|Year : 2015 | Volume
| Issue : 3 | Page : 92-97
Comparing the impact of melatonin and captopril on early effects of radiation on the heart tissue by studying glutathione, malondialdehyde, and lactate dehydrogenase enzyme activity in rats
Alireza Shirazi1, Farnaz Tabatabaie2, Mahmoud Ghazi-Khansari3, Hamidreza Mirzaei4
1 Department of Nuclear Engineering, Science and Research Branch, Islamic Azad University; Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Nuclear Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Department of Radiation and Oncology, Cancer Research Center, Shohadaye Tajrish Hospital of Shahid Beheshti, University of Medical Sciences, Tehran, Iran
|Date of Web Publication||10-Nov-2015|
Department of Radiation and Oncology, Cancer Research Center, Shohadaye Tajrish Hospital of Shahid Beheshti, University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Prevention of secondary malignancy while the patient is receiving radiotherapy for the management of primary cancer has been an enormous challenge for biological and medical safety. The aim of the study is to compare protective effects of melatonin and captopril on early effects of radiation on the heart tissue of rats. Forty-eight adult male Wistar rats weighing 180-220 g were used. The rats were divided into six groups and the rats were exposed to 8 Gy whole body dose from cobalt-60 sources. Thirty minutes prior to irradiation, six animals received melatonin (100 mg/kg body weight), and six animals received captopril (50 mg/kg body weight). All groups were sacrificed 10 days post-irradiation, and hearts were collected. Malondialdehyde (MDA), lactate dehydrogenase (LDH), and glutathione (GSH) were measured to evaluate cellular oxidative stress-induced injury. The biochemical data are presented as mean ± standard error of the mean, and the difference between the groups was analyzed using a two-way variance analysis. Treatment with captopril resulted in a significant increase in LDH and MDA, although the level of GSH was decreased (P < 0.01). MDA and LDH levels were decreased after melatonin treatment while GSH level was increased (P < 0.001). Melatonin has protective effects following radiation, while treatment with captopril post-irradiation seems to be radiosensitizing and does not have protective effects against radiation exposure.
Keywords: Captopril, melatonin, irradiation of rats, secondary malignancy, radioprotectors
|How to cite this article:|
Shirazi A, Tabatabaie F, Ghazi-Khansari M, Mirzaei H. Comparing the impact of melatonin and captopril on early effects of radiation on the heart tissue by studying glutathione, malondialdehyde, and lactate dehydrogenase enzyme activity in rats. Radiat Prot Environ 2015;38:92-7
|How to cite this URL:|
Shirazi A, Tabatabaie F, Ghazi-Khansari M, Mirzaei H. Comparing the impact of melatonin and captopril on early effects of radiation on the heart tissue by studying glutathione, malondialdehyde, and lactate dehydrogenase enzyme activity in rats. Radiat Prot Environ [serial online] 2015 [cited 2020 Jul 5];38:92-7. Available from: http://www.rpe.org.in/text.asp?2015/38/3/92/169379
| Introduction|| |
Recently, the application of radiation technology in different settings (e.g., radiotherapy, biomedical research, military, and space research) is increased. Harmful effects of radiation due to exposure to radiation is of concern. Ionizing radiation is well-known to induce oxidative stress by the production of reactive oxygen species and free radicals in irradiated tissue and cells. ,,
Due to new radiation technologies and chemotherapy, target therapy in long-term survival of cancer seems very common. Conversely, cancer treatments can cause organ injuries like cardiovascular disease, heart attack, and heart failure erythema. 
Regularly, the heart has been viewed as a radiation-safe organ that would be unaffected via cardiovascular measurements under around 30 Gy.  All through the last few years, however, confirm that radiation-related coronary disease can indeed take place at lower doses. These include investigations of patients, who got mean cardiovascular doses of 3-17 Gy when given radiotherapy after or during surgery, or investigations of survivors of the nuclear bombings of Japan who got dosages of up to 4 Gy. 
Radioprotective agents have been used to reduce injury produced by ionizing irradiation. The essential change of such agents focused on thiol synthetic mixes, for example, amifostine. Common mixes have been radioprotectors as radioprotectants, and they seem to utilize their impact through cancer prevention agent and immunostimulant exercises. 
Melatonin (N-acetyl-5-methoxytryptamine), the chief secretory product of the pineal gland in the brain, is recognized for its practical adaptability. In most of the studies, melatonin has been known as a direct free radical scavenger and an indirect antioxidant, as well as an important immune modulatory agent. The radical scavenging ability of melatonin is believed to work by electron donation to detoxify a variety of reactive oxygen and nitrogen species, containing the highly toxic hydroxyl radical. The hydroxyl radical scavenging ability of melatonin was used as a rationale to determine its radioprotective efficiency. The outcomes from many researches have been done in vitro and in vivo have confirmed that melatonin protects mammalian cells leaning on the toxic effects of ionizing radiation. 
Captopril is an angiotensin-converting enzyme (ACE) inhibitor used for the treatment of hypertension and some types of congestive heart failure. Captopril was the first ACE inhibitor developed and was considered a breakthrough both because of its novel mechanism of action and also because of the revolutionary development process. Captopril, an inhibitor of ACE I, has been shown to prevent radiation injury of normal tissue in rats and pigs. 
The aim of this study is to evaluate the melatonin and captopril effects on early effects of radiation on the heart injuries.
| Materials and methods|| |
Melatonin (N-acetyl-5-methyoxytryptamine) and captopril were obtained from Sigma-Aldrich. Melatonin was dissolved in a minimal volume of ethanol (<5%) and diluted with saline. Captopril was dissolved in distilled water and diluted with saline to confirm it's readiness for injection. All other reagents were obtained from Sigma (St. Louis, MO) and Merck (Germany) pharmaceutical companies.
Forty-eight male adult Wistar albino rats weighing 180-220 g were used for the experiment. All animals were housed in stainless steel cages and supplied with wood chips, and a 12 h light: Dark cycle in a temperature controlled room (22°C). The animals were allowed free access to tap water and a standard diet for the duration of the study. The experimental protocol was in accordance with the guidelines for care and use of laboratory animals as adopted by the Ethics Committee of the School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
The rats were divided into six groups of eight rats housed in each cage. It is divided into the following groups: (1) Control group, (2) irradiated with 8 Gy and without treatment with melatonin or captopril, (3) group treated with melatonin and without gamma radiation, (4) group treated with captopril and without gamma radiation, (5) group irradiated with 8 Gy and treated with melatonin, (6) group irradiated with 8 Gy and treated with captopril. 30 min prior to irradiation six animals received melatonin (100 mg/kg body weight), and six animals received Captopril (50 mg/kg body weight). Rats were exposed to 8 Gy whole body dose from cobalt-60 (Co-60) sources [Table 1].
In this study, we use Therateron 780°C, which is a complete Co-60 teletherapy unit. Animals were exposed to a whole-body gamma irradiation doses of 8 Gy (estimated LD50/30) at a dose rate of 0.95 Gy/min with a source surface distance of 80 cm, anteroposterior technique, and fixed field size of 30 cm × 30 cm at room temperature (22°C ± 2°C). The output of the Co-60 unit was calibrated in Gy/min by measuring the quantity of air kerma rate. During animals' set-up, all system motions were controlled from the hand control in the treatment room.  Each animal was anesthetized before exposing with an intra-peritoneal (IP) injection of ketamine (60 mg/kg) and xylazine (20 mg/kg).
Ten days after irradiation, animals were anesthetized with an IP injection of ketamine (60 mg/kg) and xylazine (20 mg/kg). The rats' heart were excised and homogenized in a 10-fold physiological saline solution in a homogenizer (Omni Accessory Pack International Homogenizer, USA). The homogenate was centrifuged at 10,000 g for 1-h to remove debris. The supernatant and sediment were taken for biochemical analysis. The clear upper supernatant was used for measuring glutathione (GSH) content, and the sediment was used for measuring the malondialdehyde (MDA) level. The samples kept in the freezer at a − 20°C temperature. Then they were used for the measurement of MDA, GSH, and lactate dehydrogenase (LDH).
The decrease of MDA level is an indication of the presence of radioprotector.  The MDA level was determined according to the thiobarbituric acid (TBA) method. 2.5 ml of 0.05 M sulfuric acid and 1.5 ml of a 0.2% solution of TBA were added to the sediment. The mixture was heated at 100°C for 30 min in a boiling water bath. Four cubic centimeters of n-butanol were added to the cooled mixture, and the sample was shaken vigorously. After centrifugation at 3500 rpm for 10 min, the organic layer was removed, and its absorbance read at 530 nm. The MDA concentration was calculated from the standard curve. 0.5 ml of standard solutions with concentrations of 1, 3, 5, 7, and 10 nmol/ml added to 2.5 ml of 0.05 M sulfuric acid and 3 ml of TBA 0.2 g/dl and then process 30 min in boiling water bath. 
The increase of GSH level is an indication of the presence of radioprotector.  GSH level was determined according to the method of Kuo and Hook given elsewhere.  Protein content was determined by the Bradford method.  Briefly, 0.5cc distilled water, 2cc of 0.3M disodium phosphate and 0.5cc of 0.04% 5,5'-dithiobis-2-nitrobenzoic acid were added to 100 μl of the supernatant and incubated for 10 min at room temperature. The absorbance of the resulting yellow color was read against the blank at 412 nm, and the GSH concentration was calculated from the standard curve. Pure GSH was used as the standard for establishing the calibration curve.
Lactate dehydrogenase analysis
The increase of MDA level is an indication of the presence of radioprotector.  LDH activities were estimated according to the methods of Szasz et al.  and Bergmeyer and Bernt.  The LDH assay involves mixing supernatant or lysates containing LDH with reconstituted dye solution and substrate buffer. The actual reaction is optimized so one part sample is to be combined with one part reconstituted dye solution and two parts substrate buffer. This reaction was measured using a spectrophotometer at 340 nm to quantify activity.
Results were expressed as mean ± standard error of the mean of at least eight animals per group. For each time point, mean values of irradiated and unirradiated animals were compared using One-Way Analysis of Variance.  Significance level was accepted at P < 0.05.
| Results|| |
[Figure 1] shows MDA levels (nmol/ml) in rat heart 10 days postirradiation in the injection of melatonin (100 mg/kg body weight) and captopril (50 mg/kg body weight). MDA levels between group control and group melatonin only and group captopril only were not significant [Figure 1]. In the radiation only group, MDA level was significantly (P < 0.01) higher than that in the melatonin only, captopril only or control group. MDA level of melatonin and radiation group was significantly (P < 0.001) lower than the radiation group.
|Figure 1: Mean malondialdehyde (nmol/ml) levels in Wistar rat hearts 10 days postirradiation (8 Gy) with the injection of melatonin and captopril (*P < 0.01 compared with control group, **P < 0.001 compared with radiation). Error bars represent standard error of the mean for six animals in each group|
Click here to view
[Figure 2] shows GSH levels (μmol/mg protein) in rat heart 10 days post-irradiation in the injection of melatonin and captopril. GSH levels between group control and group melatonin only and group captopril only were not significant [Figure 2]. The level of GSH in the group irradiated was significantly (P < 0.05) lower than that in the control and melatonin only and captopril only group. The level of GSH in the administration of captopril did not have a significant effect on GSH level after irradiation. But the level of GSH in the group irradiated was significantly (P < 0.01) lower than that in the melatonin and radiation group. GSH level in group melatonin and radiation is significantly (P < 0.01) higher than the group captopril and radiation.
|Figure 2: Mean glutathione (μmol/mg protein) levels in Wistar rat hearts 10 days postirradiation (8 Gy) with the injection of melatonin and captopril (*P < 0.05 compared with the control group, **P < 0.01compared with the group treated with radiation). Error bars represent standard error of the mean for six animals in each group|
Click here to view
Lactate dehydrogenase level
[Figure 3] shows LDH (U/L) levels in rat heart 10 days postirradiation in the injection of melatonin (100 mg/kg body weight) and captopril (50 mg/kg body weight). LDH levels between group control and group melatonin only and group captopril only were not significant [Figure 3]. In the radiation only group, LDH level was significantly (P < 0.01) higher than that in the melatonin only, captopril only or control group. LDH level of group radiation only was significantly (P < 0.001) lower than group captopril and radiation. Administration of melatonin and radiation was significantly (P < 0.001) lower than radiation Group. LDH level in captopril and radiation is significantly higher than group melatonin and radiation.
|Figure 3: Mean lactate dehydrogenase (IU/L) levels in Wistar rat hearts 10 days postirradiation (8 Gy) with the injection of melatonin and captopril (*P < 0.01 compared with control group, **P < 0.001 compared with radiation group, ***P < 0.001 compared with radiation group). Error bars represent standard error of the mean for six animals in each group|
Click here to view
| Discussion|| |
Different natural compounds have been evaluated as radioprotectants, and they seem to use their effect over antioxidant and immunostimulant activities. Although, current agents have lower effectiveness, they have lesser toxicity, extra-encouraging administration routes, and improved pharmacokinetics compared to the older thiol compounds.  On the other hand, the intrinsic toxicity of these agents at the radioprotective doses warranted further search for harmless and more effective radioprotectors. , A comprehensive dosage range for melatonin and captopril, from physiological to pharmacological concentrations, has been tested in many animal studies. The results of those studies showed that the critical and chronic toxicity of melatonin and captopril is sorely scars. ,
It has been displayed that melatonin at doses as high as 250 mg/kg is non-toxic and that high doses of melatonin are ready for use in protecting rats from lethal effects of acute whole body irradiation.  Also for captopril, the maximum non-toxix dose was estimated around 30 mg/kg/day for male but a little more than this for female rats. Body weight increase was expressively reduced in male but for the first 3 months in female rats. No death has occurred due to the toxic effect of captopril. 
Many studies proved that melatonin by antioxidant properties, appeared to ameliorate irradiation-induced injury in different organs including brain,  spinal cord, , lens,  liver, , spleen, lung, colon, and ileum. ,,
Regarding using captopril, Davis et al.  suggest that ACE inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor cell cycle. Observation and recording of time for captopril treatment in relation to radiation exposure have a different impact on the viability and repopulation capacity of spared hematopoietic stem cells and consequently, can result in either radiation protection or radiation sensitization. In addition, Haddadi and Shirazi  have shown that the radioprotective effects of melatonin against cellular damage, which are caused by oxidative stress and its low toxicity makes this molecule a potential supplement in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized.
The explanation behind using rats as a part of this investigation is that human and rats are both mammals, they share numerous similitudes in structure and function. Moreover, their little sizes, minimal effort, ease in taking care of, and capacity to breed in bondage make rats perfect for research center tests. Rats have been utilized as a part of different cancer studies on the grounds that certain strains can result in cancer spontaneously, as well as by viral and chemical induction.  Investigations of the impacts of radiations on the heart are introduced by a critical review of the clinical and experimental literature. 
The outcomes of this study indicated that whole body irradiation would cause tissue damage to rat heart as specified by increased MDA and LDH levels and decreased GSH levels. The role of oxidative mechanisms in irradiation-induced tissue damage was proved by the increase in MDA levels in the irradiated group. As previously mentioned, GSH provides major protection in oxidative injury by contributing in the cellular defense system against oxidative stress. And LDH can then determine the replacement for the amount of tissue damage; which has been shown by the increase in LDH levels in the irradiated groups.
Scientists have done many researches about the protective effects of melatonin and captopril in different organs. But it should be noted that studying the effect of these two materials against radiation on the heart tissue, is the new idea. They have lot of studies that investigate the effects of melatonin and captopril on heart failure but in the absence of radiation.
Sehirli, et al., 2013  studied the antioxidant role of melatonin (10 mg/kg) in the heart tissue. The results reveal ameliorating effect of melatonin of ischemic heart failure in rats. These observations highlight that melatonin is a promising supplement for improving protection mechanisms in the heart in contrast to oxidative stress affected by heart failure. Also, the consequences of Reiter and Tan, research in 2003  would help to explain the potential importance of the use of melatonin in situations of oxidative damage to the heart in humans.
| Conclusions|| |
Comparing the melatonin and captopril effect against radiation exposure showed that melatonin has a protective effect against radiation, but the injection of captopril post irradiation can result in radiation sensitization.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mikkelsen RB, Wardman P. Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 2003;22:5734-54.
Erol FS, Topsakal C, Ozveren MF, Kaplan M, Ilhan N, Ozercan IH, et al.
Protective effects of melatonin and vitamin E in brain damage due to gamma radiation: An experimental study. Neurosurg Rev 2004;27:65-9.
Mohseni M, Mihandoost E, Shirazi A, Sepehrizadeh Z, Bazzaz JT, Ghazi-khansari M. Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis. Mutat Res 2012;738-739:19-27.
Darby SC, Cutter DJ, Boerma M, Constine LS, Fajardo LF, Kodama K, et al.
Radiation-related heart disease: Current knowledge and future prospects. Int J Radiat Oncol Biol Phys 2010;76:656-65.
Hosseinimehr SJ. Trends in the development of radioprotective agents. Drug Discov Today 2007;12:794-805.
Vijayalaxmi, Reiter RJ, Tan DX, Herman TS, Thomas CR Jr. Melatonin as a radioprotective agent: A review. Int J Radiat Oncol Biol Phys 2004;59:639-53.
Yoon SC, Park JM, Jang HS, Shinn KS, Bahk YW. Radioprotective effect of captopril on the mouse jejunal mucosa. Int J Radiat Oncol Biol Phys 1994;30:873-8.
Vijayalaxmi, Meltz ML, Reiter RJ, Herman TS, Kumar KS. Melatonin and protection from whole-body irradiation: Survival studies in mice. Mutat Res 1999;425:21-7.
Shirazi A, Ghobadi G, Ghazi-Khansari M. A radiobiological review on melatonin: A novel radioprotector. J Radiat Res 2007;48:263-72.
Agarwal R, Chase SD. Rapid, fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples. J Chromatogr B Analyt Technol Biomed Life Sci 2002;775:121-6.
Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407-21.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
Anwar MM, Moustafa MA. The effect of melatonin on eye lens of rats exposed to ultraviolet radiation. Comp Biochem Physiol C Toxicol Pharmacol 2001;129:57-63.
Szasz G, Gruber W, Bernt E. Creatine kinase in serum: 1. Determination of optimum reaction conditions. Clin Chem 1976;22:650-6.
UV-assay with pyruvate and NADH. Methods of Enzymatic Analysis. 2 nd
ed. New York: Academic Press; 1974 . [Press release].
Neter J, Wasserman W. Applied Linear Statistical Models. Regression Analysis of Variance and Experimental Designs. Illinois: Richard D Irwin; 1974. p. 425-31.
Nair CK, Parida DK, Nomura T. Radioprotectors in radiotherapy. J Radiat Res 2001;42:21-37.
Koc M, Taysi S, Buyukokuroglu ME, Bakan N. Melatonin protects rat liver against irradiation-induced oxidative injury. J Radiat Res 2003;44:211-5.
Hashimoto K, Imai K, Yoshimura S, Ohtaki T. Twelve month studies on the chronic toxicity of captopril in rats. J Toxicol Sci 1981;6 Suppl 2:215-46.
Shirazi A, et al
. Evaluation of melatonin for prevention of radiation myelopathy in irradiated cervical spinal cord. Yakhteh Medical Journal 2009;11:43-8.
A Shirazi A, Minaee B, Haddadi G. Evaluation of melatonin for modulation of apoptosis-related genes in irradiated cervical spinal cord. Int J Low Radiat 2010;7:436-45.
Shirazi A, Haddadi GH, Asadi-Amoli F, Sakhaee S, Ghazi-Khansari M, Avand A. Radioprotective effect of melatonin in reducing oxidative stress in rat lenses. Cell J 2011;13:79-82.
Shirazi A, Fardid Reza, Mihandoost E, Protective effect of low dose melatonin on radiation-induced damage to rat liver. J Biomed Phys Eng 2012;2:6.
Taysi S, Koc M, Büyükokuroglu ME, Altinkaynak K, Sahin YN. Melatonin reduces lipid peroxidation and nitric oxide during irradiation-induced oxidative injury in the rat liver. J Pineal Res 2003;34:173-7.
Sener G, Jahovic N, Tosun O, Atasoy BM, Yegen BC. Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats. Life Sci 2003;74:563-72.
Mihandoost E, Shirazi A, Mahdavi SR, Aliasgharzadeh A. Can melatonin help us in radiation oncology treatments? Biomed Res Int 2014;2014:578137.
Davis TA, Landauer MR, Mog SR, Barshishat-Kupper M, Zins SR, Amare MF, et al.
Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation. Exp Hematol 2010;38:270-81.
Haddadi G, Shirazi A. Protective effect of melatonin: Mini review. World Congress on Medical Physics and Biomedical Engineering 2006. Proc 2007;14:2192-4.
Pour PM, Groot K, Kazakoff K, Anderson K, Schally AV. Effects of high-fat diet on the patterns of prostatic cancer induced in rats by N-nitrosobis (2-oxopropyl) amine and testosterone. Cancer Res 1991;51:4757-61.
Jones A, Wedgwood J. Effects of radiations on the heart. Br J Radiol 1960;33:138-58.
Sehirli AÖ, Koyun D, Tetik S, Özsavci D, Yiginer Ö, Çetinel S, et al.
Melatonin protects against ischemic heart failure in rats. J Pineal Res 2013;55:138-48.
Reiter RJ, Tan DX. Melatonin: A novel protective agent against oxidative injury of the ischemic/reperfused heart. Cardiovasc Res 2003;58:10-9.
[Figure 1], [Figure 2], [Figure 3]