Fd Chem. Toxic. Vol. 30, No. 6, pp. 525-531, 1992 Printed in Great Britain.All rights reserved

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A S T U D Y OF URIC ACID P R E T R E A T M E N T FOR THE P R O T E C T I O N OF RAT GASTRIC M U C O S A A G A I N S T TOXIC D A M A G E A. M. AL-BEKAIRI*,S. QURESHI*,M. M. AHMED*,M. AFZALt and A. H. SHAHt+ *Quality Control and Research Laboratory, Experimental Animal Care Centre, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11541, fCentral Laboratories, Ministry of Health, PO Box 59082, Riyadh 11525, Saudi Arabia (Accepted 8 January 1992)

Abstract--Uric acid was evaluated for its potential to protect the gastric mucosa against the injuries caused by 80% ethanol, 0.6 M-HCI and 0.2 M-NaOH in rats. Uric acid at doses of 50, 100 or 300 mg/kg body weight provided dose-dependent protection against the ulcerogeniceffects of all three agents. Other effects caused by ethanol only were studied. Serum uric acid concentrations were statistically significantly increased by both uric acid and ethanol treatments. Treatment of rats by gavage with 1 ml 80% ethanol was found to cause depletion of stomach-wall mucus, to lower the concentrations of protein, nucleic acids and non-protein sulphhydryl groups in the stomach wall, and to cause histopathological lesions, including necrosis, erosions, congestion and haemorrhage, of the stomach wall. Treatment with uric acid, at doses of 50, 100 or 300 mg/kg body weight, by gavage, provided some measure of protection against all of these effects, and the protection was generallydose dependent. The protective effects of uric acid against damage to the gastric-wall mucosa may be mediated through its effects on mucus production and non-protein sulphhydryl concentrations, and/or its free-radical scavenging properties.

INTRODUCTION

MATERIALS AND METHODS

Several attempts have been made to identify antiulcer drugs from natural sources (Akira et al., 1986; Al-Yahya et al., 1989; Best et al., 1984; Parmar et al., 1988; Rafatullah et al., 1990). It has been proposed that some natural antioxidants might protect the gastric mucosa by scavenging free radicals (Szabo, 1989; Tariq, 1988). Uric acid is known to have antioxidant activity at physiological concentrations (Ames et al., 1981; Niki et al., 1986). Uric acid is present at high concentration in human blood (White et al., 1968) and saliva (Soronsen, 1978), and it has been postulated that in conjunction with lactoperoxidase it may be involved in the defence against cancer (Ames, 1983). Recent studies have confirmed that uric acid is a potent antimutagen (A1-Bekairi et al., 1991a), which also inhibits the biochemical changes induced by cyclophosphamide in mice (A1-Bekairi et al., 1991b). However, there are as yet no reports on the effect of oral uric acid treatment on the gastro-intestinal tract. In continuation of our work on the in vivo effects of uric acid (A1-Bekairi et al., 1991b) we now report on the action of uric acid on gastric mucosal damage induced by different necrotic agents in rats.

Male Wistar albino rats (bred at the Experimental Animal Care Centre, King Saud University) and all roughly the same age, weighing 150 to 200 g, were used in the present study. All the animals were maintained under controlled condition of temperature, humidity and light and were provided with purina chow and water ad lib. The rats were randomly assigned to control and treatment groups, and were fasted for 36 hr with access to water ad lib. The necrotic agents, 80% ethanol, 0.6 M-HCI and 0.2M-NaOH, were previously found to produce gastric lesions (Robert, 1979). An arbitrary close of 300 mg uric acid/kg body weight (Sigma Chemical Co., St Louis, MO, USA) provided significant protection against necrotic agents in an experimental trial and hence a dose range of 50 to 300 mg uric acid/kg was used in the present study. An aqueous solution of uric acid or distilled water alone was administered by garage to the fasted rats 30min before oral treatment by gavage with 1 ml 80% ethanol (0.6 M-HC1 and 0.2 M-NaOH were used only in the cytoprotection studies). The animals were killed under anaesthesia, using ether 1 hr after the treatment with the necrotic agents. Serum uric acid concentrations. Serum uric acid levels were determined in samples taken from animals in each group, by an enzymatic colorimetric method using test-combination, Urica-quant (Boehringer Mannheim GmbH, Diagnostica, Germany) reagents; the measurements were carried out in a spectrophotometer, Ultrospec II (LKB).

Abbreviations: DTNB = 5-5'-dithiobis-(2-nitrobenzoicacid); NP-SH = non-protein sulphhydryl groups; TCA = trichloroacetic acid. ~To whom correspondence should be addressed.

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Cytoprotection studies. The s t o m a c h o f each of the animals was excised a n d opened along the greater curvature. After washing with n o r m a l saline the gastric lesions were quantified using a binocular magnifier. The ulcers were scored according to the m e t h o d o f Valvaci et al. (1982). Gastric-wall mucus determination. The modified procedure o f C o r n e et al. (1974) was used to determine gastric-wall mucus. The glandular segments from the s t o m a c h s were removed a n d weighed. Each segment was transferred immediately to 1% alcian blue solution (in sucrose solution, buffered with sodium acetate, p H 5), a n d the excess dye was removed by rinsing with sucrose solution. The dye complexed with the gastric wall mucus was extracted with m a g n e s i u m chloride. A 4-ml sample o f blue extract was then shaken with an equal volume o f diethyl ether. The resulting emulsion was centrifuged a n d the a b s o r b a n c e o f the aqueous layer was recorded at 580 nm. The q u a n t i t y o f alcian blue extracted/g (net) o f glandular tissue was then calculated. Estimation o f protein and nucleic acids. The levels of proteins a n d nucleic acids in the s t o m a c h were determined according to the following procedure: the s t o m a c h s were rapidly dissected from the animals, frozen in liquid nitrogen and stored at - 2 0 ° C until they were analysed for total proteins and nucleic acids ( R N A , D N A ) . Total protein was determined by the m e t h o d of Lowry et al. (1951). The m e t h o d described by B r e g m a n (1983) was used to determine the levels of nucleic acids. Tissues were h o m o g e n i z e d a n d the h o m o g e n a t e was susp e n d e d in ice-cold TCA. After centrifugation, the pellet was extracted with ethanol. D N A was determined by treating the nucleic acid extract with diphenylamine reagent a n d m e a s u r i n g the intensity o f the blue colour at 6 0 0 n m . F o r quantification of R N A , the nucleic acid extract was treated with orcinol a n d the intensity of the green colour was m e a s u r e d at 660 nm. Nucleic acids were quantified using the s t a n d a r d curves. The d a t a were analysed using Student's t-test. Estimation o f non-protein sulphhydryl groups (NP-SH). Gastric mucosal N P - S H was m e a s u r e d according to the m e t h o d of Sedlek a n d Lindsay (1968). The glandular s t o m a c h was removed a n d h o m o g e n i z e d in ice-cold 0.02M-ethylenediaminetetraacetic acid. The h o m o g e n a t e was mixed with

Table 1. Effects of treatment by gavage with uric acid and ethanol (80%) on serum uric acid concentrations in rats Treatment and Serum uric uric acid dose No. of acid concn (mg/kg body weight) rats (mg %) Control (distilled water) 6 0.84 + 0.06 Uric acid (50) 6 1.12 + 0.12 Uric acid (100) 5 1.14 + 0.10" Uric acid (300) 5 1.21 +0.11" Ethanol, 80%, 1 ml 5 1.06 + 0.02* Uric acid (50) + ethanol, 80%, 1 ml 5 1.28 + 0.08"t Uric acid (100) + ethanol, 80%, 1 ml 5 1.29 + 0.08t Uric acid (300) + ethanol, 80%, 1 ml 5 1.57 + 0.14~: Values are means + SEM; those marked with superscripts differ significantly (Student's t-test) from the value for the group indicated (*P < 0.05 compared with the distilled water control group; tP(

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Gastric mucosal protection by uric acid Bregman A. A. (Editor) (1983) Spectrophotometric analysis of DNA and RNA. In Laboratory Investigations and Cell Biology. pp. 51-60. John Wiley, New York. Come S. J., Morrisey S. M. and Woods R. J. (1974) A method for the quantitative estimation of gastric barrier mucus. Journal of Physiology 2,42, 116-117. Halliwell B. (1991) Introduction to free radicals in human disease. Saudi Medical Journal 12, 13-19. Henagan J. M., Smith G. S., Miller T. A. and Schmidt K. L. (1986) N-acetyl-cystein and prostaglandin: comparable protection against experimental ethanol injury in the stomach independent of mucus thickness. Annals of Surgery 204, 698-704. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Meadows J. and Smith R. C. (1986) Uric acid protection of nucleobases from ozone-induced degradation. Archives of Biochemistry and Biophysics 246, 838-845. Miller T. A. and Lid K. Y. J. (1985) Nonprotein sulfhydryl compounds in canine gastric mucosa: effect of PGE 2 and ethanol. American Journal of Physiology 12, 137-144. Niki E., Sait M., Yashikawa Y., Yamamoto Y. and Kamiya Y. (1986) Oxidation of lipids XII. Inhibition of oxidation of soybean phosphatidylcholine and methyl linoleate in aqueous dispersions by uric acid. Bulletin of the Chemical Society of Japan 59, 471-477. Parmar N. S., Tariq M. and Ageel A. M. (1988) Gastric antiulcer and cytoprotective effect of selenium in rats. Toxicology and Applied Pharmacology 91, 122 130. Rafatullah S., Tariq M., A1-Yahya M. A., Mossa J. S. and Ageel A. M. (1990) Evaluation of turmeric (Curcuma longa) for gastric and duodenal anti.ulcer activity in rats. Journal of Ethnopharmacology 29, 25-34. Robert A. (1979) Cytoprotection by prostaglandins. Gastroenterology 77, 761-767. Robert A., Bottcher W., Golanska E. and Kauffman G. L., Jr. (1985) Lack of correlation between mucus gel thickness and gastric cytoprotection in rats. Gastroenterology 86, 670-674.

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Rogers C., Brown A. and Szabo S. (1988) Gastric mucosal protection by new aryl sulfhydryl drugs. Digestive Diseases and Sciences 33, 324-329. Sadlak J. and Lindsay R. H. (1968) Estimation of total protein-bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry 25, 192-195. Sorensen L. B. (1978) Uric acid. In Handbook of Experimental Pharmacology. Vol. 51. Edited by W. N. Kelley and I. M. Weiner. pp. 325 336. Springer, New York. Szabo S. (1989) Role of radicals in aspirin-induced gastric mucosal lesions. Abstract 3rd Interscience World Conference on Inflammation, Monte Carlo, Principality of Monaco, 15 18 March. p. 414. Szabo S., Trier J. S. and Frank P. W. (1981) Sulfhydryl compounds may mediate gastric protection. Science, New York 214, 200-202. Tariq M. (1988) Gastric antiulcer and cytoprotective effect of vitamin E. Research Communications in Chemical Pathology and Pharmacology 60, 87 88. Terao J., Lim B. P., Murakami H. and Matsushita S. (1987) Quinone formation from carcinogenic benzo-a-pyrene mediated by lipid peroxidation in phosphatidylcholine liposomes. Archives of Biochemistry and Biophysics 254, 472-481. Terao J. and Matsushita S. (1989) The mechanisms of quinone formation from carcinogenic benzo-a-pyrene mediated by lipid peroxidation in liposomal suspension. In Oxygen Radicals in Biology and Medich~e. Edited by M. G. Simic, K. A. Taylor, J. F. Ford and C. Von Sonntag. pp. 769-773, Plenum, New York. Valcavi U., Caponi R., Brambilla A., Palmira F., Minoja F., Bernini F., Mustani R. and Fumagall R. (1982) Gastric antisecretory, antiulcer and cytoprotective properties of 9-hydroxy-19,20-bis-nor-prostanic acid in experimental animals. Arzneimittel-Forschung 32, 657-663. Wallace J. L. and Whittle B. J. R. (1986) Role of mucus in the repair of gastric epithelial damage in the rat. Gastroenterology 91, 603-611. White A., Handler P, and Smith E. L. (1968) Principles of Biochemistry. pp. 628-631. McGraw-Hill, New York.

A study of uric acid pretreatment for the protection of rat gastric mucosa against toxic damage.

Uric acid was evaluated for its potential to protect the gastric mucosa against the injuries caused by 80% ethanol, 0.6 m-HCl and 0.2 M-NaOH in rats. ...
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