- Email: [email protected]
Mechanism of the beneficial effects of dantrolene sodium on ethanol-induced acute gastric mucosal injury in rats
Pharmacological Research, Vol. 45, No. 5, 2002 doi:10.1006/phrs.2002.0951, available online at http://www.idealibrary.com on
MECHANISM OF THE BENEFICIAL EFFECTS OF DANTROLENE SODIUM ON ETHANOL-INDUCED ACUTE GASTRIC MUCOSAL INJURY IN RATS a,∗ , SEYITHAN TAYSIb , FEVZI POLATc and FATMA GOC ˘ ¨ ¸ ERa ¨ UKOKURO ¨ MEHMET EMIN BUY GLU
Departments of a Pharmacology, b Biochemistry, Medical Faculty, and c Biotechnology Application and Research Centre, Atat¨urk University, Erzurum, Turkey Accepted 28 January 2002
In our study, we examined anti-ulcerogen and antioxidant effects of dantrolene sodium on ethanolinduced gastric lesions in rats. Dantrolene sodium was administered intraperitoneally (i.p.) in several doses, and famotidine was used at a dose of 20 mg kg−1 . It was found that pretreatment with dantrolene sodium at doses of 1, 5 and 10 mg kg−1 significantly reduced ethanol-induced gastric damage and malondialdehyde levels, and significantly increased antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. We conclude that dantrolene sodium clearly has antioxidant properties and that the protective effect of dantrolene sodium against ethanol-induced gastric mucosal lesion, at least in part, depends upon the reduction in the lipid peroxidation and an increase in the activity of antioxidant enzymes SOD and GSH-Px. c 2002 Elsevier Science Ltd. All rights reserved.
K EY WORDS : dantrolene sodium, ethanol, rat, ulcer, antioxidant.
INTRODUCTION Reactive oxygen species (ROS) are continuously produced during normal physiologic events, and are removed by antioxidant defence mechanisms [1]. In pathological conditions, ROS are over produced and result in lipid peroxidation and oxidative damage. The imbalance between ROS and antioxidant defence mechanisms leads to oxidative modification in the cellular membrane or intracellular molecules [2]. Some studies have revealed that ROS and lipid peroxidation are implicated in the pathogenesis of ethanol-induced gastric lesions and gastrointestinal damage, and calcium is a very important mediator in these events [3–5]. Lipid peroxide content and oxygen-derived free radicals increase in hypoxic tissue. This situation results in severe changes at the cellular level and causes plasma membrane damage, intracellular calcium accumulation (which plays a role in free radical mediated lipid peroxidation), cell death, exfoliation and epithelial erosion [6, 7]. An increase in the intracellular calcium level activates the some calcium-dependent enzymes and worsens the oxidative damage [5]. Malondialdehyde (MDA) is an indicator of lipid peroxidation, and its level increases in tissues when ∗ Corresponding author. Department of Pharmacology, Medical Faculty,
Atat¨urk University, TR-25240, Erzurum, Turkey. E-mail: [email protected] 1043–6618/02/$ - see front matter
they are exposed to oxidative stress. Various endogenous antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) play an important role in the elimination of oxygen free radicals (OFR) and lipid peroxides, and protect cells against the toxic effects of OFR and lipid peroxides [1, 8]. Dantrolene sodium, a peripherally acting skeletal muscle relaxant, is used in the treatment of muscle spasticity, malignant hyperthermia and neuroleptic malignant syndrome and depresses excitation– contraction coupling in the muscle fibre by inhibiting calcium release from the sarcoplasmic reticulum [9]. It has no effect on neuromuscular transmission and does not affect the electrical properties of the skeletal muscle membrane [10]. Additionally, it is suggested that the dantrolene sodium affects the membranal calcium channel of smooth muscle cells and inhibits calcium influx [11, 12]. On the other hand, it is shown that the dantrolene sodium has a cytoprotective effect on the neuron or liver cells, but there is no information about its effect on antioxidant enzyme activity [13–15]. This study aimed to examine anti-ulcerogen and antioxidant effects of dantrolene sodium on ethanolinduced gastric lesions in rats. For this purpose, we studied the effects of dantrolene sodium on SOD and GSH-Px activity, and changes in lipid peroxidation which were determined by measuring MDA level. c 2002 Elsevier Science Ltd. All rights reserved.
422
MATERIALS AND METHODS
Animals Forty eight albino Sprague–Dawley male rats with a weight of 190–225 g were used for the experiment. The rats were fed with standard laboratory chow and water before the experiment. The laboratory was windowless with automatic temperature (22 ± 1 ◦ C) and lighting controls (14 h light/10 h dark). Rats were divided into six equal groups (n = 8) and housed in cages. Twenty-four hours before the experiment, the rats were fasted and allowed access to water ad libitum.
Ulcer study The anti-ulcerogenic effect of dantrolene sodium was investigated with the ethanol-induced ulcer model. On the day of the experiment, groups 1–3 were injected with 1, 5 and 10 mg kg−1 dantrolene sodium, group 4 was injected with 20 mg kg−1 famotidine and group 5 was injected with saline solution. All of the drugs were administered intraperitoneally (i.p.) and at the same volume (0.5 ml). Following a 30 min period, all the animals were given 1 ml of ethanol (70%) by gavage. One hour after the administration of ethanol, the animals were killed by decapitation. The stomach was removed and opened along the greater curvature and washed in physiological saline solution. For the measurement of the gross gastric mucosal lesions, the freshly excised stomach was laid flat and the mucosal lesions were traced on clear acetate paper. Gross mucosal lesions were recognized as haemorrhage or linear breaks (erosions) with damage to the mucosal surface. The area of stomach and gross lesions were approximately calculated by planimetry using a simple magnifier. The results were translated to the term of ‘total ulcer area/total gastric area’ and these were expressed as an ulcer index (%). After this evaluation, gastric tissue MDA, SOD and GSH-Px levels were determined. Group 6 was used as control for biochemical analysis.
Biochemical analysis After the macroscopic evaluation, stomachs were kept in an ice bath until homogenization. They were homogenized in 10-fold physiological saline solution by using homogenizer (Omni Accessory Pack International Homogenizer, USA). The homogenate was centrifuged at 10 000 g for 1 h to remove debris. Clear upper supernatant was taken and enzymatic assays were carried out in this fraction. All the procedures were performed at +4 ◦ C throughout the experiments. SOD activity was detected according to Sun and coworkers [16]. In this method, xsantine–xsantine oxidase complex produces the superoxide radicals, which react with nitrobluetetrazolium (NBT) to form the farmasone compound. SOD activity is measured at 560 nm by detecting the inhibition of this reaction. By using a blank study of all the reagents present except for the supernatant sample and determining the absorbance
Pharmacological Research, Vol. 45, No. 5, 2002 Table I Variability (%CV) of the parameters measured in the study % CV Within Between
MDA
SOD
GSH-Px
7.6 8.5
2.3 3.1
3.9 4.7
of the sample and blank, activity is calculated. One SOD unit was defined as the enzyme amount causing 50% inhibition in the NBTH2 reduction rate. SOD activity was also expressed as U mg−1 protein of stomach tissue sediment. GSH-Px activity was measured according to the Paglia and Valentina method [17]. In this method, GSH-Px catalyses the oxidation of glutathione in the presence of tert-butyl hydroperoxide (tBH). Oxidized glutathione is converted into the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. The reduction in the absorbance of NADPH at 340 nm is measured. By measuring the absorbance change per minute and by using the molar extinction coefficient of NADPH, GSH-Px activity of stomach tissue is calculated. GSH-Px activities were expressed as mU mg−1 protein of stomach tissue sediment. MDA levels were determined according to Ohkawa et al. [18]. In this method, to samples less than 0.2 ml of 10% (w/v) tissue homogenate were added 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH, and 1.5 ml of 0.8% aqueous solution thiobarbituric acid (TBA). The mixture was made up to 4.0 ml with distilled water, and heated at 95 ◦ C for 60 min. After cooling, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15 : 1, v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance at 532 nm was measured. Total TBAreactive materials were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 × 105 cm−1 M−1 . MDA level was expressed as nmol g−1 . The amount of protein was determined by using the Lowry method [19]. Biochemical measurements were carried out at room temperature by using a spectrophotometer (CEC˙IL CE, 3041, UK). The precision analysis results (within, 10 analyses with the same sample on the same day, and between, one analyses with the same sample on consecutive 10 days, batch analyses) were given in Table I.
Chemicals Dantrolene sodium, reduced glutathione (GSH), NADPH, glutathione disulfide reductase, H2 O2 , xanthine, xanthine oxidase, nitrobluetetrazolium, trichloroacetic acid and TBA n-butanol were purchased from Sigma Chemical Co. (St Louis, MO, USA). Famotidine was obtained from ‘Mustafa Nevzat A.S¸.’
Pharmacological Research, Vol. 45, No. 5, 2002
423 30
Table II Effects of datrolene sodium and famotidine on ethanol-induced gastric mucosal injury
Ethanol (n = 8) Dantrolene 1 mg kg−1 + ethanol (n = 8) Dantrolene 5 mg kg−1 + ethanol (n = 8) Dantrolene 10 mg kg−1 + ethanol (n = 8) Famotidine 20 mg kg−1 + ethanol (n = 8)
1.96 ± 0.07 0.42 ± 0.02∗ 0.54 ± 0.02∗ 0.09 ± 0.006∗ 1.05 ± 0.06∗
nmol /g tissue
Group
25
Mean ± SEM (ulcer index, %)
20 15 10
Ethanol: 70%, 1 ml/animal; ∗ P< 0.001, vs ethanol. 5
All values were expressed as mean ± SEM. Comparison of means between groups were analysed by Student’s t-test or one way analysis of variance (ANOVA). A P value of less than 0.05 was considered significant.
Effects on gastric tissue lipid peroxidation in ethanol-administered rats The administration of ethanol increased the MDA level in rat gastric tissue. In contrast, pretreatment with dantrolene sodium significantly decreased the MDA levels in a dose dependent manner, as compared with ethanol alone and there was a statistical significance between the effects of dantrolene sodium doses used on MDA level (by the Tukey test, F = 28.675, P < 0.001). Additionally, famotidine was found to prevent the rise in MDA level. MDA levels of stomach tissue are shown in Fig. 1.
in e
0 fa m
ot id
-1 da nt
-5 da nt
-1 da nt
an ol et h
12 10 8 6 4 2
e fa
m
ot
id in
0 da nt -1
t-5 da n
da n
l
t-1
0 l
Per-oral administration of 70% ethanol produced multiple mucosal lesions in the rat stomach. Pretreatment with dantrolene sodium and famotidine were found to inhibit the ethanol-induced gastric mucosal injury in rats. Preventive effects of 1, 5 and 10 mg kg−1 dantrolene sodium were in a dose-dependent manner (percent decreases were 78.4, 72.5 and 95.3, respectively, compared to ethanol) and there was a statistical significance between the effects of dantrolene sodium doses used (by Kruskal–Wallis one way ANOVA, H = 28.198, P < 0.001). Famotidine also significantly inhibited the ethanol-induced gastric lesion (percent decrease was 46.2, compared to ethanol). Ulcer indices (UI) are shown in Table II.
14
co nt ro
Effects on acute gastric mucosal lesions induced by ethanol
16
U/mg protein
RESULTS
18
ha no
Statistical analysis
Fig. 1. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue MDA level in ethanol administered rats. N = 8, ∗ P< 0.05, ∗∗ P< 0.01, ∗∗∗ P< 0.001, compared to ethanol group, by Student’s t-test.
et
(Turkey). All other chemicals and reagents were obtained from the stores of the Department of Biochemistry, Medical Faculty, Atat¨urk University.
co
nt ro l
0
Fig. 2. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue SOD level in ethanol administered rats. N = 8, ∗ P< 0.01, ∗∗ P< 0.001, compared to ethanol group, by Student’s t-test.
Effects on gastric tissue SOD and GSH-Px enzyme activity SOD activity was found to be decreased in the ethanol-administered group. In contrast, SOD activity significantly increased in 1, 5 and 10 m kg−1 doses of dantrolene sodium administered rats and there was a statistical significance between the effects of dantrolene sodium doses used on SOD level (by Kruskal–Wallis one way ANOVA, H = 20.905, P < 0.001). It was found to be elevated significantly in famotidine administered rats (Fig. 2). GSH-Px activity was found to decrease in the gastric tissue after ethanol administration, but pretreatment with 1, 5 and 10 m kg−1 doses of dantrolene sodium resulted in a dose-dependent increase in the GSH-Px activity and there was a statistical
424
Pharmacological Research, Vol. 45, No. 5, 2002 1.2
mU/mg protein
1 0.8 0.6 0.4 0.2
ot id in e
0 fa
m
t-1 da n
-5 da nt
-1 da nt
ha no l et
co
nt ro l
0
Fig. 3. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue GSH-Px level in ethanol administered rats. N = 8, ∗ P< 0.05, ∗∗ P< 0.01 compared to ethanol group, by Student’s t-test.
significance between the effects of dantrolene sodium doses used on the GSH-Px level (by the Tukey test, F = 3.713, P < 0.05). Famotidine also increased the GSH-Px activity of gastric tissue, compared to ethanol (Fig. 3).
DISCUSSION Several factors such as increased vascular permeability, gastric motility and vagal activity, decreased gastric blood flow and protective prostaglandin levels play an important role in gastric ulcer pathogenesis. Chemicals (ethanol or HCl) and drugs (indomethacin or acetylsalicylic acid) are used for the peptic ulcer model in rats, some of them, such as ethanol or aspirin, cause gastric mucosal damage and promote the generation of ROS in this tissue [20, 21]. Some ROS scavenger or inhibitor drugs such as melatonin have protective effects on indomethacin- or ethanol-induced acute gastric injury in rats [22, 23]. In some studies, it was revealed that dantrolene sodium prevented lipid peroxidation in hippocampal neurons and enterocytes [24, 25]. Again, it was shown that pretreatment with nifedipine and verapamil prevented the lipid peroxidation and increased the antioxidant enzyme [26, 27]. Results of this study showed that dantrolene sodium prevented the gastric tissue damage against ethanol-induced stress in a dose dependent manner. Furthermore, it protected the gastric tissue against lipid peroxidation and increased the antioxidant enzymes’ (SOD and GSH-Px) activity. According to these, there are similarity and accordance between our study and the aforementioned studies [4, 24–27]. GSH is an important compound in cytoplasm against oxidative stress. GSH depletion results in enhanced lipid peroxidation and excessive lipid peroxidation can cause GSH consumption. GSH-Px activity is coupled with the
oxidation of GSH to GSSG, which can subsequently be reduced by GSH-reductase, with NADPH as the reducing agent [28]. Lipid peroxide accumulation is believed to be the cause of destruction and cell membrane damage. MDA is an index of lipid peroxidation, its level increases in gastric mucosa after ethanol-induced injury [23]. The present study demonstrated that ethanolinduced damage to the gastric mucosa is associated with an increase in MDA levels in rats. But, pretreatment with 1, 5 and 10 mg kg−1 doses of dantrolene sodium or 20 mg kg−1 dose of famotidine inhibited the increase of gastric tissue MDA levels. Furthermore, MDA levels in the 10 mg kg−1 dose of dantrolene sodium administered group were very close to the normal gastric tissue MDA level (non-ethanol or drugs). This result revealed that dantrolene sodium has clearly reduced the lipid peroxidation in gastric mucosal tissue damage induced by ethanol. SOD catalyses the conversion of the superoxide anion radical into O2 and H2 O2 . H2 O2 is highly toxic to cells, it causes DNA and membrane damage, and Ca2+ release from intracellular stores [1]. GSH-Px is an important enzyme that plays a role in elimination of H2 O2 and lipid hydroperoxides. GSH-Px also reduces peroxides by using reduced GSH. In the present study, an increase in ethanol-induced gastric lesions paralleled with a decrease of SOD and GSH-Px activity. On the other hand, these enzyme activities were increased by pretreatment with dantrolene sodium. 10 mg kg−1 of dantrolene sodium almost resulted in an increase of SOD activity up to the normal (non-ethanol) level. Dantrolene sodium not only increased the SOD and GSH-Px but also decreased the MDA formation. In the present study, famotidine, a histamine H2 receptor antagonist, also prevented the ethanol-induced gastric mucosal lesion formation, decreased the lipid peroxidation and increased the SOD and GSH-Px enzyme activity. In a previous study, we had shown that some calcium channel blockers and dantrolene sodium have a protective effect in ethanol-induced gastric mucosal injury [29]. Again, in a previous study, we had speculated that the protective effect of dantrolene sodium may be partly due to its blocker effect on internal calcium release from the endoplasmic reticulum, as well as external calcium influx via the membranal calcium channel. Also the results of our present study showed that dantrolene sodium prevented gastric mucosal damage and decreased lipid peroxidation induced by ethanol in rat stomach tissue, in a dose dependent manner. Recently, we revealed that dantrolene has in vitro antioxidant properties and we speculated that the cytoprotective effect of dantrolene sodium, at the same time, may be due to its antioxidant activity [30]. This protective effect of dantrolene sodium against ethanol-induced gastric mucosal lesions, besides being the inhibitor effect on intracellular calcium release from the endoplasmic reticulum or extracellular calcium influx, at least in part, depends upon the reduction in the lipid peroxidation and increase in the activity
Pharmacological Research, Vol. 45, No. 5, 2002
of antioxidant enzymes SOD and GSH-Px, and its antioxidant properties. In conclusion, dantrolene sodium clearly has antioxidant properties and it may be a valuable drug for peptic ulcer disease therapy and/or an antioxidant against oxidative stress. Further studies are required to clarify the anti-ulcer and antioxidant action of dantrolene sodium.
ACKNOWLEDGEMENT This study was supported by Atat¨urk University Research Foundation (project no 2000/45).
REFERENCES 1. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants, and human disease: where are we now. J Lab Clin Med 1992; 119: 598–620. 2. El-Habit OH, Saada HN, Azab KS, Abdel-Rahman M, El-Malah DF. The modifying effect of β-carotene on gamma radiationinduced elevation of oxidative reactions and genotoxicity in male rats. Mutat Res 2000; 466: 179–86. 3. Cho CH, Pfeiffer CJ, Misra HP. Ulcerogenic mechanism of ethanol and the activation of sulphanilyl on the rat stomach in vivo. J Pharm Pharmacol 1991; 43: 495–8. 4. Lutnicki K, Wrobel J, Ledwozyw A, Trebas-Pietras E. The effect of calcium ions on the intensity of peroxidation processes and the severity of ethanol-induced injury to the rat’s gastric mucosa. Arch Vet Pol 1992; 32: 125–32. 5. Bast A, Haenen GRMM, Doelman CJA. Oxidant and antioxidant: state of the art. Am J Med 1991; 91(Suppl. 3C): 2S–13S. 6. Sakaguchi S, Yokota K. Role of Ca2+ on endotoxin-sensitivity by galactosamine challenge: lipid peroxide formation and hepatotoxicity in zymosan-primed mice. Pharmacol Toxicol 1995; 77: 81–6. 7. Castilho RF, Kowaltowski AJ, Meinicke AR, Vercesi AE. Oxidative damage of mitochondria induced by Fe(II)citrate or t-butyl hydroperoxide in the presence of Ca2+ : effect of coenzyme Q redox state. Free Radic Biol Med 1995; 18: 55–9. 8. Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 1991; 91(Suppl. 3C): 14C–22C. 9. Miller RD. Skeletal muscle relaxant. In: Basic and clinical pharmacology. Katzung BG, ed. USA: Appleton and Lange, 1989: 323–33. 10. Kayaalp SO. Rasyonel Tedavi Y¨on¨unden Tıbbi Farmakoloji. 9th edn, Ankara: Hacettepe-Tas¸ Kitapc¸ılık, 2000: 822–7 (in Turkish). 11. Nasu T, Oosaka H, Shibata H. Dantrolene blocks the tonic contraction and calcium influx evoked by K+ in ileal longitudinal smooth muscle. Gen Pharmacol 1996; 27: 513–7. 12. Nasu T, Oosaka H. Dantrolene blocks the tonic contractions to carbacol and histamine with smaller effects on the phasic due to release of intracellular Ca2+ in ileal longitudinal muscle. Gen Pharmacol 1995; 26: 1591–6.
425 13. Wei H, Perry DC. Dantrolene is cytoprotective in two models of neuronal cell death. J Neurochem 1996; 67: 2390–8. 14. Yamagishi F, Komoda T, Ohnishi K, Itoh S. Protective effect of dantrolene sodium on carbon tetrachloride induced liver injury in the rat. Res Commun Chem Pathol Pharmacol 1993; 82: 237–40. ¨ ¸ 15. Gepdiremen A, D¨uzenli S, Hacım¨uft¨uoglu A, S¨uleyman H, Oztas S. The effects of dantrolene alone or combination with nimodipine in glutamate-induced neurotoxicity in cerebellar granular cell cultures of rat pups. Pharmacol Res 2001; 43: 241–4. 16. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497–500. 17. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158–69. 18. Ohkawa H, Ohishi N, Yagi K. Reaction of linoleic acid hydroperoxide with thiobarbituric acid. J Lipid Res 1978; 19: 1053–7. 19. Lowry O, Roscnbraugh N, Farr L, Randall R. Protein measurement with folin phenol reagent. J Biol Chem 1951; 182: 265–75. 20. Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol- or aspirin-induced gastric mucosal damage in rats. Dig Dis Sci 1987; 32: 1395–401. 21. Szelenyi I, Brune K. Possible role of oxygen free radicals in ethanol-induced gastric mucosal damage in rats. Dig Dis Sci 1988; 33: 865–71. 22. Brzozowski T, Konturek PC, Konturek SJ, Pajdo R, Bielanski W, Brzozowska I, Stachura J, Hahn EG. The role of melatonin and Ltryptophan in prevention of acute gastric lesions induced by stress, ethanol, ischemia, and aspirin. J Pineal Res 1997; 23: 79–89. 23. Alarcon de la Lastra C, Motilva V, Martin MJ, Nieto A, Barranco MD, Cabeza J, Herrerias JM. Protective effect of melatonin on indomethacin-induced gastric injury in rats. J Pineal Res 1999; 26: 101–7. 24. Gorowara S, Sapru S, Ganguly NK. Role of intracellular second messengers and reactive oxygen species in the pathophysiology of V. Cholera O139 treated rabbit ileum. Biochim Biophys Acta 1998; 1407: 21–30. 25. Yu Z, Luo H, Fu W, Mattson MP. The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis. Exp Neurol 1999; 155: 302–14. 26. Suzuki Y, Ishihara M, Segami T, Ito M. Anti-ulcer effects of antioxidants, quercetin, alpha-tocopherol, nifedipine and tetracycline in rats. Jpn J Pharmacol 1998; 78: 435–41. 27. Alican I, Toker F, Arbak S, Yegen BC, Yalcin AS, Oktay S. Gastric lipid peroxidation, glutathione and calcium channel blockers in the stress-induced ulcer model in rats. Pharmacol Res 1994; 30: 123–35. 28. La Casa C, Villegas I, Alarcon de la Lastra C, Motilva M, Martin Calero MJ. Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. J Ethnopharmacol 2000; 71: 45–53. 29. S¨uleyman H, B¨uy¨ukokuro˘glu ME, Gepdiremen A. Verapamil, diltiazem, nifedipine and dantrolene versus famotidine in prevention of ethanol-induced gastric ulcers in rats. Int Med J 2000; 7: 139–44. ¨ ˙I. In vitro 30. B¨uy¨ukokuro˘glu ME, G¨ulc¸in ˙I, Oktay M, K¨ufrevioglu O antioxidant properties of dantrolene sodium. Pharmacol Res 2001; 44: 491–4.
MECHANISM OF THE BENEFICIAL EFFECTS OF DANTROLENE SODIUM ON ETHANOL-INDUCED ACUTE GASTRIC MUCOSAL INJURY IN RATS a,∗ , SEYITHAN TAYSIb , FEVZI POLATc and FATMA GOC ˘ ¨ ¸ ERa ¨ UKOKURO ¨ MEHMET EMIN BUY GLU
Departments of a Pharmacology, b Biochemistry, Medical Faculty, and c Biotechnology Application and Research Centre, Atat¨urk University, Erzurum, Turkey Accepted 28 January 2002
In our study, we examined anti-ulcerogen and antioxidant effects of dantrolene sodium on ethanolinduced gastric lesions in rats. Dantrolene sodium was administered intraperitoneally (i.p.) in several doses, and famotidine was used at a dose of 20 mg kg−1 . It was found that pretreatment with dantrolene sodium at doses of 1, 5 and 10 mg kg−1 significantly reduced ethanol-induced gastric damage and malondialdehyde levels, and significantly increased antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. We conclude that dantrolene sodium clearly has antioxidant properties and that the protective effect of dantrolene sodium against ethanol-induced gastric mucosal lesion, at least in part, depends upon the reduction in the lipid peroxidation and an increase in the activity of antioxidant enzymes SOD and GSH-Px. c 2002 Elsevier Science Ltd. All rights reserved.
K EY WORDS : dantrolene sodium, ethanol, rat, ulcer, antioxidant.
INTRODUCTION Reactive oxygen species (ROS) are continuously produced during normal physiologic events, and are removed by antioxidant defence mechanisms [1]. In pathological conditions, ROS are over produced and result in lipid peroxidation and oxidative damage. The imbalance between ROS and antioxidant defence mechanisms leads to oxidative modification in the cellular membrane or intracellular molecules [2]. Some studies have revealed that ROS and lipid peroxidation are implicated in the pathogenesis of ethanol-induced gastric lesions and gastrointestinal damage, and calcium is a very important mediator in these events [3–5]. Lipid peroxide content and oxygen-derived free radicals increase in hypoxic tissue. This situation results in severe changes at the cellular level and causes plasma membrane damage, intracellular calcium accumulation (which plays a role in free radical mediated lipid peroxidation), cell death, exfoliation and epithelial erosion [6, 7]. An increase in the intracellular calcium level activates the some calcium-dependent enzymes and worsens the oxidative damage [5]. Malondialdehyde (MDA) is an indicator of lipid peroxidation, and its level increases in tissues when ∗ Corresponding author. Department of Pharmacology, Medical Faculty,
Atat¨urk University, TR-25240, Erzurum, Turkey. E-mail: [email protected] 1043–6618/02/$ - see front matter
they are exposed to oxidative stress. Various endogenous antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) play an important role in the elimination of oxygen free radicals (OFR) and lipid peroxides, and protect cells against the toxic effects of OFR and lipid peroxides [1, 8]. Dantrolene sodium, a peripherally acting skeletal muscle relaxant, is used in the treatment of muscle spasticity, malignant hyperthermia and neuroleptic malignant syndrome and depresses excitation– contraction coupling in the muscle fibre by inhibiting calcium release from the sarcoplasmic reticulum [9]. It has no effect on neuromuscular transmission and does not affect the electrical properties of the skeletal muscle membrane [10]. Additionally, it is suggested that the dantrolene sodium affects the membranal calcium channel of smooth muscle cells and inhibits calcium influx [11, 12]. On the other hand, it is shown that the dantrolene sodium has a cytoprotective effect on the neuron or liver cells, but there is no information about its effect on antioxidant enzyme activity [13–15]. This study aimed to examine anti-ulcerogen and antioxidant effects of dantrolene sodium on ethanolinduced gastric lesions in rats. For this purpose, we studied the effects of dantrolene sodium on SOD and GSH-Px activity, and changes in lipid peroxidation which were determined by measuring MDA level. c 2002 Elsevier Science Ltd. All rights reserved.
422
MATERIALS AND METHODS
Animals Forty eight albino Sprague–Dawley male rats with a weight of 190–225 g were used for the experiment. The rats were fed with standard laboratory chow and water before the experiment. The laboratory was windowless with automatic temperature (22 ± 1 ◦ C) and lighting controls (14 h light/10 h dark). Rats were divided into six equal groups (n = 8) and housed in cages. Twenty-four hours before the experiment, the rats were fasted and allowed access to water ad libitum.
Ulcer study The anti-ulcerogenic effect of dantrolene sodium was investigated with the ethanol-induced ulcer model. On the day of the experiment, groups 1–3 were injected with 1, 5 and 10 mg kg−1 dantrolene sodium, group 4 was injected with 20 mg kg−1 famotidine and group 5 was injected with saline solution. All of the drugs were administered intraperitoneally (i.p.) and at the same volume (0.5 ml). Following a 30 min period, all the animals were given 1 ml of ethanol (70%) by gavage. One hour after the administration of ethanol, the animals were killed by decapitation. The stomach was removed and opened along the greater curvature and washed in physiological saline solution. For the measurement of the gross gastric mucosal lesions, the freshly excised stomach was laid flat and the mucosal lesions were traced on clear acetate paper. Gross mucosal lesions were recognized as haemorrhage or linear breaks (erosions) with damage to the mucosal surface. The area of stomach and gross lesions were approximately calculated by planimetry using a simple magnifier. The results were translated to the term of ‘total ulcer area/total gastric area’ and these were expressed as an ulcer index (%). After this evaluation, gastric tissue MDA, SOD and GSH-Px levels were determined. Group 6 was used as control for biochemical analysis.
Biochemical analysis After the macroscopic evaluation, stomachs were kept in an ice bath until homogenization. They were homogenized in 10-fold physiological saline solution by using homogenizer (Omni Accessory Pack International Homogenizer, USA). The homogenate was centrifuged at 10 000 g for 1 h to remove debris. Clear upper supernatant was taken and enzymatic assays were carried out in this fraction. All the procedures were performed at +4 ◦ C throughout the experiments. SOD activity was detected according to Sun and coworkers [16]. In this method, xsantine–xsantine oxidase complex produces the superoxide radicals, which react with nitrobluetetrazolium (NBT) to form the farmasone compound. SOD activity is measured at 560 nm by detecting the inhibition of this reaction. By using a blank study of all the reagents present except for the supernatant sample and determining the absorbance
Pharmacological Research, Vol. 45, No. 5, 2002 Table I Variability (%CV) of the parameters measured in the study % CV Within Between
MDA
SOD
GSH-Px
7.6 8.5
2.3 3.1
3.9 4.7
of the sample and blank, activity is calculated. One SOD unit was defined as the enzyme amount causing 50% inhibition in the NBTH2 reduction rate. SOD activity was also expressed as U mg−1 protein of stomach tissue sediment. GSH-Px activity was measured according to the Paglia and Valentina method [17]. In this method, GSH-Px catalyses the oxidation of glutathione in the presence of tert-butyl hydroperoxide (tBH). Oxidized glutathione is converted into the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. The reduction in the absorbance of NADPH at 340 nm is measured. By measuring the absorbance change per minute and by using the molar extinction coefficient of NADPH, GSH-Px activity of stomach tissue is calculated. GSH-Px activities were expressed as mU mg−1 protein of stomach tissue sediment. MDA levels were determined according to Ohkawa et al. [18]. In this method, to samples less than 0.2 ml of 10% (w/v) tissue homogenate were added 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH, and 1.5 ml of 0.8% aqueous solution thiobarbituric acid (TBA). The mixture was made up to 4.0 ml with distilled water, and heated at 95 ◦ C for 60 min. After cooling, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15 : 1, v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance at 532 nm was measured. Total TBAreactive materials were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 × 105 cm−1 M−1 . MDA level was expressed as nmol g−1 . The amount of protein was determined by using the Lowry method [19]. Biochemical measurements were carried out at room temperature by using a spectrophotometer (CEC˙IL CE, 3041, UK). The precision analysis results (within, 10 analyses with the same sample on the same day, and between, one analyses with the same sample on consecutive 10 days, batch analyses) were given in Table I.
Chemicals Dantrolene sodium, reduced glutathione (GSH), NADPH, glutathione disulfide reductase, H2 O2 , xanthine, xanthine oxidase, nitrobluetetrazolium, trichloroacetic acid and TBA n-butanol were purchased from Sigma Chemical Co. (St Louis, MO, USA). Famotidine was obtained from ‘Mustafa Nevzat A.S¸.’
Pharmacological Research, Vol. 45, No. 5, 2002
423 30
Table II Effects of datrolene sodium and famotidine on ethanol-induced gastric mucosal injury
Ethanol (n = 8) Dantrolene 1 mg kg−1 + ethanol (n = 8) Dantrolene 5 mg kg−1 + ethanol (n = 8) Dantrolene 10 mg kg−1 + ethanol (n = 8) Famotidine 20 mg kg−1 + ethanol (n = 8)
1.96 ± 0.07 0.42 ± 0.02∗ 0.54 ± 0.02∗ 0.09 ± 0.006∗ 1.05 ± 0.06∗
nmol /g tissue
Group
25
Mean ± SEM (ulcer index, %)
20 15 10
Ethanol: 70%, 1 ml/animal; ∗ P< 0.001, vs ethanol. 5
All values were expressed as mean ± SEM. Comparison of means between groups were analysed by Student’s t-test or one way analysis of variance (ANOVA). A P value of less than 0.05 was considered significant.
Effects on gastric tissue lipid peroxidation in ethanol-administered rats The administration of ethanol increased the MDA level in rat gastric tissue. In contrast, pretreatment with dantrolene sodium significantly decreased the MDA levels in a dose dependent manner, as compared with ethanol alone and there was a statistical significance between the effects of dantrolene sodium doses used on MDA level (by the Tukey test, F = 28.675, P < 0.001). Additionally, famotidine was found to prevent the rise in MDA level. MDA levels of stomach tissue are shown in Fig. 1.
in e
0 fa m
ot id
-1 da nt
-5 da nt
-1 da nt
an ol et h
12 10 8 6 4 2
e fa
m
ot
id in
0 da nt -1
t-5 da n
da n
l
t-1
0 l
Per-oral administration of 70% ethanol produced multiple mucosal lesions in the rat stomach. Pretreatment with dantrolene sodium and famotidine were found to inhibit the ethanol-induced gastric mucosal injury in rats. Preventive effects of 1, 5 and 10 mg kg−1 dantrolene sodium were in a dose-dependent manner (percent decreases were 78.4, 72.5 and 95.3, respectively, compared to ethanol) and there was a statistical significance between the effects of dantrolene sodium doses used (by Kruskal–Wallis one way ANOVA, H = 28.198, P < 0.001). Famotidine also significantly inhibited the ethanol-induced gastric lesion (percent decrease was 46.2, compared to ethanol). Ulcer indices (UI) are shown in Table II.
14
co nt ro
Effects on acute gastric mucosal lesions induced by ethanol
16
U/mg protein
RESULTS
18
ha no
Statistical analysis
Fig. 1. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue MDA level in ethanol administered rats. N = 8, ∗ P< 0.05, ∗∗ P< 0.01, ∗∗∗ P< 0.001, compared to ethanol group, by Student’s t-test.
et
(Turkey). All other chemicals and reagents were obtained from the stores of the Department of Biochemistry, Medical Faculty, Atat¨urk University.
co
nt ro l
0
Fig. 2. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue SOD level in ethanol administered rats. N = 8, ∗ P< 0.01, ∗∗ P< 0.001, compared to ethanol group, by Student’s t-test.
Effects on gastric tissue SOD and GSH-Px enzyme activity SOD activity was found to be decreased in the ethanol-administered group. In contrast, SOD activity significantly increased in 1, 5 and 10 m kg−1 doses of dantrolene sodium administered rats and there was a statistical significance between the effects of dantrolene sodium doses used on SOD level (by Kruskal–Wallis one way ANOVA, H = 20.905, P < 0.001). It was found to be elevated significantly in famotidine administered rats (Fig. 2). GSH-Px activity was found to decrease in the gastric tissue after ethanol administration, but pretreatment with 1, 5 and 10 m kg−1 doses of dantrolene sodium resulted in a dose-dependent increase in the GSH-Px activity and there was a statistical
424
Pharmacological Research, Vol. 45, No. 5, 2002 1.2
mU/mg protein
1 0.8 0.6 0.4 0.2
ot id in e
0 fa
m
t-1 da n
-5 da nt
-1 da nt
ha no l et
co
nt ro l
0
Fig. 3. Effects of 1, 5 and 10 mg kg−1 doses of dantrolene sodium (dant-1, dant-5, dant-10) and famotidine (20 mg kg−1 ) on gastric tissue GSH-Px level in ethanol administered rats. N = 8, ∗ P< 0.05, ∗∗ P< 0.01 compared to ethanol group, by Student’s t-test.
significance between the effects of dantrolene sodium doses used on the GSH-Px level (by the Tukey test, F = 3.713, P < 0.05). Famotidine also increased the GSH-Px activity of gastric tissue, compared to ethanol (Fig. 3).
DISCUSSION Several factors such as increased vascular permeability, gastric motility and vagal activity, decreased gastric blood flow and protective prostaglandin levels play an important role in gastric ulcer pathogenesis. Chemicals (ethanol or HCl) and drugs (indomethacin or acetylsalicylic acid) are used for the peptic ulcer model in rats, some of them, such as ethanol or aspirin, cause gastric mucosal damage and promote the generation of ROS in this tissue [20, 21]. Some ROS scavenger or inhibitor drugs such as melatonin have protective effects on indomethacin- or ethanol-induced acute gastric injury in rats [22, 23]. In some studies, it was revealed that dantrolene sodium prevented lipid peroxidation in hippocampal neurons and enterocytes [24, 25]. Again, it was shown that pretreatment with nifedipine and verapamil prevented the lipid peroxidation and increased the antioxidant enzyme [26, 27]. Results of this study showed that dantrolene sodium prevented the gastric tissue damage against ethanol-induced stress in a dose dependent manner. Furthermore, it protected the gastric tissue against lipid peroxidation and increased the antioxidant enzymes’ (SOD and GSH-Px) activity. According to these, there are similarity and accordance between our study and the aforementioned studies [4, 24–27]. GSH is an important compound in cytoplasm against oxidative stress. GSH depletion results in enhanced lipid peroxidation and excessive lipid peroxidation can cause GSH consumption. GSH-Px activity is coupled with the
oxidation of GSH to GSSG, which can subsequently be reduced by GSH-reductase, with NADPH as the reducing agent [28]. Lipid peroxide accumulation is believed to be the cause of destruction and cell membrane damage. MDA is an index of lipid peroxidation, its level increases in gastric mucosa after ethanol-induced injury [23]. The present study demonstrated that ethanolinduced damage to the gastric mucosa is associated with an increase in MDA levels in rats. But, pretreatment with 1, 5 and 10 mg kg−1 doses of dantrolene sodium or 20 mg kg−1 dose of famotidine inhibited the increase of gastric tissue MDA levels. Furthermore, MDA levels in the 10 mg kg−1 dose of dantrolene sodium administered group were very close to the normal gastric tissue MDA level (non-ethanol or drugs). This result revealed that dantrolene sodium has clearly reduced the lipid peroxidation in gastric mucosal tissue damage induced by ethanol. SOD catalyses the conversion of the superoxide anion radical into O2 and H2 O2 . H2 O2 is highly toxic to cells, it causes DNA and membrane damage, and Ca2+ release from intracellular stores [1]. GSH-Px is an important enzyme that plays a role in elimination of H2 O2 and lipid hydroperoxides. GSH-Px also reduces peroxides by using reduced GSH. In the present study, an increase in ethanol-induced gastric lesions paralleled with a decrease of SOD and GSH-Px activity. On the other hand, these enzyme activities were increased by pretreatment with dantrolene sodium. 10 mg kg−1 of dantrolene sodium almost resulted in an increase of SOD activity up to the normal (non-ethanol) level. Dantrolene sodium not only increased the SOD and GSH-Px but also decreased the MDA formation. In the present study, famotidine, a histamine H2 receptor antagonist, also prevented the ethanol-induced gastric mucosal lesion formation, decreased the lipid peroxidation and increased the SOD and GSH-Px enzyme activity. In a previous study, we had shown that some calcium channel blockers and dantrolene sodium have a protective effect in ethanol-induced gastric mucosal injury [29]. Again, in a previous study, we had speculated that the protective effect of dantrolene sodium may be partly due to its blocker effect on internal calcium release from the endoplasmic reticulum, as well as external calcium influx via the membranal calcium channel. Also the results of our present study showed that dantrolene sodium prevented gastric mucosal damage and decreased lipid peroxidation induced by ethanol in rat stomach tissue, in a dose dependent manner. Recently, we revealed that dantrolene has in vitro antioxidant properties and we speculated that the cytoprotective effect of dantrolene sodium, at the same time, may be due to its antioxidant activity [30]. This protective effect of dantrolene sodium against ethanol-induced gastric mucosal lesions, besides being the inhibitor effect on intracellular calcium release from the endoplasmic reticulum or extracellular calcium influx, at least in part, depends upon the reduction in the lipid peroxidation and increase in the activity
Pharmacological Research, Vol. 45, No. 5, 2002
of antioxidant enzymes SOD and GSH-Px, and its antioxidant properties. In conclusion, dantrolene sodium clearly has antioxidant properties and it may be a valuable drug for peptic ulcer disease therapy and/or an antioxidant against oxidative stress. Further studies are required to clarify the anti-ulcer and antioxidant action of dantrolene sodium.
ACKNOWLEDGEMENT This study was supported by Atat¨urk University Research Foundation (project no 2000/45).
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