732 Gut 1993; 34: 732-737 Role of active oxygen, lipid peroxidation, and antioxidants in the pathogenesis of gastric mucosal Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. Downloaded from injury induced by indomethacin in rats T Yoshikawa, Y Naito, A Kishi, T Tomii, T Kaneko, S linuma, H Ichikawa, M Yasuda, S Takahashi, M Kondo Abstract role of reactive oxygen species in mediating the The roles of active oxygen, lipid peroxidation, microvascular disturbance that preceded gastric and the antioxidative defence mechanism in mucosal injury induced by several kinds of stress gastric mucosal injury induced by treatment and ischaemia-reperfusion." 12 Furthermore, with indomethacin in rats were investigated. lipid peroxidation mediated by oxygen free The total area of gastric erosions and concen- radicals is believed to be an important cause of tration of lipid peroxides in the gastric mucosa destruction and damage to cell membranes, increased with time after administration of because polyunsaturated fatty acids of the indomethacin (20 mg/kg, orally). The a- cellular membranes are degraded by the lipid tocopherol:total cholesterol ratio in serum was peroxidation with consequent disruption of significantly decreased and the activity of membrane integrity.'3 Membrane peroxidation glutathione peroxidase, an important enzyme can lead to changes in membrane fluidity and to scavenger of lipid peroxides, was inhibited permeability, enhanced rates ofprotein degrada- by the administration of indomethacin. Treat- tion, and ultimately, cell lysis. We have already ments with superoxide dismutase and catalase reported that lipid peroxidation plays a signifi- inhibited the increases in gastric mucosal cant part in the pathogenesis of gastric mucosal erosions and lipid peroxides in the gastric lesions induced by water immersion restraint mucosa, and the reduction of serum stress, burn shock, and ischemia-reperfusion.I'l6 a-tocopherol. Treatment with these scav- The present study was undertaken in rats to engers did not improve the decreased measure changes in lipid peroxides and anti- glutathione peroxidase activity. These findings oxidants in serum samples and gastric mucosa http://gut.bmj.com/ suggest that active oxygen species and lipid after administration of indomethacin. Also, we peroxidation play an important part in the investigated the effects of a superoxide radical pathogenesis ofgastric mucosal injury induced scavenger (superoxide dismutase (SOD)), a by indomethacin, and that the decreased hydrogen peroxide scavenger (catalase), and a glutathione peroxidase activity aggravated the hydroxyl radical scavenger (dimethylsulphoxide injury due to accelerated accumulation of (DMSO)) on gastric mucosal injury and lipid hydrogen peroxide and lipid peroxides in the peroxide formation induced by giving indo- on September 30, 2021 by guest. Protected copyright. gastric mucosal cell. methacin to rats, and the effects ofa combination (Gut 1993; 34: 732-737) of SOD and catalase on indomethacin induced changes in antioxidants. Indomethacin, a non-steroidal anti-inflamma- tory drug (NSAID), is known to induce erosions Materials and methods and ulcers in the gastrointestinal tract.' 2 Male Sprague-Dawley rats, weight range 190- Although it has been proposed that a deficiency 210 g from Keari Co Ltd, Osaka, were used. The of endogenous prostaglandins due to inhibition animals were deprived of food but allowed free ofcyclooxygenase by indomethacin is involved in access to water for 24 hours before the experi- these effects, the exact pathogenic mechanism ment. Gastric haemorrhagic damage was First Department of oral Medicine, Kyoto remains to be elucidated.-' Recent studies induced by administration of indomethacin Prefectural University of showed that a low dose of indomethacin or (Sigma Chemical Co, St Louis, MO) at a dose of Medicine, Kamigyo-ku, aspirin inhibited cyclooxygenase activity in the 20 mg/kg, suspended in 0*5% carboxymethyl- Kyoto 602, Japan cellulose solution with a few drops ofTween 80 in T Yoshikawa gastric mucosa of rats by 90% without causing Y Naito any haemorrhagic erosions.6 Takeuchi et al7 a volume of 0 5 ml/I00 g body weight. In the A Kishi postulated that the enhanced gastric motility control groups, the rats received an equivalent T Tomii induced by indomethacin causes microcircula- volume ofthe vehicle. T Kaneko S Iinuma tory disturbances that lead to increased micro- H Ichikawa vascular permeability and cellular damage. M Yasuda Rainsford'0 also noted, with transmission EXPERIMENTAL PROCEDURE S Takahashi M Kondo electron microscopy, that microvascular injury Time course indomethacin induced gastric Correspondence to: was present during gastric mucosal injury pro- study of Dr T Yoshikawa, First duced by indomethacin. These reports sug- mucosal injury Department of Medicine, killed Kyoto Prefectural University gested that inhibition of prostaglandin synthesis Groups of indomethacin treated rats were of Medicine, Kawaramachi- was unlikely to be the sole mechanism respon- three or six hours after administration of indo- Hirokoji, Kamigyo-ku, Kyoto the methacin. Animals were killed by exsanguina- 602, Japan. sible for gastric damage induced by Accepted for publication indomethacin. tion via the abdominal aorta under inhaled ether 28 September 1992 Much recent attention has been focused on the anaesthesia. The stomachs were removed, Role ofactive oxygen, lipidperoxidation, andantioxidants in thepathogenesis ofgastric mucosal injuty induced by indomethacin in rats 733 opened along the greater curvature, and exam- cribed earlier, serum samples were collected, ined under a dissecting microscope with a square and the activities or the concentrations of anti- grid for lesions developed in the glandular oxidants were measured by the methods des- portion. The extent of the gastric damage was cribed next. Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. Downloaded from expressed as the total area (mm2) of haemor- rhagic erosion. The gastric mucosa was scraped off by means of two glass slides on ice, and ASSAYS homogenised with 1-5 ml of 10 mM potassium Concentrations of thiobarbituric acid (TBA)- phosphate buffer (pH 7 8) containing 30 mM reactive substances, an index of lipid peroxida- KCI in a Potter-Elvehjem homogeniser, to tion, were measured in serum samples by the measure concentrations of lipid peroxides and method of Yagi," and the concentrations in tocopherols. To measure the activities of SOD tissue homogenates were measured according to and glutathione peroxidase, the homogenates Ohkawa et al."9 The concentration of the TBA- were sonicated over ice for two minutes. The reactive substances were expressed as nmol sonicated samples were centrifuged at 20000 g malodialdehyde. Thiobarbituric acid (BDH for 20 minutes and the supernatants were stored Chemicals, Poole, England) and 1,1,3,3- frozen at - 80°C until assay. trimethoxypropane (Tokyo Kasei Co, Tokyo) were used for the TBA assay, and all other chemicals were ofreagent grade. Protein concen- Effects ofscavengers ofactive oxygen on tration in the gastric mucosal homogenates was indomethacin induced gastric mucosal injury measured by the method of Lowry et al.'0 The Activity of SOD in serum reached a maximum of concentration of a-tocopherol in serum samples about 100 U/ml after 150 minutes when SOD and gastric mucosa was measured by the method was subcutaneously injected at a dose of 50 000 of Abe et al2' with a high speed LC-6A liquid U/kg.'7 Therefore, the interval of injection is chromatograph (Shimazu Co, Kyoto). To important to maintain effective plasma SOD eliminate the influence of lipids, the ratio of activity. To assess the effect of SOD and a-tocopherol:total cholesterol in serum samples catalase, recombinant human Cu, Zn-SOD was determined. The serum cholesterol concen- (Nippon Kayaku Co Ltd, Tokyo) at a dose of tration was assayed according to the method of 50 000 U/kg, or catalase from bovine liver (Sigma Richmond.22 The activity of SOD was measured Chemical Co, St Louis, MO) at a dose of 90 000 by a recently developed chemiluminescence U/kg dissolved in 1 ml of physiological saline, assay,23 which involved inhibition of a Cypridina or both were injected subcutaneously one hour luciferin analog with chemiluminescence before and three hours after giving indo- dependent on superoxide generated by the http://gut.bmj.com/ methacin. The same amount of physiological hypoxanthine-xanthine oxidase system. Recom- saline was injected in the same manner into the binant human Cu,Zn-SOD (a gift from Nippon control rats. Dimethylsulphoxide (Wake Pure Kayaku Co, Tokyo) was used as a standard and Chemical Co, Osaka) at a dose of 550 mg/kg its activity was determined by the cytochrome c diluted in 0 5 ml of physiological saline was method. Activity ofSOD in tissue was expressed administered by intraperitoneal injection 30 as U/mg protein. The activity of glutathione minutes before and three hours after indo- peroxidase in the gastric mucosa was assayed on September 30, 2021 by guest. Protected copyright. methacin to keep DMSO at a sufficiently high spectrophotometrically by the method of concentration to force efficient scavenging of Ginzler et al24 with t-butyl hydroperoxidase as hydroxyl radicals. Control animals received 0-5 the substrate. This assay is based on the oxida- ml of physiological saline in the same manner. tion of reduced glutathione by glutathione Six hours after indomethacin treatment the peroxidase coupled to the oxidation of NADPH rats