Research Paper / Ara ştırma Makalesi Ege Tıp Dergisi 46.(1) : 27 - 32 , 2007 THE EFFECT OF A MONOAMINE OXIDASE (MAO)-B INHIBITOR; PARGYLINE ON OXIDANT STRESS/ ANTIOXIDANT STATUS IN AGING RAT TISSUES YA ŞLANAN SIÇAN DOKULARINDA OKS ĐDAN STRES VE ANT ĐOKS ĐDAN SAVUNMA DURUMUNA MONOAM ĐNOKS ĐDAZ (MAO)-B ĐNH ĐBĐTÖRÜ PARJ ĐLĐNĐN ETK ĐSĐ Gülinnaz ALPER Mert ÖZGÖNÜL Ferhan SA ĞIN Seda V. ĐRER Biltan ERSÖZ Ege University Medical School, Department of Biochemistry 35100 Bornova Izmir Turkey Anahtar sözcükler: MAO, ya şlanma, SOD, katalaz, MDA, dien konjugatları, lipid peroksidasyonu, antioksidant enzimler, oxidan stres Key words: MAO, aging, SOD, catalase, MDA, diene conjugates, lipid peroxidation, antioxidant enzymes, oxidant stress SUMMARY Aging is the accumulation of changes responsible for the progressive increases in the chance of disease and death. Among the theories proposed to account for this process; “the free radical theory of aging” has been widely accepted. Meanwhile, the increase in catecholamine metabolism is another physiological alteration related to this process, predisposing the organism to enhanced H2O2 production. The aim of this study was to assess the role of oxidant stress and antioxidant status in the aging process by determining MAO activity, lipid peroxidation products (LPPs) – diene conjugates and MDA levels, antioxidant enzymes – SOD and CAT in young and aging rat tissues and to investigate the effect of pargyline, a MAO-B inhibitor in reducing oxidant stress via inhibition of MAO. In general, significant increases in tissue MAO activitiy and LPPs were noted in aging rats, positively correlated wıth each other and significantly decreased after pargyline administration. In conclusion, the inhibition of MAO activity may attenuate the process of aging by reducing increased lipid peroxidation and concomitant oxidant stress. ÖZET Ya şlanma, hastalık ve ölüm riskinde ilerleyen artı ştan sorumlu de ğişikliklerin bir toplamıdır. Bu olguyu açıklamaya çalı şan teoriler içinde ‘ya şlanmanın serbest radikal teorisi’ oldukça çok kabul görmü ştür. Kate şolamin metabolizma- sında artı ş bu olguya e şlik eden fizyolojik bir de ğişimdir ve organizmada artmı ş hidrojen peroksit (H2O2) üretimine neden olur. Bu çalı şmanın amacı; oksidan stres ve antioksidan savunma durumunun ya şlanma üzerindeki etkisini; genç ve ya şlı sıçan dokularında MAO aktivitesi, lipid peroksidasyon ürünleri (LPPs) – dien konjugatları ve MDAdüzeyleri, antioksidan enzimler – SOD ve CAT enzimlerinin ölçümü yoluyla belirlemek ve MAO-B inhibitörü olan parjilinin MAO inhibisyonu yolu ile oksidan stressi azaltıcı etkisini ara ştırmaktır. Genel olarak ya şlı sıçanlarda doku MAO aktivitelerinde ve LPP’lerinde birbirleri ile korele bir artma ve parjilin uygulaması sonrası belirgin bir dü şme görülmü ştür. Sonuç olarak MAO aktivitesinin inhibisyonu, artmı ş lipid peroksidasyonu ve oksidan stresi azaltarak ya şlanma sürecini yava şlatabilmektedir. Yazı şma adresi: Gülinnaz ALPER, Ege University Medical School, Department of Biochemistry 35100 Bornova Izmir Turkey Makalenin geli ş tarihi : 10.02.2005 ; kabul tarihi : 15.12.2006 27 INTRODUCTION With advancing age, sequential alterations leading to a Vindis et al. and Bianchi et al.also showed that progressive increase in the chance of disease and death dopamine induces oxidative stress, apoptotic cascade occur and hence, this is defined as the process of aging. and cell apoptosis exclusively by mechanisms involving Till today, many theories have been put forward to H2O2 production by MAO and pargyline prevented account for this process (1, 2). Tissue damage due to apoptosis of proximal tubule cells (16, 17) Based on free radical reactions has been considered as “the free these findings, we believed that there is a need to radical theory of aging” and widely accepted as one of investigate pargyline’s effects on various tissues the most popular theories (3). influenced by aging in vivo by using a rodent model. The ultimate goal of geriatric research is to identify the Therefore, the objective of this study was to assess the mechanisms underlying the aging process and to role of MAO with respect to alterations related to oxidant increase the average life span and its quality (4, 5). An stress and antioxidant status during aging and to abundant number of reports imply the involvement of investigate the role of pargyline, a selective irreversible oxidative stress where increased formation of free MAO-B inhibitor in the prevention or amelioration of radicals lead to random deleterious effects in aging aging in various rat tissues. tissues. The increase noted in lipid peroxidation parallel to the concomitant decrease in antioxidant status MATERIAL AND METHODS supports this theory (3, 6, 7). Reagents With respect to the process of aging, increased All chemicals were analytical grade. Kynuramine, catecholamine metabolism and altered tissue distribution thiobarbituric acid, epinephrine, bovine erythrocyte SOD of monoamine oxidase (MAO [EC 1.4.3.4.;amine: standard and bovine serum albumin standard were oxygen oxidoreductase; deaminating, flavin containing]); purchased from Sigma, cyclohexane from Riedel de a key enzyme in this metabolism have been extensively Haens, and the remaining chemicals ( KH2PO4, investigated and reported to accompany this process K2HPO4, Na2HPO4, 4-hydroxyquinoline, NaOH, TCA, (8, 9, 10, 11). MAO is an enzyme responsible for the methanol, chloroform, Na2CO3, NaHCO3, Na-K metabolism of biologically active amines and oxidative tartarate, hydrogen peroxide , CuSO4 and reactives of deamination of these amines produce ammonia (NH3) Folin-Ciocalteau) from Merck Darmstadt. and hydrogen peroxide (H2O2) with established or Animals potential toxicity (5,12). The importance of H2O2 is due to the fact that it is a mediator in the production of the As shown in Table I, four groups, each consisting of most potent free oxygen radical, namely the hydroxyl young (3 months old) and aging (16-18 months old) male radical (OH •) (6). Untill now most studies have focused Swiss Albino rats (n=10 in each group) were formed. mainly on the role of oxidant stress and antioxidant Animals were housed in a temperature controlled room status related to the pathogenesis of aging (13, 14). Only (20-25˚C), on a 12 h dark / 12 h light cycle and fed with few studies have investigated the role of MAO related to standard laboratory animal chow ad libitum and water. oxidant stress and antioxidant status and the possible Rats were used in the study according to the permission beneficial effects of MAO inhibitory agents during the and rules of the Ege University Animal Research Ethics aging process (5, 15, 16). Committee. Zhang and Piantadosi showed that inhibition of MAO with pargyline protected rats from convulsions by decreasing H2O2 production in the brain (12). Recently, Table 1. Animal properties and treatment protocol of the experimental study groups (n=10 animals in each group) (the experimental procedure used in the study) .(IP=intraperitoneal) Young Control 2-3 months old 1 ml isotonic saline, IP, 90 minutes before decapitation Young Pargyline 2-3 months old 25 mg/kg pargyline in 1 ml isotonic saline, IP, 90 minutes before decapitation Aging Control 16-18 months old 2 ml isotonic saline, IP, 90 minutes before decapitation Aging Pargyline 16-18 months old 25 mg/kg pargyline in 2 ml isotonic saline, IP, 90 minutes before decapitation 28 Cilt 46 Sayı 1, Ocak-Nisan-2007 Preparation of the tissues The animals were decapitated; their livers, hearts, brains Kraml (18); results were expressed as nmol/mg prote- and kidneys immediately excised and washed in/hour. For diene conjugates, Bueger and Yust’s thoroughly in ice cold saline. Brain tissue specimens spectrophotometric method was used (19); results were were prepared according to the method in our previous given as nmol hydroperoxide/mg protein. MDA study (5 ). The brain as a whole was homogenized in determinations were conducted by Omar’s method; the phosphate buffer (KH2PO4, Na2HPO4.2 H2O, 50 mM, concentration of MDA being calculated applying an pH=7.0) (1/10, w/v). The other tissues, namely the livers, extinction coefficient of 153 000/Mol/cm and expressed hearts and kidneys were minced to three fractions; for as nmol/g ww (gram wet weıght) (20). SOD activities MAO activity a homogenate with EDTA/saccharose were determined by the method of Misra and Fridovich (1/10, w/v), for diene conjugates, superoxide dismutase (21); results expressed as U/mg protein. CAT activities (SOD) and catalase (CAT) a homogenate with were determined by a modified method of Aebi (22) and phosphate buffer (KH2PO4, Na2HPO4.2 H2O, 50 mM, Luck (23); results given as U/mg protein. Protein pH=7.0) (1/10, w/v) were prepared. For malondialdehyde determinations were performed by the method of Lowry (MDA) analysis, the remaining tissue was homogenized et al (24). in distilled water (2/1, w/v) followed by 5% TCA Statistical analysis precipitation. Supernatants were obtained from all homogenates, after centrifugation at 1500 g for 10 Statistical analysis was performed by MICROSTA and minutes. All procedures were carried on ice , the ANOVA programs. For comparison of the means, the analyses being performed on the day of decapitation. student’s t test, for intergroup comparisons, analysis of variance followed by the Newman Keuls multiple range MAO activity was determined fluorometrically (Aminco- test were used. The correlations between the Bowman spectrofluorometer) by the modified method of parameters were determined