Gut 1996; 38: 826-831

Sulphydryl blocker induced gastric damage isameliorated by scavenging of free radicals

F Karmeli, E Okon, D Rachmilewitz

AbstractBackground-Sulphydryl compounds andnitric oxide are essential in maintaininggastric mucosal integrity.Aims-To characterise the gastric damageinduced by a sulphydryl blocker, to evalu-ate the role of nitric oxide in its patho-genesis, and to reveal its possibleprevention by scavenging of free radicals.Methods-Gastritis was induced in rats byaddition of iodoacetamide (0. 1%) to thedrinking water, with and without dailyintragastric administration of TEMPOL.After death, the stomach was resected,washed, lesion area assessed, and mucosalinflammatory mediators, myeloperoxi-dase and nitric oxide synthase activitieswere determined.Results-Administration of iodoacetamideinduced gastric mucosal erosions presentfor up to two weeks. Myeloperoxidaseactivity was increased for up to seven daysand nitric oxide synthase activity wassignificantly decreased for up to 14 days.Treatment for seven days with the freeradical scavenger, TEMPOL, decreasedby 68% the damage induced by iodo-acetamide.Conclusions-Gastric damage induced byiodoacetamide, a sulphydryl alkylator,accompanied by inhibition of nitric oxidesynthase activity shows the importantcontribution of sulphydryl compoundsand nitric oxide to the maintenance ofgastric mucosal integrity. Nitric oxidedonation and scavenging of free radicalsmay be a novel approach to preventgastric damage.(Gut 1996; 38: 826-831)

Keywords: nitric oxide, TEMPOL, iodoacetamide,free radicals, gastric damage.

Departments ofMedicine andPathology, HadassahUniversity Hospital,Mount Scopus,Hebrew UniversityHadassah MedicalSchool, Jerusalem,IsraelF KarmeliE OkonD Rachmilewitz

Correspondence to:Professor D Rachmilewitz,Hadassah UniversityHospital, Mount Scopus,PO Box 24035, Jerusalem91240, Israel.

Accepted for publication7 December 1995

Glutathione exists in a combination of itsreduced form (GSH) and its oxidised dimer inalmost all mammalian cells. GSH is a nucle-ophilic scavenger of superoxide and also acts asa cofactor in the GSH peroxidase mediatedreduction of H202.1 GSH has an importantrole in maintaining mucosal integrity in thegastrointestinal tract. GSH is essential for theprotection of gastric epitheliall and chief cells2against oxidative stress. Moreover, calciuminduced gastric mucosal injury is mediated byreduction in GSH content.3 In rats, thesulphydryl (SH) blocker, iodoacetamide, waspreviously shown to induce diffuse gastritis4and, also, significant colonic injury,5 indicatingthe important contribution of SH compounds

to the maintenance of gastrointestinal integrity.In this study the model in iodoacetamideinduced gastric damage and its possible preven-tion by scavenging of free radicals was evalu-ated. This model was further characterisedmacroscopically and histologically, with specialfocus on the involvement of inflammatorymediators and nitric oxide (NO) in its patho-genesis.NO has an important role in maintaining

gastric mucosal integrity. Inhibition of gastricNO formation decreases gastric blood flow,deprives the tissue of oxygen, and increasesmucosal vulnerability to intragastric adminis-tration of irritants that mildly damage thegastric mucosa. Moreover, inhibition of NOformation also increases gastric mucosalhaemodynamic changes, such as those inducedby ethanol administration.6 Oxygen derivedradicals and non-radical species have beenimplicated previously in the pathogenesis ofgastric ulceration78 and TEMPOL, a freeradical, which is also a potent scavenger ofother free radicals, was shown by us to preventgastric mucosal lesions in rats.9 In this studythe role of gastric NO formation in iodo-acetamide induced gastric damage and itspossible prevention by scavenging of freeradicals was evaluated.

Methods

MaterialsL-arginine, L-citrulline, L-NAME, D-NAME,NADPH, valine, aminoguanidine, dithiothre-itol, phenylmethylsulphonyl fluoride, EDTA,EGTA, and 4-hydroxy-2,2,6,6-tetramethyl-peperidine-1-oxyl (TEMPOL) were purchasedfrom Sigma Chemical Co, St Louis, MO,USA; Dowex AG5OW-X8 (Na form) 100-200mesh, and TRIS base (electrophoresis grade)were purchased from Bio-Rad Labs, Richmond,CA, USA; aquasol-2 was purchased fromDuPont Co/NEN Research Products, BadHamburg, Germany.

AnimalsMale, Sprague-Dawley rats, weighing 200-250g and fed ad libitum were used in all thestudies.

Iodoacetamide induced gastritisGastritis was induced by the addition ofiodoacetamide (0/1%) to the drinking waterfor a period of 1-14 days. Control rats wereprovided with tap water without iodoace-tamide. In several experiments, in addition to

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SH blocker induced gastritis

300

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oLm0g gControl 1 2 3 7 14

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Figure 1: Gastric lesion area in iodoacetamide treated rats. Rats were treated withiodoacetamide (0 1%) added to the drinking water. Rats were killed after 1-14 days, thestomach was removed, washed, lesion area assessed, as described in Methods. Results aremean (SEM) of 4-20 rats in each time interval. *Significantly differentfrom control,p

Karmeli, Okon, Rachmilewitz

Effect of TEMPOL on iodoacetamide induced gastritis

Treatment L4 IA+ TEMPOL

Days of treatment 2 7 2 7Number 10 9-19 5 10Lesions (mm2/rat) 27-0 (2.2) 71-0 (14-7) 36-8 (9.0) 22-8 (3.2)*MPO (U/g) 2.5 (0.4) 1-8 (0.2) 1-0 (0.2) 1-6 (0.3)NOS (nrmollg/min) 3-2 (0.4) 4-2 (0.4) 4-2 (0-1) 4-0 (0-1)

Rats were treated with iodoacetamide (0-1O%) added to the drinking water with or without dailyintragastric treatment with TEMPOL (50 mg/100 g). Rats were killed after two and seven days,the stomach removed, lesion area assessed, and mucosal MPO and NOS activities determined,as described in Methods. Results are mean (SEM). IA=iodoacetamide. *Significantly differentfrom rats treated with iodoacetamide, p

SH blocker induced gastritis

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60 H

50 F-

40 F-

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lodoacetamide treatment (days)Figure 5: Gastric PGE2 generation in iodoacetamide treated rats. Rats were treciodoacetamide (0-%1°o) added to the drinking water. Rats were killed after 1-14stomach was removed, washed, the mucosa scraped, and mucosal PGE2 generaldetermined, as described in Methods. Results are mean (SEM) of 9-20 rats ineinterval. *Significantly different from control, p

Karmeli, Okon, Rachmilewitz

7

6 -

5

4

3

2

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7

/

T T

*

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Control 1 2 3 7 14

lodoacetamide treatment (days)Figure 7: Gastric NOS activity in iodoacetamide treated rats. Rats were treated withiodoacetamide (0%1 ) added to the dninking water. Rats were killed after 1-14 days, thestomach was removed, washed, gastric NOS activity was determined as described inMethods. Results are mean (SEM) of 7-16 rats in each time interval. *Significantlydifferentfrom control, p

SH blocker induced gastritis 831

its vascular effect.'7 On the other hand, in thestomach NO regulation of the mucosal haemo-dynamics, including blood flow and haemo-globin oxygen saturation, was shown to beresponsible for its important contribution tothe maintenance of mucosal integrity.6 Theseaspects of NO deprivation may certainly con-tribute to the severity of the gastric injuryinduced by iodoacetamide. Inhibition of NOformation was shown to aggravate gastricmucosal injury induced by ethanol. Decreasein the availability of NO induces a decrease inthe resting mucosal blood flow, resulting intissue hypoxia.6 The cumulative data thusshow that endogenous NO is an essential pro-tective factor in the pathogenesis of gastricinjury induced by agents such as ethanol andiodoacetamide.TEMPOL, the potent free radical scav-

enger, was found to effectively decrease theseverity of gastric mucosal injury induced byiodoacetamide. We have reported that TEM-POL is a powerful protective agent againstinjury induced by various other irritants, indi-cating the important contribution of freeradicals to the pathogenesis of gastric injury.9These results suggest that scavenging of freeradicals counteracts the deleterious effectsof NO depletion, which contributes to theincreased free radical generation by thehypoxic tissue. TEMPOL was found to haveno effect on gastric NOS activity. However, asTEMPOL removes free radicals, which rapidlyinteract with NO and lower its concentration,TEMPOL may indirectly induce an increase oftissue NO concentrations. Increase in tissueNO concentrations may be an additionalmechanism to explain the protective effect ofTEMPOL. Potent scavengers, like TEMPOL,may therefore be useful for prevention ofgastric mucosal injury induced by any irritantand, especially, by those that induce genera-tion of free radicals. The importance of SHcompounds in counteracting the deleteriouseffect of free radicals is also indicated byN-ethylmaleimide enhancement of gastricmucosal cell injury induced by oxygen freeradicals generated by the xanthine oxidase-xanthine system, an effect which is reduced byglutathione and cystamine.20

In conclusion, the results reported in thisstudy show the importance of SH compounds

and of NO to the maintenance of gastricmucosal integrity. NO donation, scavenging offree radicals, and provision of SH compoundsmay, therefore, be useful for prevention ofgastric injury.The authors are grateful to Dr S Szabo, who inspired the study.

1 Meister A, Anderson ME. Glutathione. Ann Rev Biochem1983; 52: 711-60.

2 Olson CE. Glutathione modulates toxic oxygen metaboliteinjury of canine chief cell monolayers in primary culture.AmJfPhysiol 1984; 254: G49-56.

3 Wong HM, Tepperman BL. Reduced glutathione modu-lates Ca2+-mediated damage to rabbit isolated gastricmucosal cells. Am J7 Physiol 1994; 267: G1-9.

4 Szabo S, Trier JS, Brown A, Schnoor J. Sulphydryl blockersinduce severe inflammatory gastritis in rats [Abstract].Gastroenterology 1984; 86: 1271.

5 Rachmilewitz D, Karmeli F, Okon E. Sulphydryl blockercolonic inflammation is ameliorated by inhibition of nitricoxide synthase activity. Gastroenterology 1995; 109: 98-106.

6 Masuda E, Kawano S, Nagano K, Tsuji S, Takei Y, TsujiiM, et al. Endogenous nitric oxide modulates ethanol-induced gastric mucosal injury in rats. Gastroenterology1995; 108: 58-64.

7 Pihan G, Regilo C, Szabo S. Free radicals and lipid peroxi-dation in ethanol- or aspirin-induced gastric mucosalinjury. DigDis Sci 1987; 32: 1395-401.

8 Vaananen PM, Meddings JB, Wallace JL. Role of oxygen-derived free radicals in indomethacin-induced gastricinjury. AmJrPhysiol 1991; 261: G470-5.

9 Rachmilewitz D, Karmeli F, Okon E, Samuni A. A novelantiulcerogenic stable radical prevents gastric mucosallesions in rats. Gut 1994; 35: 1181-8.

10 Bradley PP, Priebat DA, Christensen RD, Rothstein G.Measurement of cutaneous inflammation: estimation ofneutrophil content with an enzyme marker. J7 InvestDermatol 1982; 78: 206-9.

11 Bush PA, Gonzalez NE, Griscavage JM, Ignarro JL. Nitricoxide synthase from cerebellum catalyzes the formation ofequimolar quantities of nitric oxide and citrulline from L-arginine. Biochem Biophys Res Commun 1992; 185: 960-6.

12 Sharon P, Ligumsky M, Rachmilewitz D, Zor U. Role ofprostaglandins in ulcerative colitis: enhanced productionduring active disease and inhibition by sulphasalazine.Gastroenterology 1978; 65: 638-40.

13 Szabo S, Trier JS, Frankel PW. Sulphydryl compoundsmay mediate gastric cytoprotection. Science 1981; 214:220-2.

14 Hauser J, Szabo S. Extremely long protection by pyrazolederivatives against chemically induced gastric mucosalinjury. J Pharmacol Exp Ther 1991; 256: 592-8.

15 Yamada T, Marshall S, Specian RD, Grisham MB. A com-parative analysis of two models of colitis in rats.Gastroenterology 1992; 102: 1524-34.

16 Rachmilewitz D, Stamler JS, Bachwich D, Karmeli F,Ackerman Z, Loscalzo J, et al. Enhanced colonic nitricoxide generation and stimulated nitric oxide synthaseactivity in experimental colitis and in active inflammatorybowel disease. Gastroenterology 1993; 104: A766.

17 Rachmilewitz D, Karmeli F, Okon E, Bursztyn M.Experimental colitis is ameliorated by inhibition of nitricoxide synthase activity. Gut 1995; 37: 247-55.

18 Salter M, Knowels RG, Moncada S. Widespread tissue dis-tribution, species distribution and changes in activity ofCa2'-dependent and Ca2'-independent nitric oxide syn-thases. FEBS Lett 1991; 291: 145-9.

19 Gardiner SM, Compton AM, Bennett T, Palmer BMJ,Moncada S. Control ofregional blood flow by endotheliumderived nitric oxide. Hypertension 1990; 15: 486-92.

20 Li T, Zhang XJ. Role of sulphydryl compounds in theoxygen radical induced injury of isolated gastric mucosalcells. Sheng Li Jsueh Pao 1992; 44: 386-93.

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