The use of X-ray examinations for diagnostic purposes has considerably increased with a potential risk of carcinogenesis. Despite the application of sophisticated techniques toward dose reduction, increased utilization of new innovations in interventional radiology, as well as computed tomography (CT) has contributed to radiation exposure. Ionizing radiation (IR) interaction with tissues leads to the production of reactive oxygen species, which attack DNA to cause double-strand breaks (DSBs). DSB is a highly cytotoxic type of DNA lesion and results in cellular damage. These DNA lesions can be repaired. If not accurately repaired, it can lead to genomic instability, chromosomal aberration, mutations, as well as cancers in the long run. The estimated cancer risk as a result of diagnostic X-ray ranges from 0.6% to 3% in developed countries.
Phosphorylation H2AX is one of the best markers of DNA damage and DSB. Its quantity is equal to the number of foci of phospho-H2AX (γH2AX) whose formation and disappearance has been evaluated for 1 mGy. Foci yields have been shown to increase linearly with dose. Previous studies have proven that the dose received by tissues during CT coronary angiography (CTCA) ranges between 5 and 100 mGy, while this method (γH2AX) can be used for investigating the radiation effects.
Presently, enumerating foci is the most sensitive procedure for γH2AX analysis. Immunofluorescence microscopy has been used to detect foci induced by CT. As a way of decreasing the effect of IR, CT doses should be as low as possible although this could unfortunately lead to lesser image quality as well as more noise. An alternative is the use of antioxidants which have limited side effects.
Melatonin (N-acetyl-5-methoxytryptamine) is a natural hormone synthesized chiefly by the pineal gland in the human body. Its antioxidant effects have been proven in the range of therapeutic doses. Although its radioprotective effect in range of therapeutic dose is clear, its effect in range of diagnostic dose is yet unrecognized, which if proven to be effective, could be worthwhile for patients undergoing CT examinations. Thus, the aim of our study was to investigate the effect of administering 100-mg melatonin on γH2AX foci induced by 10 mGy and 100 mGy doses of X-ray.
MATERIALS AND METHODS
This experimental protocol was approved by the Ethics Committee of Tehran University of Medical Sciences and Health Services. Written informed consent was obtained from five male volunteers who are nonsmokers, nonathlete, and aged between 25 and 35 years without any history of radiation exposure. At 9 a.m., while they had been fasting, each volunteer ingested two pills (each containing 50 mg) of melatonin (Swati Spentose Pvt. Ltd, Gujarat, India). Blood samples were collected in sterile heparin tubes (for isolation of lymphocytes as well as in vitro exposure to X-ray) 5–10 min before and at 1 and 2 h after melatonin administration. All samples collected from each volunteer before administering melatonin were divided into three parts and poured into 5-ml tubes with heparin anticoagulant; one part was used as personal baseline quantity of γH2AX foci (nonirradiated nontreated), while the others as control for low-dose (CT) and high-dose group CTCA (irradiated pretreated). The other samples from each volunteer, 1 and 2 h after ingestion, were aliquot into 5 parts and then poured into heparin tubes.
Each tube of low dose was irradiated with a dose of 10 mGy at room temperature (50 kV, 0.4 mA, 2-mm aluminum filtration, 10-cm distance between anode and samples), while every tube of high dose was irradiated with a dose of 100 mGy. Before irradiation, dosimetry was carried out using a Piranha dosimeter (RTI Group, Flöjelbergsgatan, Mölndal, Sweden). To deposit doses of 10 and 100 mGy, respective irradiation times of 12 and 120 s were necessary.
Isolation of lymphocytes
At every collection time, for each donor, the blood samples in the tubes after irradiation were incubated for 30 min in a humidified incubator (with 95% air and 5% CO2 at 37°C) in order for the number of foci to reach maximum levels. Peripheral blood lymphocytes were isolated by Ficoll-Hypaque using the protocol suggested by Cheki et al. About 5 ml of the blood was diluted using 1:1 phosphate-buffered saline (PBS) carefully layered on 3 ml of Ficoll-Hypaque and emptied into a Ficoll tube. All tubes were centrifuged at 3000 rpm for 30 min at 4°C (a temperature at which reduction of foci is minimum) and then the interface layer containing the lymphocyte was removed and washed twice with PBS by centrifugation at 2000 rpm for 10 min at 4°C. The number of viable cells was counted by staining the cells with trypan blue using a hemocytometer lam (Neobar lam).
This method has been illustrated in detail. Approximately 0.5 million lymphocytes were dropped softly into clean microscope slides for 10 min at room temperature to dry slightly. Afterward, they were fixed in 4% paraformaldehyde for 7–10 min at room temperature then washed with PBS for 3–10 min. To ensure permeabilization, cells were placed in Triton X-100 for 10 min as well as for 3–10 min in blocking solution (100 ml PBS with 1% bovine serum albumin [BSA] and 0.1% Tween 20) at room temperature. The cells were stained for 2 h, while applying the specific γH2AX antibody (1:500 diluted in blocking solution, Anti-H2AX-Phosphorylated, Ser 139, BioLegend, Uithoorn, The Netherlands) and washed afterward for 4–10 min in PBS with 1% BSA. Lymphocytes were incubated for 45 min in the second antibody (1:500 diluted in blocking solution) in a dark chamber at room temperature and subsequently washed in PBS for 4–10 min. Samples were mounted by applying VECTASHIELD mounting medium (Vector Laboratories, Burlingame, USA) with 4, 6 diamidino-2-phenylindole. Enumeration of γH2AX foci was fulfilled using a fluorescent microscope (Olympus microscope, Germany) equipped with a ×100 magnification objective. DM6000 B confocal microscope (Leica, Wetzlar, Germany) equipped with a ×100 magnification objective was used in order to verify the existence of foci within the cells. In every microscope slide, whole cells were counted until at least 40 foci were detected. The amount of excess foci was estimated by subtracting nonirradiated from irradiated samples, thereby representing the level of DSBs induced by X-ray.
All data values were reported as mean ± standard deviation of mean. All statistical analyses were performed using SPSS v. 24.0 (IBM Corp., Armonk, NY, USA). Dunnett's T3 test was performed to compare the foci between the nonirradiated pretreated and control samples. It was also used to compare the excess foci irradiated 1 and 2 h after melatonin ingestion with control samples. For this study, P < 0.05 was considered statistically significant.
We observed no evidence of side effects after single oral administration of 100-mg melatonin. In all volunteers, the γH2AX foci rate of baseline (Non-irradiated and untreated samples with melatonin) samples ranged between 0.0527 and 0.123 per cell (mean ± standard deviation: 0.096 γH2AX-foci/cell ± 0.0272). The microscopic image of γH2AX foci for 10 mGy is shown in Figure 1a in which the tiny dot depicts foci of γH2AX, while Figure 1b represents cells in baseline which no γH2AX foci was observed.
γH2AX foci levels induced by irradiation (excess foci), enumerated 30 min after, for an exposure of 10 mGy ranged between 0.1 and 0.22/cell (mean ± standard deviation: 0.131 γH2AX-foci/cell ± 0.044) without antioxidant, 0.056–0.122/cell (mean ± standard deviation: 0.08 γH2AX-foci/cell ± 0.0245) at 1 h, and 0.066–0.121/cell (mean ± standard deviation: 0.083 γH2AX-foci/cell ± 0.0218) at 2 h after pretreatment with ingestion of melatonin pills.
Dunnett's T3 test revealed significantly higher foci levels induced by 10 mGy compared with baseline. Furthermore, reduction in excess foci after ingestion of 100-mg melatonin for both 1 h (39% excess foci reduction; P < 0.05) and 2 h (35% excess foci reduction; P < 0.05) before irradiation were significant in comparison with the control (10 mGy) induced without treatment with antioxidant. However, there was no significant difference between 1 and 2 h pretreatments as shown in Figure 2.
Figure 3 shows the micrograph (confocal microscope) of lymphocyte exposed to 100 mGy X-ray. We observed tiny dots in each layer, showing γH2AX foci. For better counting, we merged all the layers (FITC filter image) on the DAPI filter image. The number of excess foci induced, enumerated 30 min after, for an exposure of 100 mGy ranged between 0.74 and 1.26 per cell (mean ± standard deviation: 0.913 γH2AX-foci/cell ± 0.23) without radioprotectants, 0.514–0.882/cell (mean ± standard deviation: 0.61 γH2AX-foci/cell ± 0.152) at 1 h, and 0.536–0.96/cell (mean ± standard deviation: 0.66 γH2AX-foci/cell ± 0.171) at 2 h after pretreatment with antioxidant pills as shown in Figure 4.
Significant increase in X-ray-induced foci levels was observed when compared with baseline (P = 0.002) and with 10 mGy (P = 0.003) [Figure 5]. Decreased foci levels in samples pretreated with 100-mg melatonin before 100 mGy irradiation for both 1 h (33% excess foci reduction; P < 0.05) and 2 h (28% excess foci reduction; P < 0.05) were significant compared with the control values (100 mGy) without pretreatment with antioxidant. Furthermore, no significant difference was observed between 1 and 2 h pretreatments [Figure 4].
Several reports have shown that melatonin could reduce radiation effects in various organs such as lens, brain, liver, and spleen. The ability of melatonin to penetrate and accumulate in the nucleus, as well as its potency to scavenge hydroxyl radicals, could serve as an effectual and executable remedy to reduce the possibility of creating DSB (which could increase the risk of cancer) in lymphocytes.
In our study, immunofluorescence measurements have shown that pretreatment with 100-mg melatonin can reduce γH2AX foci levels following doses of 10 mGy and 100 mGy X-ray irradiation. Previous studies have dealt with different amounts of melatonin antioxidants for in vitro or animal studies. Vijayalaxmi et al. showed the protective effect of melatonin in 61% reduction of micronuclei after a 2 h pretreatment. However, in our study, we observed a DSB reduction of up to 39% for 10 mGy and 32% for 100 mGy following 1 h pretreatment. In contrast to previous study, our study used standard doses of diagnostic and interventional radiology (10 mGy and 100 mGy vs. 150 cGy) and different amount of melatonin (100 mg vs. 300 mg). Furthermore, we obtained our data using a different methodology (immunofluorescence microscopy vs. micronuclei). Similar to a recent study, the best time for melatonin ingestion was 1 h before exposure.
DSBs are one of the most interesting radiation-induced DNA lesions. Immunofluorescence microscopy, which relies on early phosphorylation of the histone variant H2AX, can be utilized for detecting signal of foci (which shows DSBs) within cell nuclei. In the past two decades, studies have reported that the enumerated γH2AX foci levels correlate significantly with the radiation-induced DSBs even though a linear relationship exists between a low-dose radiation and foci levels. Löbrich et al. reported that the best time after CT scan for enumeration of γH2AX is about 30 min when the foci per cell have attained maximum levels. Accordingly, it provides an accurate and sensitive method for the detection of radiation-induced DSB. In our study, we applied this method to demonstrate the antioxidant effect of melatonin. After irradiation with 10 mGy without pretreatment with melatonin, the mean excess foci observed was 0.131 foci/cell, which is similar to recent reports, while for 100 mGy, 0.913 foci/cell was obtained, indicating an almost linear relationship between foci and dose.
We used 10 and 100 mGy since they are standard doses in CT scans as well as in other radiographic imaging modalities. The choice of melatonin as a radioprotector was based on results of previous studies which showed no side effect after its oral administration. We observed that the percentage of foci reduction after the ingestion of 100 mg melatonin 1 and 2 h before irradiation with 100 mGy was less than that of 10 mGy. This could be due to an insufficient amount of melatonin and probably with more amount of melatonin; a further reduction will be induced. Hence, further research is necessary to determine the optimal amount of melatonin.
Our research had some limitations in some aspects. First and foremost, the number of examined volunteers was small; however, the observed effects were large enough such that statistical significance was achieved. Nevertheless, the sample size proposed in this report should be seen as a preliminary research. Further studies with more volunteers will give new insights.
Second, we carried out only in vivo/in vitro experiments, simulating the in vivo condition accordingly. The data were not precisely transmutable to patients undergoing CT examinations. Consequently, a performance study to survey the effect of melatonin in patients who will be exposed to X-rays (especially those referred for CT scans) should be considered in the next step.
Third, in our research, the concentration of melatonin in the serum and lymphocyte was not surveyed because of data reported by Vijayalaxmi et al., in which the γH2AX foci levels were least at 1 h pretreatment before exposure (in both 10 mGy and 100 mGy). This could be due to the maximum concentration of melatonin in the blood at this time.
Finally, despite the fact that lymphocytes of peripheral blood are differentiated cells with no possibility of cancer induction, several studies have used these cells as a biomarker. Meanwhile, it has been previously reported that the induction of DSBs is comparable between various cell types.
This study has shown that 100-mg melatonin significantly reduced the induction of γH2AX foci after 10 mGy and 100 mGy irradiation with X-ray when ingested 2 or 1 h (best time to consume) before irradiation. Therefore, our observations have shown that ingestion of 100-mg melatonin before exposure to IR can be useful for a patient set to undergo CT scan.
Financial support and sponsorship
This research was supported by grant number 31742 from the Vice-chancellor of research at Tehran University of Medical Sciences and Health Services.
Conflicts of interest
There are no conflicts of interest.
The authors gratefully acknowledge Novin Medical Radiation Institute, Tehran, Iran, for providing the medical linear accelerator necessary to carry out our research work.
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