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Indomethacin, a Non-steroidal Anti-inflammatory Drug, Develops Gastropathy by Inducing Reactive Oxygen Species-mediated Mitochondrial Pathology and Associated Apoptosis in Gastric Mucosa

2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês

10.1074/jbc.m805329200

ISSN

1083-351X

Autores

Pallab Maity, Samik Bindu, Sumanta Dey, Manish Goyal, Athar Alam, Chinmay Pal, Kalyan Mitra, Uday Bandyopadhyay,

Tópico(s)

Renal function and acid-base balance

Resumo

We have investigated the role of mitochondria on the development of indomethacin (a non-steroidal anti-inflammatory drug)-induced gastric mucosal apoptosis and associated gastropathy in rat. Transmission electron microscopic studies indicate that indomethacin damages mitochondrial ultrastructure and causes mitochondrial dysfunction as evident from decreased stage-3 respiration, dehydrogenase activity, and transmembrane potential (ΔΨm). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O2·¯, H2O2 and ·OH, leading to oxidative stress. O2·¯ is the most effective to damage mitochondrial aconitase, leading to the release of iron from its iron-sulfur cluster. The released iron, by interacting with intra-mitochondrial H2O2, forms ·OH. Immunoprecipitation of mitochondrial aconitase and subsequent Western immunoblotting indicate carbonylation of aconitase along with the loss of activity in vivo after indomethacin treatment. The release of iron has been documented by fluorescence imaging of mucosal cells by using Phen Green SK, a specific probe for chelatable iron. Interestingly, intra-mitochondrial ·OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of ·OH by dimethyl sulfoxide or α-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of ΔΨm, and mitochondrial dysfunction. The scavengers also restore indomethacin-induced activation of caspase-9 and caspase-3 to block mitochondrial pathway of apoptosis and gastric mucosal damage. This study, thus, reveals the critical role of O2·¯-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ·OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment. We have investigated the role of mitochondria on the development of indomethacin (a non-steroidal anti-inflammatory drug)-induced gastric mucosal apoptosis and associated gastropathy in rat. Transmission electron microscopic studies indicate that indomethacin damages mitochondrial ultrastructure and causes mitochondrial dysfunction as evident from decreased stage-3 respiration, dehydrogenase activity, and transmembrane potential (ΔΨm). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O2·¯, H2O2 and ·OH, leading to oxidative stress. O2·¯ is the most effective to damage mitochondrial aconitase, leading to the release of iron from its iron-sulfur cluster. The released iron, by interacting with intra-mitochondrial H2O2, forms ·OH. Immunoprecipitation of mitochondrial aconitase and subsequent Western immunoblotting indicate carbonylation of aconitase along with the loss of activity in vivo after indomethacin treatment. The release of iron has been documented by fluorescence imaging of mucosal cells by using Phen Green SK, a specific probe for chelatable iron. Interestingly, intra-mitochondrial ·OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of ·OH by dimethyl sulfoxide or α-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of ΔΨm, and mitochondrial dysfunction. The scavengers also restore indomethacin-induced activation of caspase-9 and caspase-3 to block mitochondrial pathway of apoptosis and gastric mucosal damage. This study, thus, reveals the critical role of O2·¯-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ·OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment. Non-steroidal anti-inflammatory drugs (NSAIDs) 2The abbreviations used are: NSAID, non-steroidal anti-inflammatory drugs; PBN, α-phenyl-n-tert-butylnitrone; RCR, Respiratory control ratio; DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; TEM, transmission electron microscopy; HBSS, Hanks' balanced salt solution.2The abbreviations used are: NSAID, non-steroidal anti-inflammatory drugs; PBN, α-phenyl-n-tert-butylnitrone; RCR, Respiratory control ratio; DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; TEM, transmission electron microscopy; HBSS, Hanks' balanced salt solution. are one of the most commonly prescribed drugs in the world to treat pain and inflammation (1Regula J. Butruk E. Dekkers C.P. de Boer S.Y. Raps D. Simon L. Terjung A. Thomas K.B. Luhmann R. Fischer R. Am. J. Gastroenterol... 2006; 101: 1747-1755Google Scholar). Everyday about 30 million people consume NSAIDs (1Regula J. Butruk E. Dekkers C.P. de Boer S.Y. Raps D. Simon L. Terjung A. Thomas K.B. Luhmann R. Fischer R. Am. J. Gastroenterol... 2006; 101: 1747-1755Google Scholar). These drugs are gaining enormous interest for cancer therapy as well (2Cha Y.I. Dubois R.N. Annu. Rev. Med... 2007; 58: 239-252Google Scholar, 3Chan J.M. Feraco A. Shuman M. Hernandez-Diaz S. Hematol. Oncol. Clin. North Am.. 2006; 20: 797-809Google Scholar), as they are potent inducers of apoptosis and inhibitors of cell proliferation (4Eli Y. Przedecki F. Levin G. Kariv N. Raz A. Biochem. Pharmacol... 2001; 61: 565-571Google Scholar). However, long term use of NSAIDs is associated with severe gastropathy (1Regula J. Butruk E. Dekkers C.P. de Boer S.Y. Raps D. Simon L. Terjung A. Thomas K.B. Luhmann R. Fischer R. Am. J. Gastroenterol... 2006; 101: 1747-1755Google Scholar, 5Wallace J.L. Best Pract. Res. Clin. Gastroenterol... 2001; 15: 691-703Google Scholar) that may arise from induction of gastric mucosal cell apoptosis (6Fujii Y. Matsura T. Kai M. Matsui H. Kawasaki H. Yamada K. Proc. Soc. Exp. Biol. Med... 2000; 224: 102-108Google Scholar, 7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar, 8Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem... 2005; 280: 31059-31067Google Scholar, 9Chiou S.K. Tanigawa T. Akahoshi T. Abdelkarim B. Jones M.K. Tarnawski A.S. Gastroenterology.. 2005; 128: 63-73Google Scholar). NSAIDs induce apoptosis in vitro in varieties of cancer cells such as esophageal (10Aggarwal S. Taneja N. Lin L. Orringer M.B. Rehemtulla A. Beer D.G. Neoplasia.. 2000; 2: 346-356Google Scholar) and gastric adenocarcinoma cells (11Gu Q. Wang J.D. Xia H.H. Lin M.C. He H. Zou B. Tu S.P. Yang Y. Liu X.G. Lam S.K. Wong W.M. Chan A.O. Yuen M.F. Kung H.F. Wong B.C. Carcinogenesis.. 2005; 26: 541-546Google Scholar), lung carcinoma cells (12de Groot D.J. Timmer T. Spierings D.C. Le T.K. de Jong S. de Vries E.G. Br. J. Cancer.. 2005; 92: 1459-1466Google Scholar), myeloid leukemia cells (13Zhang G. Tu C. Zhang G. Zhou G. Zheng W. Leuk. Res... 2000; 24: 385-392Google Scholar), and prostate carcinoma cells (3Chan J.M. Feraco A. Shuman M. Hernandez-Diaz S. Hematol. Oncol. Clin. North Am.. 2006; 20: 797-809Google Scholar). NSAIDs also induce apoptosis in normal gastric mucosal cells (7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar, 9Chiou S.K. Tanigawa T. Akahoshi T. Abdelkarim B. Jones M.K. Tarnawski A.S. Gastroenterology.. 2005; 128: 63-73Google Scholar), hepatocytes (14Ong M.M. Wang A.S. Leow K.Y. Khoo Y.M. Boelsterli U.A. Free Radic. Biol. Med... 2006; 40: 420-429Google Scholar), and chondrocytes (15Chang J.K. Wu S.C. Wang G.J. Cho M.H. Ho M.L. Toxicology.. 2006; 228: 111-123Google Scholar). Up-regulation of proapoptotic Bax, Bak, and down-regulation of antiapoptotic Bcl-2, BclxL, are found to occur in NSAID-induced gastric mucosal apoptosis (7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar). Up-regulation of Bax as well as down-regulation of Bcl-2 are also observed in indomethacin-induced chronic myeloid leukemic cell apoptosis (13Zhang G. Tu C. Zhang G. Zhou G. Zheng W. Leuk. Res... 2000; 24: 385-392Google Scholar). Indomethacin in vitro induces apoptosis in gastric mucosal cell line through the release of cytochrome c and activation of Ca2+ signaling (6Fujii Y. Matsura T. Kai M. Matsui H. Kawasaki H. Yamada K. Proc. Soc. Exp. Biol. Med... 2000; 224: 102-108Google Scholar, 8Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem... 2005; 280: 31059-31067Google Scholar). Studies claim that activation of mitochondrial death pathway contributes significantly in the apoptotic death of gastric mucosal cells by NSAIDs (6Fujii Y. Matsura T. Kai M. Matsui H. Kawasaki H. Yamada K. Proc. Soc. Exp. Biol. Med... 2000; 224: 102-108Google Scholar, 7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar, 8Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem... 2005; 280: 31059-31067Google Scholar, 16Redlak M.J. Power J.J. Miller T.A. Am. J. Physiol. Gastrointest. Liver Physiol... 2005; 289: 731-738Google Scholar, 17Tarnawski A.S. Dig. Dis. Sci... 2005; 50: 24-33Google Scholar). The mitochondrial death pathway is initiated by the up-regulation of Bcl-2 family of proapoptotic proteins such as Bax, Bak, and/or down-regulation of antiapoptotic Bcl-2, BclxL, (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar) to induce activation and mitochondrial translocation of Bax, where it oligomerizes (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar, 19Zimmermann K.C. Bonzon C. Green D.R. Pharmacol. Ther... 2001; 92: 57-70Google Scholar) to open mitochondrial permeability transition pores in the mitochondrial membrane (20Gulbins E. Dreschers S. Bock J. Exp. Physiol... 2003; 88: 85-90Google Scholar). Opening of mitochondrial permeability transition pores releases some apoptosis promoting factors, such as cytochrome c, Smac/DIABLO, apoptosis-inducing factor, and endonuclease G into the cytosol (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar, 20Gulbins E. Dreschers S. Bock J. Exp. Physiol... 2003; 88: 85-90Google Scholar, 21Jiang X. Wang X. Annu. Rev. Biochem... 2004; 73: 87-106Google Scholar). The role of mitochondria in the regulation of cell death is now well established (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar, 20Gulbins E. Dreschers S. Bock J. Exp. Physiol... 2003; 88: 85-90Google Scholar). The generation of reactive oxygen species (ROS) (22Orrenius S. Gogvadze V. Zhivotovsky B. Annu. Rev. Pharmacol. Toxicol... 2007; 47: 143-183Google Scholar) and the release of proteins from the mitochondria lead to the activation of different pathways of cell death (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar, 19Zimmermann K.C. Bonzon C. Green D.R. Pharmacol. Ther... 2001; 92: 57-70Google Scholar, 20Gulbins E. Dreschers S. Bock J. Exp. Physiol... 2003; 88: 85-90Google Scholar, 22Orrenius S. Gogvadze V. Zhivotovsky B. Annu. Rev. Pharmacol. Toxicol... 2007; 47: 143-183Google Scholar). At present, it seems that a combination of proteins released from the mitochondria and maintenance of a sizable intracellular ATP pool are required for the execution of the suicide program and that mitochondrial protein release is associated with enhanced ROS production by this organelle (22Orrenius S. Gogvadze V. Zhivotovsky B. Annu. Rev. Pharmacol. Toxicol... 2007; 47: 143-183Google Scholar). Mitochondria are not only a major source of ROS in aerobic cells but are also a sensitive target for the damaging effects of ROS. Increased ROS generated by mitochondria can cause oxidative damage of cellular macromolecules, including nucleic acids, lipids, and proteins along with depletion of cellular antioxidants, leading to cellular injury (22Orrenius S. Gogvadze V. Zhivotovsky B. Annu. Rev. Pharmacol. Toxicol... 2007; 47: 143-183Google Scholar).Many aspects of mitochondrial death pathway for the initiation of gastric mucosal apoptosis during NSAID-induced gastropathy have been evident (6Fujii Y. Matsura T. Kai M. Matsui H. Kawasaki H. Yamada K. Proc. Soc. Exp. Biol. Med... 2000; 224: 102-108Google Scholar, 7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar, 8Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem... 2005; 280: 31059-31067Google Scholar, 16Redlak M.J. Power J.J. Miller T.A. Am. J. Physiol. Gastrointest. Liver Physiol... 2005; 289: 731-738Google Scholar). It is now fairly established that the release of cytochrome c to the cytosol initiates the execution step of apoptosis (18Kroemer G. Galluzzi L. Brenner C. Physiol. Rev... 2007; 87: 99-163Google Scholar, 19Zimmermann K.C. Bonzon C. Green D.R. Pharmacol. Ther... 2001; 92: 57-70Google Scholar, 20Gulbins E. Dreschers S. Bock J. Exp. Physiol... 2003; 88: 85-90Google Scholar, 21Jiang X. Wang X. Annu. Rev. Biochem... 2004; 73: 87-106Google Scholar, 22Orrenius S. Gogvadze V. Zhivotovsky B. Annu. Rev. Pharmacol. Toxicol... 2007; 47: 143-183Google Scholar). However, no studies have been reported yet on how cytochrome c, which is strongly associated with the cardiolipin, is released from the mitochondria during indomethacin-induced gastropathy and the role of intra-mitochondrial ROS thereon. In this study we have shown that indomethacin (NSAID) in vivo induces mitochondrial pathology by promoting mitochondrial oxidative stress through intra-mitochondrial iron mobilization from the iron-sulfur cluster of aconitase and the subsequent ·OH generation to activate the mitochondrial pathway of apoptosis in gastric mucosal cells. Scavenging of ·OH significantly prevents indomethacin-induced mitochondrial pathology, mitochondrial pathway of gastric mucosal apoptosis, and the associated gastropathy.EXPERIMENTAL PROCEDURESMaterials—Indomethacin, thiobarbituric acid, 5,5′-dithiobis(nitrobenzoic acid), reduced glutathione (GSH), DMSO, α-phenyl-n-tert-butylnitrone (PBN), oligomycin, ADP, collagenase, hyaluronidase, paraformaldehyde, glutaraldehyde, mitoisolation kit, caspase-3 assay kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit, trichloroacetic acid, and aconitase were purchased from Sigma. Rabbit IgG against aconitase was obtained from Abgent (San Diego, CA). Horseradish peroxidase-coupled anti-rabbit IgG was procured from Santa Cruz. ECL-based chemiluminescence kit was procured from GE Healthcare. JC-1 (5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide), MitoSOX, Mitotracker Red, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), and Phen Green SK were purchased from Invitrogen. Caspase-9 assay kit was obtained from Biovision (Biovision, Mountain View, CA). Quantichrom Iron assay kit was procured from Bioassay Systems (Hayward, CA). All other reagents were of analytical grade purity.Animals Used—Sprague-Dawley rats (180–220 gm) were used throughout the experiments. Each group (control or experimental or ROS scavenger-pretreated) of animals (n = 6–8) was maintained at 24 ± 2 °C with 12-h light and dark cycles. The animals were fasted for 24 h with water ad libitum before the start of the experiments to avoid food-induced increased acid secretion and its aggravating effect on gastric lesions. All the in vivo studies were done in accordance with the guidelines of institute animal ethical committee.Indomethacin-induced Gastric Damage—Indomethacin-induced acute gastric mucosal damage was performed as described earlier (23Bandyopadhyay U. Biswas K. Chatterjee R. Bandyopadhyay D. Chattopadhyay I. Ganguly C.K. Chakraborty T. Bhattacharya K. Banerjee R.K. Life Sci... 2002; 71: 2845-2865Google Scholar, 24Biswas K. Bandyopadhyay U. Chattopadhyay I. Varadaraj A. Ali E. Banerjee R.K. J. Biol. Chem... 2003; 278: 10993-11001Google Scholar, 25Chattopadhyay I. Bandyopadhyay U. Biswas K. Maity P. Banerjee R.K. Free Radic. Biol. Med... 2006; 40: 1397-1408Google Scholar). Gastric ulcer was induced in the fasted animals (n = 6–8) with oral administration of indomethacin at the doses from 6 to 48 mg kg-1 body weight. Control group received only vehicle instead of indomethacin. In the ROS scavenger-pretreated groups, the animals were administered with DMSO (500 μl of 25% dry DMSO) or PBN (200 mg kg-1 body weight) intraperitoneally 30 min before ulcer induction with indomethacin. The optimum dose of each of the scavengers was selected from the dose-response curve. After 4 h of indomethacin treatment, the animals were sacrificed under proper euthanasia, and stomachs were dissected out. The mucosal injury was scored as ulcer index as described earlier: 0 = no pathology, 1 = one pinhead ulcer, and 2–5 for thread-like lesions of 2–5 mm length (25Chattopadhyay I. Bandyopadhyay U. Biswas K. Maity P. Banerjee R.K. Free Radic. Biol. Med... 2006; 40: 1397-1408Google Scholar). The sum of the total scores divided by the number of animals gave the ulcer index.Mitochondrial Morphology by Transmission Electron Microscopy (TEM)—Mitochondrial morphology was detected by TEM analysis (7Maity P. Bindu S. Choubey V. Alam A. Mitra K. Goyal M. Dey S. Guha M. Pal C. Bandyopadhyay U. J. Biol. Chem... 2008; 283: 14391-14401Google Scholar). The animals were anesthetized with ketamine hydrochloride (12 mg kg-1) and perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 m sodium phosphate buffer (pH 7.4). The gastric mucosal tissue from control and indomethacin-treated (at different ulcer index) and scavenger-pretreated were dissected out, washed with sodium phosphate buffer, and then cut into small pieces (1 mm3) followed by fixation in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 m sodium phosphate buffer (pH 7.4) for 4 h at room temperature (24 °C). The tissues were then postfixed in 2% osmium tetroxide in 0.1 m sodium phosphate buffer (pH 7.4) for 2 h at room temperature and dehydrated in an ascending grades of ethanol followed by embedding in Epon 812 and polymerized at 60 °C for 24 h. Ultra-thin sections (50–70 nm) were obtained using an Ultra cut Ultra-microtome (Leica Microsystems GmbH, Wetzlar, Germany) and picked up onto 200 mesh copper grids. The sections were double-stained with uranyl acetate and lead citrate and analyzed under a FEI Tecnai-12 Twin Transmission Electron Microscope equipped with a SIS Mega View II CCD camera at 80kV (FEI Co., Hillsboro, OR).Isolation of Mitochondria—Mitochondria from gastric mucosal cells were isolated using commercially available kit (Sigma) (26Guha M. Kumar S. Choubey V. Maity P. Bandyopadhyay U. FASEB J.. 2006; 20: 1224-1226Google Scholar, 27Guha M. Maity P. Choubey V. Mitra K. Reiter R.J. Bandyopadhyay U. J. Pineal. Res... 2007; 43: 372-381Google Scholar). In brief, gastric mucosa was scrapped, suspended in mitochondria extraction buffer, and minced finely. This was homogenized in a Ultra-Turrax T-25 homogenizer. The homogenate was subjected to differential centrifugation first at 800 × g for 10 min to remove nuclei and unbroken cells and finally at 12,000 × g for 15 min to get the mitochondrial fraction. It was finally suspended in mitochondrial storage buffer in a ratio of 40 μl per 100 mg tissue initially taken.Assessment of Mitochondrial Function—Mitochondrial functional status was analyzed in terms of mitochondrial oxygen consumption and mitochondrial dehydrogenase activity using an MTT reduction assay. Mitochondrial oxygen consumption was measured by using a Clark-type electrode in a Liquid-Phase Oxygen Measurement System (Oxygraph, Hansatech, Norfolk, UK) with a thermoregulated chamber set at 24 °C (28Rousou A.J. Ericsson M. Federman M. Levitsky S. McCully J.D. Am. J. Physiol. Heart Circ. Physiol... 2004; 287: 1967-1976Google Scholar). Oxygen consumption by complex I (Stage 3) was initiated by the addition of glutamate and malate (5 mm each) to 1 ml of respiratory medium (250 mm sucrose, 5 mm KH2PO4, 5 mm MgCl2, 0.1 mm EDTA, 0.1% bovine serum albumin in 20 mm HEPES (pH 7.2)). The basal respiration (state 2) was recorded after the addition of mitochondria suspension (20 μg). State 3 respiration was initiated by the addition of 1 mm ADP to the respiration medium. State 4 respiration was initiated by the addition of 15 μm oligomycin, which blocks the ATP synthase of complex V. Respiratory control ratio (RCR) was calculated from the ratio of State 3 respiration (nmol of O2 consumed) and State 4 respiration (nmol of O2 consumed). Mitochondrial metabolic function was studied by observing the ability of mitochondrial dehydrogenases to reduce MTT into formazan dye. Equal amounts of mitochondrial protein (25 μg) from control or experimental groups was incubated with MTT (0.1% final concentration) solution for 3 h at 37°C. After the incubation, the insoluble formazan dye was solubilized with MTT solubilization solution containing 10% Triton X-100 plus 0.1 n HCl in anhydrous isopropanol. The absorbance of formazan dye developed as a result of MTT reduction was measured at 570 nm.Measurement of Mitochondrial Transmembrane Potential (ΔΨm)—Mitochondrial transmembrane potential was measured as described earlier (26Guha M. Kumar S. Choubey V. Maity P. Bandyopadhyay U. FASEB J.. 2006; 20: 1224-1226Google Scholar). In brief, mitochondria (20 μg) isolated from gastric mucosa of different groups of rats were incubated with JC-1 (300 nm) in the dark for 10 min at 37 °C in JC-1 assay buffer. The fluorescence of each sample was measured in a PerkinElmer Life Sciences LS50B spectrofluorometer (excitation, 490 nm; slit, 5 nm; emission, 590 nm for J-aggregate and 530 nm for J-monomer; slit, 7.2 nm).Assay of Caspase-9 and Caspase-3 Activities—Caspase-9 activity was measured from cytosolic fraction of gastric mucosal homogenate obtained by subcellular fractionation using a commercially available kit and according to the manufacturer's protocol (Biovision). Caspase-3 activity was measured from cytosolic fraction of gastric mucosa using a commercially available kit and according to manufacturer's protocol (Sigma) as described earlier (26Guha M. Kumar S. Choubey V. Maity P. Bandyopadhyay U. FASEB J.. 2006; 20: 1224-1226Google Scholar).Measurement of Mitochondrial Oxidative Stress—Mitochondrial oxidative stress was measured in terms of GSH depletion, lipid peroxidation (23Bandyopadhyay U. Biswas K. Chatterjee R. Bandyopadhyay D. Chattopadhyay I. Ganguly C.K. Chakraborty T. Bhattacharya K. Banerjee R.K. Life Sci... 2002; 71: 2845-2865Google Scholar, 24Biswas K. Bandyopadhyay U. Chattopadhyay I. Varadaraj A. Ali E. Banerjee R.K. J. Biol. Chem... 2003; 278: 10993-11001Google Scholar), and protein carbonyl formation. Mitochondrial GSH content was measured as described (23Bandyopadhyay U. Biswas K. Chatterjee R. Bandyopadhyay D. Chattopadhyay I. Ganguly C.K. Chakraborty T. Bhattacharya K. Banerjee R.K. Life Sci... 2002; 71: 2845-2865Google Scholar). In brief, gastric mucosal scraping was homogenized using an Ultra-Turax T25 homogenizer to get the mitochondrial fraction using a commercially available mitochondria isolation kit (Sigma) as already described. The mitochondrial fraction was again sonicated in 20 mm ice-cold EDTA. Proteins present in the mitochondrial lysate were removed by trichloroacetic acid precipitation. The protein-free lysate (1 ml) was then added to 2 ml of 0.8 m Tris-Cl (pH 9) containing 20 mm EDTA. GSH content was finally determined by its reaction with 2,4-dithionitrobenzoic acid to yield the yellow chromophore of thionitrobenzoic acid, which was measured at 412 nm. GSH was used as standard (23Bandyopadhyay U. Biswas K. Chatterjee R. Bandyopadhyay D. Chattopadhyay I. Ganguly C.K. Chakraborty T. Bhattacharya K. Banerjee R.K. Life Sci... 2002; 71: 2845-2865Google Scholar). For mitochondrial lipid peroxidation, mitochondrial fraction (20 μg) was homogenized in ice-cold 0.9% saline. 1 ml of this homogenate was mixed with 2 ml of a thiobarbituric acid-trichloroacetic acid mixture (0.375% w/v, 15% w/v, respectively) in 0.25 n HCl followed by boiling for 15 min. The solution was then cooled, and after centrifugation the absorbance of the supernatant was read at 535 nm. Tetraethoxypropane was used as standard. Mitochondrial protein carbonyl content was measured using indirect enzyme-linked immunosorbent assay-based protocol (Cell Biolabs, San Diego, CA). Proteins were first adsorbed onto microtiter plate. The protein carbonyl present in the sample were first derivatized with 2,4-dinitrophenylhydrazine. The derivatized carbonyl-dinitrophenol was then measured with anti-dinitrophenol antibody followed by horseradish peroxidase-conjugated secondary antibody and o-phenyldiamine.Isolation of Gastric Mucosal Cells—Gastric mucosal cells from control and indomethacin-treated rats were isolated as described earlier (29Terano A. Ivey K.J. Stachura J. Sekhon S. Hosojima H. McKenzie Jr., W.N. Krause W.J. Wyche J.H. Gastroenterology.. 1982; 83: 1280-1291Google Scholar). Briefly, mucosa was scrapped in HBSS containing 100 units/ml penicillin and 100 μg/ml streptomycin. The scrapped mucosa was then resuspended and finely minced in HBSS containing 0.1% collagenase and 0.05% hyaluronidase. The suspension was incubated in the same solution for 30 min at 37 °C and filtered through a sterile nylon mess. The filtrate was centrifuged at 500 × g for 5 min to get the cell pellet. The pellet was washed and suspended in HBSS for using in the following studies.Detection of Intramitochondrial-reactive Oxygen Species—The isolated cells were used for detection of intramitrochondrial ROS. Intramitochondrial ROS was measured using two specific probes, MitoSOX (to detect mitochondrial O2·¯) and DCF-DA (general ROS). Cells were stained with the respective fluorescent probe in HBSS (pH 7.4) and incubated for 15 min at 37 °C in the dark as described in the manufacture's protocols. Mitotracker Red was used to visualize mitochondria. After the incubation, cells were washed with HBSS three times and continued for fluorescence microscopy (Leica, DM-2500, Leica Microsystems). Staining of MitoSOX and Miotracker Red was visualized using Red filter, and DCF-DA staining was visualized with a green filter. Hydroxyl radical (·OH) generated in the mitochondria of the gastric mucosa cells during indomethacin treatment was measured using DMSO as the ·OH scavenger (30Babbs C.F. Steiner M.G. Methods Enzymol.. 1990; 186: 137-147Google Scholar). Rats from both control and indomethacin-treated groups received 500 μl of 25% DMSO/100 g intraperitoneally, 30 min before indomethacin treatment. The animals were sacrificed after 4 h of indomethacin treatment, stomachs were dissected out, and mitochondria were purified using mitoisolation kit as described earlier (26Guha M. Kumar S. Choubey V. Maity P. Bandyopadhyay U. FASEB J.. 2006; 20: 1224-1226Google Scholar, 27Guha M. Maity P. Choubey V. Mitra K. Reiter R.J. Bandyopadhyay U. J. Pineal. Res... 2007; 43: 372-381Google Scholar). The mitochondrial pellet was sonicated in ice-cold triple-distilled water and processed for the extraction of methanesulfinic acid formed by the reaction of ·OH with DMSO. The extracted methanesulfinic acid was allowed to react with Fast Blue BB salt, and the intensity of the resulting yellow chromophore was measured at 425 nm using benzene-sulfinic acid as standard.Measurement of Intramitochondrial Chelatable Iron—Intramitochondrial free (chelatable) iron was quantified using QuantiChrom™ Iron assay kit (Bioassay Systems). Mitochondria were lysed with lysis buffer, and protein-free lysate was used for estimation of free iron. Mitochondrial protein-free lysate (50 μl) was mixed with 200 μl of reaction mixture, provided with the kit, and incubated for 40 min at room temperature, and optical density was measured at 590 nm in a microtiter plate reader (SpectraMax, Molecular Devices, Sunnyvale, CA). The concentration of free iron was measured from a standard curve using iron standard provided with the kit.Intramitochondrial Free Iron Localization—Isolated mucosal cells from both control and indomethacin-treated rat stomach was used for free iron localization using Phen Green SK, an iron-sensitive fluorescence probe using the protocol as described in the product catalogue. Cells were first incubated with Mitotracker Red (250 nm) at 37 °C for 20 min followed by washing with HBSS and further incubation with Phen Green SK (20 μm) for 15 min at 37 °C in the dark. After the incubation, cells were washed with HBSS and used for fluorescence microscopy (Leica, DM-2500, Leica Microsystems).Detection of Protein Carbonyl in Mitochondrial Aconitase by Immunoprecipitation and Western Immunoblotting—The mitochondria isolated from control and indomethacin-treated mucosa were lysed in RIPA radioimmune precipitation assay lysis buffer (50 mm Tris-Cl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor mixture (Sigma), and the lysate was used for immunoprecipitation using protein A-Sepharose conjugated with rabbit antibody against aconitase. To 30 μl of 50% (v/v) protein-A slurry (in phosphate-buffered saline (pH 7.4)), 5 μl of rabbit polyclonal antibody against aconitase was added. This was allowed to incubate at 4 °C for 2 h to prepare protein-A-antibody conjugate.

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