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EFFECTS OF PARACETAMOL AND PROPACETAMOL ON GASTRIC MUCOSAL DAMAGE AND GASTRIC LIPID PEROXIDATION CAUSED BY ACETYLSALICYLIC ACID (ASA) IN RATS
Pharmacological Research, Vol. 46, No. 2, 2002 doi:10.1016/S1043-6618(02)00083-X, available online at http://www.idealibrary.com on
EFFECTS OF PARACETAMOL AND PROPACETAMOL ON GASTRIC MUCOSAL DAMAGE AND GASTRIC LIPID PEROXIDATION CAUSED BY ACETYLSALICYLIC ACID (ASA) IN RATS B. GALUNSKA a,∗ , K. MARAZOVA b , T. YANKOVA a , A. POPOV b , P. FRANGOV b , I. KRUSHKOV b and A. DI MASSA c a Department
of Biochemistry, Medical University of Varna, Bulgaria, b Department of Pharmacology and Toxicology, Medical Faculty of Sofia, Sofia, Bulgaria, c Department of Pain Therapy, University of Siena, Siena, Italy Accepted 30 April 2002
We have studied the effect of paracetamol and its pro-drug propacetamol on gastric mucosal damage induced by acetylsalicylic acid (ASA) and its possible relation to changes in gastric lipid peroxidation status in rats. Paracetamol or propacetamol were administered intragastrically 1 h before ASA (300 mg kg−1 ) in the following equivalent doses: 62.5, 125.0 and 250.0 mg kg−1 or 125.0, 250.0 and 500.0 mg kg−1 , respectively. The effects of the tested agents were compared to that of prostaglandin E2 (PGE2) 15, 30 and 60 mg kg−1 . Gastric ulcer formation was estimated morphometrically 4 h after ASA administration. Malondialdehyde (MDA), glutathione (reduced, GSH, and oxidized, GSSG) and uric acid (UA) were determined in gastric mucosa and blood plasma and used as biochemical markers of the oxidative status. The results showed that paracetamol (250, 125, 62.5 mg kg−1 ) and propacetamol (500, 250, 125 mg kg−1 ) diminished the area of ASA-induced gastric lesions. The effect of propacetamol was more pronounced than that of paracetamol and similar to that of PGE2. Gastric MDA increased 3-fold in the ASA-group. The tested agents reduced it by a range of 30–70%. In all pretreated groups gastric glutathione and UA levels were found higher than that of control group and lower than that of ASA-group. Paracetamol and propacetamol, as well as PGE2, diminished the lipid peroxidation in plasma to a lesser extent than in gastric mucosa, but maintained elevated levels of the selective plasma antioxidant UA. These results show that the ASA-induced gastric mucosal damage is accompanied by the development of oxidative stress, evidenced by the accumulation of MDA, and concomitant initial activation of cell antioxidant defences. As paracetamol and propacetamol tend to decrease gastric lesions caused by ASA and alter gastric mucosal MDA, glutathione and UA values in a favorable manner, it could be suggested that their effects on the gastric mucosa could be related to interference with oxidative stress development. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: paracetamol, propacetamol, acetylsalicylic acid, gastric mucosal damage, gastric lipid peroxidation.
INTRODUCTION It is widely accepted that the pathogenesis of peptic ulcer is complex and still not completely understood. Increased acid secretion and pepsin activity, reduced mucus and bicarbonate secretion, enhanced contractility of the gastric wall and reduced gastric mucosal blood flow represent some of the established pathogenic factors of gastric ∗ Corresponding
author. Department of Biochemistry, Medical University of Varna, 55 Marin Drinov Street, 9002 Varna, Bulgaria. E-mail: [email protected]
1043-6618/02/$ – see front matter
ulceration. Recent data point the attention on stressors of physical and chemical origin that can damage gastric mucosa through enhanced production of oxygen species and oxidative stress. This mechanism has been shown to be involved in the gastric ulceration caused by stress [1–3], ethanol [4–7], pyloric ligation [8], Helicobacter pylori [9] or ischemia/hypotensive shock [10]. Non-steroidal anti-inflammatory drugs (NSAID) are known to induce gastric mucosal damage including bleeding, ulceration and perforation in animals and humans. Although the inhibition of cyclooxygenase (COX) and deficiency in endogenous prostaglandins (PGs) is accepted © 2002 Elsevier Science Ltd. All rights reserved.
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as a main mechanism implicated in NSAID-induced gastropathy, an increasing body of evidence suggests the involvement of oxidative stress in this pathology, as it has been demonstrated for indomethacin [11–13] and acetylsalicylic acid [4, 14]. The para-aminophenolic analgesic–antipyretic paracetamol is generally considered relatively safe for the gastric mucosa, mostly due to its central COX-inhibiting effects [15]. In experimental and clinical studies it has been demonstrated that paracetamol protects the gastric mucosa against ASA [16], water-immersion [17] and ethanol-induced stress [18], and does not induce mucosal damage in humans under regular dosage regimens [19]. The effects of propacetamol on gastric mucosa, however, are poorly investigated. In order to elicit the effect of propacetamol on gastric mucosa and further to investigate the gastroprotective effect of paracetamol in the light of a possible interference with the gastric oxidative status, in the present work we studied the effects of paracetamol and propacetamol on rat gastric mucosa damaged by acetylsalicylic acid (ASA). Gastric mucosal and blood plasma concentrations of malondialdehyde, glutathione and uric acid were used as biochemical markers of the oxidative stress.
MATERIALS AND METHODS
Ulcer induction Male Wistar rats weighing 240–270 g were used. The animals were housed in controlled environmental conditions and fed a standard food diet. They were fasted 24 h before the experiment, but had free access to water. The test-substances were administered 30 min before ASA (300 mg kg−1 ). Propacetamol was applied in doses of 125, 250 and 500 mg kg−1 , corresponding to paracetamol 62.5, 125 and 250 mg kg−1 [20], the latter shown to exert a gastroprotective effect against ulcers induced by ethanol [18], water immersion stress [17] and pyloric ligation [21]. Since prostaglandin E2 (PGE2) is gastroprotector and the ASA model is prostaglandin-deficit dependent, we compared the effect of paracetamol and propacetamol on gastric mucosa with those of PGE2 by treating a series of animals with PGE2 15, 30 or 60 mg kg−1 . All the substances were given intragastrically in a volume of 0.2 ml/100 g. Control animals were given distilled water instead of test-substances in the same test schedule and conditions. Each experimental group consisted of at least eight animals. The animals were sacrificed by rapid decapitation and exsanguination 4 h after ASA administration. The stomach was removed immediately, opened along the greater curvature, gently washed in physiological salt solution, spread over the pad and observed macroscopically for appearance of mucosal lesions. The length of each lesion was measured. In the case of petechia, five of them were considered as a 1-mm lesion. Mean ulcer area (mm2 ) was calculated. The experimental procedure was approved by the Home Office for Care and
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Use of Laboratory Animals and performed with a strong consideration for ethics of animal experimentation.
Histopathological study Pieces of the stomach were immersed in 8% formalin solution, embedded in paraffin and stained with hematoxilin–eosin. Histomorphological examination was performed under light microscopy and documented by microphotocamera Jena Lumar.
Biochemical examination Blood was taken from the femoral vein and heparinized. Plasma was separated by centrifugation at 800 × g rpm for 10 min and aliquots were stored at −80 ◦ C until analysis. Gastric mucosa was gently separated from the underlying tissue, homogenized in 1:5 w/v 50 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA, at 4000 rpm for 10 min. The homogenate was centrifuged at 800 × g rpm/15 min to discard the sediment, and the supernatant was frozen until analysis. All manipulations were performed at 4–8 ◦ C. Analysis was performed immediately after thawing of the samples. Membrane lipid peroxidation was monitored by malondialdehyde (MDA) measured by its thiobarbituric acid (TBA) reactivity in plasma or gastric mucosa homogenates using the method of Porter et al. [22]. MDA values in nM ml−1 plasma and nM mg−1 protein of gastric mucosal homogenate were determined using the extinction coefficient of the MDA–TBA complex at 532 nm = 1.56×10−5 cm−1 M−1 solution. Glutathione (GSH and GSSG) content was assayed according to the method of Hissin and Hilf [23], using o-phthaldialdehyde as the fluorescent agent. Standard solutions of GSH and GSSG were applied to calculate the glutathione content in gastric mucosal homogenates. Uric acid (UA) in plasma and in gastric mucosal homogenate was determined by the method of Bergmeyer [24], based on the ability of urate to reduce phosphowolframic acid in alkaline solution to a blue colored product. Standard solutions of uric acid were used to calculate the content in plasma and gastric mucosal homogenates.
Statistical analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) and expressed as mean ±sem. A value of P < 0.05 was considered statistically significant. The statistical procedure was performed with GraphPad InStat software.
RESULTS
Effect of paracetamol and propacetamol on ASA-induced gastric mucosal damage The macroscopic observation showed that ASA induced multiple gastric mucosal lesions, most often 1–2 mm in size or petechial, bleeding at the moment of the observation. The area of involvement was confined to the glandular part of the stomach. The mean ulcer area was
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Fig. 1. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal lesions in rats with ASA-induced ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: # P < 0.05; ### P < 0.001 versus ASA-group.
9.10 ± 0.82 mm2 (Fig. 1). Pretreatment with paracetamol 62.5, 125 or 250 mg kg−1 changed the ulcer area by −28.6, −13.0 and −4.4%, respectively. The pretreatment with propacetamol 125, 250 or 500 mg kg−1 resulted in a more pronounced and dose-dependent decrease in ulcer
area: −22.0, −38.5 and −52.9% (Fig. 1). PGE2 15, 30 or 60 mg kg−1 dose-dependently decreased the ulcer area by 20.3, 42.3 and 78.0%. There was no significant difference in ulcer area when paracetamol, propacetamol or PGE2 were applied.
Fig. 2. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of MDA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
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Fig. 3. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of UA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05 versus controls; # P < 0.05; ## P < 0.01 versus ASA-group.
Histopathological study The histomorphological studies showed that the experimental lesions evoked by ASA were manifested by acute superficial erosive defects which were filled with blood coagulates and haematin materials. The cytoplasm of the
superficial epithelium in adjacent areas showed signs of mucous dysplasia. In the groups pretreated with paracetamol or propacetamol as well as PGE2, the gastric mucosa appeared almost normal. In some cases only bending of the superficial epithelium and focal desquamation was found.
Fig. 4. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of reduced glutathione (GSH) in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
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Fig. 5. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of oxidized glutathione (GSSG) in rats with ASA-induced gastric ulceration. Results are the mean±sem of at least eight animals per group. Statistical significance: ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
Biochemical examination In the ASA-group gastric mucosal MDA increased 4-fold versus controls (Fig. 2). In the groups pretreated with paracetamol 62.5, 125.0 and 250 mg kg−1 MDA level markedly decreased up to 54.6, 46.6 and 28.9%, respectively, but still remained higher in comparison with controls. MDA level was reduced by propacetamol 125, 250 or 500 mg kg−1 by 48.4, 56.3 and 66.9%, respectively. After PGE2 15, 30, 60 mg kg−1 MDA decreased by 62.1, 61.0 and 68.7%, respectively.
ASA decreased gastric mucosal UA by 33.3% (Fig. 3). When rats were pretreated with paracetamol 250 mg kg−1 , propacetamol 500 or 250 mg kg−1 and PGE2 (all doses) UA values were enhanced above control values and significantly higher than in the ASA-group. ASA strongly increased gastric GSH and GSSG concentrations (6- and 3-fold, respectively). The increased values of GSH remained significantly high compared to controls in all pretreated groups and decreased to a different extent in comparison with the ASA-group (Fig. 4).
Fig. 6. Effects of paracetamol, propacetamol and PGE2 on plasma values of MDA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01 versus controls.
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Fig. 7. Effects of paracetamol, propacetamol and PGE2 on plasma values of UA in rats with ASA-induced gastric ulceration. Results are the mean±sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls.
In the groups pretreated with paracetamol, propacetamol and PGE2, GSSG values were found similar to that in the control group (Fig. 5). Plasma MDA in the ASA-group was increased 1.5-fold compared to controls (P < 0.05) (Fig. 6). MDA levels remained significantly elevated compared to the control group after paracetamol 125, 62.5 mg kg−1 , propacetamol 500, 250, 125 mg kg−1 or PGE2 pretreatment 15, 30 mg kg−1 . A slight not significant decrease in plasma MDA levels compared to the ASA-group values was registered with paracetamol 250 mg kg−1 (−8.7%), propacetamol 500 mg kg−1 (−1.5%) and PGE2 60 mg kg−1 (−7.5%). ASA strongly increased plasma uric acid by 2.6-fold (P < 0.001) (Fig. 7). In all pretreated groups UA remained significantly increased in comparison with controls. After propacetamol 250 and 500 mg kg−1 and PGE2 60 mg kg−1 the elevation was above the values of the ASA group (19.8, 26.7 and 6.9%, respectively).
DISCUSSION It is generally accepted that NSAIDs exert pro-ulcerogenic activity related to their ability to inhibit endogenous prostaglandin synthesis. The latter results in an increase in gastric acid secretion and a decrease in the non-parietal components of the gastric secretion such as sodium bicarbonate and mucus, as well as in reduction of the mucosal blood flow [25–27]. The focal ischemic areas, which subsequently develop into erosions and inadequate perfusion, may disturb cell metabolism with local release of tissue-damaging mediators such as oxygen-derived free radicals. The relationship between NSAID-evoked gastric mucosal lesions and lipid peroxidation in gastric
tissue evidenced by MDA production, reduced activity of glutathione peroxidase [12], superoxide dismutase [28], catalase, and decreased glutathione concentrations [29], has already been established. Altered glutathione homeostase and enzymatic antioxidant defense, together with decrease in PGE2 levels, were found also for the preferential COX-2 inhibitor meloxicam [30]. On the other side, antioxidants and free radical scavengers protect the gastric mucosa against NSAID-induced injury, as it has been shown for allopurinol and dimethylsulfoxide [4, 11], superoxide dismutase and catalase [12, 14], and alpha-tocopherol [31]. In the present work, we found that paracetamol and propacetamol tended to reduce the morphometrical and histomorphological signs of gastric mucosal damage induced by ASA. A dose-dependent decrease in ulcer area was found after propacetamol. The involvement of extensive lipid peroxidation in ASA-induced gastric mucosal damage was evidenced by the strong accumulation of MDA in gastric mucosa associated with an adaptive rise in glutathione values. As can be seen from Figure 2, lower doses of paracetamol were more effective to inhibit the generation of MDA than the higher doses. This could be explained with the ability of paracetamol to modulate oxidation through two mechanisms, either as a co-substrate for the peroxidase of cyclooxygenase or by conversion to the free radical NAPQI. All tested substances decreased MDA level, but propacetamol as well as PGE2 diminished MDA in a manner corresponding to the reduction of the lesion area and maintained high values of glutathione and UA. These findings allow suggesting that the tested agents diminished the lipid peroxidation and supported the defense reaction in response to oxidative stress. As glutathione is accepted as a major mechanism for cellular protection against agents which generate oxidative stress
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[32], keeping gastric mucosal levels of glutathione high could be assumed as an important effect of paracetamol, propacetamol and PGE2. Plasma MDA level is a complex marker of the tissue lipid peroxidation. Its elevation after ASA was smaller than those registered in gastric mucosa. The water soluble antioxidant uric acid (urate at pH 7.4) is an important part of plasma antioxidant defense. Since its concentration in plasma is very high (almost 10-fold higher than other antioxidants such as Vitamin C and E), UA contributes significantly to plasma antioxidant capacity [33–38]. Urate has been proved to be an excellent scavenger of hypochlorous acid, hydroxyl radicals and peroxynitrite [38]. As generally plasma antioxidant levels are modulated by radical overload, it may be suggested that ASA-induced lipid peroxidation was associated with an adaptive rise in plasma antioxidant UA. Paracetamol and propacetamol as well as PGE2 reduced to a lesser extent lipid peroxidation in plasma than in gastric mucosa, but maintained elevated levels of the selective plasma antioxidant UA. On the other hand, urate is found to prevent oxidative inactivation of COX [37]. This effect arises from the ability of urate to serve as an electron-donor co-substrate for the hydroperoxidase activity of COX and even to stimulate the production of PGE from arachidonic acid [39]. In that sense UA concentrations are related to PG levels. Gastroprotective effect of paracetamol has been demonstrated in different models of acute ulcerations but the underlying mechanism(s) remains unclear: in waterimmersion stressed rats paracetamol significantly reduced gastric mucosal damage, associated with promoted production of PGE2 in gastric mucosa and decreased gastric and plasma glutathione levels [17]. On the other hand, in human gastric and duodenal mucosa paracetamol was reported significantly to reduce PG production, without mucosal injury [40], ulceration or bleeding [19, 41]. Protection against ischemia/reperfusion-induced gastric injury and inhibition of gastric lipid peroxidation was reported by Nakamoto et al. [42]. Our previous investigations showed that paracetamol and its pro-drug propacetamol protected gastric mucosa against cold/restraint stress and prevented gastric lipid peroxidation [43]. In conclusion, we found that paracetamol and propacetamol reduced ASA-induced gastric musocal injury. Propacetamol revealed more pronounced gastroprotective effect, similar to that exhibited by PGE2. The gastroprotective effect of these compounds was comparable to the protective effect of PGE2 on gastric mucosa and was accompanied by significant decrease of the gastric lipid peroxidation (evaluated as MDA-generation) and support to the adaptive mechanisms against oxidative stress (by means of glutathione and uric acid). Taking into consideration the role of lipid peroxidation in NSAID-gastric injury, the interference with gastric oxidative status could be suggested as a possible way through which paracetamol and propacetamol exert protective effect against ASA-induced gastric ulceration.
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REFERENCES 1. Simmons HF, James RC, Harbison RD, Roberts SM. Depression of glutathione by cold restraint in mice. Toxicology 1990; 161: 59–71. 2. Yegen B, Dedeoglu A, Aycac I, Oktay S, Yalcin AS. Effect of cold restraint stress on glutathione and lipid peroxide level in the liver and glandular stomach of rat. Pharmacol Res 1990; 22: 45–8. 3. Das KD, Banerjee RK. Effect of stress on the antioxidant enzymes and gastric ulceration. Mol Cell Biochem 1993; 125: 115–25. 4. Salim AS. Use of scavenging oxygen-derived free radicals to protect the rat against aspirin- and ethanol-induced erosive gastritis. J Pharmacol Sci 1992; 81: 943–6. 5. Hirokawa M, Miura S, Yoshida H, Kurose I, Shigematsu T, Hokari R, Higuchi H, Watanabe N, Yakoyama Y, Kimura H, Sato S, Ishii H. Oxidative stress and mitochondrial damage precedes gastric mucosal cell death induced by ethanol administration. Alcohol Clin Exp Res 1998; 22(Suppl 3): 111S–4S. 6. Schiorff EC, Husain K, Somani SM. Dose-and time-dependent effects of ethanol on plasma antioxidant system in rat. Alcohol 1999; 17: 97–105. 7. Hernandez-Munoz R, Montiel-Ruiz C, Vazquez-Martinez O. Gastric mucosal cell proliferation in ethanol-induced chronic mucosal injury is related to oxidative stress and lipid peroxidation in rats. Lab Invest 2000; 80: 1161–9. 8. Tanaka J, Yuda Y. Role of lipid peroxidation in gastric mucosal lesions induced by ischemia-reperfusion in the pylorus-ligated rat. Biol Pharm Bull 1993; 16: 29–32. 9. Farinati F, Della Libera G, Cardin R, Molari A, Plebani M, Rugge M, Di Mario F, Naccarato R. Gastric antioxidant, nitrites, and mucosal lipoperoxidation in chronic gastritis and Helicobacter pylori infection. J Clin Gastroenterol 1996; 22: 275–81. 10. Esplugues JV, Whittle BR. Gastric damage following local intra-arterial administration of reactive oxygen metabolites in the rat. Br J Pharmacol 1989; 97: 1085–92. 11. Takeuchi K, Ueshima K, Hironaka Y, Fujioka Y, Matsumoto J, Okabe S. Oxygen free radicals and lipid peroxidation in the pathogenesis of gastric mucosal lesions induced by indomethacin in rats. Relation to gastric hypermotility. Digestion 1991; 49: 175–84. 12. Yoshikawa T, Naito Y, Kishi A, Tomii T, Kaneko T, Iinuma S, Ishikawa H, Yasuda H, Takahashi S, Kondo M. Role of active oxygen, lipid peroxidation, and antioxidants in the pathogenesis of gastric mucosal injury induced by indomethacin in rats. Gut 1993; 34: 732–7. 13. Naito Y, Yoshikawa T, Yoshida N, Kondo M. Role of oxygen radical and lipid peroxidation in indomethacin-induced gastric mucosal injury. Dig Dis Sci 1998; 43: 30S–4S. 14. Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol- and aspirin-induced gastric mucosal injury. Dig Dis Sci 1987; 32: 1395–401. 15. Flower RJ, Vane JR. Inhibition of prostaglandin synthetase in brain explains the antipyretic activity of acetaminophen. Nature 1972; 240: 410–1. 16. Konturek SJ, Brzozowski T, Piastucki I, Radecki T. Prevention of ethanol and aspirin-induced gastric mucosal lesions by paracetamol and salicylate in rats: role of endogenous prostaglandins. Gut 1982; 23: 536–40. 17. Omura H, Kamasaki Y, Kawasaki H, Itoh T. Effect of acetaminophen on stress-induced gastric mucosal lesions in rats. Res Commun Mol Pathol Pharmacol 1994; 86: 297–310. 18. Poon YK, Cho CH, Ogle CW. The protective mechanisms of paracetamol against ethanol-induced gastric mucosal damage in rats. J Pharm Pharmacol 1989; 41: 563–5. 19. Lanza FL, Codispoti JR, Nelson EB. An endoscopic comparison of gastroduodenal injury with over-the-counter doses of ketoprofen and acetaminophen. Am J Gastroenterol 1998; 93: 1051–4. 20. Farkas JC, Larrouturou P, Morin JP, Laurian C, Hichet J, Cormier JM, Boccard E. Analgesic efficacy of an injectable acetaminophen versus a dipyrone plus pitofenone plus fenpiverinium association after abdominal aortic repair. Curr Therap Res 1992; 51: 19–26. 21. Bhattacharya SK, Goel RK, Bhattacharya SK, Tandon R. Potentiation of gastric toxicity of ibuprofen by paracetamol in the rat. J Pharm Pharmacol 1991; 43: 520–1.
148 22. Porter N, Norton J, Ramdas J. Cyclic peroxidase and thiobarbituric assay. Biochem Biophys Acta 1976; 441: 596–9. 23. Hissin P, Hilf A. A fluorimetric method for determination of oxidized and reduced glutathione in tissues. Ann Biochem 1976; 74: 214– 26. 24. Bergmeyer HU. Methods of enzymatic analysis. Beartfield Beach, FL: Verlag Chemie Int., 1981. 25. Whittle BR. Relationship between prevention of rat gastric erosions and the inhibition of acid secretion by prostaglandins. Eur J Pharmacol 1976; 40: 223–39. 26. Whittle BR. The defensive role played by the gastric microcirculation. Methods Findings Exp Clin Pharmacol 1989; 11(Suppl 1): 35–43. 27. Bolton JP, Palmer D, Cohen M. Stimulation of mucus and nonparietal secretion by the E2 prostaglandins. Dig Dis Sci 1982; 27: 359–64. 28. Tanaka J, Yuda Y. Lipid peroxidation in gastric mucosal lesions induced by indomethacin in rats. Biol Pharm Bull 1996; 19: 716– 20. 29. Hassan A, Martin E, Puig-Parellada P. Role of antioxidants in gastric mucosal damage induced by indomethacin in rats. Methods Findings Exp Clin Pharmacol 1998; 20: 849–54. 30. Villegas I, Martin MJ, LaCasa C, Motilva V, Alarcon de la Lastra C. Effects of meloxicam on oxygen radical generation in rat gastric mucosa. Inflamm Res 2000; 49: 361–6. 31. Sugimoto N, Yoshida N, Yoshikawa T, Nakamuara Y, Ichikawa H, Naito Y, Kondo M. Effect of Vitamin E on aspirin-induced mucosal injury in rats. Dig Dis Sci 2000; 45: 599–605. 32. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. Free Radic Res 1999; 31: 273–300. 33. Keniia MV, Shkurat TP, Lukash AI, Guskov EP. Changes of uric acid levels in rat tissues as a systemic reaction to hyperoxia. Aviakosm Ekolog Med 1993; 27: 37–43.
Pharmacological Research, Vol. 46, No. 2, 2002 34. Becker BF. Towards the physiological function of uric acid. Free Radic Biol Med 1993; 6: 615–31. 35. Siems WG, van-Kuijk FJ, Maass R, Brenke R. Uric acid and glutathione levels during short-term whole body cold exposure. Free Radic Biol Med 1994; 16: 299–305. 36. Schrod L, Neuhaus T, Speer CP, Girschick H. Possible role of uric acid as an antioxidant in premature infants. Biol Neonate 1997; 72: 102–11. 37. Ghiselli A, Serafini M, Natella F, Scacci C. Total antioxidant capacity as a tool to assess redox status: critical review and experimental data. Free Radic Biol Med 2000; 29: 1106–14. 38. Kopprasch S, Richter K, Leonahardt W, Pietzsch J, Grassler J. Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes. Mol Cell Biochem 2000; 206: 51–6. 39. Gale PH, Egan RW. Prostaglandin endoperoxide synthase-catalyzed oxidation reactions. In: Pryor WA, ed. Free radicals in biology. Orlando: Academic Press, 1984: 1–34. 40. Konturek SJ, Obtlowicz W, Sito E. Distribution of prostaglandins in gastric and duodenal mucosa of healthy subjects and duodenal ulcer patients: effect of aspirin and paracetamol. Gut 1981; 22: 283–9. 41. Jerussi TP, Caubet JF, McCray JE, Handley DA. Clinical endoscopic evaluation of the gastroduodenal tolerance to (R)-ketoprofen, (R)-flurbiprofen, racemic ketoprofen and paracetamol: a randomized, single-blind, placebo-controlled trial. J Clin Pharmacol 1998; 38(Suppl 2): 19S–24S. 42. Nakamoto K, Kamisaki Y, Wada K, Kawasaki H, Itoh T. Protective effect of acetaminophen against acute gastric mucosal lesions induced by ischemia-reperfusion in the rat. Pharmacology 1997; 54: 203–9. 43. Galunska B, Frangov P, Marazova K, Yankova T. Paracetamol and propacetamol in stressed rats: changes in lipid peroxidation in blood red cells and plasma and stomach tissue. IMRA 1999; 3: 208.
EFFECTS OF PARACETAMOL AND PROPACETAMOL ON GASTRIC MUCOSAL DAMAGE AND GASTRIC LIPID PEROXIDATION CAUSED BY ACETYLSALICYLIC ACID (ASA) IN RATS B. GALUNSKA a,∗ , K. MARAZOVA b , T. YANKOVA a , A. POPOV b , P. FRANGOV b , I. KRUSHKOV b and A. DI MASSA c a Department
of Biochemistry, Medical University of Varna, Bulgaria, b Department of Pharmacology and Toxicology, Medical Faculty of Sofia, Sofia, Bulgaria, c Department of Pain Therapy, University of Siena, Siena, Italy Accepted 30 April 2002
We have studied the effect of paracetamol and its pro-drug propacetamol on gastric mucosal damage induced by acetylsalicylic acid (ASA) and its possible relation to changes in gastric lipid peroxidation status in rats. Paracetamol or propacetamol were administered intragastrically 1 h before ASA (300 mg kg−1 ) in the following equivalent doses: 62.5, 125.0 and 250.0 mg kg−1 or 125.0, 250.0 and 500.0 mg kg−1 , respectively. The effects of the tested agents were compared to that of prostaglandin E2 (PGE2) 15, 30 and 60 mg kg−1 . Gastric ulcer formation was estimated morphometrically 4 h after ASA administration. Malondialdehyde (MDA), glutathione (reduced, GSH, and oxidized, GSSG) and uric acid (UA) were determined in gastric mucosa and blood plasma and used as biochemical markers of the oxidative status. The results showed that paracetamol (250, 125, 62.5 mg kg−1 ) and propacetamol (500, 250, 125 mg kg−1 ) diminished the area of ASA-induced gastric lesions. The effect of propacetamol was more pronounced than that of paracetamol and similar to that of PGE2. Gastric MDA increased 3-fold in the ASA-group. The tested agents reduced it by a range of 30–70%. In all pretreated groups gastric glutathione and UA levels were found higher than that of control group and lower than that of ASA-group. Paracetamol and propacetamol, as well as PGE2, diminished the lipid peroxidation in plasma to a lesser extent than in gastric mucosa, but maintained elevated levels of the selective plasma antioxidant UA. These results show that the ASA-induced gastric mucosal damage is accompanied by the development of oxidative stress, evidenced by the accumulation of MDA, and concomitant initial activation of cell antioxidant defences. As paracetamol and propacetamol tend to decrease gastric lesions caused by ASA and alter gastric mucosal MDA, glutathione and UA values in a favorable manner, it could be suggested that their effects on the gastric mucosa could be related to interference with oxidative stress development. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: paracetamol, propacetamol, acetylsalicylic acid, gastric mucosal damage, gastric lipid peroxidation.
INTRODUCTION It is widely accepted that the pathogenesis of peptic ulcer is complex and still not completely understood. Increased acid secretion and pepsin activity, reduced mucus and bicarbonate secretion, enhanced contractility of the gastric wall and reduced gastric mucosal blood flow represent some of the established pathogenic factors of gastric ∗ Corresponding
author. Department of Biochemistry, Medical University of Varna, 55 Marin Drinov Street, 9002 Varna, Bulgaria. E-mail: [email protected]
1043-6618/02/$ – see front matter
ulceration. Recent data point the attention on stressors of physical and chemical origin that can damage gastric mucosa through enhanced production of oxygen species and oxidative stress. This mechanism has been shown to be involved in the gastric ulceration caused by stress [1–3], ethanol [4–7], pyloric ligation [8], Helicobacter pylori [9] or ischemia/hypotensive shock [10]. Non-steroidal anti-inflammatory drugs (NSAID) are known to induce gastric mucosal damage including bleeding, ulceration and perforation in animals and humans. Although the inhibition of cyclooxygenase (COX) and deficiency in endogenous prostaglandins (PGs) is accepted © 2002 Elsevier Science Ltd. All rights reserved.
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as a main mechanism implicated in NSAID-induced gastropathy, an increasing body of evidence suggests the involvement of oxidative stress in this pathology, as it has been demonstrated for indomethacin [11–13] and acetylsalicylic acid [4, 14]. The para-aminophenolic analgesic–antipyretic paracetamol is generally considered relatively safe for the gastric mucosa, mostly due to its central COX-inhibiting effects [15]. In experimental and clinical studies it has been demonstrated that paracetamol protects the gastric mucosa against ASA [16], water-immersion [17] and ethanol-induced stress [18], and does not induce mucosal damage in humans under regular dosage regimens [19]. The effects of propacetamol on gastric mucosa, however, are poorly investigated. In order to elicit the effect of propacetamol on gastric mucosa and further to investigate the gastroprotective effect of paracetamol in the light of a possible interference with the gastric oxidative status, in the present work we studied the effects of paracetamol and propacetamol on rat gastric mucosa damaged by acetylsalicylic acid (ASA). Gastric mucosal and blood plasma concentrations of malondialdehyde, glutathione and uric acid were used as biochemical markers of the oxidative stress.
MATERIALS AND METHODS
Ulcer induction Male Wistar rats weighing 240–270 g were used. The animals were housed in controlled environmental conditions and fed a standard food diet. They were fasted 24 h before the experiment, but had free access to water. The test-substances were administered 30 min before ASA (300 mg kg−1 ). Propacetamol was applied in doses of 125, 250 and 500 mg kg−1 , corresponding to paracetamol 62.5, 125 and 250 mg kg−1 [20], the latter shown to exert a gastroprotective effect against ulcers induced by ethanol [18], water immersion stress [17] and pyloric ligation [21]. Since prostaglandin E2 (PGE2) is gastroprotector and the ASA model is prostaglandin-deficit dependent, we compared the effect of paracetamol and propacetamol on gastric mucosa with those of PGE2 by treating a series of animals with PGE2 15, 30 or 60 mg kg−1 . All the substances were given intragastrically in a volume of 0.2 ml/100 g. Control animals were given distilled water instead of test-substances in the same test schedule and conditions. Each experimental group consisted of at least eight animals. The animals were sacrificed by rapid decapitation and exsanguination 4 h after ASA administration. The stomach was removed immediately, opened along the greater curvature, gently washed in physiological salt solution, spread over the pad and observed macroscopically for appearance of mucosal lesions. The length of each lesion was measured. In the case of petechia, five of them were considered as a 1-mm lesion. Mean ulcer area (mm2 ) was calculated. The experimental procedure was approved by the Home Office for Care and
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Use of Laboratory Animals and performed with a strong consideration for ethics of animal experimentation.
Histopathological study Pieces of the stomach were immersed in 8% formalin solution, embedded in paraffin and stained with hematoxilin–eosin. Histomorphological examination was performed under light microscopy and documented by microphotocamera Jena Lumar.
Biochemical examination Blood was taken from the femoral vein and heparinized. Plasma was separated by centrifugation at 800 × g rpm for 10 min and aliquots were stored at −80 ◦ C until analysis. Gastric mucosa was gently separated from the underlying tissue, homogenized in 1:5 w/v 50 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA, at 4000 rpm for 10 min. The homogenate was centrifuged at 800 × g rpm/15 min to discard the sediment, and the supernatant was frozen until analysis. All manipulations were performed at 4–8 ◦ C. Analysis was performed immediately after thawing of the samples. Membrane lipid peroxidation was monitored by malondialdehyde (MDA) measured by its thiobarbituric acid (TBA) reactivity in plasma or gastric mucosa homogenates using the method of Porter et al. [22]. MDA values in nM ml−1 plasma and nM mg−1 protein of gastric mucosal homogenate were determined using the extinction coefficient of the MDA–TBA complex at 532 nm = 1.56×10−5 cm−1 M−1 solution. Glutathione (GSH and GSSG) content was assayed according to the method of Hissin and Hilf [23], using o-phthaldialdehyde as the fluorescent agent. Standard solutions of GSH and GSSG were applied to calculate the glutathione content in gastric mucosal homogenates. Uric acid (UA) in plasma and in gastric mucosal homogenate was determined by the method of Bergmeyer [24], based on the ability of urate to reduce phosphowolframic acid in alkaline solution to a blue colored product. Standard solutions of uric acid were used to calculate the content in plasma and gastric mucosal homogenates.
Statistical analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) and expressed as mean ±sem. A value of P < 0.05 was considered statistically significant. The statistical procedure was performed with GraphPad InStat software.
RESULTS
Effect of paracetamol and propacetamol on ASA-induced gastric mucosal damage The macroscopic observation showed that ASA induced multiple gastric mucosal lesions, most often 1–2 mm in size or petechial, bleeding at the moment of the observation. The area of involvement was confined to the glandular part of the stomach. The mean ulcer area was
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Fig. 1. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal lesions in rats with ASA-induced ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: # P < 0.05; ### P < 0.001 versus ASA-group.
9.10 ± 0.82 mm2 (Fig. 1). Pretreatment with paracetamol 62.5, 125 or 250 mg kg−1 changed the ulcer area by −28.6, −13.0 and −4.4%, respectively. The pretreatment with propacetamol 125, 250 or 500 mg kg−1 resulted in a more pronounced and dose-dependent decrease in ulcer
area: −22.0, −38.5 and −52.9% (Fig. 1). PGE2 15, 30 or 60 mg kg−1 dose-dependently decreased the ulcer area by 20.3, 42.3 and 78.0%. There was no significant difference in ulcer area when paracetamol, propacetamol or PGE2 were applied.
Fig. 2. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of MDA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
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Fig. 3. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of UA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05 versus controls; # P < 0.05; ## P < 0.01 versus ASA-group.
Histopathological study The histomorphological studies showed that the experimental lesions evoked by ASA were manifested by acute superficial erosive defects which were filled with blood coagulates and haematin materials. The cytoplasm of the
superficial epithelium in adjacent areas showed signs of mucous dysplasia. In the groups pretreated with paracetamol or propacetamol as well as PGE2, the gastric mucosa appeared almost normal. In some cases only bending of the superficial epithelium and focal desquamation was found.
Fig. 4. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of reduced glutathione (GSH) in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
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Fig. 5. Effects of paracetamol, propacetamol and PGE2 on gastric mucosal values of oxidized glutathione (GSSG) in rats with ASA-induced gastric ulceration. Results are the mean±sem of at least eight animals per group. Statistical significance: ∗∗∗ P < 0.001 versus controls; # P < 0.05; ## P < 0.01; ### P < 0.001 versus ASA-group.
Biochemical examination In the ASA-group gastric mucosal MDA increased 4-fold versus controls (Fig. 2). In the groups pretreated with paracetamol 62.5, 125.0 and 250 mg kg−1 MDA level markedly decreased up to 54.6, 46.6 and 28.9%, respectively, but still remained higher in comparison with controls. MDA level was reduced by propacetamol 125, 250 or 500 mg kg−1 by 48.4, 56.3 and 66.9%, respectively. After PGE2 15, 30, 60 mg kg−1 MDA decreased by 62.1, 61.0 and 68.7%, respectively.
ASA decreased gastric mucosal UA by 33.3% (Fig. 3). When rats were pretreated with paracetamol 250 mg kg−1 , propacetamol 500 or 250 mg kg−1 and PGE2 (all doses) UA values were enhanced above control values and significantly higher than in the ASA-group. ASA strongly increased gastric GSH and GSSG concentrations (6- and 3-fold, respectively). The increased values of GSH remained significantly high compared to controls in all pretreated groups and decreased to a different extent in comparison with the ASA-group (Fig. 4).
Fig. 6. Effects of paracetamol, propacetamol and PGE2 on plasma values of MDA in rats with ASA-induced gastric ulceration. Results are the mean ± sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01 versus controls.
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Fig. 7. Effects of paracetamol, propacetamol and PGE2 on plasma values of UA in rats with ASA-induced gastric ulceration. Results are the mean±sem of at least eight animals per group. Statistical significance: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus controls.
In the groups pretreated with paracetamol, propacetamol and PGE2, GSSG values were found similar to that in the control group (Fig. 5). Plasma MDA in the ASA-group was increased 1.5-fold compared to controls (P < 0.05) (Fig. 6). MDA levels remained significantly elevated compared to the control group after paracetamol 125, 62.5 mg kg−1 , propacetamol 500, 250, 125 mg kg−1 or PGE2 pretreatment 15, 30 mg kg−1 . A slight not significant decrease in plasma MDA levels compared to the ASA-group values was registered with paracetamol 250 mg kg−1 (−8.7%), propacetamol 500 mg kg−1 (−1.5%) and PGE2 60 mg kg−1 (−7.5%). ASA strongly increased plasma uric acid by 2.6-fold (P < 0.001) (Fig. 7). In all pretreated groups UA remained significantly increased in comparison with controls. After propacetamol 250 and 500 mg kg−1 and PGE2 60 mg kg−1 the elevation was above the values of the ASA group (19.8, 26.7 and 6.9%, respectively).
DISCUSSION It is generally accepted that NSAIDs exert pro-ulcerogenic activity related to their ability to inhibit endogenous prostaglandin synthesis. The latter results in an increase in gastric acid secretion and a decrease in the non-parietal components of the gastric secretion such as sodium bicarbonate and mucus, as well as in reduction of the mucosal blood flow [25–27]. The focal ischemic areas, which subsequently develop into erosions and inadequate perfusion, may disturb cell metabolism with local release of tissue-damaging mediators such as oxygen-derived free radicals. The relationship between NSAID-evoked gastric mucosal lesions and lipid peroxidation in gastric
tissue evidenced by MDA production, reduced activity of glutathione peroxidase [12], superoxide dismutase [28], catalase, and decreased glutathione concentrations [29], has already been established. Altered glutathione homeostase and enzymatic antioxidant defense, together with decrease in PGE2 levels, were found also for the preferential COX-2 inhibitor meloxicam [30]. On the other side, antioxidants and free radical scavengers protect the gastric mucosa against NSAID-induced injury, as it has been shown for allopurinol and dimethylsulfoxide [4, 11], superoxide dismutase and catalase [12, 14], and alpha-tocopherol [31]. In the present work, we found that paracetamol and propacetamol tended to reduce the morphometrical and histomorphological signs of gastric mucosal damage induced by ASA. A dose-dependent decrease in ulcer area was found after propacetamol. The involvement of extensive lipid peroxidation in ASA-induced gastric mucosal damage was evidenced by the strong accumulation of MDA in gastric mucosa associated with an adaptive rise in glutathione values. As can be seen from Figure 2, lower doses of paracetamol were more effective to inhibit the generation of MDA than the higher doses. This could be explained with the ability of paracetamol to modulate oxidation through two mechanisms, either as a co-substrate for the peroxidase of cyclooxygenase or by conversion to the free radical NAPQI. All tested substances decreased MDA level, but propacetamol as well as PGE2 diminished MDA in a manner corresponding to the reduction of the lesion area and maintained high values of glutathione and UA. These findings allow suggesting that the tested agents diminished the lipid peroxidation and supported the defense reaction in response to oxidative stress. As glutathione is accepted as a major mechanism for cellular protection against agents which generate oxidative stress
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[32], keeping gastric mucosal levels of glutathione high could be assumed as an important effect of paracetamol, propacetamol and PGE2. Plasma MDA level is a complex marker of the tissue lipid peroxidation. Its elevation after ASA was smaller than those registered in gastric mucosa. The water soluble antioxidant uric acid (urate at pH 7.4) is an important part of plasma antioxidant defense. Since its concentration in plasma is very high (almost 10-fold higher than other antioxidants such as Vitamin C and E), UA contributes significantly to plasma antioxidant capacity [33–38]. Urate has been proved to be an excellent scavenger of hypochlorous acid, hydroxyl radicals and peroxynitrite [38]. As generally plasma antioxidant levels are modulated by radical overload, it may be suggested that ASA-induced lipid peroxidation was associated with an adaptive rise in plasma antioxidant UA. Paracetamol and propacetamol as well as PGE2 reduced to a lesser extent lipid peroxidation in plasma than in gastric mucosa, but maintained elevated levels of the selective plasma antioxidant UA. On the other hand, urate is found to prevent oxidative inactivation of COX [37]. This effect arises from the ability of urate to serve as an electron-donor co-substrate for the hydroperoxidase activity of COX and even to stimulate the production of PGE from arachidonic acid [39]. In that sense UA concentrations are related to PG levels. Gastroprotective effect of paracetamol has been demonstrated in different models of acute ulcerations but the underlying mechanism(s) remains unclear: in waterimmersion stressed rats paracetamol significantly reduced gastric mucosal damage, associated with promoted production of PGE2 in gastric mucosa and decreased gastric and plasma glutathione levels [17]. On the other hand, in human gastric and duodenal mucosa paracetamol was reported significantly to reduce PG production, without mucosal injury [40], ulceration or bleeding [19, 41]. Protection against ischemia/reperfusion-induced gastric injury and inhibition of gastric lipid peroxidation was reported by Nakamoto et al. [42]. Our previous investigations showed that paracetamol and its pro-drug propacetamol protected gastric mucosa against cold/restraint stress and prevented gastric lipid peroxidation [43]. In conclusion, we found that paracetamol and propacetamol reduced ASA-induced gastric musocal injury. Propacetamol revealed more pronounced gastroprotective effect, similar to that exhibited by PGE2. The gastroprotective effect of these compounds was comparable to the protective effect of PGE2 on gastric mucosa and was accompanied by significant decrease of the gastric lipid peroxidation (evaluated as MDA-generation) and support to the adaptive mechanisms against oxidative stress (by means of glutathione and uric acid). Taking into consideration the role of lipid peroxidation in NSAID-gastric injury, the interference with gastric oxidative status could be suggested as a possible way through which paracetamol and propacetamol exert protective effect against ASA-induced gastric ulceration.
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REFERENCES 1. Simmons HF, James RC, Harbison RD, Roberts SM. Depression of glutathione by cold restraint in mice. Toxicology 1990; 161: 59–71. 2. Yegen B, Dedeoglu A, Aycac I, Oktay S, Yalcin AS. Effect of cold restraint stress on glutathione and lipid peroxide level in the liver and glandular stomach of rat. Pharmacol Res 1990; 22: 45–8. 3. Das KD, Banerjee RK. Effect of stress on the antioxidant enzymes and gastric ulceration. Mol Cell Biochem 1993; 125: 115–25. 4. Salim AS. Use of scavenging oxygen-derived free radicals to protect the rat against aspirin- and ethanol-induced erosive gastritis. J Pharmacol Sci 1992; 81: 943–6. 5. Hirokawa M, Miura S, Yoshida H, Kurose I, Shigematsu T, Hokari R, Higuchi H, Watanabe N, Yakoyama Y, Kimura H, Sato S, Ishii H. Oxidative stress and mitochondrial damage precedes gastric mucosal cell death induced by ethanol administration. Alcohol Clin Exp Res 1998; 22(Suppl 3): 111S–4S. 6. Schiorff EC, Husain K, Somani SM. Dose-and time-dependent effects of ethanol on plasma antioxidant system in rat. Alcohol 1999; 17: 97–105. 7. Hernandez-Munoz R, Montiel-Ruiz C, Vazquez-Martinez O. Gastric mucosal cell proliferation in ethanol-induced chronic mucosal injury is related to oxidative stress and lipid peroxidation in rats. Lab Invest 2000; 80: 1161–9. 8. Tanaka J, Yuda Y. Role of lipid peroxidation in gastric mucosal lesions induced by ischemia-reperfusion in the pylorus-ligated rat. Biol Pharm Bull 1993; 16: 29–32. 9. Farinati F, Della Libera G, Cardin R, Molari A, Plebani M, Rugge M, Di Mario F, Naccarato R. Gastric antioxidant, nitrites, and mucosal lipoperoxidation in chronic gastritis and Helicobacter pylori infection. J Clin Gastroenterol 1996; 22: 275–81. 10. Esplugues JV, Whittle BR. Gastric damage following local intra-arterial administration of reactive oxygen metabolites in the rat. Br J Pharmacol 1989; 97: 1085–92. 11. Takeuchi K, Ueshima K, Hironaka Y, Fujioka Y, Matsumoto J, Okabe S. Oxygen free radicals and lipid peroxidation in the pathogenesis of gastric mucosal lesions induced by indomethacin in rats. Relation to gastric hypermotility. Digestion 1991; 49: 175–84. 12. Yoshikawa T, Naito Y, Kishi A, Tomii T, Kaneko T, Iinuma S, Ishikawa H, Yasuda H, Takahashi S, Kondo M. Role of active oxygen, lipid peroxidation, and antioxidants in the pathogenesis of gastric mucosal injury induced by indomethacin in rats. Gut 1993; 34: 732–7. 13. Naito Y, Yoshikawa T, Yoshida N, Kondo M. Role of oxygen radical and lipid peroxidation in indomethacin-induced gastric mucosal injury. Dig Dis Sci 1998; 43: 30S–4S. 14. Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol- and aspirin-induced gastric mucosal injury. Dig Dis Sci 1987; 32: 1395–401. 15. Flower RJ, Vane JR. Inhibition of prostaglandin synthetase in brain explains the antipyretic activity of acetaminophen. Nature 1972; 240: 410–1. 16. Konturek SJ, Brzozowski T, Piastucki I, Radecki T. Prevention of ethanol and aspirin-induced gastric mucosal lesions by paracetamol and salicylate in rats: role of endogenous prostaglandins. Gut 1982; 23: 536–40. 17. Omura H, Kamasaki Y, Kawasaki H, Itoh T. Effect of acetaminophen on stress-induced gastric mucosal lesions in rats. Res Commun Mol Pathol Pharmacol 1994; 86: 297–310. 18. Poon YK, Cho CH, Ogle CW. The protective mechanisms of paracetamol against ethanol-induced gastric mucosal damage in rats. J Pharm Pharmacol 1989; 41: 563–5. 19. Lanza FL, Codispoti JR, Nelson EB. An endoscopic comparison of gastroduodenal injury with over-the-counter doses of ketoprofen and acetaminophen. Am J Gastroenterol 1998; 93: 1051–4. 20. Farkas JC, Larrouturou P, Morin JP, Laurian C, Hichet J, Cormier JM, Boccard E. Analgesic efficacy of an injectable acetaminophen versus a dipyrone plus pitofenone plus fenpiverinium association after abdominal aortic repair. Curr Therap Res 1992; 51: 19–26. 21. Bhattacharya SK, Goel RK, Bhattacharya SK, Tandon R. Potentiation of gastric toxicity of ibuprofen by paracetamol in the rat. J Pharm Pharmacol 1991; 43: 520–1.
148 22. Porter N, Norton J, Ramdas J. Cyclic peroxidase and thiobarbituric assay. Biochem Biophys Acta 1976; 441: 596–9. 23. Hissin P, Hilf A. A fluorimetric method for determination of oxidized and reduced glutathione in tissues. Ann Biochem 1976; 74: 214– 26. 24. Bergmeyer HU. Methods of enzymatic analysis. Beartfield Beach, FL: Verlag Chemie Int., 1981. 25. Whittle BR. Relationship between prevention of rat gastric erosions and the inhibition of acid secretion by prostaglandins. Eur J Pharmacol 1976; 40: 223–39. 26. Whittle BR. The defensive role played by the gastric microcirculation. Methods Findings Exp Clin Pharmacol 1989; 11(Suppl 1): 35–43. 27. Bolton JP, Palmer D, Cohen M. Stimulation of mucus and nonparietal secretion by the E2 prostaglandins. Dig Dis Sci 1982; 27: 359–64. 28. Tanaka J, Yuda Y. Lipid peroxidation in gastric mucosal lesions induced by indomethacin in rats. Biol Pharm Bull 1996; 19: 716– 20. 29. Hassan A, Martin E, Puig-Parellada P. Role of antioxidants in gastric mucosal damage induced by indomethacin in rats. Methods Findings Exp Clin Pharmacol 1998; 20: 849–54. 30. Villegas I, Martin MJ, LaCasa C, Motilva V, Alarcon de la Lastra C. Effects of meloxicam on oxygen radical generation in rat gastric mucosa. Inflamm Res 2000; 49: 361–6. 31. Sugimoto N, Yoshida N, Yoshikawa T, Nakamuara Y, Ichikawa H, Naito Y, Kondo M. Effect of Vitamin E on aspirin-induced mucosal injury in rats. Dig Dis Sci 2000; 45: 599–605. 32. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. Free Radic Res 1999; 31: 273–300. 33. Keniia MV, Shkurat TP, Lukash AI, Guskov EP. Changes of uric acid levels in rat tissues as a systemic reaction to hyperoxia. Aviakosm Ekolog Med 1993; 27: 37–43.
Pharmacological Research, Vol. 46, No. 2, 2002 34. Becker BF. Towards the physiological function of uric acid. Free Radic Biol Med 1993; 6: 615–31. 35. Siems WG, van-Kuijk FJ, Maass R, Brenke R. Uric acid and glutathione levels during short-term whole body cold exposure. Free Radic Biol Med 1994; 16: 299–305. 36. Schrod L, Neuhaus T, Speer CP, Girschick H. Possible role of uric acid as an antioxidant in premature infants. Biol Neonate 1997; 72: 102–11. 37. Ghiselli A, Serafini M, Natella F, Scacci C. Total antioxidant capacity as a tool to assess redox status: critical review and experimental data. Free Radic Biol Med 2000; 29: 1106–14. 38. Kopprasch S, Richter K, Leonahardt W, Pietzsch J, Grassler J. Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes. Mol Cell Biochem 2000; 206: 51–6. 39. Gale PH, Egan RW. Prostaglandin endoperoxide synthase-catalyzed oxidation reactions. In: Pryor WA, ed. Free radicals in biology. Orlando: Academic Press, 1984: 1–34. 40. Konturek SJ, Obtlowicz W, Sito E. Distribution of prostaglandins in gastric and duodenal mucosa of healthy subjects and duodenal ulcer patients: effect of aspirin and paracetamol. Gut 1981; 22: 283–9. 41. Jerussi TP, Caubet JF, McCray JE, Handley DA. Clinical endoscopic evaluation of the gastroduodenal tolerance to (R)-ketoprofen, (R)-flurbiprofen, racemic ketoprofen and paracetamol: a randomized, single-blind, placebo-controlled trial. J Clin Pharmacol 1998; 38(Suppl 2): 19S–24S. 42. Nakamoto K, Kamisaki Y, Wada K, Kawasaki H, Itoh T. Protective effect of acetaminophen against acute gastric mucosal lesions induced by ischemia-reperfusion in the rat. Pharmacology 1997; 54: 203–9. 43. Galunska B, Frangov P, Marazova K, Yankova T. Paracetamol and propacetamol in stressed rats: changes in lipid peroxidation in blood red cells and plasma and stomach tissue. IMRA 1999; 3: 208.