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The effect of etodolac on bile salt and histamine-mediated gastric mucosal injury in the rat
Journal of Physiology - Paris 95 (2001) 43–49 www.elsevier.com/locate/jphysparis
The effect of etodolac on bile salt and histamine-mediated gastric mucosal injury in the rat Omar M.E. Abdel Salama, Siham El-Shenawya, Seham El-Batrana, Mahmoud S. Arbida, Gyula Mo´zsikb,* a Department of Pharmacology, National Research Centre, PO Box 12311, El-Tahrir St., Dokki, Cairo, Egypt First Department of Medicine, Medical Faculty, University of Pe´cs, H-7624 Ifju´sa´g Str. 13., PO Box: 99 Pe´cs, Hungary
b
Abstract The effect of the selective cyclo-oxygenase-type-2 (COX-2) inhibitor etodolac on gastric mucosal integrity and gastric acid secretion was investigated in the rat. Etodolac was given in doses comparable with those being used in man for therapy of rheumatic conditions. The effect of etodolac was studied in the presence of a mild barrier breaker and in the presence of increased rates of endogenous acid secretion. In conscious pylorus-ligated rats, etodolac given intragastrically in 16 or 32 mg /kg for 3 h did not by itself give rise to visible gastric mucosal injury. Etodolac, however, exacerbated gastric mucosal injury evoked by intragastric application of acidified sodium taurocholate (5 mM in 150 mM HCl) in a dose-dependent manner. This effect of edotolac was independent of changes in gastric acid secretory responses. In rats whose gastric acid secretion was stimulated by intraperitoneal histamine (5 mg/kg), and etodolac (given i.g. in doses of 16 or 32 mg/kg) also increased gastric mucosal injury caused by histamine dose-dependently in the 3-h pylorus-ligated rats. Etodolac decreased gastric mucus in the saline- and in the sodium taurocholatetreated rats. In urethane-anaesthetized acute gastric fistula rats, intragastric etodolac (32 mg/kg) did not modify basal gastric acid secretion. Our data suggest that etodolac, a selective COX-2 inhibitor, impairs gastric mucosal resistance and can exacerbate gastric mucosal injury caused by other mucosal barrier breaking agents. Cyclooxygenase type-2 thus contributes to the gastric mucosal defences. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Etodolac; COX-2; Gastric ulcer; Gastric acid; Rat stomach
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are widely prescribed agents for the treatment of different rheumatic conditions. In addition, accumulating evidence suggest that these drugs might be of benefit in the chemoprevention of colon cancer [10,13,34]. Asprin is widely used in secondary prevention of stroke and myocardial infarction [21,38]. These drugs however, possess serious and even life threatening gastrointestinal complications, such as bleeding, perforated gastric ulcer or intestinal and colonic ulceration and increased intestinal permeability [4,12,16,18,22,33]. Most of these side effects have been attributable to the effect of these agents on the cyclo-oxygenase enzyme, the key-limiting
* Corresponding author. Fax: +36-72-536147. E-mail addresses: [email protected] (O.M.E. Abdel Salam), [email protected] (G. Mo´zsik).
step in prostaglandin synthesis and the consequent inhibition of prostaglandin synthesis [35,41]. The latter were shown to possess protective functions in the stomach, the so called cytoprotective property of prostaglandins [31]. The recent discovery that the enzyme cyclo-oxygenase exists in two isoforms, COX-1, a house-keeping isoform and an inducible COX-2 isoform, being expressed by inflammation has paved the way towards the synthesis of a new class of NSAIDs, that are selective inhibitors of COX-2 [36,37]. In other words, hopes were raised that it is now possible to alleviate inflammation with drugs that have safe gastrointestinal and renal profile. The aim of this work is to test the hypothesis that selective COX-2 inhibitors are safe with regard to the gastric mucosa. Etodolac, a recent selective COX-2 inhibitor, reported to possess a safer GI side effect profile [7] was investigated in the present paper. The effect of the drug on the rat gastric mucosa under different experimental conditions and on gastric acid secretion was studied.
0928-4257/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00008-0
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2. Materials and methods 2.1. General Sprague–Dawley strain rats of either sex, 180–200 g of body weight were used throughout the experiments. Animals were housed under standardized conditions for light and temperature and kept in cages with wide meshed floors to help prevent coprophagy. Animals were fasted for 24 h prior to the experiments, but allowed free access to tap water. Animals were randomly assigned to different experimental groups (n=6– 7 per group). Pylorus-ligation was performed under light ether anaesthesia, care being taken not to interfere with the blood supply to the stomach and duodenum. Drugs were given through soft orogastric tube immediately after pylorus-ligation.
itoneal (i.p.) administration of histamine in a dose of 5 mg/kg. Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was ligated, the abdominal wall closed, and histamine was given i.p. in 0.5 ml volume of saline. Rats then received i.g. saline (1 ml; control group) or i.g. etodolac in 16 or 36 mg/kg in 1 ml of saline. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage.
4. Assessment of gastric mucosal damage 4.1. Gross gastric mucosal damage
3. Ulcer studies 3.1. Etodolac alone Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was then ligated and the abdominal wall closed in layers. In order to assess the effect of etodolac on the normal stomach, the drug was intragastrically (i.g.) given in 32 mg/kg in 1 ml volume of saline. Control rats received i.g. saline (1 ml) instead. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage. 3.2. Bile salt-mediated injury Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was ligated, the abdominal wall closed in layers, then acidified sodium taurocholate (5 mM in 150 mM HCl) with or without i.g. etodolac in 16 or 36 mg/kg in 1 ml volume. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage. 3.3. Histamine-mediated injury The effect of etodolac was evaluted in rats whose rates of gastric acid secretion were stimulated by the intraper-
The number and severity of mucosal lesions were noted. Lesions were scaled as follows: petechial lesions=1, lesions less than 1 mm=2, lesion between 1 and 2 mm=3, lesions between 2 and 4 mm=4, and lesions more than 4 mm=5. A total lesion score for each animal is calculated as the total number of lesions multiplied by the respective severity scores. Results are expressed as the severity of lesions/rat [26]. 4.2. Histology In some experiments, after macroscopic evaluation of mucosal lesions, the stomachs were pinned flat on cardboard and immersed in 10% formalin solution and later embedded in paraffin. From the paraffin embedded tissue blocks, haematoxylin and eosin stained sections were coded. Sections were evaluated qualitatively under light microscopy. Sections were evaluated for three grades of histological injury, namely, damage to the surface epithelium, superficial mucosal damage involving the upper one third of the gastric mucosa, and deep mucosal damage extending for more than the upper one third of the mucosa. 4.3. Determination of mucosal mucus content Mucus secretion was determined by the method of Corne et al. [5]. Briefly, the glandular portion of the stomach was excised, weighed and immersed for 2 h in 10 ml of 0.1% w/v Alcian blue 8 GX dissolved in 0.16 mol/l sucrose solution buffered to pH 5.8 with 0.05 mol/ l sodium acetate and adjusted to pH 5.8 with HCl. The excess dye was removed by two successive rinses of 15 min each in 0.25 mol/l sucrose. The mucus-bound dye was extracted by immersing the gastric tissue in 0.5 mol/l MgCl2 solution, which was intermittently shaken
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for 1 min at 30-min intervals of a 2-h period. The blue extract thus obtained was shaken with equal volume of diethylether for 10 min and the resulting emulsion was centrifuged at 3600 rpm for 10 min and the optical density of the aqueous phase was measured at 600 nm in a spectrophotometer. The quantity of Alcian blue extracted per gram of wet glandular tissue was then calculated from standard curves.
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5.4. Analysis of data Results were expressed as means S.E.M. Data were analyzed using analysis of variance and Student’s t test, P values < 0.05 were considered as significant. The Mann Whitney’s test was applied for mathematical analysis of non-parametric results (ulcer severity). The number of rats used in experiments is presented in the text in parenthesis.
5. Gastric secretory studies 6. Results 5.1. Shay rat 6.1. Gastric mucosal damage In the ulcer studies involving pyloric-ligation, after rats have been killed, the oesophagus was ligated, stomachs excised and gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature. The volume of gastric secretion was noted and gastric acid output determined by automatic titration to pH 7.0 with 0.01 N NaOH and and H+ output expressed as mmol/rat/3 h. 5.2. Anaesthetized acute gastric fistula rat Fasted rats were anaesthetized with urethane 1.25 g/ kg, given intraperitoneally (i.p.). Laparotomy was performed, the abdomen then opened through a midline incision, and a double lumen plastic cannula inserted through a small incision in the forestomach and pylorus ligated. Rats received subcutaneous saline 1 ml/h to correct for possible dehydration during the experiment. Prior to collecting gastric acid secretion, the stomach was flushed with saline to clear any solid particles present. A period of at least 30-min was allowed for stabilization of gastric acid secretion. The stomach was then flushed with 2 ml saline followed by air bolus every 15 min, gastric effluent collected, H+ output determined by titration to pH 7 with 0.01 N NaOH and expressed as mmol/15 min. Basal gastric acid was collected over 60 min, then etodolac (32 mg/kg) was introduced into the stomach (1 ml volume, for 30 min) and gastric acid collected for another 60 min. Rats were then killed by cervical dislocation, their stomachs opened and inspected for the presence of gastric mucosal lesions. Results of gastric acid output were expressed as mmol/15 min or total gastric acid output was calculated for the hour before and following etodolac administration and expressed as mmol/h.
6.1.1. Etodolac alone The i.g. administration of etodolac alone in 32 mg/kg for 3 h to pylorus-ligated rats did not result in visible or histologic gastric mucosal damage. Gastric acid output in control rats 3 h after pylorus-ligation was 143.3 13.9 mmol/rat/3 h. Intragastrically given etodolac had no significant effect on gastric acid secretion which averaged 166 20.8 mmol/rat/3h (Fig. 1). Etodolac, on the other hand, decreased mucosal mucus content by 32.5% relative to the saline-treated control group (P < 0.01; Fig. 2). 6.1.2. Bile salt-mediated injury The number and severity of gastric mucosal lesions following the i.g. administration of acidified sodium taurocholate were 7.2 1.8 and 11.8 2.1. Etodolac
5.3. Drugs Etodolac (Napliac, Global Napi Pharmaceuticals, Egypt), Alcian blue 8 GX (Sigma, USA), histamine dihydrochloride (BDH Chemicals, England) and urethane (Sigma, USA) were used.
Fig. 1. The effect of intragastric (i.g.) etodolac (32 mg/kg) on gastric acid secretion in the 3 h pylorus-ligated rat. Saline (1 ml; control group) or etodolac (in 1 ml of saline) was i.g. given immediately after pylorus-ligation and animals killed 3 h later. Results are means S.E.M. n=6–7 per group; NS, not significant vs. saline-treated control group.
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given i.g. in 16 mg/kg had no significant effect on the lesions with the number and severity of lesions being 7.7 1.3 and 15.3 2.2. The lesion scores were intensified after i.g. etodolac administration in a dose of 32 mg/kg, with the number and severity of lesions being 16.2 1.7 and 28.8 3.1, respectively (P < 0.01 and P < 0.01, respectively vs. control values; Fig. 3). Gastric acid output in the acidified sodium taurocholate control rats 3 h after pylorus-ligation was 146 12.0 mmol/rat/3 h. This was not significantly changed by co-administration of etodolac in either dose. Gastric acid output was 190 19.2 and 184 16 mmol/rat/3 h for etodolac in 16 and 32 mg/kg, respectively. Mucosal mucus content was decreased by i.g. etodolac in a dose-dependent manner, with the 16 mg/kg dose inducing a 21.1% and the 32 mg/kg dose inducing 39.7% reduction, the latter being of statistical significance (P < 0.05; Fig. 4). 6.1.3. Histamine-mediated injury Histamine given i.p. in a dose of 5 mg/kg to pylorusligated rats, did evoked a relatively minor degree of gastric mucosal damage 3 h after administration, with the number and severity of gastric mucosal lesions being 2.2 0.7 and 3.3 1.1, in which 66.7% of the animals contained lesions. The histamine-induced gastric mucosal damage was also increased by i.g. etodolac in a dosedependent manner. The number and severity of lesions being 3.3 0.4 and 4.3 0.4 for etodolac in 16 mg/kg and 5.1 0.5 and 6.4 0.6 for the dose of 32 mg/kg, respectively (Fig. 5). Mean histamine-stimulated gastric
Fig. 2. The effect of intragastric (i.g.) etodolac (32 mg/kg) on gastric mucosal mucus content of saline-treated 3 h pylorus-ligated rat. Saline (1 ml; control group) or etodolac (in 1 ml of saline) was i.g. given immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. Asterisks indicate significant change from control values. **, P<0.01 vs. saline-treated control group.
Fig. 3. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on the severity of gastric mucosal injury produced by i.g. acidified sodium taurocholate (NTC) in the 3 h pylorus-ligated rat. Etodolac was i.g. given with or without NTC (5 mM in 150 mM HCl, 1 ml ) immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS=not significant, *, P< 0.05 vs. the sodium taurocholate control group.
Fig. 4. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on gastric mucosal mucus content of acidified sodium-taurocholate (NTC)-treated 3 h pylorus-ligated rat. Etodolac was i.g. given with or without acidified taurocholate (5 mM in 150 mM HCl, 1 ml) immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS, not significant; *, P <0.05 vs. the sodium taurocholate control group.
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Fig. 5. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on the severity of gastric mucosal injury produced by I.p. histamine 5 mg/kg in plyorus-ligated rats. Rats received 1 ml of saline peres (p.o.) (control group) or etodolac in 16 or 36 mg/kg in 1 ml of saline p.o. and killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS, not significant, *, P<0.05 vs. histamine control group.
acid secretion was 224 23.2 mmol/rat/3 h. This was not significantly changed with the i.g. administration of etodolac, with gastric acid output being 202 25.7 mmol/rat/3 h for etodolac in 16 mg/kg and 189 21.5 mmol/rat/3 h for etodolac in 32 mg/kg. On the other hand, no significant differences were noted in the mucosal mucus content of the histaminetreated rats between the control group and rats that received etodolac in 16 or 32 mg/kg. 6.1.4. Urethane-anaesthetized rats Basal gastric acid secretion in anaesthetized rats was 4.3 0.4 mmol/15 min and 17.3 1.7 mmol/h (n=7). Etodolac given orally in 32 mg/kg had no significant effect on gastric acid output which averaged 3.4 0.2 mmol/15 min (n=7). The total acid response in 1-h following the drug was 13.7 0.9 mmol/h (n=7). These responses were not significantly different from values obtained before the administration of the drug (Fig. 6).
7. Discussion and conclusion The present study provided evidence that etodolac, a selective COX-2 inhibitor, although by itself did not give rise to gastric mucosal injury, did exacerbated gastric mucosal injury caused by the mild barrier breaker sodium taurocholate in acid . Etodolac in addition exacerbated gastric damage caused by histamine. Such
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Fig. 6. The effect of intragastric (i.g.) etodolac (32 mg/kg) on basal gastric acid secretion in urethane-anesthetized acute gastric fistula rat. Gastric acid secretion was measured by flushing of the gastric lumen with 2 ml saline every 15 min. Each column represents a meanSEM of 1 h collection of gastric acid secretion of seven rats. NS, not significant vs values obtained before the administration of the drug.
effect of the drug on gastric mucosal injury was not associated with changes in gastric acid secretory responses, but it involved a decrease in gastric mucosal mucus content of the rats. Non-steroidal anti-inflammatory drugs are well known to cause gastric mucosal damage both in experimental animals [15,28] and in man [2,4,11,12,16]. These effects have been attributed mainly to their inhibition of the enzyme cyclooxygenase (COX), also known as prostaglandin H synthase, which catalyzes the rate-limiting step in the formation of prostaglandins (PGs) [28,35,36,41,42]. Several lines of evidence support this notion. First, the degree of gastric mucosal damage evoked by NSAIDs correlated with their potency to inhibit prostaglandin synthesis [28,39,41]. On the other hand, when introduced into the rat stomach in doses devoid of antisecretory activity, PGs prevented gastric mucosal damage caused by a variety of necrotizing agents, a phenomenon termed cytoprotection [31]. Rabbits immunized with 6-keto PGF1a or PGI2 or both exhibited severe gastric and duodenal ulcerations [27,29]. PGs were shown to modulate gastrointestinal defense mechanisms [23]. Thus, PGs increase gastric mucosal blood flow [14,17,40], stimulate secretion of gastric mucus gel [1], secretion of gastric [8] and duodenal mucosal bicarbonate [9], increase gastric mucosal sulfhydryl compounds [24], increase surface active phospholipid [19,32], and increase the hydrophobicity of the luminal surface of the gastric mucosa [6]. In man, misoprostol, a synthetic PG analog have been used successfully in the treatment and prophylaxis of NSAIDsinduced gastroduodenal ulcers [3].
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Then came a remarkable achievement, that is the discovery that two isoforms of cyclooxygenase exist, COX1 and COX-2. The former being expressed normally in the gastrointestinal tract, the kidneys, and platelets and plays major roles in their normal function, whereas, COX-2 is expresssed primarily in response to inflammation [36]. The original hypothesis thus was that selective COX-2 inhibitors, by sparing COX-1 isoenzyme that is responsible for the production of cytoprotective PGs in the gastric mucosa, would be much safer than the commonly used NSAIDs, that are inhibitors of both COX-1 and COX-2 [37]. This was considered a major advance, for it provides a solution for those patients who are in need for NSAIDs and are at the same time at risk of developing gastric ulceration or bleeding. The original hypothesis, however, did not last long and was later shown to be an oversimplification, for a critical role played by COX-2 in a number of physiologic processes have been discovered [20,25]. The present study was thus designed to test the hypothesis that selective inhibitors of COX-2 are safe with regard to gastric mucosa. Etodolac is a highly selective COX-2 inhibitor [7,30] and thus is not expected to provoke gastric mucosal damage. In fact, being introduced alone into the rat stomach in 32 mg/kg, the drug did not cause gross or histologic gastric mucosal damage. When the drug was introduced into the rat stomach after challenge with a mild barrier breaker such as the bile salt sodium taurocholate in acid or in the presence of increaed rates of gastric acid secretion as under condtions of histamine stimulation, etodolac, however, did exacerbated the degree of gastric mucosal damage under such circumstances. It was also found that the drug led to a reduction in gastric mucosal mucus content, which might be a factor contributing to its lesion enhancing effects. In conclusion, the present study showed that selective COX-2 inhibition, although do not result in gastric mucosal damage, increases the susceptibility of the rat gastric mucosa to noxious challenge. Selective COX-2 inhibition thus impairs the gastric mucosal defence mechanism. Further investigations are necessary to delineate the role of COX-2 in maintaining gastric mucosal integrity. References [1] M. Bickel, G.L. Kuaffman, Gastric mucus gel thickness: effect of distension, 16, 16-dimethyl prostaglandin E2 and carbenoxolone, Gastroenterology 80 (1981) 770–775. [2] I. Bjarnason, A. Macpherson, The changing gastrointestinal side effect profile of non-steroidal anti-inflammatory drugs. A new approach for the prevention of a new problem, Scand J Gastroenterol 24 (Suppl 163) (1989) 56–64. [3] D.L. Brand, W.M. Roufail, A.B.R. Thomson, E.J. Tapper, Misoprostol, a synthetic PGE1 analog, in the treatment of duodenal ulcers. A multicenter double-blind study, Dig. Dis. Sci. 30 (1985) 147S–158S.
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The effect of etodolac on bile salt and histamine-mediated gastric mucosal injury in the rat Omar M.E. Abdel Salama, Siham El-Shenawya, Seham El-Batrana, Mahmoud S. Arbida, Gyula Mo´zsikb,* a Department of Pharmacology, National Research Centre, PO Box 12311, El-Tahrir St., Dokki, Cairo, Egypt First Department of Medicine, Medical Faculty, University of Pe´cs, H-7624 Ifju´sa´g Str. 13., PO Box: 99 Pe´cs, Hungary
b
Abstract The effect of the selective cyclo-oxygenase-type-2 (COX-2) inhibitor etodolac on gastric mucosal integrity and gastric acid secretion was investigated in the rat. Etodolac was given in doses comparable with those being used in man for therapy of rheumatic conditions. The effect of etodolac was studied in the presence of a mild barrier breaker and in the presence of increased rates of endogenous acid secretion. In conscious pylorus-ligated rats, etodolac given intragastrically in 16 or 32 mg /kg for 3 h did not by itself give rise to visible gastric mucosal injury. Etodolac, however, exacerbated gastric mucosal injury evoked by intragastric application of acidified sodium taurocholate (5 mM in 150 mM HCl) in a dose-dependent manner. This effect of edotolac was independent of changes in gastric acid secretory responses. In rats whose gastric acid secretion was stimulated by intraperitoneal histamine (5 mg/kg), and etodolac (given i.g. in doses of 16 or 32 mg/kg) also increased gastric mucosal injury caused by histamine dose-dependently in the 3-h pylorus-ligated rats. Etodolac decreased gastric mucus in the saline- and in the sodium taurocholatetreated rats. In urethane-anaesthetized acute gastric fistula rats, intragastric etodolac (32 mg/kg) did not modify basal gastric acid secretion. Our data suggest that etodolac, a selective COX-2 inhibitor, impairs gastric mucosal resistance and can exacerbate gastric mucosal injury caused by other mucosal barrier breaking agents. Cyclooxygenase type-2 thus contributes to the gastric mucosal defences. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Etodolac; COX-2; Gastric ulcer; Gastric acid; Rat stomach
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are widely prescribed agents for the treatment of different rheumatic conditions. In addition, accumulating evidence suggest that these drugs might be of benefit in the chemoprevention of colon cancer [10,13,34]. Asprin is widely used in secondary prevention of stroke and myocardial infarction [21,38]. These drugs however, possess serious and even life threatening gastrointestinal complications, such as bleeding, perforated gastric ulcer or intestinal and colonic ulceration and increased intestinal permeability [4,12,16,18,22,33]. Most of these side effects have been attributable to the effect of these agents on the cyclo-oxygenase enzyme, the key-limiting
* Corresponding author. Fax: +36-72-536147. E-mail addresses: [email protected] (O.M.E. Abdel Salam), [email protected] (G. Mo´zsik).
step in prostaglandin synthesis and the consequent inhibition of prostaglandin synthesis [35,41]. The latter were shown to possess protective functions in the stomach, the so called cytoprotective property of prostaglandins [31]. The recent discovery that the enzyme cyclo-oxygenase exists in two isoforms, COX-1, a house-keeping isoform and an inducible COX-2 isoform, being expressed by inflammation has paved the way towards the synthesis of a new class of NSAIDs, that are selective inhibitors of COX-2 [36,37]. In other words, hopes were raised that it is now possible to alleviate inflammation with drugs that have safe gastrointestinal and renal profile. The aim of this work is to test the hypothesis that selective COX-2 inhibitors are safe with regard to the gastric mucosa. Etodolac, a recent selective COX-2 inhibitor, reported to possess a safer GI side effect profile [7] was investigated in the present paper. The effect of the drug on the rat gastric mucosa under different experimental conditions and on gastric acid secretion was studied.
0928-4257/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00008-0
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2. Materials and methods 2.1. General Sprague–Dawley strain rats of either sex, 180–200 g of body weight were used throughout the experiments. Animals were housed under standardized conditions for light and temperature and kept in cages with wide meshed floors to help prevent coprophagy. Animals were fasted for 24 h prior to the experiments, but allowed free access to tap water. Animals were randomly assigned to different experimental groups (n=6– 7 per group). Pylorus-ligation was performed under light ether anaesthesia, care being taken not to interfere with the blood supply to the stomach and duodenum. Drugs were given through soft orogastric tube immediately after pylorus-ligation.
itoneal (i.p.) administration of histamine in a dose of 5 mg/kg. Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was ligated, the abdominal wall closed, and histamine was given i.p. in 0.5 ml volume of saline. Rats then received i.g. saline (1 ml; control group) or i.g. etodolac in 16 or 36 mg/kg in 1 ml of saline. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage.
4. Assessment of gastric mucosal damage 4.1. Gross gastric mucosal damage
3. Ulcer studies 3.1. Etodolac alone Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was then ligated and the abdominal wall closed in layers. In order to assess the effect of etodolac on the normal stomach, the drug was intragastrically (i.g.) given in 32 mg/kg in 1 ml volume of saline. Control rats received i.g. saline (1 ml) instead. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage. 3.2. Bile salt-mediated injury Rats were lightly anaesthetized with ether and laparatomy was performed. The pylorus of each rat was ligated, the abdominal wall closed in layers, then acidified sodium taurocholate (5 mM in 150 mM HCl) with or without i.g. etodolac in 16 or 36 mg/kg in 1 ml volume. Three hours later, rats were killed by cervical dislocation, after being lightly anaesthetized with ether. The oesophagus was then ligated and the stomach excised. Gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal damage. 3.3. Histamine-mediated injury The effect of etodolac was evaluted in rats whose rates of gastric acid secretion were stimulated by the intraper-
The number and severity of mucosal lesions were noted. Lesions were scaled as follows: petechial lesions=1, lesions less than 1 mm=2, lesion between 1 and 2 mm=3, lesions between 2 and 4 mm=4, and lesions more than 4 mm=5. A total lesion score for each animal is calculated as the total number of lesions multiplied by the respective severity scores. Results are expressed as the severity of lesions/rat [26]. 4.2. Histology In some experiments, after macroscopic evaluation of mucosal lesions, the stomachs were pinned flat on cardboard and immersed in 10% formalin solution and later embedded in paraffin. From the paraffin embedded tissue blocks, haematoxylin and eosin stained sections were coded. Sections were evaluated qualitatively under light microscopy. Sections were evaluated for three grades of histological injury, namely, damage to the surface epithelium, superficial mucosal damage involving the upper one third of the gastric mucosa, and deep mucosal damage extending for more than the upper one third of the mucosa. 4.3. Determination of mucosal mucus content Mucus secretion was determined by the method of Corne et al. [5]. Briefly, the glandular portion of the stomach was excised, weighed and immersed for 2 h in 10 ml of 0.1% w/v Alcian blue 8 GX dissolved in 0.16 mol/l sucrose solution buffered to pH 5.8 with 0.05 mol/ l sodium acetate and adjusted to pH 5.8 with HCl. The excess dye was removed by two successive rinses of 15 min each in 0.25 mol/l sucrose. The mucus-bound dye was extracted by immersing the gastric tissue in 0.5 mol/l MgCl2 solution, which was intermittently shaken
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for 1 min at 30-min intervals of a 2-h period. The blue extract thus obtained was shaken with equal volume of diethylether for 10 min and the resulting emulsion was centrifuged at 3600 rpm for 10 min and the optical density of the aqueous phase was measured at 600 nm in a spectrophotometer. The quantity of Alcian blue extracted per gram of wet glandular tissue was then calculated from standard curves.
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5.4. Analysis of data Results were expressed as means S.E.M. Data were analyzed using analysis of variance and Student’s t test, P values < 0.05 were considered as significant. The Mann Whitney’s test was applied for mathematical analysis of non-parametric results (ulcer severity). The number of rats used in experiments is presented in the text in parenthesis.
5. Gastric secretory studies 6. Results 5.1. Shay rat 6.1. Gastric mucosal damage In the ulcer studies involving pyloric-ligation, after rats have been killed, the oesophagus was ligated, stomachs excised and gastric juice was carefully collected in graduated tubes after removal of the oesophageal ligature. The volume of gastric secretion was noted and gastric acid output determined by automatic titration to pH 7.0 with 0.01 N NaOH and and H+ output expressed as mmol/rat/3 h. 5.2. Anaesthetized acute gastric fistula rat Fasted rats were anaesthetized with urethane 1.25 g/ kg, given intraperitoneally (i.p.). Laparotomy was performed, the abdomen then opened through a midline incision, and a double lumen plastic cannula inserted through a small incision in the forestomach and pylorus ligated. Rats received subcutaneous saline 1 ml/h to correct for possible dehydration during the experiment. Prior to collecting gastric acid secretion, the stomach was flushed with saline to clear any solid particles present. A period of at least 30-min was allowed for stabilization of gastric acid secretion. The stomach was then flushed with 2 ml saline followed by air bolus every 15 min, gastric effluent collected, H+ output determined by titration to pH 7 with 0.01 N NaOH and expressed as mmol/15 min. Basal gastric acid was collected over 60 min, then etodolac (32 mg/kg) was introduced into the stomach (1 ml volume, for 30 min) and gastric acid collected for another 60 min. Rats were then killed by cervical dislocation, their stomachs opened and inspected for the presence of gastric mucosal lesions. Results of gastric acid output were expressed as mmol/15 min or total gastric acid output was calculated for the hour before and following etodolac administration and expressed as mmol/h.
6.1.1. Etodolac alone The i.g. administration of etodolac alone in 32 mg/kg for 3 h to pylorus-ligated rats did not result in visible or histologic gastric mucosal damage. Gastric acid output in control rats 3 h after pylorus-ligation was 143.3 13.9 mmol/rat/3 h. Intragastrically given etodolac had no significant effect on gastric acid secretion which averaged 166 20.8 mmol/rat/3h (Fig. 1). Etodolac, on the other hand, decreased mucosal mucus content by 32.5% relative to the saline-treated control group (P < 0.01; Fig. 2). 6.1.2. Bile salt-mediated injury The number and severity of gastric mucosal lesions following the i.g. administration of acidified sodium taurocholate were 7.2 1.8 and 11.8 2.1. Etodolac
5.3. Drugs Etodolac (Napliac, Global Napi Pharmaceuticals, Egypt), Alcian blue 8 GX (Sigma, USA), histamine dihydrochloride (BDH Chemicals, England) and urethane (Sigma, USA) were used.
Fig. 1. The effect of intragastric (i.g.) etodolac (32 mg/kg) on gastric acid secretion in the 3 h pylorus-ligated rat. Saline (1 ml; control group) or etodolac (in 1 ml of saline) was i.g. given immediately after pylorus-ligation and animals killed 3 h later. Results are means S.E.M. n=6–7 per group; NS, not significant vs. saline-treated control group.
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given i.g. in 16 mg/kg had no significant effect on the lesions with the number and severity of lesions being 7.7 1.3 and 15.3 2.2. The lesion scores were intensified after i.g. etodolac administration in a dose of 32 mg/kg, with the number and severity of lesions being 16.2 1.7 and 28.8 3.1, respectively (P < 0.01 and P < 0.01, respectively vs. control values; Fig. 3). Gastric acid output in the acidified sodium taurocholate control rats 3 h after pylorus-ligation was 146 12.0 mmol/rat/3 h. This was not significantly changed by co-administration of etodolac in either dose. Gastric acid output was 190 19.2 and 184 16 mmol/rat/3 h for etodolac in 16 and 32 mg/kg, respectively. Mucosal mucus content was decreased by i.g. etodolac in a dose-dependent manner, with the 16 mg/kg dose inducing a 21.1% and the 32 mg/kg dose inducing 39.7% reduction, the latter being of statistical significance (P < 0.05; Fig. 4). 6.1.3. Histamine-mediated injury Histamine given i.p. in a dose of 5 mg/kg to pylorusligated rats, did evoked a relatively minor degree of gastric mucosal damage 3 h after administration, with the number and severity of gastric mucosal lesions being 2.2 0.7 and 3.3 1.1, in which 66.7% of the animals contained lesions. The histamine-induced gastric mucosal damage was also increased by i.g. etodolac in a dosedependent manner. The number and severity of lesions being 3.3 0.4 and 4.3 0.4 for etodolac in 16 mg/kg and 5.1 0.5 and 6.4 0.6 for the dose of 32 mg/kg, respectively (Fig. 5). Mean histamine-stimulated gastric
Fig. 2. The effect of intragastric (i.g.) etodolac (32 mg/kg) on gastric mucosal mucus content of saline-treated 3 h pylorus-ligated rat. Saline (1 ml; control group) or etodolac (in 1 ml of saline) was i.g. given immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. Asterisks indicate significant change from control values. **, P<0.01 vs. saline-treated control group.
Fig. 3. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on the severity of gastric mucosal injury produced by i.g. acidified sodium taurocholate (NTC) in the 3 h pylorus-ligated rat. Etodolac was i.g. given with or without NTC (5 mM in 150 mM HCl, 1 ml ) immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS=not significant, *, P< 0.05 vs. the sodium taurocholate control group.
Fig. 4. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on gastric mucosal mucus content of acidified sodium-taurocholate (NTC)-treated 3 h pylorus-ligated rat. Etodolac was i.g. given with or without acidified taurocholate (5 mM in 150 mM HCl, 1 ml) immediately after pylorus-ligation and animals killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS, not significant; *, P <0.05 vs. the sodium taurocholate control group.
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Fig. 5. The effect of intragastric (i.g.) etodolac (16 or 32 mg/kg) on the severity of gastric mucosal injury produced by I.p. histamine 5 mg/kg in plyorus-ligated rats. Rats received 1 ml of saline peres (p.o.) (control group) or etodolac in 16 or 36 mg/kg in 1 ml of saline p.o. and killed 3 h later. Results are meansS.E.M. n=6–7 per group. NS, not significant, *, P<0.05 vs. histamine control group.
acid secretion was 224 23.2 mmol/rat/3 h. This was not significantly changed with the i.g. administration of etodolac, with gastric acid output being 202 25.7 mmol/rat/3 h for etodolac in 16 mg/kg and 189 21.5 mmol/rat/3 h for etodolac in 32 mg/kg. On the other hand, no significant differences were noted in the mucosal mucus content of the histaminetreated rats between the control group and rats that received etodolac in 16 or 32 mg/kg. 6.1.4. Urethane-anaesthetized rats Basal gastric acid secretion in anaesthetized rats was 4.3 0.4 mmol/15 min and 17.3 1.7 mmol/h (n=7). Etodolac given orally in 32 mg/kg had no significant effect on gastric acid output which averaged 3.4 0.2 mmol/15 min (n=7). The total acid response in 1-h following the drug was 13.7 0.9 mmol/h (n=7). These responses were not significantly different from values obtained before the administration of the drug (Fig. 6).
7. Discussion and conclusion The present study provided evidence that etodolac, a selective COX-2 inhibitor, although by itself did not give rise to gastric mucosal injury, did exacerbated gastric mucosal injury caused by the mild barrier breaker sodium taurocholate in acid . Etodolac in addition exacerbated gastric damage caused by histamine. Such
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Fig. 6. The effect of intragastric (i.g.) etodolac (32 mg/kg) on basal gastric acid secretion in urethane-anesthetized acute gastric fistula rat. Gastric acid secretion was measured by flushing of the gastric lumen with 2 ml saline every 15 min. Each column represents a meanSEM of 1 h collection of gastric acid secretion of seven rats. NS, not significant vs values obtained before the administration of the drug.
effect of the drug on gastric mucosal injury was not associated with changes in gastric acid secretory responses, but it involved a decrease in gastric mucosal mucus content of the rats. Non-steroidal anti-inflammatory drugs are well known to cause gastric mucosal damage both in experimental animals [15,28] and in man [2,4,11,12,16]. These effects have been attributed mainly to their inhibition of the enzyme cyclooxygenase (COX), also known as prostaglandin H synthase, which catalyzes the rate-limiting step in the formation of prostaglandins (PGs) [28,35,36,41,42]. Several lines of evidence support this notion. First, the degree of gastric mucosal damage evoked by NSAIDs correlated with their potency to inhibit prostaglandin synthesis [28,39,41]. On the other hand, when introduced into the rat stomach in doses devoid of antisecretory activity, PGs prevented gastric mucosal damage caused by a variety of necrotizing agents, a phenomenon termed cytoprotection [31]. Rabbits immunized with 6-keto PGF1a or PGI2 or both exhibited severe gastric and duodenal ulcerations [27,29]. PGs were shown to modulate gastrointestinal defense mechanisms [23]. Thus, PGs increase gastric mucosal blood flow [14,17,40], stimulate secretion of gastric mucus gel [1], secretion of gastric [8] and duodenal mucosal bicarbonate [9], increase gastric mucosal sulfhydryl compounds [24], increase surface active phospholipid [19,32], and increase the hydrophobicity of the luminal surface of the gastric mucosa [6]. In man, misoprostol, a synthetic PG analog have been used successfully in the treatment and prophylaxis of NSAIDsinduced gastroduodenal ulcers [3].
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Then came a remarkable achievement, that is the discovery that two isoforms of cyclooxygenase exist, COX1 and COX-2. The former being expressed normally in the gastrointestinal tract, the kidneys, and platelets and plays major roles in their normal function, whereas, COX-2 is expresssed primarily in response to inflammation [36]. The original hypothesis thus was that selective COX-2 inhibitors, by sparing COX-1 isoenzyme that is responsible for the production of cytoprotective PGs in the gastric mucosa, would be much safer than the commonly used NSAIDs, that are inhibitors of both COX-1 and COX-2 [37]. This was considered a major advance, for it provides a solution for those patients who are in need for NSAIDs and are at the same time at risk of developing gastric ulceration or bleeding. The original hypothesis, however, did not last long and was later shown to be an oversimplification, for a critical role played by COX-2 in a number of physiologic processes have been discovered [20,25]. The present study was thus designed to test the hypothesis that selective inhibitors of COX-2 are safe with regard to gastric mucosa. Etodolac is a highly selective COX-2 inhibitor [7,30] and thus is not expected to provoke gastric mucosal damage. In fact, being introduced alone into the rat stomach in 32 mg/kg, the drug did not cause gross or histologic gastric mucosal damage. When the drug was introduced into the rat stomach after challenge with a mild barrier breaker such as the bile salt sodium taurocholate in acid or in the presence of increaed rates of gastric acid secretion as under condtions of histamine stimulation, etodolac, however, did exacerbated the degree of gastric mucosal damage under such circumstances. It was also found that the drug led to a reduction in gastric mucosal mucus content, which might be a factor contributing to its lesion enhancing effects. In conclusion, the present study showed that selective COX-2 inhibition, although do not result in gastric mucosal damage, increases the susceptibility of the rat gastric mucosa to noxious challenge. Selective COX-2 inhibition thus impairs the gastric mucosal defence mechanism. Further investigations are necessary to delineate the role of COX-2 in maintaining gastric mucosal integrity. References [1] M. Bickel, G.L. Kuaffman, Gastric mucus gel thickness: effect of distension, 16, 16-dimethyl prostaglandin E2 and carbenoxolone, Gastroenterology 80 (1981) 770–775. [2] I. Bjarnason, A. Macpherson, The changing gastrointestinal side effect profile of non-steroidal anti-inflammatory drugs. A new approach for the prevention of a new problem, Scand J Gastroenterol 24 (Suppl 163) (1989) 56–64. [3] D.L. Brand, W.M. Roufail, A.B.R. Thomson, E.J. Tapper, Misoprostol, a synthetic PGE1 analog, in the treatment of duodenal ulcers. A multicenter double-blind study, Dig. Dis. Sci. 30 (1985) 147S–158S.
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