Mutagenesis, 2015, 30, 651–661

doi:10.1093/mutage/gev026
Original Article
Advance Access publication 11 April 2015

Original Manuscript

Sesamol attenuates genotoxicity in bone

marrow cells of whole-body γ-irradiated mice
Arun Kumar, Tamizh G. Selvan, Akanchha M. Tripathi,
Sandeep Choudhary, Shahanshah Khan, Jawahar S. Adhikari and
Nabo K. Chaudhury*
Chemical Radioprotector and Radiation Dosimetry Research Group, Division of Radiation Biosciences, Institute of
Nuclear Medicine and Allied Sciences, Brig. SK Mazumdar Marg, Timarpur, Delhi 110054, India

*To whom correspondence should be addressed. Tel: +91 112 390 5131; Fax: +91 112 391 9509; Email: nkcinmas@rediffmail.com
Received 13 December 2014; Revised 17 January 2015; Accepted 5 March 2015.

Abstract
Ionising radiation causes free radical–mediated damage in cellular DNA.This damage is manifested
as chromosomal aberrations and micronuclei (MN) in proliferating cells. Sesamol, present in
sesame seeds, has the potential to scavenge free radicals; therefore, it can reduce radiation-
induced cytogenetic damage in cells. The aim of this study was to investigate the radioprotective
potential of sesamol in bone marrow cells of mice and related haematopoietic system against
radiation-induced genotoxicity. A comparative study with melatonin was designed for assessing
the radioprotective potential of sesamol. C57BL/6 mice were administered intraperitoneally
with either sesamol or melatonin (10 and 20 mg/kg body weight) 30 min prior to 2-Gy whole-
body irradiation (WBI) and sacrificed after 24 h. Total chromosomal aberrations (TCA), MN and
cell cycle analyses were performed using bone marrow cells. The comet assay was performed
on bone marrow cells, splenocytes and lymphocytes. Blood was drawn to study haematological
parameters. Prophylactic doses of sesamol (10 and 20 mg/kg) in irradiated mice reduced TCA and
micronucleated polychromatic erythrocyte frequency in bone marrow cells by 57% and 50%,
respectively, in comparison with radiation-only groups. Sesamol-reduced radiation-induced
apoptosis and facilitated cell proliferation. In the comet assay, sesamol (20 mg/kg) treatment
reduced radiation-induced comets (%  DNA in tail) compared with radiation only (P  <  0.05).
Sesamol also increased granulocyte populations in peripheral blood similar to melatonin. Overall,
the radioprotective efficacy of sesamol was found to be similar to that of melatonin. Sesamol
treatment also showed recovery of relative spleen weight at 24 h of WBI. The results strongly
suggest the radioprotective efficacy of sesamol in the haematopoietic system of mice.

Introduction dose of a radioprotector is envisaged to provide protection against

whole-body exposure. Because of the complex nature of the response
Ionising radiation exposure is harmful and consequences on health
to radiation in cells and tissues, the much desired radioprotector is
depend on the extent of damage to DNA. The haematopoietic sys-
still not available. The only compound approved by Food and Drug
tem is the most radiosensitive and in the case of whole-body expo-
Administration, USA, as cytoprotectant for head and neck cancer radi-
sure can lead to haematopoietic syndrome with increasing radiation
otherapy patients is amifostine, developed at Walter Reed Institute,
dose. Damage to DNA, genotoxicity and related short- and long-term
USA (4). Different approaches are currently being pursued in various
effects on health are well documented (1–3). Antioxidants that have
institutes (5–9). Antioxidants have gained importance because of their
the ability to scavenge free radicals, can protect DNA and facilitate
strong free radical scavenging properties, low toxicity and multiple
cell functioning are considered for investigating radioprotection
roles at cellular level. In general, most of the studies in animal models
and underlying mechanisms in animal models. A single prophylactic

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651
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652
have been performed at lethal radiation doses and have focused on
efficacy in term of survival along with related mechanisms (10,11).
Although antioxidants require high doses for radioprotective efficacy,
they have remained the major focus due to their lesser toxicity, but
these doses are difficult to extrapolate to human doses for radiopro-
tector applications. A radioprotector is required for persons undertak-
ing planned exposure, for example, first responders in the event of
radiation accidents, radiation workers in industry, health and research
institutes and patients undergoing cancer radiotherapy. In these dif-
ferent applications areas, low-dose radiation is usually expected and
therefore investigations with potential radioprotectors in animal mod-
els at low radiation doses are necessary for developing strategies.
Sesamol (3,4-methylenedioxyphenol) is a nutritional phenolic anti-
oxidant compound present in sesame seeds and has medicinal proper-
ties (12,13). Few studies are reported with sesamol from the perspective
of radioprotection (14–17). Prasad et  al. (15) showed a protective
effect on radiation-induced DNA damage in human lymphocytes (15).
A subsequent report of radioprotection in Swiss albino mice supported
with histopathological investigations of jejunum and biochemical
parameters by Parihar et al. (16) was an important step in this direc-
tion. Sesamol was investigated for antioxidant properties using pulse
radiolysis and its mechanism was revealed (17). In view of these prom-
ising results, detailed antiradical characterisation of sesamol along with
other antioxidants was carried out by our group. Antiradical assays
have shown higher antioxidant properties of sesamol in comparison
with many standard reference antioxidants, including melatonin (18).
Further in vitro studies in V79 cells have shown a higher protection
factor for sesamol in comparison with melatonin (19).
This study was undertaken to investigate the protective effects
of sesamol on radiation-induced cytogenetic damage in bone mar-
row cells of C57BL/6 mice exposed to 2-Gy whole-body irradia-
tion (WBI). Sesamol is a relatively new antioxidant molecule having
potential for radioprotection. Melatonin is a multitasking molecule,
whose free radical scavenging properties were discovered in 1993.
Melatonin has been classified as a broad spectrum antioxidant due to
its chemical structure and properties (20). A detailed mechanism of
antioxidant properties of melatonin was studied by Mahal et al. (21).
Melatonin, a strong antioxidant, has been recommended in various
reviews for development of a radioprotector and therefore this was
included in all experimental groups for the purpose of comparison.
It is an endogenous molecule synthesised in pineal gland and other
organs, and further distributed in all organs through blood circula-
tion. Melatonin, apart from its various physiological roles (22–25),
it is a strong antioxidant with high scavenging properties for various
free radicals (26). The radioprotective effect of melatonin was first
demonstrated in an animal survival study in WBI mice by Vijayalaxmi
et al. (27). In subsequent studies, the radioprotective efficacy has been
demonstrated in ex vivo and in vivo models. The pharmacokinetics of
melatonin in small animals and humans has been determined and it
appears that poor bioavailability and low efficacy are the two major
limiting factors for its development as a radioprotector, thereby
requiring high drug doses. Therefore, more efficacious antioxidants
with better bioavailability are needed for radioprotector applications.
Radiation targets DNA and causes damage in the form of single-
strand and double-strand breaks leading to formation of cytogenetic
end points [micronuclei (MN) and chromosomal aberrations (CA)]
in cells, hence these endpoints have been adopted for evaluation of
efficacy in various in vitro and in vivo studies (28–30). CAs and the
micronucleus (MN) assay are widely used for genotoxicity detec-
tion of various chemical and physical agents including ionising
radiation (31–34). WBI cause damage to all organs; the degree of
damage depends on the extent of radiation dose and sensitivity of
A. Kumar et al., 2015, Vol. 30, No. 5
the organ. Bone marrow is a key constituent of the haematopoietic
system, which is very sensitive to radiation exposure. Its protection
and recovery are necessary for survival and quality of life. WBI of
low-dose exposure is likely to be the scenario for radiation work-
ers in radiation environment in the event of radiation accidents. In
addition, there might be overexposure situations depending upon the
nature of work. The sensitivities of human and mouse bone marrow
cells are similar at low radiation doses of 2 Gy (35). In view of these
scenarios, we have carried out this study to investigate the radiopro-
tective efficacy of sesamol and melatonin in WBI mice against low-
dose (2 Gy) radiation-induced early damage in bone marrow cells.
Materials and Methods
Chemicals and reagents
Melatonin, sesamol, colchicine, Giemsa powder, soybean oil, potas-
sium chloride, DPX mountant solution, normal-melting-point aga-
rose, low-melting-point agarose and propidium iodide were procured
from Sigma Aldrich Chemicals, USA. Phosphate-buffered saline (PBS),
May–Grunewald stain, EDTA disodium, sodium chloride, sodium
hydroxide pellets and N-sodium lauryl sarcosinate were obtained from
Hi-media, India. Foetal bovine serum (FBS) from Invitrogen (USA) was
used. Sodium phosphate monobasic and dibasic buffers were obtained
from Calbiochem (India). Methanol, acetic acid, dimethyl sulphoxide
(DMSO) and Triton X-100 were of analytical grade purchased from
Spectrochem, India. Tris base and ethanol were used from Merck, India.
Animals
Male C57BL/6 mice of 8–10 weeks old were obtained from the exper-
imental animal facility of the Institute of Nuclear Medicine & Allied
Sciences (INMAS), Delhi, India. Mice were housed in cages under opti-
mum conditions of temperature (25°C ± 2°C), humidity (50–60%)
and light (14 h of light and 10 h of dark), and provided with standard
food and water ad libitum. After acclimatisation (5–6 days), mice were
weighed and the average body weight was 25.0 ± 2 g. All study proto-
cols were approved by the institutional animal ethics committee.
Experimental design
Mice were divided into groups of five animals each for CA and MN
assays separately. The alkaline comet assay was also performed in
selected groups each of three animals after the analysis of above
assays. Mice of all groups were sacrificed 24 h after various treat-
ments. The description of each group is as mentioned below:
Group I (control): unirradiated or without any treatment.
Group II (sesamol 20 mg/kg): mice were administered an intraperi-
toneal (i.p.) injection of 0.2-ml sesamol (20 mg/kg body weight).
Group III (melatonin 20 mg/kg): mice were administered an i.p.
injection of 0.2-ml melatonin (20 mg/kg body weight).
Group IV (2-Gy radiation): mice were exposed to 2-Gy WBI.
Group V (sesamol 10 mg/kg + 2 Gy): mice were administered an
i.p. injection of 0.2-ml sesamol (10 mg/kg body weight) 30 min
prior to WBI.
Group VI (sesamol 20 mg/kg + 2 Gy): mice were administered an
i.p. injection of 0.2-ml sesamol (20 mg/kg body weight) 30 min
prior to WBI.
Group VII (melatonin 10 mg/kg + 2 Gy): mice were administered
an i.p. injection of 0.2-ml melatonin (10 mg/kg body weight)
30 min prior to WBI.
Group VIII (melatonin 20 mg/kg + 2 Gy): mice were administered
i.p. injection of 0.2-ml melatonin (20 mg/kg body weight) 30 min
prior to WBI.
Radiation-induced DNA damage protection, 2015, Vol. 30, No. 5 653

Preparation and administration of antioxidants
Sesamol and melatonin solutions were freshly prepared in the
required volume of soybean oil to obtain final dose of 10 (200 µl)
and 20 mg/kg (200  µl) for each animal. The required quantities of
these antioxidants were dissolved in 2% DMSO, and made up to
the final volume with soybean oil and mixed thoroughly. Mice were
administered sesamol or melatonin i.p. at different doses 30 min
prior to γ irradiation.
γ-Irradiation
Animals were exposed to whole-body γ- rays (60Co) with 2-Gy radia-
tion dose using the Cobalt Tele-therapy Unit, Bhabhatron-II (Panacea
Biotech India) at a dose rate of 1 Gy/min, source to sample distance
(SSD) 103.9 cm and field size 20 × 20 cm2. The dose rate is routinely
calibrated as a part of quality assurance by the radiation safety officer
of the institute and the system was operated by the trained operator.
Bone marrow micronucleus assay
Animals were sacrificed by cervical dislocation 24-h post-irradiation.
Both femurs of the animals were dissected and the bone marrow
immediately flushed out with chilled PBS. For the MN assay, bone
marrow cell suspension was processed according to the method of
Schmid with minor modifications (36). Briefly, after centrifugation
at 1000 r.p.m. for 10 min, the supernatant was discarded and cell
pellet was resuspended in 2–3 drops of FBS. The cell suspension was
smeared on cleaned dry slides, air dried and fixed with methanol.
The slides were stained with a cocktail of May–Grunewald and
Giemsa (1:1) diluted in Sorensen’s phosphate buffer (pH 6.8) at 1:4
ratio for 20–25 min followed by washing with the same buffer. All
slides were coded by a person not involved in the study and scored
under light microscope with ×100 objectives (Axio Imager M2,
Carl Zeiss, Germany) and analysed. From each mouse, a minimum
of 2000 cells was scored for polychromatic erythrocytes (PCE) and
normochromatic erythrocytes (NCE). The frequency of micronu-
cleated polychromatic erythrocytes (mnPCE) and normochromatic
erythrocytes (mnNCE) were calculated with respect to total PCE
and NCE scored. The ratio of PCE and NCE (P/N) was also cal-
culated. Representative images of PCE, NCE and mnPCE, mnNCE
are shown in Figure 1. All slides were decoded after completion of
scoring and analysis.
Bone marrow CA assay
The procedure adopted has been reported earlier by different groups
(37–40). The necessary procedure was standardised for CA assay
and then used to assess the total chromosomal aberration (TCA) for
radioprotection by sesamol as well as melatonin in bone marrow
cells. Animals were injected i.p. with 0.2 ml of a metaphase arresting
agent (0.05% colchicine) 3 h prior to sacrifice by cervical dislocation
(24-h post-irradiation) (41) and cells were processed as described by
Preston et al. (42). Briefly, bone marrow cells were flushed out using
chilled PBS in 15-ml centrifuge tube and centrifuged at 1000 r.p.m.
for 10 min. The cell pellet was treated with pre-warmed hypotonic
solution (KCl 0.075 M) and incubated at 37°C for 40 min. The fixa-
tion was done after centrifugation at 1000 r.p.m. for 10 min. The cell
suspension was dropped on pre-chilled slides, air dried and stained
with 4% Giemsa solution. The slides were mounted by DPX with
cover slips. All slides were coded as mentioned previously to avoid
scorer bias. At least 100 metaphases per animal were scored using
×100 objective (oil) of light microscope (Primo star, Carl Zeiss,
Germany) to record various aberrations in score sheets and TCAs in
bone marrow cells were calculated. Representative images of normal
metaphase and aberrant metaphases are shown in Figure 2.

Figure 1.  Bone marrow smears stained with May–Grunewald Giemsa stain and observed under ×100 objective (oil) of light microscope. (A) Differential staining
pattern of PCE and NCE. (B and C) mnPCE. (D) NCEs containing micronuclei (mnNCE). (E) The frequency of mnPCE are shown for control, radiation (2 Gy),
sesamol 20 mg/kg (Ses 20 mg/kg), melatonin 20 mg/kg (Mel 20 mg/kg), sesamol 10 mg/kg with 2 Gy (Ses 10 mg/kg + 2 Gy), melatonin 10 mg/kg with 2 Gy (Mel
10 mg/kg + 2 Gy), sesamol 20 mg/kg with 2 Gy (Ses 20 mg/kg + 2 Gy) and melatonin 20 mg/kg with 2 Gy (Mel 20 mg/kg + 2 Gy) groups. Radiation-alone group
was compared with control (**P < 0.001), whereas sesamol 10 and 20 mg/kg plus 2 Gy significantly reduced the mnPCE frequency. Melatonin also reduced
the mnPCE frequency similar to sesamol. *P < 0.05, **P < 0.001. Both sesamol and melatonin at 20 mg/kg alone groups did not show any increase in mnPCE
frequency. Error bars on data points represent the SD of the mean (n = 5 mice/group).
654 A. Kumar et al., 2015, Vol. 30, No. 5

Figure 2.  Representative images of metaphase bone marrow cells observed under ×100 objective (oil) in light microscope; (A) normal metaphase, (B) metaphase
with chromatid-type aberrations, fragment and inter-exchange, (C) chromosome break and (D) chromosome ring and (E) the frequency of TCAs in control,
radiation (2 Gy), sesamol 10 mg/kg with 2-Gy radiation (Ses 10 mg/kg + 2 Gy), melatonin 10 mg/kg with 2-Gy radiation (Mel 10 mg/kg + 2 Gy), sesamol 20 mg/
kg with 2 Gy (Ses 20 mg/kg + 2 Gy), melatonin 20 mg/kg with 2-Gy radiation (Mel 20 mg/kg + 2 Gy) groups. Radiation-alone group increased TCA significantly
as compared with control **(P < 0.001); and pre-treatment of sesamol or melatonin at 10- and 20-mg/kg drug doses significantly decreased TCA frequency
*P < 0.05,**P < 0.001, respectively. Error bars on data points represent the SD of the mean (n = 5 mice/group).

Alkaline comet assay Cell cycle analysis

In order to quantify the DNA damage, the comet assay was per-
formed under alkaline conditions by the method described by
Singh et al. (43) with minor modifications. Briefly, one-end frosted
slides were precoated with 700  μl of 1% normal-melting-point
agarose before the experiment. Single cell suspensions were pre-
pared for bone marrow and for spleen by mincing on a bed of
ice. The cell suspension was passed through 100-µ mesh filters to
remove debris and doublets. In the case of lymphocytes, the blood
was layered on prefilled tubes containing HiSep LSM 1084 from
Hi-media India for isolation of lymphocytes according to the man-
ufacturer’s protocol. Cell count was performed using a haemocy-
tometer and the cell number was maintained at 1 × 106 cells/ml.
A  mixture of 650  μl of low-melting-point agarose (0.7%) and
50 μl of bone marrow cells suspension was coated as the second
layer. The comet slides were immersed in cold fresh lysis solution
(2.5 M NaCl, 1% N-sodium lauryl sarcosinate, 30 mM Na2EDTA,
10 mM Tris, 1% Triton X-100, 10% DMSO) for 1 h at 4°C. After
lysis, slides were placed in alkaline buffer for 20 min to allow
DNA unwinding. Slides were placed on a horizontal electrophore-
sis tank filled with freshly prepared alkaline electrophoresis buffer
(300 mM NaOH, 1 mM Na2EDTA, pH ≥ 13.0). Electrophoresis
was then carried out at 0.7 V/cm, 300 mA for 20 min. The slides
were then washed gently with 0.4 M Tris base buffer (pH 7.4)
to remove excess salts. The slides were stained with propidium
iodide (25  µg/ml) for 10 min and visualised at ×20 magnifica-
tion using automated scoring florescence microscope Metafer 4
(Zeiss, Germany) to determine the DNA migration in each groups.
Automated analysis of the comet assay was performed by the
MetaCyte Comet Scan system of Metafer 4 microscope. At least
1000 nuclei were scanned to calculate tail length, tail movement,
olive tail movement and % DNA in tail.
Bone marrow cells of animals sacrificed 24  h after irradiation
were fixed with methanol overnight. After fixing, 1 × 106 cells were
washed with PBS, and then treated with 200 µg/ml RNase at 37°C
for 30 min. Cells were washed with PBS and 50 µg/ml of propidium
iodide was added. After 20 min, cells were analysed using BD FACS
Callibur 3CB (BD Biosciences, India) for different phases of cell cycle
measurement.
Relative spleen weight measurement
Individual mice of different groups were weighed prior to sacrifice.
The spleen was excised, washed with chilled PBS, wiped with blot-
ting paper to soak surface water and weighed immediately. Relative
spleen weight (mg/g), that is, weight of spleen/body weight, was
calculated.
Haematology of mouse blood
Blood samples of different groups were collected in K2EDTA-coated
microvette tubes via cardiac puncture after cervical dislocation of
mice. Complete blood cell counts were obtained within 30 min of
blood collection using automated haematology analyser MEK-6450
Nihon Kohden, USA. The cell counts included white blood cells
(WBCs), red blood cells (RBCs), haemoglobin, platelets, % lympho-
cytes and % granulocytes.
Statistical analysis
Data presented are mean ± standard deviation (SD) of all samples
in the experiment. The TCA and MN frequencies as well as all
four parameters of alkaline comet assay of different groups were
compared using Student’s t-test and P  <  0.05 was considered as
significant.
Radiation-induced DNA damage protection, 2015, Vol. 30, No. 5 655

Results was reported by several research groups (44–46). Sesamol (10 mg/

The first part of this study was focused on assessment of cytogenetic
damage (MN and CAs) in bone marrow cells in all experimental
groups. Subsequent studies concentrated on DNA damage using the
alkaline comet assay in bone marrow cells, spleen and blood lym-
phocytes in these groups.
Sesamol-reduced radiation-induced MN in bone
marrow cells
The micronucleus frequency was determined in PCE and NCE and
P/N ratio was determined for PCE and NCE in all experimental
groups (Table 1). The results show an increase in the frequency of
mnPCE/1000 PCE (42.6 ± 1.67) in WBI group compared with the
control group (7.48 ± 1.26), whereas pre-administration of sesamol
(10 and 20 mg/kg) followed by WBI reduced this elevated frequency
to 30.5 ± 3.24 and 21.1 ± 3.43, respectively. A  similar trend was
observed with melatonin viz., mnPCE/1000 PCE were 33.8 ± 5.46
and 24.9 ± 3.46 at 10 and 20 mg/kg body weight, respectively. The
higher antioxidant dose provided additional protection. Most
importantly, neither sesamol nor melatonin caused any abnormal-
ity in bone marrow cells at these low doses as the frequency of
mnPCE/1000 PCE remained low at 6.6 ± 1.15 and 6.4 ± 1.09, respec-
tively (Table  1). Sesamol at 10 and 20 mg/kg significantly reduced
the mnPCE frequency per 1000 PCE scored in the radiation group
(P < 0.05 and P < 0.001, respectively). Melatonin also reduced the
frequency of mnPCE similar to sesamol and the difference was non-
significant (P > 0.05) as shown in Table 1. The plot in Figure 1 of
mnPCE per 1000 PCE for different groups shows the protective
effect of sesamol and melatonin.
A radiation dose of 2 Gy lowered P/N ratio from 1.17 ± 0.07 to
0.59 ± 0.02. A similar decrease in P/N ratio in γ-irradiated rodents
kg) reduced frequency of mnPCE to 30.5 ± 3.24 in comparison
with 42.6 ± 1.67 in radiation group, but the P/N ratio showed
only a marginal increase to 0.61 ± 0.03. However, sesamol at the
higher dose of 20 mg/kg has recovered P/N ratio to 0.70 ± 0.02 in
comparison with radiation group, 0.59 ± 0.02 (Table 1). An almost
similar trend was observed with melatonin. Therefore, the results
suggest that these doses of sesamol or melatonin provide protec-
tion in bone marrow cells
TCAs decreased by sesamol
Radiation induces two types of aberrations in bone marrow cells of
mice: chromatid type and chromosomal type. In Table 2, the detailed
observations of different aberrations are shown for different groups.
Twenty-four hours post-WBI with 2 Gy increased the break for-
mation and fragments in chromatids of bone marrow cells, 0.085
break/cell and 0.482 fragment/cell, in comparison with the control
group, 0.016 break/cell and 0.087 fragment/cell, respectively. Pre-
treatment with sesamol (20 mg/kg), followed by WBI, reduced both
chromatid-type aberrations, 0.067 break/cell and 0.214 fragment/
cell. Similarly, melatonin (20 mg/kg) also reduced radiation-induced
chromatid-type aberrations, 0.041 break/cell and 0.297 fragment/
cell. WBI increased chromosomal-type aberrations; 0.059 ring/cell
and 0.103 acentric/cell, compared with the control (0.002 ring/cell
and 0.054 acentric/cell). Pre-treatment with sesamol (10 and 20 mg/
kg) followed by radiation lowered this to 0.038 and 0.020 ring/cell
as well as 0.073 and 0.071 acentric/cell, respectively. Melatonin has
also shown comparable protection against radiation: 0.042 and
0.033 ring/cell and 0.106 and 0.088 acentric/cell, at 10 and 20 mg/
kg, respectively (Table 2). Both sesamol and melatonin significantly
reduced radiation-induced TCA. Sesamol (20 mg/kg) significantly
lowered TCA/cell up to 0.515 ± 0.032 from 0.889 ± 0.047 in the

Table 1.  Micronucleus frequency in PCEs and NCEs in bone marrow of different grouped C57Bl/6 mice presented as mean ± SD

Groups PCE mnPCE/1000 PCE ± SD NCE mnNCE/1000 NCE ± SD P/N ratio ± SD

Control 6276 7.48 ± 1.26 5369 5.8 ± 0.60 1.17 ± 0.07

Ses 20 mg/kg 5161 6.6 ± 1.15 8719 7.1 ± 0.39 1.17 ± 0.06
Mel 20 mg/kg 4201 6.4 ± 1.09 6970 7.5 ± 1.29 1.14 ± 0.04
2 Gy 5503 42.6 ± 1.67 7562 9.9 ± 2.29 0.59 ± 0.02
Ses 10 mg/kg + 2 Gy 5247 30.5 ± 3.24 8599 10.3 ± 1.14 0.61 ± 0.03
Mel 10 mg/kg + 2 Gy 4306 33.8 ± 5.46 6103 9.3 ± 0.13 0.60 ± 0.13
Ses 20 mg/kg + 2 Gy 5460 21.1 ± 3.43 4770 7.9 ± 1.76 0.70 ± 0.02
Mel 20 mg/kg + 2 Gy 6073 24.9 ± 3.46 5196 9.1 ± 1.67 0.72 ± 0.04

Table 2.  Distribution of various chromosomal and chromatid aberrations and TCAs in bone marrow of C57Bl/6 mice in different groups

Groups Cells Chromatid-type aberrations/cell Chromosome-type aberrations/cell TCA/cell ± SD

scored
Break Inter- Frag- Ring Break Acen- Double
exchange ment tric minute

Control 551 0.016 0.018 0.087 0.002 0.025 0.054 0.007 0.211 ± 0.020
2 Gy 506 0.085 0.076 0.482 0.059 0.024 0.103 0.067 0.889 ± 0.047
Ses 10 mg/kg 576 0.076 0.071 0.356 0.038 0.045 0.073 0.073 0.733 ± 0.036
+ 2 Gy
Mel 10 mg/ 639 0.061 0.066 0.388 0.042 0.039 0.106 0.036 0.739 ± 0.034
kg + 2 Gy
Ses 20 mg/kg 505 0.067 0.071 0.214 0.020 0.034 0.071 0.038 0.515 ± 0.032
+ 2 Gy
Mel 20 mg/ 512 0.041 0.027 0.297 0.033 0.027 0.088 0.055 0.568 ± 0.033
kg + 2 Gy
656
radiation-alone group (P  <  0.001; Figure  2). Interestingly, sesamol
and melatonin showed similar extents of reduction of TCA in bone
marrow. This trend remained the same at the lower dose of 10 mg/
kg. These reductions in TCA with reference to the radiation group
were significant (P < 0.05; Figure 2).
Cell cycle analysis
The analysis of cell cycle proliferation has shown a reduction of
radiation-induced % apoptotic cell population in bone marrow cells
suggesting the lowering in population of apoptotic cells in sesamol-
administered radiation groups. Both the antioxidants sesamol and
melatonin 20 mg/kg groups shown higher cell percentage in S-phase
compared with control group animals facilitating cell proliferations.
Radiation reduced the G0/G1 population, whereas pre-administra-
tion of sesamol or melatonin protected to reduce these populations
(Figure 3).
Alkaline comet assay
γ Radiation causes both DNA double-strand and single-strand
breaks leading to DNA fragments in bone marrow cells of mice, and
on electrophoresis, these migrate towards the positive electrode and
acquire a shape of a comet. The results of the comet assay in bone
marrow cells, splenocytes and lymphocytes in different groups are
represented by % DNA in tail along with other parameters. These are
tail length, tail movement and olive tail moment. Radiation alone has
increased % DNA in tail (40.5 ± 5.1) compared with untreated con-
trol group (2.7 ± 0.8) in bone marrow cells (Figure 4). Pre-treatment
of sesamol (20 mg/kg) lowered the radiation-induced % DNA in tail
to 7.7 ± 1. Similar protective effects of sesamol (20 mg/kg) were also
observed in splenocytes and lymphocytes. The % DNA in tail in sple-
nocytes and lymphocytes was 13.8 ± 1.0 and 8.9 ± 0.5 compared with
the radiation group 29.3 ± 5.2 and 16.3 ± 4.6, as shown in Figures 5
Figure 3.  Histogram representation of cell distribution in mice bone marrow of different groups. Sesamol attenuated the radiation-induced alteration in cell
cycles in bone marrow cells of mice after 24 h of irradiation. Data represent percentage of apoptotic cells, G0/G1 cells, S phase cells and G2/M cells. **P < 0.001
(n = 5 mice/group).
A. Kumar et al., 2015, Vol. 30, No. 5
and 6, respectively. The values of other parameters (tail length,
tail moment and olive tail moment) also showed similar patterns.
Sesamol treatment has shown protection in all these systems studied,
namely bone marrow, spleen and lymphocytes. Statistical analysis
showed that reductions in all four parameters of the comet assay
when compared with radiation group were significant (P  <  0.05).
Melatonin has also shown a similar level of protection to sesamol
(P < 0.05; Figures 4–6). Therefore, the results strongly suggest that
both antioxidant molecules protect against DNA damage in bone
marrow cells as well as spleen and lymphocytes.
Relative spleen weight
Organ weight is an important marker for primary screening of toxic-
ity of molecules. This parameter is an important indicator of func-
tionality of organ. Spleen is the important haematopoietic organ after
bone marrow and also the largest immunological organ where matu-
ration of cells takes place. Radiation is known to decrease the survival
of splenocytes that lead to decrease the size and weight of spleen (47–
49).The relative spleen weight was calculated by using spleen weight
and body weight of the each animal of different groups. Radiation
(2 Gy) significantly decreased the relative spleen weight compared
with control group (P < 0.001), whereas both sesamol and melatonin
(20 mg/kg) facilitated significant recovery of radiation-induced spleen
weight (P < 0.05) expressed as relative spleen weight (Figure 7).
Haematology
Drastic decrease was observed in WBCs count of mice
1.49 ± 0.5 × 103/µl after 24  h of WBI compared with control
5.80 ± 0.9 × 103/µl, whereas pre-administration of sesamol 20 mg/kg
aided to recover radiation-induced decrease in WBCs. A significant
increase was observed in % granulocytes in sesamol and melatonin
20 mg/kg alone groups compared with control group (P < 0.05). This
Radiation-induced DNA damage protection, 2015, Vol. 30, No. 5 657

Figure 4.  Effect of sesamol and melatonin on DNA damage in bone marrow cells of mice exposed to 2-Gy whole-body γ radiation in terms of decrease in comet
parameters: tail length, tail moment, olive tail moment and % DNA in tail are significantly differentiated, **P < 0.001. Error bars on data points represent the SD
(n = 3 mice/group).

Figure 5.  Effect of sesamol and melatonin in DNA damages on splenocytes of mice exposed to 2-Gy whole-body γ radiation in terms of decrease in comet
parameters: tail length, tail moment, olive tail moment and % DNA in tail are significantly differentiated (*P < 0.05, **P < 0.001). Error bars on data points
represent the SD (n = 3 mice/group).

increase was also observed in sesamol 20 mg/kg + 2-Gy group. No
difference was observed in counts of RBCs, haemoglobin and plate-
lets after irradiation at 24 h (Figure 8).
Discussion
Whole-body exposure of low ionising radiation cause damages
in bone marrow cells (50,51). Bone marrow is responsible for
peripheral blood cell lineages derived from their haematopoietic
stem cells. Its protection and recovery after damage is very important
for survival and quality of life. Single dose of radioprotector prior to
planned exposure is envisaged to reduce radiation-induced toxicities
in organs and systems. Radiation injuries to haematopoietic system
are of concern for efficacy of effective radioprotector. Damages to
genetic material at sublethal radiation dose can have radiobiological
consequences including genotoxicity in bone marrow. Antioxidant
658 A. Kumar et al., 2015, Vol. 30, No. 5

Figure  6.  Effect of sesamol and melatonin in DNA damages on peripheral blood lymphocytes of mice exposed to 2-Gy whole-body γ radiation in terms of
decrease in comet parameters: tail length, tail moment, olive tail moment and % DNA in tail are significantly differentiated (*P < 0.05, **P < 0.001). Error bars
on data points represent the SD (n = 3 mice/group).

Figure 7.  Relative spleen weight of radiation group decreased significantly relative to control group (**P < 0.001); sesamol or melatonin 20 mg/kg with radiation
groups increased the relative spleen weight significantly than radiation-alone group (*P < 0.05). The different groups were the same as in Figure 1. Error bars on
data points represent the SD (n = 5 mice/group).

treatment is known to increase antioxidant capacity of organs, a
number of studies have demonstrated radioprotective efficacy in bone
marrow of irradiated mice. The radioprotective effect of melatonin
was first suggested by Blickenstaff et al. (52) in 1994. Melatonin as
radioprotector was first investigated by Vijayalaxmi et al. using ex
vivo human peripheral blood and in vivo survival studies. The obser-
vation on ability of melatonin to reduce CA and MN frequency in
cultured human blood lymphocytes and animal survivals have been
remarkable landmark for radioprotector research (53,54). Studies
in animal models with melatonin and other antioxidants have been
reported using high doses, and therefore, toxicity becomes a major
impediment in translating leads into radioprotector development.
In this study, the radioprotective effects of sesamol in bone mar-
row cells using cytogenetic assays for CAs, MN and alkaline comet
assay at low dose in C57Bl/6 male mice were investigated. Sesamol
at 20 mg/kg body weight reduced radiation-induced MN frequency
and CAs in bone marrow cells by 50% and 57%, respectively, in
comparison with radiation-alone group (Figures 1 and 2). In an ear-
lier report by Badr et al. (44), melatonin at 10 mg/kg exhibited an
increase in MN frequency in PCE of bone marrow and CA in sper-
matocytes. No such increase was observed in this study with either
sesamol or melatonin even at relatively higher dose of 20 mg/kg sug-
gesting no genotoxicity in bone marrow cells. DNA strand breaks if
not repaired can lead to cell death via apoptosis and other modes.
Analysis of cell cycle proliferation of bone marrow cells suggests that
both sesamol and melatonin protect the radiation-induced apoptosis
and facilitate cell proliferation (Figure 3.). The relative contribution
from S phase was found to be more in sesamol- and melatonin-alone
Radiation-induced DNA damage protection, 2015, Vol. 30, No. 5
Figure 8.  Haematological parameters of mice after 24 h of 2-Gy γ-irradiation. Error bars on data points represent the SD (n = 6 mice/group).
groups. In comet assay, the decrease in percent of DNA in tail of
bone marrow cells, spleen as well as lymphocytes clearly shows the
radioprotective effect of sesamol in irradiated mice. On analysis of
the comet data, it has been observed that the sesamol dose of 20 mg/
kg reduced radiation-induced DNA damage in all comet parameters
and extent of protection in near about unirradiated control. Further
comparison with melatonin, the extent of radioprotective efficacy
of sesamol was found to be similar or marginal better in bone mar-
row, spleen and lymphocytes. Pre-administration of sesamol 20 mg/
kg reduced radiation-induced % DNA in tail and other comet assay
parameters nearly up to control values, suggesting that the amount
of dose used was sufficient to protect bone marrow, spleen and lym-
phocytes from radiation-induced genotoxicity. Hence, the results
suggests that sesamol is effective against radiation-induced cytoge-
netic damages in bone marrow and other organs at low dose.
Sesamol and melatonin treatments have increased granulocyte
counts at 24-h post-irradiation. Earlier studies have reported similar
increase in granulocytes by melatonin treatments (55). Melatonin
has role in regulating granulocyte and immune response and act as
immunomodulator. Melatonin appears to have, in general, a positive
role in regulating granulocytes and B lymphocytes in mice (56,57).
Thus, similar observations of sesamol on enhancement of granulocyte
count are important (Figure 8). Sesamol is an exogenous molecule and
therefore may not have intracellular functions similar to melatonin,
but the observation of almost equal extent of protection in bone mar-
row cells and other organs of haematopoietic relevance suggest the
possibility of direct scavenging of free radicals as major role. Sesamol
also facilitates proliferation of bone marrow cells as observed in cell
659
cycle analysis (Figure 3). Melatonin receptors exists in cells and help in
a variety of physiological responses through these receptors in mem-
brane and nucleus. Melatonin is known to regulate the antioxidant
enzymes and immune system through binding with nuclear receptor
(58), whereas, no such process has been reported for sesamol. Despite
this, sesamol showed similar efficacy. Therefore, similar observations
with melatonin are important and need to be investigated further.
Recovery of spleen weight is another marker for radioprotective
efficacy in the haematopoietic system. Thus, observation of recovery
of spleen weight comparable with melatonin is an important aspect.
Besides radioprotective properties, sesamol also has pro-apoptotic
properties in cancer cells and therefore can be exploited further
for development as a radioprotector using low drug dose for can-
cer radiotherapy applications. This study also clearly showed that a
single dose of sesamol is distributed in these organs and conferred
radioprotection. These findings are important and will be useful for
the development of a radioprotector for countering radiation effects.
Funding
The work was supported by Defence Research & Development
Organisation, Government of India (NBC 1.29).
Acknowledgements
A.K. is thankful to DRDO for a fellowship. The authors are thankful to Dr
P.K. Aggarwal for providing technical guidance in micronuclei assay and to
Dr B.G. Roy and Ms Anjali Sharma for providing experimental animals and
660
γ-irradiation facility. Mrs Namita Kalra is acknowledged for flow cytometry
measurements.
Conflict of interest statement: None declared.
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