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Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury
Life Sciences 67 (2000) 101Ð112
Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury Shinji Kanekoa,*, Kenji Okumuraa, Yasushi Numaguchia, Hideo Matsuia, Kichiro Murasea, Shinji Mokunoa, Itsuro Morishimaa, Kenji Hiraa, Yukio Tokia, Takayuki Itob, Tetsuo Hayakawaa a
Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan b Health Sciences, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Abstract During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (?OH) in the efßuent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for ?OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantiÞed using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 mM MEL1 SAL had signiÞcantly reduced amounts of 2,5- and 2,3DHBA by multiple folds, compared to the SAL group. TBARS was signiÞcantly decreased in the 100 mM MEL group (1.20 6 0.36 vs 1.85 6 0.10 mmol/g of drug-free group, p,0.001). More importantly, the 100 mM MEL group signiÞcantly recovered in LV function (LV developed pressure, 1dp/dt, and 2dp/dt; 63.0%, 60.3%, and 59.4% in the 100 mM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p,0.05). Duration of ventricular tachycardia or ventricular Þbrillation signiÞcantly decreased in the 100 mM MEL group (100 mM MEL, 159 6 67 sec; drug-free, 1244 6 233 sec; p,0.05). As a result of scavenging ?OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; Isolated rat heart; Reperfusion injury; DHBA; Salicylate method; TBARS, Hydroxyl radical
Introduction Oxygen-derived free radicals lead to deleterious effects in postischemic reperfusion of rat hearts (1,2). Recent studies have revealed that free radicals may be produced by mitochon* Corresponding author. Tel.: 181-52-744-2168; fax: 181-52-744-2175. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 0 7 -X
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dria of postischemic reperfused myocardium, leading to lipid peroxidation, and then to damage of the myocardial tissue (3,4,31). Hydroxyl radicals (?OH) are formed via a superoxidederived (Haber-Weiss reaction) and iron-catalyzed Fenton reaction (38). The hydroxyl radical has notable characteristics in taking part in ischemia/reperfusion injury. It displays much greater chemical reactivity than superoxide radicals (O2?2) or hydrogen peroxide (H2O2) and plays a signiÞcant role in postischemic reperfusion injury (1,2,19,20,31,32). Evidence has been presented to indicate that free radical scavengers such as superoxide dismutase (SOD) or catalase (CAT) attenuate postischemic reperfusion injury of myocardium (5Ð7,34). The pineal secretory production melatonin (MEL, 5-methoxy-N-acetyl-tryptamine) has recently been conÞrmed to be a potent scavenger of hydroxyl (8) and peroxyl radicals (11,12). Generally in humans melatonin regulates sleep and circadian rhythms (30). Since it is well known that MEL is both a lipophilic and hydrophilic compound (13,14), MEL freely permeates all morphophysiological barriers of cells in any organs (15). MEL may protect all tissues, including myocardium, from free radical mediated damage caused by adriamycin (16), paraquat (17), and postischemic reperfusion injury (7,28,29). However no serious side effects have been reported in people who take large doses for prolonged periods of time (30). Electron paramagnetic resonance (ESR) has been well known as a powerful tool in studying the relationship between ?OH and ischemic reperfusion injury. Nevertheless, ESR also has its limitations in the spin-trapping process. Firstly, concentration of the spin trap reagent is toxic to isolated rat heart. Secondly, the sensitivity of ESR is lower than the salicylate (SAL) method (23,31,34,35). In recent years, some investigators (Floyd et al., Grootvelt and Halliwell, and Pritsos et al.) have introduced high-performance liquid chromatography (HPLC) methods to detect ?OH (3,21Ð23,25,31,34). We used the same method. If salicylate molecules are attacked by ?OH, three products (2,5-dihydroxybenzoic acid [DHBA], 2,3-DHBA, catechol) are formed under physiological conditions. The percentages of 2,5-DHBA, 2,3DHBA, and catechol are 40%, 49%, and 11%, respectively (22,25). These high sensitive measurements of the amounts of 2,5-DHBA and 2,3-DHBA are evidence consistent with ?OH formation during postischemic reperfusion. Because MEL has been shown to have ?OH scavenging properties, we evaluated the scavenging effect of MEL by indirect measure of ?OH and the extent of lipid peroxidation, and the protective effects against postischemic reperfusion injury. Methods SAL was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). MEL, 2,5DHBA, 2,3-DHBA, and all other reagent were purchased from Sigma Chemical (St. Louis, MO, USA). Heart preparation The investigation was conducted in full compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Sprague-Dawley rats (Japan SLC, Inc.) weighing 250 to 300 g were anesthetized with pentobarbital sodium (50mg/kg) by intraperitoneal injection. After intravenous heparin injection (1000U/kg), hearts were rapidly excised and put into 48C Krebs-Henseleit solution. Aortas were cannulated for isolated perfusion (Langendorff tech-
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nique) within 60 seconds afer excision. Hearts were perfused at a constant pressure of 70mmHg with a modiÞed Krebs-Henseleit (KH) solution (mmol/L:NaCl 118, NaHCO3 25, NaH2PO4 1.18, KCl 5.8, CaCl2 2.5, MgSO4 1.2, glucose 10) gassed with 95% O2/5% CO2, achieving a pH of 7.40 and a temperature of 37.08C. A ßuid-Þlled latex balloon was inserted into the left ventricular (LV) cavity via the left auricle and mitral valve to measure the LV isovolumic pressure. The LV catheter was connected to a pressure transducer (TB-611T, Nihon Koden, Japan), and the LV isovolumic pressure and 6dp/dt were continuously recorded on a polygraph recorder (Recticorder, Nihon Kohden, Japan). The volume of the LV balloon was adjusted to obtain a LV end diastolic pressure of 5 mmHg during the equilibration period, and was not changed thereafter. Electrical activity (ECG) was monitored using two electrodes attached directly to the heart. The duration of ventricular Þbrillation (VF) or ventricular tachycardia (VT) was recorded from the ECG. The diagnostic criteria for VF and VT were consistent with the recommendations of the Lambeth Conventions (41). The rate-pressure product (RPP) was calculated by multiplying heart rate (HR) and LV developed pressure (LVDP, LV systolic pressure minus LV end diastolic pressure). Timed coronary efßuent was collected for measurement of coronary ßow (CF) every one minute. Experimental protocols The experimental protocol is shown in Fig. 1. The hearts were divided into 6 groups as follows. 1. Drug-free group (n57); Hearts were equilibrated with KH solution for the Þrst 30 min and then subjected to normothermic (378C) global ischemia for 30 min, followed by 30 min of reperfusion by turning off the aortic inßow line. 2. Ischemia/reperfusion with 1mM SAL (SAL, n57); Group 2 was similar to the control group except in that hearts were perfused with KH solution containing 1 mM SAL. 3. Ischemia/reperfusion with 1mM SAL and MEL treatment (n57); Hearts were perfused with KH solution containing 1 mM SAL and MEL. The concentrations of MEL were 10 (SAL110mM MEL), 30 (SAL130mM MEL), and 100 mM (SAL1100mM MEL). Ischemia/reperfusion time was the same as the time used for the control group. 4. Ischemia/reperfusion with MEL treatment (100mM MEL, n57); Hearts were perfused with KH solution containing 100 mM MEL. Ischemia/reperfusion time was the same as the time used for the control group. LVDP and LV 6dp/dt were measured during pacing in an atrial mode at 300 beats/min (Cardiac Stimulator, Nihon Kohden, Japan) after 30 min of reperfusion to evaluate postischemic reperfused myocardial function. At the end of the experiments, hearts were weighed and the LV free wall was immediately frozen in liquid nitrogen, stored at 2808C, and made dry with lyophil method. Measurement of ?OH In a separate group of experiments, isolated rat hearts were subjected to 30 min of aerobic perfusion with KH solution containing 1 mM SAL and 0, 10, 30, and 100 mM MEL, followed by 30 min of normothermic global ischemia and 30 min of reperfusion. Samples of ef-
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Fig. 1. This Þgure shows the protocol of the experiment. Group 1, KH solution (drug free) Group 2, KH solution 1 1 mM SAL (SAL) Group 3, KH solution 1 1 mM SAL 1 10 mM MEL (SAL 1 10 mM MEL) Group 4, KH solution 1 1 mM SAL 1 30 mM MEL (SAL 1 30 mM MEL) Group 5, KH solution 1 1 mM SAL 1 100 mM MEL (SAL 1 100 mM MEL) Group 6, KH solution 1 100 M mMEL (100 mM MEL)
ßuent were collected after 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, and 30 min of reperfusion and were stored at 2208C until use. The presence of hydroxylated benzoic acid in the perfusate was determined as previously described (23,30,33,34). Brießy, the perfusate was Þltered through a Supelco 0.45 mm pore size Nylon sample Þlter, and a 25 mL volume of the sample was injected into the HPLC apparatus. The HPLC apparatus consisted of a Model L-6000 (Yanaco) and a Voltammetric detector (Yanaco VMD-3000). The column used was a Supelcosil LC18-DB (3mm ODS, 7534.6mm) column (Supelco) attached to a Supelguard LC-18-DB precolumn (Supelco). The mobile phase was 0.03 mol/L sodium acetate and 0.03 mol/L citric acid (pH53.6) at a ßow rate of 1 mL/min. The detector potential was maintained at 10.6 V, employing a glassy carbon working electrode and an Ag/AgCl reference electrode. Retention times for the peaks of 2,5-DHBA and 2,3-DHBA were veriÞed by injecting authentic standards and all samples were measured against these standards. Measurement of the extent of lipid peroxidation To evaluate the extent of lipid peroxidation, the amount of thiobarbituric acid reactive substances (TBARS) in myocardial tissue of lyophilization, a measurement of the extent of lipid peroxidation, was assayed by a modiÞcation of the thiobarbituric acid (TBA) method (42,43). Each sample was homogenized in 1.15% KCl solution containing 10 mM deferoxamine, 0.04% butylated hydroxytoluene (BHT), and 2% ethanol. Each homogenate was incubated for 60 min at 958C in an oil bath with stock TCA-TBA-HCl reagent (15% [w/v] trichloroacetic acid, 0.375% [w/v] thiobarbituric acid, 0.25N hydrochloric acid) and 2% BHT. After cooling, the ßocculent precipitate was removed by centrifugation for 10 min at 1000 g, and the extinction coefÞcient of the supernatant at 535 nm was determined spectrophotometrically and compared with a known TBARS standard.
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Table 1 Comparison of the parameter of cardiac function in each group during equilibration
Drug-free SAL SAL 110 mM MEL SAL130 mM MEL SAL1100 mM MEL 100 mM MEL
Pre-efßuent (ml)
HR (beats/min)
Pre-LVDP (mmHg)
Pre-RPP
Pre-LV1dp/dt (mmHg/sec)
Pre-LV-dp/dt (2mmHg/sec)
10.8 6 0.3 11.6 6 0.4 11.5 6 0.4 10.9 6 0.3 11.2 6 0.2 10.3 6 0.2
249 6 10 255 6 19 245 6 9 241 6 19 262 6 7 263 6 9
106 6 3 103 6 2 102 6 2 104 6 4 102 6 2 105 6 2
26131 6 627 26067 6 1568 25036 6 1009 24763 6 1163 26600 6 517 27509 6 478
2697 6 90 2753 6 26 2807 6 50 2824 6 68 2831 6 40 2838 6 60
2256 6 78 2034 6 56 2132 6 66 2019 6 63 2169 6 31 2254 6 79
All parameters did not show signiÞcant differences among 6 groups (n57 in all groups). Data are expressed as mean 6 SEM.
Statistical analysis Data were expressed as means 6 SEM. Group comparisons were made by analysis of variance with multiple comparisons (Bonferroni corrected t test). A difference criteria for a value was set at p , 0.05 in order to determine whether the result was signiÞcant. Results Cardiac functions during equilibration Addition of SAL and MEL in KH solution had no deleterious effects on any of the parameters determined including CF, HR, LVDP, RPP, and contractility (6LVdp/dt). No effect of SAL and MEL on electrical activity of the heart was observed during equilibration (see Table 1).
Fig. 2. Effect of MEL on LVDP after 30 min of reperfusion. The heart was paced at 300 beats/min. LVDP of the 100mM MEL and SAL1100mM MEL groups signiÞcantly recovered compared to that of the drug-free and SAL groups. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups.
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Recovery of cardiac functions Fig. 2 shows the recovery of the LVDP after 30 min reperfusion. The recovery of the LVDP in the 100 mM MEL 1 SAL and 100 mM MEL groups were signiÞcantly greater than that of the SAL and drug-free groups (p , 0.05). The LVDP of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 32 6 4, 32 6 2, 39 6 8, 42 6 6, 60 6 4, and 63 6 5 mmHg, respectively. The recovery of 6LVdp/dt in the 100 mM MEL 1 SAL and 100 mM MEL groups after 30 min of reperfusion was also superior to that of the SAL and drug-free groups (p , 0.05) (Fig. 3). The 1LVdp/dt of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 1785 6 82, 1801 6 85, 1941 6 220, 1985 6 173, 11534 6 108, and 11710 6162 mmHg/sec, respectively. The -LVdp/dt of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 2629 6 82, 2711 6 62, 2829 6 196, 2838 6 141, 21269 6 102, and 21339 6 111 mmHg/sec, respectively. There were no signiÞcant differences in CF among the groups during reperfusion (data are not shown). Generation of ?OH during reperfusion The efßuent of the reperfused heart was subjected to HPLC for the detection of ?OH by measuring 2,5-DHBA and 2,3-DHBA. The retention times for 2,5-DHBA and 2,3-DHBA
Fig. 3. LV1dp/dt of the drug-free and SAL groups after 30 min of reperfusion were signiÞcantly lower than that of the 100mM MEL and SAL1100mM MEL groups. LV-dp/dt of the drug-free and SAL groups after 30 min of reperfusion also were signiÞcantly lower than that of the 100mM MEL and SAL1100mM MEL groups. The heart was paced at 300 beats/min. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups, ** p , 0.01 vs drug-free and SAL groups.
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Fig. 4. Time courses of 2,5-DHBA (A) and 2,3-DHBA (B) during reperfusion in each group. (A) All groups showed peaks at about 3Ð8 min from the onset of reperfusion. Addition of 100mM MEL and 1mM SAL to KH solution signiÞcantly reduced production of 2,5-DHBA compared with SAL group, especially during the early reperfusion period. (B) All groups also showed peaks at about 3Ð8 min from the onset of reperfusion. Isolated rat hearts of 100mM MEL 1 SAL group signiÞcantly produced lower levels of 2,3-DHBA than hearts of 1mM SAL group. Data are expressed as means 6 SEM. * p , 0.05, ** p , 0.01, p , 0.005, p , 0.001 vs SAL group.
were determined to be 7.3 and 9.5 min, respectively, by standard reagent. As shown in Fig. 4, DHBAs reached peaks at 3Ð8 min after the onset of reperfusion, and leveled off after 10 min in all groups. MEL reduced the amounts of 2,5-DHBA and 2,3-DHBA in a dose-dependent manner (p , 0.05; vs SAL group). The amounts of DHBAs of the early reperfusion period in
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the 100 mM MEL 1 SAL group were depressed compared with those of the SAL group. The values of 3Ð8 min were especially worthy of notice. The extent of lipid peroxidation in the myocardium The levels of TBARS in the 100 mM MEL 1 SAL and 100 mM MEL groups were signiÞcantly lower than those of the drug-free and SAL groups (Fig. 5). The TBARS of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups were 1.68 6 0.07, 1.85 6 0.10, 1.69 6 0.04, 1.66 6 0.08, 1.34 6 0.08, and 1.20 6 0.04 mmol/g, respectively. Ventricular arrhythmia The incidence of reperfusion-induced VT or VF is shown in Fig. 6. A reduction in the incidence of VT or VF during reperfusion was dose-dependently observed. The duration of VT or VF of the SAL, drug free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups were 1364 6 248, 1244 6 233, 805 6 284, 810 6 216, 209 6 68, and 159 6 67 sec, respectively. A signiÞcant dose of MEL was conÞrmed in the 100 mM group (p , 0.05; vs drug free and SAL groups). Discussion The mitochondrial, microsomal, and nuclear membranes contain electron transport systems which may produce free radicals (38). Under conditions of hyperbolic oxygen or at the onset of postischemic reperfusion, the mitochondrial respiratory rate will increase markedly and greater quantities of free radicals are generated. These radicals may exceed the capacity
Fig. 5. MEL signiÞcantly decreased TBARS in the myocardial tissue, compared to non-MEL groups. TBARS in the 100 mM MEL group was lower than in the drug-free, SAL, SAL 1 10 mM MEL and SAL 1 30 mM MEL groups. TBARS in the SAL 1 100 mM MEL group was lower than in the drug-free and SAL groups. Data are expressed as means 6 SEM. * p , 0.005 vs drug-free and SAL groups, p , 0.001 vs drug-free, SAL, SAL 1 10 mM MEL, and SAL 1 30 mM MEL group.
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Fig. 6. Duration of ventricular arrhythmia signiÞcantly decreased in the 100mM MEL and SAL1100mM MEL groups, compared to the drug-free and SAL groups during reperfusion. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups
of the cellular intrinsic free radical scavenging systems, and be cytotoxic to cells by attacking fatty acids. The reactions caused by exceeding radicals leads to lipid peroxidation of membranes, and that destroys and oxidizes amino acids and their polypeptide chains. These results lead to LV dysfunction and reperfusion arrhythmia in the postischemic reperfused heart. In this study, we used the SAL method, originally reported by Floyd et al. (25), to detect DHBA species produced from SAL hydroxylation. Although the ?OH has an extremely short half-life, DHBA species are extremely stable and can be used to evaluate various treatments in order to study the deleterious effects of the ?OH (31). In many cases, in the presence of ?OH, one of the most reactive radical species, the protection of the tissue by some radical scavenger (i.e., SOD, CAT, or histidine) was observed (5,6,24). Evidence has been shown that MEL prevents postischemic reperfusion injury in hamster cheek pouches (19), liver (28), stomach (29), and postischemic reperfusion arrhythmia (44). Tan et al. reported MEL prevented ischemia/reperfusion-induced arrhythmias. To further study the mechanism of the MEL effect, we measured DHBAs and TBARS in this experiment. No previous study, however, has implicated the efÞcacy of MEL against ischemia/reperfusion injury in isolated rat heart. The present results demonstrate for the Þrst time that MEL scavenges ?OHs and still has beneÞcial effects in postischemic myocardial dysfunction. In this study, MEL signiÞcantly inhibited the formation of 2,5- and 2,3-DHBA in a dosedependent manner. Addition of 100 mM melatonin in buffer clearly suppressed the production of 2,5- and 2,3-DHBA approximately by one-fourth to one-thirteenth times higher than the SAL group during reperfusion. The amount of TBARS in the myocardial tissue was also reduced by administration of 100 mM MEL. Addition of 100 mM MEL had recovering effects on the subject output, including LVDP and 6 LV dp/dt after 30 minutes of reperfusion, and reduced the duration of VT or VF. Free radicals are thought to be implicated in ventricular arrhythmia and deleterious effects on LV function after postischemic reperfusion (1,2,32). Although various free radical scavengers indicate beneÞcial effects on postischemic myo-
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cardial function and ventricular arrhythmia (5,6,7,24,33,34), Takemura et al. conÞrmed that deferoxamine effectively reduced the amount of 2,5-DHBA and failed in the protection of postischemic reperfusion injury in 15 min of ischemia (35). The parameter of 2,5-DHBA is a nonspeciÞc derivative produced by cytochrome P450 without SAL hydroxylation (34). Therefore, they should have evaluated 2,3-DHBA. Other investigators also reported that radical scavengers fail to protect against reperfusion injury under speciÞc conditions. Sunnergren and Rovelto reported that SOD, CAT, and mannitol failed to ameliorate LVDP and prevent increased permeability in ischemia/reperfused isolated rat heart (36). Although Gallagher et al. evaluated the size of infarction after postischemic reperfusion with or without SOD and CAT in in vivo dogs, SOD and CAT failed to reduce the size of infarction (37). Since many differences are observed among those studies in experimental models, protocols, and parameters to evaluate reperfusion injuries, different results may follow in association with the efÞcacy of radical scavengers reported in those studies. MEL is believed to work via electron donation to directly detoxify free radicals such as the highly toxic ?OH with no enzymatical; reaction. Tan et al. have conÞrmed MELÕs free radical scavenging ability in vitro (8,9,10). In their system, 5,5-dimethyl-1-pyrroline N-oxide, a trapping reagent, is hydroxylated by ?OH and the product of reaction was quantiÞed by HPLC and ESR. It has been proven that MEL, in the presence of ?OH, detoxiÞes it by electron donation with resultant production of the indolyl cation radical. The indolyl cation radical reacts directly, without requiring another catalyst, with an additional superoxide anion (26,27,40). Because MEL is both lipophilic and hydrophilic, it can enter all cells with ease. It is obvious that compared to other known free radical scavengers (i.e., glutathione and mannitol), MEL is certainly more effective as a ?OH scavenger in physiologically tolerable concentrations. The particular capacity of MEL as a radical scavenger is, therefore, due to its extraordinarily high chemical afÞnity for the ?OH. MEL may also protect cells from being damaged by acting synergistically with other antioxidants. Poeggeler et al. conÞrmed in their study that the radical trapping reagent ABTS (2,29-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) is incubated with H2O2 and FeSO4, derivative of the Fenton reaction, to generate the ABTS cation radical (?ABTS1), which is detected at 420nm of absorbance in a spectrophotometer. MEL acted synergically in preventing the formation of ?ABTS1 in combination with chain-braking antioxidants, including ascorbic acid, Trolox (vitamin E), and glutathione. Several other structurally-related indole compounds (i.e., 5-methoxytryptophol, 5-methoxyindole acetic acid, and 5-methoxytryptamine) also reduced ?ABTS1 formation by electron donation (39). Thus MEL protect DNA, protein, and lipid from oxidative damage (12,40) and have possible protective effect of aging by decreasing cell damage (30). In conclusion, ?OHs produced by postischemic reperfusion have deleterious effects on reperfused heart tissue. MEL showed signiÞcant protective effects against postischemic myocardial dysfunction and reperfusion arrhythmia by decreasing lipid peroxidation and scavenging ?OH. References 1. J.M. MCCORD, New Eng. J. Med. 312 159Ð163 (1985). 2. R.A. KLONER, K. PRZYKLENK and P. WHITTAKER, Circulation. 80 1115Ð1127 (1989).
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3. D.K. DAS, A. GEORGE, X. LIU and P.S. RAO, Biochem. Biophys. Res. Commun. 165 1004Ð1009 (1989). 4. H. OTANI, H. TANAKA, T. INOUE, M. UMEMOTO, K. OMOTO, K. TANAKA, T. SATO, T. OSAKO, A. MASUDA, A. NONOYAMA and T. KAGAWA, Circ. Res. 55 168Ð175 (1984). 5. G.J. GROSS, N.E. FARBER, H.F. HARDMAN and A.D.C. WARLTIER, Am. J. Physiol. 250 H372Ð377 (1986). 6. M.O. JEROUDI, F.J. TRIANA, B.S. PATEL and R. BOLLI, Am. J. Physiol. 259 H889Ð901 (1990). 7. M.L. MYERS, R. BOLLI, R.F. LEKICH, C.J. HARTLEY and R. ROBERTS, Circulation 72 915Ð921 (1985). 8. D.X. TAN, L.D. CHEN, B. POEGGELER, L.C. MANCHESTER and R.J. REITER, Endocr. J. 1 57Ð60 (1993). 9. Z. MATUSZEK, K. RESZKA, and C.F. CHINGNELL, Free Radical Biol. Med. 23 367Ð372 (1997). 10. D.X. TAN, L,C, MANCHESTER, R.J. REITER, B.F. PLUMMER, L.J. HARDIES, S.T. WEINTRAUB, VIJAYALAXMI and A.M.M. SHEPHERD, Biochem. Biophys. Res. Commun. 253 614Ð620 (1998). 11. C. PIERI, M. MARRA, F. MORONI, R. RECCHIONI and F. MARCHESELLI, Life Sci. 55 pl271Ð276 (1994). 12. O. IANÌS, R. OLINESCU and I. BÌDESCU, Rom. J. Endocrinol. 29 147Ð153 (1991). 13. C.S. SHIDA, A.M.L. CASTRUCCI and M.T. LAMY-FREUND, J. Pineal Res. 16 198Ð201 (1994). 14. R.J. REITER, Molec. Cell. Endocrinol. 79 C153Ð158 (1991). 15. R.J. REITER, Endocrinol. Diabetes 104 10Ð16 (1996). 16. I. MORISHIMA, H. MATSUI, H. MUKAWA, K. HAYASHI, Y. TOKI, K. OKUMURA, T. ITO and T. HAYAKAWA, Life Sci. 63 511Ð521 (1998). 17. D. MELCHIORRI, R.J. REITER, A.M. ATTIA, M. HARA, A. BURGOS and G. NISTICO, Life Sci. 56 83Ð 89 (1995). 18. S. BERTUGLIA, P.L. MARCHIAFAVA and A. COLANTUONI, Cardiovas. Res. 31 947Ð952 (1996). 19. R.E. WILLIAMS, J.L. ZWEIER and J.T. FLAHERTY, Circulation 83 1006Ð1014 (1991). 20. G. AMBROSIO, J.L. ZWEIER, W.E. JACOBUS, M.L. WEISFELDT and J.T. FLAHERTY, Circulation 76 906Ð915 (1987). 21. R.A. FLOYD, R. HENDERSON, J.J. WATSON and P.K. WONG, Free Radical Biol. Med. 2 13Ð18 (1986). 22. M. GROOTVELD and B. HALLIWELL, Biochem. J. 237 499Ð504 (1986). 23. S.R. POWELL and D. HALL, Free Rad. Biol. Med. 9 133Ð141 (1990). 24. Q. CAI, G. TAKEMURA and M. ASHRAF, J. Cardiovasc. Pharmacol. 25 147Ð155 (1995). 25. R.A. FLOYD, J.J. WATSON and P.K. WONG, J. Biochem. Biophys. Methods 10 221Ð235 (1984). 26. R. HARDELAND, R.J. REITER, B. POEGGELER and D.X. TAN, Neurosci. Biobehav. Rev. 17 347Ð357 (1993). 27. B. POEGGELER, S. SAARELA, R.J. REITER, D.X. TAN, L.D. CHEN, L.C. MANCHESTER and L.R. BARLOW-WALDEN, ANnals. NY. Acad. Sci. 738 419Ð420 (1994). 28. E. SEWERYNEK, R.J.REITER, D. MELCHIORRI, G.G. ORTIZ and A. LEWINSKI, Hepatogastroenterology 43 898Ð905 (1996). 29. C.A. DE LA LASTRA, J. CABEZA, V. MOTILVA and M.J. MARTIN, J. Pineal Res. 23 47Ð52 (1997). 30. A. BRZEZINSKI, New Eng. J. Med. 336 186Ð195 (1997). 31. D.K. DAS, G.A. CORDIS, P.S. RAO, X. LIU and S. MAITY, J. Chromato. 536 273Ð282 (1991). 32. J.T. FLAHERTY and M.L. WEISFELDT, Free Radical Biol. Med. 5 409Ð419 (1988). 33. M.S. ALAM, K. KU, M. HASHIMOTO, S. NOSAKA, Y. SAITOH, M. YAMAUCHI, S. MASUMURA, K. NAKAYAMA and K. TAMURA, Cardiovasc. Res. 33 686Ð692 (1997). 34. A. TOSAKI, D. BAGCHI, T. PALI, G.A. CORDIS and D.K. DAS, Biochem. Pharmacol. 45 961Ð969 (1993). 35. G. TAKEMURA, T. ONODERA and M. ASHRAF, Circ. Res. 71 96Ð105 (1992). 36. K.P. GALLAGHER, A.J. BUDA, D. PACE, R.A. GREEN and M. SHLAFER, Circulation 73 1065Ð1076 (1986). 37. K.P. SUNNERGREN and M.J. ROVETTO, Am. J. Physiol. 252 H1211Ð1217 (1987). 38. B.P. YU, Physiol. Rev. 74 139Ð162 (1994). 39. B. POEGGELER, R.J. REITER, R. HARDELAND, E. SEWERYNEK, D. MELCHIORRI and L.R. BARLOWWALDEN, Neuroendocrinol. Lett. 2 87Ð92 (1995).
112
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40. R. REITER, L. TANG, J.J. GARCIA and A. MU„OZ-HOYOS, Life Sci. 60 2255Ð2271 (1997). 41. M.J.A. WALKER, M.J. CURTIS, D.J. HEARSE, R.W.F. CAMPBELL, M.J. JANSE, D.M. YELLON, S.M. COBBE, S.J. COKER, J.B. HARNESS, D.W.G. HARRON, A.J. HIGGINS, D.G. JULIAN, M.J. LAB, A.S. MANNING, B.J. NORTHOVER, J.R. PARRATT, R.A. RIEMERSMA, E. RIVA, D.C. RUSSELL, D.J. SHERIDAN, E. WINSLOW and B. WOODWARD, Cardiovas. Res. 22 447Ð455 (1988). 42. J.A. BUEGE and S.D. AUST, Meth. Enzymol. 55 302Ð310 (1978). 43. G. AMBROSIO, J.T. FLAHERTY, C. DUILIO, I. TRITTO, G. SANTORO, P.P. ELIA, M. CONDORELLI and M. CHIARIELLO, J. Clin. Invest. 87 2056Ð2066 (1991). 44. D.X. TAN, L.C. MANCHESTER, R.J. REITER, W. QI, S.J. KIM and G.H. EL-SOKKARY, J. Pineal Res. 25 184Ð191 (1998).
Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury Shinji Kanekoa,*, Kenji Okumuraa, Yasushi Numaguchia, Hideo Matsuia, Kichiro Murasea, Shinji Mokunoa, Itsuro Morishimaa, Kenji Hiraa, Yukio Tokia, Takayuki Itob, Tetsuo Hayakawaa a
Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan b Health Sciences, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Abstract During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (?OH) in the efßuent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for ?OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantiÞed using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 mM MEL1 SAL had signiÞcantly reduced amounts of 2,5- and 2,3DHBA by multiple folds, compared to the SAL group. TBARS was signiÞcantly decreased in the 100 mM MEL group (1.20 6 0.36 vs 1.85 6 0.10 mmol/g of drug-free group, p,0.001). More importantly, the 100 mM MEL group signiÞcantly recovered in LV function (LV developed pressure, 1dp/dt, and 2dp/dt; 63.0%, 60.3%, and 59.4% in the 100 mM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p,0.05). Duration of ventricular tachycardia or ventricular Þbrillation signiÞcantly decreased in the 100 mM MEL group (100 mM MEL, 159 6 67 sec; drug-free, 1244 6 233 sec; p,0.05). As a result of scavenging ?OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; Isolated rat heart; Reperfusion injury; DHBA; Salicylate method; TBARS, Hydroxyl radical
Introduction Oxygen-derived free radicals lead to deleterious effects in postischemic reperfusion of rat hearts (1,2). Recent studies have revealed that free radicals may be produced by mitochon* Corresponding author. Tel.: 181-52-744-2168; fax: 181-52-744-2175. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 0 7 -X
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dria of postischemic reperfused myocardium, leading to lipid peroxidation, and then to damage of the myocardial tissue (3,4,31). Hydroxyl radicals (?OH) are formed via a superoxidederived (Haber-Weiss reaction) and iron-catalyzed Fenton reaction (38). The hydroxyl radical has notable characteristics in taking part in ischemia/reperfusion injury. It displays much greater chemical reactivity than superoxide radicals (O2?2) or hydrogen peroxide (H2O2) and plays a signiÞcant role in postischemic reperfusion injury (1,2,19,20,31,32). Evidence has been presented to indicate that free radical scavengers such as superoxide dismutase (SOD) or catalase (CAT) attenuate postischemic reperfusion injury of myocardium (5Ð7,34). The pineal secretory production melatonin (MEL, 5-methoxy-N-acetyl-tryptamine) has recently been conÞrmed to be a potent scavenger of hydroxyl (8) and peroxyl radicals (11,12). Generally in humans melatonin regulates sleep and circadian rhythms (30). Since it is well known that MEL is both a lipophilic and hydrophilic compound (13,14), MEL freely permeates all morphophysiological barriers of cells in any organs (15). MEL may protect all tissues, including myocardium, from free radical mediated damage caused by adriamycin (16), paraquat (17), and postischemic reperfusion injury (7,28,29). However no serious side effects have been reported in people who take large doses for prolonged periods of time (30). Electron paramagnetic resonance (ESR) has been well known as a powerful tool in studying the relationship between ?OH and ischemic reperfusion injury. Nevertheless, ESR also has its limitations in the spin-trapping process. Firstly, concentration of the spin trap reagent is toxic to isolated rat heart. Secondly, the sensitivity of ESR is lower than the salicylate (SAL) method (23,31,34,35). In recent years, some investigators (Floyd et al., Grootvelt and Halliwell, and Pritsos et al.) have introduced high-performance liquid chromatography (HPLC) methods to detect ?OH (3,21Ð23,25,31,34). We used the same method. If salicylate molecules are attacked by ?OH, three products (2,5-dihydroxybenzoic acid [DHBA], 2,3-DHBA, catechol) are formed under physiological conditions. The percentages of 2,5-DHBA, 2,3DHBA, and catechol are 40%, 49%, and 11%, respectively (22,25). These high sensitive measurements of the amounts of 2,5-DHBA and 2,3-DHBA are evidence consistent with ?OH formation during postischemic reperfusion. Because MEL has been shown to have ?OH scavenging properties, we evaluated the scavenging effect of MEL by indirect measure of ?OH and the extent of lipid peroxidation, and the protective effects against postischemic reperfusion injury. Methods SAL was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). MEL, 2,5DHBA, 2,3-DHBA, and all other reagent were purchased from Sigma Chemical (St. Louis, MO, USA). Heart preparation The investigation was conducted in full compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Sprague-Dawley rats (Japan SLC, Inc.) weighing 250 to 300 g were anesthetized with pentobarbital sodium (50mg/kg) by intraperitoneal injection. After intravenous heparin injection (1000U/kg), hearts were rapidly excised and put into 48C Krebs-Henseleit solution. Aortas were cannulated for isolated perfusion (Langendorff tech-
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nique) within 60 seconds afer excision. Hearts were perfused at a constant pressure of 70mmHg with a modiÞed Krebs-Henseleit (KH) solution (mmol/L:NaCl 118, NaHCO3 25, NaH2PO4 1.18, KCl 5.8, CaCl2 2.5, MgSO4 1.2, glucose 10) gassed with 95% O2/5% CO2, achieving a pH of 7.40 and a temperature of 37.08C. A ßuid-Þlled latex balloon was inserted into the left ventricular (LV) cavity via the left auricle and mitral valve to measure the LV isovolumic pressure. The LV catheter was connected to a pressure transducer (TB-611T, Nihon Koden, Japan), and the LV isovolumic pressure and 6dp/dt were continuously recorded on a polygraph recorder (Recticorder, Nihon Kohden, Japan). The volume of the LV balloon was adjusted to obtain a LV end diastolic pressure of 5 mmHg during the equilibration period, and was not changed thereafter. Electrical activity (ECG) was monitored using two electrodes attached directly to the heart. The duration of ventricular Þbrillation (VF) or ventricular tachycardia (VT) was recorded from the ECG. The diagnostic criteria for VF and VT were consistent with the recommendations of the Lambeth Conventions (41). The rate-pressure product (RPP) was calculated by multiplying heart rate (HR) and LV developed pressure (LVDP, LV systolic pressure minus LV end diastolic pressure). Timed coronary efßuent was collected for measurement of coronary ßow (CF) every one minute. Experimental protocols The experimental protocol is shown in Fig. 1. The hearts were divided into 6 groups as follows. 1. Drug-free group (n57); Hearts were equilibrated with KH solution for the Þrst 30 min and then subjected to normothermic (378C) global ischemia for 30 min, followed by 30 min of reperfusion by turning off the aortic inßow line. 2. Ischemia/reperfusion with 1mM SAL (SAL, n57); Group 2 was similar to the control group except in that hearts were perfused with KH solution containing 1 mM SAL. 3. Ischemia/reperfusion with 1mM SAL and MEL treatment (n57); Hearts were perfused with KH solution containing 1 mM SAL and MEL. The concentrations of MEL were 10 (SAL110mM MEL), 30 (SAL130mM MEL), and 100 mM (SAL1100mM MEL). Ischemia/reperfusion time was the same as the time used for the control group. 4. Ischemia/reperfusion with MEL treatment (100mM MEL, n57); Hearts were perfused with KH solution containing 100 mM MEL. Ischemia/reperfusion time was the same as the time used for the control group. LVDP and LV 6dp/dt were measured during pacing in an atrial mode at 300 beats/min (Cardiac Stimulator, Nihon Kohden, Japan) after 30 min of reperfusion to evaluate postischemic reperfused myocardial function. At the end of the experiments, hearts were weighed and the LV free wall was immediately frozen in liquid nitrogen, stored at 2808C, and made dry with lyophil method. Measurement of ?OH In a separate group of experiments, isolated rat hearts were subjected to 30 min of aerobic perfusion with KH solution containing 1 mM SAL and 0, 10, 30, and 100 mM MEL, followed by 30 min of normothermic global ischemia and 30 min of reperfusion. Samples of ef-
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Fig. 1. This Þgure shows the protocol of the experiment. Group 1, KH solution (drug free) Group 2, KH solution 1 1 mM SAL (SAL) Group 3, KH solution 1 1 mM SAL 1 10 mM MEL (SAL 1 10 mM MEL) Group 4, KH solution 1 1 mM SAL 1 30 mM MEL (SAL 1 30 mM MEL) Group 5, KH solution 1 1 mM SAL 1 100 mM MEL (SAL 1 100 mM MEL) Group 6, KH solution 1 100 M mMEL (100 mM MEL)
ßuent were collected after 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, and 30 min of reperfusion and were stored at 2208C until use. The presence of hydroxylated benzoic acid in the perfusate was determined as previously described (23,30,33,34). Brießy, the perfusate was Þltered through a Supelco 0.45 mm pore size Nylon sample Þlter, and a 25 mL volume of the sample was injected into the HPLC apparatus. The HPLC apparatus consisted of a Model L-6000 (Yanaco) and a Voltammetric detector (Yanaco VMD-3000). The column used was a Supelcosil LC18-DB (3mm ODS, 7534.6mm) column (Supelco) attached to a Supelguard LC-18-DB precolumn (Supelco). The mobile phase was 0.03 mol/L sodium acetate and 0.03 mol/L citric acid (pH53.6) at a ßow rate of 1 mL/min. The detector potential was maintained at 10.6 V, employing a glassy carbon working electrode and an Ag/AgCl reference electrode. Retention times for the peaks of 2,5-DHBA and 2,3-DHBA were veriÞed by injecting authentic standards and all samples were measured against these standards. Measurement of the extent of lipid peroxidation To evaluate the extent of lipid peroxidation, the amount of thiobarbituric acid reactive substances (TBARS) in myocardial tissue of lyophilization, a measurement of the extent of lipid peroxidation, was assayed by a modiÞcation of the thiobarbituric acid (TBA) method (42,43). Each sample was homogenized in 1.15% KCl solution containing 10 mM deferoxamine, 0.04% butylated hydroxytoluene (BHT), and 2% ethanol. Each homogenate was incubated for 60 min at 958C in an oil bath with stock TCA-TBA-HCl reagent (15% [w/v] trichloroacetic acid, 0.375% [w/v] thiobarbituric acid, 0.25N hydrochloric acid) and 2% BHT. After cooling, the ßocculent precipitate was removed by centrifugation for 10 min at 1000 g, and the extinction coefÞcient of the supernatant at 535 nm was determined spectrophotometrically and compared with a known TBARS standard.
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Table 1 Comparison of the parameter of cardiac function in each group during equilibration
Drug-free SAL SAL 110 mM MEL SAL130 mM MEL SAL1100 mM MEL 100 mM MEL
Pre-efßuent (ml)
HR (beats/min)
Pre-LVDP (mmHg)
Pre-RPP
Pre-LV1dp/dt (mmHg/sec)
Pre-LV-dp/dt (2mmHg/sec)
10.8 6 0.3 11.6 6 0.4 11.5 6 0.4 10.9 6 0.3 11.2 6 0.2 10.3 6 0.2
249 6 10 255 6 19 245 6 9 241 6 19 262 6 7 263 6 9
106 6 3 103 6 2 102 6 2 104 6 4 102 6 2 105 6 2
26131 6 627 26067 6 1568 25036 6 1009 24763 6 1163 26600 6 517 27509 6 478
2697 6 90 2753 6 26 2807 6 50 2824 6 68 2831 6 40 2838 6 60
2256 6 78 2034 6 56 2132 6 66 2019 6 63 2169 6 31 2254 6 79
All parameters did not show signiÞcant differences among 6 groups (n57 in all groups). Data are expressed as mean 6 SEM.
Statistical analysis Data were expressed as means 6 SEM. Group comparisons were made by analysis of variance with multiple comparisons (Bonferroni corrected t test). A difference criteria for a value was set at p , 0.05 in order to determine whether the result was signiÞcant. Results Cardiac functions during equilibration Addition of SAL and MEL in KH solution had no deleterious effects on any of the parameters determined including CF, HR, LVDP, RPP, and contractility (6LVdp/dt). No effect of SAL and MEL on electrical activity of the heart was observed during equilibration (see Table 1).
Fig. 2. Effect of MEL on LVDP after 30 min of reperfusion. The heart was paced at 300 beats/min. LVDP of the 100mM MEL and SAL1100mM MEL groups signiÞcantly recovered compared to that of the drug-free and SAL groups. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups.
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Recovery of cardiac functions Fig. 2 shows the recovery of the LVDP after 30 min reperfusion. The recovery of the LVDP in the 100 mM MEL 1 SAL and 100 mM MEL groups were signiÞcantly greater than that of the SAL and drug-free groups (p , 0.05). The LVDP of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 32 6 4, 32 6 2, 39 6 8, 42 6 6, 60 6 4, and 63 6 5 mmHg, respectively. The recovery of 6LVdp/dt in the 100 mM MEL 1 SAL and 100 mM MEL groups after 30 min of reperfusion was also superior to that of the SAL and drug-free groups (p , 0.05) (Fig. 3). The 1LVdp/dt of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 1785 6 82, 1801 6 85, 1941 6 220, 1985 6 173, 11534 6 108, and 11710 6162 mmHg/sec, respectively. The -LVdp/dt of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups when paced at 300 beats/min were 2629 6 82, 2711 6 62, 2829 6 196, 2838 6 141, 21269 6 102, and 21339 6 111 mmHg/sec, respectively. There were no signiÞcant differences in CF among the groups during reperfusion (data are not shown). Generation of ?OH during reperfusion The efßuent of the reperfused heart was subjected to HPLC for the detection of ?OH by measuring 2,5-DHBA and 2,3-DHBA. The retention times for 2,5-DHBA and 2,3-DHBA
Fig. 3. LV1dp/dt of the drug-free and SAL groups after 30 min of reperfusion were signiÞcantly lower than that of the 100mM MEL and SAL1100mM MEL groups. LV-dp/dt of the drug-free and SAL groups after 30 min of reperfusion also were signiÞcantly lower than that of the 100mM MEL and SAL1100mM MEL groups. The heart was paced at 300 beats/min. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups, ** p , 0.01 vs drug-free and SAL groups.
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Fig. 4. Time courses of 2,5-DHBA (A) and 2,3-DHBA (B) during reperfusion in each group. (A) All groups showed peaks at about 3Ð8 min from the onset of reperfusion. Addition of 100mM MEL and 1mM SAL to KH solution signiÞcantly reduced production of 2,5-DHBA compared with SAL group, especially during the early reperfusion period. (B) All groups also showed peaks at about 3Ð8 min from the onset of reperfusion. Isolated rat hearts of 100mM MEL 1 SAL group signiÞcantly produced lower levels of 2,3-DHBA than hearts of 1mM SAL group. Data are expressed as means 6 SEM. * p , 0.05, ** p , 0.01, p , 0.005, p , 0.001 vs SAL group.
were determined to be 7.3 and 9.5 min, respectively, by standard reagent. As shown in Fig. 4, DHBAs reached peaks at 3Ð8 min after the onset of reperfusion, and leveled off after 10 min in all groups. MEL reduced the amounts of 2,5-DHBA and 2,3-DHBA in a dose-dependent manner (p , 0.05; vs SAL group). The amounts of DHBAs of the early reperfusion period in
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the 100 mM MEL 1 SAL group were depressed compared with those of the SAL group. The values of 3Ð8 min were especially worthy of notice. The extent of lipid peroxidation in the myocardium The levels of TBARS in the 100 mM MEL 1 SAL and 100 mM MEL groups were signiÞcantly lower than those of the drug-free and SAL groups (Fig. 5). The TBARS of the SAL, drug-free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups were 1.68 6 0.07, 1.85 6 0.10, 1.69 6 0.04, 1.66 6 0.08, 1.34 6 0.08, and 1.20 6 0.04 mmol/g, respectively. Ventricular arrhythmia The incidence of reperfusion-induced VT or VF is shown in Fig. 6. A reduction in the incidence of VT or VF during reperfusion was dose-dependently observed. The duration of VT or VF of the SAL, drug free, 10 mM MEL 1 SAL, 30 mM MEL 1 SAL, 100 mM MEL 1 SAL, and 100 mM MEL groups were 1364 6 248, 1244 6 233, 805 6 284, 810 6 216, 209 6 68, and 159 6 67 sec, respectively. A signiÞcant dose of MEL was conÞrmed in the 100 mM group (p , 0.05; vs drug free and SAL groups). Discussion The mitochondrial, microsomal, and nuclear membranes contain electron transport systems which may produce free radicals (38). Under conditions of hyperbolic oxygen or at the onset of postischemic reperfusion, the mitochondrial respiratory rate will increase markedly and greater quantities of free radicals are generated. These radicals may exceed the capacity
Fig. 5. MEL signiÞcantly decreased TBARS in the myocardial tissue, compared to non-MEL groups. TBARS in the 100 mM MEL group was lower than in the drug-free, SAL, SAL 1 10 mM MEL and SAL 1 30 mM MEL groups. TBARS in the SAL 1 100 mM MEL group was lower than in the drug-free and SAL groups. Data are expressed as means 6 SEM. * p , 0.005 vs drug-free and SAL groups, p , 0.001 vs drug-free, SAL, SAL 1 10 mM MEL, and SAL 1 30 mM MEL group.
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Fig. 6. Duration of ventricular arrhythmia signiÞcantly decreased in the 100mM MEL and SAL1100mM MEL groups, compared to the drug-free and SAL groups during reperfusion. Data are expressed as means 6 SEM. * p , 0.05 vs drug-free and SAL groups
of the cellular intrinsic free radical scavenging systems, and be cytotoxic to cells by attacking fatty acids. The reactions caused by exceeding radicals leads to lipid peroxidation of membranes, and that destroys and oxidizes amino acids and their polypeptide chains. These results lead to LV dysfunction and reperfusion arrhythmia in the postischemic reperfused heart. In this study, we used the SAL method, originally reported by Floyd et al. (25), to detect DHBA species produced from SAL hydroxylation. Although the ?OH has an extremely short half-life, DHBA species are extremely stable and can be used to evaluate various treatments in order to study the deleterious effects of the ?OH (31). In many cases, in the presence of ?OH, one of the most reactive radical species, the protection of the tissue by some radical scavenger (i.e., SOD, CAT, or histidine) was observed (5,6,24). Evidence has been shown that MEL prevents postischemic reperfusion injury in hamster cheek pouches (19), liver (28), stomach (29), and postischemic reperfusion arrhythmia (44). Tan et al. reported MEL prevented ischemia/reperfusion-induced arrhythmias. To further study the mechanism of the MEL effect, we measured DHBAs and TBARS in this experiment. No previous study, however, has implicated the efÞcacy of MEL against ischemia/reperfusion injury in isolated rat heart. The present results demonstrate for the Þrst time that MEL scavenges ?OHs and still has beneÞcial effects in postischemic myocardial dysfunction. In this study, MEL signiÞcantly inhibited the formation of 2,5- and 2,3-DHBA in a dosedependent manner. Addition of 100 mM melatonin in buffer clearly suppressed the production of 2,5- and 2,3-DHBA approximately by one-fourth to one-thirteenth times higher than the SAL group during reperfusion. The amount of TBARS in the myocardial tissue was also reduced by administration of 100 mM MEL. Addition of 100 mM MEL had recovering effects on the subject output, including LVDP and 6 LV dp/dt after 30 minutes of reperfusion, and reduced the duration of VT or VF. Free radicals are thought to be implicated in ventricular arrhythmia and deleterious effects on LV function after postischemic reperfusion (1,2,32). Although various free radical scavengers indicate beneÞcial effects on postischemic myo-
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cardial function and ventricular arrhythmia (5,6,7,24,33,34), Takemura et al. conÞrmed that deferoxamine effectively reduced the amount of 2,5-DHBA and failed in the protection of postischemic reperfusion injury in 15 min of ischemia (35). The parameter of 2,5-DHBA is a nonspeciÞc derivative produced by cytochrome P450 without SAL hydroxylation (34). Therefore, they should have evaluated 2,3-DHBA. Other investigators also reported that radical scavengers fail to protect against reperfusion injury under speciÞc conditions. Sunnergren and Rovelto reported that SOD, CAT, and mannitol failed to ameliorate LVDP and prevent increased permeability in ischemia/reperfused isolated rat heart (36). Although Gallagher et al. evaluated the size of infarction after postischemic reperfusion with or without SOD and CAT in in vivo dogs, SOD and CAT failed to reduce the size of infarction (37). Since many differences are observed among those studies in experimental models, protocols, and parameters to evaluate reperfusion injuries, different results may follow in association with the efÞcacy of radical scavengers reported in those studies. MEL is believed to work via electron donation to directly detoxify free radicals such as the highly toxic ?OH with no enzymatical; reaction. Tan et al. have conÞrmed MELÕs free radical scavenging ability in vitro (8,9,10). In their system, 5,5-dimethyl-1-pyrroline N-oxide, a trapping reagent, is hydroxylated by ?OH and the product of reaction was quantiÞed by HPLC and ESR. It has been proven that MEL, in the presence of ?OH, detoxiÞes it by electron donation with resultant production of the indolyl cation radical. The indolyl cation radical reacts directly, without requiring another catalyst, with an additional superoxide anion (26,27,40). Because MEL is both lipophilic and hydrophilic, it can enter all cells with ease. It is obvious that compared to other known free radical scavengers (i.e., glutathione and mannitol), MEL is certainly more effective as a ?OH scavenger in physiologically tolerable concentrations. The particular capacity of MEL as a radical scavenger is, therefore, due to its extraordinarily high chemical afÞnity for the ?OH. MEL may also protect cells from being damaged by acting synergistically with other antioxidants. Poeggeler et al. conÞrmed in their study that the radical trapping reagent ABTS (2,29-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) is incubated with H2O2 and FeSO4, derivative of the Fenton reaction, to generate the ABTS cation radical (?ABTS1), which is detected at 420nm of absorbance in a spectrophotometer. MEL acted synergically in preventing the formation of ?ABTS1 in combination with chain-braking antioxidants, including ascorbic acid, Trolox (vitamin E), and glutathione. Several other structurally-related indole compounds (i.e., 5-methoxytryptophol, 5-methoxyindole acetic acid, and 5-methoxytryptamine) also reduced ?ABTS1 formation by electron donation (39). Thus MEL protect DNA, protein, and lipid from oxidative damage (12,40) and have possible protective effect of aging by decreasing cell damage (30). In conclusion, ?OHs produced by postischemic reperfusion have deleterious effects on reperfused heart tissue. MEL showed signiÞcant protective effects against postischemic myocardial dysfunction and reperfusion arrhythmia by decreasing lipid peroxidation and scavenging ?OH. References 1. J.M. MCCORD, New Eng. J. Med. 312 159Ð163 (1985). 2. R.A. KLONER, K. PRZYKLENK and P. WHITTAKER, Circulation. 80 1115Ð1127 (1989).
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3. D.K. DAS, A. GEORGE, X. LIU and P.S. RAO, Biochem. Biophys. Res. Commun. 165 1004Ð1009 (1989). 4. H. OTANI, H. TANAKA, T. INOUE, M. UMEMOTO, K. OMOTO, K. TANAKA, T. SATO, T. OSAKO, A. MASUDA, A. NONOYAMA and T. KAGAWA, Circ. Res. 55 168Ð175 (1984). 5. G.J. GROSS, N.E. FARBER, H.F. HARDMAN and A.D.C. WARLTIER, Am. J. Physiol. 250 H372Ð377 (1986). 6. M.O. JEROUDI, F.J. TRIANA, B.S. PATEL and R. BOLLI, Am. J. Physiol. 259 H889Ð901 (1990). 7. M.L. MYERS, R. BOLLI, R.F. LEKICH, C.J. HARTLEY and R. ROBERTS, Circulation 72 915Ð921 (1985). 8. D.X. TAN, L.D. CHEN, B. POEGGELER, L.C. MANCHESTER and R.J. REITER, Endocr. J. 1 57Ð60 (1993). 9. Z. MATUSZEK, K. RESZKA, and C.F. CHINGNELL, Free Radical Biol. Med. 23 367Ð372 (1997). 10. D.X. TAN, L,C, MANCHESTER, R.J. REITER, B.F. PLUMMER, L.J. HARDIES, S.T. WEINTRAUB, VIJAYALAXMI and A.M.M. SHEPHERD, Biochem. Biophys. Res. Commun. 253 614Ð620 (1998). 11. C. PIERI, M. MARRA, F. MORONI, R. RECCHIONI and F. MARCHESELLI, Life Sci. 55 pl271Ð276 (1994). 12. O. IANÌS, R. OLINESCU and I. BÌDESCU, Rom. J. Endocrinol. 29 147Ð153 (1991). 13. C.S. SHIDA, A.M.L. CASTRUCCI and M.T. LAMY-FREUND, J. Pineal Res. 16 198Ð201 (1994). 14. R.J. REITER, Molec. Cell. Endocrinol. 79 C153Ð158 (1991). 15. R.J. REITER, Endocrinol. Diabetes 104 10Ð16 (1996). 16. I. MORISHIMA, H. MATSUI, H. MUKAWA, K. HAYASHI, Y. TOKI, K. OKUMURA, T. ITO and T. HAYAKAWA, Life Sci. 63 511Ð521 (1998). 17. D. MELCHIORRI, R.J. REITER, A.M. ATTIA, M. HARA, A. BURGOS and G. NISTICO, Life Sci. 56 83Ð 89 (1995). 18. S. BERTUGLIA, P.L. MARCHIAFAVA and A. COLANTUONI, Cardiovas. Res. 31 947Ð952 (1996). 19. R.E. WILLIAMS, J.L. ZWEIER and J.T. FLAHERTY, Circulation 83 1006Ð1014 (1991). 20. G. AMBROSIO, J.L. ZWEIER, W.E. JACOBUS, M.L. WEISFELDT and J.T. FLAHERTY, Circulation 76 906Ð915 (1987). 21. R.A. FLOYD, R. HENDERSON, J.J. WATSON and P.K. WONG, Free Radical Biol. Med. 2 13Ð18 (1986). 22. M. GROOTVELD and B. HALLIWELL, Biochem. J. 237 499Ð504 (1986). 23. S.R. POWELL and D. HALL, Free Rad. Biol. Med. 9 133Ð141 (1990). 24. Q. CAI, G. TAKEMURA and M. ASHRAF, J. Cardiovasc. Pharmacol. 25 147Ð155 (1995). 25. R.A. FLOYD, J.J. WATSON and P.K. WONG, J. Biochem. Biophys. Methods 10 221Ð235 (1984). 26. R. HARDELAND, R.J. REITER, B. POEGGELER and D.X. TAN, Neurosci. Biobehav. Rev. 17 347Ð357 (1993). 27. B. POEGGELER, S. SAARELA, R.J. REITER, D.X. TAN, L.D. CHEN, L.C. MANCHESTER and L.R. BARLOW-WALDEN, ANnals. NY. Acad. Sci. 738 419Ð420 (1994). 28. E. SEWERYNEK, R.J.REITER, D. MELCHIORRI, G.G. ORTIZ and A. LEWINSKI, Hepatogastroenterology 43 898Ð905 (1996). 29. C.A. DE LA LASTRA, J. CABEZA, V. MOTILVA and M.J. MARTIN, J. Pineal Res. 23 47Ð52 (1997). 30. A. BRZEZINSKI, New Eng. J. Med. 336 186Ð195 (1997). 31. D.K. DAS, G.A. CORDIS, P.S. RAO, X. LIU and S. MAITY, J. Chromato. 536 273Ð282 (1991). 32. J.T. FLAHERTY and M.L. WEISFELDT, Free Radical Biol. Med. 5 409Ð419 (1988). 33. M.S. ALAM, K. KU, M. HASHIMOTO, S. NOSAKA, Y. SAITOH, M. YAMAUCHI, S. MASUMURA, K. NAKAYAMA and K. TAMURA, Cardiovasc. Res. 33 686Ð692 (1997). 34. A. TOSAKI, D. BAGCHI, T. PALI, G.A. CORDIS and D.K. DAS, Biochem. Pharmacol. 45 961Ð969 (1993). 35. G. TAKEMURA, T. ONODERA and M. ASHRAF, Circ. Res. 71 96Ð105 (1992). 36. K.P. GALLAGHER, A.J. BUDA, D. PACE, R.A. GREEN and M. SHLAFER, Circulation 73 1065Ð1076 (1986). 37. K.P. SUNNERGREN and M.J. ROVETTO, Am. J. Physiol. 252 H1211Ð1217 (1987). 38. B.P. YU, Physiol. Rev. 74 139Ð162 (1994). 39. B. POEGGELER, R.J. REITER, R. HARDELAND, E. SEWERYNEK, D. MELCHIORRI and L.R. BARLOWWALDEN, Neuroendocrinol. Lett. 2 87Ð92 (1995).
112
S. Kaneko et al. / Life Sciences 67 (2000) 101Ð112
40. R. REITER, L. TANG, J.J. GARCIA and A. MU„OZ-HOYOS, Life Sci. 60 2255Ð2271 (1997). 41. M.J.A. WALKER, M.J. CURTIS, D.J. HEARSE, R.W.F. CAMPBELL, M.J. JANSE, D.M. YELLON, S.M. COBBE, S.J. COKER, J.B. HARNESS, D.W.G. HARRON, A.J. HIGGINS, D.G. JULIAN, M.J. LAB, A.S. MANNING, B.J. NORTHOVER, J.R. PARRATT, R.A. RIEMERSMA, E. RIVA, D.C. RUSSELL, D.J. SHERIDAN, E. WINSLOW and B. WOODWARD, Cardiovas. Res. 22 447Ð455 (1988). 42. J.A. BUEGE and S.D. AUST, Meth. Enzymol. 55 302Ð310 (1978). 43. G. AMBROSIO, J.T. FLAHERTY, C. DUILIO, I. TRITTO, G. SANTORO, P.P. ELIA, M. CONDORELLI and M. CHIARIELLO, J. Clin. Invest. 87 2056Ð2066 (1991). 44. D.X. TAN, L.C. MANCHESTER, R.J. REITER, W. QI, S.J. KIM and G.H. EL-SOKKARY, J. Pineal Res. 25 184Ð191 (1998).