|Year : 2018 | Volume
| Issue : 12 | Page : 1070-1075
Radioprotective effect of melatonin on expression of Cdkn1a and Rad50 genes in rat peripheral blood
Hamed Rezaeejam1, Alireza Shirazi1, Pantea Izadi2, Javad Tavakkoly Bazzaz2, Mahmoud Ghazi-Khansari3, Majid Valizadeh4, Ghasem Azizi Tabesh2
1 Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Department of Medical Physics, Faculty of Medicine, Zabol University of Medical Sciences, Zabol, Iran, Iran
|Date of Web Publication||11-Dec-2018|
Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Objective: Ionizing radiation is a critical threat to biomolecules, especially DNA. Various combinatorial compounds have been studied to protect this biomolecule. Melatonin has been reported as a direct and indirect free radical scavenger, but in this study, we explored the effect of melatonin on assisting in DNA repair by expression of Cdkn1a and Rad50; both of these genes are involved in DNA repair signaling, induced by radiation in rat peripheral blood.
Materials and Methods: Rats were irradiated with single whole-body linear accelerator X-ray radiation doses of 2 and 8 Gy with or without melatonin (100 mg/kg body weight) pretreatments. The rats were randomly divided into nine groups and given an intraperitoneal injection of melatonin or the same volume of vehicle alone 1 h before radiation. Blood samples were taken 8, 24, and 48 h postradiation to measure gene expression of Cdkn1a and Rad50 using quantitative reverse transcription polymerase chain reaction technique.
Results: Melatonin pretreatment increased the expression of Cdkn1a and Rad50 in 8 and 24 h postradiations (2 and 8 Gy) (P < 0.05), and there was no significant difference in 48 h postradiation compared to the radiation-only and vehicle plus radiation (2 and 8 Gy) groups.
Conclusions: Based on our results, pretreatment with melatonin (100 mg/kg) may ameliorates injurious effects of 2 and 8 Gy ionization radiation by increasing the expression level of Cdkn1a and Rad50 in rat peripheral blood and assist in DNA double-strand breaks repair, especially during the early postradiation.
Keywords: DNA repair, melatonin, peripheral blood, quantitative reverse transcription polymerase chain reaction, X-ray radiation
|How to cite this article:|
Rezaeejam H, Shirazi A, Izadi P, Bazzaz JT, Ghazi-Khansari M, Valizadeh M, Tabesh GA. Radioprotective effect of melatonin on expression of Cdkn1a and Rad50 genes in rat peripheral blood. J Can Res Ther 2018;14, Suppl S5:1070-5
|How to cite this URL:|
Rezaeejam H, Shirazi A, Izadi P, Bazzaz JT, Ghazi-Khansari M, Valizadeh M, Tabesh GA. Radioprotective effect of melatonin on expression of Cdkn1a and Rad50 genes in rat peripheral blood. J Can Res Ther [serial online] 2018 [cited 2019 Sep 20];14:1070-5. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/1070/196758
| > Introduction|| |
Ionizing radiation leads to multiple types of DNA damage such single-strand break and double-strand break (DSBs), DNA-protein cross-links, oxidized bases, and abasic sites., Among these, DNA DSBs are the most lethal lesion induced by ionizing radiation and their repair is more difficult than that of other types of DNA damage., Following DNA DSBs induced by ionizing radiation, DNA repair and cell cycle checkpoints are the main mechanisms of maintenance of genomic stability. DSBs in mammalian cells are repaired by either nonhomologous end joining (NHEJ) pathway, which links together the two ends of broken DNA by direct ligation or involves the majority of DSB repair or homologous recombination (HR) pathway, which is only available S and G2 phases when a sister chromatid can be used as a template for the repair reaction.,, Furthermore, cells have several checkpoints that function at various phases of the cell cycle. When these checkpoints detect DNA damage at each phase, they induce cell cycle arrest and make time for repair of DNA damage. Therefore, DNA repair and cell cycle checkpoints must cooperate closely to repair DNA damage and maintain genomic stability. The hematopoietic system is well-known to be radiosensitive, and its damage may be life-threatening. Therefore, agents which protect the hematopoietic system from radiation-induced DNA damage need to be identified. To achieve this, many synthetic compounds have been investigated as potential radioprotective agents. However, the development of chemical compounds has been unsuccessful due to the various side effect problems. Therefore, natural products are being extensively studied as potentially safe alternatives.,,, Since 1993, when melatonin (N-acetyl-5-methoxytryptamine), the hormone of pineal gland was first identified as a free radical scavenger, numerous papers have been published supporting the ability of this radioprotective agent to protect against ionizing radiation-induced DNA damage.,,,,,, Koc et al. showed that melatonin administration before radiation protected the rat peripheral blood cells against radiation-induced damage. Vijayalaxmi et al. indicated that melatonin reduces radiation-induced chromosome damage, micronuclei and primary DNA damage in human peripheral blood lymphocytes in their observations. Our earlier study was investigated the capability of melatonin in the modification of radiation-induced apoptosis in rat peripheral blood. Results obtained from this study suggest that melatonin (100 mg/kg) may provide modulation of Bax and Bcl-2 expression as well as Bax/Bcl-2 ratio and has protective effects against radiation-induced apoptosis. However, these findings indirectly suggest that melatonin may have protective effects against ionizing radiation-induced DNA damage in peripheral blood.
The aim of the present study was to investigate the modulating effect of melatonin (100 mg/kg i.p.) as a radioprotective agent in expression of Cdkn1a (cyclin-dependent kinase inhibitor 1A) that plays a regulatory role in S phase DNA replication and DNA damage repair and Rad50, a component of the Mre11-Rad50-Nbs1 (MRN) complex which plays a central role in DSB repair, DNA recombination and cell cycle checkpoints,, by radiation in rat peripheral blood.
| > Materials and Methods|| |
Rats and maintenance
8–10-week-old male albino Wistar rats, each weighing 180–220 g, were obtained from the Faculty of Pharmacy Experimental Animal Laboratory, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. They were housed in animal facility, with room temperature maintained at 20–22°C, relative humidity of 50%–70% and an airflow rate of 15 exchange/h. In addition, a time-controlled system provided 08:00–20:00 h light and 20:00–08:00 h dark cycles. All rats were given standard rodent chow diet and water from sanitized bottle fitted with stopper and sipper tubes.
Experimental design and irradiation
After 2 week acclimatization period, a randomized block design based on the animal body weights was used to divide the rats into nine different groups [Table 1]. One hour before the start of experiment, all rats were transferred to a laboratory near the linear accelerator (Elekta, Compact™) facility. The experiment was executed using a total of 162 rats with 18 rats in each group.
Melatonin (Sigma-Aldrich Co., St. Louis, MO, USA) was first dissolved in a small amount of absolute ethanol (25 μl) and then diluted with phosphate-buffered saline (475 μl) in final ethanol concentration 5%. One hour after the injections, all rats were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (20 mg/kg), and then the rats in groups 2, 5, and 8 were exposed to a sublethal whole-body X-ray radiation dose of 2 Gy and the rats in groups 3, 6, and 9 were exposed to a lethal whole-body X-ray radiation dose of 8 Gy with a source surface distance of 100 cm and fixed field size of 35 cm × 35 cm at room temperature (20°C ± 2°C). The selection of 1 h interval between melatonin injection and exposure, the melatonin concentrations and the dose of X-ray radiation were based on the experience from the studies performed by our laboratory.,
Under ketamine (50 mg/kg) and xylazine (10 mg/kg) anesthesia, blood sample was taken on ethylenediaminetetraacetic acid sterile tubes from each group at each of the collection times of 8, 24, and 48 h postradiation and each blood sample was used for measurement of Cdkn1a and Rad50 expression levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). 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.
Quantitative reverse transcription polymerase chain reaction
qRT-PCR was used to measure the expression of DNA DSB repair-related genes: Cdkn1a and Rad50. In each sample, Total RNA was prepared with the High Pure RNA Isolation Kit (Hybrid-R™ Blood RNA, GeneAll, Korea) according to the manufacturer's protocol. RNA purity was quantified by spectrophotometry at 260/280 nm ratio and the integrity was confirmed by electrophoresis on a denaturing agarose gel. A 2 μg aliquot of the total RNA from each sample was reverse-transcribed into single-strand complementary DNA (cDNA) with HyperScript™ First Strand Synthesis Kit (GeneAll, Korea) according to the manufacturer's instruction.
All rat primers were designed using ABI Primer Express software (Applied Biosystems Inc., Foster City, CA, USA). The primers' pairs used in the present study were as follows: for Cdkn1a: 5'-CTG CTA CAG TGC CCG AGT TA-3' (forward) and 5'-ACT TTG CTC CTG TGT GGA AC-3' (reverse), for Rad50: 5'-CCG AGT GGT GAT GCT GAA GGG-3' (forward) and 5'-TGT AGG CTC ATC CAA GGC AAG G-3' (reverse) and for Hprt: 5'-CCA GTC AAC GGG GGA CAT AAA-3' (forward) and 5'-GGG GCT GTA CTG CTT GAC CAA-3' (reverse). The level of expression of Hprt gene served as an internal control.
The RT-PCR reactions were performed with the Rotor-Gene 6000 RT-PCR System using the SYBR Premix EX Taq™ II (Takara, Japan) following the manufacturer's recommendations. All samples were run two times. The qRT-PCR cycling conditions were as follows: initial denaturation at 95°C for 10 s, followed by forty cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s. The real-time PCR efficiencies were determined for target and internal control genes with the slope of a linear regression model. For target and internal control genes, PCR efficiencies were calculated by measuring the cycle threshold (CT) to a specific threshold for a serial dilution of cDNA. All PCRs displayed efficiencies between 98% and 102%. For each group at the 8, 24, and 48 h postradiation time points, three independent blood samples were assessed. For each sample, assays were performed in duplicate. The comparative 2−ΔΔCT was used for relative fold changes in expression of target genes (Cdkn1a and Rad50), normalized to an endogenous reference (Hprt) gene, and a relevant untreated and unradiated control. ΔΔCT is the difference between the mean ΔCT (treatment group) and mean ΔCT (control group), where ΔCT is the difference between the mean CT gene of interest and the mean CT internal control gene in each sample.
Each data point represents mean ± standard error of mean of at least six animals per group. The statistical significance of differences between groups was analyzed by one-way analysis of variance with a post hoc Tukey test. P < 0.05 was considered statistically significant.
| > Results|| |
As shown in [Figure 1] and [Figure 2], radiation-only groups in comparison with Con Group, Cdkn1a gene expression increased to a maximum value of approximately 48-fold for 2 Gy Rad Group (P < 0.05) and 160-fold for 8 Gy Rad Group (P < 0.05) at 8 h postradiation, and approximately 28-fold for 2 Gy Rad Group (P < 0.05) and 148-fold for 8 Gy Rad Group (P < 0.05) at 24 h postradiation. Then, Cdkn1a gene expression decreased but remained >10-fold for 2 Gy Rad Group (P < 0.05) and 50-fold for 8 Gy Rad Group than that in Con Group at 48 h postradiation.
|Figure 1: Real-time quantitative reverse transcription polymerase chain reaction analysis of the fold change of Cdkn1a at various time points after 2 Gy radiation (relative to control). Values are expressed as mean ± standard error of mean of six animals per group. *P < 0.05 compared to the control, vehicle and melatonin groups, **P < 0.05 compared to the 2 Gy and vehicle + 2 Gy groups|
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|Figure 2: Real-time quantitative reverse transcription polymerase chain reaction analysis of the fold change of Cdkn1a at various time points after 8 Gy radiation (relative to control). Values are expressed as mean ± standard error of mean of six animals per group. *P < 0.05 compared to the control, vehicle and melatonin groups, **P < 0.05 compared to the 8 Gy and vehicle + 8 Gy groups|
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The expression of Cdkn1a in Con, Veh, and Mel Groups were almost similar, and also in Veh + 2 Gy Rad and Veh + 8 Gy Rad Groups, Cdkn1a expression was almost similar to 2 Gy Rad and 8 Gy Rad Groups at different time points, respectively. However, in melatonin + radiation groups in comparison with Con Group, the expression of Cdkn1a was significantly upregulated to a value of approximately 104-fold for Mel + 2 Gy Rad Group (P < 0.05) and 398-fold for Mel + 8 Gy Rad Group (P < 0.05) at 8 h postradiation, and approximately 93-fold for Mel + 2 Gy Rad Group (P < 0.05) and 204-fold for Mel + 8 Gy Rad Group (P < 0.05) at 24 h postradiation. However, the statistically significant differences in the expression of Cdkn1a were not observed at 48 h time point after radiation in Mel + 2 Gy Rad Group (10.18 ± 2.9-fold) in comparison with 2 Gy Rad Group (10.43 ± 3.3-fold) and Veh + 2 Gy Rad Group (9.5 ± 2.8-fold) [Figure 1] and also in Mel + 8 Gy Rad Group (57.55 ± 12.22-fold) in comparison with 8 Gy Rad Groups (50.7 ± 7-fold) and Veh + 8 Gy Rad Group (44.2 ± 6.4-fold) [Figure 2].
As shown in [Figure 3] and [Figure 4], Rad50 gene expression was strongly decreased to a value of approximately 0.09-fold for 2 Gy Rad Group (P < 0.05) and 0.18-fold for 8 Gy Rad Group (P < 0.05) at the initial 8 h postradiation compared to Con Group, and approximately 0.19-fold for 2 Gy Rad Group (P < 0.05) and 0.27-fold for 8 Gy Rad Group (P < 0.05) at 24 h postradiation. Then, approximately 0.13-fold for 2 Gy Rad Group (P < 0.05) and 0.18-fold for 8 Gy Rad Group (P < 0.05) at 48 h postradiation. However, in melatonin + radiation groups, Rad50 expression was significantly upregulated compared to Con Group to a value of approximately 0.90-fold for Mel + 2 Gy Rad Group (P < 0.05) and 0.66-fold for Mel + 8 Gy Rad Group (P < 0.05) at 8 h postradiation, and approximately 0.84-fold for Mel + 2 Gy Rad Group (P < 0.05) and 0.76-fold for Mel + 8 Gy Rad Group (P < 0.05) at 24 h postradiation. In 48 h postradiation, the expression of Rad50 was not statistically differences in Mel + 2 Gy Rad Group (0.26 ± 0.02-fold) in comparison with 2 Gy Rad Group (0.13 ± 0.06-fold) and Veh + 2 Gy Rad Group (0.11 ± 0.06-fold) [Figure 3] and also in Mel + 8 Gy Rad Group (0.15 ± 0.05-fold) in comparison with 8 Gy Rad Groups (0.18 ± 0.11-fold) and Veh + 8 Gy Rad group (0.17 ± 0.08-fold) [Figure 4].
|Figure 3: Real-time quantitative reverse transcription polymerase chain reaction analysis of the fold change of Rad50 at various time points after 2 Gy radiation (relative to control). Values are expressed as mean ± standard error of mean of six animals per group. *P < 0.05 compared to the control, vehicle and melatonin groups, **P < 0.05 compared to the 2 Gy and vehicle + 2 Gy groups|
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|Figure 4: Real-time quantitative reverse transcription polymerase chain reaction analysis of the fold change of Rad50 at various time points after 8 Gy radiation (relative to control). Values are expressed as mean ± standard error of mean of six animals per group. *P < 0.05 compared to the control, vehicle and melatonin groups, **P < 0.05 compared to the 8 Gy and vehicle + 8 Gy groups|
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| > Discussion|| |
DSBs are the most serious type of DNA damage by ionizing radiation and barely repaired. To minimize the tortious effects of ionizing radiation, a number of combinational agents such as amifostine and other chemical compounds have been investigated as a potential radioprotective drug., Although amifostine is used in clinical trials, it has many side effects including nausea, vomiting, flushes, mild somnolence, hypocalcemia, and hypotension. Melatonin as a natural radioprotector has been studied in several experiments and shown to be an immune simulator, antioxidant and acts as a direct free radical scavenger and indirect antioxidant through its stimulatory actions on antioxidant enzymes activity and inhibitory actions on pro-oxidant enzymes activity.,,,,, In this study, for the first time, we investigated the effect of melatonin as a radioprotective agent in the expression level of Cdkn1a and Rad50 genes related to DNA DSBs repair induced by radiation in rat peripheral blood.
Cdkn1a (P21, Cip1) is known to be an important effector of the p53-mediated G1 arrest in response to many stresses.Cdkn1a belongs to the family of cyclin-dependent kinase regulators and plays an important role in the regulation of the cell cycle and proliferation. In our study, Cdkn1a expression in radiation rat peripheral blood was substantially upregulated across all radiation doses (2 and 8 Gy) and time points (8, 24, and 48 h postradiation). In our study, the maximum expression of Cdkn1a gene appeared at 8 h postradiation for every two radiation doses (2 and 8 Gy), which is well in agreement with the cell cycle arrest necessity for DNA repair postradiation. The more serious DNA damage caused by high dose (8 Gy compared to 2 Gy), the more Cdkn1a needed to satisfy the request of DNA repair.
Melatonin pretreatment led to enhanced expression of this gene in all radiation doses (2 and 8 Gy) and just in 8 and 24 h postirradiation. It means that melatonin is effective in blocking the G1/S transition in the cell cycle, which leads to limiting cellular damage by radiation because arrested cells have more time for repair of their damaged DNA. In 48 h postradiation, there was no significant difference in Cdkn1a expression compared to the radiation-only and vehicle plus irradiation groups, which could be due to the lack of melatonin concentration compared to early hours (8 and 24 h postradiation) in the rat peripheral blood. However, this explanation requires further investigation.
Rad50 is a member of MRN complex. The MRN complex plays important roles in signal transduction related to DNA repair and cell cycle checkpoints. The MRN complex, which contains exo- and endo-nuclease and helicase activities, may also function in NHEJ, particularly if the DNA ends require processing before ligation and also, in the HR repair process, the MRN complex serves as a primary damage sensor and is involved in the early steps of HR repair, which include processing of the broken DNA ends.,
Our study showed that 2 and 8 Gy radiation exposure of rat peripheral blood, resulting in significant decreased expression of Rad50 in all time points (8, 24, and 48 h) postradiation. Reduction in Rad50 gene expression can refer to this topic that MRN complex as a DNA damage response migrates to sites of damage within 30 min after DNA DSB, so the expression of MRN complex is reduced in a long time after radiation. 100 mg/kg of melatonin pretreatment prior to radiation exposure has increased significantly the Rad50 expression in 8 and 24 h postradiation. Likewise, our results showed that there was no significant difference in Rad50 expression in 48 h postradiation compared to the radiation-only and vehicle plus radiation groups which could be due to the lack of melatonin concentration compared to early hours (8 and 24 h) postradiation in the rat peripheral blood. Nevertheless, this explanation requires further investigation.
| > Conclusions|| |
Melatonin has been reported as a direct and indirect free radical scavenger, but to the best of our knowledge, this is the first in vivo study to show the effect of melatonin as a radioprotective agent in expression of Cdkn1a and Rad50 genes related to DNA damage repair induced by radiation in rat peripheral blood. This study reveals that melatonin in 100 mg/kg may improve repair of radiation-induced DNA DSBs by increasing the Cdkn1a and Rad50 expression in rat peripheral blood, especially during the early postradiation. Nevertheless, further investigations are needed to validate this effect.
The authors gratefully acknowledge Department of Medical Genetics, Tehran University of Medical Sciences for their great advice during this study.
Financial support and sponsorship
This research has been supported by the Tehran University of Medical Sciences and Health Services grant 25111.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]