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Regional antioxidant status in the gastrointestinal tract and the possible role of reactive oxygen-derived… Moghadasian, Mohammed H. 1994

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Regional antioxidant status in the gastrointestinal tract and the possible role of reactive oxygen-derived substances in peptic ulcer disease by Mohammed H. Moghadasian D. V. M., Shiraz University, Shiraz, Iran, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICS FACULTY OF MEDICINE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA © Mohammed H. Moghadasian, 1994 In presentin g thi s thesi s i n partia l fulfilmen t o f th e requirement s fo r a n advance d degree a t th e Universit y o f Britis h Columbia , I  agre e tha t th e Librar y shal l mak e i t freely availabl e fo r referenc e an d study . I  furthe r agre e tha t permissio n fo r extensiv e copying o f thi s thesi s fo r scholarl y purpose s ma y b e grante d b y th e hea d o f m y department o r b y hi s o r he r representatives . I t i s understoo d tha t copyin g o r publication o f thi s thesi s fo r financia l gai n shal l no t b e allowe d withou t m y writte n permission. (Signature) Department o f f ^ ^ c l p ^ ^  V ^ / . ' c J The Universit y o f Britis h Columbi a Vancouver, Canad a Date 0^  'h  $H DE-6 (2/88 ) ii Abstract 1. Enzymati c an d non-enzymati c antioxidan t profile s i n th e variou s segments o f th e gastrointestina l trac t o f mal e an d femal e rat s hav e bee n investigated and found to exhibit significant differences (P<0.05). In both sexes, the level s o f basa l glutathion e i n th e gastri c an d coloni c mucos a ar e comparable, bu t lowe r than those i n the proxima l an d dista l segment s o f the small intestine . Th e activitie s o f glutathion e reductas e i n variou s portion s o f gastrointestinal tract were similar. Glutathione peroxidase showed higher activity in the gastric mucos a than that i n other part s o f the gastrointestina l tract . N o significant differences were found in the activity of superoxide dismutase among the various segments examined. 2. Enzymati c an d non-enzymati c antioxidan t profile s i n th e gastri c an d duodenal mucos a of rabbit , quail , cat, pig and rat have been investigate d and found to exhibit significant differences (P<0.05) . The levels of basal glutathione were highest in the rat gastric and duodenal mucosa when compared with those in other species. In the duodenal mucosa of all species investigated, the activity of glutathion e reductas e wa s highe r tha n tha t i n th e correspondin g gastri c mucosa. The activity of glutathione peroxidase was higher in the gastric mucosa than tha t i n othe r specie s examined . Superoxid e dismutas e showe d highe r activity in quail duodenal mucosa than in any of the other species studied. 3. Th e effect s o f 8 % and undilute d ethano l administere d b y gavage o n lesion formatio n an d antioxidan t component s o f th e gastri c an d duodena l iii mucosa o f mal e an d femal e rat s hav e bee n examined . Undilute d ethano l produced macroscopi c lesion s i n the bod y o f the stomach i n association wit h decreases i n th e activit y o f glutathion e reductas e an d i n th e leve l o f basa l glutathione an d a n increas e i n th e activit y o f glutathion e peroxidase . Eigh t percent ethanol produced a small but significant increas e (12%) in the leve l of basal glutathione in rat gastric mucosa whe n compared with controls. 4. Th e effect s o f chroni c intermitten t stres s o n th e appearanc e an d antioxidant components of the gastric and duodenal mucosa of rats have been examined. No differences in the antioxidant profiles or evidence of macroscopic lesion formation were found. TABLE OF CONTENTS CHAPTER PAGE Abstract i i Table of Contents i v List of Tables vi i List of Figures vii i List of Abbreviations xi i Acknowledgements x v Dedication xv i 1. INTRODUCTION 1 1.1. Reactive oxygen-derived sustances  in  pathological and physiological conditions  1 1.2. Pathophysiology of  peptic ulcer  disease 3 1.2.1. Species 6 1.2.2. Regional tissue differences 7 1.2.3. Sex 7 1.2.4. Genetics 7 1.2.5. Age 8 1.3. Factors involved in  the pathogenesis of  peptic ulcer  disease 8 1.3.1. Gastric acid secretion 1.3.2. Pepsin activity 9 1.3.3. Helicobacter pylori 9 1.3.4. Reactive oxygen-derived substances 1 0 1.4. The role of oxidative stress in gastrointestinal disorders 1 7 1.5. Hypothesis 2 2 1.6. Rationale and Objectives 2 2 1.6.1. Experiment I 2 3 1.6.2. Experiment II 2 3 1.6.3. Experiment II I 2 3 1.6.4. Experiment IV 2 3 2. EXPERIMENT I 2 4 2.1. Materials and Methods 2 4 2.2. Results 2 6 3. EXPERIMENT II 5 2 3.1. Materials and Methods 5 2 3.2. Results 4. EXPERIMENT III 4.1. Materials and Methods 4.2. Results 5. EXPERIMENT IV 5.1. Materials and Methods 5.2. Results 6. GENERAL DISCUSSIO N 7. SUMMARY AND CONCLUSIONS 8. REFERENCES VI 62 62 63 99 99 100 109 119 121 VII LIST OF TABLES Table Pag e Table 1 Lesio n development and antioxidant changes i n the 8 0 gastric and duodenal mucosal components of control and ethanol treated rats. viii LIST OF FIGURES Figure Pag e Figure 1 Th e levels of basal glutathione in various 2 9 portions of the gastrointestinal mucosa of male rats. Figure 2 Th e activity of glutathione reductase in various 3 1 portions of the gastrointestinal mucosa of male rats. Figure 3 Th e activity of glutathione peroxidase in various 3 3 portions of the gastrointestinal mucosa of male rats. Figure 4 Th e activity of superoxide dismutase in various 3 5 portions of the gastrointestinal mucosa of male rats. Figure 5 Th e levels of basal glutathione in various 3 7 portions of the gastrointestinal mucosa of female rats. Figure 6 Th e activity of glutathione reductase in various 3 9 portions of the gastrointestinal mucosa of female rats. Figure 7 Th e activity of glutathione peroxidase in various 4 1 portions of the gastrointestinal mucosa of female rats. Figure 8 Th e activity of superoxide dismutase in various 4 3 portions of the gastrointestinal mucosa of female rats. Figure 9 Compariso n of basal glutathione levels in 4 5 various regions of the gastrointestinal tract of male and female rats. Figure 10 Compariso n of the activity of glutathione 4 7 reductase in various regions of the gastrointestinal tract of male and female rats. Figure 11 Compariso n of the activity of glutathione 4 9 peroxidase in various regions of the gastrointestinal tract of male and female rats. IX Figure Pag e Figure 12 Compariso n of the activity of superoxide 5 1 dismutase in various regions of the gastrointestinal tract of male and female rats. Figure 13 Basa l glutathione levels in the gastric and 5 5 duodenal mucosa of various species. Figure 14 Activit y of glutathione reductase in the gastric 5 7 and duodenal mucosa of various species. Figure 15 Activit y of glutathione peroxidase in the gastric 5 9 and duodenal mucosa of various species. Figure 16 Activit y of superoxide dismutase in the gastric 6 1 and duodenal mucosa of various species. Figure 17 Basa l glutathione levels in the gastric mucosa of 6 5 male and female control and ethanol treated rats. Figure 18 Th e activity of glutathione reductase in the 6 7 gastric mucosa of male and female control and ethanol treated rats. Figure 19 Th e activity of glutathione peroxidase in the 6 9 gastric mucosa of male and female control and ethanol treated rats. Figure 20 Th e activity of superoxide dismutase in the 7 1 gastric mucosa of male and female control and ethanol treated rats. Figure 21 Basa l glutathione levels in the duodenal mucosa 7 3 of male and female control and ethanol treated rats. Figure 22 Th e activity of glutathione reductase in the 7 5 duodenal mucosa of male and female contro l and ethanol treated rats. Figure 23 Th e activity of glutathione peroxidase in the 77 gastric mucosa of male and female control and ethanol treated rats. X Figure Pag e Figure 24 Th e activity of superoxide dismutase in the 7 9 gastric mucosa of male and female control and ethanol treated rats. Figure 25 Gros s appearance of the stomach and 8 2 duodenum in the abdomen of a male rat treated with 8% ethanol. Figure 26 Distende d stomach and duodenum with 8 4 increased luminal fluid volume in the abdomen of a male rat treated with undiluted ethanol. Figure 27 Compariso n of appearance of gastric and 8 6 duodenal mucosa of male rats treated with water, ethanol (8%) and ethanol (undiluted) . Figure 28 Histolog y of gastric mucosa of a male rat 8 8 (longitudinal section, gastric glands and pits). Figure 29 Appearanc e of gastric mucosa of a male rat 9 0 (transverse section). Figure 30 Superficia l necrosis of gastric epithelium of a 9 2 male rat treated with undiluted ethanol. Figure 31 Gastri c epithelial necrosis surrounded by 9 4 normal looking mucosa in a female rat treated with undiluted ethanol. Figure 32 Dee p gastric epithelial necrosis in a female rat 9 6 treated with undiluted ethanol. Figure 33 Sever e gastric mucosal necrosis following 9 8 administration of undiluted ethanol to a male rat. Figure 34 Level s of basal glutathione in the gastric and 10 2 duodenal mucosa of control and stressed male rats. XI Figure Pag e Figure 35 Activit y of glutathione reductase in the gastric 10 4 and duodenal mucosa of control and stressed male rats. Figure 36 Activit y of glutathione peroxidase in the gastric 10 6 and duodenal mucosa of control and stressed male rats. Figure 37 Activit y of superoxide dismutase in the gastric 10 8 and duodenal mucosa of control and stressed male rats. xii LIST OF ABBREVIATIONS ANOVA Analysi s of variance A. M. ant e meridiem 5-ASA 5-aminosalicyli c acid a Alf a cm CAT Cu DDC DEM DMSO dsi DNA DTNB EDTA e g -Fe GPX GRD GSH H202 H+ HCI centimeter catalase copper diethyldithiocarbamate diethylmaleate dimethyl sulfoxide distal part of small intestine deoxyribonucleic acid 5,5'-dithiobis-2-thiobenzoicacid ethylendiaminetetraacetic aci d for example iron glutathione peroxidase glutathione reductas e glutathione hydrogen peroxide hydrogen ion hydrochloric acid XIII Abbreviations (cont'd ) hr i.e. i. p. kg V mg min ml mmHg mM MTDQ-DA n N NEM nm NSAID o2 02" OH PH P.M. psi RODS hour that is intrapertonially kilogram micro milligram minute milliliter millimeter of mercury milliMolar kontrad nano normal N-ethylmaleimide nanometer non-steroidal anti-inflammatory dru g oxygen superoxide radical hydroxyl radica l hydrogen ion concentration post meridiem proximal part of small intestin e reactive oxygen-derived substance s xiv Abbreviations (cont'd) SAM S-adenosylmethionin e s. c. subcutaneousl y SE standar d error SH sulfhydry l group SOD superoxid e dismutase TCA trichloroaceti c acid UBC Th e University of British Columbia VGH Vancouve r Genera l Hospita l w/v weigh t per volume Zn zin c < les s than > greate r than °C degree s Celsius ± plu s or minus % percen t XV ACKNOWLEDGEMENTS I would lik e to express m y most sincere thank s t o Dr . Davi d V. Godi n for giving me the opportunit y t o stud y unde r hi s supervisio n i n hi s laboratory , an d also for his patience in teaching me his extensive knowledge . O f course, without his continuin g advic e an d guidanc e completio n o f thi s degre e woul d no t hav e been possible. I am also grateful to Ms. Maureen E . Garnet t for he r technical assistanc e and for allowing me to tap her many years of laboratory experience. Philip' s help and assistance were also greatly appreciated. Thanks mus t als o b e extende d t o m y committe e members , (Drs . H . J . Freeman, R . A . Wal l an d T  Madden ) an d al l facult y members , staf f an d graduate students who were always helpful and nice to me. I am indebted to the Ministr y o f Healt h an d Medica l Educatio n o f Ira n for the financial support provided during the course of my graduate studies. XVI DEDICATION This thesis is dedicated to my wife and my son. 1 1. INTRODUCTIO N 1.1. Reactive  oxygen-derived  sustances  in  pathological  and physiological conditions It i s well establishe d tha t molecula r oxyge n a t increase d partia l pressur e can b e harmfu l an d partiall y reduce d oxyge n specie s ar e th e probabl e agent s responsible fo r thi s toxicit y (1) . Reactiv e oxygen-derive d substances  (RODS ) have bee n implicate d i n a  variet y o f pathologica l conditions , mos t notabl y ischemia/reperfusion injur y (2-4) , radiation therapy (5 ) and i n the toxic effects o f certain drug s an d chemicals , includin g anthracycline s (6) , mitomyci n an d bleomycin (7). Under i n viv o conditions , oxyge n usuall y undergoe s a  step-wise , four -electron reductio n to water. The three mos t importan t partiall y reduce d forms o f molecular oxyge n ar e superoxid e radica l (0 2"), hydroge n peroxid e (H 202), an d hydroxyl radica l (OH ) (8) . These reactiv e metabolite s ca n b e produce d b y th e activity o f oxidative enzyme s i n various intracellula r compartments , suc h a s the cytosol, mitochondria , lysosomes , peroxisome s an d plasm a membranes . Superoxide radical s ca n aris e durin g th e cours e o f mitochondria l electro n transport o r a s a  resul t o f the actio n o f suc h intracellula r enzyme s a s xanthin e oxidase or cytochrome P 45o-dependent oxidases. Once produced , superoxid e radical s ca n underg o dismutatio n b y th e action of superoxide dismutase (SOD) to produce hydrogen peroxide (9). 2 202~ +  2H + >  H202 +  0 2 Hydrogen peroxide, as produced either by the above reaction or directly by oxidases present in peroxisomes, can undergo either the Fenton reaction Fe~ +  H 202 •  Fe*** +  O H +  OH" or the Haber-Weiss reaction. H202 +  0 2 ' >  OH +  O H +  0 2 to produce hydroxyl radicals. The main effects of these reactive species are on unsaturated linkages of membrane lipids (10), sulfhydryl groups of proteins (11) and nucleotides of DNA (12). The peroxidation of lipids i n biological membrane s can cause damage to organelles suc h a s mitochondria , endoplasmi c reticulum , lysosome s an d microsomal components . Interaction s wit h DN A ma y resul t i n mutagenesi s o r carcinogenesis (13) . Lipi d peroxidatio n i s on e well-studie d mechanis m o f reactive oxygen metabolite injury (14-16). I t can be initiated by hydroxyl radicals which reac t wit h unsaturate d fatt y acid s withi n th e membrane s t o generat e organic free radicals, which, in turn, react rapidly with oxygen to form peroxides. Peroxides themselve s the n generat e fre e radicals , initiatin g a n autocatalyti c chain reaction , resultin g i n furthe r los s o f unsaturate d fatt y acid s an d i n extensive membrane damage (17). Once th e reactiv e metabolite s ar e formed , the y ma y spontaneousl y decay. For example, superoxide radicals can decay spontaneously int o oxygen and hydrogen peroxide. There are, however, severa l systems that are involved in th e direc t inactivatio n o f thes e reactiv e substances . Enzymati c an d non -enzymatic antioxidan t system s suc h a s vitami n E , glutathione , ceruloplasmin , glutathione peroxidase (GPX), glutathione reductase (GRD), catalase (CAT) and superoxide dismutas e (SOD ) ca n scaveng e an d detoxif y reactiv e oxyge n substances (18). 02- S0 D >  H 202 2H202 CAr  >  02 +  2H 20 20H +  2GS H GPX  >  2H 20 +  GSS G H202 +  2GS H GPX  >  2H 20 +  GSS G GSSG GRD  >  GS H In many pathologic processes, the final effects of stimulus-inducing free radicals depend on the net balance between free radical formation and inactivation (19). 1.2. Pathophysiology  of peptic ulcer disease Peptic ulce r diseas e ca n b e generall y define d a s a  disorde r o f th e gastrointestinal trac t i n whic h a n unfavorabl e imbalanc e betwee n damagin g agents and defense mechanisms is present. This imbalance eventually results in injury to the mucosal tissue and development of peptic ulceration. Not all peptic ulcers share the same pathophysiology. Fo r example, acute and chronic ulcers can arise under different clinical circumstances, follow a different clinica l course and respond differently to treatment. However, all peptic ulcers share a common feature, i.e., their occurrence in mucosa exposed to acid and pepsin. 4 Recent studie s hav e demonstrate d tha t th e notio n "n o acid-n o ulcer " i s not alway s applicabl e t o gastri c ulcers . Chroni c gastri c ulcer s ar e usuall y associated with low acid secretion and the role of acid in the pathophysiology o f acute gastri c ulcers i s unresolved . Patient s with duodena l ulcer s almost alway s show a  marke d increas e i n both.aci d secretio n an d pepsi n activit y compare d with control subjects (20) . One might suggest that the enterogastric reflexes may be impaired due to duodenal ulceration. The mai n defens e mechanis m i s th e so-calle d "mucus-bicarbonate " barrier, acros s whic h bicarbonat e diffuse s toward s th e lume n an d neutralize s acid that i s slowly diffusing towards the epithelium. Several  factors ar e involve d in this protectiv e mechanism . Among them, one can mentio n the existence o f a viscoid mucus gel a t the surface o f epithelia l cells , the secretion o f bicarbonat e and additiona l buffe r (so-calle d "alkalin e tide" ) an d th e abilit y o f th e gastri c mucosa t o increas e mucosa l bloo d flo w t o buffe r and/o r t o remov e excessiv e intramucosal acid . Several condition s ma y predispos e t o th e developmen t o f acut e gastri c mucosal lesions . These includ e severe illnesses , burns , centra l nervous syste m trauma, acut e and severe stressfu l condition s an d ingestio n o f certai n drug s o r alcohol. Injur y t o th e centra l nervou s syste m usuall y cause s acut e gastri c mucosal lesion s referre d t o a s "Cushing' s ulcers" . Endoscopi c studie s hav e demonstrated that the incidence of acute gastric mucosal lesions in patients with head injurie s was  50-75% , whil e tha t i n patient s wit h spina l cor d injur y i t was 5 reported to b e about 3 % .  I n such patient s with damage t o the centra l nervou s system, a n increas e i n gastri c aci d secretio n an d hig h level s o f seru m gastri n were reported . Patients with burn s to 35% or mor e o f the bod y surfac e hav e a tendency t o develo p acut e gastri c mucosa l lesions , thes e bein g referre d t o a s "Curling's ulcers" (21). Selye (22 ) ha s defined stress a s a "nonspecific respons e o f the bod y t o any demand  upo n it. " Thi s conditio n i s considere d t o involv e a  comple x interaction involvin g the centra l nervou s system , the endocrine syste m an d th e immune system o f the body . Stres s ma y affec t th e limbi c system , mos t notabl y the hypothalamus, which is the regulatory center for both the autonomic nervou s system and the neuroendocrine system. It ha s bee n postulate d tha t ther e ar e three pathway s involve d i n stress -induced ulceration : th e anteriohypothalamu s throug h th e vagu s nerve , th e posteriohypothalamus vi a th e sympatheti c axi s an d the posterio r hypothalamo -pituitary-adrenal system . Stres s ma y cause som e alteration s i n gastri c motility , gastric secretio n an d gastri c mucosa l bloo d flow . Thes e kind s o f alteration s could result i n erosions or ulcers, so that the more severe the stress , the highe r the incidence of lesions. Certain kind s o f drugs , suc h a s non-steroida l anti-inflammator y drugs , usually afte r long-term/hig h dos e therapy , a s i n th e treatmen t o f rheumatoi d arthritis, can cause acute gastric mucosa l lesions . I t has bee n reporte d that the gastric toxicity o f the following non-steroida l anti-inflammatory drug s is : aspirin > 6 indomethacin >  phenylbutazon e >  ibuprofe n (23) . Thes e drug s d o inhibi t cyclooxygenase, a  ke y enzym e i n th e biosynthesi s o f prostaglandins ; prostaglandin E 2 plays an importan t rol e in gastric mucosa l protection agains t a variety o f damaging agents . Thi s prostaglandi n ca n protec t th e gastri c mucos a by a  numbe r o f mechanisms , suc h a s stimulatio n o f mucu s an d bicarbonat e secretion and/or increase in mucosal blood flow. Other possible mechanisms for ulcer formatio n b y thi s categor y o f drug s coul d involv e associate d change s i n gastric motility and/or i n gastric mucosal blood flow, as Takeuchi et al. (24) hav e suggested for indomethacin-induced gastric lesions. Alcohol i s another damagin g agent . Administration o f lo w concentration s (8-10%) o f alcoho l ca n preven t gastri c lesion s b y processe s referre d t o a s "adaptive cytoprotection" . However , administratio n o f hig h concentration s o f alcohol are definitely ulcerogenic. This damaging behavior is probably due to the ability of alcohol to alter the integrity of the mucus barrier . A variet y o f factor s ma y significantl y affec t th e incidence , severit y an d localization o f gastrointestina l mucosa l lesions . Amon g thes e factor s on e ca n consider: species, regional tissue differences, sex , genetics and age. 1.2.1. Species Butterfield (25 ) reported differences i n incidence rates of lesion formation in si x differen t specie s o f experimenta l animals . Th e incidenc e o f gastri c ulceration induce d b y restrain t fo r 2 4 hour s i n mice , rats , guine a pig s an d 7 hamsters wa s reported t o b e 92%, 86%, 46% and 4%, respectively. Gastri c lesions could not be induced in monkeys or rabbits under these conditions. 1.2.2. Regional  tissue differences Gastric and intestinal mucos a may differ i n their susceptibilitie s t o lesio n formation. I n experimenta l models , duodenu m i s mor e resistan t t o stres s ulceration, so that cold-restrain t stres s did not produce duodena l ulcer s i n rat s (23). Also , Kapla n (26 ) has reported tha t ther e i s no direct evidenc e tha t non -steroidal anti-inflammator y drug s o r alcoho l ca n produc e duodena l ulcer s i n humans. 12.3. Sex In humans, duodenal ulcers have a male:female ratio of 4:1 while the ratio for gastri c ulcer s i s 3 or 4:1 (27) . Another sourc e (28 ) claims tha t one in every ten men (10%) and one in every 25 women (4%) may have a peptic ulcer durin g the course of his or her life. Pregnant women and nursing mothers are somehow resistant to ulcer formation. Peptic ulcer is rare in females prior to menopause. 1.2.4. Genetics Peptic ulcer s ar e 2 to 3 times mor e commo n i n first degre e relative s of patients wit h ulcers . Twent y t o fift y percen t o f patient s wit h a  duodena l ulce r have a positive family history of duodenal ulcers . Individual s with blood group 0 have about a 30% increase in risk of duodenal ulcer s compared with persons of other bloo d type s (29) . Duodenal ulceratio n i s mor e commo n i n monozygoti c 8 than dizygotic twins. This suggests a role of genetics i n influencing susceptibilit y to ulcer formation. 12.5. Age Age i s anothe r importan t facto r determinin g susceptibilit y t o lesio n formation. Mos t gastrointestina l lesion s appea r fo r th e firs t tim e i n th e thir d t o fourth decade o f lif e i n man (27) . I n children, gastri c ulcer s ar e extremel y rare , except i n th e cas e o f seriou s brai n injury . Th e estimate d annua l incidenc e o f gastric lesion s i s reporte d t o b e 0.002-0.005 % i n childre n unde r ag e 15 , a s compared with 0.2% in adults (28). 1.3. Factors  involved in the pathogenesis of  peptic ulcer  disease 1.3.1. Gastric acid secretion Gastric aci d i s necessar y fo r ulce r formation . Ther e i s a n increas e i n gastric aci d secretio n i n patient s wit h duodena l ulcer , bu t i t i s norma l o r decreased in patients with gastric ulcer. Patient s admitted into a critical care unit are a t greatl y increase d ris k o f gastri c ulceration . On e stud y o f gastri c aci d secretion i n such individual s ha s show n i t to b e norma l o r belo w normal , eve n though the initiation of gastric mucosal injuries might be apparent endoscopicall y (28). Therefore , on e can conclud e tha t fo r productio n o f acut e gastri c mucosa l lesions a n increas e i n gastri c aci d secretio n doe s no t pla y a  role , althoug h hydrochloric acid is necessary for induction of these lesions. 9 1.3.2. Pepsin  activity Pepsinogen i s a polypeptide secrete d by chie f cell s and has a molecula r weight o f 42,500. The mai n stimulu s fo r pepsinoge n secretio n i s acetylcholine . Hydrochloric aci d ma y indirectl y stimulat e pepsinoge n secretio n a s well . I n th e presence of hydrochloric acid , pepsinogen i s converted int o pepsin, which has a molecular weight of 35,000 and has proteolytic activity. I n patients with duodenal ulcer, th e mea n activit y o f pepsi n i n endoscopicall y obtaine d biops y sample s from gastri c an d duodena l mucos a wa s foun d t o b e increase d (29) . Also , i t i s reported that al l patients with endoscopically prove n active duodenal ulcer s ha d a highe r pepsi n outpu t tha n contro l subject s (30) . Several  differen t type s o f pepsinogens (e.g. , pepsinogen I  and pepsinogen II ) are secreted by peptic an d mucous cell s o f the gastric glands. Kolste r e t al . (31) reporte d highl y significan t differences betwee n serum pepsinogen I  levels i n patients with duodena l ulcer s as compare d wit h thos e i n contro l subjects . I t i s reporte d tha t hyperpepsinogenemia I  i s a  reliabl e subclinica l marke r o f th e geneti c predisposition t o duodena l ulceratio n (32) . Thes e finding s sugges t tha t pepsi n has a significant role in the pathophysiology o f peptic ulcer disease. 1.3.3. Helicobacter  pylori Helicobacter pylori  i s a  bacteriu m foun d i n associatio n wit h gastri c mucosa. H.  pylori infectio n ha s bee n found i n the antru m o f approximatel y two -thirds of patients with gastric and/or duodenal ulcers . I t has been suggested that this bacteriu m ha s a  rol e i n pepti c ulce r diseas e b y causin g mucu s depletio n 10 (33). Th e organis m i s usuall y locate d i n th e superficia l mucu s layer , wher e i t tends t o b e attache d to the epithelium a t the sit e o f intercellula r junctions (34) . This locatio n provide s protectio n fro m aci d gastri c contents , bu t permit s hydrolytic enzymes produced by the organism to damage gastric epithelium. I t is suggested tha t th e organis m damage s th e mucosa , initiatin g a  progressiv e disease and in many cases culminating in atrophy and intestinal metaplasia (35) . It i s generall y accepte d tha t infectio n wit h thi s bacteriu m i s closel y associate d with pepti c ulceratio n (34) . Th e rol e o f thi s organis m i n th e pathogenesi s o f gastric ulceration, however, i s still controversial. 1.3.4. Reactive  oxygen-derived substances Production o f reactiv e oxygen-derive d substance s (RODS ) seem s t o b e an importan t facto r i n th e pathophysiolog y o f pepti c ulce r disease . Thes e reactive substance s ca n alte r cellula r functio n b y causin g oxidativ e damag e t o proteins, nuclei c acid s an d lipids , th e latte r resultin g i n loss  o f cel l membran e integrity. Gastri c mucosal cells, which are normally relativel y impermeabl e to H+ and Na + despit e th e presenc e o f hig h concentratio n gradients , coul d thereb y become permeabl e t o thes e ions . Ther e i s considerabl e evidenc e tha t ROD S can play a role in the induction of gastrointestinal mucosa l lesions , as discussed below. Niid a e t al . (36 ) wer e abl e t o induc e duodena l ulcer s i n rat s b y th e administration o f diethyldithiocarbamat e (DDC) , a  meta l chelato r an d SO D inhibitor, a t a  dose o f 75 0 mg/k g s.c . give n dail y fo r 4  days . The DDC-induce d lesions wer e prevente d b y eithe r allopurinol , a  xanthin e oxidas e inhibito r an d 11 hydroxyl radica l scavenger , o r superoxid e dismutas e (SOD) , a n endogenou s superoxide radica l scavenger, a t doses of 50 mg/kg s.c. and 50,000 unit/kg s.c , respectively. Since the administration of DDC was shown to cause a decrease in SOD activity in the duodenal mucosa, i t can be postulated that RODS can play a role in DDC-induced duodenal mucosal lesions i n rats. Oka et al. (37) were able to induc e antra l ulcer s i n anesthetize d pylorus-ligate d rat s b y injectio n o f DD C (800 mg/k g s.c) , followe d b y ora l administratio n o f on e millilite r o f 0. 1 N  HCI. Administration o f Cu,Zn-SOD (60,00 0 unit/kg s.c. ) significantl y reduce d the HCI-DDC- induced ulcer index. Takeuchi e t al . (24 ) demonstrate d a n increas e i n bot h amplitud e an d frequency o f gastri c contraction s 3 0 minute s afte r subcutaneou s administratio n of indomethaci n a t a  dos e o f 2 5 mg/k g i n rats . Th e author s demonstrate d a relationship o f indomethacin-induce d gastri c hypermotilit y t o mucosa l hemodynamics, lipi d peroxidatio n an d vascula r permeabilit y change s i n th e pathophysiology o f indomethacin-induce d gastri c lesions . Pretreatmen t o f th e animals wit h allopurino l (5 0 mg/kg) , SO D (15,00 0 units/kg/hr) , o r dimethy l sulfoxide (DMSO ; 3 0 mg/kg) , whic h react s wit h hydroxy l radical s t o produc e methane sulfini c acid , cause d a n attenuatio n o f th e indomethacin-induce d gastric mucosa l lesions . I t can, therefore, b e concluded tha t ROD S are likel y t o play a  rol e i n th e developmen t o f indomethacin-induce d gastri c mucosa l ulceration. 12 Salim e t al . (38 ) induce d gastri c mucosa l injur y i n rat s b y injectio n o f either reserpin e ( 5 mg/k g i.p. ) o r serotoni n (5 0 mg/k g i.p.) . Parentera l administration o f hig h dose s o f reserpin e cause s gastri c mucosa l vasoconstriction by vagal sympathetic fibers, leading to ischemic mucosal injury. Serotonin als o produce s gastri c mucosa l injur y b y vasoconstriction . Pretreatment with either 1 ml/day of 2% allopurinol or DMSO by gastric gavage for 2 days significantly reduced reserpine- or serotonin-induced gastric lesions in rats. Sinc e bot h allopurino l an d DMS O shar e th e propertie s o f scavengin g RODS an d eac h o f the m coul d individuall y preven t gastri c mucosa l lesio n formation, one can conclude that ROD S have a role i n the pathophysiology o f reserpine- or serotonin-induced gastric mucosal lesions. Zollei e t al . (39 ) instille d 1  m l o f 0. 1 N  HC I int o th e stomac h o f anesthetized rats and animals were bled t o reduce blood pressure to 30 mmHg for 20 minutes. The shed blood was re-transfuse d afte r 20 minute s and , after another 20 minutes, ra t stomachs wer e removed and gastric mucosa l lesion s were measured. In the same experiment, pretreatment with either allopurinol (50 mg/kg) or kontrad (MTDQ-DA, 6,6-methylene bis 2,2 dimethyl-4-methane sulfinic acid; 5 0 mg/k g i.p.) , a  syntheti c antioxidan t o f th e dihydroquinolin e type , protected th e animal s agains t hemorrhagi c shock-induce d gastri c mucosa l lesions. These results again suggest that ROD S may play an important rol e in the pathophysiology of gastric mucosal lesions produced under these conditions. 13 Granger e t al. (40) reported the protective effect s o f the xanthine oxidas e inhibitor, allopurinol , o n ilea l vascula r permeabilit y durin g ischemia/reperfusio n in ilea l segmen t o f cats . Th e author s conclude d tha t xanthin e oxidas e i s a source of RODS due to ischemia/reperfusion i n the small intestine in cats. Kvietys e t al . (41 ) investigate d th e effect s o f 5-aminosalicyli c aci d (5 -ASA), a  hydroxy l radica l scavenger , o n ischemia/reperfusion-induce d gastri c bleeding i n rats . Ra t stomach s were perfuse d wit h 3 0 m M 5-ASA i n 0. 1 N  HC I and systemic  blood pressure was then reduced to 25-30 mmHg by withdrawing blood from the femoral artery. After 30 minutes, the shed blood was reinfused via the femora l vein . Gastri c lesion s i n animal s receivin g 5-AS A wer e significantl y less sever e tha n thos e i n animal s tha t ha d no t receive d 5-ASA . Therefore , hydroxyl radical s appea r t o pla y a  rol e i n th e productio n o f gastri c lesion s induced by ischemia/reperfusion i n rat stomach. Boyd et al. (42) were able to induce acute gastric mucosa l lesion s i n rats by subcutanou s administratio n o f diethylmaleat e (DEM) , a  tissu e glutathion e depletor. O n thi s basis , th e author s conclude d tha t glutathion e ma y hav e a possible rol e i n maintaining the norma l homeostasi s an d integrit y o f the gastri c mucosa. Laudanno (43 ) investigated the effects o f S-adenosylmethionine (SAM ) on ethanol - aspirin - o r stress-induce d gastri c lesions . SA M significantl y increased non-protein sulfhydryl groups in gastric mucosa. SAM at a dose of 100 mg/kg s.c . protecte d gastri c mucos a agains t th e damagin g effect s o f aspirin , ethanol o r stress . Therefore , i t ca n b e conclude d tha t th e abov e mentione d 14 irritants ma y caus e gastri c lesion s b y depletin g glutathion e level s i n gastri c mucosa of rat stomach. Despite the abundance o f evidence concerning the effects of antioxidants in the prevention of peptic ulcers , there are a few studies suggesting there i s no relationship betwee n gastri c glutathion e level s an d ulce r formation . Thus , Ushima et al. (44) investigated the role of gastric mucosal sulfhydry l groups (SH) in th e pathophysiolog y o f indomethacin-induce d lesion s i n rats . The y foun d these lesion s coul d b e prevente d b y prio r s.c . administratio n o f cysteamine , glutathione o r diethylmaleate , irrespectiv e o f th e fac t tha t mucosa l S H level s were increase d b y th e forme r tw o agent s bu t reduce d b y th e latte r agent . Therefore, th e author s conclude d tha t gastri c mucosa l S H level s hav e n o relation t o th e ulcerogeni c effect s o f indomethaci n i n gastri c mucos a i n rats . Takeuchi e t al . (45 ) investigate d th e rol e o f gastri c mucosa l glutathion e o n lesions induce d b y 1  ml o f 50 % ethanol i n rats . Inductio n o f these lesion s wa s inhibited b y pretreatment  o f animal s wit h eithe r diethylmaleat e (  DEM ; 1  ml/kg, s.c.) o r cysteamine (10 0 mg/kg , s.c). Sinc e DE M decreased gastri c glutathion e levels an d cysteamin e increase d them , th e author s conclude d tha t gastri c mucosal glutathion e level s d o no t relat e t o ethanol-induce d gastri c lesion s i n rats. Tariq (46 ) studie d th e effect s o f th e endogenou s lipophili c chai n terminating antioxidant , vitami n E , on gastri c mucosa l damag e induce d b y cold-restraint stress , indomethacin , reserpine , 0.6 M HCI , hypertonic sodium  chlorid e 15 or ethanol i n rats. He reported that pretreatmen t o f 36-hr fasted animals with an oral dos e o f 10 0 mg/k g o f vitami n E  3 0 minute s befor e subjectin g animal s t o cold-restraint stress significantly reduced  the production of gastric lesions by the above mentione d agents . Th e result s sugges t a  possibl e rol e o f lipi d peroxidation i n the pathophysiology o f acute gastric mucosal lesion s induce d by the above mentioned agents. Tatsumi e t al . (47 ) reporte d tha t th e simultaneou s additio n o f bot h xanthine and xanthine oxidase to a homogenate of rat gastric mucosa cause d a significant reductio n i n the activit y o f glucosamin e synthetase . Thi s effec t wa s counteracted b y catalas e (a n endogenou s enzymati c antioxidant ) bu t no t b y SOD. Hydroge n peroxid e could also decreas e th e activit y o f this enzyme . Thi s effect of hydrogen peroxide was counteracted by either catalase or dithiothreitol. Since glucosamine synthetase i s important i n protecting the gastric mucosa, one might conclud e tha t ROD S ma y hav e a  rol e i n th e destructio n o f th e mucu s barrier resultin g in lesion formation. Schoenberg (48) induced ischemia of the small intestine i n cats by partia l occlusion o f th e superio r mesenteri c artery . Specimen s obtaine d durin g an d after th e ischemi a showe d th e presenc e o f mucosa l lesions . Administratio n o f SOD during the ischemia could prevent mucosa l damage. Therefore, i t could be concluded tha t generatio n o f ROD S du e t o loca l ischemi a migh t pla y a  rol e i n ischemia-induced lesions in the small intestine in cats. 16 Grisham e t al . (49 ) demonstrate d tha t ischemia/reperfusio n i n th e smal l intestine of cats could resul t i n a marked increase i n neutrophil infiltratio n and a concurrent decreas e i n reduce d glutathion e level s an d SO D activity . Pretreatment with allopurinol (50 mg/kg p.o. for 2 days) or administration of SOD (injected i n the superior mesentri c artery prio r to ischemia ) prevente d the influ x of neutrophils and the decreases i n reduced glutathione concentration and SOD activity. Sinc e inflammator y neutrophil s ar e potentia l source s o f reactiv e oxidants, such as superoxide an d hypochlorous acid , thes e data agai n sugges t that ROD S ma y hav e a  rol e i n ischemia/reperfusion-induce d injur y i n the smal l intestine in cats. Victor et al. (50) reported that topical administration of either a mild irritan t (8% ethanol ) o r prostaglandi n E 2 ( 1 M - g/ml) coul d increas e gastri c mucosa l glutathione level s b y 20 % compare d wit h contro l value s i n dogs . Bot h agent s individually coul d protec t gastri c mucos a agains t th e effect s o f 40 % ethanol , while administratio n o f N-ethylmaleimid e (NEM ; 5 0 mg/kg) , a  glutathion e depleting agent , prevente d th e protectiv e effect s o f bot h 8 % ethano l an d prostaglandin E2 . Therefore, one can conclude that gastri c mucosa l glutathion e levels may play a role in the process referred to as "adaptive cytoprotection". Pasechnikov e t al . (51 ) studie d th e rol e o f lipi d peroxidatio n an d th e activities o f SOD, glutathione peroxidase , glutathione reductas e and glutathion e levels i n gastri c mucos a obtaine d endoscopicall y fro m 6 0 patient s wit h pepti c ulcer disease . Th e author s conclude d tha t lipi d peroxidatio n an d a  dro p i n the 17 activities o f th e abov e mentione d antioxidant s ma y b e importan t pathogeni c factors leading to a chronic and recurrent course of peptic ulcers. 1.4. The  role of oxidative stress in gastrointestinal disorders Like other organs, the gastrointestinal trac t may be exposed to condition s leading to oxidative stress (52) . One such condition i s ischemia/reperfusion (53 , 54). A  relativel y subtl e indicato r o f ischemi c damag e t o tissu e i s enhance d capillary permeabilit y whic h ma y resul t i n edema . Mor e sever e damag e ma y cause cel l death and tissue necrosis . Granger e t al. (55) were able to show tha t feline intestinal ischemia can lead to accumulation of hypoxanthine as a result of extensive breakdow n o f ATP . Whe n oxyge n i s readmitte d durin g reperfusion , hypoxanthine i s oxidize d t o uri c aci d catalyze d b y xanthin e oxidase , wit h generation o f superoxid e radical . I t ha s bee n reporte d tha t xanthin e oxidas e inhibitors coul d decreas e ischemia-induce d tissu e damag e i n th e felin e smal l bowel (55) . Therefore, i t might be concluded that xanthine oxidase i s a potentia l source o f mucosa l damag e during reperfusio n i n intestina l ischemia . Younes e t al. (53 ) hav e observe d tha t durin g ischemi a substrate s o f xanthin e oxidas e accumulate i n th e felin e smal l intestine . Thes e ca n b e rapidl y metabolize d resulting in the generation of reactive oxidants that are capable of initiating lipi d peroxidation with resulting tissue damage. Moreover , Niid a et al . (36) were able to produc e duodena l ulcer s i n rat s b y administratio n o f diethyldithiocarbamat e (DDC 750 mg/kg), an SOD inhibitor . Thus, i t might be concluded that impairmen t 18 of antioxidan t capacit y o f th e duodena l mucos a ma y b e involve d i n th e pathogenesis of DDC-induced duodenal ulcers. There i s much evidence i n the literatur e that reactiv e oxygen metabolite s might b e involve d i n th e pathogenesi s o f anothe r gastrointestina l disorder , inflammatory bowe l disease (IBD) . Verspaget e t al . (56) hav e reviewe d the rol e of ROD S i n th e pathogenesi s o f colitis . I n general , scavengin g o f superoxid e radicals by SOD or prevention of their formation by inhibition of xanthine oxidase by allopurinol, have been found most effective i n the prevention o f experimenta l colitis. Sali m (57) has reported i n a double-blind investigatio n tha t the inclusio n of the xanthine oxidase inhibitor , allopurinol , with sulfasalazine could reduce the recurrence o f ulcerativ e coliti s attack s a s compare d wit h administratio n o f sulfasalazine alone . H e ha s conclude d tha t ROD S ma y b e involve d i n th e mechanism o f ulcerativ e coliti s an d tha t antioxidan t measure s ma y b e usefu l both in the treatment of attacks and in preventing their recurrence. RODS ma y als o b e involve d i n th e occurrenc e o f gastrointestina l malignancy. Babb s (58 ) ha s hypothesize d tha t hig h concentration s o f iro n i n feces and the presence of bacteria can lead to the production of larg e quantities of highl y reactiv e hydroxy l radical s i n th e colon . Intracoloni c fre e radica l formation, therefore , ma y b e responsibl e fo r th e productio n o f colo n cancer . These reactiv e metabolite s ma y hav e a  pivota l rol e i n carcinogenesi s b y affecting the genes and chromosomes resultin g i n abnormalities i n cel l division. Bostick et al. (59) have reported a strong inverse association between vitamin E 19 intake an d colo n cance r i n a  prospectiv e Iow a women' s healt h study . Sinc e vitamin E  i s a  lipid-solubl e chain-terminatin g antioxidan t agent , thes e dat a suggest tha t ROD S and lipi d peroxidation ma y be involve d i n the pathogenesi s of colon cancer. Cahil l et al. (60) have also reported a strong inverse correlatio n between serum concentrations o f antioxidant vitamins A and E and incidence o f colon cance r i n humans . Nomur a e t al.(61 ) hav e reporte d significantl y lowe r serum selenium concentrations i n individuals wit h gastrointestina l malignancie s compared with controls . Diet s lo w i n coppe r hav e bee n show n t o increas e th e incidence of 1,2 dimethylhydrazine-induced colo n tumors in rats when compared with diets high in copper (62). Peptic ulcer disease is another gastrointestinal disorder . This disease i s a general public health problem all over the world. Statistical informatio n indicate s that 10 % or mor e o f the western populatio n ma y b e afflicte d b y this diseas e a t some time i n their lives . About 10 % of al l adul t admission s t o genera l hospital s are patients with peptic ulcer disease. This disease accounts for about 1  % of al l deaths i n the genera l population . Th e healt h car e cos t o f pepti c ulce r diseas e was estimated to be 3,224 million dollars in the United States of America in 1977 (63). Peptic ulcer s ar e lesion s tha t ar e induce d b y gastri c aci d an d pepsin . These lesions are typically found i n the dista l third par t of the esophagus, i n the stomach an d i n th e duodenum . Tw o type s o f lesion s ca n b e distinguished , namely erosions and ulcers . B y definition, lesion s tha t involv e only mucos a ar e 20 called "erosions," and i f they fail to hea l an d tissue destructio n progresse s an d penetrates beyon d th e musculari s mucos a "ulcers " ar e formed . Gastri c an d duodenal lesions can be subclassified into two groups: acute and chronic. Acute ulcers usually occur in the body (glandular portion ) o f the stomach -less commonly i n the duodenum -  in patients with any conditio n associated with severe stress , suc h a s a  majo r surgica l procedure , sepsis , respirator y failure , uremia, o r hypotensio n (23) . Thes e acut e lesion s (whic h ma y b e erosion s o r ulcers) ar e frequentl y multipl e i n number , superficia l an d a  fe w millimeter s i n diameter. Thi s conditio n i s als o know n b y a  variet y o f name s suc h a s "stres s ulceration,"" acute stress ulceration, " "  acute gastric mucosa l lesions," " stres s ulcer syndrome,"" stress ulcer diathesis" or" acute hemorrhagic gastritis" (26). Central nervou s syste m injury-induce d ulcer s (Cushing' s ulcers) , burn -induced ulcer s (Curling' s ulcer ) an d drug-induce d ulcer s exhibi t simila r macroscopic an d microscopi c feature s a s othe r form s o f acut e ulceration . Cushing's ulcers can also be found in the esophagus and duodenum (23). Acute lesion s usuall y develo p withi n hours . I f damagin g agent s ar e removed, lesion s can heal without scar formation within a  few days by epithelia l cell regeneration an d they do not recur (64) . The main complication with these lesions is upper gastrointestinal bleeding due to congestion of submucosal blood vessels. There is usually no pain accompanying these lesions. Histologically, acut e inflammator y exudat e an d necroti c debri s ca n b e found i n th e ulce r crater;  however , ther e i s n o granulatio n tissue . Gros s an d 21 histological feature s o f acut e gastri c lesion s wer e describe d i n m y previou s histopathological stud y (65 ) o f th e gastri c mucos a i n rat s expose d t o fasting , psychological stress or physical activities. A chroni c pepti c ulce r o f th e stomac h o r th e duodenu m i s an ulce r tha t has faile d t o heal  withi n a  perio d o f tim e ( 2 week s o r more ) an d show s histological evidence of tissue repair,  i.e. , granulation tissue that will give rise to scar formation when the ulce r heals . Chroni c ulcer s ar e deeper an d large r tha n acute one s an d the y usuall y occu r singl y an d hav e a  roun d o r ova l shape . Chronic ulceration is less common in the stomach than in the duodenum and the former mos t frequently appears as a single lesio n along th e lesser curvature o f the stomach. The occurrence o f chronic gastric ulcers i s uncommon i n the bod y or the greater curvature o f the stomach. Chronic duodena l ulceratio n i s about 8 times more common than chronic gastri c ulceratio n an d it s site i s usually i n the duodenal bulb (66). Usually, when the terms "peptic ulcer, " "gastri c ulcer " o r "duodenal ulcer " are used without further qualification , a  chronic ulce r i s implied (28) . One of the most distinctiv e feature s o f chroni c pepti c ulcer s i s thei r tendenc y t o hea l an d recur months or years later . Another typ e o f pepti c ulce r migh t b e classifie d a s "hormonally-induce d ulceration". Thi s i s seen i n patient s wit h th e Zollinger-Elliso n syndrom e tha t i s associated with greatly enhanced secretion of gastrin by an Islet cell tumor. 22 1.5. Hypothesis RODS generatio n ma y pla y a n importan t rol e i n th e pathophysiolog y o f some gastrointestinal diseases . There are several lines of evidence that suppor t this hypothesis . I t is proposed that differences i n susceptibility to ulceration ma y be determine d b y species- , sex- , age- , an d tissue-relate d difference s i n gastrointestinal mucosa l antioxidants . Species-relate d antioxidan t enzym e differences i n tissues other than gastrointestinal mucos a have been reported by Godin and Garnet t (67 ) an d sex - and age-related difference s i n the plasm a o f normal person s b y Olinesc u e t al . (68 ) an d age-relate d antioxidan t capacit y i n rat tissues b y Matsu o e t al . (69) . Preliminar y wor k i n our laborator y ha s show n differences i n gastri c an d intestina l mucosa l antioxidant s o f rats , rabbit s an d quail. 1.6. Rationale  and Objectives The previousl y cite d literatur e studie s di d no t directl y measur e th e enzymatic an d non-enzymati c antioxidan t system s i n gastri c an d intestina l mucosa. Moreover , w e ar e unawar e o f an y report s studyin g th e activit y o f antioxidant system s i n gastri c an d duodena l mucos a o f differen t species . I n order to demonstrate any associated changes in the activity o f antioxidant status in normal and ulcerative gastri c and duodenal mucosa , a  number o f studies wil l be conducted. Specific objectives are: 23 1.6.1. Experiment  I To measur e th e activitie s o f superoxid e dismutase , glutathion e peroxidase, glutathione reductase and the levels of basal glutathione i n normal gastric, proxima l smal l intestin e (psi) , dista l smal l intestin e (dsi ) an d coloni c mucosa in both male and female rats. 1.6.2. Experiment  II To measure the activity of the above mentioned enzymes and the levels of basa l glutathion e i n th e gastri c an d duodena l mucos a i n severa l othe r species, namely rabbit , pig, quail and cat. These experiments wil l includ e both sexes. 1.6.3. Experiment  III To measur e th e activitie s o f th e abov e mentione d enzyme s an d basa l glutathione i n th e gastri c an d duodena l mucos a o f rat s treate d wit h 8 % o r undiluted ethanol . Th e result s o f thes e experiment s woul d indicat e i f an y possible changes in the activities o f the antioxidant system s occur i n either of the above regions following the exposure to different concentrations of ethano l when compared with those in control rats. 1.6.4. Experiment  IV To measure the activity of the above mentioned enzymes and the levels of basa l glutathion e i n the gastri c an d duodena l mucos a o f rat s expose d t o chronic intermittent stress. 24 2. EXPERIMEN T I Analysis o f th e antioxidan t statu s o f gastric , proxima l smal l intestine , distal small intestine and colonic mucosa in male and female rats. 2.1. Materials  and  Methods Eight mal e an d 8  female Spragu e Dawle y rat s weighin g 245-31 0 gram s were purchased from the animal unit, U.B.C. , an d kept for 2-3 days in the animal room unde r usua l condition s i n th e Departmen t o f Pharmacolog y an d Therapeutics. Animal s wer e anesthetize d wit h a n i.p . injectio n o f pentobarbita l (60 mg/kg) , an d kille d b y cardia c excisio n a t abou t 10:0 0 A.M . Differen t segments o f the gastrointestina l tract , includin g stomach , the firs t 10-1 5 c m o f the small bowel (proximal smal l intestine) , 10-1 5 cm of the smal l intestine abou t 30 c m proxima l t o ileo-ceca l connectio n (dista l smal l intestine ) an d th e colo n were remove d an d place d i n col d norma l saline . Th e stomac h was  opene d b y cutting through the greate r curvature . Al l intestina l segment s were flushed wit h cold norma l salin e an d opene d b y cuttin g alon g th e opposit e lin e o f th e mesentery. Al l mucosa l surface s wer e rinse d wit h col d norma l saline . Al l specimens wer e place d o n a  meta l plat e place d o n ic e an d examine d fo r an y macroscopic lesions. I f any macroscopic lesions were found in any portion of the gastrointestinal tract , al l tissu e specimen s obtaine d fro m tha t anima l wer e discarded. Th e exces s norma l salin e o n th e mucos a wa s remove d b y blottin g with a  piec e o f tissu e paper . Th e mucos a wa s gentl y scrape d usin g a  blun t scalpel. Th e mucosa l materia l obtaine d wa s weighe d (0.15-0. 3 grams ) an d 25 homogenized o n ic e i n 5 0 m M Tris-0. 1 m M EDT A (ethylenediaminetetraaceti c acid), p H 7.6 (10 % w/v) usin g tw o 1 5 second burst s o f a  Polytro n (Brinkmann , Westbury, N.Y. ) a t 25% maximal speed . Two hundre d milliliter s o f homogenat e were use d fo r th e estimatio n o f basal  glutathion e an d th e reminde r o f th e homogenate wa s centrifuge d fo r 1 5 minute s a t 12,00 0 x  g  i n a n Eppendor f microcentrifuge a t 4° C . The supernatant fractio n was assaye d for the activitie s of th e antioxidan t enzyme s glutathion e peroxidase , glutathion e reductas e an d superoxide dismutase. Basal glutathione (GSH ) level s were measured b y adding 0.2 m l of 0.9% saline/azide followe d b y 0. 1 m l o f 25 % TC A t o th e homogenat e o n ice . Th e samples were mixed and centrifuged for 5  minutes a t 12,00 0 x g . The resultin g supernatants were assayed for acid-soluble sulfhydry l groups by adding 0.04 ml of 3  m M 5,5'-dithiobis-(2-nitrobenzoic acid ) (DTNB ) an d the absorbanc e a t 412 nm was measure d a t 1 0 minutes. Since i n most tissues mor e than 90% of acid -soluble sulfhydry l group s i s glutathione , herei n th e ter m basal  glutathion e indicates acid-solubl e sulfhydry l groups . The activities o f glutathion e reductas e (GRD) glutathion e peroxidas e (GPX ) an d Cu-Zn-superoxid e dismutas e (SOD ) were measure d a s describe d b y Godi n et . al . (70) . A  Ceci l spectrophotomete r (CE 292, digital ultraviolet spectrophotometer) was employed in the measurmen t of SO D activit y whil e al l othe r assay s wer e performe d usin g a  Perkin-Elme r model Lambda 6B dual beam spectrophotometer . 26 Results were analyzed usin g single factor ANOVA a t a  significance leve l of P<0.05, followed by the application of the Tukey test to asses the significance of specific inter-group differences . 2.2. Results Figures 1-1 2 sho w level s o f endogenou s antioxidan t component s i n various segments of the gastrointestinal trac t o f male and female rats . Figures 1 and 5 show the level s o f basa l glutathione (GSH ) i n the gastric , proxima l smal l intestine (psi) , distal small intestine (dsi) and colon of male and female animals, respectively. I n bot h sexes , th e level s o f thi s non-enzymati c antioxidan t ar e comparable i n th e gastri c an d coloni c mucos a bu t statisticall y (P<0.05 ) lowe r than level s i n th e proxima l an d dista l segment s o f smal l intestin e (Figur e 9) . Figures 2  an d 6  sho w th e activitie s o f glutathion e reductas e (GRD ) i n th e gastrointestinal trac t o f mal e an d femal e rats , respectively . Differen t part s o f male gastrointestina l trac t showe d simila r activitie s o f thi s enzyme , wherea s colonic mucosa of female rats showed significantly (P<0.05 ) higher activity when compared wit h tha t i n the othe r part s o f eithe r mal e o r female animal s (Figur e 10). Figur e 3  show s th e activit y o f glutathion e peroxidas e (GPX ) i n th e gastrointestinal trac t o f mal e rats . Th e activit y o f thi s enzym e i s significantl y (P<0.05) highe r i n the gastric and distal smal l intestina l mucos a than that i n the other part s o f thi s organ . Figur e 7  show s tha t th e activit y o f GP X i n th e gastrointestinal trac t o f femal e animal s i s muc h highe r i n th e gastri c mucos a than in the other parts of this organ. Comparison o f the activity o f this enzyme in 27 males and females indicate that i t was significantly highe r i n the female gastric and colonic mucosa and male distal smal l intestine mucosa as compared with the corresponding segment of the other sex (Figure 11) . No marked differences were found in the activity of superoxide dismutase among the various segments examined or between males and females (Figures 4, 8 and 12). 28 Figure 1 : The levels of basal glutathione i n gastric, proxima l small intestin e (psi), distal smal l intestine (dsi ) and colon of male rats (mean ± SE). [In this and all subsequen t Figures , value s denote d b y differen t letter s (a , b , c , etc. ) ar e significantly different (P<0.05) from each other; n=8. ] a _o r£) d 1 1  1 1 1 f i l l 29 o o o CO • o CO •I o o LO o LO "d-o o ^t o CO o o CO o LO C\J o o CM o LO T -o o 1 — o LO o o o o snssi; §ui / S9jo]Aiu 30 Figure 2 : Th e activit y o f glutathion e reductas e i n variou s region s o f th e gastrointestinal trac t o f mal e rat s (mea n ±  SD) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] nMoles / min / mg tissue pp^^!\jj\)«cj*Aoioip)p)sscop<o(op ocnobiocnobiocnobiocnobiobnocno ooooooooooooooooooooo i—i—r i—i—r i—i—r i—i—r (Q CO p O •o C/> Q. V) 8 !•£ 32 Figure 3 : Th e activit y o f glutathion e peroxidas e i n variou s region s o f th e gastrointestinal trac t o f mal e rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] nMoles / min / mg tissue pp^^lOj\)UU|k^pipipp)>l ooiobiouiouiourobiocno ooooooooooooooo T T i—r T T to P o •a a. O o o o 3 ill- P-es 34 Figure 4 : Th e activit y o f superoxid e dismutas e i n variou s region s o f th e gastrointestinal trac t o f mal e rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significanc e an d numbe r o f animal s ar e a s i n Figur e 1 ; th e valu e denoted by b is not significantly (P<0.05) different from that of psi.] 35 Ctf <3 cd c o o o CO T3 CO Q. *v_ +-> CO CO o CO 1 — o 1 — o CM T— o o I — o 00 o o CO o o "3-o o CM O O o o stissij Sui / s\m[\ 36 Figure 5 : Th e level s o f basa l glutathion e i n variou s region s o f th e gastrointestinal trac t o f female rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] 37 CO c o o o O Q. CO w CO O) O O LO O LO O O o LO CO o o CO o LO C\j o o o LO o o o LO o o C\J i -StlSSI} §UI /  S9J0]/\[ U 38 Figure 6 : Th e activit y o f glutathion e reductas e i n variou s region s o f th e gastrointestinal trac t o f female rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] o 39 c o 8 CO • o co QL 1 O) o o C\J O CO o> O C\J r^  o 00 ^ o CN O O O onssij §m / mm / SSJOJMU 40 Figure 7 : Th e activit y o f glutathion e peroxidas e i n variou s region s o f th e gastrointestinal trac t o f female rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] nMoles / min / mg tissue o o o N> o -^ 00 o ^J o CO o ro o o CQ CO o p "D CO Q. CO O O O 13 •• cr cr o IP 42 Figure 8 : Th e activit y o f superoxid e dismutas e i n variou s region s o f th e gastrointestinal trac t o f female rat s (mea n ±  SE) . [Abbreviations , expressio n o f statistical significance and number of animals are as in Figure 1. ] Unints / mg tissue oooooooooo-*-»--»--^-^-^-»-o^how^uioj^acDb^K^wj^cnb) ooooooooooooooooo CD 0> CO o" -a CO Q. CO o o o 3 zv 44 Figure 9 : Compariso n o f o f basa l glutathion e level s i n variou s region s o f the gastrointestina l trac t o f mal e an d female rat s (mea n ±  SE) . [Abbreviations , expression of statistical significance and number of animals are as in Figure 1. ] nMoles / mg tissue o o o o CJ1 o —»• o o —»• Ol o N> o o rv> o CO o o CO o -p> o o ^ en o CJ1 o o (Q P> i—• o" •o C/> Q. V> O O ">—i—>—r i—'—r i—'—r i—i—|—i—| 9V 46 Figure 10 : Compariso n o f th e activit y o f glutathion e reductas e i n variou s regions o f th e gastrointestina l trac t o f mal e an d femal e rat s (mea n +  SE) . [Abbreviations, expressio n o f statistica l significanc e an d number o f animals ar e as in Figure 1. ] nMoles / min /mg tissue o-»-roG)*>.cno>^JootDO-*-i\) i i i I i i i I i i i I i i i I i i i I i i i l i i i l i i i I i i i I i i i I i i i l i i i I 0) o" (0 Q. 8 O 3 I P CD fir P 5" Zfr 48 Figure 11 : Comparison o f the activit y o f glutathion e peroxidas e i n variou s regions o f th e gastrointestina l trac t o f mal e an d femal e rat s (mea n ±  SE) . [Abbreviations, expressio n o f statistica l significanc e an d numbe r o f animals ar e as in Figure 1. ] nMoles / min /mg tissue o-»-roa>j!kOio>">icocoo-»-ro i i i | i i i | i i i | i i i | i i i | i i i | i i i | i i i | i i i | i i i | i i i | i i i | (Q T3 CO Q. CO O O O O CD 3 c7 6fr 50 Figure 12 : Compariso n o f th e activit y o f superoxid e dismutas e i n variou s regions o f th e gastrointestina l trac t o f mal e an d femal e rat s (mea n ±  SE) . [Abbreviations, expressio n o f statistica l significanc e an d number o f animals ar e as in Figure 1 ; the value denoted by b is not significantly (P<0.05 ) differen t fro m that of psi. ] 51 ctf cd c o 8 -2 cG CO " O c$ CO Q. cd • 2 cd cd CO co o to T -o -I—" o T— o q T ~ o CO d o CO d o d o CM d o o d snssi; Sui / sjTUf} 52 3. EXPERIMENT  II Comparative stud y o f antioxidant statu s i n the gastri c and proximal smal l intestinal mucosa of rat, rabbit, quail, cat and pig. 3.1. Materials  and Methods In this experiment, ra t data from the previous experiment were used, while those for the other species were obtained from untreated "control" animals being used for other purposes i n the Department or elswhere, as indicated below. For the data o n the gastri c an d proxima l portio n o f the smal l intestin e i n rats, result s from the female rat s i n Experimen t I  were used . The stomac h an d the proxima l portio n o f th e smal l bowe l o f 6  femal e rabbit s ar e fro m animal s being use d fo r othe r purpose s i n th e Departmen t o f Pharmacolog y an d Therapeutics, U.B.C . Th e proxima l segment s o f th e smal l bowe l o f eigh t mal e quail were from animals which had been sacrificed by decapitation. The stomach and th e duodenu m o f femal e cat s wer e obtaine d fro m th e Departmen t o f Ophthalmology, VGH . Th e stomac h an d th e duodenu m o f femal e pig s wer e obtained from the Department of Surgery, VGH. All other methods and material s were the same as described in Experiment I . 3.2. Results Figures 13-1 6 sho w th e level s o f th e non-enzymati c antioxidan t glutathione an d th e activitie s o f th e enzymati c antioxidan t component s i n th e gastric and duodenal mucos a of rabbit , cat , pi g and ra t and the proxima l par t of 53 small intestine of quail. In the gastric mucosa, the level of basal glutathione wa s highest i n the rat , whereas th e level s i n the duodena l mucos a wer e greate r i n quail. Rabbi t tissues , bot h gastri c an d duodena l mucosa , showe d significantl y higher basal  glutathione level s than those o f ca t o r pig . The gastri c mucos a o f cat an d pi g showe d simila r content s o f basa l glutathione , whil e tha t o f th e pi g duodenal mucosa was much higher than that of cat (Fig. 13). The highest activit y of GRD was found i n both tissues o f rat . Generally , th e activit y o f this enzym e was significantl y highe r i n th e duodena l mucos a whe n compare d t o tha t i n gastric mucosa in all species investigated (Fig. 14). Similarly, the activity of GPX in rat gastric mucosa was much higher when compared with that of other species. However, the activities of this enzyme in the duodenal mucos a o f rat , pi g and rabbi t were almos t th e same , and highe r tha n that o f quai l o r pi g (Fig . 15) . The activitie s o f SO D i n the gastri c an d duodena l mucosa o f pi g wer e lowe r tha n thos e i n ra t an d rabbit . Th e activit y o f thi s enzyme i n the quai l duodena l mucos a was muc h highe r tha n tha t o f an y o f th e other species studied (Fig. 16). 54 Figure 13: Basal glutathione levels in the gastric and duodenal mucosa of various specie s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistica l significance are as in Figure 1 ; n=8, 8, 6, 4,and 2 for rat , quail , rabbit, cat and pig, respectively.] cd cd I cd o 1 o o 10 o in • * • o o ^ o to CO o o CO o LO CVJ o o CM O 10 1 — o o I — o o o o o snssi; Stu / sqo]/\[u 56 Figure 14 : Activit y o f glutathion e reductas e i n th e gastri c an d duodena l mucosa o f variou s specie s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistical significance are as in Figure 1 ; n=8, 8, 6, 4,and 2 for rat , quail , rabbit , cat and pig, respectively.] 5-H I cd o 1 CO 8 3 E "(5 c (D "O O 3 •o CO 8 E o CO O) CD 00 CD LO CO CM i - O 9TISSI} §UI /  UlU I / S9J0y\[ U 58 Figure 15 : Activit y o f glutathion e peroxidas e i n th e gastri c an d duodena l mucosa o f variou s specie s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistical significance are as in Figure 1 ; n=8, 8, 6, 4,and 2 for rat , quail , rabbit , cat and pig, respectively.] rabbit mZl cat 12 11 § 10 •rt 9 S 8 i 7 e e 3 2 1 0 a gastric mucosa Pig rat quail duodenal mucosa 60 Figure 16 : Activit y o f superoxid e dismutas e i n th e gastri c an d duodena l mucosa o f variou s specie s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistical significance are as in Figure 1 ; n=8, 8, 6, 4,and 2 for rat, quail , rabbit , cat and pig, respectively.] Units / mg tissue o o o o o —»• o o —JL CXI o ro o o ro en o CO o o CO en o *. o o .&. en o CJl o o to C/> •-* 5" 3 c 8 to a. c o Q. CD 9L 3 c 8 ^3 0Q I P o* •Q c P 1.9 62 4. EXPERIMEN T III The effect s o f 8% and undiluted ethano l o n lesio n developmen t an d th e antioxidant statu s o f gastri c an d proxima l smal l intestina l mucos a o f mal e an d female rats. 4.1. Materials  and  Methods Twenty on e mal e and 21 female Spragu e Dawle y rat s weighing 220-36 5 grams were purchased from the animal unit , UBC . About eightee n hour s befor e the experiment, eac h animal was randomly assigned to one of the experimenta l groups an d place d i n a n individua l cag e an d foo d wa s removed , althoug h animals had free access to water. Ethano l was purchased from chemical stores , U.B.C., an d used as undilute d ethano l o r dilute d with doubl e distille d wate r t o make a n 8 % solution . A t 10:0 0 A. M o n th e da y o f experiment. , animal s wer e anesthetized usin g a  mixtur e o f 4 % halothan e an d oxygen . On e millilite r o f double distille d water , 1  m l o f undilute d ethano l o r 1  m l o f 8 % ethano l wa s administered t o anesthetize d animal s b y gastri c gavage . Animal s receivin g double distille d wate r wer e use d a s controls . On e hou r afte r administratio n o f water o r ethanol , animal s wer e agai n anesthetize d an d sacrifice d b y cardia c excision. Th e stomac h an d th e proxima l portio n o f th e smal l intestin e wer e removed, opene d an d rinsed , a s describe d previously . Th e mucos a wa s carefully examine d for macroscopic lesions . The mucosa was then scraped an d all assay s performe d a s describe d earlier . Th e result s obtaine d fro m mal e animals were compared with those of females. 63 4.2. Results Figures 17-2 4 show the effects o f 1  ml of 8% or undiluted o f ethanol o n the antioxidant components o f gastric and duodenal mucosa in male and female rats. Generally , administratio n o f 1  ml o f 8% ethano l t o rat s b y gavage cause d an increas e i n th e activit y o f enzymati c antioxidan t system s an d basa l glutathione level s i n bot h gastri c an d duodena l mucosa . O n th e othe r hand , administration o f 1  m l undilute d ethano l produce d a  genera l decreas e i n th e antioxidant system s i n bot h tissues , wit h th e exceptio n o f a n increas e i n th e activity of GPX. Figures 25-3 3 sho w macroscopi c an d microscopi c change s foun d i n th e stomachs o f rat s treate d wit h undilute d ethanol . Al l o f thes e rat s develope d visible gastri c mucosa l lesion s i n th e bod y o f th e stomach . Macroscopi c alterations observe d include d distentio n o f th e stomac h an d duodenum , wit h increased luminal fluid volume, as well as strips of redness on the surface of the stomach indicativ e o f gastri c mucosa l vascula r changes . Othe r stomach s an d duodenums appeare d norma l with no visible lesions . Gastri c epithelia l necrosi s was foun d i n gastri c specimen s o f bot h mal e an d femal e rat s treate d wit h undiluted ethanol . Tissu e damag e wa s mor e profoun d i n female s tha n tha t i n males. Significant alteration s i n th e activitie s o f mucosa l antioxidan t enzyme s and level s o f basa l glutathion e followin g th e administratio n o f ethano l ar e summarized in Table 1. 64 Figure 17 : Basa l glutathion e level s i n th e gastri c mucos a o f mal e an d female contro l an d treated rat s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistical significance are as in Figure 1; n=7 for both male and female.] nMoles / mg tissue o o o o CO o o o o CD o —»• o __L 01 o —*• 00 o ro o ro o ro o CO o o CO p CO 3 CD (Q 0) c/> «-* o" CD 3 CD 99 1 oo ro o o s 66 Figure 18 : The activit y o f glutathion e reductas e i n th e gastri c mucos a o f male an d femal e contro l an d treate d rat s (mea n ±  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 fo r bot h mal e an d female.] nMoles / min / mg tissue o-^row^cjiO)->ioocDO i i i l i i i l i i i I i i i I i i i I i i i l i i i l i i i I i i i I i i i I «Q CD </> 3 CD 0> W CD 3 0 Z9 O o o I CD pa S3 O o o CD o 68 Figure 19 : The activit y o f glutathione peroxidas e i n the gastri c mucos a o f male an d femal e contro l an d treate d rat s (mea n ±  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 for bot h mal e an d female.] nMoles / min / mg tissue o-'-rooo.&kCjio-^cocQO-'-ro i i i I i i i I i i i I i i i I i i i I i i i I i i i I i i i I i i i l i i 11 i i i I i i i I CQ in o" 3 CD (Q p> CO 0 3 CD 69 O O <-+ o I oo CD 3-g O O O a> s 70 Figure 20 : Th e activit y o f superoxid e dismutas e i n th e gastri c mucos a o f male an d femal e contro l an d treate d rat s (mea n ±  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 for bot h mal e an d female.] 71 o G as f O O o s CD 1 J L I •  I  t  I (0 E CD o 1 O) CO £ o *-* (0 CO o 00 O CD O O CM O O O CO O O CD O O O O CM O O O O snssij Sin / sjiun 72 Figure 21 : Basa l glutathione level s i n the duodena l mucos a o f male and female control and treated rats (mean ± SE). [Abbreviations and expression of statistical significance are as in Figure 1; n=7 for both male and female.] 73 o c J=! • * - > O o o I J__i L CO E (0 c 0 TD O =3 •a ca E, "cd c CO " O O ZJ •o o o in o •^ f o o ^t o m CO o o CO o in C\J o o CM o m T— o o T -o m o o o o StlSSI} §UI /  S9J0p\[ U 74 Figure 22: The activity o f glutathione reductas e i n the duodenal mucos a o f male an d femal e contro l an d treate d rat s (mea n ±  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 for bot h mal e an d female.] nMoles / min/ mg tissue o-±roGo.&.oio>-Jc©<oo-»-ro i 11 I 11 i | i i i | i i i | i 111 i i i | i 11 | i i i | 111 | i i i | i i i | i i i | o a. CD 9L 3 8L 0 c o Q. 0 CD CD 3 1 00 o o 9Z 76 Figure 23: The activity of glutathione peroxidase in the duodenal mucosa of male an d femal e contro l an d treate d rat s (mea n ±  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 fo r bot h mal e an d female.] 77 o § O o o •s oo I .22 CO E (D « C CD •a o 3 cd £ IB c V T3 O o CO o ^ o CM o o o 00 o CD O ^t O CVJ o O snssi; §ui /urai / s9jop\[u 78 Figure 24: The activity o f superoxide dismutase i n the duodenal mucos a of male an d femal e contro l an d treate d rat s (mea n +  SE) . [Abbreviation s an d expression o f statistica l significanc e ar e as i n Figur e 1 ; n=7 fo r bot h mal e an d female.] Units / mg tissue o o o o l\3 O O O O O) o o 00 O —»• o o -*• o -*• o —L o Q. C o Q. CD 13 3* 0 Q. C o CL 0 0 3 w 1 00 o § O o o §• 61 80 Table 1: Presence of macroscopic lesions (+) and average percent increases (+ ) or decreases (-) i n levels endogenous antioxidan t components of gastric (G) an d duodenum (D) of male (M) and female (F) rats following administration of double distilled water (A) , 8% ethano l (B ) o r undilute d ethano l (C) . [Dat a from Figure s 17-24.] Treatment Group A B C Sex M F M F M F Lesion G ----+ + D ------GSH G --+12 +13 -22 -11 D ---+6 -12 -17 GPX G ----+14 +5 D --+29 +14 -8 -16 GRD G --+6 +5 -14 -7 D --+6 +7 -16 -12 SOD G ---+24 -6 -D --+15 -+12 +7 81 Figure 25: Gross appearance of stomach and duodenum in the abdomen of a mal e ra t treate d wit h 8 % ethanol . [Th e stomac h an d duodenu m o f contro l animals were indistingushable i n appearance from those in the present Figure ]. 82ethanol (8%)83Figure 26: Distended stomach and duodenum with increased luminal fluidvolume in the abdomen of a male rat treated with undiluted ethanol.84F85Figure 27: Comparison of appearance of gastric and duodenal mucosa ofmale rats treated with double distilled water, ethanol (8%) or ethanol (undiluted).[Note the appearance of red strips in the body of the stomach of the animaltreated with undiluted ethanol 1.I-i.[r.CDICD 0OD87Figure 28: Histology of gastric mucosa of a male control rat (longitudinalsection, gastric glands and pits). [In this and all subsequent Figures tissueswere stained with Haemotoxylin and Eosin; prints are produced from slides thatwere taken of tissue sections by using Zeiss Photomicroscope II (Mag. X25).]88‘ 4- % “44\.’ $.. .catTIF,if ‘• •.-. —-‘w’4’ *‘;4,t•ve’:‘tA &iJI‘0. It‘S.‘A.&.:T% t.$ii .i4,“ti’1 I.P2?I- •W.14I.•• •Ia;4I&e19Q 43cL4.sse.t:.2tL çr4w br’ ,.&,, ‘-.2*. Ip p. . Ae4’ -.89Figure 29: Appearance of gastric mucosa of a control male rat (transversesection).0691Figure 30: Superficial necrosis of gastric epithelium of a male rat treatedwith undiluted ethanol.—i92‘U93Figure 31: Gastric epithelial necrosis surrounded by normallooking mucosa in a female rat treated with undiluted ethanol.17695Figure 32: Deep gastric epithelial necrosis in a female rat treated withundiluted ethanol.9697Figure 33: Severe gastric mucosal necrosis following administration ofundiluted ethanol to a female rat.,•;‘.•.•2.•,,--‘19699 5. EXPERIMEN T IV The effec t o f chroni c intermitten t stres s o n the antioxidan t statu s o f th e gastric and proximal small intestinal mucosa of male rats. 5.1. Materials  and Methods Eleven mal e Wista r rat s weighing 200-25 0 grams  wer e purchase d fro m the animal unit, U.B.C . Animals were kept in the animal room in the Departmen t of Pharmacolog y an d Therapeutic s a t U.B. C fo r 1 4 days . Beginnin g o n th e fifteenth da y following thei r arrival , 5 animals were selected randoml y t o remai n in thei r hom e cage s fo r th e nex t 1 4 days . Thes e animal s wer e use d a s th e control group. The remaining 6 animals were exposed to a stress protoco l which involved restrain t fo r on e hou r twic e daily , th e firs t exposur e takin g plac e between 9:00 A. M . an d 12:0 0 A. M. , an d the secon d betwee n 1:0 0 P . M . an d 4:00 P . M . I n orde r t o minimiz e habituation , th e sequenc e o f th e restrain t stressors was randomized for bot h the first 7  morning and afternoon exposures . The serie s was  repeate d durin g th e secon d wee k wit h th e exceptio n tha t th e morning and afternoon sequence s were reversed . The stressors use d included: 1.) towel wrap secured with tape; 2.) number one, placed in a supine position; 3.) restraint i n a plastic box with lid ; 4.) restrain t i n a polyvinlchloride tube closed at either end ; 5. ) secure d t o a  boar d with tape ; 6. ) numbe r 5 , place d i n a  supin e position; an d 7. ) meta l ba r cag e restrainer . Eac h stresso r exposur e wa s conducted i n a  roo m remot e fro m th e anima l facilit y an d animal s expose d t o stressors wer e returne d t o th e anima l facilit y 1 5 minute s followin g stres s 100 exposure t o minimiz e disturbanc e t o contro l animals . O n the da y followin g th e last exposur e t o stress , animal s wer e kille d b y decapitation . Th e stomac h an d proximal portio n o f the smal l intestin e were remove d and handled a s describe d in the previous sections. The mucosa l surfaces were examined carefully for an y macroscopic lesions . All othe r procedure s were performe d a s described earlier . Results were analyzed usin g a t  two-sample tes t assuming equa l varianc e a t a significance leve l of P<0.05. 5.2. Results Figures 34-37 show the activities of antioxidant systems in the gastric and duodenal mucos a o f bot h contro l an d stresse d rats . N o statisticall y significan t differences betwee n an y o f th e antioxidan t component s i n eithe r gastri c o r duodenal mucos a o f stressed rats were found when comparison was made with those o f contro l animal s a t a  significanc e leve l o f P<0.05 . N o macroscopicall y detectable lesion s were found i n the gastri c o r duodena l mucos a o f stressed o r control animals. 101 Figure 34: Levels o f basal  glutathione i n the gastric and duodenal mucos a of contro l an d stresse d rat s (mea n ±  SE) . [Abbreviation s an d expressio n o f statistical significanc e ar e a s i n Figur e 1 ; n=5 an d 6  fo r contro l an d stresse d rats, respectively. ] 1D2 T3 5 -C/3 X> 1 X> cd o o •E 15 c CD -a o 3 •o O s-+-> O o cd CO 8 E GO C D J I O O O O O O O O O O O O O O O O O O ^ • W O C O ( O ^ W O O O ( D ^ N O C O ( O ^ C \ I O 0 O 0 O 0 O C \ J C \ l < N ( M C \ J O O  O  O  O snssij Sui / S9jo]/\[u 103 Figure 35 : Activit y o f glutathion e reductas e i n th e gastri c an d duodena l mucosa of control and stressed rats (mean ± SE). [Abbreviations and expression of statistical significanc e ar e as i n Figure 1 ; n=5 and 6 for contro l an d stresse d rats, respectively. ] nMoles / min / mg tissue O O t-* -» IO M W W A A Ol Ol O) O) S ,M ffl buibcnbuibcnbcnbcnbcnbb-ib ooooooooooooooooo CO p oo o" 3 c 8 CO 0) I o o t o p Q. c o Q. CD 2L 3 c 8 CO 0) GO CD GO o WH 105 Figure 36: Activity o f glutathion e peroxidas e i n the gastric and duodenal mucosa of control and stressed rats (mean ± SE). [Abbreviations and expression of statistical significance are as in Figure 1 ; n=5 and 6 for control and stressed rats, respectively.] y /////ft control (D CO c/o s s CO O 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 a a gastric mucosa stressed "O duodenal mucosa 107 Figure 37 : Activit y o f superoxid e dismutas e i n th e gastri c an d duodena l mucosa of control and stressed rats (mean ± SE). [Abbreviations and expression of statistical significanc e ar e as i n Figure 1 ; n=5 and 6 for contro l an d stresse d rats, respectively. ] 108 c3 o O o cd CO V) O O E 15 c 0) •a o ZJ T3 CO W 8 E o +-• w CO O) o CO 1 — o T -o CM i — o o 1 — o CO O o CD O O o o CM O O O O snssx; Sui / s;iun 109 6. GENERA L DISCUSSION Peptic ulce r diseas e ha s a  multifactoral etiolog y tha t ultimatel y result s i n the productio n o f lesion s i n tissue s expose d t o gastri c aci d an d pepsin . Thi s disease ha s 3  forms tha t diffe r i n thei r pathophysiology . Gastri c an d duodena l ulcers ar e usuall y classifie d a s recurren t ulcer s an d ar e differen t fro m eac h other, an d als o fro m th e thir d for m calle d "stres s ulceration" . Th e difference s among them might be due to differences i n the activity of the antioxidant system s in th e gastri c an d duodena l mucosa l tissue . Som e factor s suc h a s species , genetics an d ag e ca n pla y importan t role s i n the incidenc e o f thi s disease , a s discussed in the Introduction. The pathophysiolog y o f "stres s ulceration " i n experimenta l animal s an d man is quite complex. A variety o f irritants and stressful conditions can result i n "stress ulcer" formation. With regard to the induction of lesions, irritants may well differ i n their mechanis m o f action . However , ther e i s a  bod y o f evidenc e tha t supports th e rol e o f ROD S i n the pathophysiolog y o f "stres s ulcer " productio n (71-73). With regard to "stress ulceration", susceptibility of the gastric mucosa to ulcer formation seems to be muc h greater than that of the duodenal mucosa. The susceptibilit y t o stress-induce d ulceratio n i n experimenta l animal s and ma n increase s wit h age . Administration o f enteric-coate d aspiri n cause s a lower incidenc e o f gastri c lesion s whe n compare d wit h conventiona l aspirin . Subjects receivin g salicylate s b y th e intravenou s rout e d o no t demonstrat e gastric mucosa l lesions , althoug h salicylat e bloo d level s ar e comparabl e t o 110 those i n subjects receivin g th e dru g orall y an d i n whom gastri c lesion s usuall y occur (30) . Th e literatur e indicate s tha t non-steroida l anti-inflammator y drug s (NSAIDs) induce gastric lesions by at least two different mechanisms. One is the well-characterized effec t o f thes e drug s t o inhibi t biosynthesi s o f prostaglandi n E2 .  The secon d mechanis m involve s "io n trapping" . I n th e non-ionize d state , which predominate s a t th e lo w p H o f th e stomach , thes e drug s ca n ente r mucosal cells by freely diffusing across the cel l membrane. Once inside the cell , these drug s ioniz e (NSAID s pK a =  3.5-4 ) an d becom e trapped , creatin g a concentration gradien t tha t favors the accumulation o f the NSAI D i n the gastri c mucosa. Cell permeability alterations are developed resulting in damage caused by hydrogen io n influx , with sodium and potassium ion s movin g int o the gastri c lumen (74). Therefore, one can postulate that i n NSAID-induced gastri c lesions , the direc t contac t o f gastri c mucos a with drug seem s to b e importan t fo r lesio n formation. Also, the fact that ulceration of the duodenum does no t usuall y occu r with the administration o f NSAID suggests tha t differences betwee n gastri c an d duodenal mucosa may be important. Results obtained i n the presen t study show that significan t difference s i n antioxidant statu s exis t amon g differen t portion s o f th e gastrointestina l tract . I t has als o bee n show n tha t antioxidan t system s i n th e gastrointestina l trac t o f different specie s ca n diffe r markedl y i n activity . Thes e result s sho w tha t th e levels o f basal  glutathione an d the activit y o f GPX i n the gastri c mucos a o f rats are significantl y differen t fro m thos e o f duodena l mucosa . However , thi s 111 information cannot answer the question as to whether or not these differences i n antioxidant statu s ar e th e mai n factor s determinin g th e resistanc e o r susceptibility o f gastri c an d duodena l mucos a t o lesio n formation . Significan t differences i n the antioxidant statu s have bee n demonstrated i n gastric mucos a of different species ;  however, there does not appear to be a parallel relationshi p between gastric antioxidant status and susceptibility of corresponding animals to "stress ulceration" . Fo r example , th e activit y o f enzymati c antioxidant s an d th e levels of basal glutathione i n the gastric mucosa of rats hav e been shown to be greater tha n thos e o f rabbits ; o n th e othe r hand , rabbit s ar e very  resistan t t o "stress ulceration" while rats are more sensitive (25) . Also, except for the activit y of GPX , whic h was highe r i n the gastri c mucos a o f femal e rat s tha n i n tha t o f male rats , gastri c an d duodena l muos a o f bot h mal e an d femal e rat s showe d similarity i n thei r antioxidan t status , bu t i t i s reporte d tha t gende r affect s th e susceptibility t o gastri c o r duodena l ulce r formatio n i n ma n s o tha t duodena l ulcers hav e a  male:female rati o o f 4:1 (27 , 28). Therefore , on e migh t conclud e that, a t leas t i n laborator y anima l models , the antioxidan t statu s i s no t the onl y (or the most important) factor determining susceptibility to ulcerogenesis. It ha s bee n foun d tha t administratio n o f 1  m l o f undilute d ethano l b y gastric gavag e lead s t o so-calle d "watermelo n stomach " (34) , suggestiv e o f gastric microcirculatio n injury , wit h a n incidenc e o f 100 % i n bot h mal e an d female Sprague Dawle y rats within one hour . These macroscopi c change s were associated with decreases o f 22% and 1 1 % in basal  glutathion e level s o f mal e 112 and femal e ra t gastri c mucosa , respectively . Lesio n formatio n was  als o associated with decreases o f 14 % and 7% i n the activit y o f GR D i n males an d females, respectively . Th e suceptibilit y o f gastri c mucos a t o undilute d ethano l might be influenced by its low reduced glutathione conten t relativ e to that o f the duodenum. I n this regard , i t should b e note d tha t th e administratio n o f 1  ml o f undiluted ethano l orall y produce d n o macroscopicall y detectabl e duodena l lesions i n either sex. I t might be postulated, therefore, that the higher conten t o f reduced glutathion e an d th e highe r activit y o f GR D migh t contribut e t o th e resistance of duodenal mucosa to ethanol-induced lesio n formation. I t should be noted, howevever , tha t duodena l mucos a di d sho w a  decreas e i n basal glutathione levels and in the activity of GRD despite the absence of macroscopic lesion formation, suggesting that antioxidan t statu s i n this regio n i s sufficient t o protect the mucosa from the damaging effects o f ethanol. Rapid development o f gastric lesions following the instillation of undiluted alcohol i n the stomach might be du e t o earl y gastri c vascula r damage . On e outcom e o f vascula r injur y i s formation of damaging RODS that might also be responsible for lesion formation in the body of the stomach (39, 75,76). Both male and female rat s developed a  large lumina l fluid volume i n the stomach within on e hou r following the administratio n o f undilute d ethanol . Thi s finding raises the question as to whether i t was a physiologic respons e to dilut e the irritan t o r merel y a n exudat e du e t o vascula r damag e induce d b y ethanol . We ar e unawar e o f th e compositio n o f thi s fluid , bu t w e observe d tha t som e 113 components o f the fluid tends to precipitat e ove r time, so that 2  hours followin g the administration o f undiluted ethanol , we were unabl e to drain the fluid with a normal syringe . Instead , we observe d a  brownish mass , indicativ e o f sloughe d mucosal cells and coagulated protein contents of the aforementioned fluid. Ethanol-induced gastri c mucosa l damag e wa s associate d wit h a significant reductio n i n reduce d glutathion e level s o f mucosa . Mille r e t al . (77 ) have also observed a  similar reductio n i n glutathione leve l i n gastric mucos a o f dogs treated with concentrate d ethanol . This reductio n ma y reflec t oxidatio n o f reduced glutathione by RODS produced by ethanol and/or binding of glutathione to acetaldehyd e generate d throug h the oxidatio n o f ethano l b y gastri c mucosa l alcohol dehydrogenase . Sinc e w e observe d increase s i n th e activitie s o f GP X and SO D i n th e gastri c mucos a o f rat s treate d wit h undilute d ethanol , th e possibility tha t th e decreas e i n glutathion e content s wa s du e t o damage d mucosa seem s t o b e unlikely . Gastri c glutathion e conten t depletio n wa s significantly different i n male and female rats. In contrast t o the simila r overal l appearanc e an d gross change s i n mal e and femal e gastri c mucosa , ligh t microscopi c examinatio n o f gastri c mucos a sections staine d wit h Haemotoxyli n an d Eosi n reveale d difference s betwee n males an d females . Epithelia l necrosi s was  deepe r an d mor e extensiv e i n females than i n males. The activit y o f gastri c alcoho l dehydrogenas e ha s bee n shown to be significantly lowe r in women as compared with that of men (78, 79). If this is also the case in rats, one might conclude that the metabolism of ethanol 114 in gastri c mucosa l cell s was  slowe r i n femal e rat s tha n i n males . Therefore , female gastri c mucosa l cell s wer e expose d t o ethano l an d othe r ethanol -mediated injurious factors for longer period of time, resulting i n more irreversibl e tissue damage. It is well established that acute and severe stress, such as that associated with major surgery, hemorrhagi c shock, massive burns or cold-restraint ca n lead to acut e gastri c ulceratio n (21 , 23, 26 , 80-83) . Restrain t i s on e o f th e mos t common experimenta l method s use d to produce stres s ulceration . The duratio n of restrain t neede d t o induc e acut e gastri c lesion s varie s fro m 2-2 4 hours , depending on the environmenta l temperature , so that the lowe r the temperatur e the more rapid the inductio n o f lesions (21 , 23, 80-87). Senay and Leviene  (82 ) showed that a  brie f tw o hou r cold-restrain t (4 ° to 7 ° C ) perio d produce d a  hig h degree of reproducibility and homogeneity o f acute gastric lesions in rats. On the other hand, a number of publications (88-93) have reported that mild and chronic irritants, includin g restrain t an d water-immersion , ca n protec t gastri c mucos a against damaging agents by a process called "adaptive cytoprotection." Uramot o and Ishihar a (94 ) showe d that restrain t an d water-immersion stres s (2 2 °  C ) i n rats reduced gastric damage induced by a combination o f 60% ethanol and 150 mM HCI . The 1 5 da y chroni c intermitten t stres s protoco l use d i n th e presen t study di d no t resul t i n an y macroscopicall y visibl e lesion s o r i n an y significan t alterations i n the antioxidan t component s o f eithe r gastri c o r duodena l mucos a of male rats. Therefore, the fact tha t we did no t find any macroscopi c lesion s i n 115 the stomach of stressed rats might indicate that either mucosal damage occurred acutely, then healed or the stress conditions resulted in "adaptive cytoprotection" that prevente d lesio n induction . Sinc e th e stres s protoco l di d no t alte r th e antioxidant statu s i n th e gastri c an d duodena l mucos a o f rats , i t ca n b e suggested tha t i f "adaptiv e cytoprotection " di d occur , i t apparentl y doe s no t involve antioxidant components. A large body of evidence (38, 43, 44 ,47, 75, 76, 95-97) suggests a role of RODS in the pathogenesis o f gastric ulceration. Since both stress- and ethanol-induced gastri c ulceratio n hav e bee n found to respond similarl y t o agent s suc h as sulfhydryl group-cotaining compounds and zinc (43 , 98), i t can be suggeste d that generation o f RODS ma y play a  role i n gastric ulceratio n induce d b y acute stress o r ethanol . O n th e othe r hand , th e fac t tha t alteration s i n antioxidan t status followin g th e administratio n o f undilute d ethanol , althoug h statisticall y significant, wer e relativel y smal l i n magnitud e suggest s tha t othe r mechanism s may be more important in the production of ethanol-induce d gastric lesions. The incidence of macroscopic lesions in the body of the stomach resulting from th e instillatio n o f 1  m l o f dilut e ethano l (8% ) was  zer o i n bot h sexes . However, analyse s o f antioxidan t system s i n animal s s o treate d showe d som e significant differences between antioxidant systems in both gastric and duodenal mucosa of both sexes relative to control . Fo r example, increase s o f 12-13 % i n basal glutathion e level s were note d i n gastric mucos a o f bot h male an d female rats. Thes e increase s i n basa l glutathion e level s wer e associate d wit h a n 116 increase of approximately 6% in the activity of GRD but no significant changes i n the activit y o f GPX . Victo r e t al . (50 ) hav e reporte d a  20% increas e i n gastri c mucosal glutathion e level s whe n 8 % ethano l wa s topicall y applie d t o gastri c epithelium o f anaesthetize d mongre l dogs . Thes e investigator s hav e als o reported that pretreatmen t o f gastri c mucos a with 8 % ethano l coul d protec t th e gastric mucos a agains t subsequen t damag e induce d b y 40 % ethanol . I n th e foregoing study , 8 % ethano l was  a s effectiv e a s PGE 2 i n protectin g th e gastri c mucosa. Moreover, Glavin and Szabo (99) have postulated that 10-20 % ethanol, given befor e a  necrotizin g agent , migh t caus e cytoprotectio n b y releasin g endogenous prostaglandins i n the stomach. Since ou r result s d o sho w som e similaritie s t o those which ar e reporte d by other investigators (50 , 99), one might conclud e that an increase in the leve l of basa l glutathion e accompanie d b y a n increas e i n th e activit y o f GR D an d possibly prostaglandi n E 2 levels migh t contribute t o the cytoprotective effect s o f low concentration s o f ethano l o n gastri c mucosa . Therefore , on e ma y sugges t that administration o f SH-containing compounds migh t be effective i n prevention of acut e gastri c ulceratio n du e t o ulcerogeni c agent s o r conditions , includin g stress. Wit h regar d t o ou r finding s concernin g antioxidan t alteration s unde r physiological an d pathologica l conditions , i t seem s tha t th e tw o abov e mentioned antioxidan t component s migh t pla y a  significan t rol e i n the defens e mechanisms of gastrointestinal tract . The mechanism by which the level of basal glutathione an d th e activit y o f GR D ar e increase d b y lo w concentration s o f 117 ethanol i s stil l unclea r and remains to be elucidated. Since ethano l i s absorbe d rapidly fro m gastri c mucos a bu t som e change s als o occu r i n antioxidan t components o f th e proxima l smal l intestine , i t migh t b e conclude d tha t centra l effects o f ethano l and/o r it s metabolite s migh t contribut e t o bot h gastri c cytoprotection and lesion formation. In addition to antioxidant systems , anothe r elemen t involve d i n protecting gastric mucos a i s the "mucus-bicarbonate" barrier . Du e to the existenc e o f thi s barrier o n th e gastri c mucosa , surfac e cell s ar e normall y relativel y protecte d from damag e b y aci d an d pepsin . Cell s withi n gastri c pits , suc h a s chie f an d parietal cells , migh t als o b e protecte d agains t th e aforementione d damagin g agents. The apica l surfac e o f chie f cell s seems to be impermeabl e t o hydroge n ions, even at hydrogen io n concentration gradient s o f about 100,00 0 fold. Tigh t junctions ma y play an importan t rol e i n this regard . I n contrast t o parieta l cells , chief cell s produce an d store pepsinoge n an d secrete i t whenever the y receiv e appropriate stimulation , mainl y fro m acetylcholine . ROD S coul d caus e damag e to membranes ,  including the tight junctions o f chief cells . The consequences o f this damag e a t th e membran e leve l woul d b e a n increase d permeabilit y t o hydrogen ions, the release of almost all stored pepsinogen and, ultimately, death of chie f cells . O n th e othe r hand , release d pepsinoge n ca n b e converte d t o pepsin and act as a potent damaging agent and produce further damage. Finally, i t seems to be a possibility tha t by stabilizing and increasin g the activities o f th e antioxidan t system s i n th e gastrointestina l mucosa , on e migh t 118 decrease the risk of formation of peptic ulcers . Therefore, mor e studies have t o be don e t o gai n mor e informatio n abou t th e rol e o f endogenou s antioxidan t systems i n gastrointestina l ulceratio n whic h migh t lea d t o th e developmen t o f improved therapeutic measure s t o treat, an d possibly preven t thi s common an d potentially life-threatening condition. 119 7. SUMMAR Y AND CONCLUSIONS Results obtaine d i n thi s stud y sho w tha t th e activitie s o f enzymati c antioxidants an d level s o f basal  glutathion e sho w significan t difference s i n various portion s o f th e gastrointestina l trac t o f bot h mal e an d female Spragu e Dawley rats . I t ha s als o bee n show n tha t variou s specie s includin g rat , rabbit , cat an d pi g exhibi t marke d differences i n antioxidant profile s i n the gastri c an d duodenal mucosa. Antioxidant status in the mucosa of the proximal portion of the small intestin e o f quai l was  differen t fro m tha t i n th e duodena l mucos a o f th e aforementioned species, as well. Administration of 1  ml of undiluted ethanol by gastri c gavage to male and female Sprague Dawle y rats caused visible lesion s i n the body of stomach in al l rats s o treated , whil e administratio n o f 1  m l o f 8 % ethano l cause d n o visibl e lesions i n the stomac h o f the an y o f the rat s examined . Bot h undilute d ethano l and dilute ethano l (8% ) did , however, produc e some changes i n the antioxidan t status o f th e gastri c an d duodena l mucos a o f mal e an d female rats . Epithelia l tissue necrosis was deeper and more extensive i n gastric mucosa of female rats than that in males. 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