Noninvasive Mapping of Reactive Oxygen Species by in Vivo Electron Spin Resonance Spectroscopy in Indomethacin-Induced Gastric Ulcers in Rats

Abstract

Reactive oxygen species (ROS) are thought to be involved in the gastric ulcer formation induced by indomethacin, a typical nonsteroidal anti-inflammatory drug. However, the location and the time course of ROS generation remain unknown. To assess the sites of ROS generation, we applied the noninvasive measurement of ROS to indomethacin-treated rats. By giving orally a membrane-permeable or impermeable probe, the spectra were collected as a function of time by in vivo 300-MHz electron spin resonance (ESR) spectroscopy. The ESR signal-decay rates of membrane-permeable probes, hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl, in the gastric mucosal region were significantly enhanced 1 h after indomethacin treatment, and they both caused the protection of ulcer formation; however, membrane-impermeable probes, carboxy-and trimethylammonium-TEMPO, which did not exhibit the enhanced signal decay, had no effect on ulcer formation. The enhanced signal decay in the gastric mucosa was suppressed by coadministration of the antioxidants tiron or dimethylthiourea with the nitroxyl probe. The results suggest that the enhanced signal-decay rates in the gastric ulcers observed by in vivo ESR are associated with protective effects. The enhanced signal decay caused by ROS generation in stomach, contributing to the ulcer formation induced by indomethacin, is also suggested to occur at the gastric mucus layer or the interface or the intracellular compartment of epithelial cells. Overall, these results show the potentials of noninvasive assessment of ROS production and the sites of damage by in vivo ESR using nitroxyl probes directed to specific subcellular regions.Nonsteroidal anti-inflammatory drugs (NSAIDs) have defervescence, analgesia, and anti-inflammatory effects, and their clinical applications to rheumatism, osteoporosis, and osteoarthritis have been increasing. Indomethacin ([1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl] acetic acid) is one of the most widely used NSAIDs in the world because the anti-inflammatory effect of indomethacin on carrageenin-induced edema is approximately 30 times higher than that of aspirin (2-acetoxybenzoic acid) (Winter et al., 1963). NSAIDs cause therapeutic effects by the suppression of prostaglandin biosynthesis via the inhibition of cyclooxygenase (COX) gene expression. This class of agents is also known to cause significant gastrointestinal damage (Vane, 1971). It is known that the COX enzymes have isozymes of three types (Chandrasekharan et al., 2002), and the inhibition of COX-1 in gastric mucosa has been thought to be related with the gastric ulcer formation induced by NSAIDs (Wallace et al., 1998), because COX-1 is involved in maintaining the integrity of gastric mucosa, the increment of

Highly reactive oxygen species: detection, formation, and possible functions

“…Besides the opportunity to use ESR for analyzing in vivo samples, e.g., by directly coupling it to a microdialysis facility [128], it allows non-invasive imaging, similar to NMR tomography, showing spatially resolved oxidative processes in the body [125,129]. As the ability of a biological system to reduce the introduced spin trap is proportional to its antioxidant activity, the disappearance of the ESR signal can be used as a marker for the condition of the antioxidant defense system.…”

Hepatic Reduction of Carbamoyl-PROXYL in Ferric Nitrilotriacetate Induced Iron Overloaded Mice: An <i>in Vivo</i> ESR Study

“…Thus, the rate of ESR signal decay of nitroxyl spin probes have been used to monitor free radical production and redox status in various animal models, including tumor inoculated in mouse footpads, 8) indomethacin-induced gastric ulcers, 9) rheumatoid arthritis, 10) hypertension, 11) cerebral ischemia-reperfusion, 12) lung injury caused by diesel exhaust particles, 13) and radiationinduced oxidative injury. 14) Phumala et al 15) have demonstrated in an acute iron overload murine model that free radical production is dependent on the availability of "chelatable iron" present transiently following a single injection of ferric citrate (0.2 µmol/g body weight).…”

Microencapsulation enhances the anti-ulcerogenic properties of Entada africana leaf extract

“…Of impressive note was the similar or better cytoprotection exhibited by the microcapsules when compared to the standard reference drug, misoprostol (40 g/kg). Indomethacin possesses pharmacological and toxicological properties primarily due to its inhibition of cyclo-oxygenase isoenzymes responsible for the production of prostaglandins (PGs), promoting haemorrhagic ulceration in rats by oxidative stress and a decline in mucosal production of PG (Utsumi et al, 2006). The protection provided by extract and formulations of Entada africana against Indomethacin induced ulceration, may be suggestive of an enhanced mucosal synthesis or mobilization of PG, consequently protecting gastric mucosa similar to misoprostol, a synthetic prostaglandin analogue.…”