Acta chir belg, 2006, 106, 68-72
The Effects of Desferrioxamine and Quercetin on Liver Injury Induced by Hepatic Ischaemia-Reperfusion in Rats Ç. Tokyol*, S. Yilmaz**, A. Kahraman***, H. Çakar***, C. Polat** Departments of Pathology*, General Surgery**, and Biochemistry***, Kocatepe University, Faculty of Medicine, Afyon, Turkey.
Key words. Ischaemia-reperfusion ; liver ; desferrioxamine ; quercetin. Abstract. Background : This study was designed to examine the effects of desferrioxamine and quercetin on hepatic ischaemia-reperfusion injury in rat. Methodology : Thirty Wistar albino rats were randomized into five groups. Group I was the control group. Group II received no treatment. Group III and group IV received intramuscular injections of desferrioxamine (100 mg/kg per day) and quercetin (50 mg/kg per day) respectively. Group V was administered desferrioxamine and quercetin in combination. After treatment for 3 days, groups II, III, IV, and V were exposed to total hepatic ischaemia for 45 minutes. Plasma alanine aminotransferase levels, malondialdehyde and reduced glutathione activities were measured after reperfusion for 1 hour. Histopathological analysis of liver tissues was carried out. Results : Our results indicated that tissue malondialdehyde levels and histopathological liver damage scores were significantly higher in the ischaemia-reperfusion group than in the control group. Administration of desferrioxamine, quercetin, and desferrioxamine+quercetin significantly decreased these parameters. Plasma alanine aminotransferase levels were also increased after ischaemia-reperfusion. Quercetin and desferrioxamine + quercetin significantly decreased the activity of this enzyme when compared to ischaemia-reperfusion group. Conclusions : The present data suggest that both desferrioxamine and quercetin may be useful to protect against ischaemia-reperfusion induced liver damage.
Introduction Oxidative stress has been implicated in the pathogenesis of many mechanisms, including ischaemia-reperfusion (IR) injury. Metals play both potentiating role in the IR injury of the liver and other organs (1-2). Today, most of the data have focused on the role of iron chelation. Desferrioxamine (Dfx) is an ironchelating agent and prevents free oxygen radical formation by inhibiting the catalyzing role of iron in the Fenton reaction. Enough data are now available to initiate and support clinical trials for the use of Dfx (1). Quercetin (Q) (3, 5, 7, 3’, 4’-pentahydroxy flavonol) is another powerful antioxidant with metal-ion such as iron and copper binding properties, as well as radical scavenging abilities (3-4). It has anti-inflammatory and antithrombic effects (5). The aim of this experimental study was to evaluate, mainly by histopathological and biochemical parameters, whether or not Dfx, Q and Dfx combined with Q protect liver tissue after total hepatic IR injury in rats. Material and Methods Experiments were performed in accordance with the 1996 revised form of the Guide for the Care and Use of
Laboratory Animals published by the United States National Institutes of Health. The study was approved by the University Ethics Committee. Animals In this experimental study, 30 Wistar albino rats weighing 250-300 g were used. They were housed three in a cage at a temperature of 22 ± 1°C with a fixed 12 hr of light-dark cycle. They were fasted 12 hr before the experiments but were allowed to drink water ad libitum. The rats were divided into five groups of six rats ; group I was the control group, group II was the IR group, group III was the IR + Dfx group, group IV was the IR + Q group, group V was the IR + Dfx + Q group. Treatment with Dfx and/or quercetin was initiated 3 days before the procedure. Pretreatments were applied at 48 hours, 24 hours, and 1 hour before the induction of ischaemia. The rats were anaesthetized by intramuscular 100 mg/kg ketamine HCl (Ketalar, Eczacıbas¸ı, Istanbul, Turkey) before the experiment. The abdominal region was shaved with a safety razor and sterilized with povidone iodine solution. The rats in the control group and IR group were given sterile distilled water as a vehicle.
The Effects of Desferrioxamine and Quercetin on Liver Injury
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Table I Histopathological description and grading score of liver samples Score 1 2 3 4 5
Description No abnormality Mild lesions affecting 10% of samples Lesions affecting 25% of samples Lesions affecting 50% of samples Lesions affecting more than 75% of samples
The rats in the IR group underwent total hepatic ischaemia induced by clamping the hepatic artery, the portal vein, and the common hepatic duct with a vascular microclip. Animals in IR + Dfx group were pretreated with intramuscular (im) injections of 100 mg/kg Dfx (Desferal 0.5 g, CIBA-Geigy, Istanbul, Turkey) in sterile distilled water, animals in IR + Q group were pretreated with intramuscular injections of 50 mg/kg Q dissolved in 1 ml of dimethylsulphoxide (DMSO) and suspended with sterile distilled water. Animals in IR + Dfx + Q group received both im Dfx (100 mg/kg) and im Q (50 mg/kg). The rats in the first three groups received an equal volume of DMSO. One rat died during the ischaemia-reperfusion in IR + Dfx + Q group and was excluded from the study. Biochemical Assays Blood and tissue samples were used for biochemical analysis. Blood samples were centrifugated at 3000 rpm, +4°C for 15 min. Plasma alanine aminotransferase (ALT)levels were measured to assess hepatic parenchymal damage using a Hitachi automatic analyzer 911 (Hitachi, Mannheim, Germany). Two markers of oxidative stress were measured in supernatants of homogenized liver tissue samples. Plasma malondialdehyde (MDA) was measured as an indicator of lipid oxidation, using the thiobarbituric acid method of OHKAWA et al. (6). Reduced glutathione (GSH) levels were measured as an indicator of tissue antioxidant capacity using the method of BEUTLER et al. (7). MDA and GSH levels were expressed as nmol/mg protein. Light Microscopy Liver samples preserved in 10% formaldehyde were embedded in paraffin, cut into 3 µm thickness and stained with haematoxylin-eosin (HE). Sections of each liver with the largest surface area were systematically analysed. Liver samples were evaluated for cellular oedema, single cell necrosis, pyknosis, congestion, sinusoidal dilation, focal haemorrhage, portal and sinusoidal mononucleated inflammatory cell infiltration using a semi quantitative scale adapted from HAUET et al.
Fig. 1 Plasma alanine aminotransferase levels in the control group and groups ischaemia-reperfusion (IR), desferrioxamine (Dfx), quercetin (Q), and Dfx + Q.
(Table I) (8). Each parameter mentioned was graded with a score between 1 to 5 according to its distribution in the liver section. The total sum of histopathological scores was obtained for all groups by combining the individual parameters. Statistical Analysis The results were decoded after being reported and expressed as the mean ± standard error of the mean. Statistical analysis of the data was done with the Kruskal-Wallis and Mann-Whitney U tests. A p value < 0.05 was considered significant. Results Plasma ALT levels : Plasma ALT levels were markedly increased in the IR group (p = 0.004). A significant decrease of ALT levels was not observed in the Dfx treated animals. In contrast, pretreatment with Q and Dfx + Q significantly suppressed the elevation of plasma ALT levels (p = 0.006 and p = 0.006) (Fig. 1). Pretreatment with combined Dfx+Q decreased ALT levels more significantly than Q alone (p = 0.028). Tissue MDA and GSH levels : Liver MDA markedly increased in the IR group (p = 0.016). Q, Dfx and Dfx + Q significantly decreased liver MDA (p = 0.006, p = 0.01 and p = 0.011 respectively) (Fig. 2). There was no statistically significant difference between MDA levels of these groups. Liver GSH levels markedly decreased in the IR group (p = 0.006). Liver GSH increased with pretreatment, but the difference did not reach statistical value (Fig. 2). No significant difference was seen between GSH levels of groups IR, IR + Dfx, IR + Q, and IR + Dfx + Q. Light Microscopy In the control group, histological findings of liver were normal. Hepatocellular disarray, hepatocellular oedema prominent in pericentral area, focal necrosis, pyknosis,
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Fig. 4 Well preserved liver architecture with minimal sinusoidal dilation in IR + Dfx group (HE, 200).
Fig. 2 Tissue malondialdehyde (MDA) and reduced glutathione (GSH) activities in the control group and groups ischaemia-reperfusion (IR), desferrioxamine (Dfx), quercetin (Q), and Dfx + Q.
Fig. 5 Total liver damage scores in the control group and groups ischaemia-reperfusion (IR), desferrioxamine (Dfx), quercetin (Q), and Dfx + Q.
Discussion
Fig. 3 Hepatocellular oedema prominent in pericentral area, focal necrosis, congestion, and sinusoidal dilation in IR group (HE, 200).
congestion with sinusoidal dilation, focal haemorrhage, portal and sinusoidal mononucleated inflammatory cell infiltration were observed in the liver of the IR group (Fig. 3). Histopathological findings were less severe in the groups receiving Dfx and/or Q (Fig. 4). IR significantly increased total liver damage scores (p = 0.003). Pretreatment with Dfx resulted in significantly decreased total liver damage scores with a well preserved liver architecture (p = 0.006). Pretreatment with Q and Dfx + Q also resulted in significantly decreased liver damage scores (p = 0.021 and p = 0.016 respectively). No significant difference was seen between the groups receiving Dfx, Q, or Dfx + Q (Fig. 5).
Oxygen-free radical injury plays an important role in the pathophysiology of the hepatic reperfusion injury following ischaemia. Restoration of blood flow to ischaemic tissues can result in recovery of cells if they are reversibly injured, or not affect the outcome if irreversible cell damage has occurred. Depending on the intensity and duration of the ischaemic insult, variable numbers of cells may proceed to cell death after blood flow resumes, by necrosis as well as apoptosis. New damage may be initiated during reperfusion by increased generation of oxygen free radicals by parenchymal and endothelial cells and infiltrating leukocytes. Superoxide anions can be produced in reperfused tissue as a result of incomplete and vicarious reduction of oxygen by damaged mitochondria or because of the action of oxidases derived from leukocytes, endothelial cells, or parenchmal cells. Cellular anti-oxidant defense mechanims may also be compromised by ischaemia, favouring the accumulation of radicals (9).
The Effects of Desferrioxamine and Quercetin on Liver Injury Previous studies have shown that many antioxidants, including N-acetylcysteine, DMSO, and vitamin E have protective effects in the IR injury of the liver (10-12). The purpose of this study was to investigate the potentially protective effect of Q used alone and in combination with Dfx, on tissue damage. Desferrioxamine, an iron chelator, has shown to protect against oxygen free radical injury occurring in association with IR in liver (13-15). Attenuated cell injury with Dfx suggest that microvascular failure and resultant cell death are mediated, at least in part, by iron dependant mechanims in reperfusion (13). COLLETTI et al. demonstrated that pretreatment with Dfx attenuates tumour necrosis factor alpha release following hepatic ischaemia-reperfusion (14). Quercetin (3, 5, 7, 3’, 4’-pentahydroxy flavonol) is another powerful antioxidant with metal-ion such as iron and copper binding properties, as well as radical scavenging abilities (3-4). Clinically relevant functions ascribed to flavonoids include antihypertensive and antiarrhythmic activity, anti-inflammatory and antiallergic properties, hypocholesterolaemic activity, platelet mast cell stabilization, and antihepatotoxic activity (5). SU et al. investigated the possible protection provided by oral Q pretreatment against hepatic ischaemia-reperfusion injury in rats. They concluded that Q can protect the liver against ischaemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity (16). We examined the effects of Dfx and Q pretreatment on liver tissue after total hepatic IR in an experimental model. Biochemical analysis revealed that pretreatment with Q and Dfx + Q significantly suppressed the elevation of plasma ALT levels. Dfx + Q decreased the ALT levels more significantly, though it was above control levels. Dfx, Q, and Dfx + Q decreased liver MDA to lower concentrations than control group. There was no significant difference between MDA levels of these groups. We showed depletion of liver tissue GSH after IR, but it was not restored by Dfx and/or Q treatment. The decreased free-radical production and decreased ALT levels in groups receiving Q match the previous findings of SU et al. (16). DRUGAS et al. showed that Dfx pretreatment decreased ALT 50% after IR, but we did not find such a correlation (13). Histopathological findings supported the protective role of Dfx and/or Q on liver tissue after IR, but no significant difference was seen between the groups receiving Dfx, Q, or Dfx + Q. Liver damage was less severe than IR group in both of them. Histopathological scores were not correlated with ALT levels. They may not exhibit a parallel degree in liver damage (17, 18). VARDARELI et al. showed the beneficial effects of alpha-tocopherol and pentoxyfilline on ischaemia-reperfusion induced liver injury in rats. They stated that MDA levels and
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histopathological scores were decreased by pretreatment, but the ALT levels were similar in both groups (19). Our results showed that both Dfx and Q play an important role in the attenuation of IR induced hepatic injury by decreasing tissue MDA content. With the exception of necrotic enzyme, ALT, the combined application of Dfx and Q did not appear to be more potent than use of Dfx and Q alone in ameliorating the biochemical and histopathological parameters. In conclusion, our data demonstrated that Dfx and Q have beneficial effects against the hepatic IR injury, but they do not seem to have synergistic effects when administered in combination. References 1. ARORA A. S., GORES G. J. The role of metals in ischemia/reperfusion injury of the liver. Semin Liver Dis, 1996, 16 : 31-8. 2. SOYBIR G., KOKSOY F., EKIZ F. et al. The effects of free oxygen radical scavenger and platelet-activating factor antagonist agents in experimental acute pancreatitis. Pancreas, 1999, 19 : 143-9. 3. SZETO Y.-T., BENZIE I. F. F. Effects of dietary antioxidants on human DNA ex vivo. Free Rad Res, 2002, 36 : 113-8. 4. INAL M. E., KAHRAMAN A. The protective effect of flavonol quercetin against ultraviolet a induced oxidative stress in rats. Toxicology, 2000, 154 : 21-9. 5. FORMICA J. W., REGELSON W. Review of the biology of quercetin and related bioflavonoids. Food Chem Toxicol, 1995, 33 : 1061-80. 6. OHKAWA H., OHISHI N., YAGI K. Assay for lipid peroxidase in animal tissues by thiobarbituric acid reaction. Anal Biochem, 1979, 95 : 351-8. 7. BEUTLER E. Red cell metabolism. In : BEUTLER E. (ed.). A manual of biochemical methods. New York : Grune & Stratton, 1970 : p. 66. 8. HAUET T., MOTHES D., GOUJON J.-M. et al.. Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia. Transplantation, 1997, 64 : 1082-6. 9. COTRAN R. S., KUMAR V., COLLINS T. (eds.). Cellular Pathology I : Cell injury and cell death. In : Robbins Pathologic Basis of Disease (6th . ed). Philadelphia : WB Saunders, 1999 : pp. 1-29. 10. DEMIR S., INAL-ERDEN M. Pentoxyfylline and N-acetylcysteine in hepatic ischemia/reperfusion injury. Clıin Chim Acta, 1998, 275 : 127-35. 11. HATIPOGLU A. R., TEMIZ E., YÜKSEL M., HOSCOSKUN Z., COSKUN I., HÜSEYINOVA G. The comparison of electron microscopy and scintigraphy in determining the protective effect of dimethylsulphoxide (DMSO) on ischemia-reperfusion injury through Pringle maneuver. Hepato-Gastroenterology, 2001, 48 : 798-802. 12. SENER G., TOSUN O., SEHIRLI A. O. et al. Melatonin and N-acetylcysteine have beneficial effects during hepatic ischemia and reperfusion. Life Sci, 2003, 72 : 2707-18. 13. DRUGAS G. T., PAIDAS C. N., YAHANDA A. M., FERGUSON D., CLEMENS M. G. Conjugated desferoxamine attenuates hepatic microvascular injury following ischemia/reperfusion. Circ Shock, 1991, 34 : 278-83. 14. COLLETTI L. M., REMICK D. G., CAMPBELL D. A. Jr. Desferal attenuates TNF release following hepatic ischemia/reperfusion. J Surg Res, 1994, 57 : 447-53. 15. BAILEY S. M., REINKE L. A. Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers. Free Radic Res, 2000, 32 : 497-506. 16. SU J. F., GUO C. J., WEI J. Y., YANG J. J., JIANG Y. G., LI Y. F. Protection against hepatic ischemia-reperfusion injury in rats by oral pretreatment with quercetin. Biomed Environ Sci, 2003, 16 : 1-8.
72 17. NISHIMURA T., YOSHIDA Y., WATANABE F. et al. Blood level of mitochondrial aspartate aminotransferase as an indicator of the extent of ischemic necrosis of the rat liver. Hepatology, 1986, 6 : 701-7. 18. SCHWARTZ S. I. Liver. In : SCHWARTZ S. I., SPENCER S., GALLOWAY D. F. (eds.). Principles of Surgery, 7th ed. USA, McGraw-Hill, 1999 : pp. 1395-436. 19. VARDARELI E., SARICAM T., KOKEN T., DEGIRMENCI I., ARAL E., ERENOGLU E. The effect of alpha-tocopherol and pentoxyphilline on ischemia-reperfusion induced liver injury in rats. HepatoGastroenterology, 1998, 45 : 1505-8.
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