The role of antioxidant (vit-A and glutamine) in ameliorating methotrexate induced hepatic toxicity in rats

The aim of this study was to investigate the status of antioxidant glutathione peroxidase (GSH-Px) during oxidative stress in blood serum of rats subjected to oral methotrexate administration at a dose 10 mg/kg B.W and administrated antioxidant therapy (vit A and glutamine) to reduced or ameliorate such stress. A total of thirty six, male Sprague-Dawley rats weighing (250 – 300) g divided equally into six groups. The first group, control, were fed only standard rat chow as their diet and water ad libitum. The second group administered orally a single dose of methotrexate at a dose of 10 mg/kg B.W. The third group, given a single dose of methotrexate 10 mg/kg B.W + Vit-A (5000 IU/kg B.W) orally by stomach tube, twice daily. The fourth group, given a single dose of methotrexate 10 mg/kg B.W + Glutamine (500 mg/kg B.W), twice daily dosing. fifth group, given vit-A at dose 5000 IU/kg B.W orally, twice daily. Sixth group, given glutamate at dose 500 mg/BW orally, twice daily, all this dosing continue for 8 days. At the end from all animal, draw blood from heart by syring to obtained serum to determine its level then sacrificed and take section from liver for histopathology resulted in. It was found that methotrexate caused a signifcant increase in GSH levels (an important marker of lipid peroxidation) when compared with the control group while the level GSH were signifcantly decreased in the methotrexate + vitamin A group, methotrexate+ glutamine group, vitamin A group and glutamine group. The results indicate that methotrexate cause oxidative stress by reducing the activities and consequently the effectiveness of the antioxidant enzyme defense system. while the antioxidant therapy reducing of oxidative stress. Histopathological examination observed was normal in methotrexate treated rats and MTX with glutamine and vitmine A . but showed hydrodegenerate in treated groups of MTX+vit A while the glutamine treated group showed a necrosis and amyliod change. Key ward: methotrexate; oxidative stress; Antioxidant enzymes. ( ةذسكلأا ثاداضملا رود هيماخيف A )هيماحولكلاو يف جيسكرحوثيملا جسكاعم ذبكلا تيمس دذحملا ناررجلاب ُٙٚص ٘ذٓي رناص حٚذؼس ٘شطٛت ةط , حفٕكنا حؼياخ , وًٕسنأ حٚٔدلأا عشف Kufa Journal For Veterinary Medical Sciences Vol. (3) No. (1) 2012 114 تصلاخلا : ي فذٓنا ٌاك حناز قٛقسرهن حساسذنا ِزْ ٍ ( جذسكلأا خاداضي GSH دآخأ للاخ ) ٙف جذسكلاا ود مصي ٌارشدنا نا نات جلاؼهن حضشؼرً ىفنا قٚشط ٍػ خاسكٚشذٕثًٛ جذسكلأن خاداضًنات جلاؼهن حضشؼرًنأ ٍٛيارٛف( A رن )ٍٛياذٕهكنا ٔ ٍٛسسذ ٔا مٛهق دآخا حػًٕدي ديذخرسا .جذسكلاا 36 ؽاشثس سٕكزنا ٍي ( آَصٔ ٌارشدنا ٙنٔاد 300 250 خاػًٕدي دس ٗنإ حًسقي ) .٘ٔاسرنات دًنا دٚزؿ حػًٕ ٗنٔلاا شطٛسنا( )ج ت , واظُ ٔ ٙئازؿ ت نا حػًٕدًنا دػشخ .ِاًٛنأ ٌارشدنا واؼطنا حَٛاث ىفنا قٚشط ٍػ جشي جذزأ ٍي دٛسكشذٕثٛي حػشدت 10 / ؾهي ىـك .ىسدنا ٌصٔ دػشخ ,حثناثنا حػًٕدًنا ٍي جذزأ حػشخ دٛسكٚشذٕثًٛنا 10 / ؾهي ىـك + ىسدنا ٌصٔ A ( ٍٛيارٛف 5000 ًٕٚف )حٛنٔد جذزٔ بٕثَأ قٚشط ٍػ ا دٛطػا .اٛيٕٚ ٍٛذشي , جذؼًنا ,حؼتاشنا حػًٕدًنا دٛسكٚشذٕثًٛنا ٍي جذزأ جشي 10 /ؾهي ىـك ىسدنا ٌصٔ ٍٛياذٕهكنا + 500 / ؾهي ىـك ٍٛيارٛف حسياخنا حػًٕدًنا دٛطػأ .اٛيٕٚ ٍٛذشي ىسدنا ٌصٔ A حػشدت 5000 /IU ىسدنا ٌصٔ ًدًنا دٛطػأ .ىفنا قٚشط ٍػ اٛيٕٚ ٍٛذشي , حػشدت ٍٛياذٕهكنا حسداسنا حػٕ 500 / ؾهي ىـك ىسدنا ٌصٔ خاػًٕدًنا ِزْ غًٛخٔ , اٛيٕٚ ٍٛذشي , غٚشدرنا شًرسا جشرفن 8 .واٚأ ٙفٔ ٖٕرسي ذٚذسرن مصي ٗهػ لٕصسهن حَشس حطسإت ةهقنا ٍي ود خاَإٛسنا مك ٍي دثسس حٚآُنا ذثكنا ٍي غطقي زخأ دزشش ىث ٌٕٚاثٕذٕهكنا صسفهن ٙدٛسُنا خٔ خإٚرسي ٙف حُٕٚؼي جداٚص خاسكشذٕثًٛنا ةثسذ َّأ ذ GSH ٍي حياْ حيلاػ( ٌْٕذنا جذسكا ) جشطٛسنا حػًٕدي غي حَساقًنات ضفخَا اًُٛت إُٚؼي ٖٕرسي ٙف GSH +دٛسكٚشذٕثًٛنا حػًٕدي ٙف ٍٛيارٛف A ٍٛيارٛف حػًٕدي , ٍٛياذٕهكنا+ خاسكٚشذٕثٛي حػًٕدي , A شٛشذٔ .ٍٛياذٕهكنا حػًٕدًنأ قٚشط ٍػ ٙنارناتٔ ذثكنا ٙف جذسكلاا دآخا خاًٚضَا جداٚص ٗنا ٘دؤٚ دٛسكٚشذٕثًٛنا كنر ٌأ ٗنإ حئارُنا جذسكلأن جداضًنا حدناؼًنا ٌأ ٍٛز ٙف .ىٚضَلاا حطشَلأا حٛناؼف ٍي ذسنا جذسكلأن خاداضًنا عافذنا واظَ خذز جذسكلاا ٍي . ظزلا ٙدٛسُنا صسفنا ٙف ا ٙف ٙؼٛثط ٌاك ذثكهن ٔ طقف دٛسكشذٕثًٛنات ددنٕػ ٙرنا حػًٕدًن كنزك ٍٛيارٛف غي ددنٕػ ٙرنا غٛيادًنا ٙف A حػًٕدي ٙف ُْٙذنا مهسذ شٓظ ٍكنٔ ٍٛياذٕهكنأ MTX+vit A .طقف ٍٛياذٕهكنات ددنٕػ ٙرنا حػًٕدًنا ٙف ذثكنا اٚلاخ ٍٛت خاُٛذٔشثنا ذاشذسأ شخُرنا شٓظ كنزكٔ Introduction: Methotrexate (MTX), as a competitive folic acid antagonist. It is an antimetabolite. Its affinity for dihydrofolate reductase is about 10 5 times higher than that for its physiological substrate dihydrofolic acid By inhibiting formation of the product tetrahydrofolic acid it disrupts synthesis of thymidine and nucleotides purines. It is currently the most common anti-rheumatic drug prescribed for the treatment of rheumatoid arthritis and other rheumatic disorders(1). Recently, MTX earned a new indication with its efficacy in the treatment of refractory inflammatory bowel disease (2). However, the efficacy of this drug is often limited by severe side effects and toxic sequelae. Since the cytotoxic effect of MTX is not selective for cancer cells, it also affects the normal tissues that have a high rate of proliferation, including the hematopoietic cells of the bone marrow and the actively dividing cells of the gut mucosa. (3) Methotrexate has also been used in the treatment of psoriasis, a nonneoplastic disease of the skin characterized by rapid proliferation of epidermal cells, as well as in allogenic bone marrow and organ transplantation.(4) Kufa Journal For Veterinary Medical Sciences Vol. (3) No. (1) 2012 115 Oxidative stress is more and more recognized by the scientific community to be an important factor in the genesis of chronic diseases, cancers, cardiovascular diseases and aging (5). represents an imbalance between the production and manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Some reactive oxidative species can even act as messengers through a phenomenon called redox signaling.(6) Oxidative stress has emerged as a key player in the pathogenesis of MTX-induced hepatotoxicity. The increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development and progression of hepatotoxicity. Furthermore, it has been found that MTX increases the number of Ito cells(fat-storing, vitamin Acontaining stellets cells). Ito cells can be transformed in to myofibroblasts, capable of secreting collagen. This collagenisation consequently leads to liver fibrosis. (7) Biological oxidative stress of free radicals is controlled by endogenous antioxidants including the scavenger enzymes superoxide dismutase (SOD) glutathione peroxidase (GSHPx) and catalase (CAT) and by exogenous dietary antioxidants vitamin E, C carotenoids and flavonoids . Briefly, antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes.(8) The organism exerts several defense mechanisms against free radicals. The anti-radical defense system comprises endogenous anti-oxidants (synthesised by the organism) and exogenous anti-oxidants (external supply). There are two classes of antioxidants, “scavenger” antioxidants trap-ping ROS (reactive oxygen species) and “preventive” antioxidants. Preventive antioxidants inhibit the synthesis of ROS (9). Taking these supplements antioxidant may support or otherwise help these medication work better and may help reduce the likelihood and/or severity of a potential side effect caused by the medication.(10). The aim of this study was to investigate the role of vit-A and glutamine, antioxidant, on methotrexate-induced liver oxidative stress in rats. Material and Method: Animal Male Sprague-Dawley rats weighing (250 – 300) g were used throughout the experiments. The animals were placed in Kufa Medical College's animal house. The rats were maintained in cages in the Kufa Journal For Veterinary Medical Sciences Vol. (3) No. (1) 2012 116 animal care facility, subjected to alternate 12-hour periods of dark and light and were allowed ad libitum intake of chow and water. Chemicals and drugs Methotrexate 2.5 mg/BW the manufacturer by Austrialia, Vitamine A 50000 IU manufacture by Egypt and glutamine 500mg manufacture by Holland were purchased from a local pharmacies , corn oil and ether. Animal group Thirty six adult male rats were equally divided into six experimental groups as follows: Group 1: ( control group 6 animals ) : were given diet and tap water, add libitum and served as control . Group 2: (methotrexate-treated group 6 animals) were given a single dose of methotrexate at dose 10 mg/BW orally by stomach tube, daily for period 8 days Group 3: (methotrexate vit A treated group, 6 animals) , were given a single dose of methotrexate 10 mg/BW + Vit-A (5000 IU) orally by stomach tube, twice daily for period 8 days . Group 4: (methotrexateglutamine treated group, 6 animals ) were given a single dose of methotrexate 10 mg/BW + Glutamine (500 mg/BW) orally by stomach tube, twice daily dosing for period 8 days. Group 5:( vitamin A group, 6 animals) were given vit-A at dose 5000IU/BW orally, twice daily for period 8 days. Group 6: ( glutamine group, 6 animals) were given glutamate at dose 500mg/BW orally, twice daily for period 8 days. At the end of the experiment, all rats were anesthetized using ether and drawed blood 4ml from heart using a 23-gauge needle syringe. The blood placed in centrifuge 3000/minute for 10 minutes to obtain serum for analyzing Oxidation parameters including serum GSH. Measurement of oxidative stress. Determination of Total Glutathione in serum(Mmol/L). A) Principle: 5, 5-Dithiobis (2-nitrobenzoic acid) (DTNB) is a disulfide chromogen that is readily reduced by sulfhydryl group of GSH to an intensely yellow compound. The absorbance of the reduced chromogen is measured at 412 nm and is directly proportional to the GSH concentration (108) : B) Reagents: 1. 4 % 5-sulfosalicylic acid. 2. 0.1 mM Ellman’s reagent was prepared from DTNB (5-5-dithiobis (2-nitrobenzoic acid) ; M.Wt = 396.3 gm/mole) in phosphate buffer pH 8. 3. Phosphate buffer pH 8 (this solution was prepared by a mixture of 0.6 M KH2PO4, and 0.8M Na2HPO4). Kufa Journal For Veterinary Medical Sciences Vol. (3) No. (1) 2012 117 C) Procedure: Two sets of tubes were prepared as follow: All tubes were mixed, and the absorbance of sample was read at 412 nm by using spectrophotometer instrument. D) Calculation: The concentration of glutathione is calculated by using the following equation: A of sample The concentration of GSH = ε x L Where: ε = Extinction coefficient (13600 M -1 cm -1 ), L = Light path (cm). Histopathological preparation : Section from different groups were prepared by routine techniques . the section of liver were fixed in neutral buffered formalin (10%). 56μm sections were stained by H&E (hemotoxylin and eosin stains), change in liver tissue were diagnosed through examination of section by light microscope. Statistic analysis Results were expressed as mean ± SD (standard deviation). Using ANOVAone way, the level of statistical significance was set at P<0.05. Result: Table showed the result concentration of reduced glutathione in the serum of different experimental groups. Reagent Sample Blank