Sesamol attenuates genotoxicity in bone marrow cells of whole-body γ-irradiated mice

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 radiationinduced 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 wholebody 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 Ionising radiation exposure is harmful and consequences on health depend on the extent of damage to DNA. The haematopoietic system is the most radiosensitive and in the case of whole-body exposure can lead to haematopoietic syndrome with increasing radiation dose. Damage to DNA, genotoxicity and related short- and long-term effects on health are well documented (1 3). Antioxidants that have the ability to scavenge free radicals, can protect DNA and facilitate cell functioning are considered for investigating radioprotection and underlying mechanisms in animal models. A single prophylactic dose of a radioprotector is envisaged to provide protection against whole-body exposure. Because of the complex nature of the response to radiation in cells and tissues, the much desired radioprotector is still not available. The only compound approved by Food and Drug Administration, USA, as cytoprotectant for head and neck cancer radiotherapy patients is amifostine, developed at Walter Reed Institute, USA (4). Different approaches are currently being pursued in various institutes (5 9). Antioxidants have gained importance because of their strong free radical scavenging properties, low toxicity and multiple roles at cellular level. In general, most of the studies in animal models The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 651

652 A. Kumar et al., 2015, Vol. 30, No. 5 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 radioprotector applications. A radioprotector is required for persons undertaking 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 different applications areas, low-dose radiation is usually expected and therefore investigations with potential radioprotectors in animal models at low radiation doses are necessary for developing strategies. Sesamol (3,4-methylenedioxyphenol) is a nutritional phenolic antioxidant compound present in sesame seeds and has medicinal properties (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 direction. Sesamol was investigated for antioxidant properties using pulse radiolysis and its mechanism was revealed (17). In view of these promising 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 marrow cells of C57BL/6 mice exposed to 2-Gy whole-body irradiation (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 circulation. 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 singlestrand 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 detection 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 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 workers 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 radioprotective efficacy of sesamol and melatonin in WBI mice against lowdose (2 Gy) radiation-induced early damage in bone marrow cells. Materials and Methods Chemicals and reagents Melatonin, sesamol, colchicine, Giemsa powder, soybean oil, potassium chloride, DPX mountant solution, normal-melting-point agarose, 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 experimental animal facility of the Institute of Nuclear Medicine & Allied Sciences (INMAS), Delhi, India. Mice were housed in cages under optimum 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 protocols 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 treatments. 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 intraperitoneal (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 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. 653 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 micronucleated 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 calculated. Representative images of PCE, NCE and mnpce, mnnce are shown in Figure 1. All slides were decoded after completion of scoring and analysis. γ-irradiation Animals were exposed to whole-body γ- rays (60Co) with 2-Gy radiation 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, 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 fixation 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 In order to quantify the DNA damage, the comet assay was performed 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 prepared 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 manufacturer s protocol. Cell count was performed using a haemocytometer and the cell number was maintained at 1 10 6 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 Na 2 EDTA, 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 electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer (300 mm NaOH, 1 mm Na 2 EDTA, 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 magnification 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. Cell cycle analysis Bone marrow cells of animals sacrificed 24 h after irradiation were fixed with methanol overnight. After fixing, 1 10 6 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 blotting 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 K 2 EDTA-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, % lymphocytes 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 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 lymphocytes 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 abnormality 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, respectively (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 nonsignificant (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 was reported by several research groups (44 46). Sesamol (10 mg/ 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 protection 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 formation 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. Pretreatment 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 scored Chromatid-type aberrations/cell Chromosome-type aberrations/cell TCA/cell ± SD Break Interexchange Fragment Ring Break Acentric Double 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/ kg + 2 Gy 512 0.041 0.027 0.297 0.033 0.027 0.088 0.055 0.568 ± 0.033

656 A. Kumar et al., 2015, Vol. 30, No. 5 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 sesamoladministered 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-administration 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 control 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 splenocytes 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 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 toxicity of molecules. This parameter is an important indicator of functionality of organ. Spleen is the important haematopoietic organ after bone marrow and also the largest immunological organ where maturation 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 10 3 /µl after 24 h of WBI compared with control 5.80 ± 0.9 10 3 /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 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).

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 platelets 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 observation 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 marrow 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 earlier 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 spermatocytes. No such increase was observed in this study with either sesamol or melatonin even at relatively higher dose of 20 mg/kg suggesting 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. 5 659 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 marrow, 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 lymphocytes from radiation-induced genotoxicity. Hence, the results suggests that sesamol is effective against radiation-induced cytogenetic 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 marrow 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 cycle analysis (Figure 3). Melatonin receptors exists in cells and help in a variety of physiological responses through these receptors in membrane 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 cancer 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

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