Hepatoprotective Effect of Taurine Against Oxidative Stress Due to Methotrexate in Rat

Problem statement: Taurine, 2-aminoethanesulphonic acid is an essentia l amino acid. It is present at high concentrations in many tissues. It plays important roles in numerous physiological functions including conjugation with bile acids, mo dulation of calcium levels and maintenance of osmolarity, antioxidation and stabilization of memb ranes. Methotrexate (MTX), as the most disease modifying anti-rheumatic drug used for Rheumatoid A rthritis (RA), has been available for clinical use since 1951. The main scope of this study was to assessment of probable Hepatop rotective effect of taurine as it antioxidant action against oxidative stress due to methotrexate in rats. Approach: Eighty male Wistar rats, weighing 200±20 gr, were obtained from the animal breeding center of Islamic Azad University. The rats were divided into 4 equal grou ps of 20 animals including: 1-normal control, 2normal rats treated with taurine, 3taurine + meth otrexate and 4-methotrexate. Results: Methotrexateinduced hepatotoxicity as reflected by elevated ser um ALT, AST and GGT levels (p<0.001) and reduced serum total protein level (p<0.001). Conclusion: Taurine has a protective action against hepatotoxins and suggest that taurine may find clin ical application against a variety of toxins where cellular damage is a consequence of reactive oxygen sp cies.


INTRODUCTION
Taurine, 2-aminoethanesulphonic acid is an essential amino acid. It is present at high concentrations in many tissues. It plays important roles in numerous physiological functions including conjugation with bile acids, modulation of calcium levels and maintenance of osmolarity, antioxidation and stabilization of membranes (Huxtable, 1992;Schrader et al., 2009). It was reported to have beneficial effects in various physiological and pathological conditions (Ahn et al., 2001;Chiba et al., 2002;Ozturk et al., 2003) by mainly diminishing production of Reactive Oxygen Species (ROS). It also can prevent DNA damage at physiological concentrations (Heibashy et al., 2008;Messina and Dawson, 2000). Taurine has also hepatoprotective effects such as inhibition of extracellular matrix accumulation in experimental liver fibrosis (Balkan et al., 2001;Chen et al., 1999) and improvement of liver function tests in fatty liver disease of children (Obinata et al., 1996). Hepatoprotective feature of taurine is attributed to its inhibitory activity on generation of ROS, which are known to play an important role in hepatic injury both in vitro and in vivo (Pietrangelo, 1996;Baroni et al., 1998). Moreover, several authors reported beneficial effects of taurine on histopathology and oxidative stress parameters in a rat model of CCl4-induced liver fibrosis (RefikMas et al., 2004;Tasci et al., 2007) where remarkable Histopathological improvement in taurine treated animals subjected to hepatotoxin was observed and this was associated with oxidative stress reduction and hepatocellular apoptosis. Taurine in addition to it's antioxidant effect has other performances such as it Implicated in Bromocriptine Induced Schizophrenia (Abdulrahim, 2010).
Methotrexate (MTX), as the most disease modifying anti-rheumatic drug used for Rheumatoid Arthritis (RA), has been available for clinical use since 1951 (Wilkens and Watson, 1982;Ralandison et al., 2012). Its widespread availability along with the high prevalence of RA which requires long-term therapy has attracted physicians' attention to the adverse reactions of MTX.
Antagonism with folic acid and The increases in aminotransferases (transaminitis) are a potential major adverse reaction seen with long-term use of MTX. Many risk factors such as age, duration of exposure to MTX and its cumulative dose, history of Non Alcoholic Steatohepatitis (NASH), diabetes and obesity, hepatitis B or C virus infection, alcohol consumption and hepatotoxic drugs can increase the hepatotoxic effect of MTX (Lorenz and Knudtson, 2007;Diouf et al., 2001;Visser et al., 2009;Jones and Patel, 2000;Kremer et al., 1995;Langman et al., 2001;Leonard et al., 1987;Malatjalian et al., 1996;Reynold and Lee, 1986;Salliot, C. and Heijde D.V.D., 2009;Walker et al., 1993).

MATERIALS AND METHODS
Experimental plan: This experimental study was carried out in Islamic Azad University Research Center. All procedures were conducted under supervision of Animal Rights Monitoring Committee of Islamic Azad University Research Center.

Chemicals:
All chemicals used in this study were of analytical grade and obtained from Nanjing Jiancheng Bioengineering Institute, Nanjing, China and Ziest Chemi Co., Iran.
Animals: Eighty male Wistar rats, weighing 200±20 gr, were obtained from the animal breeding center of Islamic Azad University. The rats were divided into 4 equal groups of 20 animals including: (1) Normal control, (2) Normal rats treated with taurine, (3) Taurine + methotrexate and (4) Methotrexate. Management and husbandry conditions were identical in all groups with 12/12 h light/dark cycle at 21±2°C. Food and water were provided ad libitum.

Biochemical factors evaluation:
At the end of the experiment, blood samples were collected from the retro-orbital plexus and the sera prepared through centrifuging at 2500×g for 15 min at 30°C After 12 h fasting, blood glucose and serum biomarkers of liver function including ALT, AST (humoller et al, 1957), LDH (Martinek, 1972), albumin, TP (Lowry et al., 1951) and total bilirubin (Malloy and Evelyn, 1936) were measured using commercially available kits.

Measurement of antioxidant activity:
All experimental rats were euthanized by cervical dislocation. The rat's Liver were removed immediately and washed in normal saline and homogenate 10% prepared in 1.15% w/v of potassium chloride. The homogenate was centrifuged in 7000×g for 10 min at 4˚C and supernatant were used for measurement of Oxidative stress by determination of Malondialdehyde (MDA) as well as Antioxidant Enzymes (AOE) such as Superoxide Dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) and glutathione reductase. MDA, SOD, CAT and GSH-PX, GR were measured by using commercially available kits according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Liver homogenate MDA levels were expressed as nmol MDA per mg protein and antioxidant activity was expressed as arbitrary units per mg protein.
Degree of lipid peroxidation in kidney tissue homogenates was determined in terms of thiobarbituric acid reactive substances (TBARSs) formation by following the protocol of Esterbauer and Cheesman (1990). SOD activity was measured by Nishikimi et al. (1972) method and was modified by Kakkar et al. (1984) method. Each unit of SOD activity was determined as required enzyme concentration for prohibition of creation color at 1 minute, under study conditions. CAT activity was measured by Greenwald (1985) method and was based on hydrogen peroxide breakdown. GPX activity was measured by Rotruck et al. (1973) method and was expressed as micromole of GSSG /minute/milligram of protein, based on blew reaction: GR activity was measured by Mohandas et al. (1984) method, based on blew reaction: Statistical analysis: The Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA), version 13.0, was used for statistical analysis. All data are presented as mean ± SEM. Before statistical analysis, all variables were checked for normality and homogeneity of variance by using the Kolmogorov-Smirnoff and Levene tests, respectively. The data obtained were tested by ANOVA followed by Tukey's post-hoc multiple comparison test. p<0.05 was considered statistically significant.

Effect of taurine on MTX-induced hepatotoxicity:
As shown in (Table 1), MTX-induced hepatotoxicity mirrored by increscent in serum ALT, AST and GGT levels (p<0.001) and diminished serum total protein level (p<0.001). After treatment by taurine, no markedly changes were obtained. On the other hand, rats treated with taurine and MTX had markedly increased serum total protein level (p<0.01) and lower serum ALT, AST and GGT levels compared with MTX group (p<0.001).  Fig. 1, TBARS concentration was increased in MTXtreated rats (164.66±9.21 nmol g −1 wet tissue, p<0.001) as compared to normal control rats (49.98±7.13 nmol g −1 wet tissue). Treatment with taurine prohibited MTX -induced lipid peroxidation and leads to significant decline in TBARS level (92.51±6.44 nmol/g wet tissue, p<0.001) as compared to MTX group alone. Fig. 2, the activity of catalase was markedly decreased in MTX group (33.67±3.19_mol/min/mg wet tissue, p<0.001) as compared to normal control group (86.39±2.12_mol/min/mg wet tissue).
Effect of taurine on GSH concentration: As showed in Fig. 4, MTX produced a decrement of hepatic GSH content (0.49±0.016_mol g −1 wet tissue, p<0.01) as compared to normal control group (1.89±0.341_mol g −1 wet tissue). Taurine alone induced a considerably increase in its level (4.249±0.116 _mol/g wet tissue, p<0.001) compared with the control group. Treatment with taurine depredated MTX induced GSH depletion and increased GSH concentration (1.072±0.056_mol/g wet tissue, p<0.05) as compared to MTX group alone.
Correlation analysis: Based on obtained data, TBARS has direct correlation with biochemical parameters related to hepatic injury. This indicates that lipid peroxidation has important role in MTXinduced hepatic injury. Contrary, TBARS has reverse correlation with internal antioxidants. Also, hepatotoxicity indications were inversely correlated with internal antioxidant enzymes. Finally can be conclude that internal antioxidants have protective effect against ROS (Table 2).

DISCUSSION
The protective capacity of taurine was attributed to free radicals scavenging. Subjects treated with placebo showed a significant increase in the number of severely damage mitochondria after reperfusion, whereas the number of damaged and necrotic myocytes also increased significantly in these subjects after infusion. No such damage to mitochondria or myocytes was observed in the taurine treated subjects (Lee et al., 1992). The use of supplemental taurine as a physiological protective against lipid peroxidation was advocated by Gordon et al. (1986) who demonstrated the protection of hamaster bronchioles from acute NO 2induced alterations. The authors outlined the mechanism through which acute ROS tissue damage is believed to act. NO 2 and its highly ROS interact directly with plasma membrane of cell products, possibly via lipid peroxidation triggering a series of events that include the release of chemotactic factors and acute phase reactions responsible for the influx of neutrophils. Activation of neutrophils results in production of superoxide, free radicals and hydrogen peroxide (H 2 O 2 ), which cause further epithelial damage. Activated neutrophils also release proteolytic enzymes that have the capacity to alter alveolar interstitial components. It was proposed by Gordon et al. (1986) and Cardin et al. (1999) that the protective activity of taurine may reside in its ability to become chlorinated in the presence of HOCL, thereby preventing the direct attack of this oxidant on cell membranes (Rock et al., 1996). Lee et al. (1992) postulated that the protective action of taurine on oxidant-induced damages of tissue components including degradation of hyaluronic acid may be attributed to both its scavenging action on hypochloriate (HOCl) and chloramine and the complex formation of taurine with HOCl or NH2Cl without scavenging action on oxygen free radicals.
El-Nahrawy et al., 2011 showed that the best ameliorating effect occurred in all hepatic markers in rats group which treated with both antioxidants (taruine and curcumin) dependent on time of administration (Walaa et al., 2011). Hagar (2004), demonstrated that Taurine administration (1% in the drinking water) for 3 days before and concurrently during CsA injections improved liver functions, as indicated by decline of serum transaminases and GGT levels and elevation of serum total protein. Moreover, taurine significantly reduced hepatic TBARS and increased GSH content and catalase and GSH-Px activities in the hepatic tissue (Hagar, 2004). Jagadeesan and Pillai (2007), reported that liver tissue was completely damaged, after mercury treatment. In another group of animals, taurine (5 mg kg −1 body wt.) was administrated for another 15 days. Taurine administration was observed to improve the liver function in mercury intoxicated animal as indicated by the decline in increased levels of AST, ALT and ALP in serum and LPO content in liver tissue. The decreased level of antioxidant system (GSH, GPx, CAT and SOD) has been promoted (Jagadeesan and Pillai, 2007).

CONCLUSION
These results indicate that taurine has a protective action against hepatotoxins and suggest that taurine may find clinical application against a variety of toxins where cellular damage is a consequence of reactive oxygen species.