Research Article Effect of thiamine pyrophosphate on ischemia-reperfusion induced oxidative damage in rat kidney Durdu Altuner, Nihal Cetin, Bahadir Suleyman, Zeynep Aslan1, Ahmet Hacimuftuoglu1, Mine Gulaboglu2, Neslihan Isaoglu3, Ismail Demiryilmaz4, Halis Suleyman ABSTRACT Department of Pharmacology, Objectives: The biochemical effects of thiamine pyrophosphate on ischemia-reperfusion Faculty of Medicine, Recep Tayyip (IR) induced oxidative damage and DNA mutation in rat kidney tissue were investigated, Erdogan University, Rize, and compared to thiamine. 1 Department of Pharmacology, Materials and Methods: Rats were divided into four groups: Renal ischemia-reperfusion Faculty of Medicine and (RIR); thiamine pyrophosphate + RIR (TPRIR); thiamine + RIR (TRIR); and sham group (SG). 2Biochemistry, Faculty of Pharmacy, Results: The results of biochemical experiments have shown that malondialdehyde (MDA) Ataturk University, Erzurum, 3Department of Anesthesia, Nene levels in rat kidney tissue after TRIR and TPRIR treatment were 7.2 ± 0.5 (P > 0.05) and 3.3 Hatun Obstetrics and Gynecology ± 0.3 (P < 0.0001) µmol/g protein, respectively. The MDA levels in the SG rat kidney tissue Hospital, Erzurum, 4Department of and in RIR group were 3.6 ± 0.2 (P < 0.0001) and 7.6 ± 0.6 µmol/g protein, respectively. General Surgery, Ibni Sina Hospital, Total glutathione (tGSH) levels in TRIR, TPRIR, SG, and RIR animal groups were 2.2 ± 0.3 Kayseri, Turkey (P > 0.05), 5.8 ± 0.4 (P < 0.0001), 6.2 ± 0.2 (P < 0.0001), and 1.7 ± 0.2 nmol/g protein, respectively. In the TRIR, TPRIR, SG, and RIR animal groups; 8-hydroxyguanine (8-OHGua)/ Received: 05-06-2012 Gua levels, which indicate mutagenic DNA, were 1.75 ± 0.12 (P > 0.05), 0.93 ± 0.1 (P < Revised: 03-02-2013 0.0001), 0.85 ± 0.08 (P < 0.0001), and 1.93 ± 0.24 pmol/L, respectively. Accepted: 26-04-2013 Conclusions: It has been shown that thiamine pyrophosphate prevents increase in mutagenic DNA in IR induced oxidative damage, whereas thiamine does not have this Correspondence to: effect. Dr. Halis Suleyman, E-mail: [email protected] KEY WORDS: DNA mutation, ischemia-reperfusion, oxidative damage, rat, thiamine pyrophosphate Introduction malondialdehyde (MDA).[4] Furthermore, it has been reported that excessive free oxygen radicals react with DNA, causing Ischemia-reperfusion (IR) process, which is performed oxidative damage of DNA. This oxidative damage is characterized during kidney transplants and various urological procedures, by DNA breaks in base chains and ribose-phosphate bonds, as [1] leads to kidney damage. IR injury is a complex condition and is well as double strand breaks. The initial damage is caused the main cause of acute renal failure. As soon as reoxygenation by formation of pyrimidine dimers due to breaks in thymine- is achieved by reperfusion, the enzyme xanthine oxidase, which adenine bonds. After reaction with free oxygen radicals, is formed during ischemia[2] converts accumulated hypoxanthine base changes in nucleic acids and chain breaks in DNA are to xanthine, resulting in a large amount of free oxygen radicals encountered. If this damage cannot be repaired, DNA mutation in the environment.[3] The free oxygen radicals, which are called will follow. 8-hydroxyguanine (8-OHGua) is a mutagenic form reperfusion mediators, oxidate the cell membrane lipids and of DNA.[5] GSH and other antioxidants prevent harmful effects cause formation of toxic products, such as aldehydes and of free oxygen radicals on cellular structures, but when the antioxidant defense mechanisms fail, serious tissue damage [6] Access this article online occurs. This suggests that treatment with antioxidants may Quick Response Code: be beneficial in IR injury. Website: www.ijp-online.com Thiamine pyrophosphate, which we have used in our study DOI: 10.4103/0253-7613.115005 for treatment of IR injury, is an active metabolite of thiamine. Thiamine pyrophosphate is formed by phosphatizing of thiamine in the liver. Thiamine increases the formation of antioxidants and nicotineamide adenine dinucleotide phosphate (NADPH) levels via pentose phosphate pathway.[7] Because IR damage Indian Journal of Pharmacology | August 2013 | Vol 45 | Issue 4 339 Altuner, et al.: Thiamine pyrophosphate on IR in rat kidney is a rapidly developing process, thiamine pyrophosphate use, of TPRIR and TRIR groups were compared with results of the that its active metabolite, thought to be more effective. The RIR and SG groups. antioxidant properties of thiamine pyrophosphate is not yet Biochemical Analysis of Renal Tissue fully known. In recent years, studies have shown the antioxidant From each kidney, a sample of renal tissue of 0.2 g was activity of thiamine pyrophosphate. In a study, it was found that obtained. The samples were homogenized in ice with 2 ml IR induced oxidative damage in rat ovary was not prevented by of 1.15% potassium chloride buffer for MDA analysis and [8] thiamine, but it was prevented by thiamine pyrophosphate. In phosphate buffer with a pH of 7.5 for other analyses. Then, addition, unlike thiamine, thiamine pyrophosphate is a cofactor they were centrifuged at 4°C, 10,000 rpm for 15 min. The for both pyruvate-2-oxoglutarate dehydrogenase complex that supernatant was used as the analysis sample. For all the is required for mitochondrial adenosine triphosphate (ATP) measurements, the tissue-protein estimation was performed synthesis and transketolase enzyme plays an important role using Bradford’s method.[10] in maintaining cellular redox status.[9] Therefore, thiamine MDA Analysis pyrophosphate is important for cellular energy production and The concentrations of renal lipid peroxidation were preservation. In literature, we have not found any information determined by estimating MDA using thiobarbituric acid test.[11] about protective effect of thiamine pyrophosphate in IR induced The rat renal tissues were rinsed with cold saline. The corpus kidney injury in rats. The aim of our study was to investigate the mucosa was scraped, weighed, and homogenized in 10 ml of biochemical effects of thiamine pyrophosphate on IR induced 100 g/L KCl. The homogenate (0.5 ml) was added to a solution oxidative damage and DNA mutation in rat kidney tissue, and containing 0.2 ml of 80 g/L sodium lauryl sulfate, 1.5 mL of to compare it to that of thiamine. 200 g/L acetic acid, 1.5 mL of 8 g/L 2-thiobarbiturate, and Materials and Methods 0.3 mL distilled water. The mixture was incubated at 98°C for 1 h. Upon cooling, 5 ml of n-butanol:pyridine (15:l) was Animals added. The mixture was vortexed for 1 min and centrifuged In this study, 24 albino Wistar male rats of 230–245 g for 30 min at 4,000 rpm. The absorbance of supernatant was were used, which were supplied from Atatürk University measured at 532 nm. The standard curve was obtained by using Medical Application and Research Center. The animals were 1,1,3,3-tetramethoxypropane. kept in room temperature (22°C) in groups and were fed. In Total Glutathione (tGSH) Analysis document B.30.2.ATA.0.23.85-57 dated 5 April 2012, Atatürk The amount of GSH in the total homogenate was University Local Ethical Committee of Experimental Animals measured according to the method of Sedlak and Lindsay (AUDHADYEK) approved that all the steps of this study were with some modifications.[12] The sample was weighed and compliant with ethical rules. homogenized in 2 mL of 50 mM tris-HCl buffer containing 20 mM Chemicals ethylenediamineteraacetic acid (EDTA) and 0.2 mM sucrose at Thiopental sodium was provided by IE Ulagay, Turkey. pH 7.5. The homogenate was immediately precipitated with 0.1 Thiamine and thiamine pyrophosphate were obtained from mL of 25% trichloroacetic acid, and the precipitate was removed Biopharma, Russia. after centrifugation at 4,200 rpm for 40 min at 4°C and the General Procedure supernatant was used to determine GSH levels. A total of 1500 The rats were divided into four groups as follows: Renal µL of measurement buffer (200 mM tris-HCl buffer containing 0.2 ischemia-reperfusion group (RIR); thiamine pyrophosphate mM EDTA at pH 7.5), 500 µL supernatant, 100 µL 5,5-dithiobis + RIR group (TPRIR); thiamine + RIR group (TRIR), and sham (2-nitrobenzoic acid) (DTNB) (10 mM), and 7900 µl methanol group (SG). The surgical interventions on rats were performed in were added to a tube and vortexed and incubated for 30 min in laboratories, under sterile conditions, using thiopental sodium 37°C. DTNB was used as a chromogen and it formed a yellow- (25 mg/kg intraperitoneal (i.p.)) anesthesia. colored complex with sulfhydryl (SH) groups. The absorbance was Performing Study measured at 412 nm using a spectrophotometer. The standard An hour before the thiopental sodium anesthesia, TPRIR curve was obtained by using reduced GSH. group (n = 6) was given thiamine pyrophosphate 25 mg/kg Isolation of DNA from Renal Tissue i.p. and TRIR group was given thiamine 25 mg/kg i.p. In the DNA was isolated from renal tissue using Shigenaga RIR and SG groups, distilled water was injected via the same et al.’s, modified method.[13] The samples (for renal tissue way. After thiopental sodium injection, rats were kept until 50 mg) were homogenized at 4°C in 1 mL of homogenization appropriate moment for surgical intervention. The moment buffer (0.1 M NaCl, 30 mM Tris, pH 8.0, 10 mM EDTA, that animals remain motionless in supine position is considered 10 mM 2-mercaptoethanol, 0.5% (v/v) Triton X-100) with the appropriate time to perform surgery. The kidneys of rats six passes of a teflon glass homogenizer at 200 rpm. The were reached through a unilateral dorsal incision on the left samples were centrifuged at 4°C for 10 min at 1,000 g to side.