Indomethacin, a Non-steroidal Anti-inflammatory Drug, Develops Gastropathy by Inducing Reactive Oxygen Species-mediated Mitochondrial Pathology and Associated Apoptosis in Gastric Mucosa A NOVEL ROLE OF MITOCHONDRIAL ACONITASE OXIDATION

We have investigated the role of mitochondria on the development of indomethacin (a non-steroidal anti-inflammatory drug)-induced gastric mucosal apoptosis and associated gas-tropathy 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 (Delta psi(m)). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as O-2(center dot-), H2O2 and center dot OH, leading to oxidative stress. O-2(center dot-) 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 center dot 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 center dot OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of center dot OH by dimethyl sulfoxide or alpha-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of Delta psi(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(center dot-)-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating center dot OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.