Indian Journal of Experimental Biology Vol. 40, June 2002, pp. 693-705

Involvement of reactive oxygen species in gastric ulceration: Protection by melatonin

Debashis Bandyopadhyay, Kaushik Biswas, Mrinalini Bhattacharyya, Russel J Rei ter' & Ranajit K Banerjee t

Department of Physiology, Indi an Institute of Chemical Biology, 4-Raja S.c. Mullick Road, Kolkata 700 032, Ind ia

*Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78284-7762, U.S.A.

Uncontrolled hydrochloric ac id secretion and ulceration in the stomach due to various factors are serious global prob­lems today. Although the mechanism of ac id secretion from the parietal cell is now fai rly known, the mechanism of gastric ulceration is still not clear today. Among various causes of gastric ulceration, lesions caused by stress, alcohol consumption , Helicobacter pylori infection and use of nonsteroidal antiinfl ammatory drugs have been shown to be mediated largely through the generation of reac tive oxygen species especially hydroxyl radical (OH). A number of excellent drugs have been proved useful in controlling hyperacidity and ulceration but their long term uses are not devoid of disturbing side-effects. Hence, the search is still on to find out a compound possessing ant isecretory, antiulcer and an tioxidant properties wh ich will serve as a powerful therapeutic agent to eurc gastric hyperacidity and ulcer. This article describes the role of reactive oxygen species in gastric ulceration, drugs controlling the m with the ir merits and demerits and, the role of melatonin, a pineal hor­mone in protec ting the gastric lesions with a fina l commentary on how melatonin research with respect to gastric patho­phys iology can be taken forward with a view to projecting thi s indole as a promising therapeutic agent to control gas tric ul ­ceration in humans.

Ulceration of the stomach mainly develops in the an­tral region due to lesions in the gastric mucosa. Epi­gastric pain is the common clinical feature and il'l se­vere cases blood appears in the vomitus. Since in ma­jority of cases it is aggravated due to pepsin­hydrochloric acid, it is also termed as peptic ulcer. It is considered as one of the major human sufferings today affecting nearly 5% of the global population. Management of this painful disease, its prevention or cure is one of the challenging problems today.

Gastric ulcer develops when a balance between some aggressive and defensive (cytoprotective) fac­tors is lost. The aggressive factors are either endoge­nous or exogenous in origin. The endogenous damag­ing factors are hydrochloric acid, pepsin, refluxed bile, leukotrienes :ond reactive oxygen species (ROS) such as 0 ; , H20 2 and OHo. The exogenous damaging

factors mainly include alcohol, steroidal and nonster­oidal antiinflammatory drugs and drugs which st imu­late gastric acid and pepsi n secretion, stress and ten­sion and Helicobacter pylori. The mucosal defense against these aggressive factors is contributed by mu-

tCorrespondent author Phone: 033-473 3491,473 3493 Fax: 033-473 0284 e-mail: ranaj ilb @yahoo.com

eus-bicarbonate barrier, surface active phospholipid, prostaglandin, mucosal blood tlow, cell renewal and migration, antioxidants and antioxidant enzymes and some growth factors.

Reactive oxygen species, generated in the cells of aerobically respiring organisms due to many factors, have been implicated in pathogenesis of many human sufferings like Parkinson 's, Alzheimer, Huntington ' s diseases and many other neurodegenerative conditions'. The role of ROS as a causative factor in certai n ischemic cardiovascular and pulmonary diseases, ca­taractogenes is and reproductive disorders have also been studied extensivelyl. Participation of ROS in inducing cancer in many cases has stormed the scien­ti fi c community in the last few yearsl.

This article presents a comprehensive view on how ROS is involved in the generation of gastric ulcera­tion. This is followed by a short account of the meri ts and demerits of a number of already marketed drugs for ameliorating the condition of gastric hyperacidity and ulceration. Finally, the role of pineal hormone, melatonin, in preventing gastric ulceration by scav­enging the endogenous . OH is described indicating the possible areas where extensive research works can be carried out to understand the critical importance of melatonin in gastroprotection with the ultimate goal of its therapeutic application.

694 INDIAN J EXP BIOL, JUN E 2002

Reactive oxygen species and gastric ulceration The seemingly paradoxical consequences of the

beneficial and harmful effects of oxygen (02) have been shown for several decades2.While more than 95% of the O2 taken in by the aerobic organisms is fully reduced to water (H20) during the process of mitochondrial respiration, a small percentage « 5%) of the O2 consumed is converted to semi reduced spe­

cies, i.e., the superoxide anion radical (0;), hydrogen

peroxide (H20 2) and the hydroxyl radical ("OH l These species are collectively referred to as reactive oxygen species (ROS) which can be highly toxic , and their interactions often with cellular macromolecules bring about oxidative damage4. The most toxic of the

ROS is the ·OH which is often formed when 0 ~ and

H20 2 are exposed to the trace transition metals iron or copper via metal-catalyzed Haber-Weiss reaction:

Fe3+ +0 ; -1 Fe2

+ + O2

Fe2+ + H20 2 -1 Fe3+ + ·OH + OH­

The net result is therefore,

0; + H20 2 -1 O2 + ·OH + OW

The mechanism of ROS formation and how the cellu­lar antioxidant systems defend against accumulating ROS have already been reviewed4-7 .

Involvement of ROS in pathogenesis of gastric ul­ceration was first evident from the studies on ische­mia-reoxygenation-induced gastric mucosal injurl· lO

•

A growing body of experimental and clinical evidence . 1,,·12 suggests that gastnc mucosal damage by ethano ,

nonsteroidal antiinflammatory drugs I3.14, and by Helicobacter pylori 15 is mediated through reactive oxygen species I6.17. Moreover ROS may play an im­portant role in gastric ulceration induced by several kinds of stress I6.17

• ROS also decreases the level of endogenous antioxidants such as GSH, a-tocopherol and ascorbate, and make the mucosa more prone to oxidative damage l6. The pathogenesis of gastric mu­cosal lesions by water immersion restraint stress and burn shock in rat is associated with increased lipid pcroxidation I7.18. Systemic administration of glu­tathione or superoxide dismutase prevents water­immersion stress-induced ulceration 19.20 Cold­restraint stress alters the level of various damaging and cytoprotective factors of rat gastric mucosa to

. I . 21 cause gastnc u cerat10n .

Although the involvement of ROS in gastric lesions caused by various types of stress has been

reported 17.18.20, detailed investigation on the role of

ROS in cold-restraint stress-induced gastric ulceration has been limited. Moreover, very few studies have been undertaken to show the causal role of any spe­cific oxygen-derived free radical in mediating gastric damage during stress. Stress-induced gastric ulcera­tion which is associated with increased lipid peroxida­tion and depletion of endogenous GSH is due to in­creased formation of 0; , activation of SOD and inac­tivation of gastric peroxidase (GPO)22-a condition suitable for generation of H20 2 and formation of more reactive ·OH which causes antioxidant depletion and lipid peroxidation. The cytoprotecti ve enzyme, pros­taglandin synthetase (PGS), has also been shown to be inactivated leading to decreased defence against the aggressive factors21 . Yoshikawa ef af. 9 reported su­pression of both lipid peroxidation and gastric muco­sal injury induced by ischemia-reperfusion, after ad­ministration of SOD and catalase, indicating the role of ROS in the damage. Lipid peroxidation caused by ·OH is increased in gastric lesions induced by etha­nol "·13, indomethacin 12.1 4, ischemia-reperfusion9.1O,

water immersion l8, and burn shock 17. Dimethyl sul­foxide (DMSO), a specific ·OH scavenger, reduces the gastric mucosa! injury produced by ischemia8, stress22 or ethanof3, indicating a critical role of ·OH in the mucosal damage. It has been shown by direct measurement using DMSO that ·OH is really gener­ated in the gastric mucosa under stress22

. Keeping the . 21 D l22 d results of Das and Banerjee and as et a . an

some reported by others, the plausible role of ·OH in the generation of stress-ulcer as shown in the Scheme 1 has been proposed. Stress causes both sympathetic and parasympathetic stimulation of the stomach, which induces an increased motility and muscular contraction leading to vascular compression and mu­cosal ischemia2o.24.25. Sympathetic stimulation also causes direct arteriolar vasoconstriction and, thus greatly reduces the blood flow to the stomach leadi ng to local hypoxia and near or actual '"ischemia". The ischemic condition increases the leakage of 0 ; from mitochondrial electron transport chain26 and facilitates the availability of "redox-active" copper or iron. In­creased 0 ; production leads to increased level of H20 2 (by the action of SOD), which, in conjugation with 0; generates ·OH via the metal catalyzed Haber-Weiss reaction. Hydroxyl radicals thus gener­ated oxidizes important cellular constituents such as structural and functional proteins, membrane lipids, and depletes glutathione. Lipid peroxidation causes

BANDYOPADHYAY el at.: REACTIVE OXYGEN SPECIES, GASTRIC ULCERATION & MELATONIN 695

Sy mpathetic Parasympathetic stimulati~n ----iSTRESS~(vagal) stimulation

~ Muscular contraction 4- tncreased gastric motility

and arteriolar vasoconstriction

! (

Gastric mucosal ischemia~ and acidosis t

Increased flux of 0 2: ,....--Liberation of 'red ox active'

SOD activityt iron and copper ! transition metal ions like

Higher level of H202 l-

PG +-PGS

HCI-Pepsin + +

fluidity, ion tran sport and membra ne integrity

Histamine

Scheme I -Mechanism of stress-induced gastric ulceration­Central role of hydroxy l radical.

loss of membrane fluidity , impaired ion transport and membrane integrity, and finally loss of cellular func-. 127 S I .. . f tIOns · . tress a so causes mactlvatIOn 0 pros-

taglandin synthetase21 leading to decreased biosynthe­sis of prostaglandin-the master molecule for gastro­protection against all forms of insults to the mucosa. These factors in conjugation playa key role in stress­induced gastric ulceration.

The in vivo production of 'OH from 0 ; and H20 2 need the presence of redox-active transitional metals like iron or copper27. The involvement of the metal

ions in 'OH-mediated oxidative damage has been sub­stantiated by earlier observation22 that both lipid per­oxidation and mucosal injury were prevented by the administration of a non-toxic metal ion chelator, des­ferrioxamine (DFO). Interestingly, iron has been re­ported to playa critical role in ROS-mediated injury to gastric mucosa27.28 . Yajima et. al. 29 have shown that DFO and phenanthroline protect the cultured gastric mucosal cells from H20 2 -mediated lipid peroxidation and cellular injury by chelating iron . However, the availability of metal ions for in vivo production of 'OH has been the subject of much debate27. There are reports that during tissue ischemia, iron and copper are liberated from their respective storage proteins and participate in H20 r mediated Fenton reaction 27.

Increased gastric motility and vascular compression during stress, as mentioned earlier, leading to mucosal

. h . 20 24 25 h h f 0 . d . ISC emla . , en ances t e rate 0 2 pro uctlon , which is taken care of by the adaptive increase of mi­tochondrial SOD22. In vivo activation of SOD by stress is completely prevented by a-amanitin22

, an established inhibitor of RNA polymerase3o, which controls the transcription of DNA to form mRNA. Thus stress causes an increased synthesi s of SOD by elevating the level of its mRNA. Presumably , the in­creased level of O2' generated during ischemic stress stimulates SOD biosynthesis through gene transcrip­tion . However, the source of the increased O2' during the stress is not clear yet. The xanthine dehydro­genase-xanthine oxidase system, which is supposed to form O2' in ischemia-reperfusion tissue injur/ I

, is not the source in the stomach, as earlier studies failed to show any change in xanthine dehydrogenase level by stress22

. Moreover, allopurinol, a potent inhibitor of xanthine oxidase, fails to inhibit gastric mucosal in­jury induced by water-immersion stress20. Mitochon­dria are known to be the alternative source of O2' within the ce1l26. During low oxygen tension, the mi­tochondrial electron transport chain may remain in a reduced state and leak electrons to increase the flux of O2.

1• This enhanced production of O2' may release

iron or copper or both from their storage proteins 1.27 and lead to the generation of 'OH via metal catalyzed Haber-Weiss reaction as described earlier. As these metal ions form complexes with the biological mole­cules, they serve as the redox-active centers for re­peated production of 'OH, leading to the oxidative damage of the macromolecules at or near the metal binding site27. 'OH generated in vivo not only peroxi­dizes membrane lipids but also oxidatively damages the critical cellular proteins32, thus impairing the vital cellular functions. In the stomach of stressed animals, an increased level of the protein carbonyl content22

was detected which is an indicator of metal-catalyzed 'OH-dependent oxidation of proteins32. Interestingly , gastric peroxidase (GPO), a major antioxidant enzyme of the gastric mucosa22 was found to be inactivated during stress and antioxidants like glutathione (GSH) , vitamin E and the metal ion chelator, DFO can not only prevent gastric ulceration and lipid peroxidation, but also protect the GP022. This suggests that metal ion-dependent generation of 'OH is involved in the inactivation of GPO and may, in part, contribute to the increased protein carbonyl content in the stomach under stress. Detailed studies using purified rat gastric peroxidase (GPO) have clearly demonstrated that ox i­dative damage of GPO has significant role in strcss-

696 [NDIAN J EXP BIOL, JUNE 2002

induced gastric ulceration33. In stress ischemia, when generation of O2- and H20 2 increases21 .22 , the latter diffuses to Cu2+ binding site and generates ·OH in a 's ite-specific' manner causing oxidative damage of the enzyme. The presence of a copper binding motif in GPO seems critical, as it makes the enzyme prone to suicidal inactivation by its own substrate, H20 2, under adverse condition like stress, which causes accumula­tion of increased intracellular H20 2 through stimula­tion of SOD33.

Ingestion of ethanol is the predisposing cause of acute hemorrhagic gastric erosions in human34. Etha­nol lowers the concentration of non-protein sulphy­dryls specially glutathione35, thereby exerting ulcero­genic effect by increasing ROS formation ll .12 . Non­steroidal antiinflammatory drugs (NSAIDs) such as aspirin, indomethacin, ibuprofen etc. , which are com­monly used as pain killer in the treatment of rheuma­toid arthritis and many other acute and chronic inflammatory conditions cause gastric mucosal dam­age36. The best studied drug, aspirin, by inhibiting prostaglandin synthesis, interferes with protective mechanism such as mucus and bicarbonate secretion, surface epithelial hydrophobicity and mucosal blood f1ow37

. These changes permit backdiffusion of acid through the breached surfaces to destroy cells, capil­laries and vein causing hemorrhagic ulcer. Enhance­ment of leukotriene synthesis by NSAIDs exhibits sufficient damaging effect. Aspirin also decreases mucosal A TP synthesis and cell turnover process36. The changes brought about by NSAIDs, as described above, in totality can induce gastric damage through the generation of ROS I3 and inhibiting cell prolifera­tionI 3.1 4.38. NSAIDs also inhibit gastric peroxidase and may increase mucosal H20 2 and ·OH level to cause oxidative mucosal damage39. Antithyroid drugs spe­cially mercaptomethylimidazole (MMI), which is used to treat hyperthyroidism, is a potent inducer of gastric ac id secretion40-42.The effect is partially medi­ated through increased liberation of histamine41 and possibly through increased level of H20 2 by irreversi­ble inactivation of gastric peroxidase43. MMI can in­duce acid secretion in vitro on isolated gastric mucosa or in gastric gland preparation where the effect can also be mimicked by the addition of micromolar con­centration of H20 2

44. Since MMI augments both acid d I . I' 404244 h' d . an umlna peps1l1 content . . , t IS rug IS poten-

tially dangerous to aggravate gastric ulcer.

Drugs currently used to treat hyperacidity and gastric ulceration-their merits and demerits

The secretion of hydrochloric acid (HCI) under normal physiological condition as and when required, does not cause any problem; but sustained hypersecre­tion is a grave condition which haums its victims day and night. In some cases, it leads to a serious inca­pacitating pathological condition, gastric ulcer when the natural cytoprotection of the gastric mucosa is lost to combat the aggressive factors including ROS among others. Various drugs are available to control gastric hyperacidity and ulcers of the stomach. Among these, Hz-receptor blockers and proton pump (H+-K+-ATPase) inhibitors are most widely used to­day though their prolonged use are not without side effects.

Cimetidine, ranitidine, famotidine and nizatidine bind with the H2 receptor of the parietal cell and block the histamine-stimulated acid secretion . These drugs, however, cannot totally block acid secretion due to other mechanisms involving cholinergic and gastrin receptors which are not directly related to Hz-receptor. Prolonged use of these drugs have been reported to affect the oxidative metabolism of different drugs45 , cause laxation, headache, dizziness, nausea, skin rashes and may exert weak anti androgenic effect, loss of libido and impotence in male patients45. Cimetidine and ranitidine have been reported to cause reversible mental confusion in sick elderly patients46 .

Omeprazole, a substituted benzimidazole, com­pletely blocks acid secretion irreversibly in animal and human by interacting with the proton-pumping H+ _K+_ATPase47-49. The other substituted benzimida­zoles have been reported to be less potent than ome­prazole5o. However, omeprazole is not devoid of side effects. Reported side effects include headache, diar­rhea and skin rash45. A report from World Health Or­ganization51 described the development of impotence and gynecomastia in some male subjects receiving omeprazole with other medications. However, long term use of omeprazole52 may cause block of acid secretion (achlorohydria) and hypergastrinemia. Mod­erate hyperplasia of gastric enterochromaffin-like cells has been observed in patients receiving omepra­zole for a long time53. In animals, omeprazole pro­duces gastric carcinoma when continued for a long time54, probably due to complete acid inhibition which stimulates gastrin production causing hyperpla­sia55 . However, no such change is observed in human cases. Omeprazole reduces the secretion, synthes is

BANDYOPADHY A Yet al.: REACTIVE OXYGEN SPECIES, GASTRIC ULCERATION & MELATONIN 697

and gene expression of pepsinogen, which may create problem in protein digestion resulting in diarrhea56

.

Omeprazole has been reported to interact with cyto­chrome P-4S0 system and may alter the metabolism of some drugs45

.

Protective role of melatonin in stress and drug­induced gastric ulceration

Despite the developments of the antisecretory­antiulcer drugs mentioned above, scientists are still in search for a drug having anti secretory , antiulcer and antioxidant properties which can be used to control the incidence of gastric ulceration in human with no or minimum toxicity. In this endeavour, an interesting observation was made suggesting that pineal hor­mone, melatonin [N-acetyl-5-methoxytryptamine] is capable of preventing stress and drug-induced gastric ulceration57

. The structure of melatonin is now known (Fig. 1). However, before describing its gas­troprotective effects it will be worth mentioning the protective effects of melatonin in various other sys­tems.

Being the principal secretory product of the pineal gland, melatonin influences circadian rythmicity by acting on the suprachiasmatic nucleus58

-60

• Addition­ally, melatonin influences various physiological ac­tivities such as neuroendocrine function58

, regulation of seasonal reproduction61

, possibly sexual matura­tion62

, immunoregulation63 and thermoregulation64

and ageing65•

Apart from the above functions, it is now widely claimed that melatonin possesses strong antioxidant activity by which it protects cells, tissues and organs from oxidative damage by reactive oxygen species, especially the ·OH which attacks DNA, proteins and lipids and causes pathogenesis 66. Melatonin can scav­enge the ·OH generated in vitro by ultraviolet irradia­tion of H20 2

67• It can quench the peroxynitrite68

, per­oxyl radical, hypochlorous acid and singlet oxygen,

Fig. I-Structure of melatonin. Melatonin (N-acetyl-5-methoxy­tryptamine) is a product of tryptophan metabolism in the pineal gland. The acetyl group on the side chain and the methoxy group at position 5 of the indole nucleus are required for maximal free radical scavenging activity of this molecule.

all of which cause cell damage69-71

. Melatonin also inhibits the production of nitric oxide (NO)72 . Mela­tonin protects against age-related oxidative damage in

66 73 'd' d f the central nervous system ' , OXI atlve amage 0

the neuroblastoma cells by amyloid ~ protein which is characteristic of Alzheimer's diseases74

, I-methyl-­phenyl-l,2,4,6-tetrahydropyridine (MPTP) and iron­induced Parkinson-like neurodegenerative changes75

,76

and free radical damage to outer hair cells in the or­gan of Corti 77 , Melatonin also prevents oxidative damage of liver caused by ischemia-reperfusion78 as well as in lung and brain damage induced by hyper­baric oxyen79

• Melatonin was shown to reduce cardiac arrythmias and this action was attributed, at least in part, to its radical scavening activitlo. Melatonin's inhibition of lipid peroxidation induced by 0-aminolevulinic acid which occurs in experimental porphyria81 as well as cataractogenesis induced by buthionine sulfoximine (an inhibitor of glutathione synthesis) in newborn rats82

, has also been docu­mented. Moreover, endogenous melatonin has also been shown to play an important protective role against carrageenan-induced local inflammation83

.

Furthermore, the protective effects of melatonin and related molecules against Cr (III)-induced carcino­genesis have been shown to be related to their direct ·OH scavenging ability which thereby reduces the formatiQIl of the damaged DNA product, 8-hydroxy-d 'd' 84 eoxy guam me .

Apart from the pineal gland, melatonin is also pre­sent in high concentrations in the gastrointestinal

. b' fl' 85-87 tract, which may e a major source 0 me atonm High levels of melatonin has been reported in bile88

.

Melatonin binding sites have also been detected and characterized in stomach, jejunum, ileum and colon89

.

Pigs with most severe ulcers exhibited significantly lower concentrations otrtrtelatonin in their stomach tissues and dietary supplementation of food with me­latonin have been reported to reduce the incidence of gastric ulcers in pigs90

. Very recently, endogenous melatonin has been shown to be physiologically in­volved in the pre- and postprandial changes of intesti­nal motility at night91

• It is of interest as to why the gastrointestinal tract and the bile contain such high concentrations of melatonin.

Interest has been focussed on the role of melatonin as an antioxidant67 and its protection of gastric mu­cosa against oxidative damage caused by ROS in dif­ferent experimental models of ulcer92

-95

. Melatonin can protect against ethanol-induced gastric damage by

698 INDIAN J EXP BIOL, JUNE 2002

preventing inflammation caused by accumulation of polymorphonuclear neutrophils, by ameliorating the decrease in GSH level and by decreasing the activity of GSH reductase92

•93 . The latter two systems are in­

timately involved in the protection of the cells against free radical-induced damage92. Melatonin also pre­vents gastric mucosal lesions caused by water­immersion restraint stress, ischemia and aspirin, and this is accompanied by an elevation in gastric level of prostaglandin93.95, an important gastroprotective fac­tor96

, and a marked fall in blood free radicals as measured by chemiluminiscence93.95 . Melatonin also prevents ischemia/reperfusion-induced gastric lesions by reducing lipid peroxidation, limiting the decrease in GSH peroxidase activity and by causing a reduc­tion in neutrophil accumulation97

• These works clearly demonstrate beyond doubt that melatonin has strong gastroprotective properties (Table 1). Detailed studies on the antiulcer effect of melatonin57 have revealed several interesting data. Table 2 shows the effect of administration of melatonin in rats on cold-restraint stress-induced gastric lesions as indicated by the ulcer index. Melatonin dose-dependently protected the mucosa from stress-induced ulceration, causing 87% inhibition of the ulcer index when given at a dose of

60 mg/kg bw (ip). The effect of melatonin was compared with known antiulcer compounds such as ranitidine and omeprazole and the potencies (ED5o, i.e. the dose required for 50% inhibition) of these compounds in preventing stress-ulcers are shown in Table 3. The results indicate that melatonin is almost three times more potent than ranitidine although it is less potent than omeprazole.

As stress ulcer is associated with oxidative membrane damage caused by ·OH-induced lipid peroxidation, the effect of melatonin on lipid peroxide level was meas-

Table 2-Effect of melatonin on restraint·cold stress·induced gastric ulceration

[Values are mean ± SE]

Treatment Number of ani mals Ulcer index

Non-stressed control Restraint-cold stress Restraint-cold stress + melatonin (20mg I kg) Restraint-cold stress + melatonin (40mg I kg) Restraint-cold stress + melatonin (60mg I kg)

P values: *<0.02; **<0.001

10 0 IS 59.3 ± 1.9

9 17.6±2.4* 14 12.9 ± 3.1**

IS 6.7 ± 1.9**

Table I-Gastric damage induced by various means and protection afforded by melatonin administered through different routes and in different dosages

Experiments Parameters measured Effect Effect after Reference carried out melatonin

treatment

Ischemia reperfusion in-Lesion area Increased Decreased 95 Basal acid secretion Decreased

duced gastric lesion Blood free radiacals Increased Decreased

Ischemia-reperfusion in- Lesion area Increased Decreased 97 duced gastric damage Gastric mucosal lipid peroxidation Increased Decreased

Neutrophil infiltrated myeloperoxidase activity in Increased Decreased gastric mucosa Glutathione peroxidase activity of gastric mucosa Decreased Increased

Ischemia-reperfusion Acid output Decreased 94 induced gastric damage Basal pepsin Decreased 93

Basal gastrin Increased Gastric epithelium Damaged Prevented

Water-restraint stress Gastric lesion Increased Decreased 94 induced damge DNA synthesis Decreased Increased 93

Gastric blood flow Reduced Increased PGE2 1eveis Decreased Increased Blood free radicals Increased Decreased

Ethanol-induced PMN infitration Increased Decreased 92 Gastric damage Glutathione level Decreased Restored

Glutathione reductase activity Decreased Restored

BANDYOPADHYAY et af.: REACTIVE OXYGEN SPECIES, GASTRIC ULCERATION & MELATONIN 699

ured. The protein carbonyl content and reduced glu­tathione level were also measured, as these parame­ters were found to change in oxidative stress . These parameters were studied in order to demonstrate the ability of this indole to prevent oxidative stress­induced changes. The results as shown in Table 4 indi­cate that stress-induced ulcers are associated with an increase in lipid peroxidation products, protein car­bonyl content and a decrease in the level of reduced glutathione. Melatonin, which caused an 87% reduc­tion in the stress-ulcer index, also brought down the lipid peroxidation, prevented oxidation of protein as well as helped keeping glutathione content of the gas­tric mucosa to normal level. All these observations naturally establish the antioxidative function of mela­tonin in preventing the stress-induced gastric ulcera­tion in rats.

As melatonin is now regarded both as a direct and an indirect antioxidant, the question that naturally arises is that whether melatonin's protective role against stress-induced gastric ulceration is a conse­quene of the indole's direct effect i.e., whether it af­fords protection by quenching the reactive oxygen species like ' OH or O2' or H20 2 or by an indirect mechanism such as altering the activities of the en­zymes responsible for the metabolism of reactive oxygen species thereby reducing the possibility of

Table 3-Potency of melatonin in comparison to ranitidine and omeprazole in protecting stress ulcer in vivo

Antiulcer compounds EDso ED8s (mg/kg)

Melatonin 15.5 60

Ranitidine 44 60

Omeprazole 0.8 8

EDso: Effective dose for 50 % inhibition of ulcer index; ED8s: Effective dose for 85 % inhibition of ulcer index.

accumulation of ROS in the face of oxidative-stress. Studies indicate that melatonin prevents stress­induced gastric ulcer in vivo by directly scavenging the ' OH (Table 5) . The imposed stress caused an al­most 6-fold increase in the ti ssue level of ' OH, while melatonin administration led to a significant reduc­tion. The result suggests that melatonin inhibits the endogenous level of 'OH during the restraint-cold stress and protects the stomach from ulceration . This observation prompted us to find out whether mela­tonin directly quenches the 'OH or it indirectly inhib­its the formation of ' OH by altering some antioxidant enzyme level. The ' OH scavenging ability of mela­tonin was studied in vitro where ' OH was generated with Cu2+/ascorbate, an established ' OH generating system5

? Table 6 indicates that melatonin can directly scavenge 'OH in a concentration dependent manner ex­hibiting 81 % scavenging activity at a concentration of 100 I..IM with an EC50 value of 40 f..lM.

Since 'OH are generated by the metal catalyzed Haber-Weiss reaction involving O2' and H20 2, the possibility exists that melatonin could reduce 'OH formation by scavenging O2', H20 2 or by chelating the redox active metal ions. Experiments have shown that

Table 5-Effect of melatonin on the scavenging of ·OH in vivo during restraint-cold stress

[Values are mean±S.E. The figures in parentheses are no. of ex­periments]

Treatment nmol ·OH generated per gram of stomach

Non-stressed control (3) 30.0 ± 10.0 Restraint- cold stress (3) 163.3 ± 13.0* Restraint- cold stress + melatonin 46.5 ± 13.3** (60mg/kg) (4)

P values: * < 0.00 1 vs non-stressed control ** <0.004 vs restraint-cold stress

Table 4-Effect of melatonin on lipid peroxidation, protein carbonyl content and reduced glutathione content

[Values are mean SE± of 10 animals in each group]

Treatment Ulcer index Lipid peroxidation Protein carbonyl GSH content content

(nmol TBARS/mg protein) (nmol / mg protein) (nmol/mg protein)

Non-stressed 0 0.43 ± 0.06 1.16±0.14 14.9 ± 0.95 control Restraint- cold stress 59.3 ± 1.9* 0.65 ± 0.02* 3.59 ± 0.25* 10.4 ± 0.58*

Restraint- Cold stress + melatonin 6.7 ± 1.9** 0.45 ± 0.01 ** 1.54 ± 0.16 ** 14.6 ± 0.78** (60mg/ kg)

P values: < 0.00 I; * vs non-stressed control ; ** vs restraint-cold stress

700 INDIAN J EXP BIOL, JUNE 2002

Table 6-Hydroxyl radical scavenging action of melatonin in vitro

[Values are mean± SE of 4 experiments]

System nmol ·OH generatediml.reaction mixture

Control 544 ± 20

"+Melatonin- 251lM 486 ± 42

501iM I 96 ± 23

100ilM 92 ± 6.4

250llM 84 ± II

melatonin neither scavenges O2' nor H20/7. Further, melatonin also does not possess any metal ion chelat­ing activiti7

. Thus melatonin protects the stomach from stress-induced ulceration by scavenging the en­dogenous ·OR Melatonin was proposed to scavenge ·OH by donating an eletron to the latter resulting in the generation of indolyl cation radical98

• This radical product was proposed to interact with O2' to produce N l-acetyl-N2 -formyl-5'-methoxykynuramine. Although scavenging of ·OH via electron donation has not been totally ruled out, recent evidence indicates the forma­tion of cyclic 3-hydroxymelatonin as a stable product which is excreted in the urine99

•IOO

• This provides di­rect evidence that melatonin under physiological con­ditions, acts as an antioxidant to detoxify the most cytotoxic endogenous ·OH. In vivo studies further indicate that in the protection against stress ulcer, me­latonin is superior to GSH and essentially equipotent to vitamin E, the established cellular antioxidants in­volved in the antioxidant defence57

. The results indi­cate that melatonin may be a more versatile radical scavenging antioxidant when compared with the clas­sical antioxidants57

.

Indomethacin (lMN), an NSAID, is known to cause gastric lesions by decreasing prostaglandin level through inhibition of prostaglandin synthesis. The experiments revealed that melatonin dose­dependently prevents IMN-induced gastric ulcera­tion57 indicating that it may be involved in maintain­ing endogenous prostaglandin level which offers gas­troprotection by inhibiting acid secretion, increasing mucus and bicarbonate secretion, elevating blood flow and by other gastroprotective mechanisms96

.

Similar effects of melatonin on IMN-induced gastric damage have also been reported 101.

IMN is an established inhibitor of the cyclo­oxygenase, a component of prostaglandin synthetase, involved in prostaglandin biosynthesis. How mela­tonin prevents IMN action is not known yet. Further studies are required to clarify this . Future perspectives

Modem state-of-the-art researches on gastropatho­physiology have documented some exciting observa­tions. The maintenance of gastric mucosal integrity depends on the interplay between epithelial cell pro­liferation and apoptosis (i.e. , programmed cell death). The BcI-2 family of proteins plays a central role in the regulation of apoptotic cell death by suppressing the apoptosis, while some others such as Bax proteins promote this process lO2 . Therefore, it is conceivable that stress-induced gastric ulcerations are accompa­nied by the fall in gastric mucosal cell proliferation. Studies by Konturek et.al. 102 have yielded information about the influence of the stress on the apoptosis in gastric mucosa. Through their nicely designed studies, they concluded that healing of gastric lesions involves an increase in gastric mucosal blood flow and muco­sal cell proliferation, and, the enhancement in gastric epithelial apoptosis accompanies the mucosal damage induced by stress and this appears to be triggered by the shift from cell death effector Bax to the cell death repressor Bcl-2 protein lO2.

Gastric mucosal apoptosis is characterized by a se­ries of distinct biochemical and morphological changes which involve activation of caspase proteases cascade that remains under the regulatory control of nitric oxide (NO). The results of Slomiany et.al. 103

implicated caspase-3 in the process of indomethacin­induced gastric epithelial cell apoptosis, and point towards participation of nitric oxide synthetase-2 (NOS-2) in the amplification of the cell death signal­ling cascade l03 . Additionally, indomethacin, sodium diclofenac, flurbiprofen, zaltoprofen, etodolac, but not mofezolac, have been found to enhance apoptotic DNA fragmentation and mRNA expression for cycloxygenase-2 in AGS cells, a cell line derived from human gastric epithelium. Further, lucigenin chemiluminescence showed that intracellular produc­tion of ROS increased in cells treated with indo­methacin. These observations thus indicate a crucial association between the generation of ROS and NSAID-induced apoptosis in gastric epithelial cells lO4 . Moreover, it has been envisaged that during ulcer healing, an array of factors compel mucosal cells to proliferate, differentiate, and migrate to the site of injury. The recognition of triggering cues requires close interaction between the regulatory proteins inte­grating the growth factor and cytokine-mediated sig­nals that propel cells through the cycle events, or to signal apoptosis. Investigations by Slomiany et. al. 105

provide strong indications that the initial phase of ul-

BANDYOPADHYAY el aJ.: REACTIVE OXYGEN SPECIES, GASTRIC ULCERATION & MELATONIN 701

cer healing involves the inhibition of apoptotic cas­pase activities by signalling events initiated by bFGF­receptor activation and propagated by the regulatory kinases that propel the cell cycle progression. Their findings strongly point towards participation of cNOS ' in the suppression of proapoptotic activities in gastric mucosalO5

.

Therefore, conceivably, when oxidative stress en­sues in the gastric mucosa, a group of cells die when their antioxidant defense system is overwhelmed by the accumulating ROS, and another group partially damaged also die, in anticipation of further onslaught, through the process of apoptosis which may be trig­gered by the ROS themselves acting as a second mes­senger. However, possibility of cells dying of necrosis always remain viable under such conditions. What­ever be the death pathway, the final outcome is the ulceration of the gastric mucosa following oxidative damage due to ROS.

The answer to the question of how melatonin af­fords gastroprotection through the scavenging of ROS seems available by this time. However, the answer to the question of how melatonin provides protection against oxidative damage due to stress through its in­direct antioxidant effects in other systems is gradually coming up although lacunae exist as regards such mechanisms by which this indole could provide gas­troprotection. Kotler et al. 106 examined the influence of melatonin on gene expression for antioxidant en­zymes in rat brain cortex. Their results clearly demon­strate that exogenously administered melatonin in­creases the levels of rnRNA for glutathione peroxi­dase, copper-zinc SOD, and Mn-SOD in this tissue. These stimulatory effects are observed after both acute and chronic treatment with this hormone, pro­ducing in the latter case a more marked increase thereby indicating that melatonin exerts an important role in providing indirect protection against free radi­cal injury by stimulating gene expression.

The role of melatonin in the regulation of apoptosis was studied by Sainz and coworkers 107. They investi­gated the role of melatonin in the cell death of thy­mus, a well known model for the study of apoptosis. Both in vivo and in vitro experiments were carried out by them. Morphometrical studies in semi thin sections of thymus and analysis of DNA fragmentation by gel electrophoresis showed that physiological apoptosis occuring in thymus of 65 days old rats, was prevented by the daily administration of melatonin beginning when the rats were 25 days old. However, they have

not ruled out the possibilities of direct interaction of melatonin with glucocorticoid receptors in the thy­mus, induction of interleukin-4 release, direct ge­nomic action modulating the expression of apoptosis inhibiting genes, an effect on nitric oxide synthase and finally the antioxidant action of melatonin. Later, these workers have, however, shown that melatonin has a major role in downregulating the glucocorticoid receptor and this downregulation is the likely media­tor of its thymocyte protection against dexamethasone­induced cell death. This effect of melatonin, given the oxidant properties of glucocorticoids, adds another mechanism to explain its antioxidant effects lO8.

The future work relating to the role of melatonin on the gastroprotection may address the following ques­tions.

Does melatonin influence the antioxidant en­zymes of the gastric mucosa directly or through enhancing their gene expression?

2 Does melatonin also exhibit the antiapoptotic ef­fect on gastric mucosal cells? It will be highly in­teresting to explore how and to what extent mela­tonin influences the balance of expression of Bcl-2 and Bax proteins of gastric mucosal cells.

3 Does melatonin enhance angiogenesis and prolif­eration of surface epithelial cells of the stomach? Because such actions will trigger healing of ulcer.

4 Does melatonin influence the expression of tran­scription factors (like NF-KB) as these under cer­tain conditions may induce mucosal apoptosis.

5 Attempts may be made to understand how and why melatonin gets concentrated in the stomach. What is the significance of gastric synthesis of melatonin because it is the stomach where the highest concentrations of melatonin are found among other portions of the gastrointestinal tract. Does this melatonin have any protective effect on the stomach against oxidative stress under physio­logical condition?

6 What are the functions of the melatonin receptors present in the stomach? Does binding of mela­tonin with its receptor in the stomach triggers any signal transduction pathway, if so, what message is transduced and what are its implications?

7 It will be interesting to investigate whether mela­tonin, after reaching the nucleus induces the ex­pression of some hither-to-unknown melatonin responsive element(s) that perhaps may bear gas­troprotective properties.

8 It will be worth investigating the influence of me-

702 INDIAN J EXP BIOL, JUNE 2002

latonin on prostaglandin biosynthesis for its gas­troprotective effect.

9 Absence of any reported toxicity of melatonin in different animal models as well as in human makes this indole more interesting and subject of intense research. Under this situation it is reason­able to argue whether melatonin can increase the efficacy of the already marketed antiulcer drugs (ranitidine, omeprazole) by reducing their dose­requirement or toxicity or bioavailability or all.

10 What will be an optimum (safe) dose that will not only protect patients from ulceration but also pre­vent damage of other organs from oxidative stress as general antioxidant?-a question easy to imagine but difficult to resolve.

The advantage of melatonin as an antioxidant lies in the fact that it is amphiphilic in nature I09-1I1 ; thus, it can reach every compartment of a cell to scavenge the ·OH generated in the face of any oxidative stress. However, its ·OH scavenging ability, even at physio­logical concentrations, and absence of any demon­strated (short- or long-term) toxicity at pharmacologi­cal concentrations 112.113 may predictably make this small indole an important therapeutic molecule in fu­ture. This contention is strengthened further by the fact that it has also been tested, in humans, most fre­quently for its ability to reduce the symptomps of jet lag l14 , adjust 24 hour rhythmicityl15 and as a sleep aid l16 . Presently, in U.S.A. and in some of the west­ern countries, melatonin is being sold as an over-the­counter drug as an antioxidant as well as a sleeping aid (personal communication with Prof. Russel J. Reiter). Protection against stress-induced ulcers in our model and the protection afforded against the ische­rnia-reperfusion, aspirin and water-restraint stress­induced gastric ulcers by melatonin 93-95, 101,117 could be instrumental in significantly reducing gastric ulcers in humans and it may have utility in other areas as well66,73 ,

Acknowledgement DB acknowledges the receipt of the Senior Re­

search Associateship of CSIR, New Delhi.

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