Koc, M., Taysi, S., Buyukokuroglu, M. E. and Bakan, N. The Effect of Melatonin against Oxidative Damage during Total-Body Irradiation in Rats. Radiat. Res. 160, 251–255 (2003).
Melatonin has been reported to participate in the regulation of a number of important physiological and pathological processes. Melatonin, which is a powerful endogenous antioxidant, may play a role in the prevention of oxidative damage. The aim of this study was to investigate the effect of pretreatment with melatonin (5 mg kg–1 and 10 mg kg–1) on γ-radiation-induced oxidative damage in plasma and erythrocytes after total-body irradiation with a single dose of 5 Gy. Total-body irradiation resulted in a significant increase in plasma and erythrocyte MDA levels. Melatonin alone increased the levels of SOD and GSH-Px. Erythrocyte and plasma MDA levels in irradiated rats that were pretreated with melatonin (5 or 10 mg kg–1) were significantly lower than those in rats that were not pretreated. There was no significant difference between the effects of 5 and 10 mg kg–1 on plasma MDA activities and CAT activities. However, erythrocyte MDA levels showed a dose-dependent decrease, while GSH-Px activities increased with dose. Our study suggests that melatonin administered prior to irradiation may protect against the damage produced by radiation by the up-regulation of antioxidant enzymes and by scavenging free radicals generated by ionizing radiation.
INTRODUCTION
Reactive oxygen species (ROS) and lipid peroxides have been implicated in the pathogenesis of a number of diseases, including cancer, diabetes mellitus, rheumatoid arthritis, infectious diseases and atherosclerosis, and in aging (1–3). Since these radicals initiate lipid peroxidation, that patients who receive total-body irradiation (TBI) might have higher levels lipid peroxidation (4, 5). Cells have developed a defense against ROS, the antioxidant system, which includes enzymatic and non-enzymatic components (2). The antioxidant system consists of low-molecular-weight antioxidant molecules such as glutathione (GSH), melatonin and various antioxidant enzymes. Superoxide dismutase (SOD), the first line of defense against oxygen-derived free radicals, catalyzes the dismutation of the superoxide anion (O2–) into H2O2. Catalase (CAT), which is present in peroxisomes in eukaryotic cells, transforms H2O2 into H2O and O2. Glutathione peroxidase (GSH-Px), a selenoprotein, reduces lipidic or nonlipidic hydroperoxides as well as H2O2 while oxidizing GSH to generate oxidized glutathione (GSSG), which is then reduced to GSH by glutathione reductase (6–9).
Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced by the pineal gland. Melatonin, which is a powerful endogenous antioxidant, stimulates GSH-Px and can play a role in the prevention of oxidative damage (10). Recent studies demonstrated that melatonin scavenges hydroxyl radicals generated in vitro by hydrogen peroxide exposed to ultraviolet light (11, 12). Melatonin is also a more efficient scavenger of peroxyl radicals than vitamin E (13).
The process of lipid peroxidation is one of oxidative conversion of polyunsaturated fatty acids to products to native aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal by a well-studied, biologically relevant free radical reaction. MDA itself, because of its high cytotoxicity and its inhibitory action on protective enzymes, has been suggested to act as a tumor promoter and a co-carcinogen (14). It is also a well-characterized mutagen that reacts with deoxyguanosine to form an important endogenous adduct found in the DNA of human liver. MDA, an end product of lipid peroxidation, has been used as index of oxidative damage (15, 16). The aim of this study was to investigate the antioxidant role of melatonin (5 mg kg–1 and 10 mg kg–1) against γ-radiation-induced oxidative damage in plasma and erythrocytes after total-body irradiation of rats with a single dose of 5 Gy.
MATERIALS AND METHODS
Animals and Experiments
Fifty albino Wistar male rats weighing 190–225 g were used for the experiment. All animals received humane care in compliance with the guidelines of the criteria of the Ataturk University Research Council. The rats were fed standard laboratory chow and water and housed in cages in a windowless laboratory with automatic temperature (22 ± 1°C) and lighting controls (14 h light/10 h dark). The rats were fasted for 24 h before the experiment but were allowed access to water ad libitum.
The rats were divided into five equal groups. Groups I and II were injected with 5 and 10 mg kg–1 melatonin (Sigma Chemical Co.). Group III was injected with isotonic NaCl solution. Melatonin was dissolved in ethanol and was diluted in isotonic NaCl. Drugs and saline solution were administered by intraperitoneal injection. Group IV was injected with 5 mg kg–1 melatonin only, and the last group (group V) served as the sham-treated control group.
Thirty minutes after injection, rats in groups I, II and III were anesthetized and irradiated with 5 Gy to the total body as a single fraction. Irradiation was performed using a cobalt-60 teletherapy unit (Picker C-9) at a source-to-surface distance of 80 cm. The dose was calculated to the central axis at a depth of 2 cm. The dose rate was 1.98 Gy/min. The animals in the melatonin-only and sham control groups animals were anesthetized but were not irradiated. All animals were killed 2 h after irradiation.
Biochemical Analysis
Two hours after irradiation (17), animals were anesthetized with 50 mg kg–1 of thiopental sodium. Two-milliliter blood samples were obtained by intracardiac puncture and the blood samples were coded. After the collection of the blood samples into glass tubes containing citrate (3.5 mg/ml blood), erythrocytes were sedimented and hemolyzed by dilution with deionized water (50-fold). Analyses were carried out in the hemolyzed supernatant fraction (18). The hemolysate and plasma of the rats were held at −80° until biochemical determinations.
Erythrocyte and plasma MDA levels were measured by the spectrophotometric methods of Cynamon et al. (19) and of Beuge and Aust (20), respectively. Briefly, the sample was mixed with two volumes of a stock solution of 15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, and 0.25 N hydrochloric acid. The combination of sample and stock solution was heated for 30 min in a boiling water bath. After cooling, the precipitate was removed by centrifugation, and the absorbance of the supernatant was determined at 535 nm. Total thiobarbituric acid-reactive substances (TBARS) were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 × 105 cm–1 M–1. Erythrocyte and plasma MDA levels were expressed as μmol/g hemoglobin and μmol/liter plasma, respectively.
SOD activity was detected by the method of Sun and coworkers (21). Xanthine-xanthine oxidase complex, which produces superoxide radicals, was reacted with nitroblue tetrazolium (NBT) to form the farmasone compound. SOD activity was measured spectrophotometrically at 560 nm based on the inhibition of this reaction. The blank contained all the reagents except the sample. One SOD unit was defined as the amount of enzyme required to cause a 50% reduction in the NBTH2 reaction rate, and SOD activity was expressed as U/g hemoglobin.
GSH-Px activity was measured as described by Paglia and Valentine (22). In this method, GSH-Px catalyses the oxidation of glutathione in the presence of tert-butyl hydroperoxide (tBH). Oxidized glutathione is converted to the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. The rate of oxidation of NADPH is determined from the rate of change in absorption at 340 nm and converted to GSH-Px activity using the molar extinction coefficient of NADPH. GSH-Px activity was expressed as U/g hemoglobin.
CAT activity was measured in hemolysates at 20°C according to the method of Aebi (23) by measuring the rate of decomposition of H2O2 using a molar extinction coefficient of 43.6 M–1 cm–1. The rate for the first 30 s was used to calculate the activity. One unit is equivalent to the decomposition of 1 μmol H2O2/min ml–1. CAT activity was expressed as U/g hemoglobin.
The hemoglobin values were measured with a GEN-S counter hematology analyzer. Biochemical measurements were carried out at room temperature using a spectrophotometer (CECIL CE 3041).
Statistical Analyses
Values are given as means ± SD for 10 measurements. All parameters were analyzed by one-way analysis of variance. The least significant difference multiple range test was used to compare the mean values. Statistical significance was defined as P < 0.05. Statistical analysis was performed with the Statistical Package for the Social Sciences for Windows (SPSS, version 10.0, Chicago).
RESULTS
Results are tabulated in Table 1. The plasma MDA levels were significantly higher in the radiation-only group the sham control group (P < 0.05). There was a significant reduction in the radiation + 5 mg kg–1 (P < 0.05), and radiation + 10 mg kg–1 (P < 0.05) when compared with radiation-only group. The MDA levels were not different in the groups receiving 5 mg kg–1 or 10 mg kg–1 melatonin groups (P > 0.05).
Erythrocyte MDA levels were significantly higher in the radiation-only group compared to the sham control group. There was a significant reduction in the groups receiving 5 mg kg–1 and 10 mg kg–1 melatonin compared to the radiation-only group. The erythrocyte MDA levels were significantly different in the groups receiving 5 mg kg–1 and 10 mg kg–1 melatonin (P < 0.05 for all group).
The SOD activity in the groups receiving 5 mg kg–1 and 10 mg kg–1 melatonin plus radiation was significantly higher than in the radiation-only group (P < 0.05). The SOD activity was not different in the 5 mg kg–1 and 10 mg kg–1 melatonin groups (P > 0.05).
The GSH-Px activities were significantly higher in the groups receiving 5 mg kg–1 and 10 mg kg–1 melatonin than in the radiation-only group. The levels of GSH-Px were significantly different in 5 mg kg–1 and 10 mg kg–1 melatonin groups (P < 0.05, for all groups).
The activities of CAT were significantly higher in the group receiving 10 mg kg–1 melatonin plus radiation than in both the sham and radiation-only groups (P < 0.05). CAT activities were significantly higher in the group receiving 5 mg kg–1 melatonin than in either the sham or the radiation-only group (P < 0.05).
DISCUSSION
Ionizing radiation causes harmful effects through the generation of free radicals (24). In the present study, when rats were exposed to a single dose of total-body γ radiation, lipid peroxidation was significantly increased in plasma and in erythrocytes.
The radioprotective effect of melatonin in vitro was first reported by Vijayalaxmi et al. (25) and was shown to occur through free radical scavenger activities (26). Melatonin may have an active role in protection against genetic damage due to endogenously produced free radicals, and it may be of use in reducing damage from physical and chemical mutagens and carcinogens that generate free radicals. Confirmation using the same genetic test systems has been reported by Vijayalaxmi et al. (27). They also reported that total-body-irradiated mice that were pretreated with melatonin exhibited a significant and dose-dependent reduction in the incidence of micronuclei. They suggest that their data indicate that melatonin has the ability to protect the cells of mice from the damaging effects of acute total-body irradiation (28). Badr et al. (29) reported that melatonin did not protect against radiation-induced chromosomal aberrations in spermatogonia 30 min after irradiation. In their opinion, this indicates that melatonin should be inside the cell at the time of exposure to radiation to confer protection. In our recent study, we showed radioprotection of peripheral blood cells, especially leukocytes and thrombocytes, by melatonin in total-body-irradiated rats (30).
Green et al. (31) reported that radiotherapy increased the level of MDA. In agreement with these results, we found that γ radiation increases both erythrocyte and plasma MDA levels. High plasma MDA levels may reflect erythrocytes and other cells that are exposed to oxidative stress.
The levels of both erythrocyte and plasma MDA in the groups receiving 5 and 10 mg kg–1 melatonin groups were significantly lower than in the γ-radiation-only group. This is in accordance with the literature on the antioxidant effects of melatonin (32, 33). There was a statistically significant difference between the effects of 5 and 10 mg kg−1 melatonin in erythrocyte MDA levels but not between the levels in plasma.
Nikishkin et al. (34) reported that levels of enzymatic and non-enzymatic antioxidants decrease after irradiation. SOD and GSH-Px each play a role in the antioxidant defense system, but their response to radiation is unclear. Green et al. (35) found that radiation did not significantly affect GSH-Px activities in the long term, while Kaya et al. (36) reported that GSH-Px activities were not decreased significantly after irradiation compared with sham controls but were significantly higher in the group receiving melatonin plus radiation. These authors suggest that their results are in accordance with those of Reiter (37), who found that melatonin exerted an antioxidant effect by increasing the GSH-Px activities. Our results demonstrate that the activities of GSH-Px are not different in the sham control and radiation-only groups. These results confirm those of Kaya et al. (36) and Reiter (37).
In this study, we found no significant difference between the effect of 5 and 10 mg kg–1 melatonin in terms of the plasma MDA level and the erythrocyte SOD and CAT activities. On the other hand, for erythrocyte MDA levels and GSH-Px activities, 10 mg kg–1 of melatonin was more effective than 5 mg kg–1 melatonin. Vijayalaxmi et al. (38) reported that melatonin caused a significant decrease in the percentage of polychromatic erythrocytes (PCEs) in the peripheral blood and bone marrow cells of irradiated mice. In both cases, irradiated mice pretreated with melatonin (5 or 10 mg kg–1) exhibited a dose-dependent increase in the observed incidence of PCEs relative to the expected incidence.
Melatonin stimulates the activities of SOD, CAT and GSH-Px (39, 40). By increasing the activities of antioxidant enzymes, melatonin reduces the number of free radicals or ROS generated and increases the production of molecules protecting against oxidative stress. SOD converts superoxide anion radical (O2–·) to H2O2, thus decreasing the amount of O2–· and the formation of peroxynitrite anion (ONOO–), a highly destructive product of the interaction between O2–· and nitric oxide (NO·). Melatonin increases tissue mRNA levels of two isoforms (manganese and copper) of SOD (41). Thus melatonin may decrease the quantity of O2–· in two ways: directly, by stimulating SOD, and indirectly, when the melatonyl cation radical scavenges it (42). Melatonin also stimulates the activity of GSH-Px, which transforms H2O2 to O2. (43). Finally, melatonin was recently found to stimulate CAT (44), thereby further reducing H2O2 levels and ·OH generation.
Administration of melatonin may reduce the effects of radiation when it is given prior to irradiation through up-regulation of antioxidant enzymes and through scavenging of radiation-induced free radicals. Further experimental and clinical studies are needed.
