the effect of aminoguanidine against cholestatic liver injury in rats

cell biochemistry and function

Cell Biochem Funct 2007; 25: 625–632.

Published online 7 August 2006 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1027/cbf.1359

The effect of aminoguanidine against cholestatic liver injuryin rats

Mehmet Yilmaz1, Cengiz Ara1*, Burak Isik1, Nese Karadag2, Sezai Yilmaz1, Alattin Polat3,Sacit Coban1 and Halil Duzova3

1Department of General Surgery, Inonu University School of Medicine, Turkey2Department of Pathology, Inonu University School of Medicine, Turkey3Department of Physiology, Inonu University School of Medicine, Turkey

We investigated the protective role of aminoguanidine (AG) in rat liver injury induced by chronic biliary obstruction.Secondary biliary cirrhosis was induced by bile duct ligation for 14 days. Swiss albino rats were divided into three groups:Common bile duct ligated (CBDL) rats; Group A, CBDL rats treated with AG as Group B and simple laparotomy groupknown as the Sham group; Group C. Group B received 200 mg/kg of AG intraperitoneally daily throughout 14 days. Thepresent data showed decreased gama glutamyl transferase (GGT), aspartate aminotransferase (AST), bilirubin and alanineaminotransferase (ALT) levels in the AG treated rats, when compared with CBDL rats (p< 0.05). In the AG treated rats,tissue levels of malondialdehyde (MDA) were significantly lower than that in CBDL rats (p< 0.001). Although the levels ofglutathione (GSH) in AG treated rats were higher and myeloperoxidase (MPO) were lower than that in CBDL rats, thedifference was not statistically significant (p> 0.05). The levels of interleukin-1a (IL-1a) and tumor necrosis factor-a (TNF-a) were significantly lower and although the levels of interleukin-6 (IL-6) were lower in AG treated rats than that in CBDLrats, the difference was not statistically significant. Administration of AG in the rats with biliary obstruction resulted ininhibition of ductular proliferation and portal inflammation. The present study demonstrates that intraperitoneal admin-istration of AG in CBDL rats maintains antioxidant defenses, reduces liver oxidative and cytokine damage and ductularproliferation and portal inflammation. This effect of AG may be useful in the preservation of liver injury in cholestasis.Copyright # 2006 John Wiley & Sons, Ltd.

key words— bile duct ligation; aminoguanidine; oxidative stress; cytokine

INTRODUCTION

Repair after cholestatic liver injuries is oftencomplicated by progressive hepatic damage despiteadequate technical decompression of the biliary tree.1

This progressive hepatic damage accompanies anincreased morbidity and mortality observed in biliaryatresia, choledochal cyst, sclerosing cholangitis, andother biliary obstructive pathology.2,3 The pathogen-esis of liver injury during extrahepatic cholestasisinduced by bile duct ligation is poorly understood.There are many experimental and clinical reports that

* Correspondence to: Cengiz Ara, Department of General Surgery,Inonu University Turgut Ozal Medical Center, Malatya, Turkey.Tel: þ90 422 3410660/3710. Fax: þ90 422 3410728.E-mail: [email protected]

Copyright # 2006 John Wiley & Sons, Ltd.

show the important role of reactive oxygen species inthe pathogenesis of hepatic damage and cholestasisproduced by biliary obstruction.4–6 Bile acid concen-trations increase in rats after bile duct ligation andinduce lipid peroxidation, probably related to thestimulation of phagocytic activity in polymorpho-nuclear phagocytes and inflammatory cells.7

AG, a compound structurally similar to L-arginine,inhibits inducible nitric oxide synthase (iNOS) in aselective and competitive manner, leading todecreased production of NO.8 Also, AG has anti-oxidant and free radical scavenger actions especiallyon peroxynitrite (ONOO�) production.9,10 Addition-ally, the beneficial effects of AG in variousexperimental models of inflammation have beenreported.11 To date, there is no study regarding theprotective effect of AG on liver damage in common

Received 4 January 2006Revised 6 June 2006

Accepted 28 June 2006

626 m. yilmaz ET AL.

bile duct ligated (CBDL) rats. The aim of this studywas to evaluate whether AG administration protectsagainst cholestatic liver injury in rats with bile ductligation. To assess the protective ability of AG inCBDL rats, we measured the activities of tissuereduced glutathione (GSH), malondialdehyde (MDA),myeloperoxidase (MPO), interleukin-1a (IL-1a),interleukin-6 (IL-6), and tumor necrosis factor-a(TNF-a) in the liver tissue plus bilirubin, aspartateaminotransferase (AST), alanine aminotransferase(ALT), alkaline phosphatase (AP), and gama glutamyltransferase (GGT) activities in blood. Also weexamined histopathological changes in liver.

MATERIALS AND METHODS

Experimental conditions

A total of 27, 3-month-old male Swiss-albino ratsweighing 300–350 g were used in the study which wasapproved by Inonu University Ethics Committee.

Rats were kept in stainless steel cages, allowed freeaccess to food and water ad libitum and quarantinedfor 7 days before surgery. Food was withheld 8 h priorto surgery, but free access to water was allowed. Ratswere subjected to controlled conditions of temperatureand humidity, in animal quarters with a 12-h light-dark. All surgical procedures were performed whilethe rats were under intraperitoneal ketamine (50 mg/kg) and xylazine HCl (10 mg/kg) anesthesia.

A total of 27 rats were divided into three groups:

Group A: C

Copyright # 2

BDL rats (n¼ 9)
Group B: C BDL plus AG (n¼ 9) Group C: S ham (n¼ 9)
Only laparatomy was performed on the Shamgroup. Secondary biliary cirrhosis was induced inanimals by double ligation and division of thecommon bile duct.12 Abdominal layers were closedwith appropriate suture materials. All animals weremaintained under the same conditions after surgery.For the second control group (CBDL rats), 1 ml ofdilution vehicle (NaCl) was given for 14 consecutivedays after surgery. AG (Sigma-Aldrich Chemie Gmbh,Steinheim, Germany) was dissolved in saline (0.9%NaCl w/v) to obtain a final concentration of 200 mg/ml. Because of very variable AG dosage schemesreported in the literature, we administered AG at adose of 200 mg /kg day i.p., which is reported to bemarkedly antioxidative.13

Animals in AG group were treated with 200 mg/kg,AG (i.p.) once a day throughout 14 days. To eliminate

006 John Wiley & Sons, Ltd.

complications arising from the diurnal effects, all ratswere killed under anesthesia at the same time of dayand liver was rapidly removed. The blood sampleswere taken by vena cava inferior puncture. A part ofthe liver was preserved in formalin for histologicalexamination and the remainder was stored at �808Cfor the analyses of MDA, MPO, GSH, and cytokine(IL-1a, IL-6, TNF-a) levels.

Biochemical analyses

The plasma was used to measure total bilirubin, AST,ALT, AP, and GGT as parameters indicative of hepaticfunction. The plasma activities of total bilirubin, AST,ALT, AP, and GGT were estimated by commerciallyavailable kits (Olympus Diagnostica Gmbit, Ireland).All solutions were prepared with distilled-deionizedwater. ZnSO4, NaOH, glycine, CuSO4, sulfanilamide,NaNO2, KNO3, Na2B4O7 � 10H2O, DTNB, n-butanol,TBA, and NaN3, were purchased from SigmaChemical Co., Germany; KH2PO4 and Na2HPO4,were from Merck Co., Germany, cadmium (Cd)granules were from Fluka (Chemische Fabrik AG,Bushs, Switzerland). Hexadecyl-trimethyl ammoniumbromide and o-dianisidine chloride were fromSigma.

The liver tissues were homogenized (TempestVirtishear, Model 278069; The Virtis Company,Gardiner, NY, USA) and the MDA contents ofhomogenates were determined spectrophotometri-cally.14 A 3 ml aliquot of 1% phosphoric acid and1 ml of 0.6% thiobarbituric acid solution were addedto 0.5 ml of 10% tissue homogenate. The mixture washeated in boiling water for 45 min. After cooling, thecolor was extracted into 4 ml of n-butanol. Theabsorbance was measured in a spectrophotometer(Ultraspec Plus, Pharmacia LKB Biochrom, UK) at532 nm. The amounts of lipid peroxides werecalculated as thiobarbituric acid reactive substancesof lipid peroxidation and are given as nmol/g tissue.

Myeloperoxidase activity was determined using a4-aminoantipyrine/phenol solution as the substrate forMPO-mediated oxidation by H2O2 and changes inabsorbance at 510 nm (A510) were recorded.15 Oneunit of MPO activity was defined as the amount ofprotein that degrades 1mmol H2O2 min�1 at 258C.Results were presented as mU � g�1 protein.

Results were expressed as mmol/g tissue. TissueGSH content was determined by the spectrophoto-metric method based on the use of Elman’s reagent.16

Results were expressed as nmol/g tissue.


DOI: 10.1002/cbf

aminoguanidine against oxidative stress in cholestasis 627

Tissue homogenates were prepared (2� 45 sec,08C) in 0.5 M Tris/1.5 m NaCl/50 mM CaCl2/2 mMsodium azide buffer at pH¼ 7. The homogenates werethen centrifuged at 15 000g for 15 min at þ48C andthe supernatants were used for ELISA. Rat inter-leukin-1a (rIL-1a), rat interleukin-6 (rIL-6) and rattumor necrosis factor-a (rTNF-a) (Biosource Immu-noassay Kit, California, USA) levels were measuredusing a sandwich ELISA protocol supplied by themanufacturer of the antibodies and the resultingabsorbances were determined using a microplatereader at 450 nm. Results were expressed as pg/gtissue.

Histopathological evaluation

The liver tissue samples were fixed in 10% formalinsolution and processed for routine histopathologicalexamination. Paraffin blocks were sliced into sectionsof 5mm in thickness. The sections were stained withH&E and Masson’s Trichrome. Light microscopicexaminations were made in a blind fashion by twopathologists. The slides were reviewed for degree ofductal proliferation, portal inflammation, and collagendeposition. Histopathological findings were expressedas þ1\þ3 according to the severity of lesions.

Statistical analysis

The results were statistically analyzed by theKruskal–Wallis H-test. The differences betweengroups were evaluated by the Mann–Whitney U-test

Figure 1. The liver tissue levels of MDA in the groups


followed by a t-test with Bonferroni correction whenindicated. P< 0.05 was considered significant. Theresults are expressed as the arithmetic mean� STD.

RESULTS

Twenty-four hours after the operation, the clinicalcondition of the animals of Group A and B, but notGroup C worsened with decreased activity, yellowedears and tails, darkened urine and pale faces. All of theanimals survived until the end of the experiment.Jaundice was observed in the visceral and parietalperitoneum of all animals except those of the Shamgroup. The livers were enlarged and the bile ductsabove the obstruction point were dilated. Animalsfrom the Sham group showed no alteration aftersurgery.

The results of MDA, GSH, and MPO are shown inFigures 1–3. In the AG treated rats the MDA levelswere significantly lower than that of Group A(p< 0.001). On the other hand, although the levelsof GSH in AG treated rats were higher and MPO werelower than in CBDL rats, the differences were notstatistically significant (p> 0.05). Total bilirubin,AST, ALT, ALP, and GGT levels in rats with bileduct ligation were higher than that of the Sham group(Table 1). In the AG treated rats, the levels of bilirubin,AST, ALT, and GGT were significantly lower than inthe CBDL rats (p< 0.01, p< 0.02, and p< 0.001,respectively). The results of IL-1a, IL-6, and TNF-aare shown in Figure 4. In the AG treated rats, the liverlevels of IL-1a and TNF-a were significantly lowerthan in the CBDL group (p< 0.03, p< 0.02,


DOI: 10.1002/cbf

Table 1. Plasma total bilirubin, AST, ALT, AP, and GGT levels in the groups

Groups Total bilirubin (mg/dl) AST (U/L) ALT (U/L) AP (U/L) GGT (U/L)

Sham (n¼ 9) 0.18� 0.17 122.8� 13.09 54.4� 7.5 353.5� 27.9 2.5� 0.9CBDL (n¼ 9) 9.5� 3.5 498.00� 121.4 157.5� 22.2 1663.2� 401.3 12.1� 2.7CBDLþAG (n¼ 9) 5.7� 2.3 275.7� 97.9 80.1� 13 1342.00� 365.03 4.8� 3.2p values�

I versus II 0.001 0.001 0.001 0.001 0.165II versus III 0.07 0.007 0.001 0.165 0.002

�p< 0.05 was considered to be statistically significant.CBDL, common bile duct ligation; AG, aminoguanidine; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkalinephosphatase; GGT, gama glutamyl transferase.

Figure 2. The liver tissue levels of GSH in the groups

Figure 3. The liver tissue levels of MPO activity in the groups

Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 625–632.

DOI: 10.1002/cbf


Figure 4. The liver tissue levels of TNF-a, IL-6, and IL-1a in the groups

Figure 5. Liver sections from rats that receive AG (A) showed less inflammation than bile-duct ligated rats (B) (H&E, 200�). There wasless ductular proliferation and collagen deposition in AG group when compared with CBDL rats (C and D) (Masson’s Trichrome, 200�)

Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 625–632.

DOI: 10.1002/cbf



respectively). Although levels of IL-6 in the AGinjected rats were higher than in the CBDL (p¼ 0.053)group, the difference was not statistically significant(p> 0.05).

Histopathology results

In the liver specimens, less portal inflammation wasfound in the AG treated rats (Figure 5A and B). Therewas less ductular proliferation and collagen depositionin AG group when compared with CBDL rats(Figure 5C and D). The histopathology results areshown in Table 2.

DISCUSSION

Several reports showed that oxidative stress associatedwith lipid peroxydation is involved in the developmentof liver injury with extrahepatic cholestasis in ratsafter bile duct ligation.6,17,18 The present study

Table 2. Portal inflammation, collagen deposition, ductal prolifer-ation degrees in groups

Portalinflammation

Collagendeposition

Ductalproliferation

S1 � � �S2 � � �S3 � � �S4 � � �S5 � � �S6 � � �S7 � � �S8 � � �S9 � � �CBDL 1 0/þ þþ þþCBDL 2 þþþ þþ þþþCBDL 3 þþ þþþ þþþCBDL 4 þþ þþ/þþþ þþþCBDL 5 þþ/þþþ þþþ þþþCBDL 6 þþþ þþþ þþþCBDL 7 þþ/þþþ þþ þþþCBDL 8 þþþ þþ þþþCBDL 9 þþ þþ/þþ þþþAG1 þþ þþ þþAG2 þþ þþþ þþþAG3 þ þþ þþAG4 þþ þþ/þþþ þþþAG5 þþ þþ/þþþ þþAG6 þþ þþþ þþþAG7 þþ þþ þþAG8 þþ þþ þþAG9 þþ þþ þþ

S, Sham group; CBDL, common bile duct ligation group; AG,aminoguanidine group.


indicates that intraperitoneal administration of AGat a dose of 200 mg/kg/day reduced plasma bilirubin,AST, ALT, and GGT levels after bile duct ligation.Additionaly, AG decreased oxidative stress, the levelsof interleukin-1a, interleukin-6 and inhibited ductularproliferation and collagen deposition. There areseveral reports indicating that levels of MPO increaseafter bile duct ligation in rats.19,20 Our results are inagreement with previous works reporting high levelsof MPO. In the present study, levels of MPO in the AGtreated rats were lower than in the CBDL group.Although tissue MPO levels were clearly decreased byAG, its exact mechanism is not known. Reductions inMPO levels in the AG treated rats could be due to itsantioxidant, free-radical-scavenging effect,11,12 andinhibition of neutrophil infiltration.21 This suggeststhat intraperitoneally administered AG could exert apreventive effect on an enhancement of liverneutrophil infiltration during the progression ofcholestatic liver injury in rats with BDL.

MDA is a secondary product of oxidative stressformed during lipid peroxidation and there are severalreports indicating that levels of MDA increase afterbile duct ligation in rats.18,22–24 The present resultsare consistent with previous works reporting highlevels of MDA. Our present study showed that levelsof MDA in the AG treated rats were significantly lowerthan in the CBDL rats. Reductions in MDA levels inthe AG treated rats may be due to its antioxidant andfree-radical-scavenging actions.11,12 The simplestexplanation is that AG directly scavenged thehydroxyl radicals and thereby inhibited lipid peroxi-dation and MDA formation.25 It is possible that theinterference of AG with free radical generation may berelated to a decline in oxidative stress in cholestatic rats.

In the current study, levels of GSH in the CBDLgroup were significantly lower than in the AG treatedgroup. The increased GSH level in the AG treated ratsmay be related to its antioxidant and free-radical-scavenging effects. Another explanation of thissignificant increase in GSH levels seen in the AGtreated rats is the effect of AG upon the enzymesinvolved in glutathione synthesis, whereby AG maymaintain the levels of glutathione during oxidativestress.26

In common bile duct ligated rats, phospholipase isactivated as a result of retention of bile salts, which aretoxic for cells. Arachidonic acid products triggerductal proliferation that is the initial phase of hepaticinflammation.27,28 Both TNF-a and IL-6 are proin-flammatory and have been studied specifically withrespect to the pathophysiology of liver disease becausemost of their biological effects parallel the clinical


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and biochemical abnormalities found in patients withchronic liver disease.29 Several cytokines such asTNF-a are secreted from Kupffer cells and IL-6 andIL-1a are secreted from hepatocytes.30,31 In thecurrent study, levels of IL-6, IL-1a, and TNF-a in theCBDL group were higher than in the AG treatedgroup. The protective effect of AG may be due toinhibition of IL-6,32 IL-1a, and TNF-a.33

CONCLUSION

The present study demonstrates that intraperitonealadministration of AG maintains antioxidant defensesand reduces liver oxidative damage, cytokine pro-duction, and focal and portal inflammation in commonbile ligated rats. This effect of AG may be useful topreserve liver function in patients with biliaryobstruction. However, more investigations arerequired to evaluate AG’s antioxidant, anti-inflamma-tory hepatoprotective effects in clinical and exper-imental models.

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the effect of aminoguanidine against cholestatic liver injury in rats

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