Arjona, Álvaro

Cruz, Adolfo

Montilla, Pedro

Muntané Relat, Jordi

Padillo Ruiz, Francisco Javier

Ramírez, Luz María

Segura, J.

Tasset Cuevas, Inmaculada

Túnez, Isaac

Revista Espanola de Enfermedades Digestivas

idUS. Depósito de Investigación Universidad de Sevilla

ABSTRACTObjective: melatonin has been demonstrated to have activeantioxidant properties in different tissues during experimentalcholestasis. The aim of this research was to study myocardial ox-idative stress on obstructive jaundice, and to analyze the effect ofmelatonin on myocardial oxidative lesions.Material and methods: we achieved cholestasis by ligatureand sectioning of the main bile duct. Melatonin was administeredintraperitoneally (500 µg/kg/day). We measured malondialdehyde(MDA), reduced glutathione (GSH), catalase (CAT), superoxidedismutase (SOD) and glutathione peroxydase (GPx) antioxidantenzyme levels in the heart tissue.Results: obstructive cholestasis increased MDA and decreasedGSH as well as all antioxidant enzymes. Melatonin administrationsignificantly decreased MDA values, and increased GSH and an-tioxidant enzymes on the icteric animal myocardium.Conclusions: melatonin treatment prevents oxidative stressin the cardiac tissue as induced by experimental cholestasis.Key words: Antioxidants. Cholestasis. Cardiac dysfunction.Melatonin. Oxidative stress.INTRODUCTIONBiliary obstruction has an important effect both on theliver and extrahepatic organs (1). Obstructive jaundice(OJ) increases oxidative stress, characterized by a rise ofsystemic malondialdehyde (MDA) and a decrease in cel-lular antioxidant defenses, such as reduced glutathione(GSH) and antioxidant enzymes (2). The rise of differentparameters of oxidative stress during experimental OJhas been shown in serum (2), hepatic (2) and renal (3)parenchyma, and on the central nervous system, specifi-cally on the hypothalamus, bulb and cerebral cortex (4).Oxidative stress has been related to serious complicationsin liver (5-7) and renal dysfunction (3). OJ has beenshown to induce hypotension and fluid depletion (8).Moreover, it has also been suggested that biliary obstruc-tion causes depression mainly in myocardial contractilityof the left ventricle (9). Melatonin is a hormone withgreat antioxidant capacity (10). Its beneficial effectsagainst oxidative stress have also been demonstrated inliver cirrhosis (11) and acute pancreatitis (12). The aimsof our study were firstly to measure oxidative stress inthe myocardium tissue after biliary obstruction, and sec-ondly to demonstrate the antioxidant properties of mela-tonin in myocardial tissue during obstructive cholestasis.METHODSAnimalsMale Wistar rats (250-330 g) were subjected to con-trolled conditions of temperature (about 22-24 ºC) and il-lumination (12-h light: 12-h dark cycle), and were pro-vided with food (Purina®, Barcelona, Spain) and water adlibitum. Animals were treated according to institutionalguidelines, and the study was approved by the ResearchCommittee of the Reina Sofía University Hospital.Effect of melatonin on myocardial oxidative stress induced byexperimental obstructive jaundiceA. Cruz1*, I. Tasset2, L. M. Ramírez3, A. Arjona1, J. Segura4, I. Túnez2, P. Montilla2,J. Muntané3* and F. J. Padillo1*1Departments of General Surgery, 3Research Unit and 4Cardiology. Reina Sofía University Hospital. Córdoba, Spain.2Department of Biochemistry and Molecular Biology. School of Medicine. University of Córdoba. Spain. *Centro deInvestigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd)1130-0108/2009/101/7/460-463REVISTA ESPAÑOLA DE ENFERMEDADES DIGESTIVASCopyright © 2009 ARÁN EDICIONES, S. L.REV ESP ENFERM DIG (Madrid)Vol. 101. N.° 7, pp. 460-463, 2009Received: 03-03-09.Accepted: 12-03-09.Correspondence: Francisco J. Padillo. Unidad de Cirugía Pancreática yPrograma de Trasplante Pancreático. Departamento de Cirugía. HospitalUniversitario Reina Sofía. Avda. Menéndez Pidal, s/n. 14004 Córdoba,Spain. e-mail: javierpadiruiz@hotmail.comCruz A, Tasset I, Ramírez LM, Arjona A, Segura J, Túnez I,Montilla P, Muntané J, Padillo FJ. Effect of melatonin onmyocardial oxidative stress induced by experimental obstructi-ve jaundice. Rev Esp Enferm Dig 2009; 101: 460-463.ORIGINAL PAPERS05. A. CRUZ:Maquetación 1  28/7/09  16:35  Página 460Experimental designAnimals (n = 21, 7 in each group) were distributed ineach of the following groups: sham operated (SO), OJ,and OJ plus melatonin (OJ + MEL). All the surgicalprocedures were done in animals anesthetized with ket-amine (60 mg/kg i.p.) and midazolam (4 mg/kg i.p.).Cephazoline (17 mg/kg i.m.) was used as antibiotic pro-phylaxis. SO animals were submitted to laparotomy andabdominal closure without bile duct intervention. Theprocedure for OJ was started by a midline ventral inci-sion with exposure of the extra-hepatic bile duct. A dou-ble ligature with silk suture was done and the bile ductwas sectioned. A two-layer running suture was used forabdominal closure with polyglycolic acid and silk.Melatonin (500 µg/kg/day i.p.; Sigma-Aldrich, StLouis, MO) was diluted in 5% ethanol in 0.9% NaCl.The administration of the solvent did not change the pa-rameters (data not shown). Melatonin was injected dailyat 10 h starting the day before the bile duct ligature wasmade, and until the day preceding the sacrifice. The ani-mals were sacrificed under anesthesia 7 days after OJ.Blood was collected from the abdominal aorta andserum was frozen at -80 ºC until the measurement of al-kaline phosphatase (AP) and total bilirubin. The heartwas dissected, aliquot and immediately frozen at -80 ºCuntil the measurements could be taken. Tissue was ho-mogenized in ice-cold phosphate-EDTA buffer (10 mMphosphate buffer [pH 7.2], containing 1 mM EDTA and1 mM BHA diluted in 0.15% ethanol) using a glass-glass Potter-Elvehjem homogenizer.Biochemical assaysTotal bilirubin and AP were measured in serum us-ing an Axon analyzer (Bayer Diagnostics, Tarrytown,NY). MDA was determined in tissue according to theprocedure described in the assay (LPO-586, Bioxytec,Portland, OR). A GSH-400 assay commercial kit(Bioxytec, Portland, OR) was used for measuring GSHin tissue. Catalase (CAT) and superoxide dismutase(SOD) activities were measured according to Aebi (13)and Sun (14), respectively. Glutathione peroxidase(GPx) was measured according to Flohe and Gunzler(15).Statistical analysisThe results are expressed as means with their corre-sponding standard errors. Homogeneity of variance wasassessed with Levene’s test. Comparisons were made us-ing the two-tailed, unpaired Student’s t test and ANOVAtest. Statistical significance was set at p < 0.05.RESULTSThe biliary obstruction model caused acute cholestasisin OJ animals, characterized by a significant increase inboth total bilirubin and AP in comparison with SO ani-mals (Table I). Regarding this effect, animals which un-derwent bile-duct obstruction had a significant increasein the MDA oxidative stress marker in the myocardium(Fig. 1). Moreover, a positive correlation between serumAP and MDA concentration has been shown in myocar-dial tissue (Fig. 2). In addition to increased lipoperoxides,the concentration of antioxidant defenses was drasticallyreduced in the myocardium of OJ animals. In this respect,OJ induced a significant decrease in GSH (Fig. 3) and inantioxidant enzymes SOD, CAT and GPx (Fig.4).On the other hand, melatonin administration to ictericanimals determined a significant restoration of oxidativestress markers in the myocardium. Melatonin decreasedMDA concentration and increased GSH (Figs. 1 and 3),and likewise recovered the activity of all the enzymaticantioxidants studied (Fig. 4).DISCUSSIONThe study of the involvement of oxidative stress duringOJ has identified several potential agents that exert a ben-eficial effect against tissue injury induced by biliary ob-struction. Oxidative stress by OJ is characterized by a riseof lipoperoxides and a reduction of cellular antioxidantstatus. A rise in MDA in the liver parenchyma and serumhas been observed in animals with biliary obstruction (2).The reduction in blood antioxidant concentration mayhave a relevant repercussion on different tissues duringOJ. In addition, free radicals may originate in extrahepaticorgans or in organs not related to the biliary tree duringOJ. We have proven that there is increased oxidativestress in the kidney (3) and central nervous system (4) thatVol. 101. N.° 7, 2009 EFFECT OF MELATONIN ON MYOCARDIAL OXIDATIVE STRESS INDUCED 461BY EXPERIMENTAL OBSTRUCTIVE JAUNDICEREV ESP ENFERM DIG 2009; 101 (7): 460-463Table I. Cholestatic markers in the serum of rats with obstructive jaundiceSO OJ p OJ-MEL p*Total bilirubin (mg/dL) 0.12 ± 0.030 9.5 ± 0.80 < 0.0001 7.7 ± 0.10 < 0.05AP (U/L) 86.5 ± 21.79 378 ± 26.0 < 0.0001 354 ± 13.2 n.s.*In relation to the OJ group. SO: Sham operated; OJ: Obstructive jaundice group; OJ-MEL: Animals treated with melatonin after biliary obstruction.05. A. CRUZ:Maquetación 1  28/7/09  16:35  Página 461may explain various complications during cholestasis. Onthe lines of the impact of biliary obstruction on extrahep-atic organs, this study proves for the first time an increasein MDA and a decrease in GSH and antioxidant enzymesin the myocardium from OJ rats. It has been recognizedthat bile-duct obstruction has a detrimental effect on car-diovascular physiology (16). A more recent study hasshown a reverse correlation between the left ventricle sys-tolic work and bilirubin in obstructive jaundice patients,suggesting that bile-duct obstruction has a depressor ef-fect on myocardial contractility, mainly in the left ventri-cle (8). This fact could help explain the pathophysiologyof shock in patients suffering from severe cholangitis, andcould be important for icteric patients with previous heartdisease. In addition to that, in the above-mentioned study,462 A. CRUZ ET AL. REV ESP ENFERM DIG (Madrid)REV ESP ENFERM DIG 2009; 101 (7): 460-463Fig. 1. Reduction of malondialdehyde (MDA) content by melatonin incardiac tissue from obstructed animals. Obstructive jaundice (OJ) wasinduced in control animals (SO) by bile duct ligation. The OJ-MEL groupwas submitted to OJ and received melatonin (500 µg/kg) daily untiltheir sacrifice 7 days after the surgical intervention. MDA was determi-ned in myocardial tissue following the procedure described in “Ani-mals” (Methods). Statistical significance was set at p ≤ 0.0001; ªsignifi-cantly different from the SO group.Fig. 3. Recovery of reduced glutathione (GSH) content by melatonin inthe myocardium tissue from obstructed animals. Obstructive jaundice(OJ) was induced in control animals (SO) by bile duct ligation. The OJ-treated animals (OJ-MEL) received melatonin (500 µg/kg) daily untiltheir sacrifice 7 days after the surgical intervention. GSH was determi-ned in myocardium tissue following the procedure described in “Ani-mals” (Methods). Statistical significance was set at p ≤ 0.0001; ªsignifi-cantly different from the SO group.Fig. 4. Recovery of antioxidant status by melatonin in cardiac tissuefrom obstructed animals. Obstructive jaundice (OJ) was induced in con-trol animals (SO) by bile duct ligation. The OJ-MEL group was submit-ted to OJ and received melatonin (500 µg/kg) daily until their sacrifice7 days after the surgical intervention. Superoxide dismutase (SOD), ca-talase (CAT) and glutathione peroxidase (GSH-Px) were measured inmyocardium tissue following the procedure described in “Animals”(Methods). Statistical significance was set at p ≤ 0.0001; ªsignificantlydifferent from the SO group.Fig. 2. Correlation analysis between alkaline phosphatase (AP) and ma-londialdehyde (MDA) in animals submitted to bile duct ligation (OJ).05. A. CRUZ:Maquetación 1  28/7/09  16:35  Página 462Vol. 101. N.° 7, 2009 EFFECT OF MELATONIN ON MYOCARDIAL OXIDATIVE STRESS INDUCED 463BY EXPERIMENTAL OBSTRUCTIVE JAUNDICEREV ESP ENFERM DIG 2009; 101 (7): 460-463biliary drainage allowed a normalization of hemodynamicparameters (8). The existence of a higher redox state inpatients suffering from cardiac insufficiency has beenproven, this being correlated with functional parameters;so there is a reverse connection between MDA figures andventricular ejection fraction (17). Although we have notevaluated hemodynamic dysfunction in our study, wehave clearly demonstrated an increase in myocardial ox-idative stress, which could be involved in myocardial dys-function in OJ rats. Moreover, a positive correlation be-tween AP and MDA in the myocardium has been proven,which suggests an increase in myocardium oxidativedamage when a worsening of cholestasis occurs. Al-though surgical procedures for cholestasis, such as biliarydrainage, are suitable for the treatment of OJ, we havesuccessfully tested antioxidant therapies to reduce the ox-idative damage associated with OJ. Melatonin has demon-strated hepatoprotective properties on experimentalcholestasis (5) and drastically improves oxidative stressparameters in the renal parenchyma (3). The discovery ofmelatonin as a direct scavenger of free radicals and as anindirect antioxidant stimulating antioxidant enzymes hasraised interest in its potential cardioprotective properties(18). Our study suggests that the cardioprotection of mela-tonin was related to a reduction of MDA and a restorationof the antioxidant status in the myocardium. Melatoninhad beneficial effects on other myocardial dysfunctionssuch as ischemia reperfusion (19), reducing areas withheart congestion (20). On the basis of experimental re-sults, we suggest that melatonin could have a protectiveeffect on myocardial dysfunction generated in OJ thatmay be relevant in order to decrease the systemic compli-cations of these patients. Melatonin has demonstrated an-tihypertensive properties (21) that would have a beneficialimpact on ventricular function. In addition, melatonin haspharmacological advantages such as oral administration,excellent tissue distribution both in fat-soluble and water-soluble environments, and its capacity to be highly concen-trated in every section of the cell, especially in mitochon-dria, which is very important for myocardial cells (18).In conclusion, myocardial biochemical oxidative dys-function induced by biliary obstruction may be counter-acted upon by melatonin. However, we should carry outnew clinical trials to prove the benefits of antioxidanttherapy with melatonin, and to help decrease mortalityrates among patients with obstructive cholestasis.ACKNOWLEDGEMENTSThis study has been supported by the Instituto deSalud Carlos III (FIS 02/0181, FIS 05/0703) and the Con-sejería de Salud (SAS 50/03). CIBERehd is funded by theInstituto de Salud Carlos III.REFERENCES1. Pérez-Fernández T, López-Serrano P, Tomás E, Gutiérrez ML, LledóJL, Cacho G, et al. Diagnostic and therapeutic approach to cholestat-ic liver disease. Rev Esp Enferm Dig 2004; 96: 60-73.2. Montilla P, Cruz A, Padillo FJ, Tunez I, Gascon F, Muñoz MC, et al.Melatonin versus vitamin E as protective treatment against oxidativestress after extrahepatic bile duct ligation in rats. J Pineal Res 2001;31: 138-44.3. Cruz A, Padillo FJ, Túnez I, Muñoz C, Granados J, Pera-Madrazo C,et al. Melatonin protects against renal oxidative stress after obstruc-tive jaundice in rats. Eur J Pharmacol 2001; 425: 135-9.4. Cruz A, Túnez I, Martínez R, Muñoz-Castañeda JR, Ramírez LM,Recio M, et al. Melatonin prevents brain oxidative stress induced byobstructive jaundice in rats. J Neurosci Res 2007; 85: 3652-6.5. Padillo FJ, Cruz A, Navarrete C, Bujalance I, Briceño J, Gallardo JI, etal. Melatonin prevents oxidative stress and hepatocyte cell death in-duced by experimental cholestasis. Free Radic Res 2004; 38: 697-704.6. Conde de la Rosa L, Moshage H, Nieto N. Hepatocyte oxidant stressand alcoholic liver disease. Rev Esp Enferm Dig 2008; 100: 156-63.7. Muntané J, González R, Ranchal I, Collado JA, López-Sánchez L,Herencia C, et al. Mechanisms of liver injury. Rev Esp Enferm Dig2007; 99: 405-10.8. Padillo FJ, Rodríguez M, Gallardo JM, Andicoberry B, Naranjo A,Miño G, et al. Preoperative assessment of body fluid disturbances inpatients with obstructive jaundice. Word J Surg 1999; 23: 681-7.9. Padillo J, Puente J, Gómez M, Dios F, Naranjo A, Vallejo JA, et al.Improved cardiac function in patients with obstructive jaundice afterinternal biliary drainage. Hemodynamic and hormonal assessment.Ann Surg 2001; 234: 652-6.10. Reiter RJ, Tan Dx, Terron MP, Flores LJ, Czarnocki Z. Melatoninand its metabolites: new findings regarding their production and theirradical scavenging actions. Acta Biochim Pol 2007; 54: 1-9.11. Cruz A, Padillo FJ, Torres E, Navarrete CM, Muñoz-Castañeda JR,Caballero FJ, et al. Melatonin prevents experimental liver cirrhosisinduced by thioacetamide in rats. J Pineal Res 2005; 39: 143-50.12. Muñoz-Casares FC, Padillo FJ, Briceño J, Collado JA, Muñoz-Cas-tañeda JR, Ortega R, et al. Melatonin reduces apoptosis and necrosisinduced by ischemia/reperfusion injury of the pancreas. J Pineal Res2006; 40: 195-203.13. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121-6.14. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of su-peroxide dismutase. Clin Chem 1988; 34: 479-500.15. Flohe L, Gunzler WA. Assays of glutathione peroxidase. MethodsEnzymol 1984; 105: 114-21.16. Better OS, Bomzon A. Effects of jaundice on the renal and cardio-vascular systems. In: Epstein M, editor. The kidney in liver disease.3rd ed. Baltimore: Willians & Wilkins; 1988. p. 508-34.17. Sam F, Kerstetter DL, Pimental DR, Mulukutla S, Tabaee A, BristowMR, et al. Increased reactive oxygen species production and func-tional alterations in antioxidant enzymes in human mailing my-ocardium. Congest Heart Fail 2005; 11: 2130-44.18. Tengattini S, Reiter RJ, Tan DX, Terron P, Rodella LF, Rezani R.Cardiovascular diseases: protective effects of melatonin. J Pineal Res2008; 44: 16-25.19. Kaneko S, Okumura K, Numaguchi Y, Matsui H, Murase K, MokunoS, et al. Melatonin scavenges hydroxyl radical and protects isolated rathearts from ischemic reperfusion injury. Life Sci 2000; 67: 101-12.20. Lagneux C, Joyeux M, Demente P, Ribuot C, Godin-Ribuot D. Pro-tective effects of melatonin against ischemia-reperfusion injury in theisolated rat heart. Life Sci 2000; 66: 503-9.21. Simko P, Paulis L. Melatonin as a potential antihypertensive treat-ment. J Pineal Res 2007; 42: 319-22.05. A. CRUZ:Maquetación 1  28/7/09  16:35  Página 463

Revista Española de Enfermedades Digestivas

Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice

http://scielo.isciii.es/pdf/diges/v101n7/original1.pdf?origin=publication_detail

Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice

Abstract

Similar works

Full text

Available Versions

idUS. Depósito de Investigación Universidad de Sevilla