Pretreatment Hepatoprotective Effect of Regular Aerobic Training Against Hepatic Toxicity Induced by Doxorubicin In Rats

Cancer is a major public health problem for many people around the world in the industries countries (Siegel et al., 2012). Conventional drug therapy for many common and chronic disorders, including cancers, has limited efficacy and potentially lifethreatening side effects (Mahmud et al., 2012). Doxorubicin (DOX) is a powerful and highly efficacious drug and shows a broad range of antitumor activity in many kinds of cancers (Vishwanatha et al., 2012). However, the clinical use of DOX is often limited because of its undesirable serious toxic side effects on various organs such as cardiac, liver, and etc. (Ashrafi and Dabidi Roshan, 2012). Several mechanisms have been proposed to account for the DOX-induced cardiotoxic and hepatotoxic side effects including free radical, lipid peroxidation, mitochondrial damage and cellular toxicity (Ashrafi and Dabidi Roshan, 2012). In contrast, several researchers have reported that an organism is generally


Introduction
Cancer is a major public health problem for many people around the world in the industries countries (Siegel et al., 2012). Conventional drug therapy for many common and chronic disorders, including cancers, has limited efficacy and potentially life-threatening side effects (Mahmud et al., 2012). Doxorubicin (DOX) is a powerful and highly efficacious drug and shows a broad range of antitumor activity in many kinds of cancers (Vishwanatha et al., 2012). However, the clinical use of DOX is often limited because of its undesirable serious toxic side effects on various organs such as cardiac, liver, and etc. . Several mechanisms have been proposed to account for the DOX-induced cardiotoxic and hepatotoxic side effects including free radical, lipid peroxidation, mitochondrial damage and cellular toxicity . In contrast, several researchers have reported that an organism is generally RESEARCH ARTICLE

Pretreatment Hepatoprotective Effect of Regular Aerobic Training Against Hepatic Toxicity Induced by Doxorubicin In Rats
Fatemeh Zolfagharzadeh 1 , Valiollah Dabidi Roshan 2 * protected from damage caused by free radicals by means of its antioxidant defense system and administration of DOX modulates glutathione and glutathione-dependent antioxidant systems . The cellular antioxidant defense system operates mainly via antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and apart from nonenzymatic antioxidants. Previous studies showed that one of the major functions of antioxidant is the detoxification of reactive species and toxic oxygen metabolites generated by the endogenous and exogenous metabolism pathways (Choi et al., 2013). For example, Glutathione peroxidase mediates scavenging of intermediates such as hydrogen peroxide and hydroperoxides. Therefore, up-regulation of antioxidant and down-regulation of oxidants is increasingly important in oxidative stress related diseases and their care.
On the other hand, drug-induced hepatotoxicity is continuously emerging as a significant cause of liver disorders, representing today a leading cause of acute liver failure in economically developed countries (Mrzljak et al., 2013). During DOX therapy, the liver receives, accumulates and metabolizes high concentrations of DOX. Hence, it is expectable that the liver is one of the most affected organs by DOX therapy. Indeed, almost 40% of patients suffered liver injury after DOX treatment (Carvalho et al., 2009). Thus, protection of normal tissue and organs during acute and chronic DOX exposure is still a matter of special concern. Therefore, it is necessary to search for complementary and alternative strategies against DOX-induced hepatotoxicity. Although, there is evidence that acute exercise resulted in oxidative stress and cardiac damage , it seems probable that regular endurance exercise training could constitute an excellent tool either to prevent and/or to treat several diseases. Also, while most recent studies have focused on the treatment effect of endurance exercise in induce DOX cardiotoxicity (Wonders et al., 2009;Shirinbayan and Dabidi Roshan., 2012), To the best of our knowledge, we are the first to investigate the hepatoprotective effects of prior (pretreatment) short-term treadmill running aerobic exercise on doxorubicin-induced hepatotoxicity with various dosages (10 and 20 mg/kg) of the DOX drug in liver tissue. These new insights would consist in the recognition of regular training as a non-drug therapeutics protective strategy against DOX treatment. The hypothesis proposed was that if DOX hepatotoxicity is related to free radical formation and oxidative stress, an enhancement in antioxidant/oxidation ratio after regular endurance exercise may protect against DOX-induced toxicity in the liver tissue. Therefore, the purpose of this study was to determine the pretreatment effects of 6 weeks treadmill running on malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in rats that have been acutely exposed to DOX-induced hepatotoxicity with various dosages (10 and 20 mg/kg).

Experimental design and laboratory environment
The experimental protocol of the current study approved by department of physiology, university of Mazandaran were performed according to guiding procedures in the care and use of animals, prepared by the Council of the American Physiological Society. The experiments were carried out with forty-eight Wistar male rats, (8-weeks-old, initially weighing 269±4 g), which were obtained from the laboratory of animal bearing and multiplying at the Pasture institute of Iran. Rats were housed in standard cages of polycarbonate (20×15×15 cm), made at the Pasture institute of Iran, in a large air-conditioned room with a controlled temperature of 22±2°C, light-dark cycles of 12: 12 h and humidity of 50±5%. The pollutant standard index (PSI) was in the acceptable range as determined by the Iranian meteorological organization. Rats were fed with a standard rat chow provided by Pars Institute for animals and poultry with a daily regimen of 10 g per 100 g body weight for each rat. Water was available ad libitum.

Familiarization and aerobic exercise protocols
Animals were habituated to treadmill running for 5 days (10 min exercise/day at 10 m/min, 0% grade). Because rats are more active in darkness, the front portion of the treadmill lines was covered with a dark thick paper to darken this area. At the rear of the lines, an electric grid provided a stimulus for running. An electric stimulus (30 V and 0.5 A) was manually turned on for less than 2 s when the animals stayed on the electric grid for longer than 10 s. Rats quickly learned to stay on the belt and avoid shock, except for one rat, which would not stay on the moving belt, and thus was quickly removed from familiarization process. Following this familiarization period, they were randomly assigned into sedentary and trained groups. Exercise training protocol was performed on treadmill with zero slopes between 25 to 54 min/session and 15 to 20 m/min, 5 days/week for 6 weeks. We replicated the aforesaid exercise training protocol that was previously reported by .
Doxorubicin treatment DOX was obtained from EBEWE Pharma Ges.m.b.H.Nfg. KG (A-4866 unterach, Austria) as a vial of ph. Eur. In order to bring the drug concentration of 10 and 20 mg/ kg, it was dissolved in 0.9% saline for administration. The dose 20 mg.kg -1 of DOX is human clinical doses that are pharmacologically scaled for use in rats (Ogawa et al., 2011). Saline was used as the vehicle and the placebo treatment and was used to form saline solution (0.9% NaCl ip).

Liver tissue collection and preparation
All groups were anesthetized with ketamine and xylozine and decapitated after 10 to 12 h overnight fasting. Abdominal cavity was opened and the liver was quickly excised from the hiatus of liver. Liver tissue were weighed and placed into Petri dishes containing cold isolation medium (0.1 mol/L K2HPO4, 0.15 mol/L NaCl, pH 7.4) to remove the blood and were frozen immediately in liquid nitrogen and stored at -80ºC for subsequent analysis of MDA, NO, SOD and GPx. Liver tissue was squashed in liquid nitrogen, homogenized in a lysis buffer (5 ml/g of tissue) with a protease inhibitor cocktail for mammalian cell and tissue extracts (Sigma-Aldrich, St. Louis, U.S.A) 100 ul/1 ml, and 10 m Mtris base (Sigma-Aldrich, St. Louis, U.S.A), pH 7.4 and centrifuged at 1500 g at 4ºC for 15 min. The homogenates were diluted with cold 20 mM Tris-HCl and centrifuged (10 min at 58C, 3000 g). Biochemical measurements on activity of the GPx enzyme were conducted using GPx-Pretreatment Hepatoprotective Effect of Regular Aerobic Training 340 kit (OXIS, Portland, OR, USA). In the supernatants, activity of GPx was estimated by spectrophotometry. All samples were processed in the same assay to avoid interassay variations. Superoxide dismutase (SOD) activity was determined spectrophotometrically using the method described by . In brief, for total SOD (tSOD) activity the adequate amount of protein (2 mg tissue wet weight) was incubated at 258C with 1 mM N,Nbis(2-(bis(carboxymethyl)amino)-ethyl) glycine (DTPA) in 50 mM Tris_HCl, pH 8.2, in 1 ml final volume. Reaction was started with 0.3 mM pyrogallol, in which the auto-oxidation rate was recorded at 420 nm. Furthermore, the NO concentration was determined by first reducing the nitrate to nitrite using nitrate reductase (Sigma-Aldrich, St. Louis, USA). Lipid peroxidation levels in the homogenate tissue were measured with the thiobarbituric acid reaction using the method of Ohkawa et al. (1979). The Thiobarbituric acid-reactive substances (TBARS) were quantified at 532 nm by comparing the absorption to a standard curve of malondialdehyde (MDA) equivalents generated by acid catalysed hydrolysis of 1,1,3,3 tetramethoxypropane.

Statistical analysis
All data have been expressed as mean±SD. Statistical analysis was performed using a commercial software package (SPSS version 20.0 for Windows). Data of the biomarkers related to the liver oxidative stress were normally distributed after log-transformation. A one-way analysis of variance (Statistics software, Stat Soft, Inc., Tulsa, OK) was used to detect statistical differences between groups. A post-hoc test (Tukey test) was performed to determine differences in the various biomarkers between groups. Differences were considered statistically significant at p-value<0.05.

Results
Mean Values from malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in rats that have been acutely exposed to DOX-induced hepatotoxicity with various dosages (10 and 20 mg/kg), are shown in Tables 1 and 2. After DOX10 mg/kg administration, there was a significant increase (33.8%) in MDA levels, an insignificant increase (20%) in NO levels and an insignificant decrease in SOD and GPX levels (7.39% and 9.21%, respectively) as compared to control+saline group. However, DOX20 mg/kg treatment groups was showed a significant increase in MDA and NO levels (70.99% and 35%, respectively) and a significant decrease (24.85%) in SOD levels and an insignificant decrease (23.56%) in GPx levels, in comparison with control+saline group.
In contrast, the 6 weeks of treadmill running aerobic training led to an insignificant decrease (17.45 %) in

NO(nm/mg protein)
control exercise levels, and a decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPX) values in liver tissue. This result indicates that there is a potent relationship between oxidative stress and DOX-induced hepatotoxicity.
The main findings of our study was that pretreatment of treadmill running training prevented the Dox-induced hepatotoxicity in the aforesaid biomarkers levels. In the present study, a significant decrease in the MDA and No levels and a significant increase in SOD and GPx values in rats treated with training+DOX treated groups, suggests the protective and pretreatment effect of aerobic exercise in DOX-induced oxidative stress in liver tissue. These findings are consistent with data from randomized controlled trials that reported increase in DOX-induced cardiotoxicity and hepatotoxicity (Injac et al., 2008;Ana et al., 2009;Rašković et al., 2011;Henninger et al., 2012;Mahmud et al., 2012). Furthermore, the current study provided additional support to understand how regular physical exercise, particularly treadmill running training, could contribute to improve of liver resistance against free radical-induced oxidative stress induced by DOX administration.
The liver is a vital organ that plays a major role in metabolism and has a number of important functions in the body including glucose storage in the form of glycogen, plasma protein synthesis and detoxification. During DOX therapy, the liver receives, accumulates and metabolizes high concentrations of DOX. Hence, it is expectable that the liver is one of the most affected organs by DOX therapy. Despite, this effect, the most important cause of DOX toxicity in the liver seems to be oxidative stress (Carvalho et al., 2009). Two different ways of free radical formation by DOX have been described. The first way implicates the formation of a semiquinone free radical by the action of several NADPH-dependent reductases that produce a one-electron reduction of the DOX to the corresponding DOX semiquinone. In the presence of oxygen, redox cycling of DOX -derived quinonesemiquinone yields superoxide radicals. In the second way, DOX free radicals come from a non-enzymatic mechanism that involves reactions with iron (Kalender et al., 2005). DOX not only increases free radical production in the tissue, but also decreases its ability to detoxify reactive oxygen species (Kalender et al., 2005). In the present study, decrease in SOD and GPx values and an increase in MDA and No levels in liver tissue indicate that DOX have hepatotoxic effects. We believe that the changes observed in the antioxidant defenses are a result of DOX induced hepatotoxicity. In contrast, in this study, we showed that the regular exercise training can limited DOX-induced oxidant/antioxidant imbalanced and aerobic regular exercise before administration of DOX may be considered as a potentially useful candidate to protect liver tissue against oxidative stress. These antioxidant enzymes are involved in the reduction of reactive oxygen species (ROS) and peroxides produced in the living organism thus play a vital role in the maintenance of a balanced redox status (Mahmud et al., 2012). The restoration of the SOD activity toward a normal value indicates that the pretreatment of aerobic training can help in cellular defense mechanisms by preventing cell MDA levels in T+DOX 10 mg.kg -1 group and a significant decrease (17.76%) in T+DOX 20 mg.kg -1 group as comparison to similar control group (Figure 1). However, the 6 weeks of treadmill running aerobic training led to a significant increase in NO (20.83% and 18.52%) and SOD (21.40% and 39.96%) levels in T+DOX 10 mg.kg -1 and T+DOX 20 mg.kg -1 , as comparison to similar control groups (Figure 2 and 3). Also, after of the prior (pretreatment) short-term aerobic exercise, GPx value has a significant increase (45.58%) in T+ DOX10 mg.kg -1 group, as comparison to the control+DOX 10 mg.kg -1 group ( Figure 4). However, pretreatment of treadmill running exercise with DOX20 mg.kg -1 administration resulted in an insignificant increase (46.85%) in GPx value, as comparison to the control+DOX 20 mg.kg -1 group. While, after the 6 weeks training, there was no significant difference between the training+doxorubicin 10 mg/kg and training+doxorubicin 20 mg/kg groups in GPx, SOD and NO levels, a significant difference was detected in MDA levels between the aforesaid groups ( Figure 4).

Discussion
The present study was designed to determine pretreatment effects of moderate-term aerobic training before the various dosages (10 and 20 mg/kg) of doxorubicin (DOX) on biomarkers related to oxidative stress in liver tissue. The primary novel finding in present study was that, administration of DOX 20 mg/kg induced hepatotoxicity in rats, which was detected by an increase in malondialdehyde (MDA) and nitric oxide (NO) membrane oxidation. Therefore, in the present study, the hepatoprotection of the regular aerobic training against DOX-induced hepatotoxicity in rats could be attributed to its ability to restore the antioxidant enzymes SOD and GPx of liver cytosol or to the free radical scavenging activity of the pretratment. Liver plays an important role during exercise through glucose release to the bloodstream and gluconeogenesis, and mitochondria are clearly important in exercise performance due to aerobic energy production (Lima et al., 2013). While, current data demonstrate that exercise training protects the liver against Dox-induced oxidative stress, the mechanism(s) by which exercise training protects oxidative stress remain unclear. There were three possible pathways to explain the protective effects of regular endurance exercise against DOX-induced oxidative stress. At present, the principal mechanism of Dox-induced oxidative stress is believed to be increased oxidant production by the mitochondria. In addition, mitochondria are also the major sites for the production of ROS. (Ascensao et al., 2005;Kavazis et al., 2010). ROS can also activate signal-transduction pathways to induce a stress-resistance response that protects against some of the toxic outcomes of ROS generation (Powers et al., 2010). In contrast, exercise seems to increase the oxygen consumption rate by 10-20 fold as reported earlier and might have released the above factors and thereby induced oxidant activity (Husain, 2002). Indeed, exercise training has been reported to produce adaptive responses to oxidative stress, as studied primarily on skeletal muscles, but also in the liver (Lima et al., 2013).
The metabolic adaptations to exercise are not restricted to the working muscle; exercise is also a major challenge to other organs such as cardiac muscle, stomach or brain (Lima et al., 2013). This is particularly relevant to the liver due to its central role in the maintenance of energy supply to the exercising muscle (Hoene et al., 2010). Studies aiming to evaluate the effects of the acute exercise on oxidative stress in the liver have shown increased lipid peroxidation (Korivi et al., 2012), and decreased antioxidant defenses (Lima et al., 2013). Additionally, it is known that strenuous exercise causes a number of marked metabolic changes that may impair mitochondrial function in several ways, one major factor being mitochondrial ROS formation (Radak et al., 2004). Interestingly, mitochondrial dysfunction appears to be a key issue during exhaustive exercise, and may cause oxidative damage and tissue injury to liver, among others organ (Lima et al., 2013).
In conclusion, the results of the present study indicate that pretreatment of aerobic exercise induces positive adaptations in liver tissue of rats, characterized by increases in the antioxidant defense and decrease in the oxidants biomarkers. Therefore, it has a potential hepatoprotective effect against doxorubicin (DOX)-induced hepatotoxicity with dosages 10 and 20 mg.kg. However, further studies are required to examine other mechanisms, which are responsible for the hepatoprotective efficacy of this regular exercise protocols and protect against exerciseinduced stress.