732 Gut 1993; 34: 732-737

Role of active , lipid peroxidation, and

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 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 , an important 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 and 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- 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 pathogenesis ofgastric mucosal injury induced scavenger (superoxide dismutase (SOD)), a by indomethacin, and that the decreased scavenger (catalase), and a glutathione peroxidase activity aggravated the 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, 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. 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- 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 were killed by exsanguination via the by glutathione reductase. The rate of NADPH abdominal aorta and the extent ofgastric damage oxidation was monitored photometrically. For was expressed as the total area of haemor- the glutathione assay, the stomach was perfused rhagic erosion. The concentration of lipid intraluminally with 5% sulphosalicylic acid and peroxides in the gastric mucosa was also then homogenised in 10 vol/g of the same measured. solution. The tissue homogenate was centrifuged for five minutes at 10 000 g, and then the supernatant was stored on ice until use. The Effect ofSOD plus catalase on indomethacin amount of reduced glutathione (GSH) was induced changes in antioxidants measured by the method of Griffith25 and the The effects of treatment with SOD plus catalase amount of oxidised glutathione (GSSG) was on the activities of SOD and glutathione peroxi- measured by masking GSH with N-ethyl- dase in gastric mucosa, on the concentration of malemide (NEM). a-tocopherol in gastric mucosa and serum samples and on the concentrations of reduced and oxidized glutathione after administration of STATISTICAL ANALYSIS indomethacin were investigated. Rats received Results are presented as means (SEM). For SOD (recombinant human Cu,Zn-SOD) and statistical analysis, the tests used were the catalase at the same doses as previously des- Bartlett test for homogenicity of variance, cribed, by subcutaneous injection one hour Kruskal-Wallis analysis of variance for effects in before and three hours after indomethacin time and comparison of differences between administration. Control animals received the control and groups treated with scavengers, and same amount of physiological saline. Gastric the Mann-Whitney test for individual compari- mucosal homogenates were prepared as des- son ofthe group means. Differences between the 734 Yoshikawa, Naito, Kishi, Tomii, Kaneko, Iinuma, Ichikawa, Yasuda, Takahashi, Kondo

co Figure 1: Total area of U, 30 0 gastric erosions after 0 indomethacin. Values are Co mean (SEM) ofnine E T E experiments. ***p

located on the upper part of the mucosal folds. after administration ofindomethacin the level of http://gut.bmj.com/ TBA-reactive substances in the gastric reduced glutathione was significantly decreased mucosa, an index of lipid peroxidation, (p<0-001) to 46-9% of the control value, but the increased significantly (p<0-01) from a basal level ofoxidised glutathione was not affected. concentration of mean (SEM) 0-521 (0-014) nmol/mg protein to 0-626 (0-019) nmol/mg protein and 0 663 (0 036) nmol/mg protein three EFFECTS OF ACTIVE OXYGEN SCAVENGERS ON

hours and six hours after administration of INDOMETHACIN INDUCED GASTRIC MUCOSAL on September 30, 2021 by guest. Protected copyright. indomethacin respectively. Indomethacin did INJURY (Table) not affect the level ofTBA-reactive substances in The mean (SEM) of the total area of gastric serum, however (Fig 2). a-Tocopherol in serum haemorrhagic erosions in control animals receiv- samples, but not in the gastric mucosa, ing subcutaneous physiological saline was 26-8 decreased significantly (p<0-05) with time after (4-5) mm' at six hours after indomethacin indomethacin administration. The a-tocopherol: administration. SOD and a combination of SOD cholesterol ratio in serum decreased to 4 99 and catalase significantly reduced (p<005) the (0_19)xlO-3 and 4-96 (0 26)x10-3 at three lesion area to 46-6% and 49*6% of the mean of hours and six hours after indomethacin adminis- control values. The lesion area of the group tration respectively, from 5 88 (0 24) x 10-3 treated with catalase was not significantly differ- measured before the experiment (Fig 3). ent from that of the control rats. The increase in

5c 5 en .C~~~~~~~~~~. C) Cn _ 3.0 0 co CoW co 07K a) z C0 4C)o - m cn cn~ c E 4oC -14 :E 20 o0 >.T c 3czo _ ?____--? _ .> = a) 0) co >-00 06k E 10- SOD .0 2c 4o _L m -o O Glutathione 2 o 5o EE perodixase *5X 1C *GSH GSSG m ._ o > 1.0 05H 0~~1: 2~~c m "0 0 -- 1 0 JH~~~0 0 3 6 Time 0 3 6 (h) Figure 4: Activities ofsuperoxide dismutase (SOD) and Time (h) glutathione peroxidase and the amounts ofreduced and Figure 2: Thiobarbituric acid (TBA)-reactive substances in oxidised glutathione (GSH and GSSG) in gastric mucosa the gastric mucosa and serum after indomethacin. Values are after indomethacin. Values are mean (SEM) ofsix mean (SEM) ofsix experiments. **p<001 compared with experiments. *p<005 and ***p<0O001 compared with the the values before indomethacin. values before indomethacin. Role ofactive oxygen, lipidperoxidation, and antioxidants in thepathogenesis ofgastric mucosal injury induced by indomethacin in rats 735

Effects ofsuperoxide dismutase (SOD), catalase, SOD plus catalase, and dimethylsulphoxide (DMSO) on total area of haemorrhagic erosions and thiobarbituric acid (TBA) reactive the gastric mucosa at six hours after giving 4_ 400 - substances in Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. Downloaded from indomethacin o ) - a)-e "C Lesion TBA-reactive x + 300 - -IL No of area substances (nmollmg protein) animals (mm2) 0cm * Q E *-r Control 1 6 26-8 (4 5) 0-663 (0-030) a) - 200 I 085) Z) SOD 6 12-5 (1-6)* 0 589(0 .0O E Catalase 6 18-5 (4-7) 0 575 (0-097) r- SOD plus catalase 6 13-3 (3-8)* 0-559 (0.035)* Control 2 9 22-8 (3-5) 0-715 (0 069) 100 - DMSO 9 12-1 (2-4)* 0-524(0.048)*

Values are mean (SEM) for that group. *p<0.05 by Mann- o Whitney test for the difference between presented value and that I of rats treated with physiological saline as the control. Control Disease SOD + (n = 6) control catalase (n = 6) (n = 6) at Figure 6: Superoxide dismutase (SOD) plus catalase TBA-reactive substances in the gastric mucosa treatment on glutathione peroxidase activity in the gastric six hours after indomethacin administration was mucosa six hours after indomethacin. Values are mean significantly inhibited by combination treatment (SEM). *p<005 compared with normal control group. with SOD and catalase, but not by treatment with SOD or catalase alone. was administered, its activity in the gastric The total areas of gastric erosion six hours mucosa did not increase. The level of reduced after indomethacin administration were 22-8 glutathione in the gastric mucosa was 1 *49 (0-1 1) (3 5) mm2 in the control rats receiving intra- pg/g tissue in the untreated group, 0-70 (0-15) peritoneal saline and 12-1 (2 4) mm2 in the .tg/g tissue in the control group, and 1-10 DMSO group. There was a significant difference (0 07) [ig/g tissue in the SOD plus catalase group (p<005) in the lesion area between the two respectively (Fig 7). There were significant groups. The increase in TBA-reactive sub- differences between the normal group and the stances was also significantly inhibited (p<005) control group (p<0-001), and between the by intraperitoneal treatment with DMSO. control group and the SOD plus catalase group (p<005).

ON EFFECTS OF SOD PLUS CATALASE INDOMETHACIN http://gut.bmj.com/ INDUCED CHANGES IN ANTIOXIDANTS Discussion Thedecreaseinthea-tocopherol:total cholesterol Our present studies implicate active oxygen and ratio in the serum of the control (indomethacin lipid peroxidation in the pathogenesis of gastric only) rats recovered to that of the normal mucosal injury induced by indomethacin. Itoh (untreated) rats after treatment with SOD and and Guth" first showed that SOD could signifi- catalase (Fig 5). There was no significant differ- cantly protect against gastric mucosal injury 20 minutes ence in the concentration of a-tocopherol in the induced by haemorrhageic shock for on September 30, 2021 by guest. Protected copyright. gastric mucosa among the normal, the control, and retransfusion in rats. We also produced an and the SOD plus catalase groups. Figure 6 ischaemia- model with rat shows that indomethacin significantly (p<005) stomach, and found a suppression ofboth gastric reduced the activity ofglutathione peroxidase in mucosal lesions and increased lipid peroxidation the gastric mucosa in groups treated and not by SOD and catalase."' Also, Perry et al'2 have treated with SOD and catalase. There were no shown that a hydroxyl radical scavenger, significant differences in the activity of SOD DMSO, significantly reduced both "Cr-red cell among these three groups. Even when SOD loss and the less sensitive index of gastric

70-

0 20 F _a) -a a) 6-0- IL 4.' 0 c') .2'. 1.5 F 0 ) -C x a) **-T cJ E --r o ' 50 F 1 0 F 0- *** 0. .' 'a) 0 E 0 0 8 0-5 40 : F

0 Control Disease SOD + V (n = 6) control catalase Control Disease SOD + (n = 6) (n = 6) (n = 6) control catalase Figure 5: Superoxide dismutase (SOD)plus catalase (n = 6) (n = 6) treatment on the a-tocopherol:total cholesterol ratio in serum Figure 7: Superoxide dismutase (SOD) plus catalase six hours after indomethacin. Values are mean (SEM). treatment on reduced glutathione concentration in the gastric *p

mucosal injury, '25I-albumin loss produced by of a-tocopherol in the serum was returned to the ischaemia. Cochran et al26 and Terano et al2' also normal range by concomitant treatment with reported that DMSO inhibited gastric mucosal SOD and catalase. Our previous study showed injury induced by stress or ethanol. Rats that subcutaneously injected SOD was pre- Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. Downloaded from received intraperitoneal DMSO at a dose of 550 dominantly localised in extracellular compart- mg/kg, because previous work had shown that ments,'7 and our present study showed that the peak concentrations occur within 30 minutes and SOD activity in intracellular compartments of that the average halflife is about six hours at that the rat stomach did not increase after the sub- dose and route ofadministration.27 In the present cutaneous injection of human SOD. Thus we study, indomethacin produced gastric haemor- speculated that a free radical reaction occurred rhagic erosions and increased TBA-reactive first in the after indomethacin substances, which are indicators of lipid peroxi- administration, and SOD and catalase after the dation, in the gastric mucosa with time after concentration of ct-tocopherol by their actions as administration. Also, lipid peroxide accumula- scavengers of and inhibitors of the tion closely paralleled the development ofgastric production ofhydroxyl radicals. mucosal injury. This indicates that lipid peroxi- Glutathione is an important constituent of dation plays a significant part in the pathogenesis intracellular protective mechanisms against a of the gastric mucosal lesions induced by indo- number of noxious stimuli including oxidative methacin, as well as gastric injuries produced by stress. Reduced glutathione is known as a major burn shock,'5 water immersion restraint stress,'4 low molecular weight scavenger of free radicals and ischaemia-reperfusion.'6 Also, the treatment in . On the other hand, the with SOD, SOD plus catalase, and DMSO activity of GSH peroxidase is coupled with the significantly inhibited the pathological changes oxidation of GSH to GSSG, which can subse- induced by indomethacin. As is well known, the quently be reduced by GSH reductase with superoxide radical can interact with hydrogen NADPH as the reducing agent. Concentration of peroxide in the presence of to generate the glutathione is exceedingly high in the glandular hydroxyl radical, which is thought to be the most stomach compared with concentrations in other toxic reactant and to abstract methylene hydro- portions of the gastrointestinal tract or in most gen atoms from polyunsaturated fatty acids, other organs.34 Boyd et al35 reported that deple- which initiates lipid peroxidation. The protec- tion of gastric glutathione by diethyl maleate tion by radical scavengers, therefore, suggests produced gastric ulceration. Mutoh et a136 that lipid peroxidation mediated by oxygen showed that intracellular glutathione was mainly radicals, especially hydroxyl radicals, plays an responsible for protecting against gastric cell important part in the formation and develop- injury induced by ethanol. These findings http://gut.bmj.com/ ment of the gastric mucosal lesions induced by suggest that in the gastric mucosa free radicals or indomethacin. lipid peroxides are injurious offensive factors The source of oxygen radicals in gastric and GSH is a protecting defensive factor. Deple- mucosal injury induced by indomethacin in rats tion of glutathione results in enhanced lipid is not clear. Recent studies, however, suggest a peroxidation37 and excessive lipid peroxidation possible role of in the early patho- can cause increased GSH consumption.38 The genic process. The severity of gastric damage decrease in GSH in our study was accompanied on September 30, 2021 by guest. Protected copyright. induced by indomethacin can be significantly by an increase in lipid peroxides measured as a reduced by prior depletion ofcirculatory neutro- TBA-reactive substance in the gastric mucosa. phils and by treatment with a monoclonal anti- The content of GSSG, however, showed no body against the CD18 leucocyte adhesion significant increases in response to the depletion molecule.2829 Both SOD and DMSO have been of GSH and the activity of GSH peroxidase was shown to diminish leucocyte adherence in micro- significantly inhibited by indomethacin admin- vessels.303' Therefore, the protective action of istration, which indicates that GSH was con- these scavengers in this model is consistent with sumed during the reaction with oxygen radicals, the proposal by Wallace et al.2829 organic radicals, and peroxide radicals. Vitamin E, a lipid soluable antioxidant, inter- Glutathione peroxidase is important in the acts with oxygen and lipid radicals and prevents elimination of hydrogen peroxide and lipid the propagation of free radical lipid peroxida- hydroperoxides in the gastric mucosal cell. Thus tion. Our previous work showed that the concen- inhibition of this enzyme may result in the trations ofa-tocopherol decreased both in serum accumulation of hydrogen peroxide with subse- samples and in gastric mucosa during ischaemia- quent oxidation of lipids. The GSH peroxidase reperfusion injury in rats,32 which suggests that actvity ofthis study was decreased six hours after a-tocopherol was consumed in the process of indomethacin administration and its activity lipid peroxidation mediated by oxygen radicals could not be recovered by the elimination of in ischemia-reperfusion to prevent the develop- oxygen radicals. These findings suggest that ment of tissue damage. When the radicals are indomethacin itself, or depletion of prosta- formed initially in the aqueous phase of whole glandins, inhibits GSH peroxidase activity of the blood, the water soluble antioxidants in the gastric mucosa. Our findings are in line with plasma such as vitamin C and plasma vitamin E those of Banerjee,39 who recently showed the participate in the primary defence and later inhibition of peroxidase activity in the mito- vitamin E in the erythrocyte membrane chondrial fraction of rat gastric mucosa by decreases.33 In the present study, a-tocopherol in indomethacin. the serum but not that in the gastric mucosa In summary, the results indicate that excessive significantly decreased with time after indo- generation of oxygen radicals in the extracellular methacin administration, and also the decrease space and the depletion of GSH in conjunction Role ofactive oxygen, lipidperoxidation, andantioxidants in thepathogenesis ofgastric mucosal injury inducedby indomethacin in rats 737

with inhibition of GSH peroxidase activity 20 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem is responsible for the oxidative tissue damage 1951; 193: 265-75. of gastric mucosa after administration of 21 Abe KY, Yuguchi Y, Katsui G. Quantitative determination of

tocopherols by high-speed liquid chromatography. J Nutr Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. Downloaded from indomethacin. Sci Vitaminol (Tokyo) 1975; 21: 183-8. 22 Richmond W. Preparation and properties of a cholesterol 1 Djahanguiri B. The production of acute gastric ulceration by oxidase from Nacardia sp and its application to the enzy- indomethacin in the rat. Scand J Gastroenterol 1969; 4: matic assay oftotal cholesterol in serum. Clin Chem 1973; 19: 265-7. 1350-6. 2 Fries JF, Miller SR, Spitz BW, Williams CA, Hubert HB, 23 Nakano M, Kimura H, Hara M, Kuroiwa M, Kato M, Block DA. Toward an epidemiology of gastropathy associ- Totsune K, Yoshikawa T. A highly sensitive method for ated with nonsteroidal antiinflammatory drug use. Gastro- determining both Mn- and Cu-Zn superoxide dismutase enterology 1989; %: 647-55. activities in tissue and blood cells. Anal Biochem 1990; 187: 3 Whittle BJR. Temporal relationship between cyclooxygenase 277-80. inhibition, as measured by prostaglandin biosynthesis, and 24 Gunzler WA, Flohe L. Glutathione peroxidase. In: Greenwald the gastrointestinal damage induced by indomethacin in the RA, ed. CRC handbook of methods for oxygen radical rat. Gastroenterology 1981; 80: 94-8. research. Boca Raton: CRC Press 1985: 285-90. 4 Rainsford KD, Willis C. Relationship of gastric mucosal 25 Griffith OW. Determination of glutathione and glutathione damage induced in pigs by antiinflammatory drugs to their disulfide using glutathione reductase and 2-vinylpyridine. effects on prostaglandin production. Dig Dis Sci 1982; 27: Anal Biochem 1980; 106: 207-12. 624-35. 26 Cochran T, Stefanko J, Moore C. Dimethylsulfoxide protec- 5 Kobayashi K, Arakawa T, Satoh H, Fukuda T, Nakamura H. tion against gastric stress ulceration. Current Surg 1983; Effect of indomethacin, and dicronfenac on Nov-Dec: 435-7. rat gastric mucosal damage and content of prostacyclin and 27 Terano A, Hiraishi H, Ota S, Shiga J, Sugimoto T. Role of prostaglandin E2. Prostaglandins 1985; 30: 609-18. superoxide and hydroxyl radicals in rat gastric mucosal 6 Ligmusky M, Golanska EM, Hansen DG, Kauffman GL. injury induced by ethanol. GastroenterolJ3pn 1989; 24: 488- Aspirin can inhibit gastric mucosal cyclooxygenase without 93. causing lesions in rats. Gastroenterology 1983; 84: 756-61. 28 Wallace JL, Kennan CM, Granger DN. Gastric ulceration 7 Takeuchi K, Ueki S, Okabe S. Importance of gastric motility induced by non-steroidal anti-inflammatory drugs is a in the pathogenesis of indomethacin-induced gastric lesions -dependent process. Am J Physiol 1990; 259; inrats. DigDisSci 1986; 31: 1114-21. G462-7. 8 Ueki S, Takeuchi K, Okabe S. Gastric motility is an important 29 Wallace JL, Arfors K-E, McKnight W. A monoclonal anti- factor in the pathogenesis of indomethacin-induced gastric bodyagainst the CD18 leukocyte adhesion molecule prevents mucosal lesions in rats. Dig Dis Sci 1988; 33: 209-16. indomethacin-induced gastric damage in the rabbit. Gastro- 9 Takeuchi K, Okada M, Ebara S, Osano H. Increased micro- enterology 1991; 100: 878-83. vascular permeability and lesion formation during gastric 30 Suzuki M, Inauen W, Kvietys PR, Grisham MB, Meininger C, hypermotility caused by indomethacin and 2-deoxy-D- Schelling ME, et al. Superoxide mediates reperfusion- glucose in the rat.J Clin Gastroenterol 1990; 12: S76-84. induced leukocyte-endothelial cell interactions. Am J 10 Rainsford KD. Microvascular injury during gastric mucosal Physiol 1989; 257: H1740-5. damage by antiinflammatory drugs in pigs and rats. Agents 31 Sekizuka E, Benoit JN, Grisham MB, Granger DN. andActions 1983; 13: 5-6. Dimethylsulfoxide prevents chemoattractant-induced 11 Ito M, Guth PH. Role of oxygen-derived free radicals in leukocyte adherence. Am J Physiol 1989; 256: hemorrhagic shock-induced gastric lesions in the rat. Gastro- H594-7. enterology 1985; 88: 1162-7. 32 Yoshikawa T, Yasuda M, Ueda S, Naito Y, Tanigawa T, 12 Perry MA, Wadhwa S, Parks DA, Pickard W, Granger DN. Oyamada H, et al. Vitamin E in gastric mucosal injury Role ofoxygen radicals in ischemia-induced lesions in the cat induced by ischemia-reperfusion. AmJ Clin Nutr 1991; 53: stomach. Gastroenterology 1986; 90: 362-7. 210S-4S. 13 Tappel AL. Lipid peroxidation damage to cell components. 33 Niki E, Komura E, Takahashi M, Urano S, Ito E, Terao K. Fed Proc 1973; 32: 1870-4. Oxidative hemolysis of erythrocytes and its inhibition http://gut.bmj.com/ 14 Yoshikawa T, Miyagawa H, Yoshida N, Sugino S, Kondo M. by free radical scavengers. J Biol Chem 1988; 263: 19809- Increase in lipid peroxidation in rat gastric mucosal lesions 14. induced by water-immersion restraint stress. J Clin Biochem 34 Boyd SC, Sasame HA, Boyd MR. High concentration of Nutr 1986; 1: 271-7. glutathione in glandular stomach: possible implications for 15 Yoshikawa T, Yoshida N, Miyagawa H, Takemura T, carcinogenesis. Science 1979; 205: 1010-2. Tanigawa T, Sugino S, Kondo M. Role oflipid peroxidation 35 Boyd SC, Sasame HA, Boyd MR. Gastric glutathione deple- in gastric mucosal lesions induced by burn shock in rats. tion and acute ulcerogenesis by diethylmaleate given sub- J Clin Biochem Nutr 1987; 2: 163-70. cutaneously to rats. LifeSci 1981; 28: 2987-92. 16 Yoshikawa T, Ueda S, Naito Y, Takahashi S, Oyamada H, 36 Mutoh H, Hiraishi H, Ota S, Yoshida H, Ivey KJ, Terano A,

Morita Y, et al. Role of oxygen-derived free radicals in et al. Protective role of intracellular glutathione against on September 30, 2021 by guest. Protected copyright. gastric mucosal injury induced by ischema or ischemia- ethanol-induced damage in cultured rat gastric mucosal reperfusion in rats. Free Radic Res Comms 1989; 7: 285-91. cells. Gastroenterology 1990; 98: 1452-9. 17 Miyagawa H, Yoshikawa T, Tanigawa T, Yoshida N, Sugino 37 Younes M, Siegers CP. Mechanistic aspects of enhanced lipid S, Kondo M. Measurement of serum superoxide dismutase peroxidation following glutathione depletion in vivo. Chem activity by electron spin resonance. J Clin Biochem Nutr Biol Interact 1981; 34: 257-66. 1988; 5: 1-7. 38 Comporti M. Biology of disease: lipid peroxidation and 18 Yagi K. A simple fluorometric assay for lipid peroxides in cellular damage in toxic liver injury. Lab Invest 1985; 53: blood plasma. Biochemical Medicine 1976; 15: 212-6. 599-623. 19 Ohkawa H, Ohnishi N, Yagi K. Assay for lipid peroxides for 39 Banerjee RK. Nonsteroidal anti-inflammatory drugs inhibit animal tissues by thiobarbituric acid reaction. Anal Biochem gastric peroxidase activity. Biochim BiophysActa 1990; 1034: 1979; 95: 351-8. 275-80.