose treated group (G₂-G₄) as compared to control group, meanwhile smooth total lipid showed non significant change as compared to G₁. These changes were increased with the increase the boldenone dose injection from G₂ and G₄, respectively.

As represented in Table 3, there was a significant increase in skeletal total protein; total lipid, SOD, MDA and GSH in G₂ as compared to control group, meanwhile skeletal NO showed non significant change as compared to G₁. In G₃, there was a significant increase in skeletal total protein, SOD, MDA, NO and GSH as compared to control group, meanwhile skeletal total lipid showed non significant change as compared to G₁. On the other hand there was a significant increase in skeletal total protein, SOD, MDA and GSH and a non significant change in skeletal total lipid and NO as compared to G₂. In G₄, there was a significant increase in skeletal total protein, total lipid, SOD, MDA and GSH as compared to G₁ and G₂ except for skeletal NO which show a non significant change as compared to G₁ and G₂. These changes in total antioxidant capacity and the oxidative stress parameters were increased with the increase the boldenone dose injection from one dose to three doses (G₂ and G₄), respectively.

DISCUSSION

The abuse of anabolic-androgenic steroids to enhance physical performance is widespread in sport communities despite their reported side effects. Biochemical analysis of the rabbit’s muscles tissues after boldenone injection with different doses showed a significant increase in total protein, total lipid, NO, SOD, GSH and MDA comparing with the control group. In the present study, there is a significant increase in the total NO_x (NO metabolites) in rabbits that injected with boldenone when compared to control group. This finding may be due to increased vascular oxidative burden associated with homocysteinaemia that induces NADPH oxidase and inducible nitric oxide synthase activity, contributing to increased superoxide radicals production in rabbit vessels which react with nitric oxide to form peroxynitrite radicals, leading to low NO bioavailability and endothelial dysfunction (Ungvari et al., 2003). Fahey (1998) reported that the mechanism of action of boldenone undecylenate steroid hormones work by stimulation of receptors molecule in muscle cells, which activate specific genes to produce proteins. They also affect the activation rate of enzyme system involved in protein mechanism. Thus enhancing protein synthesis and inhibiting protein degradation. Effectiveness of anabolic steroids is dependent upon unbound receptor sites in muscle.

Injection of the anabolic steroid boldenone (5 mg kg^-1 body weight) induced changes in oxidative stress biomarker levels and antioxidant defense systems in skeletal, cardiac and smooth muscles. Boldenone treatment can cause an oxidative stress situation in muscles as indicated by enhanced MDA, SOD and GSH extent. Our results showed that, significant increases in the activity of MDA, SOD and GSH in muscles tissues after boldenone injections in G₂, G₃ and G₄ comparing with the control (p<0.05). These changes in total antioxidant capacity and the oxidative stress parameters were increased with the increase the boldenone dose injection from one dose to three doses (G₂ and G₄) respectively. Mitochondria might be an important cellular target for oxidative damage since the mitochondrial membrane is rich in polyunsaturated fatty acids; alteration in the lipid environment of respiratory chain complexes may cause a decrease in their activity leading, in turn, to a perturbation of energetic metabolism. Our results are in agreement with Pey et al. (2003) who reported that the anabolic-androgenic steroids induced changes in oxidative stress. Our results are in agreement with Urhausen et al. (2003) in athletes abusing anabolic androgenic steroids and not agreed with Istasse et al. (1988) who, reported that, estradiol 17β increased nitrogen retention and decreased blood urea nitrogen concentrations. Our results are in agreement with Gabr et al. (2009) who reported that in addition to the growth promoting effects, anabolic steroids have been shown to adversely affect the cardiovascular and endocrine systems. Also, Gabr et al. (2009) and Tousson et al. (2013) reported that the total protein concentrations in male lambs were significantly increased after boldenone injections. Oxidative stress has been also recently implicated in hormone-induced prostate carcinogenesis (Tam et al., 2003). Thus, it is tempting to speculate with the possibility that the observed changes in prooxidant/antioxidant status could be causally related with the adverse effects of Anabolic-androgenic steroids on muscle tissues.

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CONCLUSION

Using boldenone while preparing for a bodybuilding contest may cause an alteration in the biochemical parameters of the rabbit muscles. These findings explain the common phenomena in athletics and bodybuilders who suffer from muscles damage (rhabdomyolysis), atherosclerotic heart disease and myocardial hypertrophy as they injected with some drugs as steroids (boldenone) to build muscles.