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Keywords:

  • free radicals;
  • irradiation;
  • lipid peroxidation;
  • melatonin;
  • oxidative stress

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Abstract:  Radiation therapy is a popular and useful tool in the treatment of cancer. Melatonin participates in the regulation of a number of important physiological and pathological processes. Melatonin, a powerful endogenous antioxidant, plays a role in the reduction of oxidative damage. Thirty adult rats were divided into five equal groups. On the day of the experiment, groups I and II were injected with 5 or 10 mg/kg melatonin, respectively, while group III received isotonic NaCl solution. Thirty minutes later, groups I, II and III were exposed to 6.0 Gy whole body ionizing radiation in a single fraction. Group IV was injected with 5 mg/kg melatonin but was not irradiated. The final group was reserved as sham treated. Liver malondialdehyde (MDA) and nitric oxide (NOċ) levels were measured in all groups. Whole body irradiation caused a significant increase in liver MDA and NOċ levels. Hepatic MDA and NOċ levels in irradiated rats that were pretreated with melatonin (5 or 10 mg/kg) were significantly decreased. Malondialdehyde and NOċ levels were reduced in a dose-related manner by melatonin. The data show that melatonin reduces liver damage inflicted by irradiation when given prior to the exposure to ionizing radiation. The radioprotective effect of melatonin is likely achieved by its ability to function as a scavenger for free radicals generated by ionizing radiation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Radiation therapy is a common and important tool for cancer treatment. The radiosensitivity of normal tissues adjacent to the tumor limits therapeutic gain [1]. For example, the combination of chemotherapy and irradiation produces hepatic toxicity when radiation is used in the treatment of intrahepatic tumors, Hodgkin's disease, or ovarian cancers and after bone marrow transplantation [2]. Radiation-induced liver disease (RILD) is a dose-limiting complication of liver irradiation and the treatment options for RILD are limited, and in severe cases, liver failure and death can occur [2, 3].

There is growing evidence that nitric oxide (NOċ), as well as its derivatives, produced by activated phagocytes may also play a role in multistage carcinogenesis [4]. Nitric oxide is known, together with reactive oxygen species, to induce cytotoxicity and cytostasis. Several studies using NO- and H2O2 -induced oxidative damage have shown to induce similar cytotoxicity [5].

Nitric Oxide is an inorganic free radical gas produced from l-arginine by a family of isoenzymes called NO synthases. Two of them are constitutively expressed and a third is inducible by immunological stimuli. It is NOċ induced by the constitutive enzymes which acts as an important signaling molecule in the cardiovascular and nervous systems, and when NOċ induced by the inducible NO synthase (iNOS) is generated for prolonged periods by cells of the immune system, it is cytostatic/cytotoxic for tumor cells and a variety of microorganisms [6].

It is well known that NOċ possesses both antioxidant and pro-oxidant properties. The concentrations of NOċ, under non-pathological conditions, are in the nanomolar and under conditions of oxidant injury in the micromolar range [7]. NOċ reacts rapidly with the superoxide anion (Oinline image) to form peroxynitrite (ONOO), which in itself is cytotoxic and readily decomposes into the highly reactive and toxic hydroxyl radical (ċOH) and nitrogen dioxide (NO2) [4]. ONOO is much more reactive than NOċ or Oċinline image which causes diverse chemical reactions in biological systems including nitration of tyrosine residues of proteins, triggering of lipid peroxidation, inactivation of aconitases, inhibition of the mitochondrial electron transport, and oxidation of biological thiol compounds [8].

Radiation-induced lipid peroxidation is a free radical process [9]. The process of lipid peroxidation is one of oxidative conversion of polyunsaturated fatty acids to several products including as malondialdehyde (MDA) and lipid peroxides. Malondialdehyde, because of its high cytotoxicity and inhibitory actions on protective enzymes, acts as a tumor promoter and a co-carcinogenic agent [10]. Also, lipid hydroperoxides decompose to yield reactive aldehydes, including MDA and 4-hydroxynoneal. Malondialdehyde is a well-characterized mutagen that reacts with deoxyguanosine to form a major endogenous adduct found in the DNA of human liver. Malondialdehyde is the end product of lipid peroxidation and serves as an index of oxidative damage [11, 12].

Melatonin is the chief secretory product of the pineal gland but it is also produced in other organs. Melatonin participates in the regulation of a number of physiological and pathological processes [13–20]. Melatonin also scavenges the ċOH [21, 22] and possibly peroxyl radicals [23] as well as the ONOO [24]; it is a powerful antioxidant [22, 25]. In vitro investigations of human peripheral blood lymphocytes, which were pretreated with melatonin, were found to exhibit a significantly reduced (60–70%) incidence of gamma radiation-induced chromosomal damage as compared with irradiated cells not pretreated with melatonin [26, 27]. Also, melatonin reduced (60–65%) genetic damage in lymphocytes taken from volunteers who had ingested a single oral dose of 300 mg of melatonin (1 hr prior to collection of blood). These results suggest that melatonin functions as an important protective agent against the insults of whole-body irradiation [16, 28–29].

The aim of this study was to investigate the antioxidant role of different doses of melatonin (5 mg/kg and 10 mg/kg) in animals subjected to whole body irradiation at a single dose of 6.0 Gy.

Chemicals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Melatonin, sodium dodecyl sulfate (SDS), acetic acid, NaOH, thiobarbituric acid (TBA), nicotinamide adenine dinucleotide, n-butanol, sulphanilamide and N-(1-napthyl) ethylenediamine were purchased from Sigma Chemical Co (St Louis, MO, USA). All other chemicals were of analytical grade.

Animal experiment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Thirty albino Wistar male rats (190–215 g) were used for the experiment. All animals received humane care in compliance with the guidelines of Atatürk University Research Council's criteria. The rats were fed standard laboratory chow and water. The animal room was windowless with automatic temperature (22 ± 1°C) and lighting controls (14 hr light/10 hr dark). Rats were divided into five equal groups. Twenty-four hours before the experiment, the rats were starved and allowed access to water ad libitum.

On the day of the experiment, rats of groups I and II were injected with 5 and 10 mg/kg melatonin, respectively, while rats of group III were injected with isotonic NaCl solution. Equal volume of ethanol, dissolved in melatonin, was added to the NaCl solution. Melatonin was initially dissolved in ethanol diluted with isotonic NaCl. The drugs and isotonic NaCl were administered intraperitoneally. Group IV rats were injected with 5 mg/kg melatonin only and the last group (group V) of rats was reserved as untreated controls.

Thirty minutes after the injections, groups I, II and III were anesthetized and irradiated with 6.0 Gy whole body radiation in a single fraction. Irradiation was performed using a cobalt-60 teletherapy unit (Picker C-9, Maryland, NY, USA) at 80 cm skin source distance. The dose was calculated at the central axis at a depth of 2 cm. The dose rate was 196.4 cGy/min. Melatonin treated group and untreated control group rats were anesthetized, but were not irradiated. All groups were killed 2 hrs after the irradiation.

Biochemical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Two hours after the irradiation, rats were anesthetized with 50 mg/kg of thiopenthal sodium. Livers were excised and homogenized in physiological saline solution (Omni Accessory Pack International Homogenizer, Warrenton, VA, USA). The homogenate was centrifuged at 10,000 g for 1 hr to remove debris. The clear upper supernatant was collected and enzymatic assays were carried out on this fraction. All the procedures were performed at +4°C.

Malondialdehyde levels were determined according to the method of Ohkawa et al. [30]. Samples less than 0.2 mL of 10% (w/v) tissue homogenate were added 0.2 mL of 8.1% SDS, 1.5 mL of 20% acetic acid solution (pH was adjusted to 3.5 with NaOH), and 1.5 mL of 0.8% aqueous solution of TBA. The mixture was brought up to 4.0 mL with distilled water, and heated at 95°C for 60 min. After cooling, 1.0 mL of distilled water and 5.0 mL of mixture of n-butanol and pyridine (15:1, v/v) were added and shaken vigorously. After centrifugation at 3500 g for 10 min, the organic layer was taken and its absorbance at 532 nm was measured. Total TBA-reactive materials were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 × 105/cm/m. Malondialdehyde level was expressed as nmol/mg protein.

Liver NOċ (nitrite + nitrate) levels were measured using the Griess reagent as previously described [31, 32]. Griess reagent consists of sulphanilamide and N-(1-napthyl) ethylenediamine. First, nitrate is converted to nitrite using nitrate reductase. The second step is the addition of Griess reagent, which converts nitrite to a deep purple azocompound; photometric measurement of the absorbance of 540 nm determines the nitrite concentration (sodium nitrate is used as a standard). Nitric Oxide levels are expressed as μmol/mg protein. The protein was determined using the Bradford method [33]. Biochemical measurements were carried out at room temperature using a spectrophotometer (CECIL CE 3041, Cambridge, UK).

Statistical analyses

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

Results are presented as mean ± S.D. All parameters were analyzed using a one-way variance analysis. Least significant difference multiple range test was used to compare the mean values. Acceptable significance was recorded when P values were <0.05. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows (SPSS, version 10.0, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

In the irradiation only group liver tissue, MDA levels were significantly higher compared with either the melatonin only group or the control levels (Fig. 1, P < 0.001). Melatonin at either 5 mg/kg or 10 mg/kg significantly reduced MDA levels in the livers of rats subjected to whole body irradiation (P < 0.05). Whole body irradiation also increased hepatic levels of NOċ (measured as nitrate/nitrite) and these values were significant (Fig. 1, P < 0.001) reduced when melatonin was given.

image

Figure 1. Malondialdehyde (MDA) (a) and nitric oxide (NOċ); (b) levels in the liver of rats exposed the whole body irradiation (WBI, 6 Gy) and given melatonin (MEL) either at a dose of 5 or 10 mg/kg body weight. Control (CON) animals received only isotonic NaCl solution. Equal volume of ethanol was added the NaCl solution. a, P < 0.05; b, P < 0.001 versus CON; c, P < 0.05; d, P < 0.001 versus WBI; e, 0.05 versus MEL-5 + WBI.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References

There are numerous studies concerning the in vitro and in vivo antioxidant properties of melatonin [15, 20, 24, 34]. Antioxidant effects of melatonin can occur by at least two mechanisms. In one case, melatonin itself exerts direct antioxidant effects via free radical scavenging and/or by inhibiting their generation [35, 36]. Additionally, melatonin alters the activities of enzymes, which improve the endogenous antioxidant defense capacity of the organisms. These enzymes include superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase [20, 37, 38].

Some studies reported that irradiation increased the formation of MDA, the main product of lipid peroxidation, in the liver [38–40]. In the present study, when rats exposed to single dose (6.0 Gy) total body γ-irradiation, lipid peroxidation significantly increased in the liver. Our results are in agreement with the published literature. The levels of liver MDA in the γ-irradiation-plus 5 and 10 mg/kg melatonin groups significantly decreased when compared with the γ-irradiation-only group. This is in accordance with the literature relevant to the antioxidant effect of melatonin. These results revealed that melatonin clearly decreased the lipid peroxidation in liver induced by total body γ-irradiation and the decrease in the MDA level was dose-related. When comparing the liver MDA levels, there was a statistically significant difference between the groups that were administered 5 and 10 mg/kg melatonin. In our recent study, we demonstrated that the use of melatonin before irradiation, as a radioprotector of peripheral blood, protected the cells, especially leukocytes and thrombocytes in total body irradiated rats [41].

The mechanisms of inhibition of lipid peroxidation by melatonin probably include the direct scavenging of the initiating radicals, especially ċOH and ONOO. High-energy ionizing radiation is known to cause the hemolytic scission of H2O to generate the ċOH, which in turn, attacks DNA. Nuclear DNA damage sustained as a consequence of ionizing radiation has been shown in numerous studies to be blunted in the presence of melatonin [42].

The production of large amounts of NOċ, a free radical produced by the iNOS, has been implicated as a cytotoxic factor in a variety of pathophysiological processes [24]. Bettahi et al. [43] in their study carried out in rat hypothalamus reported that melatonin partially inhibited NOS activity at physiological concentrations. Agrawal et al. [1] reported that NOċ levels induced by radiation were significantly higher in the liver of tumor-bearing animals as compared with the non-tumor-bearing control group of animals. In the present investigation, when rats exposed to single dose (6.0 Gy) total body γ-irradiation, the NOċ levels significantly increased in the liver. Our NOċ results are in agreement with the study carried out by Agrawal et al. [1] in rat liver tissue.

Under conditions of oxidative stress induced by irradiation, NOċ is often produced; its cytotoxicity is primarily because of the production of ONOO, a toxic oxidant, generated when the NOċ couples with Oinline image [44]. The processes triggered by ONOO include initiation of lipid peroxidation, inhibition of mitochondrial respiration, inhibition of membrane pumps, depletion of glutathione, and damage to DNA [24].

Melatonin's protective effects in some subcellular compartments may be due to its indirect antioxidative actions, e.g. stimulation of enzymes that either promotes the synthesis of other antioxidants [44, 45] or metabolize reactive species to non-radical products [46]. Melatonin is reported to scavenge ONOO both in vitro and in vivo [24, 47] and to inhibit iNOS activity [48] thereby reducing NOċ generation.

Based on our results, as NOċ seems to have a double-edged role in tumor progression, the high concentrations of NOċ for long periods could result in damage in DNA leading to mutation and cancer. It may be concluded that melatonin administration decreases lipid peroxidation and NOċ generation in radiation-treated cancer patients and alleviates the radiation toxicity to liver.

In conclusion, melatonin has clear antioxidant properties and is likely to be a valuable drug for protection against γ-irradiation and/or be used as an antioxidant against oxidative stress. In addition, melatonin may enable the use of higher doses of radiation during therapy and may therefore allow higher dose rates in some patients with cancer.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Chemicals
  6. Animal experiment
  7. Biochemical analysis
  8. Statistical analyses
  9. Results
  10. Discussion
  11. References
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