Effect of Thiamine Pyrophosphate on Ischemia-Reperfusion Induced Oxidative Damage in Rat Kidney

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. Subsequently, except for the SG group, a vascular clip was pellet nuclei. The supernatant was discarded, and the crude placed on lower part of renal artery and vein, and ischemia was nuclear pellet resuspended and rehomogenized in 1 mL of maintained for 1 h. After this period, in the RIR, TPRIR, and extraction buffer (0.1 M Tris, pH 8.0, 0.1 M NaCl, 20 mM TRIR groups; vascular clip was removed in order to provide EDTA) and recentrifuged as above for 2 min. The washed reperfusion for 6 h. Afterwards, all the animals were terminated pellet was resuspended in 300 µl of extraction buffer with by high-dose anesthesia, both kidneys were removed and a wide orifice 200 µl Pipetman tip. The resuspended pellet biochemical studies were performed. The biochemical results was subsequently incubated at 65°C for 1 h with presence of

340 Indian Journal of Pharmacology | August 2013 | Vol 45 | Issue 4 Altuner, et al.: Thiamine pyrophosphate on IR in rat kidney

0.1 ml of 10% sodium dodecyl sulfate (SDS), 40 µL proteinase Effect of thiamine pyrophosphate and thiamine on MDA levels K, and 1.9 mL leukocyte lysis buffer. Then, ammonium acetate in kidney tissue after IR was added to crude DNA sample to give a final concentration As seen in Figure 1, MDA levels in TPRIR and TRIR groups of 2.5 mol/L and centrifuged in a microcentrifuge for 5 min. were 3.3 ± 0.3 and 7.2 ± 0.5 µmol/g protein, while 3.6 ± 0.2 and The supernatant was removed and mixed with two volumes 7.6 ± 0.6 µmol/g protein in the SG and RIR groups; respectively. of ethanol to precipitate DNA fraction. After centrifugation, MDA level is lower in the SG (P < 0.0001) and TPRIR groups than the pellet was dried under reduced pressure and dissolved in in the RIR group (P < 0.0001) It does not show any statistically sterile water. The absorbance (A) of this fraction was measured significant difference of MDA levels between the TRIR and RIR at 260 and 280 nm. Purification of DNA was determined as A groups (P > 0.05) [Figure 1]. 260/280 ratio 1.8. Effect of thiamine pyrophosphate and thiamine on tGSH levels DNA Hydrolysis with Formic Acid in kidney tissue after IR Approximately 50 mg of DNA was hydrolyzed with 0.5 mL The tGSH levels in TPRIR, TRIR, SG, and RIR groups were of formic acid (60%, v/v) for 45 min at 150°C. The tubes were 5.8 ± 0.4, 2.2 ± 0.3, 6.2 ± 0.2, and 1.7 ± 0.2 nmol/g protein, allowed to cool. The contents were then transferred to Pierce respectively. tGSH level is higher in SG (P < 0.0001) and TPRIR microvials, covered with Kleenex tissues cut to size (secured groups than in RIR group (P < 0.0001). tGSH levels of TPRIR and in place using a rubber band), and cooled in liquid nitrogen. RIR groups were statistically not different (P > 0.05) [Figure 2]. Formic acid was then removed by freeze drying. Before analysis Effect of thiamine pyrophosphate and thiamine on 8-OHGua/ by high performance liquid chromatography (HPLC), they were Gua levels in kidney tissue after IR redissolved in the eluent (final volume 200 µL). 8-OHGua/Gua levels in TPRIR, TRIR, SG, and RIR groups Measurement of 8-hydroxy-2 deoxyguanine (8-OH Gua) were 0.93 ± 0.1, 1.75 ± 0.12 pmol/L, 0.85 ± 0.08 pmol/L, and The amount of 8-OH Gua and Gua were measured by using 1.93 ± 0.24 pmol/L, respectively. 8-OHGua/Gua level was a HPLC system equipped with an electrochemical detector (HP lower in SG and TPRIR groups than in RIR group (P < 0.0001). Agilent 1100 module series, E.C.D. HP 1049 A), as described 8-OH Gua/Gua levels of TPRIR and RIR groups did not differ > previously.[14] The amount of 8-OH Gua and Gua were analyzed statistically (P 0.05) [Figure 3]. on a 250 4.6 mm Supelco LC-18-S reverse-phase column. Discussion The mobile phase was 50 mM potassium phosphate, pH 5.5, In this study, the biochemical effects of thiamine with acetonitrile (97 volumes of acetonitrile and 3 volumes of pyrophosphate on the IR induced oxidative stress and DNA potassium phosphate), and the flow rate was 1.0 mL/min. The mutation in rat kidney were investigated, and were compared detector potential was set at 0.80 V for measuring oxidized to that of thiamine. It is known that tissue damage of various base. Gua and 8-OH Gua (25 pmol) were used as standards. degrees occur after IR of the kidneys. The oxygen radicals such The 8-OH Gua levels were expressed as number of 8-OH Gua as superoxide anion (O −), hydrogen peroxide (H O ), hydroxyl molecules/105 Gua molecules.[15] 2 2 2 radical (OH), and peroxynitrite anion (ONOO−) are thought to Statistical Analysis be responsible for tissue damage associated with IR.[16] During All data were subjected to one-way analyze of variance IR, the excessive free oxygen radicals increased the lipid (ANOVA) using Statistical Package for the Social Sciences peroxidation, impairing integrity of cellular structure.[17] In our (SPSS) 18.0 software. The differences among groups were study, MDA levels in kidney tissue in RIR group were significantly analyzed using the least significant difference (LSD) option and higher than the SG group. This indicates the presence of a P-value < 0.05 was considered to be statistically significant. oxidative stress in rat kidney tissue in our experiment. The results are presented as the mean ± standard error of It is known that serious imbalance between free radical mean (SEM). formation and antioxidant defense mechanisms during oxidative stress leads to tissue damage.[18] There are studies reporting Results an increase in MDA and a decrease in GSH levels in damaged MDA, GSH and 8-OHGua levels in SG, TRIR, TPRIR and RIR kidney tissue after IR.[19] In tissues, there are enzymatic and groups were seen in Table 1. nonenzymatic antioxidant mechanisms against oxidative damage.

Table 1:

Effect of thiamine pyrophosphate and thiamine on amount of 8-hydroxyguanine (8-OHGua), malondialdehyde (MDA), and total glutathione (tGSH) levels in kidney tissue after ischemia-reperfusion

Groups MDA Significance tGSH Significance 8-OHGua Significance (µmol/g protein) P-value (nmol/g protein) P-value (pmol/L) P-value SG 3.6 ± 0.2* <0.0001 6.2 ± 0.2* <0.0001 0.85 ± 0.08 <0.0001 TRIR 7.2 ± 0.5 >0.05 2.2 ± 0.3 >0.05 1.75 ± 0.12 >0.05 TPRIR 3.3 ± 0.3* <0.0001 5.8 ± 0.4* <0.0001 0.93 ± 0.13 <0.001 RIR 7.6 ± 0.6 – 1.7 ± 0.2 – 1.93 ± 0.24 – Significance was evaluated according to ANOVA and LSD tests. Results are mean ± standard error of mean (n = 6,). *P < 0.0001 according to RIR group RIR = Renal ischemia-reperfusion group, TRIR = Thiamine received RIR group, TPRIR = Thiamine pyrophosphate received RIR group, SG = sham group

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Figure 1: Effect of thiamine pyrophosphate and thiamine on Figure 2: Effect of thiamine pyrophosphate and thiamine on glutathione malondialdehyde levels in kidney tissue after ischemia-reperfusion (IR) levels in kidney tissue after ischemia-reperfusion

*P < 0.0001 * P < 0.0001 according to RIR according to RIR group group RIR-Renal ischemia- RIR-Renal reperfusion group ischemia- TRIR-thiamine reperfusion group received RIR group TRIR-thiamine TPRIR-thiamine received RIR group pyrophosphate TPRIR-thiamine received RIR group pyrophosphate received RIR group

of a large number of phosphate groups with a negative load, it Figure 3: Effect of thiamine pyrophosphate and thiamine on 2+/3+ 1+/2+ [23] 8-hydroxyguanine/Guanine levels in kidney tissue after ischemia- is bounded to positive metal ions such as Fe and Cu . reperfusion (IR) These DNA bonded metal ions react with H2O2 in the nucleus (on the DNA), and form toxic radicals such as OH leading to oxidative damage of DNA.[22] It was shown in tissue cultures, by increasing Fe3+and Cu2+ concentrations, oxidative DNA base

damage increases; and in the cells exposed to H2O2, the use of copper and/or iron chelators prevents oxidative damage to DNA.[24] This indicates the importance of use of antioxidants together with iron chelators in prevention of DNA damage. In our study, we have shown that thiamine pyrophosphate *P < 0.0001 according to prevents increase in mutagenic DNA (8-OHGua) levels in case of RIR group oxidative damage induced by IR in rat kidney. Although Lukienko RIR-Renal ischemia- [25] reperfusion group et al., demonstrated the antioxidant activity of thiamine, in TRIR-thiamine received our study, thiamine could not prevent oxidative damage and RIR group TPRIR-thiamine associated increase in mutagenic DNA levels after IR in kidney pyrophosphate received tissue. RIR group As a result, we have found an increase in mutagenic DNA levels in rat kidney tissue, associated with oxidative stress, after Reduced GSH is the most important endogenous nonenzymatic IR injury. It was found that thiamine pyrophosphate prevents antioxidant. Reduced GSH plays a role in protection of cells increase in mutagenic DNA levels in case of oxidative damage. − from superoxide anion (O2 ), hydrogen peroxide (H2O2), hydroxyl Thiamine, on the other hand, does not have this effect. The (OH), and peroxynitrite anion (ONOO−) radicals.[20] In some protective effect of thiamine pyrophosphate on IR damage of experimental kidney failure models, it was reported that reduced kidney can be due to its antioxidant activity. GSH decreases in case of kidney damage.[21] In our study, there was no statistically significant difference in MDA and GSH levels References in kidney tissue between TPRIR and the SG group. However, 1. Krˇen V, Walterová D. Silybin and silymarin—new effects and applications. the difference in MDA and GSH levels in kidney tissue was Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005;149:29-41. statistically significant between TRIR and RIR groups. These 2. Lindsay TF, Liauw S, Romaschin AD, Walker PM. The effect of ischemia/ reperfusion on adenine nucleotide metabolism and xanthine oxidase production results indicate that thiamine pyrophosphate prevents oxidative in skeletal muscle. J Vasc Surg 1990;12:8-15. stress induced by IR in kidney tissue. 3. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N The excessive free oxygen radicals do not only cause Engl J Med 1985;312:159-63. damage to the lipids, but also to DNA. The damage to DNA 4. Del Maestro RF. An approach to free radicals in medicine and biology. Acta Physiol Scand Suppl 1980;492:153-68. can be in the form of single and double strain breaks, abasic 5. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361-70. areas, base modifications, or formation of crosslinks between 6. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol the proteins.[22] OH, which is a free radical, can lead to DNA 2001;54:176-86. damage. OH has to be very close to DNA in order to be effective 7. Bomser J, Madhavi DL, Singletary K, Smith MA. In vitro anticancer activity of and cause damage. Nevertheless, OH radicals cannot penetrate fruit extracts from Vaccinium species. Planta Med 1996;62:212-6. 8. Demiryilmaz I, Cetin N, Altuner D, Akcay F, Suleyman H. A comparative the cell nucleus. However, OH radicals, which come free as a investigation of biochemical and histopathological effects of thiamine and result of the reaction of H2O2 with Fe-Cu ions in the nucleus, thiamine pyrophosphate on ischemia-reperfusion induced oxidative damage do induce DNA damage. Furthermore, because DNA consists in rat ovarian tissue. Arch Pharm Res. [In press]

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