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

Abstract

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 (⌬ m ). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O 2 . , H 2 O 2 and ⅐OH, leading to oxidative stress. O 2 . 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 H 2 O 2 , forms ⅐OH. 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 ⅐OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of ⅐OH by dimethyl sulfoxide or ␣-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of ⌬ m , 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 O 2 . -mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ⅐OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.Non-steroidal anti-inflammatory drugs (NSAIDs) 2 are one of the most commonly prescribed drugs in the world to treat pain and inflammation (1). Everyday about 30 million people consume NSAIDs (1). These drugs are gaining enormous interest for cancer therapy as well (2, 3), as they are potent inducers of apoptosis and inhibitors of cell proliferation (4). However, long term use of NSAIDs is associated with severe gastropathy (1, 5) that may arise from induction of gastric mucosal cell apoptosis (6 -9). NSAIDs induce apoptosis in vitro in varieties of cancer cells such as esophageal (10) and gastric adenocarcinoma cells (11), lung carcinoma cells (12), myeloid leukemia cells (13), and prostate carcinoma cells (3). NSAIDs also induce apoptosis in normal gastric mucosal cells (7, 9), hepatocytes (14), and chondrocytes (15). Up-regulation of proapoptotic Bax, Bak, and down-regulation of antiapoptotic Bcl-2, BclxL, are found to occur in NSAID-induced gastric mucosal apoptosis (7). Up-regulation of Bax as well as down-regulation of Bcl-2 are also observed in indomethacin-induced chronic myeloid leukemic c...

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“…The excess O 2 . , if not dismutated, offers toxic insult by oxidatively damaging and inactivating mitochondrial aconitase, resulting in the release of iron from its Fe-S cluster (6,17). Again, the released iron in presence of H 2 O 2 generates ⅐ OH through the Fenton reaction (17).…”

“…The overproduction of ROS develops mitochondrial pathology (22,24,28,29), as indicated by the defect in electron transport chain and ATP synthesis, opening of mitochondrial permeability transition pore (MPTP), fall in transmembrane potential (⌬⌿ m ), oxidative damage of mitochondrial DNA, proteins, and phospholipids (30), and finally the activation of the mitochondrial pathway of apoptosis (6,31). Thus, mitochondrial dysfunction triggers the mitochondrial pathway of apoptosis (6,(32)(33)(34)(35)(36)(37)(38). Mitochondrial dysfunction and concurrent apoptosis play an important role in NSAID-induced gastropathy (4, 6 -7, 12).…”

“…generates highly reactive hydroxyl radical ( ⅐ OH) through the Haber-Weiss reaction (6). The excess O 2 .…”

“…Equal numbers of gastric mucosal primary cultured cells from control and experimental groups were used for the detection of intramitochondrial O 2 . using MitoSOX, a superoxide-sensitive fluorescence probe, following the protocol as described in the product catalogue (6,43). Cells were stained with the fluorescent probe in HBSS (pH 7.4) and incubated for 15 min at 37°C in the dark.…”

“…The animals, which were not treated with SEGA (3a) and received only indomethacin, served as the autohealing group. Starting from 0 h, the stomach was dissected out from all groups at intervals of 2, 4, 8, and 24 h, respectively, for measuring injury index as described (6,12) and histological studies.…”