Biomedical

Communication

Biosci. Biotech. Res. Comm. 9(4): 821-827 (2016)

Effects of melatonin on repair of DNA double strand

breaks caused by ionizing radiation in rat peripheral

blood

Majid Valizadeh

, Alireza Shirazi*

, Pantea Izadi

and Javad Tavakkoli Bazzaz

Hamed Rezaeejam

and Ghasem Azizi Tabesh

1,2,5

Department of Medical, Physics and Biomedical Engineering, Tehran University of Medical Sciences,

Tehran, Iran

3,4,6

Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran

ABSTRACT

The present study aimed to evaluate the effect of melatonin on DNA Double-Strand Breaks repair using gene expres-

sion change of Ku70 and Xrcc4 in rat peripheral blood. One hundred eight male rats were randomly divided in six

different groups of control, vehicle-only, melatonin alone, irradiation-only, vehicle plus irradiation, and melatonin

plus irradiation. Rats were given an intraperitoneal (IP) injection of melatonin (100 mg/kg) 1 hr prior to irradiation.

Irradiation was done with 2Gy whole-body radiation by linear accelerator. Peripheral blood samples were collected

at 8, 24 and 48 h after irradiation and then RNA was immediately extracted from the peripheral blood lymphocytes

and the mRNA transcriptional changes of Ku70 and XRCC4 were evaluated by real-time quantitative polymerase

chain reaction (Real-time PCR). Irradiation-only group showed an increase in gene expression change of Ku70 and

XRCC4, which was signi cantly different from control group (P<0.01) in 24 h post-irradiation, while in melatonin

plus irradiation group, Ku70 and XRCC4 genes were upregulated signi cantly compared to control group (P<0.01) at

all three post-irradiation times. It is concluded that melatonin may provide modulation of Ku70 and XRCC4 expres-

sion to protect rat peripheral blood lymphocytes against ionizing radiation.

KEY WORDS: IONIZING RADIATION, DOUBLE-STRAND BREAK, MELATONIN, DNA REPAIR

821

ARTICLE INFORMATION:

*Corresponding Author: shirazia@tums.ac.ir

Received 30

Oct, 2016

Accepted after revision 10

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

822 EFFECTS OF MELATONIN ON REPAIR OF DNA DOUBLE STRAND BREAKS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Majid Valizadeh et al.

INTRODUCTION

Ionizing radiation (IR) is applied constructively in a wide

variety of  elds such as medicine, research, manufac-

turing, construction especially in medical diagnostic for

cancer treatment (Jobert et al. 2011; Thoms and Bristow,

2010), but presents a health hazard if proper measures

against undesired exposure aren’t followed. Exposure to

ionizing radiation causes damage to living tissue, and

can result in mutation, radiation sickness, cancer, and

death. The absorption of ionizing radiation by living

cells can directly disrupt atomic structures, producing

chemical and biological changes. It can also act indi-

rectly through radiolysis of water, thereby generating

reactive chemical species that may damage nucleic acids,

proteins and lipids. DNA damage of exposed tumour tis-

sue leading to cell death is one of the detrimental effects

of ionizing radiations, (Hall and Giaccia, 2006, Azzam

et al.2012; Lomax et al. 2013 an d Rezaeejam et al 2015).

There are many different forms of IR-induced DNA

damages such as single-strand breaks (SSBs), double-

strand breaks (DSBs), base and sugar damage, DNA-

DNA cross-links and DNA-protein cross-links. DSBs are

lesions formed when both strands of the DNA duplex are

broken. DSBs are highly toxic and are the most impor-

tant IR-induced DNA damages in chromosomes after

exposure which leads to cells death, mutation or car-

cinogen. They must be repaired to protect the genome

and cells survival, (Brandsma and Gent, 2012; Ciccia and

Elledge, 2010 Chapman et al. 2012).

These kinds of damages are mainly repaired by

homologous recombination (HR) and non-homologous

end joining (NHEJ) pathways (Lieber, 2010). HR leads to

accurate repair, while NHEJ is intrinsically mutagenic.

Mao et al (2008) suggested that NHEJ is a faster and

more ef cient DSB repair pathway than HR in human

cells. According to Guirouilh-Barbat et al. (2004), NHEJ

is the predominant DSB repair pathway in mammalian

cells. NHEJ is the major DSB repair pathway in eukary-

otes and is utilized in the cellular response of mamma-

lian cells to the repair of IR-induced DSBs. NHEJ can

take place throughout the cell cycle. NHEJ modi es the

broken DNA ends, and ligates them together with no

regard for homology, generating deletions or insertions

(Lieber, 2008), while HR uses an undamaged DNA tem-

plate to repair the break, leading to the reconstitution of

the original sequence (Thompson and Schild, 2001).

The proteins that participate in NHEJ pathway include

XRCC4, XRCC5 (Ku80), XRCC6 (Ku70), DNA-PKcs,

DNA ligase IV, Artemis, and XLF (Bassing et al. 2002;

Shrivastav et al. 2008). First, both ends of the break are

joined by the Ku70/80 heterodimer which protects the

DNA ends from degradation. Then Ku70/80 recruits the

catalytic subunit of the DNA dependent protein kinase

(DNA-PKcs) to DNA ends to form the active DNA-PK;

the ends can be trimmed or  lled in by nucleases and

polymerases. Finally the DNA-PKcs complex stimulates

the end processing for subsequent ligation by XRCC4/

DNA ligase IV (Ahnesorg et al. 2006; San Filippo et

al.2008; Schulte-Uentrop et al. 2008; Zhang et al. 2013).

The radio protective agents protects against the dam-

aging effects of IR with various mechanisms. Many

studies have reported the ability of melatonin (N-acetyl-

5-methoxytryptamine), a pineal gland hormone, to pro-

tect against IR-induced damages (Reiter, 1991; Undeger

et al. 2004; Shirazi et al. 2011; Shirazi, 2011). It scaven-

gers free radicals, directly and indirectly, especially the

highly toxic hydroxyl radicals. Melatonin is also an anti-

oxidant agent by increasing antioxidant enzyme activ-

ity and inhibiting pro-oxidative enzyme activity (Koc

et al. 2003; Rodriguez et al. 2004; Parihar et al. 2007).

Many in vitro and in vivo investigations have con rmed

that melatonin protects mammalian cells from the toxic

effects of ionizing radiation. Furthermore, several clini-

cal reports indicate that melatonin administration, either

alone or in combination with traditional radiotherapy,

results in a favorable ef cacy (toxicity ratio) during the

treatment of human cancers (Vijayalaxmi et al. 2004).

There are many studies which have examined mela-

tonin as a radio protector in radiobiology but there is

a serious lack of information about its impact on the

DNA repair with NHEJ pathway. In this regard, in this

study we have investigated the effect of melatonin on

the repair of IR-induced DNA DSB in the peripheral

blood of rat when NHEJ pathway is used. We studied

the expression change of Ku70 and XRCC4 genes under

2Gy whole-body gamma irradiations to show the radio-

protective effect of 100 mg/kg administered melatonin

on DNA DSB repair.

MATERIALS AND METHOD

In this in vivo study, all experiments were in accord-

ance with the guidelines for care and use of labora-

tory animals as adopted by the Ethics Committee of the

School of Medicine, Tehran University of Medical Sci-

ences (TUMS), Tehran, Iran. One hundred eight 70-day

old male Wistar rats with a body weight range of 180 to

220 g were used for the study from pharmacy faulty of

TUMS. They were kept in a room temperature and main-

tained at 20-22° C and light-controlled environment

with a 12-hour light/dark cycle. All rats were given

standard diet with no additives.

EXPERIMENTAL DESIGN AND IRRADIATION

After one week acclimatization period, animals were

randomly divided into six different groups:

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF MELATONIN ON REPAIR OF DNA DOUBLE STRAND BREAKS 823

Majid Valizadeh et al.

Group 1. Control (CON): In this group, rates

received no melatonin or irradiation

but received both an intraperitoneal (IP)

injection of 500 μl of phosphate-buff-

ered saline (PBS) and sham-irradiation;

Group 2. Melatonin (MEL): In this group, one hour

before irradiation, all anesthetized rats

received 100 mg/kg melatonin with IP

injection of 500 μl PBS and  nal ethanol

concentration 5% and then they went

under and sham-irradiation. It should

be noted that all rats were anesthetized

with an IP injection of ketamin (100 mg/

kg) and xylazin (5 mg/kg);

Group 3. Irradiation (IR): In this group rats went

under 2 Gy whole body gamma radia-

tion and received the same volume of

PBS 1 h prior to irradiation;

Group 4. Vehicle (VEH): In this group rats received

5% absolute ethanol with IP injection of

500 μl PBS;

Group 5. Vehicle + irradiation (VEH+IR): In this

group rats received 5% absolute ethanol

with IP injection of 500 μl PBS plus 2 Gy

whole body gamma radiation;

Group 6. Melatonin + irradiation (MEL+IR): In

this group rats received 100 mg/kg mel-

atonin with 5% absolute ethanol and

an IP injection of 500 μl PBS plus 2 Gy

whole body gamma radiation.

It should be mentioned that melatonin  rst was dissolved

in a small amount of absolute ethanol (25μl) and then

diluted by PBS (475μl) in  nal ethanol concentration 5%

based on previous studies like Cassatt et al. (2002). Also,

all rats were anesthetized with ketamin (100 mg/kg) and

xylazin (5 mg/kg) by an IP injection before any inter-

vention based on previous studies like Prasad (1995).

Rats were irradiated with a 6 MV X-ray linear accelera-

tor machine (Elekta Compact 6 MV, China) with a  xed

 eld size of 35cm×35cm at room temperature (22 ± 2ºC).

Before irradiation, to ensure the output of the accelera-

tor, dosimetry and calibration were performed by using

an ionizing chamber based on International Atomic

Energy Agency (IAEA) TRS-398 standard.

BLOOD SAMPLE PREPARATION, RNA

ISOLATION AND CDNA SYNTHESIS

Each study group includes 18 rats and divided in three

subgroups containing six rats. From all of these sub-

groups peripheral blood sample was taken on EDTA ster-

ile tubes at 8, 24 and 48 h after irradiation. Total RNA

was isolated from whole blood by Hybrid-R blood RNA

mini 315-150 kit (GeneAll Biotechnology, Seoul, South

Korea) according to the manufacturer’s instructions. The

extracted RNA was quanti ed and its purity quali ed

by using a Nanodrop-2000 spectrophotometer (Thermo

Scienti c, Wilmington, USA) respectively at 260/280

nm and 260/230 nm ratios. The integrity of isolated

RNA was con rmed with Agarose gel electrophoresis.

The representative samples were stained with ethidium

bromide to visualize the 18S and 28S RNA subunits by

band size discrimination under UV transillumination.

For cDNA synthesis, a 2-μg aliquot of the total RNA was

reverse transcribed in a total volume of 20μl by using

the Hyperscript TM  rst strand synthesis Kit (GeneAll

Biotechnology, Seoul, South Korea).

QUANTITATIVE REAL TIME RT-PCR

Quantitative real time PCR was used to measure the

expression of Ku70 and XRCC4 genes. After RNA isola-

tion and cDNA synthesis, Ku70 and XRCC4 primers were

were determined by using HPRT as an internal control.

The sequences of forward and reverse primers were as

follows: Ku70, forward primer: GCT TGT CTT CCT CCC

TTA CG, reverse primer: CGA AAC TGT CGC TCC TGT

ATG; XRCC4, forward primer: CTG AGG AGG ATG GGC

TTT ATG AT, reverse primer: CAA GAT TTG TCT GCA

TTC GGT GT; and HPRT, forward primer: CCA GTC AAC

GGG GGA CAT AAA, reverse primer: GGG GCT GTA

CTG CTT GAC CAA. Basic Local Alignment Search Tool

(BLAST) searches were also conducted to verify primer

speci city in the absence of DNA ampli cation. The

primers were synthesized by Takapouzist laboratory in

Tehran, Iran.

The real time polymerase chain reactions (real time

PCR) were carried out by the Rotor-gene Q system

(QIAGEN), based on the SYBR green method using the

SYBR Premix Ex Taq No. RR820L (TaKaRa) following

the manufacturer’s instructions. The PCR reaction mix-

ture contained all reactions running in duplicate and the

real time PCR cycling conditions were as follows: ini-

tial denaturation at 95ºc for 10 min, followed by cycles

of denaturation at 95ºc for 10s, annealing at 60ºc for

20s. In real-time PCR studies, relative quanti cation or

relative gene expression is the parameter used for rela-

tive fold changes in expression of target genes (Ku70

and XRCC4), normalized to an internal reference (HPRT

gene) and a relevant untreated and unirradiated control.

This parameter is calculated according to

ΔΔCT

formula.

ΔΔCT is the difference between the mean ΔCT (treatment

group) and mean ΔCT (control group) and ΔCT is the

difference between the mean CT gene of interest and the

mean CT

of internal control gene in each sample. CT is

the threshold cycle, i.e. the cycle number at which the

824 EFFECTS OF MELATONIN ON REPAIR OF DNA DOUBLE STRAND BREAKS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Majid Valizadeh et al.

PCR product crosses the threshold of detection. For each

group at 8, 24 and 48 h post-irradiation, six independent

blood samples were assessed. Assays were performed in

duplicate for each sample.

STATISTICAL ANALYSIS

For analyzing data the mean ± SEM of experiments per

group were presented and one-way analysis of variance

(ANOVA) was performed in order to compare the differ-

ences among groups, followed by Tukey’s test for mul-

tiple comparison. The signi cance level was set at 0.05.

RESULTS

The expression change of Ku70 and XRCC4 genes in the

rat peripheral blood at 8, 24 and 48 h after irradiation of

sublethal dose of 2Gy whole body radiation were ana-

lyzed by real-time quantitative PCR. The results have

been summarized in Table 1.

The obtained results show that in comparison with

the control group, the mRNA levels of Ku70 and XRCC4

genes (normalized against HPRT) have signi cant

change in some groups. Gene expression increased sig-

ni cantly in IR and VEH + IR groups only at 24 h after

irradiation compared to control group or both genes.

In IR group these changes were 5.9 (P<0.01) and 7.67

fold (P<0.05) for Ku70 and XRCC4, respectively; and in

VEH + IR group the fold changes were 5.39 (P<0.01) and

7.01 (P<0.05) for Ku70 and XRCC4, respectively. Expres-

sion of these two genes also increased signi cantly in

MEL+IR group at all three times after irradiation in com-

parison with control group (Fig. 1). Expression changes

for XRCC4 gene at 8, 24 and 48 h were 14.42, 41.93, and

5.39 fold respectively (P<0.01), whiles these results for

Ku70 gene were 7.36, 29.65, and 4.47 fold, respectively

(P<0.01). We found no signi cant differences in the gene

expression of Ku70 and XRCC4 in other groups com-

pared to control group.

Results showed that regulation changes of Ku70 and

XRCC4 genes were signi cantly different in MEL + IR

group at three times after irradiation compared to IR

group as well. We calculated a ratio (fraction) in order to

understand the effect of melatonin on the NHEJ repair

pathway in the current study. Thus relative expression

of MEL+ IR group was divided by relative expression of

IR group. This ratio is going to show the role of mela-

tonin in regulating Ku70 and XRCC4 genes. Based on

the results for Ku70 and XRCC4 genes, it can be said

that melatonin upregulates Ku70 gene as 6.94, 5.02 and

5.02 fold higher and XRCC4 gene as 5.55, 5.47 and 6.91

fold higher at 8, 24 and 48 post- irradiation, respectively

(Fig.2). It can be seen that melatonin upregulates Ku70

more than XRCC4 gene at 8 h and upregulates XRCC4

gene more than Ku70 gene at 48 h after irradiation.

In other words, with the increase of post- irradiation

time, melatonin decreases gene expression for Ku70 and

increases it for XRCC4. Results indicate that melatonin

not only prevents cell death by apoptosis but also can

repair damaged cells in NHEJ pathway.

DISCUSSION

Some studies have been conducted on the protective

role of melatonin against oxidative DNA damage and

the effects of melatonin on cell cycle and apoptosis.

Before conducting our study, it was very important to

know if damaged cells which must be killed have been

previously repaired or not. The effect of melatonin on

apoptosis has been studied previously by Mohseni et

al. (2012), Shirazi et al. (2010), and Rezaeejam et al.

(2015) whose results showed that melatonin decreases

relative expression of pro-apoptotic BAX and increases

anti-apoptotic Bcl-2 genes. In this basis, current study

Table 1: Real-time quantitative RT-PCR analysis of the fold change of Ku70 and

XRCC4 at various time points after irradiation relative to control group.

Group

8 h 24 h 48 h

Ku70 XRCC4 Ku70 XRCC4 Ku70 XRCC4

CON 1 1 1 1 1 1

VEH 0.97 0.98 0.93 0.93 0.93 0.95

MEL 1.02 1.07 1.03 1.07 1.04 1.10

IR 1.06 2.60 5.90 7.67 0.89 0.78

VEH + IR 0.97 2.46 5.39 7.01 0.95 0.75

MEL + IR 7.36 14.42 29.65 41.93 4.47 5.39

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF MELATONIN ON REPAIR OF DNA DOUBLE STRAND BREAKS 825

Majid Valizadeh et al.

FIGURE 1. Temporal response of Ku70 and XRCC4 genes expression at untreated and pretreated

groups. Points denote the mean of responses in six different rats.

FIGURE 2. Manifold effect of melatonin on the

regulation of genes.

investigated the in uence of melatonin on DNA DSBs

repair-related important genes because of sublethal dose

of ionizing radiation in the NHEJ pathway in peripheral

blood rat. According to many previous studies, XRCC4,

XRCC5 and XRCC6 (KU70) genes are the core of DNA

DSBs repair-related genes involved in NHEJ pathway. In

this study, the gene expression of Ku70 and XRCC4 were

examined using real-time quantitative PCR.

We used 2Gy of 6MV irradiation because it is a stand-

ard fraction that is administrated to patients daily in

a fractionation regimen and the highest sensitivity for

human DNA is obtained for this dose of radiation. The

selection of one hour interval between melatonin injec-

tion and exposure to gamma radiation was largely based

on previous studies (Vijayalaxmi et al. 1999; Hussein

et al. 2005). The melatonin concentration was selected

based on the experience from the performed studies by

other researchers (e.g. Yurtcu et al. 2007) and our pre-

vious studies (Mohseni et al. 2012; Shirazi et al. 2010;

Rezaeejam et al. 2015) where it was found out this con-

centration doesn’t have any toxicity.

Based on the results in this study, since VEH and MEL

groups showed no signi cant difference with control

group, we found out that ethanol and melatonin do not

have any effects on the expression change of Ku70 and

XRCC4 genes alone. On the other hand, IR group had

no signi cant difference with VEH + IR group, so we

can say that the injection of ethanol before irradiation

doesn’t affect the regulation of Ku70 and XRCC4 genes.

Furthermore, results illustrated that DBS damages was

repaired at 24 h and there was no repair at 8 and 48 h

after irradiation in the NHEJ pathway, while the DSBs

was repaired at 8, 24 and 48 h post-irradiation when

melatonin was injected before irradiation. The repair

process was also accelerated and strengthened in this

condition at 24 h post-irradiation.

CONCLUSION

In this study we concluded that melatonin injection

before ionizing radiation can increase the expres-

sion levels of Ku70 and XRCC4 genes, and melatonin

has positive effect on repair in NHEJ pathway. Further

inestigation is recommended on the other doses, post-

irradiation times and genes related to HR and NHEJ

pathways.

Majid Valizadeh et al.

826 EFFECTS OF MELATONIN ON REPAIR OF DNA DOUBLE STRAND BREAKS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENT

This research was supported by Tehran University of

Medical Sciences in Iran with health services grant

No.28688.

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